WO2002102976A2 - Mutations in the bcr-abl tyrosine kinase associated with resistance to sti-571 - Google Patents

Mutations in the bcr-abl tyrosine kinase associated with resistance to sti-571 Download PDF

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WO2002102976A2
WO2002102976A2 PCT/US2002/018729 US0218729W WO02102976A2 WO 2002102976 A2 WO2002102976 A2 WO 2002102976A2 US 0218729 W US0218729 W US 0218729W WO 02102976 A2 WO02102976 A2 WO 02102976A2
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abl
bcr
wherem
kmase
residue
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WO2002102976A3 (en
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Charles L. Sawyers
Mercedes E. Gorre
Neil Pravin Shah
John Nicoll
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Priority to AU2002345670A priority Critical patent/AU2002345670A1/en
Priority to AT02744318T priority patent/ATE431417T1/en
Priority to DE60232370T priority patent/DE60232370D1/en
Priority to CA2450473A priority patent/CA2450473C/en
Priority to EP02744318A priority patent/EP1466174B1/en
Priority to JP2003506431A priority patent/JP4444651B2/en
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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Definitions

  • the invention described herein relates to novel genes and their encoded proteins, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express diem
  • Cancers are characterized by multiple oncogenic events that collectively contribute to the phenotype of advanced stage disease. With the advent of new drugs that target specific molecular abnormalities, it is important to know whether the initial oncogenic event continues to play a functional role at later stages of tumor progression and at relapse with the development of chemodierapy resistance This question has been addressed in transgenic mice through regulated expression of the initial oncogene In three models testing different oncogenes in different tissues, the primary oncogene was required to maintain the tumor phenotype, despite the presence of numerous additional oncogene and tumor suppressor mutations (see, e.g. L. Chin et al, Nature 400, 468 (1999); D. W. Felsher et al., Mol.
  • CML is a pluripotent hematopoietic stem cell disorder characterized by the Philadelphia (Ph) chromosome translocation (see, e.g. C. L Sawyers, JV. Engl. f. Med. 340, 1330 (1999); and S. Fade et al, N. Engl. J. Med. 341, 164 (1999)).
  • the resulting BCR- ABL fusion gene encodes a cytoplasmic protein with constitutive tyrosine kinase activity (see, e g. J. B. Konopka et al., Proc. Natl. Acad. S t. U.SA. 82, 1810 (1985) and NCBI Accession NP__067585).
  • BCR-ABL is an oncogene and is sufficient to produce CML-like disease in mice (see, e.g. G. Q. Daley et al , Science 247, 824 (1 90); and N Heisterkamp et al., Nature 344, 251 (1990)).
  • CML progresses through distinct clinical stages. The earliest stage, termed chronic phase, is characterized by expansion of terminally differentiated neutrophils. Over several years die disease progresses to an acute phase termed blast crisis, characterized by maturation arrest with excessive numbers of undifferentiated myeloid or lymphoid progenitor cells.
  • the BCR-ABL oncogene is expressed at all stages, but blast crisis is characterized by multiple additional genetic and molecular changes.
  • STI-571 is a 2-phenylam ⁇ no pyrimidine that targets the ATP- binding site of the kinase domain of ABL (see, e.g. B. J.
  • the disclosure provided herein characterizes specific events associated widi such drug resistance by identifying specific domains within protein l ⁇ nases where amino acid mutations occur d at impart resistance to the kinase inhibitor yet allow the kinase to retain its biological activity.
  • the disclosure provided herein further identifies diese regions as domains shown to be highly conserved among families of protein ktnases (e.g the c-Abl tyrosine kinase activation loop). Consequendy dus disclosure identifies those specific regions in protein l ⁇ nases that are to be analyzed in a variety of diagnostic protocols which examine drug resistance.
  • the invention described herein further includes novel genes and their encoded proteins expressed in cancer cells d at are associated with resistance to STI-571.
  • disease STI-571 resistant genes and their encoded proteins are mutants of Bcr- Abl, an oncogene that is expressed in chrome myeloid leukemias.
  • the invention described herein discloses a number of Bcr-Abl Mutants Associated with Resistance to STI-571 (hereinafter these mutants are collectively described using the acronym "MARS”), as well as diagnostic and dierapeutic methods and compositions useful in the management of cancers that express these mutants.
  • MARS Bcr-Abl Mutants Associated with Resistance to STI-571
  • a typical example of a MARS is a Bcr-Abl mutant having a single amino acid substitution in a Thr residue at position 315 of the Abl kinase (termed T315I Bcr-Abl)
  • T315I Bcr-Abl a residue in the Abl kinase domain known to form a critical hydrogen bond with this drug.
  • Biochemical analyses of this mutant show that the Thr— »Ile change is sufficient to confer STI-571 resistance in a reconstitution experiment. Additional MARS are identified in Tables I provided below. The disclosure provided herein presents evidence that genetically complex cancers retain dependence on an initial oncogenic events and provides a strategy for identifying inhibitors of STI-571 resistance.
  • the disclosure provided herein further provides for a variety of diagnostic methods for examining the characteristics of cancers such as chronic myeloid leukemia
  • All poor knowledge of the Bcr-Abl tyrosine kinase is based on published sequence that has been in the public domain for >15 years.
  • the invention provides novel sequences of DNA of die Bcr-Abl tyrosine kinase fusion protein that causes chrome myeloid leukemia (CML), which is present in a high fraction of patients who develop resistance to d e drug STI-571, which is soon to become standard of care for the treatment of CML.
  • CML chrome myeloid leukemia
  • T315I Bcr-Abl provides a representative example of the inventions provided by the MARS disclosed herein.
  • the T315I Bcr-Abl mutant disclosed herein contains an amino acid change in the kinase domain of Bcr-Abl that inhibits ST1571 binding to Bcr- Abl.
  • the T315I Bcr-Abl embodiment of the invention has been tested in a number of patient samples and confirmed at the sequence level.
  • This mutant Bcr-Abl protein has been expressed in cells and shown to be resistant to STI-571. Therefore, patients develop resistance to the drug because it can no longer inhibit its kinase activity.
  • the invention provided herein provides diagnostic tests for early relapse in CML as well as for drug development in the field of tyrosine kinase inhibitors.
  • the disclosure provided herein allows one to detect the presence of drug resistant cells in CML patients prior to relapse, using, for example, PCR based assays.
  • Representative embodiments of d e invention include PCR and analogous assays that are used to detect resistant cells in patient blood samples.
  • the invention can also be practiced as a tool to identify molecules which bind and/or inhibit the mutant tyrosine kinases.
  • a typical embodiment of this aspect of d e invention is a method of identifying a compound which specifically binds to a mutant protein kinase such as a Bcr-Abl mutant shown in Table I by contacting the mutant with a test compound under conditions favorable to binding; and then determining whether said test compound binds to the mutant so d at a compound wliich binds to the mutant is identified.
  • a mutant protein kinase such as a Bcr-Abl mutant shown in Table I
  • the invention provides polynucleotides corresponding or complementary to all or part of the MARS genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding MARS proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to the MARS genes or mRNA sequences or parts thereof, and polynucleotides or ohgonucleotides that hybridize to die MARS genes, mRNAs, or to MARS-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding MARS.
  • MARS proteins and polypeptide fragments thereof are also provided.
  • the invention further provides MARS proteins and polypeptide fragments thereof.
  • the invention further provides antibodies that bind to MARS proteins and polypeptide fragments diereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled widi a detectable marker.
  • the invention furdier provides methods for detecting the presence and status of
  • MARS polynucleotides and proteins in various biological samples as well as methods for identifying cells that express MARS.
  • a typical embodiment of this invention provides methods for monitoring MARS gene products in a tissue sample having or suspected of having some form of growd dysregula ⁇ on such as cancer.
  • One preferred embodiment of the invention is a method of identifying a mutant
  • Abelson tyrosine kinase expressed by a cell by determining a nucleotide sequence of a portion of d e catalytic domain of the Abelson tyrosine kinase expressed by the cell and then comparing the nucleotide sequence so determined to that of the wild type sequence of the catalytic domain of the Abelson protem tyrosine kmase to identify the presence of a mutation within the catalytic domam, wherein the mutation so identified has d e characteiistics of occurrmg at a amino acid residue located widim the polypeptide sequence of the Abelson protein tyrosine kinase at a amino acid residue diat has homology to an amino acid position in a Bcr-Abl kmase shown m SEQ ID NO: 1 diat is associated with a resistance to an inhibition of tyrosine kmase activity by a 2- phenylarmnopy ⁇ rnidine, wherem
  • Anod er embodiment of d e invention is an isolated Bcr-Abl polypeptide comprising an ammo acid sequence which differs from the sequence of the Bcr-Abl of SEQ ID NO:l and has one or more amino acid substitutions at the residue position(s) m SEQ ID NO:l selected from the group consisting of: D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291 , E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311 , 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328,
  • a related embodiment of the invention is an isolated nucleic acid compnsmg a nucleotide sequence encoding the Bcr-Abl polypeptide.
  • Other embodiments of the invention is a vector compnsmg this nucleic acid sequence, a host cell compnsmg such vectors (e.g. E.
  • Bcr-Abl polypeptide variant polypeptide compnsmg the steps of: providmg a host cell compnsmg such a vector; (b) providmg culture media; (c) culturmg the host cell m the culture media under conditions sufficient to express die Bcr-Abl polypeptide vanant polypeptide; (d) recovering the Bcr- Abl polypeptide variant polypeptide from the host cell or culture media; and (e) purifying die Bcr-Abl polypeptide vanant polypeptide.
  • Yet another embodiment of the invention is a Bcr-Abl polypeptide vanant polypeptide that is chemically modified or con j ugated or linked to a matrix or a heterologous protem.
  • the invention furdier provides vanous therapeutic compositions and strategies foi treating cancers that express MARS, including mediods for identifying molecules (e.g STI- 571 analogs) which inhibit the biological activities (e.g. kmase activity) of vanous MARS.
  • identifying molecules e.g STI- 571 analogs
  • biological activities e.g. kmase activity
  • FIG. 1 Clinical relapse of STI-571-treated patients is associated with persistent Bcr-Abl kmase activity.
  • A Immunoblot analyses of one CML patient's bone marrow cells after a 2-hour incubation with different concentrations of STI-571 in vitro Whole cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with Crkl (top panel), phosphotyrosme (middle panel), and Abl (bottom panel) antibodies.
  • Ph-positive cell l ne, K562 was used as a positive control for autophosphorylated Bcr-Abl.
  • D Crkl lrnmunoblots of cell lysates from relapse patients taken prior to (pre-Tx) and during the course of (Tx and relapse) STI-571 therapy. Densitometnc analyses of Crkl lmmunoblots (expressed as percentage of phosphorylated Crkl over total Crkl protem) are presented in bar graphs.
  • Figure 2 Altered sensitivity of relapsed patient cells to STI-571.
  • FIG. 3 BCR-ABL amplification m patients who relapsed after an initial response to STI-571.
  • B BCR-ABL FISH analyses of interphase nuclei from blast cnsis patient M14 pnor to, durmg, and after removal from STI-571 dierapy showmg BCR-ABL-amph.&ed phenotype and reversion to non-amphfied phenotype upon removal from STI-571 therapy.
  • Bar 10 ⁇ .
  • FIG. 4 Pomt mutation m die ATP-binding pocket of d e Abl kinase domam confers STI-571 resistance in relapsed patients.
  • A Schematic of PCR strategy to determine the sequence of a 578 base pair region of BCR-ABL that corresponds to the ATP-binding pocket and activation loop of the kinase domain m patient samples. Ammo acid sequence of the region of Abl analyzed is shown m black. Residues predicted to form hydrogen bonds with STI-571, based on crystal structure data, are m boldface and are numbered from the first ammo acid of c-Abl (GenBank accession number- Ml 4752, shown in Table II) (SEQ ID NO: 1).
  • Corresponding nucleotide sequence (shown in red) was aligned with sequences obtamed from nine patient cDNAs.
  • the C— T mutation at ⁇ LB nucleotide 944 (detected in six patients at relapse and m no pre-treatment samples) is shown in blue.
  • Sequence of wild-type ABL exon 3 (GenBank accession number: NT008338.2) was aligned with sequences obtamed from patient genomic DNA pnor to treatment and at relapse. Examples of primary sequence data (represented as chromatographs) from wdd-type BCR-ABL (left) and BCR-ABL widi the C— T pomt mutation (right).
  • Ba/F3 cells expressmg wild-type, T315I, or E255K BCR-ABL were mcubated m the presence of mcreasmg concentrations of geldanamycm (GA) for 24 hours Immunoblottmg of cell lysates was performed with anti-ABL (Ab3, Oncogene) (upper panels), ant ⁇ -RAF-1 (Santa Cruz) (middle panels), and anti-actin (ac-15, Sigma) as a control for protem loadmg (lower panels).
  • CRKL when tyrosine- phosphorylated, migrates more slowly on SDS-PAGE resulting m an upper band representing phosphorylated CRKL (P-CRKL) and a lower band representing non- phosphorylated CRKL.
  • D Densitometnc analysis of CRKL immunoblot shown m
  • C usmg ImageQuant software (Molecular Dynamics). Quantified CRKL phosphorylation is expressed as percentage of phosphorylated CRKL over total CRKL protem (% P- CRKL).
  • Abl cDNA is represented with Bcr sequences stippled, and Abl sequences m black. Horizontal arrows represent PCR primers.
  • Initial PCR results in amplification of a 1.3 kb Bcr-Abl subfragment which serves as template for a second round PCR of the kmase domam which is then subcloned.
  • Ten mdependent clones per patient time pomt were sequenced. Sequence deviations from wild-type Bcr-Abl observed m at least two of ten clones were considered mutations.
  • Bcr-Abl kinase domam mutants exhibit varying degrees of biochemical and biologic resistance to STI-571.
  • Western blot usmg an anti- phosphotyrosine antibody (4G10) of lysates prepared from Ba/F3 populations mfected with retroviruses expressmg the Bcr-Abl isoforms mdicated and grown m the absence of IL-3 were exposed to varying concentrations of STI-571 for two hours are shown.
  • Biochemical IC-50s for each of the mutations is shown.
  • Biologic IC-50s were determined by viable cell count of cells after 48 hours of STI-571 exposure.
  • FIG. 11 Summary of STI-571 -resistant Bcr-Abl kmase domam mutations. Each letter represents a patient with m whom the corresponding mutation was detected. Chrome phase patients are represented by the letter “C.” Relapsed myeloid blast crisis patients are mdicated by the letter “M.” Patients with relapsed lymphoid blast cnsis are represented by the letter “L.” “R” indicates mutations pnor to STI-571 treatment m patients with myeloid blast cnsis who were refractory to treatment. Note that kmase domam is not drawn to scale
  • polynucleotide means a polymeric form of nucleotides of at least about 10 bases or base pairs m length, either nbonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include smgle and double stranded forms of DNA
  • polypeptide means a polymer of at least about 6 ammo acids. Throughout the specification, standard three letter or smgle letter designations for ammo acids are used. As used herem, a polynucleotide is said to be “isolated” when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than, for example, the MARS genes or that encode polypeptides other than
  • MARS gene product or fragments thereof As used herem, a polypeptide is said to be
  • isolated when it is substantially separated from contaminant polypeptide that correspond to polypeptides odier than the MARS polypeptides or fragments thereof.
  • a skilled artisan can readily employ polynucleotide or polypeptide isolation procedures to obtain an isolated polynucleotides and polypeptides.
  • d e terms “hybndize”, “hybndizmg”, “hybndizes” and the like, used m d e context of polynucleotides are meant to refer to conventional hybndization conditions, preferably such as hybndization m 50% formam ⁇ de/6XSSC/0.1% SDS/100 ⁇ g/ml ssDNA, m which temperatures for hybridization are above 37 degrees C and temperatures for washing m 0.1X SSC/0.1% SDS are above 55 degrees C, and most preferably to stringent hybndization conditions.
  • “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, wlender shorter probes need lower temperatures.
  • Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present m an environment below their melting temperature.
  • the higher d e degree of desired homology between the probe and hybridizable sequence the higher the relative temperature that can be used. As a result, it follows d at higher relative temperatures would tend to make d e reaction conditions more stringent, while lower temperatures less so.
  • stringency of hybridization reactions see Ausubel et al, Current Protocols m Molecular Biology, Wiley Interscience Publishers, (1995).
  • “Stringent conditions” or “high stringency conditions”, as defined herem, may be identified by d ose that. (1) employ low lomc strength and high temperature for washing, for example 0.015 M sodium chlonde/0.0015 M sodium c ⁇ trate/0.1% sodium dodecyl sulfate at 50°C; (2) employ durmg hybndization a denaturing agent, such as formamide, for example, 50% (v/v) formamide widi 0 1% bovme serum albumm/0.1% Ficoll/0.1% polyvmylpyrrokdone/50mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chlonde, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0 1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated
  • Modely stringent conditions may be identified as descnbed by Sambrook et al , 1989, Molecular Clomng: A Laboratory Manual, New York- Cold Spring Harbor Press, and mclude the use of washing solution and hybndization conditions (e.g., temperature, ionic strength and %SDS) less stringent d an those descnbed above.
  • washing solution and hybndization conditions e.g., temperature, ionic strength and %SDS
  • moderately stringent conditions is overnight incubation at 37 ⁇ C m a solution compnsmg: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters i x SSC at about 37-50°C.
  • the skilled artisan will recognize how to adjust the tempeiature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
  • identity is used to identify and express the percentage of ammo acid residues at the same relative positions that are the same
  • homoology is used to identify and express the percentage of ammo acid residues at the same relative positions that are either identical or are similar, usmg d e conserved ammo acid cntena of BLAST analysis, as is generally understood m the art.
  • identity and homology values may be generated by WU-BLAST-2 (Altschul et al., Methods Enzymology, 266: 460-480 (1996): http://blast.wusd/edu/blast/ README.html).
  • Percent (%) ammo acid sequence identity with respect to the sequences identified herem is defined as the percentage of amino acid residues in a candidate sequence that are identical with the am o acid residues m the BCR-ABL sequence, after aligning the sequences and introducing gaps, if necessary, to achieve die maximum percent sequence identity. Alignment for purposes of determining percent ammo acid sequence identity can be achieved m various ways that are within d e skill m d e art can determine appropriate parameters for measuring alignment, including assigning algorithms needed to achieve maximal alignment over the full-length sequences bemg compared.
  • percent ammo acid identity values can also be obtamed usmg the sequence companson computer program, ALIGN-2, d e source code of which has been filed widi user documentation m the US Copyright Office, Washington, DC, 20559, registered under the US Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, CA. All sequence comparison parameters are set by the ALIGN-2 program and do not vary
  • the terms "cancer", “cancerous”, or “malignant” refer to or describe the physiological condition m mammals that is typically characterized by unregulated cell growth.
  • cancer examples include but are not limited to, leukemia, lymphoma, blastoma, carcmoma and sarcoma. More particular examples of such cancers mclude chrome myeloid leukemia, acute lymphoblastic leukemia, squamous cell carcmoma, small-cell lung cancer, non-small cell lung cancer, ghoma, gastrointestinal cancer, renal cancer, ovanan cancer, liver cancer, colorectal cancer, endometnal cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, ghoblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcmoma, and head and neck cancer.
  • treating refers to curative therapy, prophylactic therapy, and preventative therapy.
  • subject selected for treatment refer to an individual who has been identified as having a condition diat artisans understand can respond to a specific therapy and, consequentially is being considered for treatment (or bemg treated with) d at dierapy (e.g. an individual suffering from chrome myelogenous leukemia who is bemg treated with STI-571).
  • mammal refers to any mammal classified as a mammal, mcludmg humans, cows, horses, dogs and cats In a preferred embodiment of the mvention, the mammal is a human.
  • the mvention descnbed herem relates to novel genes and their encoded protems, termed Mutants Associated with Resistance to STI-571 (e.g., T315I Bcr-Abl), and to diagnostic and therapeutic methods and compositions useful m the management of various cancers d at express MARS.
  • Mutants Associated with Resistance to STI-571 e.g., T315I Bcr-Abl
  • Embodiments of the mvention provided herem are illustrated by studies of the Bcr-Abl protem kinase m STI-571 -treated patients. To charactenze the mechamsm of relapse m STI-57 -treated patients, we first assessed the status of Bcr-Abl signalmg m primary leukemia cells.
  • Bcr-Abl dependent versus Bcr-Abl mdependent mechanisms of relapse If Bcr-Abl remains critical for proliferation of the leukemia clone, then the Bcr-Abl signalmg pathway should be reactivated. Alternatively, if expansion of the leukemia clone is mdependent of Bcr-Abl, d en signalmg through d e Bcr-Abl pathway should remam impaired by STI-571.
  • the most direct measure of signaling through Bcr-Abl pathway is the enzymatic activity of Bcr-Abl protem itself (see, e.g. J B. Konopka et al, Proc. Natl. Acad. Set.
  • Crkl migrates witih altered mobility m SDS-PAGE gels and can be quantified usmg densitometry
  • Crkl phosphorylation in primary CML patient cells was mhibited in a dose-dependent manner when exposed to STI-571 and correlated widi dephosphorylation of Bcr-Abl (Fig. 1A). This Crkl assay allows for an assessment of the enzymatic activity of Bcr-Abl protem m a reproducible, quantitative fashion in clinical matenals.
  • STI-571 resistance m patients is due to a host- mediated response agamst the drug
  • resistance might be mediated by a cell-autonomous event m a leukemia subclone that allows escape from kinase inhibition by STI-571.
  • a cell-autonomous event m a leukemia subclone that allows escape from kinase inhibition by STI-571.
  • STI-571 resistance is a consequence of a host response, pretreatment and relapse leukemia cells should be equally sensitive to ex vivo STI-571 treatment. However, if STI-571 is cell-intrinsic, leukemia cells obtamed at relapse should be less sensitive to STI-571 dian pretreatment cells. In those patients for whom we had sufficient matched clinical material, a 10-fold or greater shift m sensitivity to STI-571 was observed at relapse (Fig. 2A). Aggregate analysis of 11 samples confirmed that higher concentrations of STI-571 are required to inhibit Crkl phosphorylation in patients cells obtamed at relapse versus pre-treatment (Fig. 2B).
  • IA-IF provide a summary of patient data.
  • Example 1 we also considered the possibility that mutations in BCR-ABL might confer resistance to STI- 571.
  • Consequendy a 579 base pair region corresponding to d e ATP-binding pocket and the activation loop of the kinase domam of Bcr-Abl was sequenced m die 9 patients for whom RNA was available at the time of relapse (Fig 4A).
  • Fig 4A A smgle, identical C— >T nucleotide change was detected at ABL nucleotide 944 m six of nine cases examined (F ⁇ g.4A).
  • a smgle nucleotide C— »T change results m a threonine to isoleucine substitution at position 315 of c-Abl.
  • the recendy-solved crystal structure of the catalytic domam of Abl complexed widi a variant of STI-571 identified the ammo acid residues within the ATP-binding site and activation loop of c- Abl that are required for STI-571 bmdmg and thus inhibition of Abl kmase activity (see, e.g. T. Schmdler et al, Science 289, 1938 (2000)).
  • Thr 315 is among those that form critical hydrogen bonds with STI-571.
  • T315I Bcr-Abl is discussed as a representative embodiment of the MARS disclosed herem (e.g. those described m Table IA below).
  • embodiments of a smgle gene are used (T315I Bcr-Abl, for example) to illustrate typical embodiments of the mvention that apply to all of die MARS disclosed herem (e.g. E255K, Q252H, V304D, M351T, E355G etc. as shown m Table I)
  • artisans understand that discussing a typical embodiment directed to a smgle species (e.g. T315I) when the embodiments are commonly applicable to the other species disclosed herem (e.g. E255K, Q252H, V304D, M351T, E355G etc.) eliminates unnecessary redundancy m the descriptions of d e mvention.
  • the T315I mutation is shown to preserve kmase activity and, based upon the crystal structure of the kinase domam when bound to STI-571, is predicted to result in ineffective bmdmg of STI-571 to BCR/ABL.
  • kmase domam mutations m a larger sample size, we sequenced the BCR/ABL kmase domam m 18 patients with CML m myeloid blast crisis.
  • T315I mutation has also been detected m subsequent studies of 9 of 28 patients (6/25 myeloid blast crisis, 3/3 with lymphoid blast cnsis or Ph+ ALL). Two other mutations, M351T and E255K, were also found m 4 patients and 3 patients respectively. Additional mutations were also found but did not always represent the dominant subclone at time of relapse. These fmdmgs mdicate that BCR/ABL kmase domam mutations occur commonly m CML blast cnsis and can be detected, m some cases, prior to STI-571 treatment.
  • a fragment containing the C to T mutation at ABL nucleotide 944 was made by PCR and swapped with the corresponding sequence m pSRaMSVtkNeo p210 Bcr-Abl wild-type to create the pSRaMSVtkNeo p210 Bcr-Abl T315I mutant.
  • the resulting construct was confirmed by sequencmg.
  • Cells were transfected with wild-type or T315I p210 Bcr-Abl and cultured in the presence of mcreasmg concentrations of STI-571. Briefly, the transient transfection of 293T cells was performed usmg CaCi2 (see, e.g. A. J. Muller et al, Mol Cell.
  • the kmase activities of wild-type Bcr-Abl and the T315I mutant appear comparable m the absence of STI-571
  • the T315I mutant retained high levels of phosphotyrosme at all concentrations of inhibitor tested (Fig. 4C, top panels).
  • STI-571 resistance can occur through at least two distinct mechanisms. Some patients develop chromosomal amplification of the genomic region encodmg Bcr-Abl, resulting presumably m levels of Bcr-Abl protem that overcome the lntracellular concentration of STI-571 (Gorre et al, Science 293:876- 880 (2001)).
  • a second mechamsm mvolves pomt mutations m the kinase domam that presumably mterfere with drug-protein bmdmg without compiomising kmase activity
  • the best characterized of these mvolves a substitution of isoleucine for direonine at ammo acid position 315 (T315I) which alters the shape of the drug-bmdmg pocket based on a crystallographic-based model (Gorre et al., Science 293:876-880 (2001)).
  • Phenylalanine is highly conserved at diis position m the Src-family of tyrosine l ⁇ nases, and when engmeered m to c-Abl, this mutation has been demonstrated to impart oncogenicity as reflected by cellular transformation assays (Allen et al, / Biol Chem 275:19585-19591 (1996)). We have recendy found d e sensitivity of Y253F to STI-571 to be intermediate between wild-type Bcr-Abl and die T315I mutant.
  • Bcr-Abl kinase domam mutations can be detected m chrome phase patients who fail to achieve cytogenetic remission or lose an established ma j or cytogenetic response and are associated with disease progression and decreased survival.
  • Cells from chrome phase patients who failed to obtam cytogenetic remission or who lost a previously achieved cytogenetic remission were subjected to sequence analysis of the Bcr-Abl kmase domam. Analysis was performed on samples obtamed at the time of sustained hematologic response. A number of patients were found to harbor mutations. Three of these mutations were also observed m cases of relapsed myeloid blast cnsis described above (E255K, F317L, F359V).
  • F317L was recendy descnbed m a smgle patient (Branford et al. Blood 99:3472-3475 (2002)) with chronic phase disease and cytogenetic persistence who subsequendy suffered progressive disease.
  • the last mutation, V379I has not been documented in any other patient to date.
  • the patient harboring the V379I mutation continues to have a complete hematologic remission m response to STI-571 in the absence of a cytogenetic response.
  • kmase domam mutations occur m chrome phase patients who lose cytogenetic or hematologic responses to STI-571, and in a subset of chrome phase patients who have persistence of the Philadelphia chromosome m the setting of complete hematologic response.
  • Bcr-Abl kinase domam mutations can be detected prior to STI-571 treatment m patients with myeloid blast crisis that exhibit de novo resistance, but not in patients with STI-571 -sensitive myeloid blast crisis or chrome phase CML.
  • To determine whether Bcr-Abl kmase domam mutations may play a role m de novo resistance to STI-571 we analyzed pre-treatment samples from four patients with MBC who failed to achieve even a transient response to STI-571. One patient exhibited T315I prior to initiation of therapy. Also detected m the same patient was a Bcr-Abl allele that contamed two mutations, M343T and F382L. A second patient had the E255K mutation prior to STI- 571 treatment.
  • Bcr-Abl kmase domam mutations retam catalytic activity, and are capable of conferrmg STI-571 resistance in vitro.
  • eight of the observed mutations (G250E, Q252H, Y253F, E255K, T315I, F317L, M351T, and E355G) were mdependendy introduced mto pSRalphaP210Bcr-Abl. While these mutants are provided as preferred embodiments of the mvention descnbed herem, those skilled m the art can generate comparable mutants of any one of the MARS desc ⁇ bed herem such as those identified m Table I.
  • the eight mutations were each transiendy transfected mto 293-T cells, and found to exhibit varying degrees of sensitivity to STI-571, with IC-50 for enzymatic mliibition m cells rangmg from 1.27 uM to 5.63 uM as documented by phosphotyrosine-containing Bcr-Abl (see Figure 9).
  • the murine hematopoietic cell line Ba/F3 requires exogenous IL-3 m the absence of Bcr-Abl.
  • Stable Ba/F3 cell lines capable of growing m the absence of interleukin-3, were derived for each of d e eight mutant isoforms, demonstrating diat each of the eight mutant isoforms retams biologic activity m this assay.
  • concentrations of STI-571 on cellular viability after 48 hours was determined.
  • Agam, die eight mutant isoforms were found to exhibit varying degrees of sensitivity to STI-571.
  • Several of the mutants appeared to impart only moderate resistance, retaimng sensitivity to concentrations of STI-571 which are theoretically achievable m patients (see Figure 9).
  • Bcr-Abl kinase domam mutations can be detected m nearly all patients with relapsed myeloid blast crisis; that resistance frequendy mvolves the coexistence of cell populations containing different kinase domam mutations; that Bcr- Abl kmase domam mutations exhibit a wide range of STI-571 resistance in vitro; that kmase domam mutations occur in a subset of chrome phase CML patients with persistence of the Philadelphia chromosome, and portend a poor prognosis; and that some STI-571 -resistant kmase domam mutations can be occasionally detected in advanced phase cases CML prior to STI-571 treatment, and therefore may contribute to the leukemic drive in cells that harbor them.
  • Bcr-Abl kmase domam mutations may thus contribute to the natural progression of CML from chrome to advanced phases m some cases. Given our findings, we believe routine sensitive sequence analysis of the Bcr-Abl kmase domam in patients bemg treated with STI-571 is warranted.
  • die disclosure provided herem supports kmase domam mutation as the primary mechamsm for STI-571 failure.
  • Previous studies of kmase domam mutations have been performed largely on isolated cases of Philadelphia chromosome- positive ALL and CML in lymphoid blast cnsis.
  • Our finding of Bcr-Abl kmase domam mutations in nearly all cases of relapsed myeloid blast cnsis was not expected based upon previous reports. Because the complete cytogenetic remission rate is lower in myeloid blast crisis patients treated with Gleevec, it is possible that resistance to Gleevec m patients with lymphoid blast crisis CML and Ph+ ALL more commonly represents greater genetic homogeneity.
  • Less sensitive methods of mutation detection may therefore adequately demonstrate the presence of nucleotide substitutions m these cases, yet fail to reliably detect mutations m relapsed myeloid blast crisis cases.
  • kmase domam mutations m cases of myeloid blast cnsis prior to treatment with STI-571. While it is formally possible that such mutants merely reflect genomic instability, the finding of such mutants at a frequency of twenty percent is more suggestive of a significant clonal expansion of these cells. It is possible that certain kmase domam mutations may confer a growth advantage m affected cells.
  • the viral oncogene v-abl is known to contam pomt mutations in addition to alternative N-terminal coding sequences when compared with murine c-abl In this study we detected T315I pnor to treatment m a patient whose disease subsequendy failed to respond to STI-571.
  • v-src differs from its cellular counterpart by substitution of isoleucine for threonine.
  • MARS A number of specific sequences of MARS are identified m Table I below.
  • One aspect of the mvention provides polynucleotides corresponding or complementary to all or part of a MARS gene, mRNA, and/or coding sequence, preferably in isolated form, mcludmg polynucleotides encodmg a MARS protem and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a MARS gene or mRNA sequence or a part thereof, and polynucleotides or ohgonucleotides that hybridize to a MARS gene, mRNA, or to a MARS encodmg polynucleotide (coUectively, "MARS polynucleotides").
  • the MARS gene and protem is meant to mclude the MARS genes and protems specifically described herem and the genes and protems corresponding to MARS protems.
  • Typical embodiments of the mvention disclosed herem mclude MARS polynucleotides containing specific portions of the MARS mRNA sequence (and those which are complementary to such sequences), for example, those that encode the T315I codon sequence.
  • one specific aspect of the mvention provides polynucleotides corresponding or complementary to all or part of a T315I Bcr-Abl gene, mRNA, and/or coding sequence, preferably in isolated form, mcludmg polynucleotides encodmg a T315I Bcr-Abl protem and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or ohgonucleotides complementary to a T315I Bcr-Abl gene or mRNA sequence or a part diereof, and polynucleotides or ohgonucleotides that hybridize to a T315I Bcr-Abl gene, mRNA, or to a T315I Bcr-Abl encoding polynucleotide (collectively, "T315I Bcr-Abl polynucleotides").
  • the T315I Bcr-Abl gene and protem is meant to mclude the T315I Bcr-Abl genes and protems specifically described herem and the genes and protems corresponding to T315I Bcr-Abl protems.
  • Typical embodiments of the mvention disclosed herem mclude T315I Bcr-Abl polynucleotides containing specific portions of the T315I Bcr-Abl mRNA sequence (and those which are complementary to such sequences), for example, those that encode the T315I codon.
  • the MARS polynucleotides of the mvention are useful for a vanety of purposes, mcludmg but not limited to their in the detection of the MARS gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of cancers; as coding sequences capable of directing the expression of MARS polypeptides; as tools for modulating or inhibiting the function of the MARS protem.
  • probes and primer pairs which allow the specific amplification of the MARS polynucleotides of the mvention or of any specific parts thereof, and probes that selectively or specifically hybndize to nucleic acid molecules of the mvention or to any part thereof.
  • Probes may be labeled with a detectable marker, such as, for example, a rachoisotope, fluorescent compound, bioluminescent compound, a chemilummescent compound, metal chelator or enzyme.
  • a detectable marker such as, for example, a rachoisotope, fluorescent compound, bioluminescent compound, a chemilummescent compound, metal chelator or enzyme.
  • Such probes and primers can be used to detect the presence of a MARS polynucleotide in a sample and as a means for detecting a cell expressmg a MARS protem
  • probes and primers examples include polypeptides compnsmg all or part of a human MARS cDNA sequence shown m Table I.
  • primer pairs capable of specifically amplifying MARS mRNAs e.g. those primers disclosed herem
  • primer pairs capable of specifically amplifying MARS mRNAs are readily ascertainable by those skilled in the art.
  • a great many different primers and probes may be prepared based on the sequences provided m herem and used effectively to amplify and/or detect a MARS mRNA.
  • the mvention also provides recombmant DNA or RNA molecules containing a MARS polynucleotide, mclud g but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known m the art, and cells transformed or transfected with such recombmant DNA or RNA molecules.
  • a recombmant DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular mampulation in vitro. Methods for generating such molecules are well known (see, for example, Sambrook et al, 1989, supra).
  • the mvention further provides a host-vector system compnsmg a recombmant
  • DNA molecule containing a MARS polynucleotide within a suitable prokaryotic or eukaryotic host cell examples include a yeast cell, a plant cell, or an animal cell, such as a mammahan cell or an msect cell (e.g., a baculovirus- lnfecttble cell such as an Sf9 cell).
  • suitable mammalian cells examples include vanous cancer cell lines, odier transfectable or ttansducible cell lines, mcludmg those mammalian cells routinely used for die expression of recombmant protems (e.g., COS, CHO, 293, 293T cells etc.).
  • a polynucleotide compnsmg the coding sequence of a MARS may be used to generate MARS protems or fragments thereof usmg any number of host vector systems routinely used and widely known m the art.
  • host vector systems suitable for the expression of MARS protems or fragments thereof are available, see for example, Sambrook et al, 1989, supra; Current Protocols m Molecular Biology, 1995, supra).
  • Preferred vectors for mammalian expression m include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSR ⁇ tkneo (Muller et al, 1991, MCB 11 :1785).
  • MARS may be preferably expressed m cell Imes, mcludmg for example CHO COS, 293, 293T, rat-1, 3T3 etc.
  • the host vector systems of the mvention are useful for the production of a MARS protem or fragment thereof. Such host-vector systems may be employed to study the functional properties of MARS and MARS mutations.
  • MARS POLYPEPTIDES MARS POLYPEPTIDES
  • MARS protems and polypeptide fragments thereof are also mcluded.
  • MARS protems will be collectively referred to as the MARS protems, die protems of the mvention, or MARS.
  • MARS polypeptide refers to a polypeptide fragment or a MARS protem of at least about 6 ammo acids (e.g. a Bcr-Abl polypeptide havmg about 6 contiguous ammo acids mcludmg a MARS such as T315I, preferably at least about 10-15 amino acids)
  • Protems encoded by the MARS genes, or by fragments thereof, will have a variety of uses, mcludmg but not limited to generating antibodies and in methods for identifying ligands and odier agents (e.g. small molecules such as 2-phenylpynm ⁇ dmes) and cellular constituents that bmd to a MARS gene product.
  • Antibodies raised agamst a MARS protem or fragment thereof may be useful m diagnostic and prognostic assays, imaging methodologies (mcludmg, particularly, cancer imaging), and therapeutic methods m the management of human cancers characterized by expression of a MARS protem, mcludmg but not limited to cancer of die lymphoid lineages.
  • Vanous immunological assays useful for the detection of MARS protems are contemplated, mcludmg but not limited to vanous types of radioimmunoassays, enzyme-lmked immunosorbent assays (ELISA), enzyme-lmked lmmunofluorescent assays (ELIFA), lmmunocytochemical methods, and the like.
  • ELISA enzyme-lmked immunosorbent assays
  • ELIFA enzyme-lmked lmmunofluorescent assays
  • Such antibodies may be labeled and used as immunological imaging reagents capable of detecting leukemia cells (e.g., m radioscintigraphic imaging methods).
  • antibody is used m the broadest sense and specifically covers smgle anti-MARS monoclonal antibodies (mcludmg agonist, antagonist and neutralizing antibodies) and anti-MARS antibody compositions with polyepitopic specificity.
  • monoclonal antibody mAb as used herem refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the antibodies compnsmg die mdividual population are identical except for possible naturally-occurnng mutations diat may be present m minor amounts.
  • Another aspect of the mvention provides antibodies diat lmmunospecifically bmd to MARS protems and polypeptides
  • the most preferred antibodies will specifically bmd to a MARS protem and will not bmd (or will bmd weakly) to Bcr-Abl protems and polypeptides.
  • Anti-MARS antibodies that are particularly contemplated mclude monoclonal and polyclonal antibodies as well as fragments containing the antigen bmdmg domam and/or one or more complementanty determining regions of these antibodies.
  • an antibody fragment is defined as at least a portion of the vanable region of the immunoglobulin molecule that bmds to its target, i.e., die antigen bmdmg region.
  • antibodies which specifically react with a particular MARS protem and/or an epitope within a particular structural domain may be desirable to generate antibodies which specifically react with a particular MARS protem and/or an epitope within a particular structural domain.
  • preferred antibodies useful for diagnostic purposes are those which react with an epitope m a mutated region of the MARS protem as expressed m cancer cells.
  • Such antibodies may be generated by usmg die MARS protems descnbed herem, or usmg pep ⁇ des denved from vanous domains thereof, as an immunogen.
  • MARS antibodies of the mvention may be particularly useful in cancer (e.g chrome myelogenous leukemia) therapeutic strategies, diagnostic and prognostic assays, and imagmg mediodologies. Similarly, such antibodies may be useful m the diagnosis, and/or prognosis of other cancers, to the extent MARS is also expressed or overexpressed m other types of cancer.
  • the mvention provides vanous immunological assays useful for the detection and quantification of MARS and mutant MARS protems and polypeptides.
  • Such assays generally comprise one or more MARS antibodies capable of recogmzmg and binding a MARS or mutant MARS protem, as appropriate, and may be performed within various immunological assay formats well known m die art, mcludmg but not limited to various types of radioimmunoassays, enzyme-hnked immunosorbent assays (ELISA), enzyme-hnked lmmunofluorescent assays (ELIFA), and the like.
  • immunological imaging methods capable of detecting cancer cells are also provided by the mvention, mcludmg but limited to radioscm ⁇ graphic imagmg mediods usmg labeled MARS antibodies.
  • Such assays may be used clinically m the detection, monitoring, and prognosis of cancers, particularly chrome myeloid leukemia
  • MARS TRANSGENIC ANIMALS Nucleic acids that encode MARS can also be used to generate either transgemc animals which, m turn, are useful m die development and screening of therapeutically useful reagents.
  • a transgemc animal e.g, a mouse or rat
  • a transgene is a DNA that is mtegrated mto the genome of a cell from which a transgemc animal develops.
  • cDNA encodmg T315I Bcr-Abl can be used to clone genomic DNA encodmg T315I Bcr-Abl m accordance with estabhshed techniques and d e genomic sequences used to generate transgemc animals that contam cells diat express DNA encodmg T315I Bcr-Abl.
  • Methods for generating transgemc animals, particularly animals such as mice or rats have become conventional m the art and are descnbed, for example, m U S. Patent Nos. 4,736,866 and 4,870,009.
  • particular cells would be targeted for MARS transgene incorporation with tissue-specific enhancers.
  • Transgemc animals that mclude a copy of a transgene encodmg MARS introduced mto die germ line of the animal at an embryonic stage can be used to examine the effect of mcreased expression of DNA encodmg MARS.
  • Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its expression.
  • an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearmg the transgene, would mdicate a potential therapeutic intervention for the pathological condition.
  • Another aspect of the present mvention relates to mediods for detecting MARS polynucleotides and MARS protems, as well as mediods for identifying a cell that expresses MARS.
  • the expression profile of MARS makes them diagnostic markers for disease states.
  • the status of MARS gene products m patient samples may be analyzed by a vanety protocols that are well known m the art mcludmg lmmunohistochemical analysis, the vanety of Nordiern blotting techmques mcludmg in situ hybndization, RT-PCR analysis (for example on laser capture micro-dissected samples), western blot analysis and tissue array analysis.
  • the mvention provides assays for the detection of MARS polynucleotides m a biological sample, such as cell preparations, and the like.
  • a number of methods for amplifying and/or detecting d e presence of MARS polynucleotides are well known in the art and may be employed in the practice of dns aspect of the invention
  • a method for detecting a MARS mRNA m a biological sample compnses producmg cDNA from the sample by reverse transcnption usmg at least one primer; amplifying the cDNA so produced usmg a MARS polynucleotides as sense and antisense primers to amplify MARS cDNAs therein; and detecting the presence of the amplified MARS cDNA.
  • Any number of appropnate sense and antisense probe combmations may be designed from the nucleotide sequences provided for the MARS and used for this purpose.
  • the mvention also provides assays for detecting the presence of a MARS protem m a biological sample.
  • Methods for detecting a MARS protem are also well known and mclude, for example, lmmunoprecipitation, lmmunohistochemical analysis, Western Blot analysis, molecular bmdmg assays, ELISA, ELIFA and the like.
  • a mediod of detecting the presence of a MARS protem in a biological sample comprises first contacting the sample with a MARS antibody, a MARS-reac ⁇ ve fragment diereof, or a recombmant protem containing an antigen bmdmg region of a MARS antibody; and then detecting the bmdmg of MARS protem in the sample thereto.
  • Methods for identifying a cell that expresses MARS are also provided.
  • an assay for identifying a cell that expresses a MARS gene comprises detecting the presence of MARS mRNA m the cell.
  • Methods for the detection of particular mRNAs m cells are well known and mclude, for example, hybridization assays usmg complementary DNA probes (such as in situ hybndization usmg labeled MARS nboprobes, Nordiern blot and related techmques) and vanous nucleic acid amphfication assays (such as RT-PCR usmg complementary pnmers specific for MARS, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA
  • a significant aspect of the mvention disclosed herem is the discovery that ammo acid substitutions m the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 can produce cancer cells havmg a resistance to tyrosine kmase inhibitors such as STI-571.
  • a resistance to tyrosine kmase inhibitors such as STI-571.
  • the physiological mechanisms of drug resistance are diverse and that drug resistance typically occurs through other mechanisms such as an mcrease m the expression of protems that export the drug out of the cell (see, e.g.
  • a preferred embodiment of the mvention is a method of identifying at least one ammo acid substitution m at least one Bcr-Abl polypeptide havmg some level of tyrosine kmase activity that is expressed m a human cancer cell from an mdividual selected for treatment with a tyrosine kmase inhibitor, the mediod compnsmg determining d e polypeptide sequence of at least one Bcr-Abl polypeptide expressed by die human cancer cell and comparing the polypeptide sequence of the Bcr-Abl polypeptide expressed by the human cancer cell to the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 so diat an am o acid substitution m die Bcr-Abl polypeptide expressed by die human cancer cell can be identified.
  • an ammo acid substitution so identified confers some level of resistance to STI-571.
  • a significant aspect of the mvention disclosed herem is the delineation of a discreet region m the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 that contams mutations that can produce cancer cells havmg a resistance to tyrosine kinase inhibitors such as STI-571.
  • This discovery allows artisans to focus on this region m diagnostic protocols so as to facihtate such analyses.
  • a preferred mediod of the mvention is a method of identifying an ammo acid substitution in at least one Bcr- Abl polypeptide expressed m a human cancer cell from an mdividual selected for treatment widi a tyrosine kmase inhibitor, the method compnsmg determining the polypeptide sequence of at least one Bcr-Abl polypeptide expressed by die human cancer cell and comparing the polypeptide sequence of the Bcr-Abl polypeptide expressed by die human cancer cell to the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 so that an ammo acid substitution in the Bcr-Abl polypeptide expressed by the human cancer cell can be identified, wherem the amino acid substitution occurs m a region of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 compnsmg residue D233 through residue T406.
  • Widiout bemg bound by a specific scientific dieory the data disclosed herem provides evidence that this region defines boundaries for the structural architecture of the portions of Bcr-Abl diat are predommandy mvolved m an interaction with STI-571.
  • Another significant aspect of the mvention disclosed herem is the delineation of a discreet subregions m the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 that contams the mutations that can produce cancer cells havmg a resistance to tyrosine kmase inhibitors such as STI-571.
  • This discovery allows artisans to focus on such subregions in diagnostic protocols so as to facihtate such analyses.
  • a preferred method of die mvention is a method of identifying an amino acid substitution m at least one Bcr-Abl polypeptide expressed m a human cancer cell from an mdividual selected for treatment widi a tyrosine kinase inhibitor, the mediod compnsmg determining d e polypeptide sequence of at least one Bcr-Abl polypeptide expressed by the human cancer cell and comparing d e polypeptide sequence of d e Bcr-Abl polypeptide expressed by the human cancer cell to the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 so that an ammo acid substitution m the Bcr-Abl polypeptide expressed by die human cancer cell can be identified, wherem the amino acid substitution occurs m the P-loop (residue G249 through residue V256 of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1), helix C (residue
  • ammo acid substitution is proximal (e.g within about 10 ammo acid residues) to one of diese subregions m a manner that perturbs the function of the subregion
  • a particularly significant aspect of die mvention disclosed herem is the delineation of a discreet residue positions in the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 that, when mutated, can produce cancer cells havmg a resistance to tyrosine kmase inhibitors such as STI-571.
  • a preferred method of the mvention is a method of identifymg an ammo acid substitution m at least one Bcr-Abl polypeptide expressed m a human cancer cell from an mdividual selected for treatment with a tyrosine kmase inhibitor, d e method compnsmg determining the polypeptide sequence of at least one Bcr-Abl polypeptide expressed by die human cancer cell and comparmg die polypeptide sequence of d e Bcr-Abl polypeptide expressed by d e human cancer cell to the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 so diat an ammo acid substitution m the Bcr-Abl polypeptide expressed by the human cancer cell can be identified, wherem the amino acid substitution occurs at residue D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E
  • the amino acid substitution is D233H, T243S, M244V, G249D, G250E, G251S, Q252H, Y253F, Y253H, E255K, V256L, Y257F, Y257R, F259S, K262E, D263G, K264R, S265R, V268A, V270A, T272A, Y274C, Y274R, D276N, T277P, M278K, E282G, F283S, A288T, A288V, M290T, K291R, E292G, I293T, P296S, L298M, L298P, V299L, Q300R, G303E, ⁇ 304A, V304D, C305S, C305Y, T306A, F311L, I314V, T315A, T315I, E
  • the mediods disclosed herem can also be used to identify other mutations that are associated with resistance to tyrosine kinase inhibitors such as STI-571 such as truncations diat result from a mutation diat mtroduces a stop codon at an ammo acid residue position such as K245STOP or E334STOP.
  • tyrosine kinase inhibitors such as STI-571
  • truncations diat result from a mutation diat mtroduces a stop codon at an ammo acid residue position such as K245STOP or E334STOP.
  • Embodiments of the mvention mclude those that examine any one to all of the ammo acid positions m the Bcr-Abl polypeptide sequence (e.g. Ml, L2, E3 dirough VI 128, Q1129 and R1130) as occurs when one compares the sequence of a polypeptide expressed by a cancer cell with the polypeptide sequence shown m SEQ ID NO: 1.
  • the mven ⁇ on one can examine residue G250, Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35, K357, 1360, V379 or H396.
  • another embodiment of the mvention is a method of identifymg an ammo acid substitution m at least one Bcr-Abl polypeptide expressed m a human cancer cell from an mdividual selected for treatment with a tyrosine kmase inhibitor, the method compnsmg determining the polypeptide sequence of at least one Bcr-Abl polypeptide expressed by the human cancer cell and comparing the polypeptide sequence of the Bcr- Abl polypeptide expressed by the human cancer cell to the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 so that an ammo acid substitution m die Bcr-Abl polypeptide expressed by the human cancer cell can be identified, wherein the ammo acid substitution does not occur at residue G250, Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35, K357, 13
  • polynucleotide and/or polypeptide sequences of Bcr-Abl can be identified by any one of a wide vanety of protocols known m the art such as diose disclosed herem.
  • the Bcr-Abl polynucleotide expressed by die human cancer cell is isolated by the polymerase cham reaction.
  • methods used in the identification of one Bcr-Abl polypeptide expressed in a human cancer cell from an mdividual selected for treatment with one tyrosine kmase inhibitor can be identical to methods used m the identification of one Bcr-Abl polypeptide expressed m a human cancer cell from an mdividual selected for treatment with anodier tyrosine kmase inhibitor.
  • the kmase inhibitor is a 2-phenylammopyr ⁇ m ⁇ chne.
  • the mediods of the present mvention can be used in determmmg whether or not to treat an mdividual with a specific tyrosine kmase inhibitor such as STI-571.
  • Another embodiment of die mvention disclosed herem is a method of identifymg a mutation m a Bcr-Abl polynucleotide m a mammalian cell, wherem the mutation m a Bcr-Abl polynucleotide is associated with resistance to inhibition of Bcr- Abl tyrosine kmase activity by a 2-phenylammopynmidme, the method compnsmg determmmg the sequence of at least one Bcr-Abl polynucleotide expressed by the mammahan cell and comparing the sequence of d e Bcr-Abl polynucleotide to the Bcr- Abl polynucleotide sequence encodmg the polypeptide sequence shown
  • a Bcr-Abl polynucleotide associated widi resistance to inhibition of Bcr-Abl tyrosine kmase by a 2-phenylammopyrimichne refers to a Bcr-Abl polynucleotide that has been identified m cancer cells that exhibit some level of resistance to a 2-phenylarrunopynmidme such as STI-571 (or analogs or derivatives thereof) and which encodes a polypeptide havmg at least one ammo acid difference from die polypeptide sequence shown in SEQ ID NO- 1 (e.g those disclosed m Table IA).
  • the Bcr-Abl polynucleotide associated with resistance to inhibition of Bcr-Abl tyrosine kmase by a 2-phenylammopynmichne encodes a polypeptide that exhibits exhibit some level of resistance to a 2-phenylammopynmidme such as STI-571.
  • the mammahan cell is a human cancer cell.
  • the human cancer cell is a chrome myeloid leukemia cell.
  • the human cancer cell is obtamed from an mdividual treated with STI-571.
  • the ammo acid substitution m d e Bcr-Abl polypeptide expressed m human cancer cell confers resistance to mliibition of tyrosine kmase activity by STI-571.
  • MARS expression analysis may also be useful as a tool for identifymg and evaluating agents that modulate MARS gene expression. Identification of a molecule or biological agent that could inhibit MARS activity is of therapeutic value.
  • MARS mRNA is expressed in cancers demonstrating STI-571 resistance provides evidence that mutations in Bcr-Abl are associated with STI-571 resistance and therefore identifies diese genes and their products as targets that the skilled artisan can use to evaluate biological samples from mdividuals suspected of havmg a disease associated with MARS expression.
  • the evaluation of the status of MARS genes and their products can be used to gam information on die disease potential of a tissue sample.
  • status refers to the condition a gene and its products mcludmg, but not limited to die integnty and/or methylation of a gene mcludmg its regulatory sequences, the location of expressed gene products (mcludmg the location of MARS expressmg cells), die presence, level (e.g.
  • MARS expressmg myeloid cancer cells m a total population of myeloid cancer cells
  • biological activity of expressed gene products such as MARS mRNA polynucleotides and polypeptides
  • transcnptional and translational modifications to expressed gene products as well as associations of expressed gene products with other biological molecules such as protem bmdmg partners.
  • the status of MARS can be evaluated by a wide variety of mediodologies well known m the art, typically those discussed below.
  • the status of MARS may provide information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness.
  • the mvention provides methods and assays for determmmg MARS status and diagnosing cancers diat express MARS.
  • MARS status in patient samples may be analyzed by a number of means well known m the art, mcludmg without limitation, mimunohistochemical analysis, m situ hybndization, RT-PCR analysis on laser capture micro-dissected samples, western blot analysis of clinical samples and cell lines, and tissue array analysis Typical protocols for evaluating the status of the MARS gene and gene products can be found, for example in Ausubul et al eds, 1995, Current Protocols In Molecular Biology, Units 2 [Northern Blotting], 4 [Southern Blotting], 15 [Immunoblotting] and 18 [PCR Analysis].
  • a typical aspect of the mvention is directed to assessmg the effectiveness of STI- 571 m a therapeutic regimen.
  • a method for assessmg the effectiveness of STI-571 compnses detecting MARS mRNA or MARS protem m a tissue sample, its presence mchcating a likely resistance to STI-571, wherem the degree of MARS mRNA expression (e.g. the percentage of clones that express one or more MARS) is proportional to the likelihood of resistance to STI-571.
  • a mediod for examining a stage of cancer compnses detecting MARS mRNA or MARS protem m a tissue sample, its presence indicating susceptibility to cancer, wherem the degree of MARS mRNA expression present is proportional to the degree of susceptibility.
  • the presence of MARS m a tissue sample is examined, widi the presence of MARS m the sample providmg an indication of a stage of leukemia (or d e emergence or existence of a leukemia).
  • a method for gauging aggressiveness of a tumor compnses determining the level of MARS mRNA or MARS protem expressed by cells in a sample of the tumor, comparing the level so determined to the level of MARS mRNA or MARS protem expressed m a corresponding control tissue, wherem the degree of MARS mRNA expression present is proportional to the degree of aggressiveness.
  • aggressiveness of leukemias is evaluated by determining the extent to which MARS is expressed m the tumor cells, widi relatively higher numbers of cells expressmg one or more MARS indicating more aggressive tumors (e.g. in that they are resistant to a therapeutic agent such as STI-571)
  • Yet another related aspect of the mvention is directed to mediods for observing the progression of a malignancy in an mdividual over time.
  • methods for observing the progression of a malignancy m an mdividual over time comp ⁇ se deterrnining the level of MARS mRNA or MARS protem expressed by cells m a sample of the tumor, companng the level so determined to the level of MARS mRNA or MARS protem expressed in an equivalent tissue sample taken from the same mdividual at a different time, wherem die degree of MARS mRNA or MARS protem expression m the tumor sample over time provides information on the progression of the cancer.
  • the progression of a cancer is evaluated by determining the extent to which MARS expression m the tumor cells alters over time, with higher expression levels over time mchcating a progression of the cancer.
  • Gene amphfication provides an additional method of assessmg the status of Bcr- Abl.
  • Gene amphfication may be measured m a sample chrecdy, for example, by conventional Southern blotting, Northern blotting to quan ⁇ tate the transcription of mRNA (Thomas, 1980, Proc. Nad. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or m situ hybndization, usmg an appropnately labeled probe, based on the sequences provided herem.
  • antibodies may be employed that can recognize specific duplexes, mcludmg DNA duplexes, RNA duplexes, and DNA-RNA hybnd duplexes or DNA-protem duplexes.
  • the antibodies turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on die surface, the presence of antibody bound to the duplex can be detected.
  • Another embodiment of the mvention disclosed herem is directed to methods for observing a comcidence between the expression of MARS gene and/or MARS gene products and a factor that is associated with malignancy as a means of diagnosing and prognosticating the status of a tissue sample.
  • a wide vanety of factors associated with malignancy may be utilized such as the expression of genes otherwise associated with malignancy as well as gross cytological observations (see e.g. Bocking et al, 1984, Anal. Quant. Cytol. 6(2):74-88; Eptsein, 1995, Hum. Pathol.
  • methods for observing a comcidence between the expression of MARS gene and MARS gene products (or perturbations in MARS gene and MARS gene products) and a factor that is associated wid malignancy entails detecting the overexpression of MARS mRNA or protem m a tissue sample and then detecting the altered expression of another oncogene such RAS, or a tumor suppressor such as p53 or Rb, m a tissue sample, and observing a comcidence of MARS mRNA or protem expression and, for example, RAS mRNA or protem overexpression.
  • the expression of MARS and RAS mRNA in tissue is examined.
  • the comcidence of MARS and RAS mRNA overexpression m the sample provides an indication of leukemia stage, or the emergence or existence of a leukemia.
  • Preferred embodiments of the mvention descnbed herem m clude methods for characterizing a cancer genotype and/or phenotype such as the genotype and/or phenotype of cancers of the myeloid lineage.
  • Specific embodiments of the mvention descnbed herem m clude methods of assessmg die likelihood of resistance to a nucleotide analog such as 2-phenylam ⁇ no pynmidine.
  • Particular embodiments of die mvention descnbed herem m clude methods for specifically identifymg cells having some degree of resistance to STI-571.
  • Such methods typically mclude the step of sequencmg a target kmase such as Bcr-Abl to identify a mutation associated with a specific genotype or phenotype such as resistance to STI-571.
  • the mutation is widitn a domam shown to be associated with the cancer genotype and/or phenotype (e g. the ATP bmdmg domain of Bcr-Abl).
  • the mutation is m a Bcr-Abl residue identified m Table I below (or m an equivalent residue of a kmase having homology to Bcr-Abl).
  • the mvention disclosed herem allows artisans to examine MARS m a vanety of contexts to determme whether different mutations segregate with specific clinical phenotypes (e.g. lymphoid versus myeloid disease) or with different clinical patterns of STI-571 resistance (e.g. refractory disease; delayed relapse versus rapid relapse).
  • the mvention further allows those skilled m the art to determine whedier kmase domam mutations restricted to patients with advanced stage disease or also occur m chrome phase patients.
  • the mvention also allows those skilled in the art to determme whether one or more mutations are a manifestation of the clonal diversity and genetic instability associated with disease progression.
  • the mvention also allows those skilled m the art to determine whether such mutations are a consequence of pnor exposure to chemotherapy, or occur only m patients exposed to STI-571
  • the mvention also allows those skilled m the art to determme die biological implications for other targeted kmase inhibitors currendy in clinical development.
  • Methods for detecting and quantifying the expression of MARS mRNA or protem are descnbed herem and use standard nucleic acid and protem detection and quantification technologies well known m the art.
  • Standard methods for the detection and quantification of MARS mRNA m include m situ hybndization usmg labeled MARS nboprobes, northern blot and related techniques usmg MARS polynucleotide probes, RT-PCR analysis usmg primers specific for MARS, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like.
  • semi- quantitative RT-PCR may be used to detect and quantify MARS mRNA expression as descnbed m the Examples that follow.
  • Any number of primers capable of amplifying MARS may be used for this purpose, mcludmg but not limited to the vanous primer sets specifically descnbed herem. Standard methods for the detection and quantification of protem may be used for this purpose.
  • polyclonal or monoclonal antibodies specifically reactive widi the MARS protem may be used in an lmmunohistochemical assays of samples
  • Antibodies directed agamst MARS protem can also be used to detect MARS m a patient specimen (e.g, blood or other sample) usmg conventional tecliniques such as fluorescence-activated cell sorting (FACS) and/or ELISA.
  • FACS fluorescence-activated cell sorting
  • ELISA ELISA
  • the disclosure provided herem allows those skilled m the art to identify and characterize cells havmg a genotype and/or phenotype associated with a cancer such as a genotype and/or phenotype associated widi cancers of the myeloid lineage.
  • Specific embodiments of the mvention descnbed herem m clude methods for the identification and characterization of Bcr-Abl mutants associated resistance to a nucleotide analog such as 2- phenyla ino pyrimidine.
  • Particular embodiments of the mvention descnbed herem m clude mediods for the identification and charactenzation of cells having some degree of resistance to an inhibitor such as STI-571.
  • a first method for characterizing cells havmg a genotype and/or phenotype associated with a cancer cludes the sequencmg of Bcr-Abl m those cells to identify one or more mutations associated with a particular phenotype (e.g. resistance to STI-571) such as a mutation m a domam or region shown to be associated with a specific genotype and/or phenotype (e.g. the ATP bmdmg domam of Bcr-Abl).
  • a particular phenotype e.g. resistance to STI-571
  • the mutation is m a Bcr- Abl residue identified in Table I below.
  • a related method for charactenzmg cells havmg a genotype and/or phenotype associated with a cancer and/or cancer stage m cludes considering the location of the mutation m the context of the crystal structure of the ABL kmase domam bound to a variant STI-571 (see, e.g. Schindler et al. Science. 2000 Sep 15;289(5486).l 938-42).
  • This definition of the crystal structure allows one to evaluate whether the mutation might mterfere with the anti-leukemia activity of STI-571. Based on this analysis, one can p ⁇ ontize mutations for direct experimental analysis of ABL kmase activity, leukemogenicity and level of inhibition by STI-571.
  • Another mediod for characterizing cells havmg a genotype and/or phenotype associated with a cancer and/or cancer stage m cludes analyzmg another factor associated a genotype and/or phenotype associated with a cancer m a target cell bemg examined such as die stage of die disease progression, die relative frequency of the mutant within the population (e.g. is the clone a dommant population which provides evidence that they have a growth advantage).
  • Another method for characterizing cells havmg a genotype and/or phenotype associated with a cancer cludes engineering selected mutations mto wild-type BCR-ABL cDNA to create a mutant allele whose enzymological and biological properties can be examined direcdy.
  • Enzymology can be performed by measurmg tyrosme kinase activity m vitro or in cells usmg standard assays known m the art (see, e.g. those cited m Example 1).
  • Biological activity can be measured usmg standard oncogene transformation assays usmg growth factor dependent hematopoietic cell lines or primary mouse bone marrow cells (see, e.g. those cited m Example 1). In this way, resistance to STI-571 can be measured usmg such kmase assays and transformation assays.
  • die above descnbed assays for characte ⁇ zmg cells havmg a genotype and/or phenotype associated with a cancer can be performed mdependendy or m combination with each odier.
  • Residues shown to mutated m MARS occur m domains that are highly conserved among members of the protem kmase family (see, e.g. Hanks et al. Science 241: 42-51 (1988))
  • the finding that a highly conserved residue is mutated m cancers and d at this mutation is associated with resistance to a chemod erapeutic agent provides evidence that this domam is associated with dysregulated cell growd and therefore identifies diese domains and residue position as a targets that the skilled artisan can use to evaluate the status of related members of the tyrosme kinase family (see, e.g. those identified m Figure 1 of Hanks et al.
  • the evaluation of the status of a domam and/or residue m the tyrosme kmase family member can be used to gam information on the disease potential of a tissue sample.
  • m a syndrome in which the dysregulation of a specific tyrosme kmase family member is known or suspected preferably one that exhibits a pattern of pathology that is similar to that seen with Bcr-Abl
  • one can determine if a mutation has occurred at that residue m order to obtain evidence of genetic changes associated widi growth dysregulation e.g.
  • hybndization assays usmg complementary DNA probes (such as in situ hybndization, Northern blot and related techmques) and vanous nucleic acid amplification assays (such as RT-PCR usmg complementary pnmers specific for die mRNA of mterest, and other amphfication type detection methods, such as, for example, branched DNA, SISBA, TMA and die like).
  • hybndization assays usmg complementary DNA probes (such as in situ hybndization, Northern blot and related techmques) and vanous nucleic acid amplification assays (such as RT-PCR usmg complementary pnmers specific for die mRNA of mterest, and other amphfication type detection methods, such as, for example, branched DNA, SISBA, TMA and die like).
  • Embodiments of the mvention mclude methods for identifying a functional hotspot (e.g. a region in a protem which has significant functional importance m kmase activity and drug resistance) m a target kmase compnsmg sequencmg at least a portion of the target kmase to identify a mutation and companng the location of die mutation to the location of functional hotspots identified m a homologous kinase (e.g.
  • Bcr-Abl wherem die identification of a mutation m a target kmase that conesponds to a hotspot m a homologous kmase provides evidence that the mutation m the target kmase is m a functional hotspot.
  • the hotspot occurs m a Bcr-Abl domain havmg mutations associated with STI-571 resistance (e.g. the activation loop). More preferably the hotspot occurs m a Bcr-Abl residue identified in Table I.
  • the homologous kinase is Bcr- Abl and the homologies are compared via a BLAST analysis.
  • the target kmase may be any one of a wide vanety of kinases known m die art such as c-kit, PDGFR, EGFR and VEGFR or one of the kinases identified m Figure 1 of Hanks et al , Science 241: 42-51 (1988).
  • these methods can be used to characterize cells from patients suffering from a pathology associated with abenant expression of the target kmase.
  • mventions include mediods for assessmg the likelihood of a target kmase havmg a resistance to a nucleotide analog such as 2-phenylam ⁇ no pyrimidine compnsmg sequencmg at least a portion of the target kmase to identify a mutation, wherem the identification of a mutation in a target kmase that corresponds to a hotspot m a homologous kinase provides evidence that target kmase will be resistant to the inhibitor.
  • the hotspot occurs in a Bcr-Abl domam havmg mutations associated with STI-571 resistance.
  • the hotspot occurs m a Bcr-Abl residue identified m Table I.
  • the homologous kmase is Bcr-Abl and the homologies are compared via a BLAST analysis.
  • the target kinase may be any one of a wide vanety of kinases known m the art such as c-kit, PDGFR, EGFR and VEGFR or one of die kinases identified m Figure 1 of Hanks et al. Science 241: 42-51 (1988) which is incorporated herem by reference.
  • these methods can be used to characterize cells from patients suffering from a pad ology associated widi aberrant expression of die target kmase.
  • the mvention disclosed herem mcludes the identification of ammo acid residues m Bcr-Abl that are mutated m a manner characterized such that they retam kmase activity yet are associated with resistance to mliibition of kinase activity by a 2- phenylaminopynmichne.
  • One embodmient of an mvention provided by diis disclosure is a mediod of identifymg such a mutation m an Abelson protem kinase, wherem the mutation is associated with the resistance to an inhibition of kinase activity by a 2- phenylaminopynmidme, the method compnsmg: determining an amino acid sequence of a portion of a polynucleotide encodmg the Abelson protem kinase to determine the presence of a mutation, wherem the mutation occurs at a ammo acid residue at the same relative position as a mutation m the C-Abl protem kmase shown m SEQ ID NO: 1 that is associated with STI-571 resistance as determined usmg the homology cntena of BLAST analysis.
  • mutations m the C-Abl protem kmase shown m SEQ ID NO. 1 that are associated with STI-571 resistance include mutations m the C-Abl protem kmase which have, for example, been identified m cancer cells isolated from individuals shown to exhibit a resistance to a d erapeutic regime mvolvmg a 2-phenylammopynm ⁇ chne such as STI-571.
  • mutants of the C-Abl protem kinase shown m SEQ ID NO: 1 that are identified as bemg associated with STI-571 resistance are readily characterized by any one of a wide variety of techmques that are well known m the art in view of the extensive biological characterization of c-Abl, Bcr-Abl and/or one of die Abelson protem kinases such as ARG etc.
  • Such protocols m clude analyses based on the understanding of the biological significance of a domam or residue within these protems that has been characterized as havmg significance m kmase activity or small molecule interaction (see, e g.
  • Example 3 which identifies vanous previously identified domams as well as residues which direcdy mteract widi STI-571 via previously descnbed crystallographic analyses etc). Such protocols further mclude biological analyses of biological activity of these mutants mcludmg for example, the well known assays for charactenzmg the kmase activities and transforming abilities of Abelson protem kinases that are cited in Example 1 below.
  • a related embodiment is a method of identifymg a mutant Abelson protem tyrosme kinase expressed by a cell by determining a nucleotide sequence of a portion of a polynucleotide encodmg the kmase domam of the Abelson protem tyrosme kmase expressed by the cell and then companng the nucleotide sequence so determined to that of the wild type sequence of the Abelson protem tyrosme kmase to identify the presence of a mutation, wherem the mutation so identified has the characteristics of occurrmg at a amino acid residue located within the polypeptide sequence of die Abelson protem tyrosme kmase at the same relative position as a mutation m the C-Abl protem kmase shown m SEQ ID NO: 1 that has been identified as bemg associated with a resistance to an inhibition of tyrosme kinase activity by a 2-phenylammopyr ⁇ m ⁇
  • the cell expressmg the mutant Abelson protem tyrosme kmase is found m a population of cancer cells that has been observed m clmical populations to exhibit a resistance to an inhibition of tyrosme kmase activity by a 2-phenylam ⁇ nopyr ⁇ m ⁇ chne (e.g. STI-571).
  • a 2-phenylam ⁇ nopyr ⁇ m ⁇ chne e.g. STI-571
  • the mutation m die C-Abl protem kinase shown m SEQ ID NO: 1 that has been identified as bemg associated with a resistance to an inhibition of tyrosme kmase activity by a 2-phenylam ⁇ nopyr ⁇ m ⁇ chne is a Bcr-Abl residue identified m Table I.
  • Yet another embodmient of the mvention is a method of identifymg a mutant Abelson tyrosme kinase expressed by a cell by determmmg a nucleotide sequence of a portion of the catalytic domam of the Abelson tyrosme kmase expressed by the cell (and more preferably the nucleotide bmdmg site within the catalytic domam) and then comparmg the nucleotide sequence so determined to that of the wild type sequence of die catalytic domam of the Abelson protem tyrosme kmase to identify the presence of a mutation within the catalytic domam, wherem the mutation so identified has the characteristics of occurrmg at a ammo acid residue located within the polypeptide sequence of the Abelson protem tyrosme kmase at a amino acid residue d at has homology to an ammo acid position m a C-Abl kmase shown m SEQ ID NO
  • an Abelson tyrosme kmase refers to the family of kinases known m the art to be closely related to the c-Abl protem or have domams d at share a high degree of homology with a domam m the c-Abl protem.
  • die Philadelphia translocation is known to result in the expression of a family of chmienc protems in which a portion of the Bcr protem is fused to c-Abl protem.
  • a specific groupmg of Abelson tyrosme kmase family members are those which exhibit an amino acid sequence homology that is structurally and/or functionally related such that a 2- phenylaminopynmichne can mteract with these molecules and inhibit their kmase activities (e.g. Bcr-Abl, TEL-Abl, c-kit, PDGFR, EGFR and VEGFR).
  • ARG Abelson tyrosme kinase family
  • ARG Abelson tyrosme kinase family
  • An analysis of the ammo acid sequence of the ARG protem reveals that it is closely related to that of c-Abl (see, e.g, Kruh et al, PNAS 1990, 87(15): 5802-6 and Wang et al, Oncogene 1996, 13(7): 1379-85).
  • c-Abl and ARG are strikingly similar with regard to overall structural architecture as well as the amino acid sequences of dieir tyrosme kmase domams.
  • Additional members of the family m include for example, Dash, Nabl, and Fes/Fps (see e.g. Hunter et al. Science 241, 42-51 (1988)).
  • STI-571 also mteracts with representative members of this family such as c-kit and PDGFR (see, e g , Tuveson et al ,
  • the catalytic domams of these protem kinases have a highly conserved structural and functional architecture which allows for the interaction of compounds of the 2-phenylammopynmidme class of molecules to mteract with this domam and further provides the basis for a vanety of comparative analyses as well as rational drug design (see, e.g., Traxler et al, Med Res Rev 2001 Nov;21 (6):499-512; Traxler et al, J Med Chem. 1999 Mar 25;42(6):1018-26; and Parang et al, Nat Struct Biol.
  • the domam compnsmg the ATP bmdmg site is identified as a region that is mutated m Bcr-Abl protems exhibiting resistance to STI-571.
  • other chemical classes of TK inhibitors are known to bmd the ATP binding site including quinazohnes and pyrazolo-pyrrolo-pyndopyrimidines (see, e g, Tian et al. Biochemistry. 2001 Jun 19;40(24):7084-91; Fry et al. Science. 1994 Aug 19;265(5175):1093-5; Rewcasde et al, J Med Chem.
  • TK inhibitors which bmd an ATP bmdmg site havmg a high homology to the ATP bmdmg site of Bcr-Abl (and mutants exhibiting resistance to such inhibitors) can be analogously identified and characterized usmg the disclosure provided herem.
  • the mvention provided herem identifies specific regions within conserved protem kmase family members that impart resistance to a class of tyrosme kmase inhibitors, thereby identifymg diese regions as the targets of die diagnostic protocols described herem.
  • kmase inhibitors such as 2-phenylarrunopynm ⁇ dmes
  • the disclosure provided herem identifies specific target domams (e.g. die ATP-binding domam) within protem l ⁇ nases m which ammo acid mutations can occur that render the kmase resistant to kmase inhibitors such as 2-phenylam ⁇ nopynm ⁇ chnes yet allow the kmase to retam a biological activity that is associated with a pathological condition (e.g. chrome myeloid leukemia).
  • a pathological condition e.g. chrome myeloid leukemia
  • Typical embodiments of the mvention mclude a method of identifymg a mutation m the catalytic domam of a target protem kmase compnsmg determmmg the amino acid sequence of the catalytic domam and companng it to the wild type sequence of the target protem kmase catalytic domam to identify a mutation dierein, wherem the catalytic domam of the target protem kmase has at least about 60, 70, 80, 85, 90 or 95% homology to the catalytic domam of c-able catalytic domam shown m SEQ ID NO: 1.
  • a related embodiment is a method of identifymg a mutation m the activation loop domam of a target protem kmase compnsmg determmmg die ammo acid sequence of the activation loop domam and companng it to the wild type sequence of the target protem kmase activation loop domam to identify a mutation therein, wherem the activation loop domam of the target protem kmase has at least about 60, 70, 80, 85, 90 or 95% homology to the activation loop domam of c-able activation loop domam shown m SEQ ID NO: 1.
  • a related embodiment is a method of identifymg a mutation m d e nucleotide bmdmg pocket of a target protem kinase compnsmg determining d e amino acid sequence of the nucleotide bmdmg pocket and companng it to the wild type sequence of the target protem kmase nucleotide bmdmg pocket domam to identify a mutation therein, wherem the nucleotide bmdmg pocket domam of the target protem kmase has at least about 60, 70, 80, 85, 90 or 95% homology to the nucleotide bmdmg pocket domam of c-able catalytic domam shown m SEQ ID NO: 1.
  • a related embodiment is a method of identifymg a mutation m a target tyrosme kmase that is likely to be associated with resistance to a tyrosme kmase inhibitor compnsmg determining the ammo acid sequence of die P-loop, hehx c, activation loop or catalytic sequences as well as sequences within about 10 ammo acids of die respective domam(s), and comparmg it to the wild type sequence of the target protem kmase P-loop, he x c, activation loop or catalytic sequences as well as sequences within about 10 amino acids of die respective domam(s) to identify a mutation therein, wherem the P-loop, hehx c, activation loop or catalytic sequences as well as sequences within about 10 amino acids of the respective domam(s) of die target protem kmase has at least about 60, 70, 80, 85, 90 or 95% homology to the P-loop, hehx
  • Another related embodiment is a method of isolating a polynucleotide encodmg a mutated catalytic domam of a target protem kmase compnsmg employing PCR to amplify the catalytic domam of a target protem kinase, wherem the target protem kinase exhibits a biological activity that is associated with a pathological condition and wherem the target protem kmase exhibits a resistance to tyrosme kmase inhibitors, and wherem the catalytic domam of the target protem kmase has at least about 60, 70, 80, 85, 90 or 95% homology to die catalytic domam of c-able catalytic domam shown m SEQ ID NO: 1, companng the polynucleotide sequence encoding the amino acid sequence of the catalytic domam and companng it to die polynucleotide sequence encoding the ammo acid sequence wild type
  • At least one amino acid residue that is mutated m the domam has homology to a residue identified in Table I.
  • the target protem kmase havmg the mutation exhibits a kmase activity diat is associated with a pathological condition (e.g. cancer).
  • d e kmase activity of the target protem kinase that is associated with a pathological condition e.g. cancer
  • a pathological condition e.g. cancer
  • the kmase activity of the target protem kmase that is associated with a pathological condition e.g.
  • the target protem kmase is shown m Table 2 of Hanks et al , Science 241: 42-51 (1988).
  • the target protem kmase is a Bcr-Abl, a TEL-Abl, a c-kit, a PDGFR, an EGFR, an VEGFR.
  • a related embodiment compnses a method of charactenzmg a property of a protem tyrosme kmase, wherem the protem kmase has at least about 60, 70, 80, 85, 90 or 95% homology to c-able shown m SEQ ID NO: 1 compnsmg determining whether the protem tyrosme kmase exhibits an activity that is associated with a pathological condition (e.g.
  • determmmg whether die protem tyrosme kmase exhibits resistance to a tyrosme kmase inhibitor (e g via a procedure identified herem or citations m the art), determmmg an ammo acid sequence of the protem tyrosme kmase, determining whether the ammo acid sequence of the protem tyrosme kmase contams a mutated residue, determining whether the mutated residue occurs m the catalytic domam, the activation loop and/or the ATP bmdmg domam and/or determining whether the mutated residue has homology to a residue shown m Table I, wherem the presence of a mutated residue occurrmg m the catalytic domam, d e activation loop and/or the ATP bmdmg domain and/or wherem the mutated residue has homology to a residue shown m Table I provides
  • the kinase activity of the protem k ase that is associated widi a pathological condition is resistant to inhibition by a 2-phenylammopynmidine.
  • the protem kmase is a protem kmase shown m Table 2 of Hanks et al , Science 241: 42-51 (1988)
  • d e protem kmase is a Bcr-Abl, a TEL-Abl, a c- kit, a PDGFR, an EGFR, an VEGFR.
  • Yet another embochment of die mvention is a method of identifymg a mutant Abelson protem tyrosme kmase expressed by a mammahan cancer cell by determmmg a nucleotide sequence of a portion of a polynucleotide encodmg the kmase domam of the Abelson protem tyrosme kmase expressed by the cell and then comparmg die nucleotide sequence so determined to that of the wild type sequence of the Abelson protem tyrosme kmase to identify the presence of a amino acid substitution m the mutant Abelson protem tyrosme kmase, wherem any ammo acid substitution so identified has the charactenstics of occurrmg at a ammo acid residue located wid in d e polypeptide sequence of the Abelson protem tyrosme kmase at the same relative position as an amino acid substitution m the C-Abl protem kmase
  • the mutant Abelson tyrosme kinase expressed by the cell is a mutant c-Abl (see, e.g. NCBI Accession P00519), Bcr-Abl (see, e.g. NCBI Accession NP_067585), PDGFR (see, e.g. NCBI Accession NP002600), c-kit (see, e.g. NCBI Accession CAA29458), TEL-Abl (see, e.g. NCBI Accession CAA84815), or TEL-PDGFR (see, e.g. NCBI Accession AAA19786).
  • a related embodiment of the mvention comprises repeating steps (a) -(b) another mammahan cancer cell obtamed from a different mdividual; and dien catalogmg the mutations found m the mutant Abelson protem tyrosme l ⁇ nases present in the mammahan cancer cells.
  • the cell expressmg die mutant Abelson protem tyrosme kmase is found in a population of mammahan cancer cells that are observed to exhibit a resistance to an mhibition of tyrosme kmase activity after exposure to a 2-phenylam ⁇ nopyr ⁇ m ⁇ chne.
  • the mammalian cancer cell is can be a human cancer cell obtamed from an mdividual selected for treatment with a tyrosme kmase mhibitor compnsmg a 2-phenylammopyr ⁇ rn ⁇ dme.
  • the ammo acid substitution confers resistance to mhibition of tyrosme kmase activity by a 2- phenylammopynmidme.
  • the mutation in the C-Abl protem kmase shown m SEQ ID NO: 1 that has been identified as bemg associated with a resistance to an mhibition of tyrosme kmase activity by a 2-phenylammopynmichne occurs at the same relative position as ammo acid residue D233, T243, M244, K245, G249, G250, G251 , Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291 , E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311 , 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327
  • amino acid substitution occurs at the same relative position as ammo acid residue G250, Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35, K357, 1360, V379 or H396.
  • the disclosure provided herem allows a mutant identified by one the methods disclosed herem to be further characterized.
  • SpecificaUy by utihzmg enzymological and/or biological assays described herem as weU as diose known m the art (illustrated by those disclosed, for example, m the Examples below), a mutant that is found to occur m a conserved target domam of a protem kmase can be readdy characterized to assess the biological significance of this mutation (e.g rendering the protem kmase resistant to kmase mhibitors such as 2-phenylammopyr ⁇ m ⁇ d ⁇ nes yet aUowmg die kmase to retam a biological activity that is associated with a pathological condition).
  • scanning amino acid analysis can also be employed m comparative analyses of compounds such as 2- phenylaminopynmidmes to identify the significance of one or more amino acids which are structuraUy and/or functionaUy mvolved m the interaction between Abelson tyrosme kmases and compounds such as 2-phenylammopynmidines (see, e.g. U.S. Patent No. 6,004,931 and 5,506,107).
  • scanning ammo acids are relatively smaU, neutral amino acids. Such ammo acids mclude alanme, glycine, serme, and cysteme.
  • Alanme is typicaUy a preferred scanning ammo acid among this group because it eliminates die side-cham beyond the beta-carbon and is less likely to alter the main- cham conformation of the vanant. Alanme is also typicaUy preferred because it is the most common amino acid. Further, it is frequendy found m both buried and exposed positions [Creighton, The Proteins, (W.H Freeman & Co, N.Y.); Chothia, J. Mol. Biol, 150:1 (1976)]. If alanme substitution does not yield adequate amounts of variant, an lsostenc ammo acid can be used.
  • the MARS protem and nucleic acid sequences disclosed herem aUow a skilled artisan to identify protems, smaU molecules and other agents that mteract with MARS, as weU as pathways activated by MARS via any one of a vanety of art accepted protocols.
  • aUow a skilled artisan to identify protems, smaU molecules and other agents that mteract with MARS, as weU as pathways activated by MARS via any one of a vanety of art accepted protocols.
  • a representative embochment of this mvention comprises a method of screening for a molecule that mteracts with an MARS amino acid sequence compnsmg die steps of contacting a population of molecules with the MARS amino acid sequence, aUowmg the population of molecules and d e MARS ammo acid sequence to mteract under conditions that facihtate an interaction, determining the presence of a molecule diat mteracts with the MARS ammo acid sequence, and then separating molecules that do not mteract with the MARS amino acid sequence from molecules diat do.
  • the method furdier comprises punfymg a molecule diat mteracts with the MARS amino acid sequence.
  • the identified molecule can be used to modulate a function performed by MARS.
  • This embodiment of the mvention is weU suited to screen chemical hbranes for molecules which modulate, e.g, inhibit, antagonize, or agonize or niimic, the activity of BCR-ABL as measured by one of the assays disclosed herem.
  • the chemical hbranes can be peptide hbranes, peptidorritmetic hbranes, chemicaUy synthesized hbranes, recombmant, e.g, phage display hbranes, and m vitro translation-based libraries, other non-peptide synthetic organic hbranes (e.g.
  • hbranes of 2-phenylammopynmidines, quinazohnes or pyrazolo-pyrrolo-pyndopynmichnes and the like etc. are commerciaUy avadable from several sources (ArQule,
  • these chemical hbranes are generated usmg combmatonal strategies that encode die identity of each member of the library on a substrate to which the member compound is attached, thus aUowmg direct and immediate identification of a molecule that is an effective modulator.
  • the position on a plate of a compound specifies that compound's composition.
  • a smgle plate position may have from 1-20 chemicals d at can be screened by administration to a weU containing the interactions of mterest.
  • smaUer and smaUer pools of interacting pairs can be assayed for the modulation activity.
  • many candidate molecules can be screened.
  • Many diversity hbranes suitable for use are known m the art and can be used to provide compounds to be tested according to the present mvention.
  • hbranes can be constructed usmg standard methods. Chemical (synthetic) hbranes, recombmant expression hbranes, or polysome-based hbranes are exemplary types of hbranes that can be used.
  • peptides that bmd to a molecule such as MARS are identified by screening hbranes that encode a random or controUed coUection of amino acids.
  • Peptides encoded by the hbranes are expressed as fusion protems of bacte ⁇ ophage coat protems, the bacte ⁇ ophage particles are then screened agamst the protem of mterest.
  • peptides havmg a wide variety of uses are thus identified widiout any prior information on the structure of the expected ligand or receptor molecule
  • Typical peptide hbranes and screening methods that can be used to identify molecules that mteract widi MARS protem sequences are disclosed for example m U S. Patent Nos. 5,723,286 issued 3 March 1998 and 5,733,731 issued 31 March 1998.
  • SmaU molecules and ligands that mteract with MARS can be identified through related embodiments of such screening assays.
  • smaU molecules can be identified that mterfere with protem function, mcludmg molecules diat mterfere with a MARS's ability to mediate phosphorylation and de-phosphorylation.
  • a typical embodiment is a method of identifymg a compound which SpecificaUy bmds a MARS shown m Table I, wherem said MARS exhibits tyrosme kinase activity, compnsmg the steps of: contacting said MARS with a test compound under conditions favorable to bmdmg; and then determmmg whether said test compound bmds to said MARS so that a compound which bmds to said MARS can be identified.
  • skiUed artisans are familiar with the conditions conducive to bmdmg.
  • a specific embodiment of this aspect of the mvention mcludes the steps of transfecting ceUs with a construct encodmg the MARS, contacting said ceUs with said test compound that is tagged or labeUed with a detectable marker and then analyzmg said ceUs for the presence bound test compound.
  • a test compound which bmds said MARS may then be further screened for the mhibition of a biological activity (e.g. tyrosme kmase activity) of said MARS.
  • a biological activity e.g. tyrosme kmase activity
  • Such an embodiment mcludes, for example determining whether said test compound inhibits the tyrosme kmase activity of the MARS by utihzmg molecular biological protocols to create recombmant contracts whose enzymological and biological properties can be examined direcdy
  • a specific biological activity such as resistance to STI-571 can be measured usmg standard kmase assays and transformation assays.
  • Enzymology is performed for example, by measurmg tyrosme kinase activity m vitro or m MARS expressmg ceUs usmg standard assays (see, e.g. one of those cited m the Examples below).
  • biological activity is measured usmg standard oncogene transformation assays (see, e.g. one of those cited m the Examples below).
  • a specific embodiment of the mvention entails determining whether a test compound inhibits the biological activity of a MARS tyrosme kmase mhibitor m a procedure that is analogous for examinmg how STI-571 inhibits the tyrosme kmase activity of Bcr-Abl.
  • Yet another embodiment of the mvention is a method of identifymg a compound which SpecificaUy bmds a mutant Bcr-Abl polypeptide; wherem the Bcr-Abl polypeptide compnses an ammo acid substitution that occurs m a region of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 compnsmg residue D233 through residue T406, the method compnsmg the steps of contacting the mutant Bcr-Abl polypeptide with a test compound under conditions favorable to bmdmg; and determining whether the test compound SpecificaUy bmds to the mutant Bcr-Abl polypeptide such that a compound which bmds to the mutant Bcr-Abl polypeptide can be identified.
  • the bmdmg of the compound is typicaUy determined by any one of a wide vanety of assays known m the art such as ELISA, RIA, and/or BIAcore assay
  • the ammo acid substitution m the mutant Bcr-Abl polypeptide occurs at residue D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291, E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351, E352, E355, K357, N358, F359, 1360, L364, E373, N
  • a related embodiment of the mvention consists of the method descnbed above and further compnsmg determining whether the test compound inhibits the tyrosme kinase activity of the mutant Bcr-Abl polypeptide by transfecting mammalian ceUs with a construct encoding the mutant Bcr-Abl polypeptide, contacting the mammaban ceUs with the test compound; and then momtormg the mammahan ceUs for die tyrosme kinase activity of die mutant Bcr-Abl polypeptide, wherem an mhibition m tyrosme kmase activity m the presence of the test compound as compared to the absence of the test compound identifies the test compound as an mhibitor of the mutant Bcr-Abl polypeptide.
  • the tyrosme kmase activity of die mutant Bcr-Abl polypeptide is measured by examinmg die phosphotyrosme content of Cr
  • yet another embodiment of the mvention is a method of determining whether a test compound inhibits the tyrosme kmase activity of a mutant Bcr-Abl polypeptide, wherem the Bcr-Abl polypeptide compnses an ammo acid substitution that occurs m a region of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 compnsmg residue D233 through residue T406, the method compnsmg the steps of transfecting mammahan ceUs (e.g.
  • the ammo acid substitution occurs at residue D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291, E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311 , 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351 , E352, E355, K357, N358, F359, 1360, L364, E373, N374, K37
  • the tyrosme kmase activity of the mutant Bcr-Abl polypeptide is measured by examinmg the phosphotyrosme content of Crkl.
  • the tyrosme kmase activity of the mutant Bcr-Abl polypeptide is measured via Western blot analysis usmg an anti-phosphotyrosme antibody to examine the phosphotyrosme content of lysates of the mammahan ceUs. These mediods can be used to examine a wide vanety of compounds such as 2-phenylam ⁇ nopynm ⁇ chnes or pyndo [2,3-d]pyr ⁇ m ⁇ d ⁇ nes.
  • the ammo acid substitution does occur at one of the residues identified m Table IA (e.g residue T315) but not another of the residues identified m Table LA (e.g. residue E255).
  • kits are also provided by the mvention.
  • Such kits may compnse a carrier means bemg compartmentalized to receive m close confinement one or more contamer means such as vials, tubes, and the like, each of die contamer means compnsmg one of the separate elements to be used in the method.
  • one of the contamer means may compnse a probe that is or can be detectably labeled.
  • Such probe may be an antibody or polynucleotide specific for a MARS protem or a MARS gene or message, respectively.
  • the kit may also have contamers containing nucleotide(s) for amphfication of the target nucleic acid sequence and/or a contamer compnsmg a reporter-means, such as a biotin-binding protem, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or rachoisotope label.
  • a reporter-means such as a biotin-binding protem, such as avidin or streptavidin
  • the kit of the mvention wiU typicaUy comprise the contamer described above and one or more other contamers compnsmg matenals desirable from a commercial and user standpomt, mcludmg buffers, diluents, filters, needles, syrmges, and package mserts wid instructions for use.
  • a label may be present on the contamer to mdicate that the composition is used for a specific therapy or non-therapeutic application, and may also mdicate directions for either m vivo or m vitro use, such as diose described above EXAMPLES
  • EXAMPLE 1 ILLUSTRATIVE MATERIALS AND METHODS FOR EXAMINING BCR-ABL
  • a preferred experimental strategy is to use PCR to amplify a region of the BCR-ABL transcnpt usmg primers specific to BCR and ABL, subclone this product and sequence at least 10 mdependent clones m both directions. This strategy aUows one to quantify fluctuations m chfferent clones from d e same patient over time.
  • EXAMPLE 1A ILLUSTRATIVE METHODS FOR EXAMINING BCR-ABL POLYNUCLEOTIDE AND POLYPEP ⁇ DE SEQUENCES Blood samples were obtamed from consenting patients enroUed m clmical trials at UCLA assessmg the efficacy of STI-571 m the treatment of CML. RNA was extracted usmg TnReagent or TriAzol. CDNA synthesis was performed usmg MMTV reverse transcnptase.
  • PCR Polymerase cham reaction
  • CM10 5'-GAAGCTTCTCCCTGACATCCGT-3'
  • 3' Abl KD 5'-GCCAGGCTCTCGGGTGCAGTCC-3'
  • the resultant 1.3 kb fragment was excised from a low melting pomt agarose gel foUowing electrophoresis.
  • a second PCR was performed on the gel-purified 1.3 kb fragment to isolate the kinase domam usmg the primers 5' Abl KD (5'-GCGCAACAAGCCCACTGTCTATGG-3') (SEQ ID NO: 8) and 3' Abl KD.
  • the resultant 0.6 kb fragment was hgated mto pBluescript II KS+ digested with Eco RV Bacterial transformants were plated on media containing ampicilhn and X-gal. Ten white colonies per cDNA were inoculated mto media and miniprep DNA was isolated. Sequencmg of each clone was performed usmg Ml 3 universal forward and reverse primers Because two rounds of amphfication were employed, a mutation was considered present if it was detected on both strands of at least two mdependent clones per patient (see Figure 8).
  • mutant P210 isoforms were created as foUows.
  • Ohgonucleotides containing vanous pomt mutations were synthesized by Gibco/BRL.
  • PSRalphaP210Bcr-Abl was used as die template DNA for site-directed mutagenesis reactions utihzmg the mutant ohgonucleotides and the QuikChange mutagenesis kit (Stratagene). Successful mutagenesis was confirmed by sequence analysis of the kmase domam.
  • Other P210 abl constructs are known m the art (see, e.g. Sun et al. Cancer Res. 2002, 62(11): 3175-3183; Dugray et al. Leukemia 2001 15(10): 1658-1662; and Heisterkamp et al, Transgemc Res. 1991 1 (1): 45-53).
  • 293-T ceUs were co-transfected with mutant P210 expression vectors and a packagmg plasmid (Ecopack, kmdly provided by R. van Etten). Media containing virus was used to infect Ba/F3 ceUs. Stable lines were selected m the presence of G418 and IL-3. Subsequendy, IL-3 was removed from the media. Expression of Bcr-Abl was document by Western blot analysis. To determine the biochemical sensitivity of mutant P210 isoforms to STI-571, ceUs were mcubated m the presence of STI-571 (kmdly provided by Novartis, Switzerland) at 0, 0.5, 1, 5, and 10 micromoles per hter .
  • ceU lysates were prepared m 1% Tnton.
  • Western blot analysis usmg AB-3 (Oncogene Research Products) or 4G10 (Upstate Biochemicals) was performed.
  • Ba/F3 ceUs expressmg vanous isoforms of P210 were mcubated m the presence of STI-571 (kmdly provided by Novartis, Switzerland) at 0, 0.5, 1, 5, and 10 micromoles per hter. After 24 hours of incubation, live ceUs were quantitated by trypan blue stam exclusion.
  • EXAMPLE 1B ILLUSTRATIVE METHODS FOR EXAMINING DISCREET REGIONS IN BCR-ABL
  • a specific region m BCR-ABL such as one of the functional domams discussed herem.
  • a 579 base pair region corresponding to the ATP-binding pocket and the activation loop of the kinase domam of Bcr-Abl was sequenced m the 9 patients for whom RNA was available at the time of relapse (Fig. 4A). Briefly, RNA was extracted from purified peripheral blood or bone marrow ceUs with Tnreagent-LS (Molecular Research Center, Inc., Cincinnati, OH).
  • RNA 2 mg was subjected to RT-PCR usmg Ohgo dT primers
  • a 1327-bp cDNA fragment was amphfied by PCR with a 5' BCR-specific primer (5'- GAAGCTTCTCCCTGGCATCCGT-3') (SEQ ID NO: 6) and a 3 * ABL-specific primer (5'-GCCAGGCTCTCGGGTGCAGTCC-3') (SEQ ID NO: 7).
  • the BCR-ABL fragment could not be amphfied; therefore, a 579-bp fragment was amphfied usmg an alternative 5' ABL-specific primer (5'-GCGCAACAAGCCCACTGTCTATGG- 3') (SEQ ID NO: 8) and die same 3' ABL primer.
  • PCR products were cloned mto the pCR2.1 TA clomng vector (Invitrogen, Carlsbad, CA) Bodi strands of a 579-bp region were sequenced with the 5' ABL primer and Ml 3 forward prmier or Ml 3 forward and reverse primer set for the 1327-bp and the 579-bp fragments, respectively, on an ABI pnsm 377 automated DNA sequencer (PE Applied Biosystems, Foster City, CA) Sequence analysis was performed usmg die ClustalW alignment algorithm). A smgle, identical C— >T nucleotide change was detected at ABL nucleotide 944 m six of nine cases examined (F ⁇ g.4A).
  • genomic DNA was extracted from punfied bone marrow or penpheral blood ceUs with the QiaAMP Blood Mini Kit (Qiagen, Inc , Valencia, CA).
  • a 361 -bp DNA fragment was amphfied by PCR with two primers (5'- GCAGAGTCAGAATCCTTCAG-3' (SEQ ID NO: 2) and 5'- TTTGTAAAAGGCTGCCCGGC-3') (SEQ ID NO: 3) which are specific for lntron sequences 5' and 3' of ABL exon 3, respectively.
  • PCR products were cloned and sequenced. Analysis of RNA or genomic DNA from pre-treatment samples failed to provide evidence of the mutation pnor to STI-571 therapy; however, we cannot rule out the possibility that rare ceUs bearmg the mutation exist prior to treatment
  • EXAMPLE 2 ILLUSTRATIVE METHODS FOR MEASURING OF BCR-ABL KINASE ACTIVITY VIA THE PHOSPHOTYROSINE CONTENT OF CRKL Aldiough the enzymatic activity of Bcr-Abl protem is readily measured m ceU lines (e.g. via one of the assays discussed herem below), at times such assays can be difficult to perform m a reproducible, quantitative fashion with clmical materials because Bcr-Abl is sub j ect to rapid degradation and dephosphorylation upon ceU lysis.
  • phosphorylated Crkl migrates with altered mobility m SDS-PAGE gels and can be quantified usmg densitometry.
  • Crkl phosphorylation m primary CML patient ceUs was mhibited m a dose- dependent manner when exposed to STI-571 and correlated with dephosphorylation of Bcr-Abl (Fig. 1A).
  • ceUs are lysed m 1% Triton X-100 buffer with protease and phosphatase mhibitors (see, e.g. A. Goga et al. Cell 82, 981 (1995)).
  • Equal amounts of protem are separated by SDS-PAGE, transferred to nitroceUulose and lrnmunoblotted with phosphotyrosme antibody (4G10, Upstate Biotechnologies, Lake Placid, NY), Abl antibody (pex5, (see, e.g. A. Goga et al. Cell 82, 981 (1995)), ⁇ -actin antibody (Sigma Chemicals, St. Louis, MO) or Crkl antiserum (Santa Cruz Biotechnology, Santa Cruz, CA).
  • Immunoreactive bands are visuahzed by ECL (Amersham Pharmacia Biotech, Piscataway, NJ). Several exposures are obtamed to ensure linear range of signal intensity. Optimal exposures are quantified by densitometry usmg ImageQuant software (Molecular Dynamics, Sunnyvale, CA)).
  • Furdier cytogenetic and FISH characterization of metaphase spreads from these patients showed a unique mverted duphcate Ph- cliromosome with interstitial amphfication of the BCR-ABL fusion gene (Fig. 3C).
  • the mverted duphcate Ph-chromosome could be detected prior to the initiation of STI-571.
  • additional copies of the aberrant Ph- chromosome were observed as STI-571 treatment continued, as weU as rmg chromosomes harbormg multiple copies of the BCR-ABL.
  • Patient MB 14 was reevaluated by FISH one month after receivmg alternative treatment for her leukemia.
  • genomic DNA was extracted from purified bone marrow or peripheral blood ceUs with the QiaAMP Blood Mini Kit (Qiagen, Inc , Valencia, CA). 10 ng of total genomic DNA was subjected to real-time PCR analysis with the iCycler lQ system (Bio-Rad Laboratories, Hercules, CA).
  • a 361- bp gDNA fragment mcludmg ABL exon 3 was amphfied usmg two primers (5'- GCAGAGTCAGAATCCTTCAG-3' (SEQ ID NO- 2) and 5'- TTTGTAAAAGGCTGCCCGGC-3' (SEQ ID NO- 3)) which are specific for lntron sequences 5' and 3' of ABL exon 3, respectively
  • a 472-bp gDNA fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amphfied usmg two primers (5'-TTCACCACCATGGAGAAGGC-3' (SEQ ID NO: 4) and 5'- CAGGAAATGAGCTTGACAAA-3' (SEQ ID NO.
  • Fold mcrease m ABL copy number was determined by calculating the chfference between threshold cycle numbers of ABL and GAPDH for each sample (DCt). Usmg control LB3 as reference sample, DCt from each sample was subtracted from DCt of control to determine D(DCt). Fold mcrease was calculated as 2- D ( DCt ).
  • a vanety of assays for measurmg the enzymological properties of protem kmases such as Abl are known m the art, for example those described m Konopka et al, Mol CeU Biol. 1985 Nov;5(l l):3116-23; Davis et al, Mol CeU Biol. 1985 Jan;5(l):204-13; and Konopka et al, CeU. 1984 Jul;37(3):1035-42 the contents of winch are incorporated herem by reference. Usmg such assays the skilled artisan can measure the enzymological properties of mutant BCR-Abl protem l ⁇ nases.
  • a vanety of bioassays for measurmg the transforming activities of protem l ⁇ nases such as Abl are known m the art, for example those described m Lugo et al. Science. 1990 Mar 2;247(4946): 1079-82; Lugo et al, Mol CeU Biol. 1989 Mar;9 (3): 1263-70; Klucher et al , Blood. 1998 May 15,91 (10):3927-34; Renshaw et al, Mol CeU Biol. 1995 Mar;15(3):1286-93; Sirard et al. Blood. 1994 Mar 15;83(6):1575-85; LaneuviUe et al, Cancer Res.
  • EXAMPLE 5 ADDITIONAL ILLUSTRATIVE ANALYTICAL SCHEMES FOR CHARACTERIZING THE FUNCTIONAL IMPORTANCE OF BCR- ABL MUTATIONS
  • typical illustrative algo ⁇ thms such as those whose parameters are outlined below can be used to characterize the clmical importance of the various mutations found m the kmase domain.
  • aUustrative method one can examine the location of the mutation m the context of the crystal structure of the ABL kinase domam (winch has been solved bound to STI-571). This structure aUows one to postulate whether the mutation might mterfere with the anti-leukemia activity of STI-571. Based on this analysis, one can pnontize mutations for direct experimental analysis of ABL kmase activity, leukemogenicity and level of mhibition by STI-571.
  • Enzymology can be performed by measurmg tyrosme kmase activity m vitro or m ceUs usmg standard assays known m the art.
  • Biological activity can be measured usmg standard oncogene transformation assays usmg growth factor dependent hematopoietic ceU lines or primary mouse bone marrow ceUs.
  • Resistance to STI-571 can be measured usmg kmase assays and transformation assays.
  • EXAMPLE 6 USE OF INFORMATION REGARDING BCR-ABL DOMAINS AND CRYSTALLOGRAPHIC ANALYSIS TO CHARACTERIZE BCR-ABL MUTATIONS As d e certam domams within BCR-ABL have been characterized and the crystal structure of dns protem has been elucidated, this information can be used m con j unction with the disclosure provided herem to characterize MARS such diose shown m Table I and to illustrate their role m resistance to inhibition of tyrosme kmase activity by STI- 571. For example, from the initial inspection of diese mutations m the context of the ABL crystal structure, one can categorize the mutants, for example m the foUowmg groups:
  • Hehx C mutations e.g. ammo acid residue positions 304, 278
  • Hehx C is a key regulatory hehx m the kmase V304D, V304A. These are located at the mterface with hehx C; M278K, M278L: Surface exposed methionme is disordered (borders hehx C). The functional significance of mutations found within this region or proximal to this region (m a manner that can perturb the normal function of this region), are supported by references which characterize this aspect of BCR-ABL.
  • P loop mutations e.g. amino acid residue positions 253, 252, 250.
  • the P loop is die phosphate bmdmg loop whose conformation is thought to be induced by STI-571. These mutations could prevent the required conformation of the loop to accommodate STI-571.
  • Gly motifs m the P loop (249, 251 and 254). These are highly conserved across other l ⁇ nases (so caUed Gly-X-Gly-X- X-Gly motif) and presumably are essential for kmase function.
  • mutations m each of the X positions m the P loop The functional significance of mutations found within this region or proximal to this region (m a manner that can perturb the normal function of this region), are supported by references which characterize this aspect of BCR-ABL.
  • Y253F Direcdy stacks up agamst STI-571. -OH makes a tight H-bond with CL (or H20). Others: Q252H, Q252L, Q252R, G250E, E255K.
  • M351T van der Waal interactions widi His 361 which m turn mteracts direcdy with STI-571 piperazme group.
  • Thr mutation could disrupt d e packing here and weaken mteraction with STI-571.
  • this mutation may not affect compound 15 bmdmg (the one o ⁇ gmaUy crystallized with Abl) smce it has no piperazme group.
  • E355G at the end of the hehx that precedes the catalytic loop, which mteracts with the piperazme group of STI-571. Mutating to a Gly could make this region more flexible and weaken STI bmdmg.
  • Agam Compound 15 should be less affected by this mutation.
  • F317L direcdy stacks agamst STI-571. Leu mutation could weaken STI-571 bmdmg.
  • M290T, M290V makes direct van der Waal interactions with STI-571. Mutation to either T or V would weaken STI-571 bmdmg.
  • Activation loop mutations e.g. ammo acid residue positions 396.
  • the functional sigmficance of mutations found within this region or proximal to this region are supported by references which characterize this aspect of BCR-ABL.
  • H396K, H396R disordered part of the activation loop.
  • EXAMPLE 7 BCR-ABL POINT MUTANTS ISOLATED FROM PATIENTS WITH STI571-RESISTANT CHRONIC MYELOID LEUKEMIA REMAIN SENSITIVE TO INHIBITORS OF THE BCR-ABL CHAPERONE HEAT SHOCK PROTEIN 90
  • Heat shock protem 90 is a molecular chaperone winch affects the stability and function of multiple oncogemc protems mcludmg BCR-ABL (An WG et al, CeU Growth Differ. 2000;11 :355-360; Shiotsu et al,. Blood.
  • G is a benzoqumone ansamycm winch specificaUy mlnbits Hsp90 by competitively bmdmg to an ATP-binding pocket m the amino-terrninus of Hsp90 (Prodromou et al, CeU. 1997;90:65-75; Stebbms et al, CeU. 1997;89:239-250; Grenert et al, 1997;272:23843- 23850).
  • WUd- type and mutant P210 BCR-ABL were subsequendy subcloned mto the EcoRI site of pMSCVpuro (Clontech) for retrovirus generation.
  • Ecotropic retroviruses were generated by cotransfection of pMSCVpuro DNA and Ecopac retroviral packaging vector (kmdly provided by R. Van Etten) mto 293T ceUs usmg the CaCl 2 method (Muller AJ, et al , Mol CeU. Biol. 1991;11:1785-1792).
  • the murine hematopoietic ceU line Ba/F3 was maintained m RPMI1640 supplemented with 10% fetal bovme serum, L-glutamine, and 1 ng/ml of recombmant murine IL-3 (R&D).
  • Ba/F3 populations with stable BCR-ABL expression were derived by retroviral mfection of Ba/F3 ceUs m the presence of IL-3, and subsequent selection by puromycin.
  • IL-3-mdependent BCR-ABL-expressmg ceUs were denved by culturmg m IL-3-free media at low densities m 96-weU tissue culture plates.
  • BCR-ABL protem was also degraded m ceUs expressmg either T315I or E255K BCR- ABL, but this degradation occurred at a lower GA concentration (0.5 ⁇ M) (Figure 7A).
  • This apparendy enhanced degradation of the two mutant BCR-ABL protems was specific because degradation of another Hsp90 chent protem, RAF-1, was comparable m aU ceUs tested.
  • GA may have greater potency agamst mutant BCR-ABL protems compared to wUd-type.
  • AU BCR-ABL-expressmg ceUs were more sensitive to 17-AAG than Ba/F3 parental ceUs, and the STI571 -resistant BCR- ABL-expressmg ceUs agam displaying a heightened sensitivity to mhibition compared to wild-type BCR-ABL-expressmg ceUs (Table III).
  • EXAMPLE 8 IDENTIFICATION OF A NOVEL PYRIDOPYRIMIDINE INHIBITOR OF ABL KINASE THAT IS A PICOMOLAR INHIBITOR OF BCR-ABL DRIVEN K562 CELLS AND IS EFFECTIVE AGAINST STI571- RESISTANT BCR-ABL MUTANTS.
  • TK constitutively active Bcr-abl tyrosme kinase
  • STI571 is only transiendy effective m blast cnsis and drug resistance emerges by amphfication of or development of mutational changes m Bcr-abl.
  • TK mhibitors of the pyndo [2,3- ⁇ pyr ⁇ m ⁇ d ⁇ ne class, unrelated to STI571, and descnbe here a compound widi substantial activity agamst STI-resistant mutant Bcr-abl protems
  • This compound, PD 166326 is a dual specificity TK mhibitor and inhibits src and abl in vitro with IC50S of 6 and 8nM respectively.
  • PD 166326 inhibits the growth of K562 ceUs with IC50 of 300 picomolar, leading to apoptotic Gl arrest, while non-Bcr-abl ceU types require more dian 1000 times higher concentrations.
  • the T315I mutation widnn die ATP-binding pocket reduces the affinity of STI571 for this pocket while the structural basis for resistance of the E255K mutation is currendy unknown.
  • PD 166326 potendy inhibits the E255K mutant Bcr-abl protem and the growth of Bcr-ablE255K driven ceUs.
  • the T315I mutant Bcr-abl protem is resistant to PD166326, however the growth of Bcr-ablT315I driven ceUs is partiaUy sensitive to this compound, likely through die mhibition of Bcr-abl effector pathways.
  • CeU were cultured m RPMI medium supplemented with 100 U/ml pemcilhn, lOO ⁇ g/ml streptomycin, 4mM glutamine, 10% heat inactivated fetal bovme serum and mcubated at 37C m 5%C0 2 .
  • ceUs were seeded m 12-weU clusters at 10-20,000 ceUs per weU.
  • CeUs were placed m media containing various concentrations of the drugs with vehicle (DMSO) never contributing more than 0.1%. After 4-7 days, ceUs were counted usmg a coulter counter. AU experiments were performed m duphcate and results averaged.
  • PD 166326 was stored in a lOmg/ml DMSO solution and stored at -70C. The derivation and chemical structure of PD 166326 has been previously published (see e.g Kraker et al. Biochemical Pharmacology 60, 885-898. 2000). Cell cycle assays
  • CeUs were treated with mdicated concentrations of PD 166326 or vehicle (DMSO) for the mdicated times. For synchronization, ceUs were mcubated m media containing 5ug/ml aphidicohn for 24 hours, washed twice m PBS, and replaced m growth media. At die time of harvest, ceUs were washed once m PBS and ceU nuclei prepared by d e method of Nusse (see e.g. Nusse et al , Cytometry 1990;1 1 :813-821) and ceU cycle distribution determined by flow cytometnc analysis of DNA content usmg red fluorescence of 488nm excited edndium bromide stamed nuclei.
  • DMSO vehicle
  • CeUs were washed m PBS once and lysed m modified RIPA buffer (10 mM Na phosphate pH 7.2, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% NP-40, 1% Na deoxycholate, ImM Na Vanadate, and protease mhibitors).
  • C-abl kmase assays were performed usmg punfied recombmant c-abl and peptide substrate (New England Biolabs).
  • Kinase assays were performed m 50mM Tns-Cl pH 7.5, lOmM MgCL?, ImM ethylene glycol bis-ammoethyl ether tetraacetic acid (EGTA), 2mM dithiothreitol (DTT), 0.2% t ⁇ ton-X, lOOuM ATP, 40uM peptide substrate, m lOOul reaction volumes containing 50 units c-abl enzyme and I OUCI [ 32 P] ⁇ -ATP.
  • Reactions were aUowed to proceed for 10 minutes at 30C and stopped by addition of EDTA and boiling. Reaction products were spotted on phosphoceUulose paper, washed several times with phosphoric acid, then acetone, and counted m scintillation fluid. Pilot experiments were lnitiaUy performed to establish that these reaction conditions were m linear range. Bcr-Abl was immune precipitated from ceU lysates of K562 ceUs mamtamed m log-phase culture conditions.
  • reaction was stopped by the addition of SDS-PAGE sample buffer and heated at 100C for 10 minutes. Protems were separated on 7.5% SDS-polyacrylamide gels and gels were dned under vacuum and phosphorylation was visualized by autoradiography on x-ray film.
  • PD 166326 shows no significant activity agamst JNK l ⁇ nases, cychc AMP-dependent protem kmase (PKA), PKB- ⁇ , PKC- ⁇ , rho-dependent protem kmase, casern kmase-2, and phosphorylase kinase.
  • PKA cychc AMP-dependent protem kmase
  • PKB PKB- ⁇
  • PKC- ⁇ PKC- ⁇
  • rho-dependent protem kmase casern kmase-2
  • phosphorylase kinase phosphorylase kinase.
  • PD166326 also inhibits Bcr-abl kmase in vitro with
  • PD 166326 also inhibits Bcr-abl activity m ceUs as determined by Western blot analysis of Bcr-abl autophosphorylation m K562 ceUs. In these ceUs Bcr-abl autophosphorylation is mhibited widi IC50 of InM compared with lOOnM for STI571. Bcr-abl autophosphorylation correlates with Bcr-abl signalmg activity as shown by die paraUel decline of MAP kmase activity with inhibition of Bcr-abl m these assays.
  • Other Bcr-abl dnven ceU lines are also extremely sensitive to PD 166326 with IC50S of 0.8 and 6nM (see M07- p210 bcr ⁇ bl and BaF3-p210 b « - abl ).
  • the potent biologic activity of PD 166326 is highly specific for Bcr-abl-driven ceUs as additional hematopoetic and epithelial ceU lines are only mhibited at 2 to 3 logs higher concentrations and IC50S m d e 0.8-2uM range.
  • PD 166326 inhibits ceU prohferation specificaUy m the Gl phase of the ceU cycle.
  • concentrations that fully inhibit the growth of Bcr-abl positive ceUs but not other ceU types PD 166326 leads to accumulation of ceUs m the Gl phase accompanied by a significant mcrease m the number of apoptotic ceUs. Additional phases of the ceU cycle are not affected by this compound as shown by experiments with synchronized ceUs. K562 ceUs were synchronized at the Gl/S boundary with aphidicohn and released mto PD 166326 or vehicle and ceU cycle progression studied over die foUowmg 24 hours.
  • PD 166326 is a potent mhibitor of Bcr-abl k ase activity and inhibits Bcr-abl dnven ceU growth through mhibition of Gl progression leadmg to apoptotic ceU death.
  • Resistance to STI571 treatment is associated with mutations m the Bcr-abl oncoprotem that render it refractory to STI571 mhibition (see e.g Gorre et al. Science 2001;293:876-880). Because PD166326 inhibits both Src and Abl whereas STI571 only inhibits Abl, it may bmd Bcr-abl differendy than STI571.
  • the E255K mutation also hes within a region of Bcr-abl commonly mutated in relapsed patients, however the structural basis for STI571 resistance conferred by mutations m this region is not currendy understood.
  • BaF3 mouse hematopoietic ceU lines were stably transfected with either the wild-type p210 bcr " abl cDNA or the T315I or E255K mutant versions as previously descnbed (see e.g. Gorre et al. Science. 2001,293.876-880). Expression of Bcr-abl renders BaF3 ceUs IL-3 mdependent while control ceUs transfected with vector alone require IL-3 for growth.
  • the T315I and E255K mutant p210 bcr - abl ceUs are highly resistant.
  • resistance to STI571 does not appear to confer cross-resistance to PD 166326.
  • PD166326 inhibits the autophosphorylation of p210 Bcr - blE255K in vivo as effectively as die autophosphorylation of the wild type p210 Bcc abl , while this mutant is highly resistant to mhibition by STI571.
  • the p210 Bcr - ablT3151 mutant is resistant to PD166326 as it is to STI571.
  • PD166326 is also active agamst src l ⁇ nases and its anti-leukemic effects may be m part related to its mhibition of the src l ⁇ nases Hck and Lyn which function downstream of Bcr-abl.
  • the src l ⁇ nases Hck and Lyn are activated by Bcr-abl and may mediate some of the transforming functions of Bcr-abl.
  • Phosphorylation of tyr 416 in the catalytic domam is required for activation of src kinases, although the mechamsm by which Bcr- abl activates Hck and Lyn is not undeistood.
  • Hck activation of Hck by d e Bcr-ablTM 51 mutant is not inhibited by PD166326 and this correlates with the observed resistance of Bcr-abl T3151 activity to PD166326.
  • PD166326 inhibits die growdi of BaF3p210 Bcr - ablT3,SI ceUs with IC50 of 150nM, likely through additional mechamsms.
  • STI571 has revolutionized the treatment of CML, the problem of TK drug resistance is now emerging as a clmical reality. Resistance to STI571 appears to have a structural basis and newer TK mhibitors may also be susceptible to similar mechamsms of resistance. However TK mhibitors of a chfferent structural class may have more favorable bmdmg characteristics.
  • Dorsey et al mitiaUy reported that a src-selective TK mhibitor of the py ⁇ do [2,3- ⁇ pynm ⁇ d ⁇ ne class has substantial activity agamst Bcr-abl kmase (see e.g. Dorsey et al. Cancer Research.
  • the micromolar activity of PD166326 agamst the growth of non-Bcr-abl dnven ceUs is most likely mediated dirough mhibition of additional ceUular targets smce unlike Bcr-abl positive ceUs, the growth of Bcr-abl negative ceUs is mhibited during the S phase of the ceU cycle .
  • the picomolar potency and ceUular selectivity of PDl 66326 are sigmficandy superior to STI571 in vitro.
  • Smce Bcr-abl signalmg is known to mvolve the src family l ⁇ nases Hck and Lyn, and smce PDl 66326 is also a potent mhibitor of src family kmases, it is plausible that the biologic potency of this compound is related to dual mhibition of diese two functionaUy related tyrosme l ⁇ nases.
  • Hck associates with and phosphorylates Bcr-abl on Tyr 177 leading to recruitment of Grb2/Sos and activation of the Ras pathway (see e.g. Warmuth et al. Journal of Biological Chemistry. 1997;272:33260-33270).
  • Hck-mediated signalmg is essential for the transforming activity of Bcr-abl (see e.g. Lionberger et al. Journal of Biological Chemistry. 2000;275T8581-18585).
  • Lyn m Bcr-abl signalmg is less weU studied.
  • Lyn activity is also elevated m acute myeloid leukemia ceU lines and m these ceUs inhibition of Lyn expression usmg anti-sense molecules leads to decreased proUferative activity and mhibition of Lyn kinase activity usmg src family selective pharmacologic mhibitors leads to potent mhibition of ceU growth and colony formation (see e.g. Roginskaya et al. Leukemia. 1999;13:855-861). It is also possible that the potency of PDl 66326 is mediated through the mhibition of other, yet undiscovered ceUular protems, and our data does not exclude this possibihty. However the role of currendy unknown ceUular targets m mediating the growth inhibitory effects of this compound m Bcr-abl dnven ceUs is difficult to know until such candidate targets are identified and studied.
  • this activation loop favors an open and activating conformation which, by virtue of its amino-terminal anchor, mterferes with STI571 bmdmg to the ATP-binding pocket. Consistent with this model, die bmdmg of STI571 is selective for the mactive conformation of Abl, and this compound is unable to inhibit the catalytic activity of active phosphorylated Abl (see e g. Schmdler et al. Science 2000;289.1938-1942).
  • phosphorylation of the activation loop of Abl is catalyzed by the src family kmase Hck m Bcr-abl transformed ceUs Smce PDl 66326 also mlnbits Hck, this may prevent phosphorylation of the activation loop, destabilizing the Abl active conformation
  • This aUostenc mechamsm m addition to the direct bmdmg of PDl 66326 to the ATP-binchng pocket could provide dual mechamsms for its mhibition of Abl activation and provide the basis for its mcreased potency. Validation of these hypotheses awaits crystaUographic studies of PDl 66326 bound to Abl.
  • PDl 66326 is non-cross-resistant with STI571 and has substantial activity agamst the T315I and E255K STI571-res ⁇ stant Bcr-abl mutants This finding has important implications for the future design and use of TK mhibitors of aU lands, smce it is the first report showmg that TK-inhibitor resistance can be overcome by anodier TK-inhibitor of a different structural class. It is difficult to speculate on whether the development of resistance to PDl 66326 wiU be just as likely as with STI571, but smce these compounds are structuraUy unrelated, resistance to PDl 66326 will likely involve a different structural basis than resistance to STI571.
  • the T315I mutation seen m STI571 -resistant CML, precludes hydrogen bonding with STI571 and results m a stenc clash due to the extra hydrocarbon group m He (see e.g. Gorre et al. Science. 2001;293:876-880).
  • PD166326 does not inhibit the activity of Bcr- abl 0151 in vivo suggesting that Thr 315 is also important for its bmdmg within the ATP pocket of abl.
  • PD166326 has some activity agamst BaF3p210 T3151 ceUs and mlnbits their growdi with IC50 of 150nM.
  • Smce PDl 66326 is a potent inhibitor of src l ⁇ nases, and smce the src l ⁇ nases Hck and Lyn mediate some of the transforming activities of Bcr-abl, it is possible that PD166326 inhibits the growth of BaF3p2lO T3151 ceUs through the mhibition of Hck and Lyn.
  • Hck smce the src l ⁇ nases
  • Y 416 is a site of auto-phosphorylation m src l ⁇ nases, it may also be a substrate for phosphorylation by other l ⁇ nases. In fact m our experiments Hck Y 416 phosphorylation status paraUels Bcr-abl activity winch suggests that Hck Y 1 ⁇ may also be a substrate for Bcr-abl.
  • the ceUular IC50 of PDl 66326 agamst BaF3Bcr-abl E255K ceUs (15nM) is much lower than its activity m non-Bcr-abl dnven ceU types (0.8-2uM), and much greater than the activity of STI571 agamst this mutant. If die basis for Bcr-abl E255K resistance to STI571 is destabihzation of the mactive conformation, and if PDl 66326 m fact bmds to the active conformation, dien this would explain why PDl 66326 is effective in inhibiting Bcr-abl E255K . However validation of these hypotheses requires crystal structure data to better define the function of the Glu 255 residue and the bmdmg of PDl 66326 to Bcr-abl.
  • Tables 1A-1E identify typical MARS. The data are from analysis of patients, with an average of 10 clones sequenced per patient. These tables identify subgroups of mutations that are more likely to be significant because d ey occur m more than one patient or they are dommant (defined as bemg detected m at least 2 of 10 clones m the same patient). The observation that these mutants are showmg up so commonly provides further evidence that these mutations wiU turn out to be clmicaUy significant.
  • Table III Sensitivity of STI571-resistant BCR-ABL-transformed cells to geldanamycin and 17-AAG.
  • Table IV Detailed summary of Bcr-Abl kinase domain mutations by disease category.
  • MBC denotes relapsed myeloid blast cnsis despite STI-571.
  • LBC denotes relapsed lymphoid blast crisis.
  • CP denotes chrome phase with no cytogenetic response.
  • R-MBC denotes pre-STI-571 sample from myeloid blast cnsis patients whose disease was subsequendy refractory.

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Abstract

The invention described herein relates to novel genes and their encoded proteins, termed Mutants Associated with Resistance to STI-571 (e.g.,T315I Bcr-Abl), and to diagnostic and therapeutic methods and compositions useful in the management of various cancers that express MARS. The invention further provides methods for identifying molecules that bind to and/or modulate the functional activity of MARS.

Description

MUTATIONS IN THE BCR-ABL TYROSINE KINASE ASSOCIATED WITH
RESISTANCE TO STI-571
Statement of Government Support
This invenαon was made with Government support by a USPHS Naϋonal Research Service Award GM07185 (M.E.G.). The Government may have certain rights in tihis invention. Related Applications
This apphcauon claims priority under Section 119(e) from U S. Provisional Application Serial No. 60/298,728 filed June 14, 2001 and U S Provisional Apphcauon Serial No 60/331,709 filed November 20, 2001 , the contents of each of which are incorporated herein by reference. FIELD OF THE INVENTION
The invention described herein relates to novel genes and their encoded proteins, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express diem
BACKGROUND OF THE INVENTION Cancer is the second leading cause of human death next to coronary disease
Worldwide, millions of people die from cancer every year In the United States alone, cancer causes the death of well over a half-million people annually, widi some 1 4 million new cases diagnosed per year. Wliile deaths from heart disease have been declining significandy, those resulting from cancer generally are on the rise and are predicted to become the leading cause of death in the developed world.
Cancers are characterized by multiple oncogenic events that collectively contribute to the phenotype of advanced stage disease. With the advent of new drugs that target specific molecular abnormalities, it is important to know whether the initial oncogenic event continues to play a functional role at later stages of tumor progression and at relapse with the development of chemodierapy resistance This question has been addressed in transgenic mice through regulated expression of the initial oncogene In three models testing different oncogenes in different tissues, the primary oncogene was required to maintain the tumor phenotype, despite the presence of numerous additional oncogene and tumor suppressor mutations (see, e.g. L. Chin et al, Nature 400, 468 (1999); D. W. Felsher et al., Mol. Cell , 199 (1999); and C. S. Huettner et al., Nature Genet. 24, 57 (2000)) Recent clinical trials of die Abelson tyrosine kinase (Abl) inhibitor STI-571 in chronic myeloid leukemia (CML) allow this question to be addressed direcdy in human cancer (see, e.g. B. J. Druker et al., N. Engl. J. Med. 344, 1038 (2001); and B J. Druker et al., N Engl. ]. Med. 344, 1031 (2001)).
CML is a pluripotent hematopoietic stem cell disorder characterized by the Philadelphia (Ph) chromosome translocation (see, e.g. C. L Sawyers, JV. Engl. f. Med. 340, 1330 (1999); and S. Fade et al, N. Engl. J. Med. 341, 164 (1999)). The resulting BCR- ABL fusion gene encodes a cytoplasmic protein with constitutive tyrosine kinase activity (see, e g. J. B. Konopka et al., Proc. Natl. Acad. S t. U.SA. 82, 1810 (1985) and NCBI Accession NP__067585). Numerous experimental models have established diat BCR- ABL is an oncogene and is sufficient to produce CML-like disease in mice (see, e.g. G. Q. Daley et al , Science 247, 824 (1 90); and N Heisterkamp et al., Nature 344, 251 (1990)). CML progresses through distinct clinical stages. The earliest stage, termed chronic phase, is characterized by expansion of terminally differentiated neutrophils. Over several years die disease progresses to an acute phase termed blast crisis, characterized by maturation arrest with excessive numbers of undifferentiated myeloid or lymphoid progenitor cells. The BCR-ABL oncogene is expressed at all stages, but blast crisis is characterized by multiple additional genetic and molecular changes.
A series of inhibitors, based on die 2-phenylam opyrιmιdιne class of pharmacophores, has been identified diat have exceptionally high affinity and specificity for Abl (see, e.g., Zimmerman et al., Bloorg, Med. Chem. Lett. 7, 187 (1997). The most successful of these, STI-571 (formerly referred to as Novartis test compound CGP 57148 and also known as Gleevec and lmatinib), has been successfully tested in clinical trail a therapeutic agent for CML. STI-571 is a 2-phenylamιno pyrimidine that targets the ATP- binding site of the kinase domain of ABL (see, e.g. B. J. Druker et al., Nature Med. 2, 561 (1996)). In phase I clinical trials, STI-571 induced remissions in patients in chronic phase as well as blast crisis (see, e.g. B J. Druker et al, N. Engl. ]. Med. 344, 1038 (2001); and B. J. Druker e al, N. Engl. J. Med. 344, 1031 (2001)). While responses in chronic phase have been durable, remissions observed in blast crisis patients have usually lasted only 2-6 months, despite continued drug treatment (see, e.g. B. J. Druker et al, N. Engl. J. Med. 344, 1038 (2001)).
In view of the relapse observed in patients treated with STI-571 there is a need for an understanding of the mechanisms associated with STI-571 resistance in CML and related cancers as well as diagnostic and therapeutic procedures and compositions tailored to address dus phenomena. The invention provided herein satisfies this need.
SUMMARY OF THE INVENTION Clinical studies with the Abl tyrosine kinase inhibitor STI-571 in chrome myeloid leukemia (CML) demonstrate diat many patients with advanced stage disease respond initially but dien relapse. While, biochemical and molecular analysis of clinical materials from these patients shows that drug resistance is associated with reactivation of Bcr-Abl signal transduction, the specific events associated with this resistance have not been not well characterized.
The disclosure provided herein characterizes specific events associated widi such drug resistance by identifying specific domains within protein lαnases where amino acid mutations occur d at impart resistance to the kinase inhibitor yet allow the kinase to retain its biological activity. The disclosure provided herein further identifies diese regions as domains shown to be highly conserved among families of protein ktnases (e.g the c-Abl tyrosine kinase activation loop). Consequendy dus disclosure identifies those specific regions in protein lαnases that are to be analyzed in a variety of diagnostic protocols which examine drug resistance.
The invention described herein further includes novel genes and their encoded proteins expressed in cancer cells d at are associated with resistance to STI-571. Typically diese STI-571 resistant genes and their encoded proteins are mutants of Bcr- Abl, an oncogene that is expressed in chrome myeloid leukemias. The invention described herein discloses a number of Bcr-Abl Mutants Associated with Resistance to STI-571 (hereinafter these mutants are collectively described using the acronym "MARS"), as well as diagnostic and dierapeutic methods and compositions useful in the management of cancers that express these mutants.
A typical example of a MARS is a Bcr-Abl mutant having a single amino acid substitution in a Thr residue at position 315 of the Abl kinase (termed T315I Bcr-Abl) In clinical studies, patients exhibited STI-571 resistance associated with this mutation at residue 315, a residue in the Abl kinase domain known to form a critical hydrogen bond with this drug. Biochemical analyses of this mutant show that the Thr— »Ile change is sufficient to confer STI-571 resistance in a reconstitution experiment. Additional MARS are identified in Tables I provided below. The disclosure provided herein presents evidence that genetically complex cancers retain dependence on an initial oncogenic events and provides a strategy for identifying inhibitors of STI-571 resistance. The disclosure provided herein further provides for a variety of diagnostic methods for examining the characteristics of cancers such as chronic myeloid leukemia All poor knowledge of the Bcr-Abl tyrosine kinase is based on published sequence that has been in the public domain for >15 years. The invention provides novel sequences of DNA of die Bcr-Abl tyrosine kinase fusion protein that causes chrome myeloid leukemia (CML), which is present in a high fraction of patients who develop resistance to d e drug STI-571, which is soon to become standard of care for the treatment of CML. As disclosed herein, methods for evaluating the status of the MARS polypeptides and polynucleotides it can be used in the evaluation of cancers, for example to detect early relapse Moreover, MARS polypeptides and polynucleotides can be used to create assays to identify drugs winch inhibit the biological activity of these mutant proteins. T315I Bcr-Abl provides a representative example of the inventions provided by the MARS disclosed herein. The T315I Bcr-Abl mutant disclosed herein contains an amino acid change in the kinase domain of Bcr-Abl that inhibits ST1571 binding to Bcr- Abl. The T315I Bcr-Abl embodiment of the invention has been tested in a number of patient samples and confirmed at the sequence level. This mutant Bcr-Abl protein has been expressed in cells and shown to be resistant to STI-571. Therefore, patients develop resistance to the drug because it can no longer inhibit its kinase activity.
Knowledge of mutant sequences provide immediate utility for a number of mediods. In particular, currendy there are no methods for detecting or treating drug- resistant CML. Consequendy, the invention provided herein provides diagnostic tests for early relapse in CML as well as for drug development in the field of tyrosine kinase inhibitors. For example, the disclosure provided herein allows one to detect the presence of drug resistant cells in CML patients prior to relapse, using, for example, PCR based assays. Representative embodiments of d e invention include PCR and analogous assays that are used to detect resistant cells in patient blood samples.
The invention can also be practiced as a tool to identify molecules which bind and/or inhibit the mutant tyrosine kinases. A typical embodiment of this aspect of d e invention is a method of identifying a compound which specifically binds to a mutant protein kinase such as a Bcr-Abl mutant shown in Table I by contacting the mutant with a test compound under conditions favorable to binding; and then determining whether said test compound binds to the mutant so d at a compound wliich binds to the mutant is identified. Using such mediods one can perform structure-based drug design and/or high diroughput screening of chemical libraries to identify inhibitors of mutant tyrosine kinases. Such an inhibitor will have immediate clinical relevance. The invention provides polynucleotides corresponding or complementary to all or part of the MARS genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding MARS proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to the MARS genes or mRNA sequences or parts thereof, and polynucleotides or ohgonucleotides that hybridize to die MARS genes, mRNAs, or to MARS-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding MARS. Recombinant DNA molecules containing MARS polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of MARS gene products are also provided. The invention further provides MARS proteins and polypeptide fragments thereof. The invention further provides antibodies that bind to MARS proteins and polypeptide fragments diereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled widi a detectable marker. The invention furdier provides methods for detecting the presence and status of
MARS polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express MARS. A typical embodiment of this invention provides methods for monitoring MARS gene products in a tissue sample having or suspected of having some form of growd dysregulaαon such as cancer. One preferred embodiment of the invention is a method of identifying a mutant
Abelson tyrosine kinase expressed by a cell by determining a nucleotide sequence of a portion of d e catalytic domain of the Abelson tyrosine kinase expressed by the cell and then comparing the nucleotide sequence so determined to that of the wild type sequence of the catalytic domain of the Abelson protem tyrosine kmase to identify the presence of a mutation within the catalytic domam, wherein the mutation so identified has d e characteiistics of occurrmg at a amino acid residue located widim the polypeptide sequence of the Abelson protein tyrosine kinase at a amino acid residue diat has homology to an amino acid position in a Bcr-Abl kmase shown m SEQ ID NO: 1 diat is associated with a resistance to an inhibition of tyrosine kmase activity by a 2- phenylarmnopyπrnidine, wherem the homology between the amino acid residue located within the polypeptide sequence of the Abelson protem tyrosine kmase and die am o acid residue in the Bcr-Abl kinase shown m SEQ ID NO 1 diat is associated with a resistance to an inhibition of tyrosine kmase activity by a 2-phenylaminopyrimidme can be illustrated via a BLAST analysis. Anod er embodiment of d e invention is an isolated Bcr-Abl polypeptide comprising an ammo acid sequence which differs from the sequence of the Bcr-Abl of SEQ ID NO:l and has one or more amino acid substitutions at the residue position(s) m SEQ ID NO:l selected from the group consisting of: D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291 , E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311 , 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351, E352, E355, K357, N358, F359, 1360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396, A399, P402, and T406. A related embodiment of the invention is an isolated nucleic acid compnsmg a nucleotide sequence encoding the Bcr-Abl polypeptide. Other embodiments of the invention is a vector compnsmg this nucleic acid sequence, a host cell compnsmg such vectors (e.g. E. coli) as well as a method of making Bcr-Abl polypeptide variant polypeptide, compnsmg the steps of: providmg a host cell compnsmg such a vector; (b) providmg culture media; (c) culturmg the host cell m the culture media under conditions sufficient to express die Bcr-Abl polypeptide vanant polypeptide; (d) recovering the Bcr- Abl polypeptide variant polypeptide from the host cell or culture media; and (e) purifying die Bcr-Abl polypeptide vanant polypeptide. Yet another embodiment of the invention is a Bcr-Abl polypeptide vanant polypeptide that is chemically modified or conjugated or linked to a matrix or a heterologous protem.
The invention furdier provides vanous therapeutic compositions and strategies foi treating cancers that express MARS, including mediods for identifying molecules (e.g STI- 571 analogs) which inhibit the biological activities (e.g. kmase activity) of vanous MARS.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Clinical relapse of STI-571-treated patients is associated with persistent Bcr-Abl kmase activity. (A) Immunoblot analyses of one CML patient's bone marrow cells after a 2-hour incubation with different concentrations of STI-571 in vitro Whole cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with Crkl (top panel), phosphotyrosme (middle panel), and Abl (bottom panel) antibodies. (B) Crkl immunoblot of whole cell lysates from CML patients pnor to STI- 571 therapy (left) and from Ph-positive blast crisis patients who achieved hematological remission (<5% blasts) on STI-571 but remained 100% J3CR-.ABL-posιtive (right). (C) Crkl immunoblots of whole cell lysates from lymphoid blast crisis or Ph-positive acute lymphoid leukemia patients (top panel) and myeloid blast cnsis patients (middle panel) who relapsed after initially responding to STI-571 therapy. Phosphotyrosme immunoblot of patient cell lysates at time of relapse (bottom panel). Ph-positive cell l ne, K562, was used as a positive control for autophosphorylated Bcr-Abl. (D) Crkl lrnmunoblots of cell lysates from relapse patients taken prior to (pre-Tx) and during the course of (Tx and relapse) STI-571 therapy. Densitometnc analyses of Crkl lmmunoblots (expressed as percentage of phosphorylated Crkl over total Crkl protem) are presented in bar graphs.
Figure 2. Altered sensitivity of relapsed patient cells to STI-571. (A) STI-571 dose-response curves of Crkl phosphorylation m cells taken from blast crisis patients (LB3 and LB2) prior to STI-571 therapy (o) and at the time of relapse (•). Cells from both time pomts were exposed to mcreasmg concentrations of STI-571, harvested, and analyzed by Crkl immunoblot and densitometry. (B) IC50 values for inhibition of Crkl phosphorylation determined by exposure of cells isolated from untreated versus relapsed CML patients to mcreasmg concentrations of STI-571, and subsequent Crkl immunoblot and densitometnc analyses. Crkl phosphorylation in one relapsed patient sample (LB2) could not be mhibited with high concentrations of STI-571. (IC50 = concentration of STI-571 required to reduce CRKL phosphorylation by 50%).
Figure 3. BCR-ABL amplification m patients who relapsed after an initial response to STI-571. (A) BCR-ABL FISH analyses of lnterphase nuclei from blast crisis patient Ml 3 pnor to and durmg STI-571 dierapy. Nuclei are visualized with DAPI stam (blue), ABL probe is labeled wiui Spectrum Orange (red signal) and BCR probe is labeled with Spectrum Green (green signal) BCR-ABL gene fusions, indicated by yellow signals, show an increase in BCR-ABL gene amplification dur g STI-571 -resistant disease progression. (Bar = 10 μ. (B) BCR-ABL FISH analyses of interphase nuclei from blast cnsis patient M14 pnor to, durmg, and after removal from STI-571 dierapy showmg BCR-ABL-amph.&ed phenotype and reversion to non-amphfied phenotype upon removal from STI-571 therapy. (Bar = 10 μ. (C) Giemsa stained image (left panel; Bar = 5 μ) and dual color FISH images (middle and nght panels; Bars = 3 μ of sample from patient LB1 showmg duplicated mverted Ph-chromosome Arrows indicate BCR-ABL gene fusions. (D) Quantitative PCR analysis of genomic DNA from BCR-^ B -amphfied patients (MB13, MB14, LB1) and one non-amplified patient LB3 (control) confirming mcreased ABL gene copy number in all three patients.
Figure 4. Pomt mutation m die ATP-binding pocket of d e Abl kinase domam confers STI-571 resistance in relapsed patients. (A) Schematic of PCR strategy to determine the sequence of a 578 base pair region of BCR-ABL that corresponds to the ATP-binding pocket and activation loop of the kinase domain m patient samples. Ammo acid sequence of the region of Abl analyzed is shown m black. Residues predicted to form hydrogen bonds with STI-571, based on crystal structure data, are m boldface and are numbered from the first ammo acid of c-Abl (GenBank accession number- Ml 4752, shown in Table II) (SEQ ID NO: 1). Corresponding nucleotide sequence (shown in red) was aligned with sequences obtamed from nine patient cDNAs. The C— T mutation at ^LB nucleotide 944 (detected in six patients at relapse and m no pre-treatment samples) is shown in blue. Sequence of wild-type ABL exon 3 (GenBank accession number: NT008338.2) was aligned with sequences obtamed from patient genomic DNA pnor to treatment and at relapse. Examples of primary sequence data (represented as chromatographs) from wdd-type BCR-ABL (left) and BCR-ABL widi the C— T pomt mutation (right). (B) Model of STI-571 -binding pocket of wild-type Abl m complex with STI-571 (left panel) and predicted structure of STI-571 -binding pocket of T315I mutant Abl in complex with STI-571 (nght panel). In the molecular structures representing STI-571 and Abl residue 315, mtrogen atoms are shown in blue and oxygen atoms are shown in red. Van-der-Waals interactions are depicted m grey for STI-571 (both panels), m blue for wild-type Abl residue Thr315 (left panel), and in red for mutant Abl residue He315 (right panel). Polypeptide backbone of d e Abl kmase domam is represented m green. (C) Immunoblots of whole cell lysates isolated from transfected 293T cells (wild-type p210 BCR-ABL shown in left panels and T315I mutant shown in right panels) after a 2-hour incubation with different concentrations of STI-571 Blots were probed with phosphotyrosme (top panels) and Abl (bottom panels) antibodies. Figure 5. Graphic schematic of mutations m more than one patient. Figure 6. Bar graph schematic of total mutations m 2 or more patients. Figure 7. Geldanamycin and 17-AAG mduce degradation of wild-type and STI571 -resistant, mutant BCR-ABL protems and inhibit BCR-ABL signaling. (A) Ba/F3 cells expressmg wild-type, T315I, or E255K BCR-ABL were mcubated m the presence of mcreasmg concentrations of geldanamycm (GA) for 24 hours Immunoblottmg of cell lysates was performed with anti-ABL (Ab3, Oncogene) (upper panels), antι-RAF-1 (Santa Cruz) (middle panels), and anti-actin (ac-15, Sigma) as a control for protem loadmg (lower panels). (B) Ba/F3 cells expressmg wild-type, T315I, or E255K BCR-ABL were mcubated m the presence of mcreasmg concentrations of 17-AAG for 24 hours Immunoblotting of these lysates was performed with anti-ABL (upper panels) and anti- actin as a control for protem loadmg (lower panels). (C) Immunoblotting of the same lysates from (B) was performed with anti-CRKL (Santa Cruz). CRKL, when tyrosine- phosphorylated, migrates more slowly on SDS-PAGE resulting m an upper band representing phosphorylated CRKL (P-CRKL) and a lower band representing non- phosphorylated CRKL. (D) Densitometnc analysis of CRKL immunoblot shown m (C) usmg ImageQuant software (Molecular Dynamics). Quantified CRKL phosphorylation is expressed as percentage of phosphorylated CRKL over total CRKL protem (% P- CRKL). (E) Densitometnc analysis of CRKL immunoblotting usmg lysates from the same Ba/F3 cell lines mcubated m the presence of mcreasmg concentrations of STI571 for 24 hours. Figure 8. Schematic of Bcr-Abl kmase domam sequencing med odology. Bcr-
Abl cDNA is represented with Bcr sequences stippled, and Abl sequences m black. Horizontal arrows represent PCR primers. Initial PCR results in amplification of a 1.3 kb Bcr-Abl subfragment which serves as template for a second round PCR of the kmase domam which is then subcloned. Ten mdependent clones per patient time pomt were sequenced. Sequence deviations from wild-type Bcr-Abl observed m at least two of ten clones were considered mutations.
Figure 9. Bcr-Abl kinase domam mutants exhibit varying degrees of biochemical and biologic resistance to STI-571. Western blot usmg an anti- phosphotyrosine antibody (4G10) of lysates prepared from Ba/F3 populations mfected with retroviruses expressmg the Bcr-Abl isoforms mdicated and grown m the absence of IL-3 were exposed to varying concentrations of STI-571 for two hours are shown. Biochemical IC-50s for each of the mutations is shown. Biologic IC-50s were determined by viable cell count of cells after 48 hours of STI-571 exposure.
Figure 10. Imatmib-resistant mutations occur over a wide range of the Bcr-Abl kmase domam. The kmase domam ammo acid sequence of wild-type Bcr-Abl is shown.
Asterisks mark the conserved ammo acids of the Gly-X-Gly-X-X-Gly-X-Val consensus sequence found within the P-loop Amino acid substitutions found m STI-571 -resistant patients are mdicated beneath the wdd-type sequence
Figure 11. Summary of STI-571 -resistant Bcr-Abl kmase domam mutations. Each letter represents a patient with m whom the corresponding mutation was detected. Chrome phase patients are represented by the letter "C." Relapsed myeloid blast crisis patients are mdicated by the letter "M." Patients with relapsed lymphoid blast cnsis are represented by the letter "L." "R" indicates mutations pnor to STI-571 treatment m patients with myeloid blast cnsis who were refractory to treatment. Note that kmase domam is not drawn to scale
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise defined, all terms of art, notations and other scientific terminology used herem are intended to have the meanings commonly understood by those of skill m the art to which this mvention pertams In some cases, terms with commonly understood meanings are defined herem for clanty and/or for ready reference, and the inclusion of such definitions herem should not necessarily be construed to represent a substantial difference over what is generally understood m die art. The techniques and procedures described or referenced herem are generally well understood and commonly employed usmg conventional methodology by those skilled m the art, such as, for example, the widely utilized molecular cloning methodologies descnbed m Sambrook et al., 1989, Molecular Clomng: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y and Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995) As appropriate, procedures mvolvmg the use of commercially available kits and reagents are generally carried out in accordance wid manufacturer defined protocols and/or parameters unless od erwise noted.
As used herem, the term "polynucleotide" means a polymeric form of nucleotides of at least about 10 bases or base pairs m length, either nbonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include smgle and double stranded forms of DNA
As used herem, d e term "polypeptide" means a polymer of at least about 6 ammo acids. Throughout the specification, standard three letter or smgle letter designations for ammo acids are used. As used herem, a polynucleotide is said to be "isolated" when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than, for example, the MARS genes or that encode polypeptides other than
MARS gene product or fragments thereof As used herem, a polypeptide is said to be
"isolated" when it is substantially separated from contaminant polypeptide that correspond to polypeptides odier than the MARS polypeptides or fragments thereof. A skilled artisan can readily employ polynucleotide or polypeptide isolation procedures to obtain an isolated polynucleotides and polypeptides.
As used herem, d e terms "hybndize", "hybndizmg", "hybndizes" and the like, used m d e context of polynucleotides, are meant to refer to conventional hybndization conditions, preferably such as hybndization m 50% formamιde/6XSSC/0.1% SDS/100 μg/ml ssDNA, m which temperatures for hybridization are above 37 degrees C and temperatures for washing m 0.1X SSC/0.1% SDS are above 55 degrees C, and most preferably to stringent hybndization conditions.
"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, wliile shorter probes need lower temperatures.
Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present m an environment below their melting temperature. The higher d e degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows d at higher relative temperatures would tend to make d e reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al, Current Protocols m Molecular Biology, Wiley Interscience Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herem, may be identified by d ose that. (1) employ low lomc strength and high temperature for washing, for example 0.015 M sodium chlonde/0.0015 M sodium cιtrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ durmg hybndization a denaturing agent, such as formamide, for example, 50% (v/v) formamide widi 0 1% bovme serum albumm/0.1% Ficoll/0.1% polyvmylpyrrokdone/50mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chlonde, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0 1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chlonde/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.
"Moderately stringent conditions" may be identified as descnbed by Sambrook et al , 1989, Molecular Clomng: A Laboratory Manual, New York- Cold Spring Harbor Press, and mclude the use of washing solution and hybndization conditions (e.g., temperature, ionic strength and %SDS) less stringent d an those descnbed above. An example of moderately stringent conditions is overnight incubation at 37υC m a solution compnsmg: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM tπsodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters i x SSC at about 37-50°C. The skilled artisan will recognize how to adjust the tempeiature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
For purposes of shorthand designation of BCR-ABL variants described herem, it is noted that numbers refer to the amino acid residue position along the amino acid sequence of the BCR-ABL polypeptide Ammo acid identification uses d e single-letter alphabet of ammo acids, i.e.,
Asp D Aspartic acid He I Isoleucine
Thr T Threonine Leu L Leucme
Ser s Serme Tyr Y Tyrosine
Glu E Glutamic acid Phe F Phenylalanine
Pro P Proline His H Histidine
Gly G Glycme Lys K Lysme
Ala A Alanine Arg R Arginine
Cys C Cysteme Trp W Tryptophan
Val V Valine Gin Q Glutamine
Met M Methionme ASN N Asparagme
In die context of ammo acid sequence compansons, the term "identity" is used to identify and express the percentage of ammo acid residues at the same relative positions that are the same Also in this context, the term "homology" is used to identify and express the percentage of ammo acid residues at the same relative positions that are either identical or are similar, usmg d e conserved ammo acid cntena of BLAST analysis, as is generally understood m the art. For example, identity and homology values may be generated by WU-BLAST-2 (Altschul et al., Methods Enzymology, 266: 460-480 (1996): http://blast.wusd/edu/blast/ README.html).
"Percent (%) ammo acid sequence identity" with respect to the sequences identified herem is defined as the percentage of amino acid residues in a candidate sequence that are identical with the am o acid residues m the BCR-ABL sequence, after aligning the sequences and introducing gaps, if necessary, to achieve die maximum percent sequence identity. Alignment for purposes of determining percent ammo acid sequence identity can be achieved m various ways that are within d e skill m d e art can determine appropriate parameters for measuring alignment, including assigning algorithms needed to achieve maximal alignment over the full-length sequences bemg compared. For purposes herem, percent ammo acid identity values can also be obtamed usmg the sequence companson computer program, ALIGN-2, d e source code of which has been filed widi user documentation m the US Copyright Office, Washington, DC, 20559, registered under the US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, CA. All sequence comparison parameters are set by the ALIGN-2 program and do not vary The terms "cancer", "cancerous", or "malignant" refer to or describe the physiological condition m mammals that is typically characterized by unregulated cell growth. Examples of cancer mclude but are not limited to, leukemia, lymphoma, blastoma, carcmoma and sarcoma. More particular examples of such cancers mclude chrome myeloid leukemia, acute lymphoblastic leukemia, squamous cell carcmoma, small-cell lung cancer, non-small cell lung cancer, ghoma, gastrointestinal cancer, renal cancer, ovanan cancer, liver cancer, colorectal cancer, endometnal cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, ghoblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcmoma, and head and neck cancer. The terms "treating", "treatment" and "therapy" as used herem refer to curative therapy, prophylactic therapy, and preventative therapy. The terms "individual selected for treatment" refer to an individual who has been identified as having a condition diat artisans understand can respond to a specific therapy and, consequentially is being considered for treatment (or bemg treated with) d at dierapy (e.g. an individual suffering from chrome myelogenous leukemia who is bemg treated with STI-571).
The term "mammal" as used herem refers to any mammal classified as a mammal, mcludmg humans, cows, horses, dogs and cats In a preferred embodiment of the mvention, the mammal is a human.
Additional definitions are provided throughout the subsections that follow. The mvention descnbed herem relates to novel genes and their encoded protems, termed Mutants Associated with Resistance to STI-571 (e.g., T315I Bcr-Abl), and to diagnostic and therapeutic methods and compositions useful m the management of various cancers d at express MARS. Embodiments of the mvention provided herem are illustrated by studies of the Bcr-Abl protem kinase m STI-571 -treated patients. To charactenze the mechamsm of relapse m STI-57 -treated patients, we first assessed the status of Bcr-Abl signalmg m primary leukemia cells. As discussed m the Examples below, penpheral blood and/or bone marrow samples were obtamed with appropriate informed consent from CML and Ph-positive ALL patients at UCLA who were enrolled m multicenter clinical trials of STI-571 sponsored by Novartis Pharmaceuticals All patients had >30 percent blasts m die marrow prior to treatment. Responding patients had reduction m the percentage of bone marrow blasts to < 15 percent (partial) or < 5 percent (complete), as descnbed in B. J. Druker et al., N. Engl. J. Med. 344, 1031 (2001). Progressive disease was defined as an mcrease in percentage of blasts after an initial response, despite continued STI-571 treatment Mononuclear cells were isolated by centnfugation through Ficoll-Hypaque, washed twice in phosphate-buffered salme, counted and used immediately or cryopreserved.
A goal was to distinguish between Bcr-Abl dependent versus Bcr-Abl mdependent mechanisms of relapse. If Bcr-Abl remains critical for proliferation of the leukemia clone, then the Bcr-Abl signalmg pathway should be reactivated. Alternatively, if expansion of the leukemia clone is mdependent of Bcr-Abl, d en signalmg through d e Bcr-Abl pathway should remam impaired by STI-571. The most direct measure of signaling through Bcr-Abl pathway is the enzymatic activity of Bcr-Abl protem itself (see, e.g. J B. Konopka et al, Proc. Natl. Acad. Set. U.SA. 82, 1810 (1985); S S. Clark et al „ Science 235, 85 (1987); and S. S. Clark et al, Science 239, 775 (1988)). Although the enzymatic activity of Bcr-Abl protem is readily measured m cell lines, such assays are difficult to perform m a reproducible, quantitative fashion with clinical material because Bcr-Abl is subject to rapid degradation and dephosphorylation upon cell lysis. In a search for alternative measures of Bcr-Abl kmase activity, we found that the phosphotyrosme content of Crkl, an adaptor protem which is specifically and consαtutively phosphorylated by Bcr-Abl m CML cells (see, e.g. J. ten Hoeve et al , Blood 84, 1731 (1994); T. Oda et al, J. Biol Chem. 269, 22925 (1994); and G. L Nichols et al, Blood 84, 2912 (1994)), could be measured reproducibly and quantitatively in clinical specimens (see Example 2 below). Crkl bmds Bcr-Abl direcdy and plays a functional role in Bcr-Abl transformation by linking the kinase signal to downstream effector pathways (see, e.g. K. Senechal et al , /. Biol Chem. 271, 23255 (1996)). When phosphorylated, Crkl migrates witih altered mobility m SDS-PAGE gels and can be quantified usmg densitometry As expected, Crkl phosphorylation in primary CML patient cells was mhibited in a dose-dependent manner when exposed to STI-571 and correlated widi dephosphorylation of Bcr-Abl (Fig. 1A). This Crkl assay allows for an assessment of the enzymatic activity of Bcr-Abl protem m a reproducible, quantitative fashion in clinical matenals.
A recent preclinical study of STI-571 resistance m mice engrafted with a human blast crisis CML cell line demonstrated that l acid glycoprotem, an acute-phase reactant synthesized by the liver, can bmd STI-571 m serum and block its activity agamst Bcr-Abl (see, e g. C. Gambacorti-Passerini et al, J. Natl Cancer but. 92, 1641 (2000)). This observation raises the possibility that STI-571 resistance m patients is due to a host- mediated response agamst the drug Alternatively, resistance might be mediated by a cell-autonomous event m a leukemia subclone that allows escape from kinase inhibition by STI-571. To distinguish between these two possibilities, we determined the sensitivity of patient cells taken pnor to treatment and at the time of relapse to STI-571 by measuring inhibition of Crkl phosphorylation Briefly, punfied cells were plated at 1-10 x 106/ml m RPMI-1640 + 10% human AB serum with varying concentrations of STI-571 for 24 hours. Protems were extracted and subjected to immunoblot analysis.
If STI-571 resistance is a consequence of a host response, pretreatment and relapse leukemia cells should be equally sensitive to ex vivo STI-571 treatment. However, if STI-571 is cell-intrinsic, leukemia cells obtamed at relapse should be less sensitive to STI-571 dian pretreatment cells. In those patients for whom we had sufficient matched clinical material, a 10-fold or greater shift m sensitivity to STI-571 was observed at relapse (Fig. 2A). Aggregate analysis of 11 samples confirmed that higher concentrations of STI-571 are required to inhibit Crkl phosphorylation in patients cells obtamed at relapse versus pre-treatment (Fig. 2B).
Smce these ex vivo studies provide evidence that STI-571 resistance is cell- mtrmsic, we considered several possible mechanisms. Some CML cell lines that develop resistance to STI-571 after months of m vitro growth m sub-therapeutic doses of the drug have amplification of the BCR-ABL gene (see, e g. E. Weisberg et al , Blood 95, 3498 (2000); P. le Coutre et al, Blood 95, 1758 (2000), and F. X Mahon et al, Blood 96, 1070 (2000)) We performed dual-color fluorescence m situ hybndization (FISH) expermients to show that BCR-ABL gene amplification is similarly implicated in STI-571 resistance m human clinical samples (see Example 3 below) Through die disclosure, data from various groups of patients is discussed. Tables
IA-IF provide a summary of patient data. As illustrated m Example 1 below, we also considered the possibility that mutations in BCR-ABL might confer resistance to STI- 571. Consequendy, a 579 base pair region corresponding to d e ATP-binding pocket and the activation loop of the kinase domam of Bcr-Abl was sequenced m die 9 patients for whom RNA was available at the time of relapse (Fig 4A). A smgle, identical C— >T nucleotide change was detected at ABL nucleotide 944 m six of nine cases examined (Fιg.4A). In all six patients a mixture of wild-type and mutant cDNA clones were found, with the frequency of mutant clones ranging from 17% to 70%. The mutation was found m three of three patients with lymphoid disease and m three of six patients with myeloid blast crisis. The presence of the mutation was confirmed by analysis of genomic DNA
In the MARS designated T315I Bcr-Abl, a smgle nucleotide C— »T change results m a threonine to isoleucine substitution at position 315 of c-Abl. The recendy-solved crystal structure of the catalytic domam of Abl complexed widi a variant of STI-571 identified the ammo acid residues within the ATP-binding site and activation loop of c- Abl that are required for STI-571 bmdmg and thus inhibition of Abl kmase activity (see, e.g. T. Schmdler et al, Science 289, 1938 (2000)). Thr315 is among those that form critical hydrogen bonds with STI-571. The potential consequence of the T315I substitution on the STI-571 bmdmg pocket was modeled based on the crystal structure of the wild-type Abl kmase domam m complex with STI-571 (Fig. 4B) The absence of the oxygen atom normally provided by the side cham of Thr315 would preclude formation of a hydrogen bond with the secondary ammo group of STI-571. In addition, isoleucine contams an extra hydrocarbon group m the side cham, which would result m stenc clash with STI- 571 and presumably inhibit bmdmg. Notably, the model predicts d at the T315I mutation should not mterfere with ATP bmdmg. The structure of the kmase domam of Hck in complex widi an ATP analog (AMP-PNP) was superimposed onto the model of the Ile315 Abl kinase domam.
T315I Bcr-Abl is discussed as a representative embodiment of the MARS disclosed herem (e.g. those described m Table IA below). In certam descriptions of the mvention provided herem, embodiments of a smgle gene are used (T315I Bcr-Abl, for example) to illustrate typical embodiments of the mvention that apply to all of die MARS disclosed herem (e.g. E255K, Q252H, V304D, M351T, E355G etc. as shown m Table I) In th s context, artisans understand that discussing a typical embodiment directed to a smgle species (e.g. T315I) when the embodiments are commonly applicable to the other species disclosed herem (e.g. E255K, Q252H, V304D, M351T, E355G etc.) eliminates unnecessary redundancy m the descriptions of d e mvention.
The T315I mutation is shown to preserve kmase activity and, based upon the crystal structure of the kinase domam when bound to STI-571, is predicted to result in ineffective bmdmg of STI-571 to BCR/ABL. In an effort to define the full spectrum of kmase domam mutations m a larger sample size, we sequenced the BCR/ABL kmase domam m 18 patients with CML m myeloid blast crisis. In 13 patients, samples obtamed at the time of relapse after a partial or complete response to STI-571 (acquired resistance) were analyzed In 5 patients who did not respond to STI-571 (de novo resistance), analysis was performed on samples obtamed pnor to treatment. To ensure detection of subclones with kmase domam mutations that might account for a minority of BCR/ABL expressmg cells m the blood, we typically sequenced ten mdependent clones from each patient sample. A mutation was considered present only if it was detected by sequencmg of both cDNA strands. The previously identified T315I mutation was found in 3 additional patients. In conjunction widi our preliminary analysis of 11 patients (Gorre et al, 2001,
Science, Aug 3;293(5531):876-80), the T315I mutation has also been detected m subsequent studies of 9 of 28 patients (6/25 myeloid blast crisis, 3/3 with lymphoid blast cnsis or Ph+ ALL). Two other mutations, M351T and E255K, were also found m 4 patients and 3 patients respectively. Additional mutations were also found but did not always represent the dominant subclone at time of relapse. These fmdmgs mdicate that BCR/ABL kmase domam mutations occur commonly m CML blast cnsis and can be detected, m some cases, prior to STI-571 treatment. These mutations may be a reflection of genetic instability associated with disease progression or, possibly, prior treatment exposure. Following the protocols used to examine the T315I mutation, which we have previously shown to cause in vitro resistance to STI-571, the significance of these additional mutations m STI-571 drug resistance can be defined.
To confirm that this ammo acid substitution mterferes with STI-571 activity, we engineered the T315I mutaαon into wild-type p210 Bcr-Abl (see, e.g. Full-length p210 Bcr-Abl was subcloned into the pSRaMSVtkNeo retrovirus vector (see, e.g. A. J. Muller et al, Mol. Cell. Biol 11, 1785 (1991)). A fragment containing the C to T mutation at ABL nucleotide 944 was made by PCR and swapped with the corresponding sequence m pSRaMSVtkNeo p210 Bcr-Abl wild-type to create the pSRaMSVtkNeo p210 Bcr-Abl T315I mutant. The resulting construct was confirmed by sequencmg. Cells were transfected with wild-type or T315I p210 Bcr-Abl and cultured in the presence of mcreasmg concentrations of STI-571. Briefly, the transient transfection of 293T cells was performed usmg CaCi2 (see, e.g. A. J. Muller et al, Mol Cell. B ol 11, 1785 (1991)). After a 24-hour transfection, cells were mcubated with varying concentrations of STI-571 (provided by Novartis Pharmaceuticals, Basel, Switzerland) for 2 hours. Protems were extracted and subjected to immunoblot analysis. As shown by Abl immunoblot analysis, the expression of wild-type and T315I mutant Bcr-Abl protems was similar, and was not changed by STI-571 (Fig. 4C, bottom panels). Based on anti-phosphotyrosine immunoblot analysis, the kmase activities of wild-type Bcr-Abl and the T315I mutant appear comparable m the absence of STI-571 Whereas wild-type Bcr-Abl kmase activity was inhibited by STI-571, the T315I mutant retained high levels of phosphotyrosme at all concentrations of inhibitor tested (Fig. 4C, top panels).
In summary, our preliminary analysis of 11 patients with advanced stage CML who underwent disease progression after an initial response to STI-571 shows that reactivation of Bcr-Abl signaling occurred m all patients, despite continued STI-571 treatment. Therefore, the primary explanation for disease progression m these patients appears to be Bcr-Abl dependent proliferation rather than secondary oncogemc signals that permit Bcr-Abl mdependent growdi It is possible that studies of a larger number of patients may identify exceptions to this theme, as has been reported in transgenic mice expressmg conditional oncogenes where an occasional tumor can escape dependence on the initiating oncogene (see, e.g. L. Chin et al, Nature 400, 468 (1999); D. W. Felsher et al , Mol Cell 4, 199 (1999); and C. S. Huettner et al., Nature Genet. 24, 57 (2000)). In the majority of patients we studied, the mechamsm of resistance is a consequence of mutation or amplification of die target oncogene BCR-ABL (one patient had both events). These results provide evidence in a genetically complex human cancer diat a smgle molecular target remains relevant in late stage, relapsed disease. Interestingly for example, the identity of the Abl kmase domam mutation found m these patients bears remarkable similarity to a threonine to isoleucine change m v-Src versus c-Src at position 338, wliich corresponds to Thr315 m c-Abl. Despite d e fact that v-Src and c-Src have almost identical kmase domain sequences (98% identity), v-Src is approximately 50-fold more resistant than c-Src to kmase inhibition by the Src inhibitor PP1 (see, e.g. Y Liu et al, Chem. Biol 6, 671 (1999)
Eleven Patients described m our preliminary study obtamed complete hematologic remissions and, m some cases, complete cytogenetic remissions on STI-571, then relapsed within two to six months. This clinical scenario must be distinguished from those of patients who obtam only partial responses to STI-571 or fail to respond at all. In a phase II trial of 260 patients treated with STI-571 m myeloid blast crisis, only about 20% of patients fell into the former group. Therefore, d e 1 1 patients described m our study represented a highly select population. This distinction is important, because patients with partial hematologic responses and no cytogenetic response will have a substantial number of mature BCR-ABL expressmg hematopoietic cells diat persist durmg treatment and are not representative of the relapsmg, drug-resistant subclone. Smce the current protocols for mutation detection do not specifically isolate relapsmg, drug-resistant cells from other BCR-ABL ex-pressing blood cells, failure to detect a mutation might be explamed by an insensitive assay. In contrast, the dominant population of BCR-ABL expressmg cells m patients who relapse after a cytogenetic response will, by defimtion, be representative of the resistant subclone. Indeed, we found the T315I mutation m more than 80% of BCR-ABL expressmg cells from three such patients.
With respect to methodology, we subcloned our PCR products rather d an perform direct sequencmg, and we sequenced at least 10 mdependent clones per patient. All mutations required confirmation by sequencmg in both directions We chose d is strategy to maximize our sensitivity of detecting mutations d at may be present in a minority of BCR-ABL-expressmg cells. In addition, this method provided a rough quantitative estimate of the fraction of BCR-ABL-expressmg cells that contained the mutation, so that clonal evolution could be monitored over time. In retrospect, this method allowed us to find the T315I mutation m several patients m whom die resistant clone represented less than 20% of the BCR-ABL-expressmg cells.
Although the development of STI-571 resistance presents new dierapeutic challenges, the fact that Bcr-Abl remains active in STI-571 -resistant cells provides evidence that the chimeric oncoprotem remains a rational drug target. Because a significant fraction of the patients examined to date share an identical mutation associated with drug resistance, it may be possible to identify an inhibitor of d e mutant BCR-ABL allele that would have broad utility. In addition, knowledge of this mutation provides for d e development of a wide variety of assays to evaluate this mutation, for example to detect drug resistant clones prior to clinical relapse. See, e.g. B. J. Druker et al, N Engl J. Med. 344, 1038 (2001); A. Goga et al., Q// 82, 981 (1995); E Abruzzese et al, Cancer Genet. Cytogenet. 105, 164 (1998); J. D. Thompson et al., Nucleic. Acids Re 25, 4876 (1997); A. J. Muller et al, Mol Cell Biol 11, 1785 (1991).
As noted herem, analysis has revealed diat STI-571 resistance can occur through at least two distinct mechanisms. Some patients develop chromosomal amplification of the genomic region encodmg Bcr-Abl, resulting presumably m levels of Bcr-Abl protem that overcome the lntracellular concentration of STI-571 (Gorre et al, Science 293:876- 880 (2001)). A second mechamsm mvolves pomt mutations m the kinase domam that presumably mterfere with drug-protein bmdmg without compiomising kmase activity The best characterized of these mvolves a substitution of isoleucine for direonine at ammo acid position 315 (T315I) which alters the shape of the drug-bmdmg pocket based on a crystallographic-based model (Gorre et al., Science 293:876-880 (2001)). A limited number of odier mutations within the kmase domam has been reported at frequencies ranging from two of 44 cases (Barthe et al., Science 293:2163 (2001); Hochhaus et al., Science 293:2163 (2001)) to seven of eight m cases of acquired resistance (Von Bubnoff et al. Lancet 359:487-491 (2001); Lancet 359:487-491 (2001)). The result of one small study revealed no detectable mutations m a smgle patient with accelerated phase CML at die time of relapse, but found unique mutations, mcludmg E255K m each of five patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL), as well as m one patient with CML m lymphoid blast crisis (Von Bubnoff et al. Lancet 359:487-491 (2001)). A separate study also focused upon Philadelphia chromosome-positive ALL and detected E255K m six of nine patients at the time of relapse, as well as T315I m one of rune patients (Hofmann et al , Blood 99-1860-1862 (2002)) Most recendy, results from a heterogenous group of patients revealed the presence of Bcr-Abl kmase domain mutations in two of four cases of relapsed myeloid blast crisis (one patient was found to harbor T315I, and the second revealed evidence of a novel mutation, G250E), and m two of seven patients widi chrome phase disease who suffered progressive disease after initial hematologic response Evidence of new mutations G250E, F317L, and M351T was presented, but no biologic or biochemical assays were reported (Branford et al. Blood 99:3472-3475 (2002)). Given the reliance of leukemic cells upon Bcr-Abl activity at the time of STI-571 resistance, efforts to overcome STI-571 resistance must be equipped to deal with the most common mechanisms of resistance. Odier investigators have reported widely varying frequencies of kmase domam mutations usmg mediodology d at involved direct sequencmg of cDNA, which represents a consensus of sequences presence at the time of relapse. We sought to define the full spectrum of Bcr-Abl kinase domain mutations m cases of resistance usmg methods of mutation detection with supenor sensitivity. Here we report our sequence analyses of the Bcr-Abl kmase domam m patients treated with STI-571. These mclude cases of acquired resistance of myeloid blast crisis phase, cases of myeloid blast cnsis exhibiting de now resistance, cases of lymphoid blast cnsis, and cases of chrome phase cytogenetic refractoriness/relapse. We found evidence of Bcr- Abl kmase domain mutations m nearly all cases of acquired resistance.
Analysis of a subgroup of the more common mutations provides evidence that diese mutant isoforms retain the biologic activity of Bcr-Abl but exhibit varying degrees of resistance to STI-571 in bod biochemical and biological assays. Kinase domain pomt mutations apparendy represent a common mechamsm through which resistance to STI- 571 is acquired. Additionally, we provide the first evidence of polyclonal resistance to STI-571 m individual patients. Efforts to target Bcr-Abl m the setting of STI-571 - resistance will need to address the activities of the numerous mutant Bcr-Abl isoforms Medical management of CML patients receivmg targeted therapy will likely be facihtated by routine periodic assessment for kmase domam mutations. Lasdy, we provide evidence for pre-existing kmase domam mutations m a small number of patients with STI-571 -refractory myeloid blast cnsis pnor to institution of therapy, suggesting that the evolution of chrome phase CML to blast crisis CML may be, m some cases, facihtated by the accumulation of activating Bcr-Abl kmase domam mutations. Human malignancies, even those believed to rely upon a very small number of genetic alterations, likely comprise a significandy heterogeneous population of cells, and the developmg field of targeted therapy of malignancy appears to face daunting obstacles
In an effort to define the true mcidence and full spectrum of kmase domam mutations d at are capable of causmg resistance m cases of myeloid blast crisis, we performed sensitive sequence analysis of the Bcr-Abl kinase domam m patients whose disease relapsed after an initial response to STI-571 treatment ("acquired resistance"). We identified different mutations m patients with relapsed myeloid blast crisis m the vast majority of cases evaluated. Evidence of mutation was found m all four of the variable P-loop consensus (Gly-X-Gly-X-X-Gly-X-Val) amino acids. Mutations were found as far as 140 ammo acids away from the P-loop, and could be grouped by location to categories. Moreover, we provide evidence that resistance frequendy mvolves a polyclonal expansion of Bcr-Abl expressmg cells.
In vitro analysis of a subset of kmase domam mutations demonstrated varying degrees of STI-571 resistance relative to wild-type Bcr-Abl. We also analyzed the Bcr- Abl kmase domam m patients with chrome phase CML who had no cytogenetic response to STI-571 and found evidence of kmase domam mutations m a number of cases analyzed A subset of these kinase domam mutations were identical to those seen m relapsed myeloid blast crisis cases. Significandy, the presence of kmase domam mutation m this setting strongly correlated with disease progression and decreased overall survival. Lasdy, we found evidence of STI-571 -resistant kmase domam mutations prior to STI-571 treatment m a subset of patients with myeloid blast cnsis who subsequendy failed to respond to STI-571.
Multiple mutations m the Bcr-Abl kinase domam can be detected at the time of resistance m cases of myeloid blast crisis. Cytogenetic analysis of patients with myeloid blast cnsis whose disease initially responded to STI-571 revealed persistence of the Philadelphia chromosome m nearly 100 percent. We envisioned the possibility of resistant clones emerging from this population of Bcr-Abl containing cells, and therefore likely compnsmg a subset of die Philadelphia chromosome-containing cells at the time of relapse. By sequencmg ten mdependent PCR products per patient sample and requiring two mdependent isolates of a given mutation, we detect mutations that comprise as few as twenty percent of the population of Bcr-Abl sequences. Sequence analysis of d e Bcr- Abl kinase domam revealed evidence of pomt mutations m sixteen of seventeen cases of relapsed myeloid blast crisis (MBC) CML at the time of relapse (see Table Iλ7). The previously identified T315I mutation was detected. E255K, which has been previously described (Von Bubnoff et al. Lancet 359:487-491 (2001); Hofmann et al. Blood 99:1860-1862 (2002); Branford et al. Blood 99:3472-3475 (2002)), was also detected. M351T was also recendy reported (Branford et al. Blood 99:3472-3475 (2002)) and was also detected. The novel mutation Q252H as well as the recendy reported G250E (Branford et al. Blood 99:3472-3475 (2002)) were found at die time of relapse. Patients had one of two alternative substitutions at position Y253; mcludmg mutations which substituted histidine for tyrosine (Y253H), as previously described (Von Bubnoff et al. Lancet 359:487-491 (2001); Branford et al. Blood 99:3472-3475 (2002)). Interestingly, a novel conversion of tyrosine to phenylalanine (Y253F) was also observed. Phenylalanine is highly conserved at diis position m the Src-family of tyrosine lαnases, and when engmeered m to c-Abl, this mutation has been demonstrated to impart oncogenicity as reflected by cellular transformation assays (Allen et al, / Biol Chem 275:19585-19591 (1996)). We have recendy found d e sensitivity of Y253F to STI-571 to be intermediate between wild-type Bcr-Abl and die T315I mutant. Patients exhibited mutations within the activation loop at position H396, mvolvmg substitution to eidier proline or arginine Interestingly, several tyrosine kinases, mcludmg Hck, c-Src, v-src, lck, and Fyn all have an arginine at this position. Unique examples of die novel mutations V304D, E355G, and F359V, as well as the recendy reported F317L (Branford et al. Blood 99:3472-3475 (2002)) were each observed. Usmg our method of detection analysis, we were able to detect Bcr-Abl kmase domam mutations m the vast majority of samples obtamed from patients with relapsed myeloid blast crisis, mcludmg a number of novel mutations
In addition to offering greater sensitivity of mutation detection, our methodology afforded the ability to assess for polyclonal resistance to STI-571 , i.e. the presence of more than one resistant clone m a given patient. Indeed, a significant percentage of patients with myeloid blast crisis exhibiting acquired resistance to STI-571 were found to harbor more dian one mdependent mutation. Samples obtamed prior to treatment m cases of acquired resistance exhibited no evidence of mutation.
To address whether the surprisingly high frequency and vanety of kmase domam mutations represented artifact introduced durmg the PCR amplification process, we sequenced ten mdependent subclones of the Abl kmase domam obtamed from each of two healthy blood donors. Usmg our cntena of at least two mdependent isolates out of ten clones, we found no evidence of Abl kinase domam mutation. We therefore conclude that die Bcr-Abl kinase domam mutations descnbed here are highly unlikely to be the result of PCR-introduced error, and most probably represent accurate reflections of kmase domam sequence heterogeneity m these STI-571 -resistant patients.
Imatinib-resistant cases of lymphoid blast cnsis reveal kmase domam mutations similar to myeloid blast cnsis. Analysis of samples obtamed from four of five patients with lymphoid blast crisis (LBC) at the time of relapse revealed the presence of Bcr-Abl kmase domam mutations. Agam, clear evidence for polyclonal resistance was observed, with die coexistence of four separate mutations (Y253F, E255K, T315I, and M351T) m a smgle patient. Another patient harbored both E255K and Y253F. Two additional patients were found to harbor T315I in the absence of any odier mutations.
Bcr-Abl kinase domam mutations can be detected m chrome phase patients who fail to achieve cytogenetic remission or lose an established major cytogenetic response and are associated with disease progression and decreased survival. Cells from chrome phase patients who failed to obtam cytogenetic remission or who lost a previously achieved cytogenetic remission were subjected to sequence analysis of the Bcr-Abl kmase domam. Analysis was performed on samples obtamed at the time of sustained hematologic response. A number of patients were found to harbor mutations. Three of these mutations were also observed m cases of relapsed myeloid blast cnsis described above (E255K, F317L, F359V). F317L was recendy descnbed m a smgle patient (Branford et al. Blood 99:3472-3475 (2002)) with chronic phase disease and cytogenetic persistence who subsequendy suffered progressive disease. The last mutation, V379I, has not been documented in any other patient to date. Of the patients we studied, four have suffered progressive disease and have since discontinued STI-571. Among these, three have died and the fourth is living following subsequent allogeneic stem cell transplantation. Three of these four patients had Bcr-Abl kmase domam mutations (E255K, F317L, F359V) while one had no evidence of mutation. The patient harboring the V379I mutation continues to have a complete hematologic remission m response to STI-571 in the absence of a cytogenetic response. We conclude that kmase domam mutations occur m chrome phase patients who lose cytogenetic or hematologic responses to STI-571, and in a subset of chrome phase patients who have persistence of the Philadelphia chromosome m the setting of complete hematologic response.
Bcr-Abl kinase domam mutations can be detected prior to STI-571 treatment m patients with myeloid blast crisis that exhibit de novo resistance, but not in patients with STI-571 -sensitive myeloid blast crisis or chrome phase CML. To determine whether Bcr-Abl kmase domam mutations may play a role m de novo resistance to STI-571 , we analyzed pre-treatment samples from four patients with MBC who failed to achieve even a transient response to STI-571. One patient exhibited T315I prior to initiation of therapy. Also detected m the same patient was a Bcr-Abl allele that contamed two mutations, M343T and F382L. A second patient had the E255K mutation prior to STI- 571 treatment.
Bcr-Abl kmase domam mutations retam catalytic activity, and are capable of conferrmg STI-571 resistance in vitro. To assess whether the novel mutations observed were capable of conferring resistance to STI-571 in vitro, we performed site-directed mutagenesis of Bcr-Abl m a retroviral expression plasmid. In an illustrative embodiment of the mvention, eight of the observed mutations (G250E, Q252H, Y253F, E255K, T315I, F317L, M351T, and E355G) were mdependendy introduced mto pSRalphaP210Bcr-Abl. While these mutants are provided as preferred embodiments of the mvention descnbed herem, those skilled m the art can generate comparable mutants of any one of the MARS descπbed herem such as those identified m Table I.
Successful introduction of die expected mutations was confirmed by sequence analysis of the kinase domam. The eight mutations were each transiendy transfected mto 293-T cells, and found to exhibit varying degrees of sensitivity to STI-571, with IC-50 for enzymatic mliibition m cells rangmg from 1.27 uM to 5.63 uM as documented by phosphotyrosine-containing Bcr-Abl (see Figure 9). The murine hematopoietic cell line Ba/F3 requires exogenous IL-3 m the absence of Bcr-Abl. Stable Ba/F3 cell lines, capable of growing m the absence of interleukin-3, were derived for each of d e eight mutant isoforms, demonstrating diat each of the eight mutant isoforms retams biologic activity m this assay. The effect of
Figure imgf000029_0001
concentrations of STI-571 on cellular viability after 48 hours was determined. Agam, die eight mutant isoforms were found to exhibit varying degrees of sensitivity to STI-571. Several of the mutants appeared to impart only moderate resistance, retaimng sensitivity to concentrations of STI-571 which are theoretically achievable m patients (see Figure 9). Analysis of cells containing kmase domam mutations reveals no evidence of pomt mutation m Bcr-Abl immediately sequences 5' to d e kinase domam or m the tyrosine kinase domam of c-Kit. Genomic instability durmg advanced phase CML has been previously described. The high frequency of kmase domam mutations observed m our study, in addition to the finding of subpopulations of different mutations m individual patients, could theoretically be a reflection of a global decrease m DNA mismatch repair, or alternatively, may reflect a strong selection for these isoforms m the presence of STI-571. In an effort to address this issue, sequencmg of a 700 bp fragment of Bcr-Abl immediately 5' to the kmase domam was performed m five patients m whom several kmase domam mutations were detected. No evidence of additional mutation was found m these samples. We also assessed the kmase domam of the related tyrosine kinase c-Kit, which resides on chromosome 4 and exhibits sensitivity to STI-571 at concentrations equivalent to Bcr-Abl, in the same group of five patients No evidence of c-Kit kinase domam mutation was detected, arguing against the possibility of widespread genomic instability. We hypothesize that the increased genomic instability associated with blast crisis may result in a low background of Bcr-Abl sequence vanants, and STI- 571 strongly selects for the emergence of kinase-active STI-571 -resistant Bcr-Abl isoforms.
From the disclosure provided herem we conclude that with use of sensitive detection methods, Bcr-Abl kinase domam mutations can be detected m nearly all patients with relapsed myeloid blast crisis; that resistance frequendy mvolves the coexistence of cell populations containing different kinase domam mutations; that Bcr- Abl kmase domam mutations exhibit a wide range of STI-571 resistance in vitro; that kmase domam mutations occur in a subset of chrome phase CML patients with persistence of the Philadelphia chromosome, and portend a poor prognosis; and that some STI-571 -resistant kmase domam mutations can be occasionally detected in advanced phase cases CML prior to STI-571 treatment, and therefore may contribute to the leukemic drive in cells that harbor them. Bcr-Abl kmase domam mutations may thus contribute to the natural progression of CML from chrome to advanced phases m some cases. Given our findings, we believe routine sensitive sequence analysis of the Bcr-Abl kmase domam in patients bemg treated with STI-571 is warranted.
As noted above, die disclosure provided herem supports kmase domam mutation as the primary mechamsm for STI-571 failure. Previous studies of kmase domam mutations have been performed largely on isolated cases of Philadelphia chromosome- positive ALL and CML in lymphoid blast cnsis. Our finding of Bcr-Abl kmase domam mutations in nearly all cases of relapsed myeloid blast cnsis was not expected based upon previous reports. Because the complete cytogenetic remission rate is lower in myeloid blast crisis patients treated with Gleevec, it is possible that resistance to Gleevec m patients with lymphoid blast crisis CML and Ph+ ALL more commonly represents greater genetic homogeneity. Less sensitive methods of mutation detection may therefore adequately demonstrate the presence of nucleotide substitutions m these cases, yet fail to reliably detect mutations m relapsed myeloid blast crisis cases.
While some previous studies suggested a predominance of one to two different kmase domam mutations in the majonty of STI-571 -resistant cases, our expanded analysis of the Bcr-Abl kmase domam in resistant cases reveals a large spectrum of such mutations (see Figure 10). Inspection of the P-loop, which contams the consensus sequence Gly-X-Gly-X-X-Gly-X-Val, reveals the presence of STI-571 -resistant mutations at each of the non-conserved ammo acid sites Moreover, kinase domam mutations are exceedingly common m cases of acquired resistance.
The methodology utilized m the current study represents the only technique by wliich die sequence of mdividual mRNA molecules can be determined. We demonstrate here that cells from patients wid acquired resistance to STI-571 frequendy represent a polyclonal population, with different cells containing different Bcr-Abl kmase domam mutations. Furthermore, this methodology affords increased sensitivity by enabling the detection of mutant isoforms that comprise as litde as approximately 20 percent of the resistant population of cells. In many of our examples of polyclonal resistance, direct sequencmg would be predicted to yield a consensus wild-type kinase domam sequence, due to lack of a clonally dominant clone. Moreover, we provide the first direct evidence for the presence of two separate kmase domam mutations on a smgle strand of DNA. The method of mutation detection employed here is dius expected to be supenor to the method of direct cDNA sequencmg utilized by odier mvestigators, particularly m cases where emerging resistance is the result of polyclonal expansion.
We further document the first evidence of kmase domam mutations m cases of myeloid blast cnsis prior to treatment with STI-571. While it is formally possible that such mutants merely reflect genomic instability, the finding of such mutants at a frequency of twenty percent is more suggestive of a significant clonal expansion of these cells. It is possible that certain kmase domam mutations may confer a growth advantage m affected cells. The viral oncogene v-abl is known to contam pomt mutations in addition to alternative N-terminal coding sequences when compared with murine c-abl In this study we detected T315I pnor to treatment m a patient whose disease subsequendy failed to respond to STI-571. Interestingly, at the corresponding residue m the src gene, v-src differs from its cellular counterpart by substitution of isoleucine for threonine. Given a complete lack of kinase-domain mutations in pre-treatment samples obtamed from patients who subsequendy responded to STI-571, the presence of kmase domam mutations pnor to treatment may represent a marker for refractory disease, most likely related to mcreased genetic heterogeneity.
We detected kinase domam mutations in die majority of chrome phase patients who subsequendy suffered progressive disease, and m only one of rune patients who have a continued hematologic response on STI-571 despite the persistence of the Philadelphia chromosome. The ability to detect kinase domam mutations in this setting thus appears to serve as a strong predictor for the likelihood of hematologic relapse. Moreover, all diree patients with disease progression had evidence of their mutations prior to exhibiting clinical signs of progressive disease. Periodic mutation analysis m this setting may be warranted to facihtate alternative therapies. The only example of a detectable mutation m chronic phase CML without disease progression consisted of the V379I mutation, which has not been detected m any other patients. Given the correlation between the kmase domam mutations which have been shown to be functionally active in this study and disease progression in the chrome phase despite STI- 571 therapy, it may be useful to periodically perform kmase domam mutation analysis of patients on STI-571 who have any degree of persistence of die Philadelphia chromosome in an effort to anticipate disease progression and to facilitate the prompt mstitution of allogeneic transplantation or other treatment options.
Our finding of mutant P210 isoforms in the oveπvheltrung majority of patients at the time of acquired resistance reinforces pomt mutation m the Bcr-Abl kmase domam as a pnmary reason for STI-571 failure. The future of targeted therapy for CML is thus dependent upon overcoming STI-571 resistance mediated by Bcr-Abl kmase domam mutations. The differential sensitivity of kinase domam mutant isoforms to STI-571 deserves consideration. Given the sensitivity of some mutants, such as F317L, M351T, and E355G, to concentrations of STI-571 theoretically obtamable in humans, trials of higher doses of STI-571 may be warranted m some cases of acquired resistance. However, a few mutations, such as T315I, E255K, and G250E, clearly confer resistance to very high concentrations of STI-571. We speculate that medical management m the future of both chrome and advanced phase CML exhibiting acquired resistance to STI- 571 will necessitate mutation-specific PCR, and depending upon the presence or absence of certam mutations, dose escalation can be attempted. Should a highly resistant mutant isoform, such as T315I, E255K, or G250E subsequendy achieve clonal dominance, second generation drugs with activity agamst the most STI-571 -resistant isoforms could then be employed.
The clinical applicability of highly sensitive methods for mutation detection is most well-established m the treatment of human immunodeficiency virus (HIV), where, armed widi a number of targeted therapies, clinicians make treatment decisions periodically based upon the spectrum of retroviral mutations detected in the blood of dieir patients. Occasionally, drug-resistant mutations significandy hamper the ability of virus to replicate, and anα-retroviral agents are withdrawn in an effort to allow re- establishment of wild-type HIV. It will be important to characterize the biochemical and biological activity of each of the various mutant Bcr-Abl isoforms. If, m comparison with wild-type Bcr-Abl, some STI-571 -resistant mutations actually impart decreased growth promoting effects, intermittent STI-571 therapy could be instituted m an effort to delay disease progression toward the blast crisis stage
The development of STI-571 for the treatment of CML continues to represent a major advance toward the future of targeted therapy for human mahgnancies. Our work clearly implicates the activity of Bcr-Abl as essential to the malignant clone in nearly all acquired resistance cases studied. Imatmib is used much more commonly to treat chrome phase CML. Here we have provided examples of kmase domam mutations in four of fourteen cases of cytogenetic persistence despite STI-571 therapy. The presence of kmase domam mutation strongly correlated with subsequent development of progressive disease and decreased overall survival. The activity of Bcr-Abl therefore remains an optimal target for future therapies. In light of our fmdmgs, attempts to understand acquired resistance to other malignancies treated with STI-571, such as metastatic gastrointestinal stromal tumors, might logically begm with sensitive sequence analysis of the c-Kit kmase domam We envision the future of clinical management for CML to volve, in addition to the routine usage of sensitive kinase domam mutation detection mediods, combination molecular therapy, usmg multiple agents with the ability to target Bcr-Abl as well as kinase-active STI-571 -resistant isoforms in addition to downstream effectors. Typical embodiments of the mvention are described below.
MARS POLYNUCLEOTIDES
A number of specific sequences of MARS are identified m Table I below. One aspect of the mvention provides polynucleotides corresponding or complementary to all or part of a MARS gene, mRNA, and/or coding sequence, preferably in isolated form, mcludmg polynucleotides encodmg a MARS protem and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a MARS gene or mRNA sequence or a part thereof, and polynucleotides or ohgonucleotides that hybridize to a MARS gene, mRNA, or to a MARS encodmg polynucleotide (coUectively, "MARS polynucleotides"). As used herem, the MARS gene and protem is meant to mclude the MARS genes and protems specifically described herem and the genes and protems corresponding to MARS protems. Typical embodiments of the mvention disclosed herem mclude MARS polynucleotides containing specific portions of the MARS mRNA sequence (and those which are complementary to such sequences), for example, those that encode the T315I codon sequence.
Therefore, one specific aspect of the mvention provides polynucleotides corresponding or complementary to all or part of a T315I Bcr-Abl gene, mRNA, and/or coding sequence, preferably in isolated form, mcludmg polynucleotides encodmg a T315I Bcr-Abl protem and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or ohgonucleotides complementary to a T315I Bcr-Abl gene or mRNA sequence or a part diereof, and polynucleotides or ohgonucleotides that hybridize to a T315I Bcr-Abl gene, mRNA, or to a T315I Bcr-Abl encoding polynucleotide (collectively, "T315I Bcr-Abl polynucleotides"). As used herem, the T315I Bcr-Abl gene and protem is meant to mclude the T315I Bcr-Abl genes and protems specifically described herem and the genes and protems corresponding to T315I Bcr-Abl protems. Typical embodiments of the mvention disclosed herem mclude T315I Bcr-Abl polynucleotides containing specific portions of the T315I Bcr-Abl mRNA sequence (and those which are complementary to such sequences), for example, those that encode the T315I codon.
The MARS polynucleotides of the mvention are useful for a vanety of purposes, mcludmg but not limited to their in the detection of the MARS gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of cancers; as coding sequences capable of directing the expression of MARS polypeptides; as tools for modulating or inhibiting the function of the MARS protem.
Further specific embodiments of this aspect of the mvention mclude primers and primer pairs, which allow the specific amplification of the MARS polynucleotides of the mvention or of any specific parts thereof, and probes that selectively or specifically hybndize to nucleic acid molecules of the mvention or to any part thereof. Probes may be labeled with a detectable marker, such as, for example, a rachoisotope, fluorescent compound, bioluminescent compound, a chemilummescent compound, metal chelator or enzyme. Such probes and primers can be used to detect the presence of a MARS polynucleotide in a sample and as a means for detecting a cell expressmg a MARS protem
Examples of such probes and primers mclude polypeptides compnsmg all or part of a human MARS cDNA sequence shown m Table I. Examples of primer pairs capable of specifically amplifying MARS mRNAs (e.g. those primers disclosed herem) are readily ascertainable by those skilled in the art. As will be understood by the skilled artisan, a great many different primers and probes may be prepared based on the sequences provided m herem and used effectively to amplify and/or detect a MARS mRNA. RECOMBINANT DNA MOLECULES AND HOST-VECTOR SYSTEMS
The mvention also provides recombmant DNA or RNA molecules containing a MARS polynucleotide, mclud g but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known m the art, and cells transformed or transfected with such recombmant DNA or RNA molecules. As used herem, a recombmant DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular mampulation in vitro. Methods for generating such molecules are well known (see, for example, Sambrook et al, 1989, supra). The mvention further provides a host-vector system compnsmg a recombmant
DNA molecule containing a MARS polynucleotide within a suitable prokaryotic or eukaryotic host cell Examples of suitable eukaryotic host cells mclude a yeast cell, a plant cell, or an animal cell, such as a mammahan cell or an msect cell (e.g., a baculovirus- lnfecttble cell such as an Sf9 cell). Examples of suitable mammalian cells mclude vanous cancer cell lines, odier transfectable or ttansducible cell lines, mcludmg those mammalian cells routinely used for die expression of recombmant protems (e.g., COS, CHO, 293, 293T cells etc.). More particularly, a polynucleotide compnsmg the coding sequence of a MARS may be used to generate MARS protems or fragments thereof usmg any number of host vector systems routinely used and widely known m the art. A wide range of host vector systems suitable for the expression of MARS protems or fragments thereof are available, see for example, Sambrook et al, 1989, supra; Current Protocols m Molecular Biology, 1995, supra). Preferred vectors for mammalian expression mclude but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRαtkneo (Muller et al, 1991, MCB 11 :1785). Usmg these expression vectors, MARS may be preferably expressed m cell Imes, mcludmg for example CHO COS, 293, 293T, rat-1, 3T3 etc. The host vector systems of the mvention are useful for the production of a MARS protem or fragment thereof. Such host-vector systems may be employed to study the functional properties of MARS and MARS mutations. MARS POLYPEPTIDES
Another aspect of the present mvention provides MARS protems and polypeptide fragments thereof. The MARS protems of die mvention mclude those specifically identified herem. Fusion protems that combme parts of different MARS protems or fragments thereof, as well as fusion protems of a MARS protem and a heterologous polypeptide are also mcluded. Such MARS protems will be collectively referred to as the MARS protems, die protems of the mvention, or MARS. As used herem, the term "MARS polypeptide" refers to a polypeptide fragment or a MARS protem of at least about 6 ammo acids (e.g. a Bcr-Abl polypeptide havmg about 6 contiguous ammo acids mcludmg a MARS such as T315I, preferably at least about 10-15 amino acids)
Protems encoded by the MARS genes, or by fragments thereof, will have a variety of uses, mcludmg but not limited to generating antibodies and in methods for identifying ligands and odier agents (e.g. small molecules such as 2-phenylpynmιdmes) and cellular constituents that bmd to a MARS gene product. Antibodies raised agamst a MARS protem or fragment thereof may be useful m diagnostic and prognostic assays, imaging methodologies (mcludmg, particularly, cancer imaging), and therapeutic methods m the management of human cancers characterized by expression of a MARS protem, mcludmg but not limited to cancer of die lymphoid lineages. Vanous immunological assays useful for the detection of MARS protems are contemplated, mcludmg but not limited to vanous types of radioimmunoassays, enzyme-lmked immunosorbent assays (ELISA), enzyme-lmked lmmunofluorescent assays (ELIFA), lmmunocytochemical methods, and the like. Such antibodies may be labeled and used as immunological imaging reagents capable of detecting leukemia cells (e.g., m radioscintigraphic imaging methods).
MARS ANTIBODIES
The term "antibody" is used m the broadest sense and specifically covers smgle anti-MARS monoclonal antibodies (mcludmg agonist, antagonist and neutralizing antibodies) and anti-MARS antibody compositions with polyepitopic specificity. The term "monoclonal antibody" (mAb) as used herem refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the antibodies compnsmg die mdividual population are identical except for possible naturally-occurnng mutations diat may be present m minor amounts.
Another aspect of the mvention provides antibodies diat lmmunospecifically bmd to MARS protems and polypeptides The most preferred antibodies will specifically bmd to a MARS protem and will not bmd (or will bmd weakly) to Bcr-Abl protems and polypeptides. Anti-MARS antibodies that are particularly contemplated mclude monoclonal and polyclonal antibodies as well as fragments containing the antigen bmdmg domam and/or one or more complementanty determining regions of these antibodies. As used herem, an antibody fragment is defined as at least a portion of the vanable region of the immunoglobulin molecule that bmds to its target, i.e., die antigen bmdmg region.
For some applications, it may be desirable to generate antibodies which specifically react with a particular MARS protem and/or an epitope within a particular structural domain. For example, preferred antibodies useful for diagnostic purposes are those which react with an epitope m a mutated region of the MARS protem as expressed m cancer cells. Such antibodies may be generated by usmg die MARS protems descnbed herem, or usmg pepαdes denved from vanous domains thereof, as an immunogen.
MARS antibodies of the mvention may be particularly useful in cancer (e.g chrome myelogenous leukemia) therapeutic strategies, diagnostic and prognostic assays, and imagmg mediodologies. Similarly, such antibodies may be useful m the diagnosis, and/or prognosis of other cancers, to the extent MARS is also expressed or overexpressed m other types of cancer. The mvention provides vanous immunological assays useful for the detection and quantification of MARS and mutant MARS protems and polypeptides. Such assays generally comprise one or more MARS antibodies capable of recogmzmg and binding a MARS or mutant MARS protem, as appropriate, and may be performed within various immunological assay formats well known m die art, mcludmg but not limited to various types of radioimmunoassays, enzyme-hnked immunosorbent assays (ELISA), enzyme-hnked lmmunofluorescent assays (ELIFA), and the like. In addition, immunological imaging methods capable of detecting cancer cells are also provided by the mvention, mcludmg but limited to radioscmαgraphic imagmg mediods usmg labeled MARS antibodies. Such assays may be used clinically m the detection, monitoring, and prognosis of cancers, particularly chrome myeloid leukemia
MARS TRANSGENIC ANIMALS Nucleic acids that encode MARS can also be used to generate either transgemc animals which, m turn, are useful m die development and screening of therapeutically useful reagents. A transgemc animal (e.g, a mouse or rat) is an animal havmg cells that contam a transgene, which transgene was introduced mto the animal or an ancestor of the animal at a prenatal, e.g, an embryonic stage. A transgene is a DNA that is mtegrated mto the genome of a cell from which a transgemc animal develops. In one embodiment, cDNA encodmg T315I Bcr-Abl can be used to clone genomic DNA encodmg T315I Bcr-Abl m accordance with estabhshed techniques and d e genomic sequences used to generate transgemc animals that contam cells diat express DNA encodmg T315I Bcr-Abl. Methods for generating transgemc animals, particularly animals such as mice or rats, have become conventional m the art and are descnbed, for example, m U S. Patent Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for MARS transgene incorporation with tissue-specific enhancers. Transgemc animals that mclude a copy of a transgene encodmg MARS introduced mto die germ line of the animal at an embryonic stage can be used to examine the effect of mcreased expression of DNA encodmg MARS. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its expression. In accordance with this facet of the mvention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearmg the transgene, would mdicate a potential therapeutic intervention for the pathological condition.
METHODS FOR THE DETECTION OF MARS
Another aspect of the present mvention relates to mediods for detecting MARS polynucleotides and MARS protems, as well as mediods for identifying a cell that expresses MARS. The expression profile of MARS makes them diagnostic markers for disease states. As discussed in detail below, the status of MARS gene products m patient samples may be analyzed by a vanety protocols that are well known m the art mcludmg lmmunohistochemical analysis, the vanety of Nordiern blotting techmques mcludmg in situ hybndization, RT-PCR analysis (for example on laser capture micro-dissected samples), western blot analysis and tissue array analysis.
More particularly, the mvention provides assays for the detection of MARS polynucleotides m a biological sample, such as cell preparations, and the like. A number of methods for amplifying and/or detecting d e presence of MARS polynucleotides are well known in the art and may be employed in the practice of dns aspect of the invention In one embodiment, a method for detecting a MARS mRNA m a biological sample compnses producmg cDNA from the sample by reverse transcnption usmg at least one primer; amplifying the cDNA so produced usmg a MARS polynucleotides as sense and antisense primers to amplify MARS cDNAs therein; and detecting the presence of the amplified MARS cDNA. Any number of appropnate sense and antisense probe combmations may be designed from the nucleotide sequences provided for the MARS and used for this purpose.
The mvention also provides assays for detecting the presence of a MARS protem m a biological sample. Methods for detecting a MARS protem are also well known and mclude, for example, lmmunoprecipitation, lmmunohistochemical analysis, Western Blot analysis, molecular bmdmg assays, ELISA, ELIFA and the like. For example, m one embodiment, a mediod of detecting the presence of a MARS protem in a biological sample comprises first contacting the sample with a MARS antibody, a MARS-reacαve fragment diereof, or a recombmant protem containing an antigen bmdmg region of a MARS antibody; and then detecting the bmdmg of MARS protem in the sample thereto. Methods for identifying a cell that expresses MARS are also provided. In one embodiment, an assay for identifying a cell that expresses a MARS gene comprises detecting the presence of MARS mRNA m the cell. Methods for the detection of particular mRNAs m cells are well known and mclude, for example, hybridization assays usmg complementary DNA probes (such as in situ hybndization usmg labeled MARS nboprobes, Nordiern blot and related techmques) and vanous nucleic acid amphfication assays (such as RT-PCR usmg complementary pnmers specific for MARS, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA
Figure imgf000041_0001
A significant aspect of the mvention disclosed herem is the discovery that ammo acid substitutions m the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 can produce cancer cells havmg a resistance to tyrosine kmase inhibitors such as STI-571. Specifically those skilled m that art understand that the physiological mechanisms of drug resistance are diverse and that drug resistance typically occurs through other mechanisms such as an mcrease m the expression of protems that export the drug out of the cell (see, e.g. Suzuki et al, Curr Drug Metab 2001 Dec;2(4):367-77) Consequendy, die disclosure herem provides d e scientific evidence to confirm the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 as a target for analysis m mediods relating to identifying drug resistant cells, such as mediods of identifying an ammo acid substitution in at least one Bcr-Abl polypeptide expressed m human cancer cell from an mdividual selected for treatment with a tyrosine kinase inhibitor.
A preferred embodiment of the mvention is a method of identifying at least one ammo acid substitution m at least one Bcr-Abl polypeptide havmg some level of tyrosine kmase activity that is expressed m a human cancer cell from an mdividual selected for treatment with a tyrosine kmase inhibitor, the mediod compnsmg determining d e polypeptide sequence of at least one Bcr-Abl polypeptide expressed by die human cancer cell and comparing the polypeptide sequence of the Bcr-Abl polypeptide expressed by the human cancer cell to the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 so diat an am o acid substitution m die Bcr-Abl polypeptide expressed by die human cancer cell can be identified. In prefeired methods of the mvention, an ammo acid substitution so identified confers some level of resistance to STI-571.
A significant aspect of the mvention disclosed herem is the delineation of a discreet region m the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 that contams mutations that can produce cancer cells havmg a resistance to tyrosine kinase inhibitors such as STI-571. This discovery allows artisans to focus on this region m diagnostic protocols so as to facihtate such analyses. In d is context, a preferred mediod of the mvention is a method of identifying an ammo acid substitution in at least one Bcr- Abl polypeptide expressed m a human cancer cell from an mdividual selected for treatment widi a tyrosine kmase inhibitor, the method compnsmg determining the polypeptide sequence of at least one Bcr-Abl polypeptide expressed by die human cancer cell and comparing the polypeptide sequence of the Bcr-Abl polypeptide expressed by die human cancer cell to the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 so that an ammo acid substitution in the Bcr-Abl polypeptide expressed by the human cancer cell can be identified, wherem the amino acid substitution occurs m a region of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 compnsmg residue D233 through residue T406. Widiout bemg bound by a specific scientific dieory, the data disclosed herem provides evidence that this region defines boundaries for the structural architecture of the portions of Bcr-Abl diat are predommandy mvolved m an interaction with STI-571.
Another significant aspect of the mvention disclosed herem is the delineation of a discreet subregions m the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 that contams the mutations that can produce cancer cells havmg a resistance to tyrosine kmase inhibitors such as STI-571. This discovery allows artisans to focus on such subregions in diagnostic protocols so as to facihtate such analyses. In diis context, a preferred method of die mvention is a method of identifying an amino acid substitution m at least one Bcr-Abl polypeptide expressed m a human cancer cell from an mdividual selected for treatment widi a tyrosine kinase inhibitor, the mediod compnsmg determining d e polypeptide sequence of at least one Bcr-Abl polypeptide expressed by the human cancer cell and comparing d e polypeptide sequence of d e Bcr-Abl polypeptide expressed by the human cancer cell to the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 so that an ammo acid substitution m the Bcr-Abl polypeptide expressed by die human cancer cell can be identified, wherem the amino acid substitution occurs m the P-loop (residue G249 through residue V256 of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1), helix C (residue E279 through residue 1293 of the Bcr-Abl polypeptide sequence shown m SEQ ID NO. 1), the catalytic domam (residue H361 through residue R367 of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1) or the activation loop (residue A380 through residue P402 of die Bcr- Abl polypeptide sequence shown m SEQ ID NO: 1). Alternatively, the ammo acid substitution is proximal (e.g within about 10 ammo acid residues) to one of diese subregions m a manner that perturbs the function of the subregion A particularly significant aspect of die mvention disclosed herem is the delineation of a discreet residue positions in the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 that, when mutated, can produce cancer cells havmg a resistance to tyrosine kmase inhibitors such as STI-571. This discovery allows artisans to focus on such residue positions m diagnostic protocols so as to facihtate such analyses In this context, a preferred method of the mvention is a method of identifymg an ammo acid substitution m at least one Bcr-Abl polypeptide expressed m a human cancer cell from an mdividual selected for treatment with a tyrosine kmase inhibitor, d e method compnsmg determining the polypeptide sequence of at least one Bcr-Abl polypeptide expressed by die human cancer cell and comparmg die polypeptide sequence of d e Bcr-Abl polypeptide expressed by d e human cancer cell to the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 so diat an ammo acid substitution m the Bcr-Abl polypeptide expressed by the human cancer cell can be identified, wherem the amino acid substitution occurs at residue D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291, E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351, E352, E355, K357, N358, F359, 1360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396, A399, P402, or T406 This identification of discreet residue positions in the Bcr-Abl polypeptide sequence shown in SEQ ID NO: 1 that, when mutated, can produce cancer cells havmg a resistance to tyrosine kinase inhibitors such as STI-571 is significant m part because of art which teaches that m situations where methodical experimentation has estabhshed that the properties of a specific residue at a particular position within the polypeptide cham are crucial for mamtainmg some aspect of a protem's functional mtegnty, an alteration in the size, shape, charge, hydrogen-bonding capacity or chemical reactivity of the am o acid side cham at one of these "active" ammo acid positions is likely to affect the properties of d e protem m some way (See e.g. Rudiger et al, Peptide Hormones, University Park Press (1976)). For this reason, the skilled artisan would reasonably expect a substitution m a residue shown to be important for the inhibition of tyrosine kinase activity by STI-571 m the wild type protem to effect the ability of STI-571 to inhibit the kinase activity of the Bcr-Abl polypeptide. As disclosed herem, die specific effects of any substitution mutation (or a truncation, a deletion, a frame shift etc ) on STI-571 resistance can be examined by protocols such as those disclosed m the examples below.
In specific embodiments of the methods disclosed herem, the amino acid substitution is D233H, T243S, M244V, G249D, G250E, G251S, Q252H, Y253F, Y253H, E255K, V256L, Y257F, Y257R, F259S, K262E, D263G, K264R, S265R, V268A, V270A, T272A, Y274C, Y274R, D276N, T277P, M278K, E282G, F283S, A288T, A288V, M290T, K291R, E292G, I293T, P296S, L298M, L298P, V299L, Q300R, G303E, λ^304A, V304D, C305S, C305Y, T306A, F311L, I314V, T315A, T315I, E316G, F317L, M318T, Y320C, Y320H, G321E, D325H, Y326C, L327P, R328K, E329V, Q333L, A337V, V339G, L342E, M343V, M343T, A344T, A344V, I347V, A350T, M351T, E352A, E352K, E355G, K357E, N358D, N358S, F359V, I360K, I360T, L364H, E373K, N374D, K378R, V379I, A380T, A380V, D381G, F382L, T389S, T392A, T394A, A395G, H396K, A399G, P402T or T406A. While the identification of substitutions is a preferred embodiment of the mvention, the mediods disclosed herem can also be used to identify other mutations that are associated with resistance to tyrosine kinase inhibitors such as STI-571 such as truncations diat result from a mutation diat mtroduces a stop codon at an ammo acid residue position such as K245STOP or E334STOP.
Embodiments of the mvention mclude those that examine any one to all of the ammo acid positions m the Bcr-Abl polypeptide sequence (e.g. Ml, L2, E3 dirough VI 128, Q1129 and R1130) as occurs when one compares the sequence of a polypeptide expressed by a cancer cell with the polypeptide sequence shown m SEQ ID NO: 1. In this context, m preferred embodiments of the mvenαon, one can examine residue G250, Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35, K357, 1360, V379 or H396. In specific embodiments one can examine residue G250, Q252, Y253, E255, T315, F317, M351 or E355. As is known m the art, it may be desirable to examine one residue but not necessarily all of the ammo acid positions in the Bcr-Abl polypeptide sequence. Consequendy, another embodiment of the mvention is a method of identifymg an ammo acid substitution m at least one Bcr-Abl polypeptide expressed m a human cancer cell from an mdividual selected for treatment with a tyrosine kmase inhibitor, the method compnsmg determining the polypeptide sequence of at least one Bcr-Abl polypeptide expressed by the human cancer cell and comparing the polypeptide sequence of the Bcr- Abl polypeptide expressed by the human cancer cell to the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 so that an ammo acid substitution m die Bcr-Abl polypeptide expressed by the human cancer cell can be identified, wherein the ammo acid substitution does not occur at residue G250, Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35, K357, 1360, V379 or H396 Corresponding embodiments of the mvention mclude d ose that examine one or more amino acid mutations m a Bcr-Abl polypeptide but do not examine anod er specific amino acid position m the Bcr-Abl polypeptide sequence (e.g. methods wliich examine residue position 315 but not residue position 255).
The polynucleotide and/or polypeptide sequences of Bcr-Abl can be identified by any one of a wide vanety of protocols known m the art such as diose disclosed herem. In preferred mediods, the Bcr-Abl polynucleotide expressed by die human cancer cell is isolated by the polymerase cham reaction. In addition, methods used in the identification of one Bcr-Abl polypeptide expressed in a human cancer cell from an mdividual selected for treatment with one tyrosine kmase inhibitor can be identical to methods used m the identification of one Bcr-Abl polypeptide expressed m a human cancer cell from an mdividual selected for treatment with anodier tyrosine kmase inhibitor. In illustrative methods of the mvention, the kmase inhibitor is a 2-phenylammopyrιmιchne. As noted herem, the mediods of the present mvention can be used in determmmg whether or not to treat an mdividual with a specific tyrosine kmase inhibitor such as STI-571. Another embodiment of die mvention disclosed herem is a method of identifymg a mutation m a Bcr-Abl polynucleotide m a mammalian cell, wherem the mutation m a Bcr-Abl polynucleotide is associated with resistance to inhibition of Bcr- Abl tyrosine kmase activity by a 2-phenylammopynmidme, the method compnsmg determmmg the sequence of at least one Bcr-Abl polynucleotide expressed by the mammahan cell and comparing the sequence of d e Bcr-Abl polynucleotide to the Bcr- Abl polynucleotide sequence encodmg the polypeptide sequence shown m SEQ ID NO: 1, wherem the mutation m the Bcr-Abl polynucleotide compnses an alteration at ammo acid residue position: D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291, E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351 , E352, E355, K357, N358, F359, 1360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396, A399, P402, or T406 of the polypeptide sequence shown m SEQ ID NO: 1. As used herem, "a Bcr-Abl polynucleotide associated widi resistance to inhibition of Bcr-Abl tyrosine kmase by a 2-phenylammopyrimichne" refers to a Bcr-Abl polynucleotide that has been identified m cancer cells that exhibit some level of resistance to a 2-phenylarrunopynmidme such as STI-571 (or analogs or derivatives thereof) and which encodes a polypeptide havmg at least one ammo acid difference from die polypeptide sequence shown in SEQ ID NO- 1 (e.g those disclosed m Table IA). Preferably the Bcr-Abl polynucleotide associated with resistance to inhibition of Bcr-Abl tyrosine kmase by a 2-phenylammopynmichne encodes a polypeptide that exhibits exhibit some level of resistance to a 2-phenylammopynmidme such as STI-571.
Optionally, m die methods disclosed above, the mammahan cell is a human cancer cell. In preferred methods, the human cancer cell is a chrome myeloid leukemia cell. In highly preferred methods, the human cancer cell is obtamed from an mdividual treated with STI-571. Optionally, the ammo acid substitution m d e Bcr-Abl polypeptide expressed m human cancer cell confers resistance to mliibition of tyrosine kmase activity by STI-571.
MARS expression analysis may also be useful as a tool for identifymg and evaluating agents that modulate MARS gene expression. Identification of a molecule or biological agent that could inhibit MARS activity is of therapeutic value.
MONITORING THE STATUS OF MARS
The finding that MARS mRNA is expressed in cancers demonstrating STI-571 resistance provides evidence that mutations in Bcr-Abl are associated with STI-571 resistance and therefore identifies diese genes and their products as targets that the skilled artisan can use to evaluate biological samples from mdividuals suspected of havmg a disease associated with MARS expression. In this context, the evaluation of the status of MARS genes and their products can be used to gam information on die disease potential of a tissue sample.
The term "status" m this context is used according to its art accepted meaning and refers to the condition a gene and its products mcludmg, but not limited to die integnty and/or methylation of a gene mcludmg its regulatory sequences, the location of expressed gene products (mcludmg the location of MARS expressmg cells), die presence, level (e.g. the percentage of MARS expressmg myeloid cancer cells m a total population of myeloid cancer cells), and biological activity of expressed gene products (such as MARS mRNA polynucleotides and polypeptides), the presence or absence of transcnptional and translational modifications to expressed gene products as well as associations of expressed gene products with other biological molecules such as protem bmdmg partners. The status of MARS can be evaluated by a wide variety of mediodologies well known m the art, typically those discussed below.
The status of MARS may provide information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The mvention provides methods and assays for determmmg MARS status and diagnosing cancers diat express MARS. MARS status in patient samples may be analyzed by a number of means well known m the art, mcludmg without limitation, mimunohistochemical analysis, m situ hybndization, RT-PCR analysis on laser capture micro-dissected samples, western blot analysis of clinical samples and cell lines, and tissue array analysis Typical protocols for evaluating the status of the MARS gene and gene products can be found, for example in Ausubul et al eds, 1995, Current Protocols In Molecular Biology, Units 2 [Northern Blotting], 4 [Southern Blotting], 15 [Immunoblotting] and 18 [PCR Analysis].
A typical aspect of the mvention is directed to assessmg the effectiveness of STI- 571 m a therapeutic regimen. In a representative embodiment, a method for assessmg the effectiveness of STI-571 compnses detecting MARS mRNA or MARS protem m a tissue sample, its presence mchcating a likely resistance to STI-571, wherem the degree of MARS mRNA expression (e.g. the percentage of clones that express one or more MARS) is proportional to the likelihood of resistance to STI-571.
Another aspect of die mvention is directed to examining the stage of cancer m an mdividual. In one embodiment, a mediod for examining a stage of cancer compnses detecting MARS mRNA or MARS protem m a tissue sample, its presence indicating susceptibility to cancer, wherem the degree of MARS mRNA expression present is proportional to the degree of susceptibility. In a specific embodiment, the presence of MARS m a tissue sample is examined, widi the presence of MARS m the sample providmg an indication of a stage of leukemia (or d e emergence or existence of a leukemia). In a closely related embodmient, one can evaluate the mtegnty MARS nucleotide and am o acid sequences m a biological sample m order to identify perturbations m the structure of these molecules such as insertions, deletions, substitutions and the like, with the presence of one or more perturbations m MARS gene products m the sample providing an indication of cancer stage or susceptibility (or the emergence or existence of a cancer type or stage).
Yet another related aspect of the mvention is directed to mediods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor compnses determining the level of MARS mRNA or MARS protem expressed by cells in a sample of the tumor, comparing the level so determined to the level of MARS mRNA or MARS protem expressed m a corresponding control tissue, wherem the degree of MARS mRNA expression present is proportional to the degree of aggressiveness. In a specific embodiment, aggressiveness of leukemias is evaluated by determining the extent to which MARS is expressed m the tumor cells, widi relatively higher numbers of cells expressmg one or more MARS indicating more aggressive tumors (e.g. in that they are resistant to a therapeutic agent such as STI-571)
Yet another related aspect of the mvention is directed to mediods for observing the progression of a malignancy in an mdividual over time. In one embodiment, methods for observing the progression of a malignancy m an mdividual over time compπse deterrnining the level of MARS mRNA or MARS protem expressed by cells m a sample of the tumor, companng the level so determined to the level of MARS mRNA or MARS protem expressed in an equivalent tissue sample taken from the same mdividual at a different time, wherem die degree of MARS mRNA or MARS protem expression m the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining the extent to which MARS expression m the tumor cells alters over time, with higher expression levels over time mchcating a progression of the cancer.
Gene amphfication provides an additional method of assessmg the status of Bcr- Abl. Gene amphfication may be measured m a sample chrecdy, for example, by conventional Southern blotting, Northern blotting to quanαtate the transcription of mRNA (Thomas, 1980, Proc. Nad. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or m situ hybndization, usmg an appropnately labeled probe, based on the sequences provided herem. Alternatively, antibodies may be employed that can recognize specific duplexes, mcludmg DNA duplexes, RNA duplexes, and DNA-RNA hybnd duplexes or DNA-protem duplexes. The antibodies m turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on die surface, the presence of antibody bound to the duplex can be detected.
The above diagnostic approaches may be combmed with any one of a wide vanety of prognostic and diagnostic protocols known m the art. For example, another embodiment of the mvention disclosed herem is directed to methods for observing a comcidence between the expression of MARS gene and/or MARS gene products and a factor that is associated with malignancy as a means of diagnosing and prognosticating the status of a tissue sample. In this context, a wide vanety of factors associated with malignancy may be utilized such as the expression of genes otherwise associated with malignancy as well as gross cytological observations (see e.g. Bocking et al, 1984, Anal. Quant. Cytol. 6(2):74-88; Eptsein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al, 1998, Mod. Pathol. 11 (6):543-51 ; Baisden et al, 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a comcidence between die expression of MARS gene and MARS gene products and an additional factor that is associated with malignancy are useful, for example, because the presence of a set or constellation of specific factors that comcide provides information crucial for diagnosing and prognosticating the status of a tissue sample.
In a typical embodiment, methods for observing a comcidence between the expression of MARS gene and MARS gene products (or perturbations in MARS gene and MARS gene products) and a factor that is associated wid malignancy entails detecting the overexpression of MARS mRNA or protem m a tissue sample and then detecting the altered expression of another oncogene such RAS, or a tumor suppressor such as p53 or Rb, m a tissue sample, and observing a comcidence of MARS mRNA or protem expression and, for example, RAS mRNA or protem overexpression. In a specific embodiment, the expression of MARS and RAS mRNA in tissue is examined. In a prefened embodiment, the comcidence of MARS and RAS mRNA overexpression m the sample provides an indication of leukemia stage, or the emergence or existence of a leukemia.
Preferred embodiments of the mvention descnbed herem mclude methods for characterizing a cancer genotype and/or phenotype such as the genotype and/or phenotype of cancers of the myeloid lineage. Specific embodiments of the mvention descnbed herem mclude methods of assessmg die likelihood of resistance to a nucleotide analog such as 2-phenylamιno pynmidine. Particular embodiments of die mvention descnbed herem mclude methods for specifically identifymg cells having some degree of resistance to STI-571. Such methods typically mclude the step of sequencmg a target kmase such as Bcr-Abl to identify a mutation associated with a specific genotype or phenotype such as resistance to STI-571. Preferably the mutation is widitn a domam shown to be associated with the cancer genotype and/or phenotype (e g. the ATP bmdmg domain of Bcr-Abl). More preferably the mutation is m a Bcr-Abl residue identified m Table I below (or m an equivalent residue of a kmase having homology to Bcr-Abl). A variety of permutations of these methods are provided by the mvention disclosed herem. For example, the mvention disclosed herem allows artisans to examine MARS m a vanety of contexts to determme whether different mutations segregate with specific clinical phenotypes (e.g. lymphoid versus myeloid disease) or with different clinical patterns of STI-571 resistance (e.g. refractory disease; delayed relapse versus rapid relapse). The mvention further allows those skilled m the art to determine whedier kmase domam mutations restricted to patients with advanced stage disease or also occur m chrome phase patients. The mvention also allows those skilled in the art to determme whether one or more mutations are a manifestation of the clonal diversity and genetic instability associated with disease progression. The mvention also allows those skilled m the art to determine whether such mutations are a consequence of pnor exposure to chemotherapy, or occur only m patients exposed to STI-571 The mvention also allows those skilled m the art to determme die biological implications for other targeted kmase inhibitors currendy in clinical development.
Methods for detecting and quantifying the expression of MARS mRNA or protem are descnbed herem and use standard nucleic acid and protem detection and quantification technologies well known m the art. Standard methods for the detection and quantification of MARS mRNA mclude m situ hybndization usmg labeled MARS nboprobes, northern blot and related techniques usmg MARS polynucleotide probes, RT-PCR analysis usmg primers specific for MARS, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi- quantitative RT-PCR may be used to detect and quantify MARS mRNA expression as descnbed m the Examples that follow. Any number of primers capable of amplifying MARS may be used for this purpose, mcludmg but not limited to the vanous primer sets specifically descnbed herem. Standard methods for the detection and quantification of protem may be used for this purpose. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive widi the MARS protem may be used in an lmmunohistochemical assays of samples Antibodies directed agamst MARS protem can also be used to detect MARS m a patient specimen (e.g, blood or other sample) usmg conventional tecliniques such as fluorescence-activated cell sorting (FACS) and/or ELISA. As discussed m detail below, once a mutant sequence is identified one can then identify compounds which bmd and/or inhibit the activity of the mutant kinases.
METHODS FOR IDENTIFYING AND CHARACTERIZING MARS
The disclosure provided herem allows those skilled m the art to identify and characterize cells havmg a genotype and/or phenotype associated with a cancer such as a genotype and/or phenotype associated widi cancers of the myeloid lineage. Specific embodiments of the mvention descnbed herem mclude methods for the identification and characterization of Bcr-Abl mutants associated resistance to a nucleotide analog such as 2- phenyla ino pyrimidine. Particular embodiments of the mvention descnbed herem mclude mediods for the identification and charactenzation of cells having some degree of resistance to an inhibitor such as STI-571.
A first method for characterizing cells havmg a genotype and/or phenotype associated with a cancer mcludes the sequencmg of Bcr-Abl m those cells to identify one or more mutations associated with a particular phenotype (e.g. resistance to STI-571) such as a mutation m a domam or region shown to be associated with a specific genotype and/or phenotype (e.g. the ATP bmdmg domam of Bcr-Abl). Preferably the mutation is m a Bcr- Abl residue identified in Table I below.
A related method for charactenzmg cells havmg a genotype and/or phenotype associated with a cancer and/or cancer stage mcludes considering the location of the mutation m the context of the crystal structure of the ABL kmase domam bound to a variant STI-571 (see, e.g. Schindler et al. Science. 2000 Sep 15;289(5486).l 938-42). This definition of the crystal structure allows one to evaluate whether the mutation might mterfere with the anti-leukemia activity of STI-571. Based on this analysis, one can pπontize mutations for direct experimental analysis of ABL kmase activity, leukemogenicity and level of inhibition by STI-571. Another mediod for characterizing cells havmg a genotype and/or phenotype associated with a cancer and/or cancer stage mcludes analyzmg another factor associated a genotype and/or phenotype associated with a cancer m a target cell bemg examined such as die stage of die disease progression, die relative frequency of the mutant within the population (e.g. is the clone a dommant population which provides evidence that they have a growth advantage).
Another method for characterizing cells havmg a genotype and/or phenotype associated with a cancer mcludes engineering selected mutations mto wild-type BCR-ABL cDNA to create a mutant allele whose enzymological and biological properties can be examined direcdy. Enzymology can be performed by measurmg tyrosme kinase activity m vitro or in cells usmg standard assays known m the art (see, e.g. those cited m Example 1). Biological activity can be measured usmg standard oncogene transformation assays usmg growth factor dependent hematopoietic cell lines or primary mouse bone marrow cells (see, e.g. those cited m Example 1). In this way, resistance to STI-571 can be measured usmg such kmase assays and transformation assays.
Those skilled m the art will understand that die above descnbed assays for characteπzmg cells havmg a genotype and/or phenotype associated with a cancer can be performed mdependendy or m combination with each odier.
MUTATIONS IN RELATED MOLECULES
Residues shown to mutated m MARS occur m domains that are highly conserved among members of the protem kmase family (see, e.g. Hanks et al. Science 241: 42-51 (1988)) The finding that a highly conserved residue is mutated m cancers and d at this mutation is associated with resistance to a chemod erapeutic agent provides evidence that this domam is associated with dysregulated cell growd and therefore identifies diese domains and residue position as a targets that the skilled artisan can use to evaluate the status of related members of the tyrosme kinase family (see, e.g. those identified m Figure 1 of Hanks et al. Science 241: 42-51 (1988)), from mdividuals suspected of having a disease associated with the dysregulation of that member of the tyrosme kmase family. In this context, the evaluation of the status of a domam and/or residue m the tyrosme kmase family member can be used to gam information on the disease potential of a tissue sample. For example, m a syndrome in which the dysregulation of a specific tyrosme kmase family member is known or suspected (preferably one that exhibits a pattern of pathology that is similar to that seen with Bcr-Abl), one can determine if a mutation has occurred at that residue m order to obtain evidence of genetic changes associated widi growth dysregulation (e.g. resistance to a chemodierapeutic agent). Methods for die detection of mRNAs havmg such specific mutations m cells are well known and mclude, for example, hybndization assays usmg complementary DNA probes (such as in situ hybndization, Northern blot and related techmques) and vanous nucleic acid amplification assays (such as RT-PCR usmg complementary pnmers specific for die mRNA of mterest, and other amphfication type detection methods, such as, for example, branched DNA, SISBA, TMA and die like). As discussed below, methods for identifymg molecules that mteract wid such mutant members of the tyrosme kmase family are also provided. Embodiments of the mvention mclude methods for identifying a functional hotspot (e.g. a region in a protem which has significant functional importance m kmase activity and drug resistance) m a target kmase compnsmg sequencmg at least a portion of the target kmase to identify a mutation and companng the location of die mutation to the location of functional hotspots identified m a homologous kinase (e.g. Bcr-Abl), wherem die identification of a mutation m a target kmase that conesponds to a hotspot m a homologous kmase provides evidence that the mutation m the target kmase is m a functional hotspot. Typically the hotspot occurs m a Bcr-Abl domain havmg mutations associated with STI-571 resistance (e.g. the activation loop). More preferably the hotspot occurs m a Bcr-Abl residue identified in Table I. Preferably, the homologous kinase is Bcr- Abl and the homologies are compared via a BLAST analysis. The target kmase may be any one of a wide vanety of kinases known m die art such as c-kit, PDGFR, EGFR and VEGFR or one of the kinases identified m Figure 1 of Hanks et al , Science 241: 42-51 (1988). Optionally these methods can be used to characterize cells from patients suffering from a pathology associated with abenant expression of the target kmase. Other embodiments of the mvention mclude mediods for assessmg the likelihood of a target kmase havmg a resistance to a nucleotide analog such as 2-phenylamιno pyrimidine compnsmg sequencmg at least a portion of the target kmase to identify a mutation, wherem the identification of a mutation in a target kmase that corresponds to a hotspot m a homologous kinase provides evidence that target kmase will be resistant to the inhibitor. Preferably the hotspot occurs in a Bcr-Abl domam havmg mutations associated with STI-571 resistance. More preferably the hotspot occurs m a Bcr-Abl residue identified m Table I. Preferably, the homologous kmase is Bcr-Abl and the homologies are compared via a BLAST analysis. The target kinase may be any one of a wide vanety of kinases known m the art such as c-kit, PDGFR, EGFR and VEGFR or one of die kinases identified m Figure 1 of Hanks et al. Science 241: 42-51 (1988) which is incorporated herem by reference. Optionally these methods can be used to characterize cells from patients suffering from a pad ology associated widi aberrant expression of die target kmase.
The mvention disclosed herem mcludes the identification of ammo acid residues m Bcr-Abl that are mutated m a manner characterized such that they retam kmase activity yet are associated with resistance to mliibition of kinase activity by a 2- phenylaminopynmichne. One embodmient of an mvention provided by diis disclosure is a mediod of identifymg such a mutation m an Abelson protem kinase, wherem the mutation is associated with the resistance to an inhibition of kinase activity by a 2- phenylaminopynmidme, the method compnsmg: determining an amino acid sequence of a portion of a polynucleotide encodmg the Abelson protem kinase to determine the presence of a mutation, wherem the mutation occurs at a ammo acid residue at the same relative position as a mutation m the C-Abl protem kmase shown m SEQ ID NO: 1 that is associated with STI-571 resistance as determined usmg the homology cntena of BLAST analysis. In this context, skilled artisans understand that mutations m the C-Abl protem kmase shown m SEQ ID NO. 1 that are associated with STI-571 resistance mclude mutations m the C-Abl protem kmase which have, for example, been identified m cancer cells isolated from individuals shown to exhibit a resistance to a d erapeutic regime mvolvmg a 2-phenylammopynmιchne such as STI-571. As disclosed herem, mutants of the C-Abl protem kinase shown m SEQ ID NO: 1 that are identified as bemg associated with STI-571 resistance are readily characterized by any one of a wide variety of techmques that are well known m the art in view of the extensive biological characterization of c-Abl, Bcr-Abl and/or one of die Abelson protem kinases such as ARG etc. Such protocols mclude analyses based on the understanding of the biological significance of a domam or residue within these protems that has been characterized as havmg significance m kmase activity or small molecule interaction (see, e g. Example 3 below which identifies vanous previously identified domams as well as residues which direcdy mteract widi STI-571 via previously descnbed crystallographic analyses etc). Such protocols further mclude biological analyses of biological activity of these mutants mcludmg for example, the well known assays for charactenzmg the kmase activities and transforming abilities of Abelson protem kinases that are cited in Example 1 below.
A related embodiment is a method of identifymg a mutant Abelson protem tyrosme kinase expressed by a cell by determining a nucleotide sequence of a portion of a polynucleotide encodmg the kmase domam of the Abelson protem tyrosme kmase expressed by the cell and then companng the nucleotide sequence so determined to that of the wild type sequence of the Abelson protem tyrosme kmase to identify the presence of a mutation, wherem the mutation so identified has the characteristics of occurrmg at a amino acid residue located within the polypeptide sequence of die Abelson protem tyrosme kmase at the same relative position as a mutation m the C-Abl protem kmase shown m SEQ ID NO: 1 that has been identified as bemg associated with a resistance to an inhibition of tyrosme kinase activity by a 2-phenylammopyrιmιdme, as determined usmg the homology parameters of a BLAST analysis (e.g. c-src position 338 which corresponds to position 315 m SEQ ID NO. 1). In a specific version of diis embodiment, the cell expressmg the mutant Abelson protem tyrosme kmase is found m a population of cancer cells that has been observed m clmical populations to exhibit a resistance to an inhibition of tyrosme kmase activity by a 2-phenylamιnopyrιmιchne (e.g. STI-571). In a highly preferred embodiment the mutation m die C-Abl protem kinase shown m SEQ ID NO: 1 that has been identified as bemg associated with a resistance to an inhibition of tyrosme kmase activity by a 2-phenylamιnopyrιmιchne is a Bcr-Abl residue identified m Table I. Yet another embodmient of the mvention is a method of identifymg a mutant Abelson tyrosme kinase expressed by a cell by determmmg a nucleotide sequence of a portion of the catalytic domam of the Abelson tyrosme kmase expressed by the cell (and more preferably the nucleotide bmdmg site within the catalytic domam) and then comparmg the nucleotide sequence so determined to that of the wild type sequence of die catalytic domam of the Abelson protem tyrosme kmase to identify the presence of a mutation within the catalytic domam, wherem the mutation so identified has the characteristics of occurrmg at a ammo acid residue located within the polypeptide sequence of the Abelson protem tyrosme kmase at a amino acid residue d at has homology to an ammo acid position m a C-Abl kmase shown m SEQ ID NO: 1 that is associated with a resistance to an inhibition of tyrosme kmase activity by a 2- phenylaminopyrimidine, wherem the homology between the ammo acid residue located within the polypeptide sequence of the Abelson protem tyrosme kinase and the amino acid residue m die C-Abl kmase shown m SEQ ID NO: 1 that is associated with a resistance to an mliibition of tyrosme kmase activity by a 2-phenylaminopynmidme can be illustrated via a BLAST analysis.
As used herem, an Abelson tyrosme kmase refers to the family of kinases known m the art to be closely related to the c-Abl protem or have domams d at share a high degree of homology with a domam m the c-Abl protem. For example, die Philadelphia translocation is known to result in the expression of a family of chmienc protems in which a portion of the Bcr protem is fused to c-Abl protem. A specific groupmg of Abelson tyrosme kmase family members are those which exhibit an amino acid sequence homology that is structurally and/or functionally related such that a 2- phenylaminopynmichne can mteract with these molecules and inhibit their kmase activities (e.g. Bcr-Abl, TEL-Abl, c-kit, PDGFR, EGFR and VEGFR).
Another representative member of the Abelson tyrosme kinase family is the protem designated ARG. An analysis of the ammo acid sequence of the ARG protem reveals that it is closely related to that of c-Abl (see, e.g, Kruh et al, PNAS 1990, 87(15): 5802-6 and Wang et al, Oncogene 1996, 13(7): 1379-85). Specifically, c-Abl and ARG are strikingly similar with regard to overall structural architecture as well as the amino acid sequences of dieir tyrosme kmase domams. Additional members of the family mclude for example, Dash, Nabl, and Fes/Fps (see e.g. Hunter et al. Science 241, 42-51 (1988)).
As is known m the art, the Abelson tyrosme kmase family of protem kinases contam a catalytic domam diat has a highly conserved structural and functional architecture (see, e.g Sichen et al, Curr Opm Struct Biol. 1997 Dec;7(6):777-85; and Sichen et al. Nature 1997 Feb 13;385(6617).602-9). Understandably, because regions within the catalytic domam of these tyrosme lαnases are known to be highly conserved among members of this gene family, it is observed that STI-571 also mteracts with representative members of this family such as c-kit and PDGFR (see, e g , Tuveson et al ,
Oncogene. 2001 Aug 16;20(36):5054-8; Buchdunger et al, J Pharmacol Exp Ther. 2000
Oct;295(l):139-45; Wang et al, Oncogene. 2000 Jul 20;19(31):3521 -8; Heinnch et al.
Blood. 2000 Aug l ;96(3):925-32; and Carroll et al. Blood. 1997 Dec 15;90(12):4947-52).
As noted above, the catalytic domams of these protem kinases have a highly conserved structural and functional architecture which allows for the interaction of compounds of the 2-phenylammopynmidme class of molecules to mteract with this domam and further provides the basis for a vanety of comparative analyses as well as rational drug design (see, e.g., Traxler et al, Med Res Rev 2001 Nov;21 (6):499-512; Traxler et al, J Med Chem. 1999 Mar 25;42(6):1018-26; and Parang et al, Nat Struct Biol. 2001 Jan;8(l):37-41 Smgh et al , J Med Chem 1997 Mar 28;40(7): 1130-5 and Furet et al , J Comput Aided Mol Des. 1995 Dec;9(6) 465-72. Moreover, because the crystal structure of the catalytic domam of Abl complexed 2-phenylammopyrιmιdιnes such as variants of STI-571 has been determined, d is provides information as to how this class of molecules mteracts with these highly conserved regions within these lαnases (see, e.g, Schmdler et al. Science. 2000 Sep 15;289(5486):1938-42). Such analyses are enhanced by the fact that the crystal structures of a number of odier tyrosme kmase inhibitors have also been determined (see, e.g, Schmdler et al, Mol Cell. 1999 May;3(5):639-48; Mohammach et al, EMBO J. 1998 Oct 15;17(20):5896-904).
As disclosed herem, the domam compnsmg the ATP bmdmg site is identified as a region that is mutated m Bcr-Abl protems exhibiting resistance to STI-571. Interestingly, other chemical classes of TK inhibitors are known to bmd the ATP binding site including quinazohnes and pyrazolo-pyrrolo-pyndopyrimidines (see, e g, Tian et al. Biochemistry. 2001 Jun 19;40(24):7084-91; Fry et al. Science. 1994 Aug 19;265(5175):1093-5; Rewcasde et al, J Med Chem. 1996 Feb 16;39(4):9l8-28; Rewcasde et al, J Med Chem. 1995 Sep l ;38(18):3482-7; Toledo et al, Curr Med Chem. 1999 Sep;6(9):775-805; and Bridges et al, Curr Med Chem. 1999 Sep;6(9):825-43). Consequendy, TK inhibitors which bmd an ATP bmdmg site havmg a high homology to the ATP bmdmg site of Bcr-Abl (and mutants exhibiting resistance to such inhibitors) can be analogously identified and characterized usmg the disclosure provided herem. The mvention provided herem identifies specific regions within conserved protem kmase family members that impart resistance to a class of tyrosme kmase inhibitors, thereby identifymg diese regions as the targets of die diagnostic protocols described herem. In particular, while certam ammo acid residues known to be mvolved in an interaction with kmase inhibitors such as 2-phenylarrunopynmιdmes have been identified, it was not known whether a mutation could occur at a residue within a domam havmg a specific biological activity that would inhibit d e interaction between the kmase and the kmase inhibitor yet allow the kmase to retam a biological activity associated with a pathological condition, particularly m cases where the mutation is observed m clmical specimens. The disclosure provided herem identifies specific target domams (e.g. die ATP-binding domam) within protem lαnases m which ammo acid mutations can occur that render the kmase resistant to kmase inhibitors such as 2-phenylamιnopynmιchnes yet allow the kmase to retam a biological activity that is associated with a pathological condition (e.g. chrome myeloid leukemia). By identifymg a specific region m protem lαnases m which mutations havmg these dual characteristics occur, the disclosure provided herem allows the skilled artisan to employ diagnostic procedures that are tailored to specifically analyze polynucleotides encodmg these regions (e.g. m PCR protocols used to identify protem kmases likely to be resistant to kmase inhibitors). In this way, the disclosure provided herem can reduce the amount of experimentation necessary to characterize a mutant protem kinase that is associated with a pathological condition. Such analyses are facihtated by the fact that these target domains are so highly conserved among a vanety of protem kinases they are readily identified, and therefore easily targeted m protocols used to identify d e presence of such mutations m these domams.
Typical embodiments of the mvention mclude a method of identifymg a mutation m the catalytic domam of a target protem kmase compnsmg determmmg the amino acid sequence of the catalytic domam and companng it to the wild type sequence of the target protem kmase catalytic domam to identify a mutation dierein, wherem the catalytic domam of the target protem kmase has at least about 60, 70, 80, 85, 90 or 95% homology to the catalytic domam of c-able catalytic domam shown m SEQ ID NO: 1. A related embodiment is a method of identifymg a mutation m the activation loop domam of a target protem kmase compnsmg determmmg die ammo acid sequence of the activation loop domam and companng it to the wild type sequence of the target protem kmase activation loop domam to identify a mutation therein, wherem the activation loop domam of the target protem kmase has at least about 60, 70, 80, 85, 90 or 95% homology to the activation loop domam of c-able activation loop domam shown m SEQ ID NO: 1. A related embodiment is a method of identifymg a mutation m d e nucleotide bmdmg pocket of a target protem kinase compnsmg determining d e amino acid sequence of the nucleotide bmdmg pocket and companng it to the wild type sequence of the target protem kmase nucleotide bmdmg pocket domam to identify a mutation therein, wherem the nucleotide bmdmg pocket domam of the target protem kmase has at least about 60, 70, 80, 85, 90 or 95% homology to the nucleotide bmdmg pocket domam of c-able catalytic domam shown m SEQ ID NO: 1. A related embodiment is a method of identifymg a mutation m a target tyrosme kmase that is likely to be associated with resistance to a tyrosme kmase inhibitor compnsmg determining the ammo acid sequence of die P-loop, hehx c, activation loop or catalytic sequences as well as sequences within about 10 ammo acids of die respective domam(s), and comparmg it to the wild type sequence of the target protem kmase P-loop, he x c, activation loop or catalytic sequences as well as sequences within about 10 amino acids of die respective domam(s) to identify a mutation therein, wherem the P-loop, hehx c, activation loop or catalytic sequences as well as sequences within about 10 amino acids of the respective domam(s) of die target protem kmase has at least about 60, 70, 80, 85, 90 or 95% homology to the P-loop, hehx c, activation loop or catalytic sequences as well as sequences within about 10 ammo acids of these domams m c-Abl.
Another related embodiment is a method of isolating a polynucleotide encodmg a mutated catalytic domam of a target protem kmase compnsmg employing PCR to amplify the catalytic domam of a target protem kinase, wherem the target protem kinase exhibits a biological activity that is associated with a pathological condition and wherem the target protem kmase exhibits a resistance to tyrosme kmase inhibitors, and wherem the catalytic domam of the target protem kmase has at least about 60, 70, 80, 85, 90 or 95% homology to die catalytic domam of c-able catalytic domam shown m SEQ ID NO: 1, companng the polynucleotide sequence encoding the amino acid sequence of the catalytic domam and companng it to die polynucleotide sequence encoding the ammo acid sequence wild type ammo acid sequence of the target protem kmase catalytic domam so that a polynucleotide encodmg a mutated catalytic domam is identified. In a specific embodiment of these methods, at least one amino acid residue that is mutated m the domam has homology to a residue identified in Table I. In anodier specific embochment, the target protem kmase havmg the mutation exhibits a kmase activity diat is associated with a pathological condition (e.g. cancer). In anodier specific embodiment, d e kmase activity of the target protem kinase that is associated with a pathological condition (e.g. cancer) is resistant to inhibition by a tyrosme kmase mhibitor. In anodier specific embodiment, the kmase activity of the target protem kmase that is associated with a pathological condition (e.g. cancer) is resistant to mliibition by a 2- phenylaminopynmichne. In another specific embochment, the target protem kmase is shown m Table 2 of Hanks et al , Science 241: 42-51 (1988). In another specific embodiment, the target protem kmase is a Bcr-Abl, a TEL-Abl, a c-kit, a PDGFR, an EGFR, an VEGFR.
A related embodiment compnses a method of charactenzmg a property of a protem tyrosme kmase, wherem the protem kmase has at least about 60, 70, 80, 85, 90 or 95% homology to c-able shown m SEQ ID NO: 1 compnsmg determining whether the protem tyrosme kmase exhibits an activity that is associated with a pathological condition (e.g. via a procedure identified herem or citations m the art), determmmg whether die protem tyrosme kmase exhibits resistance to a tyrosme kmase inhibitor (e g via a procedure identified herem or citations m the art), determmmg an ammo acid sequence of the protem tyrosme kmase, determining whether the ammo acid sequence of the protem tyrosme kmase contams a mutated residue, determining whether the mutated residue occurs m the catalytic domam, the activation loop and/or the ATP bmdmg domam and/or determining whether the mutated residue has homology to a residue shown m Table I, wherem the presence of a mutated residue occurrmg m the catalytic domam, d e activation loop and/or the ATP bmdmg domain and/or wherem the mutated residue has homology to a residue shown m Table I provides evidence that the mutation so identified inhibits the interaction between the kmase and the kmase mhibitor yet allow the kmase to retam its kmase activity. In a specific embodmient, the kinase activity of the protem k ase that is associated widi a pathological condition is resistant to inhibition by a 2-phenylammopynmidine. In another specific embodiment, the protem kmase is a protem kmase shown m Table 2 of Hanks et al , Science 241: 42-51 (1988) In another specific embodiment, d e protem kmase is a Bcr-Abl, a TEL-Abl, a c- kit, a PDGFR, an EGFR, an VEGFR.
Yet another embochment of die mvention is a method of identifymg a mutant Abelson protem tyrosme kmase expressed by a mammahan cancer cell by determmmg a nucleotide sequence of a portion of a polynucleotide encodmg the kmase domam of the Abelson protem tyrosme kmase expressed by the cell and then comparmg die nucleotide sequence so determined to that of the wild type sequence of the Abelson protem tyrosme kmase to identify the presence of a amino acid substitution m the mutant Abelson protem tyrosme kmase, wherem any ammo acid substitution so identified has the charactenstics of occurrmg at a ammo acid residue located wid in d e polypeptide sequence of the Abelson protem tyrosme kmase at the same relative position as an amino acid substitution m the C-Abl protem kmase shown m SEQ ID NO: 1 that has been identified as bemg associated with a resistance to an inhibition of tyrosme kmase activity by a 2-phenylaminopyrimichne, as can be determined usmg the homology parameters of a WU-BLAST-2 analysis. In preferred embodiments of the mvention, the mutant Abelson tyrosme kinase expressed by the cell is a mutant c-Abl (see, e.g. NCBI Accession P00519), Bcr-Abl (see, e.g. NCBI Accession NP_067585), PDGFR (see, e.g. NCBI Accession NP002600), c-kit (see, e.g. NCBI Accession CAA29458), TEL-Abl (see, e.g. NCBI Accession CAA84815), or TEL-PDGFR (see, e.g. NCBI Accession AAA19786). A related embodiment of the mvention comprises repeating steps (a) -(b) another mammahan cancer cell obtamed from a different mdividual; and dien catalogmg the mutations found m the mutant Abelson protem tyrosme lαnases present in the mammahan cancer cells. Preferably m such methods, the cell expressmg die mutant Abelson protem tyrosme kmase is found in a population of mammahan cancer cells that are observed to exhibit a resistance to an mhibition of tyrosme kmase activity after exposure to a 2-phenylamιnopyrιmιchne. In such methods, the mammalian cancer cell is can be a human cancer cell obtamed from an mdividual selected for treatment with a tyrosme kmase mhibitor compnsmg a 2-phenylammopyrιrnιdme. Preferably, the ammo acid substitution confers resistance to mhibition of tyrosme kmase activity by a 2- phenylammopynmidme.
In a specific embodiment of such methods, the mutation in the C-Abl protem kmase shown m SEQ ID NO: 1 that has been identified as bemg associated with a resistance to an mhibition of tyrosme kmase activity by a 2-phenylammopynmichne occurs at the same relative position as ammo acid residue D233, T243, M244, K245, G249, G250, G251 , Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291 , E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311 , 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351 , E352, E355, K357, N358, F359, 1360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396, A399, P402, or T406. Typically, the amino acid substitution occurs at the same relative position as ammo acid residue G250, Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35, K357, 1360, V379 or H396.
The disclosure provided herem allows a mutant identified by one the methods disclosed herem to be further characterized. SpecificaUy, by utihzmg enzymological and/or biological assays described herem as weU as diose known m the art (illustrated by those disclosed, for example, m the Examples below), a mutant that is found to occur m a conserved target domam of a protem kmase can be readdy characterized to assess the biological significance of this mutation (e.g rendering the protem kmase resistant to kmase mhibitors such as 2-phenylammopyrιmιdιnes yet aUowmg die kmase to retam a biological activity that is associated with a pathological condition). Moreover, m the context of protems m which a target protem is identified, the disclosure herem of assays for the measurement of the phosphotyrosme content in an analogous fashion to the assays of Crkl, an adaptor protem which is SpecificaUy and constitutively phosphorylated by Bcr-Abl in CML ceUs (see, e.g. Figures 1 and 2).
In addition to the mutations identified in Table I, scanning amino acid analysis can also be employed m comparative analyses of compounds such as 2- phenylaminopynmidmes to identify the significance of one or more amino acids which are structuraUy and/or functionaUy mvolved m the interaction between Abelson tyrosme kmases and compounds such as 2-phenylammopynmidines (see, e.g. U.S. Patent No. 6,004,931 and 5,506,107). Among the preferred scanning ammo acids are relatively smaU, neutral amino acids. Such ammo acids mclude alanme, glycine, serme, and cysteme. Alanme is typicaUy a preferred scanning ammo acid among this group because it eliminates die side-cham beyond the beta-carbon and is less likely to alter the main- cham conformation of the vanant. Alanme is also typicaUy preferred because it is the most common amino acid. Further, it is frequendy found m both buried and exposed positions [Creighton, The Proteins, (W.H Freeman & Co, N.Y.); Chothia, J. Mol. Biol, 150:1 (1976)]. If alanme substitution does not yield adequate amounts of variant, an lsostenc ammo acid can be used.
IDENTIFICATION OF MOLECULES THAT INTERACT WITH MARS
As lUustrated m Example 8, the MARS protem and nucleic acid sequences disclosed herem aUow a skilled artisan to identify protems, smaU molecules and other agents that mteract with MARS, as weU as pathways activated by MARS via any one of a vanety of art accepted protocols. For example, usmg the disclosure provided herem, one can employ methods used m the art to evaluate the interaction between STI-571 and Bcr- Abl to evaluate interactions between test molecules and MARS.
A representative embochment of this mvention comprises a method of screening for a molecule that mteracts with an MARS amino acid sequence compnsmg die steps of contacting a population of molecules with the MARS amino acid sequence, aUowmg the population of molecules and d e MARS ammo acid sequence to mteract under conditions that facihtate an interaction, determining the presence of a molecule diat mteracts with the MARS ammo acid sequence, and then separating molecules that do not mteract with the MARS amino acid sequence from molecules diat do. In a specific embodiment, the method furdier comprises punfymg a molecule diat mteracts with the MARS amino acid sequence. The identified molecule can be used to modulate a function performed by MARS.
This embodiment of the mvention is weU suited to screen chemical hbranes for molecules which modulate, e.g, inhibit, antagonize, or agonize or niimic, the activity of BCR-ABL as measured by one of the assays disclosed herem. The chemical hbranes can be peptide hbranes, peptidorritmetic hbranes, chemicaUy synthesized hbranes, recombmant, e.g, phage display hbranes, and m vitro translation-based libraries, other non-peptide synthetic organic hbranes (e.g. hbranes of 2-phenylammopynmidines, quinazohnes or pyrazolo-pyrrolo-pyndopynmichnes and the like etc.). Exemplary hbranes are commerciaUy avadable from several sources (ArQule,
Tnpos/PanLabs, ChemDesign, Pharmacopoeia). In some cases, these chemical hbranes are generated usmg combmatonal strategies that encode die identity of each member of the library on a substrate to which the member compound is attached, thus aUowmg direct and immediate identification of a molecule that is an effective modulator. Thus, m many combmatoπal approaches, the position on a plate of a compound specifies that compound's composition. Also, m one example, a smgle plate position may have from 1-20 chemicals d at can be screened by administration to a weU containing the interactions of mterest. Thus, if modulation is detected, smaUer and smaUer pools of interacting pairs can be assayed for the modulation activity. By such methods, many candidate molecules can be screened. Many diversity hbranes suitable for use are known m the art and can be used to provide compounds to be tested according to the present mvention. Alternatively, hbranes can be constructed usmg standard methods. Chemical (synthetic) hbranes, recombmant expression hbranes, or polysome-based hbranes are exemplary types of hbranes that can be used.
In one embodmient, one can screen peptide hbranes to identify molecules that mteract with MARS protem sequences. In such methods, peptides that bmd to a molecule such as MARS are identified by screening hbranes that encode a random or controUed coUection of amino acids. Peptides encoded by the hbranes are expressed as fusion protems of bacteπophage coat protems, the bacteπophage particles are then screened agamst the protem of mterest.
Accordingly, peptides havmg a wide variety of uses, such as therapeutic, prognostic or chagnostic reagents, are thus identified widiout any prior information on the structure of the expected ligand or receptor molecule Typical peptide hbranes and screening methods that can be used to identify molecules that mteract widi MARS protem sequences are disclosed for example m U S. Patent Nos. 5,723,286 issued 3 March 1998 and 5,733,731 issued 31 March 1998.
SmaU molecules and ligands that mteract with MARS can be identified through related embodiments of such screening assays. For example, smaU molecules can be identified that mterfere with protem function, mcludmg molecules diat mterfere with a MARS's ability to mediate phosphorylation and de-phosphorylation.
A typical embodiment is a method of identifymg a compound which SpecificaUy bmds a MARS shown m Table I, wherem said MARS exhibits tyrosme kinase activity, compnsmg the steps of: contacting said MARS with a test compound under conditions favorable to bmdmg; and then determmmg whether said test compound bmds to said MARS so that a compound which bmds to said MARS can be identified. As the interaction between vanous Abelson tyrosme kmases and a variety of test compounds have been previously descnbed, skiUed artisans are familiar with the conditions conducive to bmdmg. A specific embodiment of this aspect of the mvention mcludes the steps of transfecting ceUs with a construct encodmg the MARS, contacting said ceUs with said test compound that is tagged or labeUed with a detectable marker and then analyzmg said ceUs for the presence bound test compound. In contexts where the transfected ceUs are observed to preferentiaUy bmd the test compound as compared to ceUs d at have not been transfected with a MARS construct, this mchcates that the test compounds is bmdmg to the MARS protem expressed by those ceUs
A test compound which bmds said MARS may then be further screened for the mhibition of a biological activity (e.g. tyrosme kmase activity) of said MARS. Such an embodiment mcludes, for example determining whether said test compound inhibits the tyrosme kmase activity of the MARS by utihzmg molecular biological protocols to create recombmant contracts whose enzymological and biological properties can be examined direcdy A specific biological activity such as resistance to STI-571 can be measured usmg standard kmase assays and transformation assays. Enzymology is performed for example, by measurmg tyrosme kinase activity m vitro or m MARS expressmg ceUs usmg standard assays (see, e.g. one of those cited m the Examples below). Alternatively, biological activity is measured usmg standard oncogene transformation assays (see, e.g. one of those cited m the Examples below).
A specific embodiment of the mvention entails determining whether a test compound inhibits the biological activity of a MARS tyrosme kmase mhibitor m a procedure that is analogous for examinmg how STI-571 inhibits the tyrosme kmase activity of Bcr-Abl. Such mediods typicaUy compnse d e steps of examining the kmase activity or growth potential of a MARS expressmg ceU line m the absence of a test compound and comparmg this to the kmase activity or growth potential of a MARS expressmg ceU line m the presence of a test compound, wherem an decrease m the kmase activity or growth potential of the MARS expressmg ceU line m the presence of a test compound mchcates that said compound may be an mhibitor of the biological activity of
Yet another embodiment of the mvention is a method of identifymg a compound which SpecificaUy bmds a mutant Bcr-Abl polypeptide; wherem the Bcr-Abl polypeptide compnses an ammo acid substitution that occurs m a region of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 compnsmg residue D233 through residue T406, the method compnsmg the steps of contacting the mutant Bcr-Abl polypeptide with a test compound under conditions favorable to bmdmg; and determining whether the test compound SpecificaUy bmds to the mutant Bcr-Abl polypeptide such that a compound which bmds to the mutant Bcr-Abl polypeptide can be identified. The bmdmg of the compound is typicaUy determined by any one of a wide vanety of assays known m the art such as ELISA, RIA, and/or BIAcore assays.
In preferred embodiments, the ammo acid substitution m the mutant Bcr-Abl polypeptide occurs at residue D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291, E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351, E352, E355, K357, N358, F359, 1360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396, A399, P402, or T406 In a specific embodmient of d e mvention, the ammo acid substitution is D233H, T243S, M244V, G249D, G250E, G251S, Q252H, Y253F, Y253H, E255K, V256L, Y257F, Y257R, F259S, K262E, D263G, K264R, S265R, V268A, V270A, T272A, Y274C, Y274R, D276N, T277P, M278K, E282G, F283S, A288T, A288V, M290T, K291R, E292G, I293T, P296S, L298M, L298P, V299L, Q300R, G303E, V304A, V304D, C305S, C305Y, T306A, F311L, I314V, T315A, T315I, E316G, F317L, M318T, Y320C, Y320H, G321E, D325H, Y326C, L327P, R328K, E329V, Q333L, A337V, V339G, L342E, M343V, M343T, A344T, A344V, I347V, A350T, M351T, E352A, E352K, E355G, K357E, N358D, N358S, F359V, I360K, I360T, L364H, E373K, N374D, K378R, V379I, A380T, A380V, D381G, F382L, T389S, T392A, T394A, A395G, H396K, A399G, P402T or T406A.
A related embodiment of the mvention consists of the method descnbed above and further compnsmg determining whether the test compound inhibits the tyrosme kinase activity of the mutant Bcr-Abl polypeptide by transfecting mammalian ceUs with a construct encoding the mutant Bcr-Abl polypeptide, contacting the mammaban ceUs with the test compound; and then momtormg the mammahan ceUs for die tyrosme kinase activity of die mutant Bcr-Abl polypeptide, wherem an mhibition m tyrosme kmase activity m the presence of the test compound as compared to the absence of the test compound identifies the test compound as an mhibitor of the mutant Bcr-Abl polypeptide. In preferred embodiments of the mvention the tyrosme kmase activity of die mutant Bcr-Abl polypeptide is measured by examinmg die phosphotyrosme content of Crkl.
As illustrated m the Examples below, yet another embodiment of the mvention is a method of determining whether a test compound inhibits the tyrosme kmase activity of a mutant Bcr-Abl polypeptide, wherem the Bcr-Abl polypeptide compnses an ammo acid substitution that occurs m a region of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 compnsmg residue D233 through residue T406, the method compnsmg the steps of transfecting mammahan ceUs (e.g. 293-T ceUs) with a construct encodmg the mutant Bcr-Abl polypeptide so that the mutant Bcr-Abl polypeptide is expressed by the mammahan ceUs, contactmg the mammalian ceUs with die test compound and then momtormg the mammalian ceUs for the tyrosme kmase activity of die mutant Bcr-Abl polypeptide, wherem an mhibition m tyrosme kmase activity m the presence of the test compound as compared to the absence of the test compound identifies the test compound as an mhibitor of the mutant Bcr-Abl polypeptide. In specific embodiments of the mvention, the ammo acid substitution occurs at residue D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291, E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311 , 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351 , E352, E355, K357, N358, F359, 1360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396, A399, P402, or T406.
Preferably m such methods, the tyrosme kmase activity of the mutant Bcr-Abl polypeptide is measured by examinmg the phosphotyrosme content of Crkl. Alternatively, the tyrosme kmase activity of the mutant Bcr-Abl polypeptide is measured via Western blot analysis usmg an anti-phosphotyrosme antibody to examine the phosphotyrosme content of lysates of the mammahan ceUs. These mediods can be used to examine a wide vanety of compounds such as 2-phenylamιnopynmιchnes or pyndo [2,3-d]pyrιmιdιnes.
TypicaUy the ammo acid substitution occurs at residue G250, Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35,
K357, 1360, V379 or H396. In certam embodiments of die mvention, the ammo acid substitution does occur at one of the residues identified m Table IA (e.g residue T315) but not another of the residues identified m Table LA (e.g. residue E255).
KITS
For use m the diagnostic and therapeutic applications described or suggested above, kits are also provided by the mvention. Such kits may compnse a carrier means bemg compartmentalized to receive m close confinement one or more contamer means such as vials, tubes, and the like, each of die contamer means compnsmg one of the separate elements to be used in the method. For example, one of the contamer means may compnse a probe that is or can be detectably labeled. Such probe may be an antibody or polynucleotide specific for a MARS protem or a MARS gene or message, respectively. Where the kit utilizes nucleic acid hybridization to detect the target nucleic acid, the kit may also have contamers containing nucleotide(s) for amphfication of the target nucleic acid sequence and/or a contamer compnsmg a reporter-means, such as a biotin-binding protem, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or rachoisotope label.
The kit of the mvention wiU typicaUy comprise the contamer described above and one or more other contamers compnsmg matenals desirable from a commercial and user standpomt, mcludmg buffers, diluents, filters, needles, syrmges, and package mserts wid instructions for use. A label may be present on the contamer to mdicate that the composition is used for a specific therapy or non-therapeutic application, and may also mdicate directions for either m vivo or m vitro use, such as diose described above EXAMPLES
EXAMPLE 1: ILLUSTRATIVE MATERIALS AND METHODS FOR EXAMINING BCR-ABL In an illustrative strategy for examining MARS, our laboratory has embarked on a large scale sequencmg project to identify mutations m the ABL kmase domam m patients with chrome myeloid leukemia. A preferred experimental strategy is to use PCR to amplify a region of the BCR-ABL transcnpt usmg primers specific to BCR and ABL, subclone this product and sequence at least 10 mdependent clones m both directions. This strategy aUows one to quantify fluctuations m chfferent clones from d e same patient over time. Several different groups of patients have been analyzed m order to determine if die frequency and type of ABL mutation differs wid disease stage or prior treatment. These groups mclude: chrome phase untreated with STI-571 (Gleevec), chrome phase treated with STI-571, blast crisis untreated with STI-571 and blast cnsis treated with STI- 571. Usmg diis strategy we have found over 40 such mutations Typical methodologies are for such protocols are provided below
EXAMPLE 1A: ILLUSTRATIVE METHODS FOR EXAMINING BCR-ABL POLYNUCLEOTIDE AND POLYPEPΗDE SEQUENCES Blood samples were obtamed from consenting patients enroUed m clmical trials at UCLA assessmg the efficacy of STI-571 m the treatment of CML. RNA was extracted usmg TnReagent or TriAzol. CDNA synthesis was performed usmg MMTV reverse transcnptase. Polymerase cham reaction (PCR) was performed usmg the foUowmg primers: CM10 (5'-GAAGCTTCTCCCTGACATCCGT-3') (SEQ ID NO: 6) and 3' Abl KD (5'-GCCAGGCTCTCGGGTGCAGTCC-3') (SEQ ID NO- 7). The resultant 1.3 kb fragment was excised from a low melting pomt agarose gel foUowing electrophoresis. A second PCR was performed on the gel-purified 1.3 kb fragment to isolate the kinase domam usmg the primers 5' Abl KD (5'-GCGCAACAAGCCCACTGTCTATGG-3') (SEQ ID NO: 8) and 3' Abl KD. The resultant 0.6 kb fragment was hgated mto pBluescript II KS+ digested with Eco RV Bacterial transformants were plated on media containing ampicilhn and X-gal. Ten white colonies per cDNA were inoculated mto media and miniprep DNA was isolated. Sequencmg of each clone was performed usmg Ml 3 universal forward and reverse primers Because two rounds of amphfication were employed, a mutation was considered present if it was detected on both strands of at least two mdependent clones per patient (see Figure 8). Analysis of the Abl kinase domam from two healthy blood donors was performed usmg PCR of the Abl kinase domam, foUowed by subsequent reampkfication to control for the number of amplification cycles to which patient samples were subjected Sequence analysis of a 0.7 kb portion of Bcr-Abl immediately 5' to the kmase domam was performed by amphfication of die previously described 1.3 kb fragment usmg CM10 and 5' Abl KD reverse complement (5'-CCATAGACAGTGGGCTTGTTGCGC-3') (SEQ ID NO. 9) foUowed by hgation mto pBluescript II KS+ as above The kmase domam of c-Kit was amplified usmg the foUowmg primers: (5'-TGAGGAGATAAATGGAAACAA-3') (SEQ ID NO: 10) and (5'-AACTCGTCATCCTCCATGAT-3') (SEQ ID NO: 11). To control for the number of cycles used for the Bcr-Abl k ase domam, a second amphfication was performed; the resultant 0.6 kb fragment was subcloned mto pBluescript II KS+ and ten mdependent colomes were sequenced
Expression vectors of mutant P210 isoforms were created as foUows. Ohgonucleotides containing vanous pomt mutations were synthesized by Gibco/BRL. PSRalphaP210Bcr-Abl was used as die template DNA for site-directed mutagenesis reactions utihzmg the mutant ohgonucleotides and the QuikChange mutagenesis kit (Stratagene). Successful mutagenesis was confirmed by sequence analysis of the kmase domam. Other P210 abl constructs are known m the art (see, e.g. Sun et al. Cancer Res. 2002, 62(11): 3175-3183; Dugray et al. Leukemia 2001 15(10): 1658-1662; and Heisterkamp et al, Transgemc Res. 1991 1 (1): 45-53).
293-T ceUs were co-transfected with mutant P210 expression vectors and a packagmg plasmid (Ecopack, kmdly provided by R. van Etten). Media containing virus was used to infect Ba/F3 ceUs. Stable lines were selected m the presence of G418 and IL-3. Subsequendy, IL-3 was removed from the media. Expression of Bcr-Abl was document by Western blot analysis. To determine the biochemical sensitivity of mutant P210 isoforms to STI-571, ceUs were mcubated m the presence of STI-571 (kmdly provided by Novartis, Switzerland) at 0, 0.5, 1, 5, and 10 micromoles per hter . After two hours of incubation, ceU lysates were prepared m 1% Tnton. Western blot analysis usmg AB-3 (Oncogene Research Products) or 4G10 (Upstate Biochemicals) was performed. To determine the biological sensitivity to STI-571, Ba/F3 ceUs expressmg vanous isoforms of P210 were mcubated m the presence of STI-571 (kmdly provided by Novartis, Switzerland) at 0, 0.5, 1, 5, and 10 micromoles per hter. After 24 hours of incubation, live ceUs were quantitated by trypan blue stam exclusion.
EXAMPLE 1B: ILLUSTRATIVE METHODS FOR EXAMINING DISCREET REGIONS IN BCR-ABL In certam contexts, it may be desirable to amplify a specific region m BCR-ABL such as one of the functional domams discussed herem. In this context, a 579 base pair region corresponding to the ATP-binding pocket and the activation loop of the kinase domam of Bcr-Abl was sequenced m the 9 patients for whom RNA was available at the time of relapse (Fig. 4A). Briefly, RNA was extracted from purified peripheral blood or bone marrow ceUs with Tnreagent-LS (Molecular Research Center, Inc., Cincinnati, OH). 2 mg of total RNA was subjected to RT-PCR usmg Ohgo dT primers A 1327-bp cDNA fragment was amphfied by PCR with a 5' BCR-specific primer (5'- GAAGCTTCTCCCTGGCATCCGT-3') (SEQ ID NO: 6) and a 3* ABL-specific primer (5'-GCCAGGCTCTCGGGTGCAGTCC-3') (SEQ ID NO: 7). In two patients, the BCR-ABL fragment could not be amphfied; therefore, a 579-bp fragment was amphfied usmg an alternative 5' ABL-specific primer (5'-GCGCAACAAGCCCACTGTCTATGG- 3') (SEQ ID NO: 8) and die same 3' ABL primer. PCR products were cloned mto the pCR2.1 TA clomng vector (Invitrogen, Carlsbad, CA) Bodi strands of a 579-bp region were sequenced with the 5' ABL primer and Ml 3 forward prmier or Ml 3 forward and reverse primer set for the 1327-bp and the 579-bp fragments, respectively, on an ABI pnsm 377 automated DNA sequencer (PE Applied Biosystems, Foster City, CA) Sequence analysis was performed usmg die ClustalW alignment algorithm). A smgle, identical C— >T nucleotide change was detected at ABL nucleotide 944 m six of nine cases examined (Fιg.4A). In aU six patients a mixture of wild-type and mutant cDNA clones were found, with the frequency of mutant clones rangmg from 17% to 70%. The mutation was found m three of three patients with lymphoid disease and m three of six patients widi myeloid blast crisis. The presence of the mutation was confirmed by analysis of genomic DNA (Fig. 4A). Briefly, genomic DNA was extracted from punfied bone marrow or penpheral blood ceUs with the QiaAMP Blood Mini Kit (Qiagen, Inc , Valencia, CA). A 361 -bp DNA fragment was amphfied by PCR with two primers (5'- GCAGAGTCAGAATCCTTCAG-3' (SEQ ID NO: 2) and 5'- TTTGTAAAAGGCTGCCCGGC-3') (SEQ ID NO: 3) which are specific for lntron sequences 5' and 3' of ABL exon 3, respectively. PCR products were cloned and sequenced. Analysis of RNA or genomic DNA from pre-treatment samples failed to provide evidence of the mutation pnor to STI-571 therapy; however, we cannot rule out the possibility that rare ceUs bearmg the mutation exist prior to treatment
EXAMPLE 2: ILLUSTRATIVE METHODS FOR MEASURING OF BCR-ABL KINASE ACTIVITY VIA THE PHOSPHOTYROSINE CONTENT OF CRKL Aldiough the enzymatic activity of Bcr-Abl protem is readily measured m ceU lines (e.g. via one of the assays discussed herem below), at times such assays can be difficult to perform m a reproducible, quantitative fashion with clmical materials because Bcr-Abl is subject to rapid degradation and dephosphorylation upon ceU lysis. In a search for alternative measures of Bcr-Abl kinase activity, we found that d e phosphotyrosme content of Crkl, an adaptor protem which is specificaUy and constitutively phosphorylated by Bcr-Abl m CML ceUs (see, e.g. J. ten Hoeve et al. Blood 84, 1731 (1994); T. Oda et al, J. Biol Chem. 269, 22925 (1994); and G. L Nichols et al, Blood 84, 2912 (1994)), could be measured reproducibly and quantitatively m clmical specimens. Crkl bmds Bcr-Abl direcdy and plays a functional role m Bcr-Abl transformation by linking the kmase signal to downstream effector pathways (see, e.g. K. Senechal et al, /. Biol Chem. 271, 23255 (1996)). When phosphorylated, Crkl migrates with altered mobility m SDS-PAGE gels and can be quantified usmg densitometry. As expected, Crkl phosphorylation m primary CML patient ceUs was mhibited m a dose- dependent manner when exposed to STI-571 and correlated with dephosphorylation of Bcr-Abl (Fig. 1A). This Crkl assay aUows for an assessment of the enzymatic activity of Bcr-Abl protem m a reproducible, quantitative fashion m clmical matenals Briefly, ceUs are lysed m 1% Triton X-100 buffer with protease and phosphatase mhibitors (see, e.g. A. Goga et al. Cell 82, 981 (1995)). Equal amounts of protem, as determined by die BioRad DC protem assay (Bio-Rad Laboratones, Hercules, CA), are separated by SDS-PAGE, transferred to nitroceUulose and lrnmunoblotted with phosphotyrosme antibody (4G10, Upstate Biotechnologies, Lake Placid, NY), Abl antibody (pex5, (see, e.g. A. Goga et al. Cell 82, 981 (1995)), β-actin antibody (Sigma Chemicals, St. Louis, MO) or Crkl antiserum (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands are visuahzed by ECL (Amersham Pharmacia Biotech, Piscataway, NJ). Several exposures are obtamed to ensure linear range of signal intensity. Optimal exposures are quantified by densitometry usmg ImageQuant software (Molecular Dynamics, Sunnyvale, CA)).
To establish the dynamic range of this assay m patient matenal, we measured Crkl phosphorylation m ceUs from BCR-ABL-negative individuals (»=4), untreated CML patients («=4), as weU as from patients who responded to STI-571 therapy but whose bone marrow ceUs remamed 100% Ph-chromosome-positive («=8). The mean level of Crkl phosphorylation m ceUs from CML patients pnor to STI-571 treatment was 73 ± 13.3% (Fig. IB). At die time of response die mean was 22 ± 9.9% (Fig. IB), similar to the mean level of Crkl phosphorylation in ceUs from BCR-ABL-negative individuals (22 ± 6.0%) (see, e.g. M. E. Gorre, C. L. Sawyers). We next measured levels of Crkl phosphorylation m primary leukemia ceUs from 11 patients who responded to STI-571 but subsequendy relapsed on treatment. In these cases, which mcluded one patient with lymphoid blast cnsis, three with Ph+ acute lymphoid leukemia, and seven with myeloid blast crisis, the mean level of Crkl phosphorylation at relapse was 59 ± 12.5% (Fig. IC). Anti-phosphotyrosme immunoblot analysis of a subset of diese samples confirmed that Bcr-Abl was phosphorylated on tyrosme at relapse (Fig. IC). Longitudmal analysis of blood or bone marrow samples obtamed from a subset of these patients before and throughout the course of STI-571 treatment confirmed that Crkl phosphorylation feU durmg the response to treatment, but mcreased at the time of relapse (Fig. ID). Therefore, disease progression m patients who mitiaUy respond to STI-571 is associated with failure to mamtam effective mhibition of Bcr-Abl kmase activity. EXAMPLE 3: ILLUSTRATIVE METHODS FOR EXAMINING
AMPLIFICATION OF THE BCR-ABL GENE IN MAMMALIAN CELLS
Some CML ceU lines that develop resistance to STI-571 after mond s of m vitro growth m sub-therapeutic doses of the drug have amphfication of the BCR-ABL gene (see, e.g. E. Weisberg et al. Blood 95, 3498 (2000); P. le Coutre et al, Blood 95, 1758 (2000); and F. X. Mahon et al, Blood 96, 1070 (2000)). We performed dual-color fluorescence m situ hybndization (FISH) experiments to determine if BCR-ABL gene amphfication could be similarly implicated m STI-571 resistance m human clinical samples Briefly, interphase and metaphase ceUs were prepared (see, e.g. E. Abruzzese et al, Cancer Genet. Cytogenet. 105, 164 (1998)) and examined usmg Locus Specific Identifier (LSI) BCR-ABL dual color translocation probe (Vysis, Inc., Downers Grove, IL)). Multiple copies of the BCR-ABL gene were detected m interphase nuclei m three (two myeloid blast crisis, one lymphoid blast cnsis) of the patients who relapsed after lnitiaUy responding to STI-571 (Fig. 3). Furdier cytogenetic and FISH characterization of metaphase spreads from these patients showed a unique mverted duphcate Ph- cliromosome with interstitial amphfication of the BCR-ABL fusion gene (Fig. 3C). In one patient, the mverted duphcate Ph-chromosome could be detected prior to the initiation of STI-571. In aU diree cases, additional copies of the aberrant Ph- chromosome were observed as STI-571 treatment continued, as weU as rmg chromosomes harbormg multiple copies of the BCR-ABL. Patient MB 14 was reevaluated by FISH one month after receivmg alternative treatment for her leukemia. Strikingly, BCR-ABL amphfication was no longer detectable 4 weeks after discontinuation of STI-571, raismg the possibihty that persistent STI-571 administration might select for mcreased copies of d e BCR-ABL gene m some patients.
Quantitative PCR analysis of genomic DNA obtamed from diese three patients confirmed mcreased ABL gene copy number at relapse when compared to a patient without BCR-ABL gene amphfication (Fig. 3D). Briefly, genomic DNA was extracted from purified bone marrow or peripheral blood ceUs with the QiaAMP Blood Mini Kit (Qiagen, Inc , Valencia, CA). 10 ng of total genomic DNA was subjected to real-time PCR analysis with the iCycler lQ system (Bio-Rad Laboratories, Hercules, CA). A 361- bp gDNA fragment mcludmg ABL exon 3 was amphfied usmg two primers (5'- GCAGAGTCAGAATCCTTCAG-3' (SEQ ID NO- 2) and 5'- TTTGTAAAAGGCTGCCCGGC-3' (SEQ ID NO- 3)) which are specific for lntron sequences 5' and 3' of ABL exon 3, respectively A 472-bp gDNA fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amphfied usmg two primers (5'-TTCACCACCATGGAGAAGGC-3' (SEQ ID NO: 4) and 5'- CAGGAAATGAGCTTGACAAA-3' (SEQ ID NO. 5)) which are specific for sequences m exon 5 and exon 8 of GAPDH, respectively. Fold mcrease m ABL copy number was determined by calculating the chfference between threshold cycle numbers of ABL and GAPDH for each sample (DCt). Usmg control LB3 as reference sample, DCt from each sample was subtracted from DCt of control to determine D(DCt). Fold mcrease was calculated as 2-D(DCt).
EXAMPLE 4. ART ACCEPTED METHODS FOR MEASURING
ENZYMOLOGICAL AND BIOLOGICAL PROPERTIES OF BCR-ABL MUTANTS
A vanety of assays for measurmg the enzymological properties of protem kmases such as Abl are known m the art, for example those described m Konopka et al, Mol CeU Biol. 1985 Nov;5(l l):3116-23; Davis et al, Mol CeU Biol. 1985 Jan;5(l):204-13; and Konopka et al, CeU. 1984 Jul;37(3):1035-42 the contents of winch are incorporated herem by reference. Usmg such assays the skilled artisan can measure the enzymological properties of mutant BCR-Abl protem lαnases. A vanety of bioassays for measurmg the transforming activities of protem lαnases such as Abl are known m the art, for example those described m Lugo et al. Science. 1990 Mar 2;247(4946): 1079-82; Lugo et al, Mol CeU Biol. 1989 Mar;9 (3): 1263-70; Klucher et al , Blood. 1998 May 15,91 (10):3927-34; Renshaw et al, Mol CeU Biol. 1995 Mar;15(3):1286-93; Sirard et al. Blood. 1994 Mar 15;83(6):1575-85; LaneuviUe et al, Cancer Res. 1994 Mar l ;54(5):1360-6; LaneuviUe et al. Blood. 1992 Oct 1 ;80(7):1788-97; Mandanas et al. Leukemia. 1992 Aug;6(8):796-800; and LaneuviUe et al, Oncogene. 1991 Feb;6(2):275-82 the contents of which are incorporated herem by reference. Usmg such assays the skilled artisan can measure the phenotype of mutant BCR-Abl protem kinases. Usmg protocols known m the art we have shown that T315I and E255K bod retain potent kmase activity and can confer growth factor mdependence in BaF3 murine hematopoietic ceUs. This mutant is resistant to mliibition by STI-571 m kinase assays and m growth assays. Other mutants can be similarly studied usmg such analyses.
EXAMPLE 5: ADDITIONAL ILLUSTRATIVE ANALYTICAL SCHEMES FOR CHARACTERIZING THE FUNCTIONAL IMPORTANCE OF BCR- ABL MUTATIONS
In addition to die methods descnbed above, skilled artisans can undertake additional analyses of one or more BCR-ABL mutants such as those identified m Table I.
For example, typical illustrative algoπthms such as those whose parameters are outlined below can be used to characterize the clmical importance of the various mutations found m the kmase domain.
In a first lUustrative mediod, one can examine samples from die same patient obtained at different times du mg their disease progression. Clones which become dominant over time may be presumed to have a growth advantage. This advantage could, for example be a consequence of mcreased potency of the BCR-ABL oncogene or resistance to a drug treatment such as STI-571 (as demonstrated by the T315I mutation).
In addition, mutations which appear more commonly can be given pnonty.
In a second lUustrative method, one can examine the location of the mutation m the context of the crystal structure of the ABL kinase domam (winch has been solved bound to STI-571). This structure aUows one to postulate whether the mutation might mterfere with the anti-leukemia activity of STI-571. Based on this analysis, one can pnontize mutations for direct experimental analysis of ABL kmase activity, leukemogenicity and level of mhibition by STI-571.
In yet another illustrative method, one can engmeer selected mutations mto wild- type BCR-ABL cDNA to create a mutant aUele whose enzymological and biological properties can be examined direcdy (see, e.g. Example 1 above). Enzymology can be performed by measurmg tyrosme kmase activity m vitro or m ceUs usmg standard assays known m the art. Biological activity can be measured usmg standard oncogene transformation assays usmg growth factor dependent hematopoietic ceU lines or primary mouse bone marrow ceUs. Resistance to STI-571 can be measured usmg kmase assays and transformation assays.
EXAMPLE 6: USE OF INFORMATION REGARDING BCR-ABL DOMAINS AND CRYSTALLOGRAPHIC ANALYSIS TO CHARACTERIZE BCR-ABL MUTATIONS As d e certam domams within BCR-ABL have been characterized and the crystal structure of dns protem has been elucidated, this information can be used m conjunction with the disclosure provided herem to characterize MARS such diose shown m Table I and to illustrate their role m resistance to inhibition of tyrosme kmase activity by STI- 571. For example, from the initial inspection of diese mutations m the context of the ABL crystal structure, one can categorize the mutants, for example m the foUowmg groups:
1. Hehx C mutations (e.g. ammo acid residue positions 304, 278):
Hehx C is a key regulatory hehx m the kmase V304D, V304A. These are located at the mterface with hehx C; M278K, M278L: Surface exposed methionme is disordered (borders hehx C). The functional significance of mutations found within this region or proximal to this region (m a manner that can perturb the normal function of this region), are supported by references which characterize this aspect of BCR-ABL.
2. P loop mutations (e.g. amino acid residue positions 253, 252, 250). The P loop is die phosphate bmdmg loop whose conformation is thought to be induced by STI-571. These mutations could prevent the required conformation of the loop to accommodate STI-571. Interestingly, we have found no mutations in the Gly motifs m the P loop (249, 251 and 254). These are highly conserved across other lαnases (so caUed Gly-X-Gly-X- X-Gly motif) and presumably are essential for kmase function. We do have examples of mutations m each of the X positions m the P loop. The functional significance of mutations found within this region or proximal to this region (m a manner that can perturb the normal function of this region), are supported by references which characterize this aspect of BCR-ABL.
Y253F: Direcdy stacks up agamst STI-571. -OH makes a tight H-bond with CL (or H20). Others: Q252H, Q252L, Q252R, G250E, E255K.
3. Residues which direcdy mteract with STI-571 (e.g. am o acid residue positions
315, 351, 355, 317, 290): The functional sigmficance of these residues or proximal to these residues (m a manner diat can perturb the normal function of this region), are supported by references which charactenze this aspect of BCR-ABL
M351T: van der Waal interactions widi His 361 which m turn mteracts direcdy with STI-571 piperazme group. Thr mutation could disrupt d e packing here and weaken mteraction with STI-571. Interestingly, this mutation may not affect compound 15 bmdmg (the one oπgmaUy crystallized with Abl) smce it has no piperazme group. E355G: at the end of the hehx that precedes the catalytic loop, which mteracts with the piperazme group of STI-571. Mutating to a Gly could make this region more flexible and weaken STI bmdmg. Agam Compound 15 should be less affected by this mutation.
F317L: direcdy stacks agamst STI-571. Leu mutation could weaken STI-571 bmdmg.
M290T, M290V: makes direct van der Waal interactions with STI-571. Mutation to either T or V would weaken STI-571 bmdmg.
4) Activation loop mutations (e.g. ammo acid residue positions 396). The functional sigmficance of mutations found within this region or proximal to this region (m a manner that can perturb the normal function of this region), are supported by references which characterize this aspect of BCR-ABL. H396K, H396R: disordered part of the activation loop. EXAMPLE 7: BCR-ABL POINT MUTANTS ISOLATED FROM PATIENTS WITH STI571-RESISTANT CHRONIC MYELOID LEUKEMIA REMAIN SENSITIVE TO INHIBITORS OF THE BCR-ABL CHAPERONE HEAT SHOCK PROTEIN 90
Clmical resistance to STI571 (Gleevec/imatmib mesylate) is commonly observed m patients with advanced Philadelphia chromosome-positive (Ph+) leukemias. Acquired resistance is typicaUy associated with reactivation of BCR-ABL due to kmase domam mutations or gene amphfication, mchcating that BCR-ABL remains a viable target for mhibition m these patients. Strategies for overcoming resistance can be envisioned through exploitation of other molecular features of the BCR-ABL protem, such as its dependence on d e molecular chaperone heat shock protem 90 (Hsp90). To determine whether mhibition of Hsp90 could mduce degradation of STI571 -resistant, mutant BCR- ABL protems, hematopoietic ceUs expressmg two mutant BCR-ABL protems found m STI571 -resistant patients (T315I and E255K) were examined for sensitivity to geldanamycm and 17-AAG. Both compounds mduced the degradation of wild-type and mutant BCR-ABL and mhibited ceU growdi, widi a trend mchcating more potent activity agamst mutant BCR-ABL protems. These data support clmical investigations of 17- AAG m STI571 -resistant Ph-positive leukemias. Strategies for overcoming resistance associated with kmase domam mutations wiU likely require targeting odier molecular features of the BCR-ABL protem. Heat shock protem 90 (Hsp90) is a molecular chaperone winch affects the stability and function of multiple oncogemc protems mcludmg BCR-ABL (An WG et al, CeU Growth Differ. 2000;11 :355-360; Shiotsu et al,. Blood. 2000;96:2284-2291) Geldanamycm (GA) is a benzoqumone ansamycm winch specificaUy mlnbits Hsp90 by competitively bmdmg to an ATP-binding pocket m the amino-terrninus of Hsp90 (Prodromou et al, CeU. 1997;90:65-75; Stebbms et al, CeU. 1997;89:239-250; Grenert et al, 1997;272:23843- 23850). Disruption of Hsp90 function by geldanamycm or its less toxic analogue, 17- aUylaminogeldanamycin (17-AAG), in BCR-ABL-expressmg leukemia ceUs has been shown to mduce BCR-ABL protem degradation and suppress ceU prohferation (An WG et al, CeU Growth Differ. 2000;11:355-360; Blagosklonny MV, et al. Leukemia. 2001;15:1537-1543; Nimmanapalh R, et al , Cancer Res. 2001;61 :1799-1804). 17-AAG is currendy m phase I clmical trials.
To determine whether mhibition of Hsp90 could mduce degradation of STI571- resistant, mutant BCR-ABL protems, hematopoietic ceUs expressmg two mutant BCR- ABL protems found m STI571 -resistant patients (T315I and E255K) were denved and tested for sensitivity to geldanamycm and 17-AAG We found that both compounds mduced the degradation of wild-type and mutant BCR-ABL protems as weU as mhibited ceU growth. The data also suggest a trend mchcating a greater potency agamst mutant BCR-ABL protems. These results provide a rationale for the use of 17-AAG m die cl ical setting of STI571 -resistant Ph-positive leukemia.
Chemicals. Stock solutions of GA (Sigma), 17-AAG (NSC 330507, National Cancer Institute), and STI571 (Novartis) were prepared as 10 mM dimethylsulfoxide solutions and stored at -20°C. Plasmids and ceU Unes. Full-length P210 T315I and P210 E255K BCR-ABL m pBluescript (Stratagene) were generated usmg site-directed mutagenesis and confirmed by sequencmg as descnbed previously (Gorre et al. Science. 2001;293-876-880). WUd- type and mutant P210 BCR-ABL were subsequendy subcloned mto the EcoRI site of pMSCVpuro (Clontech) for retrovirus generation. Ecotropic retroviruses were generated by cotransfection of pMSCVpuro DNA and Ecopac retroviral packaging vector (kmdly provided by R. Van Etten) mto 293T ceUs usmg the CaCl2 method (Muller AJ, et al , Mol CeU. Biol. 1991;11:1785-1792). The murine hematopoietic ceU line Ba/F3 was maintained m RPMI1640 supplemented with 10% fetal bovme serum, L-glutamine, and 1 ng/ml of recombmant murine IL-3 (R&D). Ba/F3 populations with stable BCR-ABL expression were derived by retroviral mfection of Ba/F3 ceUs m the presence of IL-3, and subsequent selection by puromycin. IL-3-mdependent BCR-ABL-expressmg ceUs were denved by culturmg m IL-3-free media at low densities m 96-weU tissue culture plates. Multiple IL-3-mdependent populations were assayed for comparable BCR-ABL protem expression by western blot. In vitro drug exposure assays. CeUs were cultured m 24-weU plates at 2X105 ceUs/ml m growth media (plus IL-3 for parental ceUs) with GA, 17-AAG, or STI571 for 24 or 48 hours Subsequent analyses of protem by western blot or ceU viability by trypan blue dye exclusion were done as previously descnbed (Gorre et al. Science. 2001;293:876-880; Goga A, et al, CeU. 1995;82:981-988). Results
Previous studies have shown d at the Hsp90 mhibitors GA and its derivative, 17- AAG, disrupt Hsp90 function and mduce BCR-ABL protem degradation (An WG et al, CeU Growth Differ. 2000;11 :355-360; Blagosklonny MV, et al. Leukemia. 2001;15:1537- 1543; NimmanapaUi R, et al. Cancer Res. 2001 ;61 :1799-1804). To determme whether GA can similarly cause the degradation of BCR-ABL protems carrying STI571 -resistant pomt mutations, populations of ιnterleukιn-3 (IL-3) dependent Ba/F3 murine hematopoietic ceUs were engineered to express either wild-type, T315I, or E255K P210 BCR-ABL and exposed to varying concentrations of mhibitor. Consistent with previous reports, both mutant BCR-ABL aUeles rendered the ceUs mdependent of IL-3, and ceUs expressmg either mutant contamed high levels of phosphotyrosme on BCR-ABL and odier substrate protems (Gorre et al. Science. 2001 ;293:876-880; von Bubnoff et al. Lancet. 2002;359:487-491). Western blot analyses usmg ABL-specific antibodies demonstrated that GA caused BCR-ABL protem levels to decrease significandy m ceUs expressmg wild-type BCR-ABL after treatment for 24 hours at a dose of 1.0 μM, as expected (An WG et al, CeU Growd Differ. 2000;11:355-360; Blagosklonny MV, et al. Leukemia. 2001 ;15:1537-1543; NimmanapaUi R, et al. Cancer Res. 2001;61:1799-1804). BCR-ABL protem was also degraded m ceUs expressmg either T315I or E255K BCR- ABL, but this degradation occurred at a lower GA concentration (0.5 μM) (Figure 7A). This apparendy enhanced degradation of the two mutant BCR-ABL protems was specific because degradation of another Hsp90 chent protem, RAF-1, was comparable m aU ceUs tested. These data suggest that GA may have greater potency agamst mutant BCR-ABL protems compared to wUd-type.
We next tested 17-AAG - a GA derivative currendy m phase I clmical trials - for its ability to mduce BCR-ABL protem degradation m the same Ba/F3 ceU lines. Western blot analyses of lysates from ceUs cultured in 17-AAG showed a similar trend to that seen with GA. Wild-type BCR-ABL protem levels feU graduaUy after 24 hour exposure to 0.5-1.0 μM 17-AAG. Although BCR-ABL protem levels m both the T315I and E255K BCR-ABL-expressmg ceUs began to decline at a similar concentration of 17-AAG as wild-type BCR-ABL (0.5 μM), the magnitude of decrease was more dramatic m ceUs expressmg the BCR-ABL mutants. VirtuaUy no BCR-ABL protem was detectable at 1.0 μM of 17-AAG for both mutants (Figure 7B). This trend was confirmed when we assessed the effect of 17-AAG on downstream BCR-ABL signalmg by measurmg the phosphorylation status of CRKL, a direct BCR-ABL substrate with functional relevance m CML (Nichols et al. Blood. 1994;84:2912-2918; Oda et al, J. Biol. Chem. 1994;269-22925-22928; Senechal et al,. J. Biol. Chem. 1996;271 :23255-23261 ; ten Hoeve J et al. Blood. 1994;84:1731-1736). Western blot analysis usmg CRKL-specific antisera on lysates from ceUs mcubated m the presence of mcreasmg concentrations of STI571 confirmed that the BCR-ABL mutants conferred resistance to STI571 (Figure 7E). CRKL western blot analysis on lysates from 17-AAG- treated ceUs revealed that lower doses of 17-AAG were needed to inhibit BCR-ABL activity m ceUs expressmg the BCR- ABL mutants when compared to wild-type BCR-ABL (Figure 7C,D). Significant changes m CRKL phosphorylation were not observed m wild-type BCR-ABL-expressmg ceUs until a 17-AAG concentration of 5 0 μM was reached, whereas CRKL phosphorylation m T315I and E255K BCR-ABL-expressmg ceUs was sigmficandy mhibited at 0.5 μM of drug (Figure 7C,D). While 17-AAG may affect another kmase which plays a role m CRKL phosphorylation m these ceUs, the fact that 17-AAG also reduced the level of BCR-ABL protem, together with previously pubhshed data showmg that constitutively elevated CRKL phosphorylation is relatively specific for CML (Nichols et al. Blood. 1994;84:2912-2918), provides strong evidence that BCR-ABL is the target.
Previous studies have also shown that GA and 17-AAG inhibit growth and mduce apoptosis of BCR-ABL-positive leukemic ceU lines (Blagosklonny MV, et al. Leukemia. 2001;15:1537-1543; NimmanapaUi R, et al. Cancer Res. 2001 ;61 :1799-1804). To determine whether GA could inhibit growth m ceUs expressmg STI571 -resistant BCR-ABL mutants, Ba/F3 ceUs transformed by wild-type, T315I, and E255K BCR-ABL were cultured m a range of GA concentrations. Trypan blue dye exclusion assessments of viability and corresponding IC50 calculations mdicated that the growth of aU three BCR-ABL-positive ceU lines was inhibited by GA at lower doses when compared to BCR-ABL-negative parental ceUs (Table III). The enhanced sensitivity of the STI-571 resistant BCR-ABL mutants compared to wild-type BCR-ABL observed m the biochemical analyses was also recapitulated m the growth mhibition assays. Similar results were observed with 17-AAG-treated ceUs. AU BCR-ABL-expressmg ceUs were more sensitive to 17-AAG than Ba/F3 parental ceUs, and the STI571 -resistant BCR- ABL-expressmg ceUs agam displaying a heightened sensitivity to mhibition compared to wild-type BCR-ABL-expressmg ceUs (Table III).
In summary, targeted mhibition of Hsp90 with either GA or 17-AAG mduced die degradation of wild-type BCR-ABL and two STI571 -resistant BCR-ABL mutants T315I and E255K. Both compounds also mhibited the growth of hematopoietic ceUs transformed by wild-type and mutant BCR-ABL. The results also suggest that the STI571 -resistant mutants are more sensitive to Hsp90 mhibition than wild-type BCR- ABL. One potential explanation could be that these two mutant protems are less stable than wild-type BCR-ABL, and therefore more dependent on molecular chaperones. A better understanding of the vanables that determine the relative dependence of chent protems on Hsp90 function is required to fully evaluate this question. Nevertheless, these data provide support for clmical investigations of 17-AAG m STI571 -resistant Ph- positive leukemia.
EXAMPLE 8: IDENTIFICATION OF A NOVEL PYRIDOPYRIMIDINE INHIBITOR OF ABL KINASE THAT IS A PICOMOLAR INHIBITOR OF BCR-ABL DRIVEN K562 CELLS AND IS EFFECTIVE AGAINST STI571- RESISTANT BCR-ABL MUTANTS.
Inhibition of the constitutively active Bcr-abl tyrosme kinase (TK) by STI571 has proven to be a highly effective treatment for chrome myelogenous leukemia (CML).
However STI571 is only transiendy effective m blast cnsis and drug resistance emerges by amphfication of or development of mutational changes m Bcr-abl. As described m this example, we have screened a family of TK mhibitors of the pyndo [2,3-^pyrιmιdιne class, unrelated to STI571, and descnbe here a compound widi substantial activity agamst STI-resistant mutant Bcr-abl protems This compound, PD 166326, is a dual specificity TK mhibitor and inhibits src and abl in vitro with IC50S of 6 and 8nM respectively. PD 166326 inhibits the growth of K562 ceUs with IC50 of 300 picomolar, leading to apoptotic Gl arrest, while non-Bcr-abl ceU types require more dian 1000 times higher concentrations. We tested the effects of PD166326 on two of the clmicaUy observed Bcr- abl mutants. The T315I mutation widnn die ATP-binding pocket reduces the affinity of STI571 for this pocket while the structural basis for resistance of the E255K mutation is currendy unknown. PD 166326 potendy inhibits the E255K mutant Bcr-abl protem and the growth of Bcr-ablE255K driven ceUs. The T315I mutant Bcr-abl protem is resistant to PD166326, however the growth of Bcr-ablT315I driven ceUs is partiaUy sensitive to this compound, likely through die mhibition of Bcr-abl effector pathways. These findings show that tyrosme kmase drug resistance is a structure-specific phenomenon and can be overcome by TK mhibitors of other structural classes, suggesting new approaches for future anti-cancer drug development. PD 166326 is a prototype of a new generation of anti-Bcr-abl compounds with picomolar potency and substantial activity agamst STI571- resistant mutants.
Cell culture and growth assays CeU were cultured m RPMI medium supplemented with 100 U/ml pemcilhn, lOOμg/ml streptomycin, 4mM glutamine, 10% heat inactivated fetal bovme serum and mcubated at 37C m 5%C02. For growth assays, ceUs were seeded m 12-weU clusters at 10-20,000 ceUs per weU. CeUs were placed m media containing various concentrations of the drugs with vehicle (DMSO) never contributing more than 0.1%. After 4-7 days, ceUs were counted usmg a coulter counter. AU experiments were performed m duphcate and results averaged. PD 166326 was stored in a lOmg/ml DMSO solution and stored at -70C. The derivation and chemical structure of PD 166326 has been previously published (see e.g Kraker et al. Biochemical Pharmacology 60, 885-898. 2000). Cell cycle assays
CeUs were treated with mdicated concentrations of PD 166326 or vehicle (DMSO) for the mdicated times. For synchronization, ceUs were mcubated m media containing 5ug/ml aphidicohn for 24 hours, washed twice m PBS, and replaced m growth media. At die time of harvest, ceUs were washed once m PBS and ceU nuclei prepared by d e method of Nusse (see e.g. Nusse et al , Cytometry 1990;1 1 :813-821) and ceU cycle distribution determined by flow cytometnc analysis of DNA content usmg red fluorescence of 488nm excited edndium bromide stamed nuclei.
Protein extraction and western blotting
CeUs were washed m PBS once and lysed m modified RIPA buffer (10 mM Na phosphate pH 7.2, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% NP-40, 1% Na deoxycholate, ImM Na Vanadate, and protease mhibitors). 50ug of total ceUular protem was separated by SDS-PAGE, transferred to membrane, and irnmunoblotted usmg antibodies to phosphotyrosme (SantaCruz), c-abl (8E9), and phospho-Hck (SantaCruz), MAP kmase (SantaCruz) and phospho-MAP kmase (Promega).
In vitro kinase assay
C-abl kmase assays were performed usmg punfied recombmant c-abl and peptide substrate (New England Biolabs). Kinase assays were performed m 50mM Tns-Cl pH 7.5, lOmM MgCL?, ImM ethylene glycol bis-ammoethyl ether tetraacetic acid (EGTA), 2mM dithiothreitol (DTT), 0.2% tπton-X, lOOuM ATP, 40uM peptide substrate, m lOOul reaction volumes containing 50 units c-abl enzyme and I OUCI [32P] γ-ATP. Reactions were aUowed to proceed for 10 minutes at 30C and stopped by addition of EDTA and boiling. Reaction products were spotted on phosphoceUulose paper, washed several times with phosphoric acid, then acetone, and counted m scintillation fluid. Pilot experiments were lnitiaUy performed to establish that these reaction conditions were m linear range. Bcr-Abl was immune precipitated from ceU lysates of K562 ceUs mamtamed m log-phase culture conditions. Complexes were coUected on protem A-sepharose and washed three times m lysis buffer and twice m abl kmase buffer (50mM ens pH 8.0, lOmM MgC12, 1 mM DTT, and 2mM p-nitrophenylphosphate, and 2 mM ATP; New England Biolabs Buffer and protocol) Kinase assays were performed with 10 mM [γ-32P] ATP/ sample for 5-60 minutes at 30C m the presence or absence of mdicated concentrations of drug The immune complexes were pre-mcubated with the drug for 10 minutes at 4C prior to addition of labeUed ATP and initiation of the reaction at 30C. The reaction was stopped by the addition of SDS-PAGE sample buffer and heated at 100C for 10 minutes. Protems were separated on 7.5% SDS-polyacrylamide gels and gels were dned under vacuum and phosphorylation was visualized by autoradiography on x-ray film.
Results
In screening a compound library for mhibitors of c-src tyrosme kinase activity, a number of pyndo[2,3-^pyπmιdιnes were previously described that are ATP-competitive inhibitors of c-src with IC50 values <20nM and varying degrees of selectivity for c-src
(see e g. Kraker et al. Biochemical Pharmacology 60, 885-898 2000). We screened dns group of compounds for activity agamst c-abl usmg purified recombmant c-abl and peptide substrate m in vitro kmase assays. The most potent compound was PD 166326 with an IC50 of 8nM (agamst c-abl) and 6nM (agamst src). The src family kmase Lck is mhibited with IC50 <5nM. This compound also has activity agamst basic-FGF, PDGF, and EGF receptor tyrosme lαnases in vitro with IC50S of 62, 139, and 80 nM respectively .
PD 166326 shows no significant activity agamst JNK lαnases, cychc AMP-dependent protem kmase (PKA), PKB-β, PKC-α, rho-dependent protem kmase, casern kmase-2, and phosphorylase kinase. In companson with PD 166326, STI571 is a weaker mhibitor of Abl in vitro with an ICso=50nM. PD166326 also inhibits Bcr-abl kmase in vitro with
IC50=lnM.
PD 166326 also inhibits Bcr-abl activity m ceUs as determined by Western blot analysis of Bcr-abl autophosphorylation m K562 ceUs. In these ceUs Bcr-abl autophosphorylation is mhibited widi IC50 of InM compared with lOOnM for STI571. Bcr-abl autophosphorylation correlates with Bcr-abl signalmg activity as shown by die paraUel decline of MAP kmase activity with inhibition of Bcr-abl m these assays.
The biologic activity and potency of PD 166326 was lnitiaUy evaluated m ceU growth assays usmg K562 ceUs This compound inhibits K562 ceU growth with IC5o=0.3nM. Other Bcr-abl dnven ceU lines are also extremely sensitive to PD 166326 with IC50S of 0.8 and 6nM (see M07- p210bcr αbl and BaF3-p210b«-abl). The potent biologic activity of PD 166326 is highly specific for Bcr-abl-driven ceUs as additional hematopoetic and epithelial ceU lines are only mhibited at 2 to 3 logs higher concentrations and IC50S m d e 0.8-2uM range.
Further analysis reveals that PD 166326 inhibits ceU prohferation specificaUy m the Gl phase of the ceU cycle. At concentrations that fully inhibit the growth of Bcr-abl positive ceUs but not other ceU types, PD 166326 leads to accumulation of ceUs m the Gl phase accompanied by a significant mcrease m the number of apoptotic ceUs. Additional phases of the ceU cycle are not affected by this compound as shown by experiments with synchronized ceUs. K562 ceUs were synchronized at the Gl/S boundary with aphidicohn and released mto PD 166326 or vehicle and ceU cycle progression studied over die foUowmg 24 hours. These data show diat PD 166326 treatment does not mterfere with progression through the S, G2 or mitotic phases of the ceU cycle, but PD166326 treated ceUs are unable to exit the Gl phase. Similar experiments with nocodazole-synchronized ceUs also confirm that PD 166326 blocks Gl progression. The inhibition of Gl progression and induction of apoptosis m K562 ceUs are similar to the effects previously reported for STI571 (see e.g Dan et al, CeU Death & Differentiation. 1998,5 710-715). These data show that PD 166326 is a potent mhibitor of Bcr-abl k ase activity and inhibits Bcr-abl dnven ceU growth through mhibition of Gl progression leadmg to apoptotic ceU death.
Resistance to STI571 treatment is associated with mutations m the Bcr-abl oncoprotem that render it refractory to STI571 mhibition (see e.g Gorre et al. Science 2001;293:876-880). Because PD166326 inhibits both Src and Abl whereas STI571 only inhibits Abl, it may bmd Bcr-abl differendy than STI571. This chfference raises die possibihty that it may be effective agamst some mutant Bcr-abl protems We compared the activities of PD166326 and STI571 agamst two such mutant Bcr-abl protems derived from patients who have relapsed on STI571 therapy. The T315I mutation is frequendy seen m relapsed patients and eliminates a critical Threonine residue within the ATP bmdmg pocket of Abl and gready reduces the bmdmg affimty of STI571. The E255K mutation also hes within a region of Bcr-abl commonly mutated in relapsed patients, however the structural basis for STI571 resistance conferred by mutations m this region is not currendy understood. BaF3 mouse hematopoietic ceU lines were stably transfected with either the wild-type p210bcr"abl cDNA or the T315I or E255K mutant versions as previously descnbed (see e.g. Gorre et al. Science. 2001,293.876-880). Expression of Bcr-abl renders BaF3 ceUs IL-3 mdependent while control ceUs transfected with vector alone require IL-3 for growth. Although STI571 inhibits the wild-type p210bcr"abl ceUs with IC50=500nM, the T315I and E255K mutant p210bcr-abl ceUs are highly resistant. However resistance to STI571 does not appear to confer cross-resistance to PD 166326. PD166326 inhibits the autophosphorylation of p210Bcr- blE255K in vivo as effectively as die autophosphorylation of the wild type p210Bcc abl, while this mutant is highly resistant to mhibition by STI571. However, the p210Bcr-ablT3151 mutant is resistant to PD166326 as it is to STI571. This is not surprising, considering the cntical role of Thr315 within the ATP bmdmg pocket. To determme whether ceU growth sensitivity to PD166326 correlates with mhibition of the mutant Bcr-abl oncoprotems, we also determined the sensitivity of the BaF3 ceUs dnven by the wild type and mutant Bcr-abl protems. BaF3P210Bcr"abl ceUs are very sensitive to PD166326 (IC5o=6nM) and the E255K mutant p210bcr abl ceUs remam relatively sensitive to this compound (IC50=15nM). The effective mhibition of p210E255 bcr-abl activity at dose ranges that inhibit the growth of these ceUs is further evidence that STI571 -resistant leukermc ceUs are dnven by persistent activity of the mutated Bcr-abl oncoprotein. In comparison, the T315I mutant ceUs are partiaUy resistant to PD166326, although not fully resistant. PD166326 inhibits BaF3P210T3151 ceUs with IC50 of 150nM. Although this is 25 fold weaker than the mhibition of the wild-type BaF3P210 ceUs, it may stiU be of therapeutic value smce it is 8-fold more potent than the mhibition of the BaF3-vector controls and non-Bcr-abl driven ceUs. Although PD166326 inhibits the growth of BaF3p210B«-ablT3151 ceUs with IC50 of 150nM, it fails to mlnbit the autophosphorylation of die T351I Bcr-abl mutant at doses up to luM, suggesting that its anti-prohferative effects are mediated m part through mechanisms other than the mhibition of Bcr-abl.
PD166326 is also active agamst src lαnases and its anti-leukemic effects may be m part related to its mhibition of the src lαnases Hck and Lyn which function downstream of Bcr-abl. The src lαnases Hck and Lyn are activated by Bcr-abl and may mediate some of the transforming functions of Bcr-abl. Phosphorylation of tyr416 in the catalytic domam is required for activation of src kinases, although the mechamsm by which Bcr- abl activates Hck and Lyn is not undeistood. Inhibition of Bcr-abl by STI571 results m a paraUel mhibition of Hck activation m K562 ceUs. In diese ceUs PD 166326 also inhibits Bcr-abl and Hck activation although at 100 fold lower doses than seen with STI571. Hck is also activated by mutant forms of Bcr-abl and m the mutant BaF3p210Bcr"ablE255 ceUs, PD 166326 inhibits Hck activation and this correlates with the observed mhibition of Bcr-ablE255K autophosphorylation and inhibition of ceU growth. In contrast, the activation of Hck by d e Bcr-abl™51 mutant is not inhibited by PD166326 and this correlates with the observed resistance of Bcr-ablT3151 activity to PD166326. However despite failure to inhibit Bcr-abl activity and die consequent activation of Hck, PD166326 inhibits die growdi of BaF3p210Bcr-ablT3,SI ceUs with IC50 of 150nM, likely through additional mechamsms.
Although STI571 has revolutionized the treatment of CML, the problem of TK drug resistance is now emerging as a clmical reality. Resistance to STI571 appears to have a structural basis and newer TK mhibitors may also be susceptible to similar mechamsms of resistance. However TK mhibitors of a chfferent structural class may have more favorable bmdmg characteristics. Dorsey et al mitiaUy reported that a src-selective TK mhibitor of the pyπdo [2,3-^pynmιdιne class has substantial activity agamst Bcr-abl kmase (see e.g. Dorsey et al. Cancer Research. 2000;60:3127-3131). We have extended this finding by screening a family of src-selective pyndo [2,3-^pyrιmιdιnes and identified a compound with the most potent activity agamst abl kmase. Here we report the characterization of this compound, PDl 66326, a novel dual specificity TK mhibitor that is more than 100 fold more potent than STI571 in vivo and inhibits K562 ceUs with IC50 of 300 picomolar. It is unlikely that the potent growth inhibitory activities of PDl 66326 are related to non-specific activities smce the potency of this compound appears to be specific for ceU types dnven by Bcr-abl kmase. While Bcr-abl-dnven ceUs are inhibited with IC50S in the 0.3-6nM range, other ceU types mcludmg the hematopoietic ceUs BaF3 and 32D as weU as epithelial cancer ceUs mcludmg MCF-7 ceUs and MDA-MB-468 ceUs, winch are driven by EGFR overactivity, are mhibited with IC50S m the 0.8-2uM range (table 2). The micromolar activity of PD166326 agamst the growth of non-Bcr-abl dnven ceUs is most likely mediated dirough mhibition of additional ceUular targets smce unlike Bcr-abl positive ceUs, the growth of Bcr-abl negative ceUs is mhibited during the S phase of the ceU cycle . The picomolar potency and ceUular selectivity of PDl 66326 are sigmficandy superior to STI571 in vitro.
Smce Bcr-abl signalmg is known to mvolve the src family lαnases Hck and Lyn, and smce PDl 66326 is also a potent mhibitor of src family kmases, it is plausible that the biologic potency of this compound is related to dual mhibition of diese two functionaUy related tyrosme lαnases. Hck associates with and phosphorylates Bcr-abl on Tyr 177 leading to recruitment of Grb2/Sos and activation of the Ras pathway (see e.g. Warmuth et al. Journal of Biological Chemistry. 1997;272:33260-33270). Kinase-defecαve Hck mutants suppress Bcr-abl mduced transformation suggesting that Hck-mediated signalmg is essential for the transforming activity of Bcr-abl (see e.g. Lionberger et al. Journal of Biological Chemistry. 2000;275T8581-18585). The role of Lyn m Bcr-abl signalmg is less weU studied. However Lyn activity is also elevated m acute myeloid leukemia ceU lines and m these ceUs inhibition of Lyn expression usmg anti-sense molecules leads to decreased proUferative activity and mhibition of Lyn kinase activity usmg src family selective pharmacologic mhibitors leads to potent mhibition of ceU growth and colony formation (see e.g. Roginskaya et al. Leukemia. 1999;13:855-861). It is also possible that the potency of PDl 66326 is mediated through the mhibition of other, yet undiscovered ceUular protems, and our data does not exclude this possibihty. However the role of currendy unknown ceUular targets m mediating the growth inhibitory effects of this compound m Bcr-abl dnven ceUs is difficult to know until such candidate targets are identified and studied.
Smce relapse on STI571 is associated with mutations m Bcr-abl that alter die bmdmg of STI571, understanding the nature of the STI571 mteraction with Abl is of fundamental importance m order to overcome drug resistance. The crystal structure of a vanant STI571 m complex with the catalytic domam of Abl was recendy solved by Schmdler et al (see e g. Schindler et al. Science. 2000;289 1938-1942). STI bmds within the ATP bmdmg pocket of Abl m its mactive conformation This mteraction is criticaUy affected by the conformation of the Abl activation loop. When phosphorylated, this activation loop favors an open and activating conformation which, by virtue of its amino-terminal anchor, mterferes with STI571 bmdmg to the ATP-binding pocket. Consistent with this model, die bmdmg of STI571 is selective for the mactive conformation of Abl, and this compound is unable to inhibit the catalytic activity of active phosphorylated Abl (see e g. Schmdler et al. Science 2000;289.1938-1942). The broader activity of PDl 66326, mcludmg activity agamst src lαnases suggests that unlike STI571, it may not bmd selectively to the mactive conformation of Abl, smce m its active conformation, Abl bears considerable structural homology to the src lαnases (see e g Schmdler et al , Science. 2000;289 1938-1942). While selectivity for the mactive conformation is postulated to confer a lngh degree of molecular specificity to STI571 , this may be at the price of potency PDl 66326 may be binding to both mactive and active conformations of Abl leadmg to the more effective mhibition of overaU enzyme activity that we see in vitro. In addition, phosphorylation of the activation loop of Abl is catalyzed by the src family kmase Hck m Bcr-abl transformed ceUs Smce PDl 66326 also mlnbits Hck, this may prevent phosphorylation of the activation loop, destabilizing the Abl active conformation This aUostenc mechamsm m addition to the direct bmdmg of PDl 66326 to the ATP-binchng pocket could provide dual mechamsms for its mhibition of Abl activation and provide the basis for its mcreased potency. Validation of these hypotheses awaits crystaUographic studies of PDl 66326 bound to Abl.
PDl 66326 is non-cross-resistant with STI571 and has substantial activity agamst the T315I and E255K STI571-resιstant Bcr-abl mutants This finding has important implications for the future design and use of TK mhibitors of aU lands, smce it is the first report showmg that TK-inhibitor resistance can be overcome by anodier TK-inhibitor of a different structural class. It is difficult to speculate on whether the development of resistance to PDl 66326 wiU be just as likely as with STI571, but smce these compounds are structuraUy unrelated, resistance to PDl 66326 will likely involve a different structural basis than resistance to STI571. This distinction creates the opportunity for strategies to prevent or overcome resistance such as sequential or combination dierapies. However understanding drug sensitivity and resistance is of fundamental importance in this regard. While additional studies will elucidate the exact structural and ceUular basis underlymg STI571 resistance and PDl 66326 sensitivity, existing data explams our findings. A number of ammo acid residues mediate the binding of STI571 within die ATP-binding pocket, and among these, Thr315 is cntical for hydrogen bond formation with the drug (see e.g. Schmdler et al. Science. 2000;289:1938-1942). The T315I mutation, seen m STI571 -resistant CML, precludes hydrogen bonding with STI571 and results m a stenc clash due to the extra hydrocarbon group m He (see e.g. Gorre et al. Science. 2001;293:876-880). Likewise PD166326 does not inhibit the activity of Bcr- abl0151 in vivo suggesting that Thr315 is also important for its bmdmg within the ATP pocket of abl. However PD166326 has some activity agamst BaF3p210T3151 ceUs and mlnbits their growdi with IC50 of 150nM. This activity is related to Bcr-abl driven growth smce growth mhibition of non-Bcr-abl dnven ceU types requires 5-15 fold higher concentrations. Smce PDl 66326 is a potent inhibitor of src lαnases, and smce the src lαnases Hck and Lyn mediate some of the transforming activities of Bcr-abl, it is possible that PD166326 inhibits the growth of BaF3p2lOT3151 ceUs through the mhibition of Hck and Lyn. However, seemingly mconsistent with this hypothesis, we fail to see mhibition of Hck Y416 phosphorylation m these ceUs at growth inhibitory concentrations. However this does not disprove the hypodiesis due to limitations m assaymg Hck activity in vivo. If PDl 66326 bmds with and inhibits the active Y416 phosphorylated conformation of Hck, dien this catalyticaUy mactive drug-Hck complex may remam stably m this phosphorylated conformation and phospho-Y416 Hck antibodies wiU be unable to demonstrate the in vivo mhibition of Hck catalytic function. In vitro kinase assays do not help in this regard either, smce durmg the process of ceU lysis and lmmunoprecipitation, the Hck-PDl 66326 mteraction is lost. Therefore, m BaF3 p210T3151 ceUs, where Bcr-abl activity is resistant to PDl 66326, inhibition of Hck activity may be responsible for the observed growth inhibitory effects at ICso=150nM despite persistent phosphorylation of Hck at these doses. In addition, although Y416 is a site of auto-phosphorylation m src lαnases, it may also be a substrate for phosphorylation by other lαnases. In fact m our experiments Hck Y416 phosphorylation status paraUels Bcr-abl activity winch suggests that Hck Y 1δ may also be a substrate for Bcr-abl. Although the activity of PDl 66326 agamst src kinases would suggest diat it inhibits BaF3 p21013151 ceUs through a src family member, these experiments do not rule out die possibihty that this ceUular sensitivity is mediated through the mhibition of odier, yet unknown, lαnases.
The structural basis for die STI571 resistance of the E255K mutated Bcr-abl is less clear smce the functional sigmficance of this residue is currendy unknown. Interestingly this mutation confers htde resistance to PD166326. PD166326 shows no loss of activity agamst Bcr-ablE255K autophosphorylation in vivo and only 2.5 fold less activity agamst the growth of BaF3Bcr-ablE255K ceUs compared with wild type Bcr-abl controls. The ceUular IC50 of PDl 66326 agamst BaF3Bcr-ablE255K ceUs (15nM) is much lower than its activity m non-Bcr-abl dnven ceU types (0.8-2uM), and much greater than the activity of STI571 agamst this mutant. If die basis for Bcr-abl E255K resistance to STI571 is destabihzation of the mactive conformation, and if PDl 66326 m fact bmds to the active conformation, dien this would explain why PDl 66326 is effective in inhibiting Bcr-ablE255K. However validation of these hypotheses requires crystal structure data to better define the function of the Glu255 residue and the bmdmg of PDl 66326 to Bcr-abl.
TABLES
Tables 1A-1E identify typical MARS. The data are from analysis of patients, with an average of 10 clones sequenced per patient. These tables identify subgroups of mutations that are more likely to be significant because d ey occur m more than one patient or they are dommant (defined as bemg detected m at least 2 of 10 clones m the same patient). The observation that these mutants are showmg up so commonly provides further evidence that these mutations wiU turn out to be clmicaUy significant.
Table IA: Residues Mutated in Individuals Treated with STI-571
D233, T243, M244, K245, G249, G250, G251 , Q252, Y253, E255, V256L Y257,F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291, E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351, E352, E355, K357, M359, N358, F359, 1360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396, A399, P402, T406.
Table IB: Typical Mutations at a Glance
D233H, T243S, M244V, G249D, G250E, G251S, Q252H, Y253F, Y253H, E255K, E255V, V256L, Y257F, Y257R, F259S, K262E, D263G, K264R, S265R, V268A, V270A, T272A, Y274C, Y274R, D276N, T277P, M278K, E282G, F283S, A288T, A288V, M290T, K291R, E292G, I293T, P296S, L298M, L298P, V299L, Q300R, G303E, V304A, V304D, C305S, C305Y, T306A, F311L, I314V, T315A, T315I, E316G, F317L, M318T, Y320C, Y320H, G321E, D325H, Y326C, L327P, R328K, E329V, Q333L, A337V, V339G, L342E, M343V, M343T, A344T, A344V, I347V, A350T, M351T, E352A, E352K, E355G, K357E, N358D, N358S, F359V, I360K, I360T, L364H, E373K, N374D, K378R, V379I, A380T, A380V, D381G, F382L, T389S, T392A, T394A, A395G, H396K, A399G, P402T, T406A.
Table IC: Mutations Occurring in More Than One Patient
Q252H, E255K, K264R, F283S, M290T, P296S, V304D, T315I, R328K, M343T, M343V, A344T, M351T, K357E, M359V, I360T.
Table ID: Dominant Mutations or Mutations with Frequencies Greater Than One Clone/Patient
G250E, Q252H, Y253F, Y253H, E255K, V270A, V304D, T315I, F317L, M343T, M351T, E355G, M359V, I360K, V379I, F382L, H396K. Table IE: Mutations Occurring in More Than One Patient With a Dominant Clone or at Least Greater Than One Clone Occurrence
Q252H, E255K, V304D, T315I, F317L, R328K, F359V, M351T.
Table IF: Attached Mutations
Y257F, Y274R, D276N, E282G, M290T, I293T, P296S, L298M, L298P, V304D, T315I, F317L, G321E, Q333L, A337V, V339G, M343T, M351T, E352A, I360T, E373K, V379I, D381G, F382L, T392A.
TABLE II: GenBank accession number M14752
MLEICLKLVGCKSKKGLSSSSSCYLEEALQRPVASDFEPQGLSEAARWNSKENLLAG PSENDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWCEAQTKNGQG VPSN YITPVNSLEKHSWYHGPVSR AAEYLLSSGINGSFLVRESESSPGQRSISLRYEGRV YHYRINTASDGKLYVSSESRFNTLAELλ HHSTVADGLITTLHYPAPKRNKPTVYGV SPNYDKWEMERTDITMΗKLGGGQYGEVYEGλ/ KKYSLTVAVKTLKEDTMEVEEFLK EAAλ KEIKHPNLVQLLGVCTREPPFYIITEF TYGNLLDYLRECNRQE'v' AWLLY MATQISSAMEYLEKK FIHRDKAARNCLVGENHLVrVADFGLSRLMTGDTYTAHAGA KFPIKWTAPESLAYNKFSIKSDV AFGVLLWEIATYGMSPYPGIDLSQVYELLEKDY RMERPEGCPEKVYELMRACWQ NPSDRPSFAEIHQAFET FQESSISDEVEKELGKQ GVRGAVSTLLQAPELPTKTRTSRRAAEHRDTTDVPEMPHSKGQGESDPLDHEPAVSP LLPRKERGPPEGGLNEDERLLPKDKKTNLFSALIKKKKKTAPTPPKRSSSFREMDGQ PERRGAGEEEGRDISNGALAFTPLDTADPAKSPKPSNGAGVPNGALRESGGSGFRSP HL KKSSTLTSSRLATGEEEGGGSSSKRFLRSCSASCVPHGAKDTEWRSVTLPRDLQ STGRQFDSSTFGGHKSEKPALPRKRAGENRSDQVTRGTVTPPPRLVKKNEEAADEVF KDIMESSPGSSPPNLTPKPLRRQVTVAPASGLPHKEEAEKGSALGTPAAAEPVTPTS KAGSGAPGGTSKGPAEESRVRRHKHSSESPGRDKGKLSRLKPAPPPPPAASAGKAGG KPSQSPSQEAAGEAVLGAKTKATSLVDAλ/NSDAAKPSQPGEGLKKPVLPATPKPQSA KPSGTPISPAPVPSTLPSASSALAGDQPSSTAFIPLISTRVSLRKTRQPPERIASGA ITKGλA^LDSTEALCLAISRNSEQMASHSAVLEAGrOMLYTFCVSYVDSIQQMRNKFAF REAINKLEN LRELQICPATAGSGPAATQDFSKLLSSVKEISDIVQR (SEQ ID NO: 1)
Table III. Sensitivity of STI571-resistant BCR-ABL-transformed cells to geldanamycin and 17-AAG.
MEAN IC50 ± S.D. (μM)
CELL LINE GA 17-AAG
Ba/F3 + IL-3 27.3 ± 14.1 12.4 ± 0.3 Ba/F3 P210 WT 4.9 ± 1.6 5.2 ± 2.4 Ba/F3 P210 T315I 1.8 ± 2.1 (P=0.03) * 2.3 ± 0.4 (P=0.04) Ba/F3 P210 E255K 2.6 ± 2.4 (P=0.05) 1.0 ± 0.2 (P=0.0T)
Representative data from at least two mdependent experiments performed m duphcate; IC50, concentration of mlnbitor required to reduce the number of viable ceUs by 50%;
*Dιfferences m the mean IC50 values between WT and mutant P210 Ba/F3 ceUs were analyzed with the unpaired Student's t-test; two-tailed P values are shown.
Table IV. Detailed summary of Bcr-Abl kinase domain mutations by disease category.
MBC denotes relapsed myeloid blast cnsis despite STI-571. LBC denotes relapsed lymphoid blast crisis. CP denotes chrome phase with no cytogenetic response. R-MBC denotes pre-STI-571 sample from myeloid blast cnsis patients whose disease was subsequendy refractory.
Patient No Duration of Treatment with Mutatιon(s) # of mdependent clones imatinib at time of analysis containing mutation
1 (MBC) 4 5 months G250E (7/10)
H396R (2/10)
2 (MBC) 13 months T315I (10/10)
3 (MBC) 8 months none N/A
4 (MBC) 3 5 months M351T (5/10)
5 (MBC) 1 month Q252H (5/10)
6 (MBC) 13 months M351T (8/10)
7 (MBC) 13 months M351T (6/10)
8 (MBC) 1 month V304D (2/10)
9 (MBC) 5 months E255K (4/10)
Y253H (2/10)
10 (MBC) 7 months E355G (5/10)
F317L (2/10)
1 1 (MBC) 6 months G250E (8/10)
12 (MBC) 3 months Y253F (3/10)
E255K (2/10)
M351T (2/10)
H396R (2/10) 13 (MBC) 1 5 months M351T (3/10)
T315I (2/10)
Y253H (2/10)
E255K (2/10)
14 (MBC) 3 months Y253F (2/10)
E255K (2/10)
T315I (2/10)
15 (MBC) 3 months E255K (2/10)
16 (MBC) 2 months E255 (2/10)
Q252H (2/10)
17 (MBC) 4 months F359V (8/10)
18 (LBC) 1 month M351T (3/10)
E255K (2/10)
T315I (2/10)
Y253F (2/10)
19 (LBC) T315I (5/10)
E255K (2/10)
Q252R (2/10)
20 (LBC) T315I (5/10)
21 (LBC) T315I (6/10)
22 (LBC) none 3 (CPNCR) V379I (7/10) 4 (CPNCR) F317L (6/10) 5 (CPNCR) E255K (7/10) 6 (CPNCR) F359V ( 10/10)
27-35 none
Patient No Duration of Treatment with Mutatιon(s) # of independent clones imatinib at time of analysis containing mutation 6 (R-MBC) T315I (2/10)
M343T (2/10)
F382L (2/10) 7 (R-MBC) none 8 (R-MBC) none 9 (R-MBC) none
Throughout dns apphcauon, various publications are referenced. The disclosures of these publications are hereby mcorporated by reference herem m their entireties The present mvention is not to be limited m scope by die embodiments disclosed herem, which are mtended as smgle lUustrations of mdividual aspects of the mvention, and any that are functionaUy equivalent are within the scope of the mvention. Vanous modifications to the models and methods of the mvention, m addition to those described herem, will become apparent to those skiUed m the art from die foregomg descnpαon and teachings, and are similarly mtended to faU within the scope of the mvention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the mvention.

Claims

CLAIMS:
1. A method of identifymg an ammo acid substitution m at least one Bcr-Abl polypeptide expressed m a human cancer ceU from an mdividual selected for treatment with a tyrosme kinase mhibitor, the method compnsmg determining the polypeptide sequence of at least one Bcr-Abl polypeptide expressed by the human cancer ceU and companng the polypeptide sequence of the Bcr-Abl polypeptide expressed by the human cancer ceU to the Bcr-Abl polypeptide sequence shown m SEQ ID NO. 1 so that an ammo acid substitution m the Bcr-Abl polypeptide expressed by the human cancer ceU can be identified.
2. The method of claim 1, wherem the amino acid substitution occurs m a region of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 compnsmg residue D233 through residue T406.
3. The method of claim 2, wherem the ammo acid substitution occurs m die P-loop (residue G249 through residue V256 of the Bcr-Abl polypeptide sequence shown m SEQ ID NO 1), hehx C (residue E279 through residue 1293 of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1), the catalytic domam (residue H361 through residue R367 of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1) or the activation loop (residue A380 through residue P402 of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1).
4. The method of claim 2, wherem the ammo acid substitution occurs at residue D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259,
K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291, E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351, E352, E355, K357, N358, F359, 1360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396, A399, P402, or T406.
5. The method of claim 4, wherem the ammo acid substitution is D233H, T243S, M244V, G249D, G250E, G251S, Q252H, Y253F, Y253H, E255K, V256L, Y257F,
Y257R, F259S, K262E, D263G, K264R, S265R, V268A, V270A, T272A, Y274C, Y274R, D276N, T277P, M278K, E282G, F283S, A288T, A288V, M290T, K291R, E292G, I293T, P296S, L298M, L298P, V299L, Q300R, G303E, V304A, V304D, C305S, C305Y, T306A, F311L, I314V, T315A, T315I, E316G, F317L, M318T, Y320C, Y320H, G321E, D325H, Y326C, L327P, R328K, E329V, Q333L, A337V, V339G, L342E, M343V, M343T, A344T, A344V, I347V, A350T, M351T, E352A, E352K, E355G, K357E, N358D, N358S, F359V, I360K, I360T, L364H, E373K, N374D, K378R, V379I, A380T, A380V, D381G, F382L, T389S, T392A, T394A, A395G, H396K, A399G, P402T or T406A.
6. The method of claim 4, wherem the ammo acid substitution occurs at residue G250, Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35, K357, 1360, V379 or H396.
7. The mediod of claim 4, wherem the ammo acid substitution occurs at residue
G250, Q252, Y253, E255, T315, F317, M351 or E355.
8. The method of claim 4, wherem the ammo acid substitution does not occur at residue E255.
9. The mediod of claim 1, wherem the kmase mhibitor is a 2- phenylaminopynmidme.
10. The method of claim 9, wherem the ammo acid substitution confers resistance to mhibition of tyrosme kinase activity by STI-571.
11. The method of claim 1, wherem the polypeptide sequence of at least one Bcr-Abl polypeptide expressed by the human cancer ceU is determined by determining the nucleotide sequence of a polynucleotide expressed by the human cancer ceU that encodes the Bcr-Abl polypeptide.
12. The method of claim 11, wherem the Bcr-Abl polynucleotide expressed by the human cancer ceU is isolated by the polymerase cham reaction.
13. A method of identifymg a mutation m a Bcr-Abl polynucleotide m a mammahan ceU, wherem the mutation m a Bcr-Abl polynucleotide is associated with resistance to mhibition of Bcr-Abl tyrosme kmase activity by a 2-phenylarmnopynmιchne, the method compnsmg determining the sequence of at least one Bcr-Abl polynucleotide expressed by d e mammahan ceU and comparmg the sequence of the Bcr-Abl polynucleotide to the Bcr-Abl polynucleotide sequence encodmg the polypeptide sequence shown m SEQ ID NO: 1 , wherem the mutation m the Bcr-Abl polynucleotide compnses an alteration at ammo acid residue position: D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291 , E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351, E352, E355, K357, N358, F359, 1360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396, A399, P402, or T406 of the polypeptide sequence shown m SEQ ID NO: 1.
14. The mediod of claim 13, wherem the mammahan ceU is a human cancer ceU.
15. The method of claim 14, wherem the human cancer ceU is a chrome myeloid leukemia ceU.
16. The method of claim 14, wherem the human cancer ceU is obtamed from an mdividual treated with STI-571.
17. The method of claim 13, wherem the substitution mutation occurs at residue G250, Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343,
M343, A344, M351T, E35, K357, 1360, V379 or H396.
18. The method of claim 17, wherem the substitution mutation does not occur at residue E255.
19. The method of claim 13, wherem the 2-phenylammopynmidme is STI-571.
20. The method of claim 19, wherem the ammo acid substitution m the Bcr-Abl polypeptide expressed m human cancer ceU confers resistance to mhibition of tyrosme kmase activity by STI-571.
21. The method of claim 13, wherem the polypeptide sequence of at least one Bcr- Abl polypeptide expressed by die human cancer ceU is determined by sequencmg a polynucleotide expressed by the human cancer ceU that encodes the Bcr-Abl polypeptide, and wherem d e Bcr-Abl polynucleotide expressed by the human cancer ceU is isolated by the polymerase cham reaction.
22. A method of identifymg a mutant Abelson protem tyrosme kinase expressed by a mammahan cancer ceU, the method compnsmg: (a) determining a nucleotide sequence of a portion of a polynucleotide encodmg the kmase domam of the Abelson protem tyrosme kinase expressed by die ceU; and
(b) comparmg the nucleotide sequence so determined to that of the wild type sequence of the Abelson protem tyrosme kmase to identify the presence of a ammo acid substitution m the mutant Abelson protem tyrosme kinase, wherem any amino acid substitution so identified has the characteristics of occurrmg at a ammo acid residue located within d e polypeptide sequence of the Abelson protem tyrosme kmase at the same relative position as an ammo acid substitution m die C-Abl protem k ase shown m SEQ ID NO: 1 that has been identified as bemg associated with a resistance to an inhibition of tyrosme kmase activity by a 2-phenylammopyrιmιchne, as can be determined usmg the homology parameters of a WU-BLAST-2 analysis.
23. The method of claim 22, wherem die ceU expressmg the mutant Abelson protem tyrosme kmase is found m a population of mammahan cancer ceUs that are observed to exhibit a resistance to an mhibition of tyrosme kmase activity after exposure to a 2- phenylammopynmichne.
24. The method of claim 22, wherem die mammahan cancer ceU is a human cancer ceU obtamed from an mdividual selected for treatment with a tyrosme kinase mhibitor comprising a 2-phenylamιnopynmιchne.
25. The method of claim 22, wherem the mutation m die C-Abl protem kmase shown m SEQ ID NO: 1 that has been identified as bemg associated with a resistance to an mhibition of tyrosme kmase activity by a 2-phenylaminopyrmndme occurs at the same relative position as ammo acid residue D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291, E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, 1314, T315, E316, F317, M318, Y320, G321 , D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351, E352, E355, K357, N358, F359, 1360, L364, E373, N374, K378, V379, A380, D381 , F382, T389, T392, T394, A395, H396, A399, P402, or T406.
26. The method of claim 25 wherem the ammo acid substitution occurs at die same relative position as ammo acid residue G250, Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35, K357, 1360, V379 or H396.
27. The method of claim 22 wherem the ammo acid substitution confers resistance to mhibition of tyrosme kmase activity by STI-571.
28. The method of cla i 22, wherem the mutant Abelson tyrosme lαnase expressed by the ceU is a mutant c-Abl, Bcr-Abl, PDGFR, c-kit, TEL-Abl or TEL-PDGFR.
29. The method of claim 22, further compnsmg repeating steps (a)-(b) another mammahan cancer ceU obtamed from a different mdividual; and (c) catalogmg the mutations found m the mutant Abelson protem tyrosme lαnases present m the mammahan cancer ceUs.
30. A mediod of identifymg a compound which specificaUy bmds a mutant Bcr-Abl polypeptide; wherem the Bcr-Abl polypeptide compnses an ammo acid substitution diat occurs m a region of the Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 compnsmg residue D233 through residue T406, the mediod compnsmg the steps of: contacting the mutant Bcr-Abl polypeptide with a test compound under conditions favorable to bmdmg; and determmmg whether die test compound specificaUy bmds to the mutant Bcr-Abl polypeptide such that a compound which bmds to the mutant Bcr- Abl polypeptide can be identified.
31. The method of chum 30, wherem the ammo acid substitution occurs at residue D233, T243, M244, K245, G249, G250, G251, Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291 , E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351, E352, E355, K357, N358, F359, 1360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396, A399, P402, or T406.
32. The method of claim 31, wherem the ammo acid substitution is D233H, T243S, M244V, G249D, G250E, G251S, Q252H, Y253F, Y253H, E255K, V256L, Y257F, Y257R, F259S, K262E, D263G, K264R, S265R, V268A, V270A, T272A, Y274C, Y274R, D276N, T277P, M278K, E282G, F283S, A288T, A288V, M290T, K291R, E292G, I293T, P296S, L298M, L298P, V299L, Q300R, G303E, V304A, V304D, C305S, C305Y, T306A, F311L, I314V, T315A, T315I, E316G, F317L, M318T, Y320C, Y320H, G321E, D325H, Y326C, L327P, R328K, E329V, Q333L, A337V, V339G, L342E, M343V, M343T, A344T, A344V, I347V, A350T, M351T, E352A, E352K, E355G, K357E, N358D, N358S, F359V, I360K, I360T, L364H, E373K, N374D, K378R, V379I, A380T, A380V, D381G, F382L, T389S, T392A, T394A, A395G, H396K, A399G, P402T or T406A.
33. The method of claim 30, wherem the ammo acid substitution occurs at residue G250, Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35, K357, 1360, V379 or H396.
34. The method of claim 30, wherem the amino acid substitution occurs at residue G250, Q252, Y253, E255, T315, F317, M351 or E355.
35. The method of claim 30, wherem the amino acid substitution does not occur at residue E255.
36. The method of claim 30, wherem the compound is a 2-phenylannnopyrιmιchne.
37. The method of Claim 30, further compnsmg deterrriining whether the test compound inhibits the tyrosme kmase activity of the mutant Bcr-Abl polypeptide compnsmg d e steps of: transfecting mammahan ceUs with a construct encodmg the mutant Bcr- Abl polypeptide; contactmg the mammalian ceUs widi die test compound, and momtormg die mammahan ceUs for the tyrosme kinase activity of the mutant Bcr-Abl polypeptide, wherem an mhibition m tyrosme kinase activity in the presence of the test compound as compared to the absence of the test compound identifies the test compound as an mhibitor of die mutant Bcr-Abl polypeptide.
38. The method of claim 37, wherem the ammo acid substitution occurs at residue T315.
39. The method of claim 37, wherem the tyrosme kinase activity of the mutant Bcr- Abl polypeptide is measured by examinmg the phosphotyrosme content of Crkl.
40. A mediod of determining whether a test compound inhibits the tyrosme kmase activity of a mutant Bcr-Abl polypeptide, wherem the Bcr-Abl polypeptide compnses an amino acid substitution that occurs m a region of die Bcr-Abl polypeptide sequence shown m SEQ ID NO: 1 compnsmg residue D233 through residue T406, the mediod compnsmg the steps of: transfecting mammahan ceUs with a construct encodmg the mutant Bcr- Abl polypeptide so that the mutant Bcr-Abl polypeptide is expressed by the mammahan ceUs, contacting the mammahan ceUs with die test compound; and momtormg the mammahan ceUs for the tyrosme kmase activity of the mutant Bcr-Abl polypeptide, wherem an inhibition m tyrosme kmase activity m d e presence of the test compound as compared to the absence of the test compound identifies the test compound as an mhibitor of the mutant Bcr-Abl polypeptide.
41. The method of claim 40, wherem the tyrosme kmase activity of the mutant Bcr- Abl polypeptide is measured by examining the phosphotyrosme content of Crkl.
42. The method of claim 40, wherem the tyrosme kinase activity of the mutant Bcr- Abl polypeptide is measured via Western blot analysis usmg an anti-phosphotyrosme antibody to examine the phosphotyrosme content of lysates of the mammahan ceUs.
43. The method of claim 40, wherem the mammahan ceUs are 293-T ceUs
44. The mediod of claim 40, wherem the amino acid substitution occurs at residue D233, T243, M244, K245, G249, G250, G251 , Q252, Y253, E255, V256L Y257, F259, K262, D263, K264, S265, V268, V270, T272, Y274, D276, T277, M278, E282, F283, A288, M290, K291, E292, 1293, P296, L298, V299, Q300, G303, V304, C305, T306, F311, 1314, T315, E316, F317, M318, Y320, G321, D325, Y326, L327, R328, E329, Q333, E334, A337, V339, L342, M343, A344, 1347, A350, M351, E352, E355, K357, N358, F359, 1360, L364, E373, N374, K378, V379, A380, D381, F382, T389, T392, T394, A395, H396, A399, P402, or T406.
45. The method of claim 44, wherem the amino acid substitution is D233H, T243S, M244V, G249D, G250E, G251S, Q252H, Y253F, Y253H, E255K, V256L, Y257F, Y257R, F259S, K262E, D263G, K264R, S265R, V268A, V270A, T272A, Y274C, Y274R, D276N, T277P, M278K, E282G, F283S, A288T, A288V, M290T, K291R, E292G, I293T, P296S, L298M, L298P, V299L, Q300R, G303E, V304A, V304D, C305S, C305Y, T306A, F311L, I314V, T315A, T315I, E316G, F317L, M318T, Y320C, Y320H, G321E, D325H, Y326C, L327P, R328K, E329V, Q333L, A337V, V339G, L342E, M343V, M343T, A344T, A344V, I347V, A350T, M351T, E352A, E352K, E355G, K357E, N358D, N358S, F359V, I360K, I360T, L364H, E373K, N374D, K378R, V379I, A380T, A380V, D381G, F382L, T389S, T392A, T394A, A395G, H396K, A399G, P402T or T406A.
46. The method of claim 40, wherem the amino acid substitution occurs at residue G250, Q252, E255, K264, V270, F283, M290, P296, V304, T315, F317, R328, M343, M343, A344, M351T, E35, K357, 1360, V379 or H396.
47. The mediod of claim 40, wherem the amino acid substitution occurs at residue G250, Q252, Y253, E255, T315, F317, M351 or E355.
48. The method of claim 40, wherem the ammo acid substitution does not occur at residue E255.
49. The method of claim 40, wherem the compound is a 2-phenylammopyrimichne.
50. The method of claim 40, wherem the compound is a pyndo [2,3-^pynmιdιne.
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