WO2009005688A2 - Activation de mutations dans notch-1 - Google Patents

Activation de mutations dans notch-1 Download PDF

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WO2009005688A2
WO2009005688A2 PCT/US2008/007958 US2008007958W WO2009005688A2 WO 2009005688 A2 WO2009005688 A2 WO 2009005688A2 US 2008007958 W US2008007958 W US 2008007958W WO 2009005688 A2 WO2009005688 A2 WO 2009005688A2
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notch
exon
mutation
gene
patient
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WO2009005688A3 (fr
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Aldolfo A. Ferrando
Maria Luisa Sulis
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Columbia University in the City of New York
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Columbia University in the City of New York
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the field of the present invention relates to methods and compositions for diagnosing, preventing, treating, and/or ameliorating the effects of various conditions in a mammal, such as cancer, for example, T-cell lymphoblastic leukemia and lymphoma.
  • the present invention further relates to screening assays as well as patient stratification methods.
  • NOTCH signaling plays a critical role in lineage specification decisions that enable multipotential precursor cells to become committed to specific cell lineages during development, and therefore has important roles in cell differentiation, proliferation and apoptosis.
  • the fundamental components of the NOTCH pathway include the DSL ligands (Delta-like 1 , 3 and 4; and Jagged 1 and 2), the NOTCH receptors (NOTCH1-4), and the CSL DNA binding protein, a transcription factor that mediates the conversion of NOTCH-activating signals at the cell surface to changes in gene expression in the nucleus.
  • the mature NOTCH 1 receptor is a heterodimeric class I transmembrane glycoprotein generated by proteolytic processing of a precursor polypeptide in the trans- Golgi network.
  • This first protease cleavage (S1) is catalyzed by a furin protease, which cuts the NOTCH 1 precursor protein approximately 70 amino acids external to the transmembrane domain to generate extracellular (N EC ) and transmembrane and intracellular (NTM) NOTCH 1 subunits.
  • N EC extracellular
  • NTM transmembrane and intracellular
  • the extracellular subunit of NOTCH1 contains 36 epidermal growth factor (EGF)-like repeats involved in ligand-receptor interaction, followed by three LIN-12/NOTCH repeats (LNR), which stabilize the interaction between the extracellular and transmembrane subunits and help keep the receptor in the resting state in the absence of ligand.
  • EGF epidermal growth factor
  • LNR LIN-12/NOTCH repeats
  • the NTM NOTCH 1 subunit consists of a short extracellular juxtamembrane peptide followed by a transmembrane sequence and a series of cytoplasmic domains, including a RAM domain, a membrane proximal to a nuclear localization signal, a series of ankyrin repeats, a distal nuclear localization signal, a transactivation domain and a carboxy-terminal PEST sequence, which together function as a ligand-activated transcription factor.
  • the final cleavage is catalyzed by the ⁇ -secretase complex at position VaI 1744 (site S3), which releases the intracellular fraction of NOTCH 1 (ICN 1) from the membrane, allowing it to translocate to the nucleus where it activates the transcription of target genes in a complex with the DNA-binding factor CSL and members of the mastermind family of coactivators.
  • site S3 the ⁇ -secretase complex at position VaI 1744
  • ICN 1 intracellular fraction of NOTCH 1
  • NOTCH signaling pathway plays a critical role in the hematopoietic system by maintaining stem cell homeostasis and participating at multiple stages of T- cell development.
  • NOTCH signaling is required for the commitment of multipotent hematopoietic progenitors to the T-cell lineage.
  • NOTCH1 is required later in T-cell development for progression through the early DN1 , DN2 and DN3 stages of thymocyte maturation; participates in the regulation of TCRB gene rearrangement; and regulates lineage decisions between ⁇ vs. ⁇ 5 lineages and, at least in some systems, between CD4 vs. CD8 lineages.
  • T-ALL T-cell lymphoblastic leukemia
  • NOTCH 1 activation was first demonstrated in T-ALL cases harboring the t(7;9)(q34;q34.3), a rare chromosomal translocation, which juxtaposes a truncated NOTCH 1 gene next to the TCRB locus, leading to the aberrant expression of an intracellular constitutively active form of NOTCH 1.
  • More recent studies have demonstrated the major role of NOTCH 1 in the pathogenesis of human leukemias by showing the presence of activating mutations in NOTCH 1 in over 50% of human T-ALLs.
  • Activating mutations in NOTCH1 in human T- ALL are concentrated in exons 26 and 27, which encode the homodimerization domain; and in the 3' end of exon 34, which encodes the PEST domain in the C-terminal region of the protein.
  • HD mutations are typically single-amino-acid substitutions and small in- frame deletions and insertions, which probably operate by inducing ligand-independent activation of NOTCH!
  • PEST mutations result in premature stop codons, the loss of the PEST domain, and increased levels of intranuclear active NOTCH1 due to impaired degradation of the activated receptor by the proteasome.
  • HD and PEST mutations are often found in cis in the same NOTCH 1 transcript. These HD- PEST-double mutant alleles result in synergistic activation of NOTCH 1 signaling and are 10-times more active than NOTCH1 alleles containing an HD or a PEST mutation alone.
  • NOTCH1 that contribute to ligand-independent activation of NOTCH1.
  • assays for identifying candidate compounds that may be used to treat or ameliorate the effects of cancer generally comprise the steps of (a) contacting cells from a cell line comprising a juxtamembrane expansion (JME) mutation with a candidate compound and (b) determining whether NOTCH 1 expression is modulated in the cell line contacted with the candidate compound relative to control cells from the same cell line that have not been contacted with the candidate compound.
  • JME juxtamembrane expansion
  • methods of making a cell line that over-expresses NOTCH1 comprise introducing a mutation into a region of DNA that encodes the extracellular juxtamembrane region of a NOTCH1 receptor.
  • assays for identifying candidate compounds that may be used to treat or ameliorate the effects of cancer are provided. These assays generally employ the steps of contacting cells from a cell line that over-expresses a NOTCH 1 receptor, which comprises a mutation in the extracellular juxtamembrane region thereof, with a candidate compound. The assays further comprise the step of determining whether NOTCH 1 expression is modulated in the cell line contacted with the candidate compound, compared to control cells from the same cell line that have not been contacted with the candidate compound.
  • isolated polypeptides are provided.
  • polypeptides comprise a mutation in the extracellular juxtamembrane region of a
  • methods of determining whether reducing or blocking NOTCH 1 activation will be effective to treat, prevent, or ameliorate the effects of a cancer in a patient generally comprise (a) determining if the patient harbors one or more mutations in exon 28 or intron 27 of a NOTCH1 gene or (b) determining if NOTCH1 comprises the amino acid sequence of SEQ ID NO:2 proximal to the transmembrane domain of NOTCH1.
  • methods of (a) determining whether reducing or blocking NOTCH1 activation will be effective to treat, prevent, or ameliorate the effects of a cancer, (b) identifying whether a patient is sensitive to one or more ⁇ -secretase inhibitors, and (c) identifying a patient population for inclusion in a clinical trial of a drug candidate for treating cancer are provided.
  • Such methods comprise carrying out a screen for mutations in exon 26, exon 27, intron 27, exon 28, and exon 34 of a NOTCH 1 gene in a sample of DNA from a patient.
  • kits for determining whether reducing or blocking NOTCH 1 activation will be effective to treat, prevent, or ameliorate the effects of a cancer comprise a set of PCR primers that may be used to generate an amplicon from patient DNA which comprises intron 27 and/or exon 28 of the NOTCH1 gene.
  • FIG. 1 Identification of internal tandem duplications in the extracellular juxtamembrane domain of NOTCH 1 in T-ALL.
  • A Western blot analysis of T-ALL cell lines lacking HD and PEST domain mutations in NOTCH1 demonstrating high levels of activated NOTCH1 in the Jurkat cell line. ICN1 levels were detected with the NOTCH1 VaM 744 antibody. Tubulin is shown as loading control.
  • B Sequence analysis of the NOTCH 1 transcripts showing the mutation identified in the Jurkat cell line. The wild type NOTCH 1 transcript sequence is shown at the bottom and inserted sequences on top. S2 and S3 indicate the sites for ADAM metalloprotease and ⁇ -secretase cleavage, respectively.
  • Duplicated nucleotides are shown in italics and underlined in the wild type sequence.
  • the common 4 amino acids (QLHF) present in all three JME mutants in this series are highlighted in yellow.
  • C PCR amplification of exon 28 of NOTCH 1 and adjacent intronic sequences.
  • Three samples corresponding to Jurkat cells and two independent primary T-ALL cases show bands of increased size compared to the control genomic DNA indicating the presence of insertions in the extracellular juxtamembrane domain of NOTCH-1.
  • NOTCH1 constructs expressing juxtamembrane insertions of 5-14 amino acids corresponding to the VSV-G epitope as well as the Jurkat JME 17 allele were tested in a CSL luciferase reporter assay. Reporter activity is shown as fold change compared with wild type NOTCH 1 (WT).
  • FIG. 1 A. Western blot analysis of activated NOTCH1 (ICN1) levels in
  • Jurkat cells treated with CompE (100 nM) for 24 hours shows inhibition of NOTCH1 processing and clearance of activated intracellular NOTCH1.
  • B Quantitative RT-PCR analysis of HES1 expression in Jurkat cells treated with CompE (100 nM) or vehicle only (DMSO).
  • C-D CSL luciferase reporter assay (C) and Western blot analysis (D) of ICN1 activity and expression in HeLa cells expressing NOTCH1 JME mutants treated with vehicle (DMSO) or a ⁇ -secretase inhibitor (CompE 100 nM).
  • E-F CSL luciferase reporter assay
  • D Western blot analysis of ICN1 activity and expression in HeLa cells expressing NOTCH1 JME mutants treated with vehicle (DMSO) or a ⁇ -secretase inhibitor
  • Luciferase reporter assay with a CSL reporter construct shows effective inhibition of NOTCH 1 signaling from the NOTCH1 Jurkat JME 17 allele (E) and the NOTCH1 VSVG JME 14 allele (F) upon mutation of the S2 cleavage site.
  • A Schematic representation of the soluble LNR-HD complex encoded by the NOTCH1 minigene. In addition to the LNR repeats (green), the N-terminal (HD-N) and C-terminal (HD-C) subunits of the HD domain (blue) and the extracellular juxtamembrane region (JM), this construct contains an N-terminal FLAG tag and a C-terminal HA tag.
  • B Schematic representation of the soluble LNR-HD complex encoded by the NOTCH1 minigene. In addition to the LNR repeats (green), the N-terminal (HD-N) and C-terminal (HD-C) subunits of the HD domain (blue) and the extracellular juxtamembrane region (JM), this construct contains an N-terminal FLAG tag and a C-terminal HA tag.
  • NOTCH 1 minigene protein products recovered from conditioned media of 293T cells transfected with empty plasmid (Control), a NOTCH 1 wild type minigene (WT), a NOTCH 1 minigene containing the Jurkat JME 17 amino acid insertion (Jurkat JME 17) and a NOTCH1 minigene containing the artificial VSVG-P JME 14 amino acid insertion.
  • NOTCH 1 minigene protein products from a NOTCH 1 wild type minigene (WT) and NOTCH1 minigene containing the Jurkat JME 17 amino acid insertion (Jurkat JME 17) incubated with increased concentrations of urea to test the stability of the LNR-HD domain complex.
  • WT NOTCH 1 wild type minigene
  • Jurkat JME 17 the Jurkat JME 17 amino acid insertion
  • FIG. 5 Schematic representation of NOTCH 1 mutations leading to increased ⁇ -secretase processing and activation in T-ALL.
  • Functional domains of NOTCH1 are annotated in the structure of wild type NOTCH-1.
  • EGF-like EGF-like repeats.
  • HD heterodimerization domain.
  • LNR LNR repeats.
  • RAM RAM domain.
  • Ankyrin ankyrin repeats.
  • TAD transactivation domain.
  • PEST PEST domain.
  • S2 metalloprotease cleavage site.
  • S3 ⁇ -secretase cleavage site. Sequences altered by the different NOTCH1 mutations are highlighted in red.
  • HD class 1 mutants disrupt the HD domain structure.
  • HD class 2 mutants displace the S2 cleavage site away from the HD-LNR complex.
  • NOTCH1 JME mutants displace the HD-LNR repeat complex away from the cell surface.
  • Figure 6 A table summarizing certain NOTCH 1 mutations and the effects thereof on the predicted amino acid sequences of the resulting polypeptides.
  • assays for identifying candidate compounds that may be used to treat or ameliorate the effects of cancer generally comprise the steps of (a) contacting cells from a cell line comprising a juxtamembrane expansion (JME) mutation with a candidate compound and (b) determining whether NOTCH 1 expression is modulated in the cell line contacted with the candidate compound relative to control cells from the same cell line that have not been contacted with the candidate compound.
  • the modulation of NOTCH1 expression may be represented by a decrease in NOTCH 1 expression in the cells of the cell line containing the JME mutation. The invention provides that such a decrease in NOTCH 1 expression is indicative that the candidate compound may be effective to treat cancer.
  • Candidate compounds identified by such assays may be used to treat or ameliorate the effects of various types of cancer, including without limitation T-cell leukemia, myeloleukemia, and solid tumors.
  • solid tumors include those implicated in neuroblastoma, breast cancer, and ovarian cancer.
  • the cancer is T-ALL leukemia.
  • the JME mutation may be, for example, an internal tandem duplication that comprises a polynucleotide that encodes at least 11 amino acids, such at least 12, and preferably at least 14 or more, and is located in a region that encodes the extracellular juxtamembrane region of a NOTCH1 receptor.
  • the mutation e.g., an internal tandem duplication, may be inserted in a region extending from about the distal part of intron 27 to about the proximal part of exon 28 of a NOTCH 1 gene and, more specifically, within about 50 nucleotides (upstream or downstream) of nucleotide 1740 of a NOTCH1 gene (SEQ ID NO:1).
  • the mutation e.g., internal tandem duplication
  • the mutation will be inserted between about nucleotide 1738 to about 1741 of SEQ ID NO:1 , and encodes from about 11 to about 36 amino acids.
  • the mutation e.g., internal tandem duplication, comprises a polynucleotide that encodes a peptide sequence comprising the amino acids: QLHF (SEQ ID NO:2).
  • QLHF SEQ ID NO:2
  • both endpoints recited in the range are included within the range.
  • the assays of the present invention may employ the use of a mammalian cell line, such as for example, a human cell line.
  • the cell line is a human T-cell line, such as for example, a Jurkat, IARC 301, PER-117, AG-F, or MT-2 cell line.
  • methods of making cell lines that over-express NOTCH1 comprise introducing a mutation into a region of DNA that encodes the extracellular juxtamembrane region of a NOTCH 1 receptor.
  • the mutation comprises an insertion that encodes at least 11 amino acids.
  • the mutation may comprise an internal tandem duplication.
  • the mutation, e.g., internal tandem duplication may be inserted from about the distal part of intron 27 to about the proximal part of exon 28 of a NOTCH1 gene.
  • the mutation e.g., internal tandem duplication
  • the mutation will be inserted within about 50 nucleotides (upstream or downstream), such as within about 25 nucleotides, such as 15 or 10 nucleotides, of nucleotide 1740 of a NOTCH 1 gene or, more specifically, between about nucleotide 1738 to about nucleotide 1741 of the NOTCH 1 gene.
  • the NOTCH 1 gene will preferably comprise the polynucleotide sequence of SEQ ID NO:1.
  • the invention provides that an internal tandem duplication encoding from about 11 to about 36 amino acids is preferred. More particularly, in certain preferred embodiments, the mutation, e.g., internal tandem duplication, comprises a polynucleotide that encodes a tetrapeptide sequence comprising the amino acids: QLHF (SEQ ID NO:2).
  • the cell lines made by the processes of the present invention may be obtained from any appropriate source.
  • the source is a mammalian cell line, such as for example, a human cell line. More preferably, the source is a human T-cell line, such as for example, a Jurkat cell line.
  • the invention further encompasses pure or substantially pure cell lines that are produced by the foregoing methods.
  • the invention is an assay for identifying candidate compounds that may be used to treat or ameliorate the effects of cancer.
  • This assay comprises contacting cells from a cell line made by any of the processes disclosed herein with a candidate compound and determining whether NOTCH 1 expression is modulated in the cell line contacted with the candidate compound compared to control cells from the same cell line that have not been contacted with the candidate compound.
  • isolated polynucleotide sequences are provided that comprise a mutation in the extracellular juxtamembrane region of a NOTCH 1 receptor.
  • the NOTCH 1 receptor is obtained from a mammal. More preferably, the NOTCH1 receptor is obtained from a human.
  • isolated polynucleotides means nucleotide sequences that are free of the genes and other sequences that flank the polynucleotide sequences in the naturally-occurring genome of the organism from which the isolated polynucleotide sequences of the invention are derived.
  • the term therefore includes, for example, a recombinant nucleotide sequence which is incorporated into a vector; into an autonomously replicating plasmid or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant nucleotide sequence which is part of a hybrid gene encoding additional polypeptide sequences.
  • the isolated polynucleotide sequences of the invention comprise an insertion of nucleotides that encode at least 11 amino acids.
  • the insertion is an internal tandem duplication.
  • the mutation e.g., internal tandem duplication, is inserted in a region extending from about the distal part of intron 27 to about the proximal part of exon 28 of a NOTCH1 gene, such as within about 50 nucleotides (upstream or downstream), as described in more detail above, of nucleotide 1740 of a NOTCH 1 gene.
  • the isolated polynucleotide sequences of the invention comprise a mutation, e.g., an internal tandem duplication, between about nucleotide 1738 to about 1741 of a NOTCH1 gene (SEQ ID NO:1).
  • the mutation, e.g., internal tandem duplication, of such isolated polynucleotide sequences will preferably encode from about 11 to about 36 amino acids, which may comprise a tetrapeptide sequence comprising the amino acids: QLHF (SEQ ID NO:2).
  • the isolated polynucleotide sequences of such embodiments will preferably include a mutation that promotes ligand-independent activation of NOTCH 1.
  • methods for determining whether reducing or blocking NOTCH 1 activation will be effective to treat, prevent, or ameliorate the effects of a cancer in a patient comprise determining if the patient harbors one or more mutations in exon 28 or intron 27 of the NOTCH1 gene.
  • NOTCH1 activation is reduced or blocked by providing the patient with one or more ⁇ -secretase inhibitors, such as [(2S)-2- ⁇ [(3,5-Difluorophenyl)acetyl]amino ⁇ -N-[(3S)1-methyl-2-oxo-5-phenyl-2,3- dihydro-1 H-1 ,4-benzodiazepin-3-yl] propanamide], N-[N-(3,5-difluorophenacetyl)-L- alanyl]-Sphenylglycine-t-butylester, [1 ,1'-Biphenyl]-4-acetic acid, 2-fluoro-alpha-methyl, NGX-555, LY-411575, Cellzome, LY-450139, E-2012, 2-Thiophenesulfonamide, 5- chloro-N-[(1S)-3,3,3-trifluoro-1-(hydroxymethyl)-2-(trifluor
  • the cancer may be, for example, T-cell leukemia, myeloleukemia, or solid tumors.
  • the solid tumors may include, e.g., neuroblastoma, breast cancer, and ovarian cancer.
  • the cancer is T-cell leukemia.
  • the one or more mutations in exon 28 or intron 27 of the NOTCH 1 gene may be detected using PCR and DNA sequencing techniques, as well as other well-known detection techniques, e.g. hybridization reactions.
  • the one or more mutations in exon 28 or intron 27 of the NOTCH 1 gene may be detected by (a) extracting DNA from a patient, (b) amplifying a portion of the DNA that comprises the NOTCH1 gene to produce an amplicon, and (c) sequencing the amplicon and determining whether the amplicon comprises one or more mutations in exon 28 or intron 27 of the NOTCH 1 gene.
  • pre-mRNA may be extracted from the patient, which may be used to generate and amplify cDNA therefrom by performing an RT-PCR reaction.
  • the cDNA may then be sequenced in order to determine whether the cDNA comprises one or more mutations in exon 28 or intron 27 of the NOTCH1 gene.
  • NOTCH 1 gene may be detected by (a) extracting DNA from the patient and (b) determining whether portions of the DNA in which exon 28 or intron 27 of the NOTCH 1 gene resides hybridizes to one or more polynucleotides that are complementary to mutated forms of exon 28 or intron 27 under standard conditions, preferably high stringency conditions.
  • standard conditions refers to the conditions that are generally used by a person skilled in the art to detect specific hybridization signals, or preferably so-called stringent hybridization conditions used by a person skilled in the art.
  • stringent hybridization conditions means that hybridization will occur if there is 85% and preferably at least 90% or 95%, such as for example, 96%, 97%, 98% or 99% identity between the sequences.
  • Stringent hybridization conditions may include, e.g., overnight incubation at 42°C using, e.g., a digoxygenin (DIG)-labeled DNA probe (constructed by using a DIG labeling system; Roche Diagnostics GmbH, 68298 Mannheim, Germany) in a solution comprising 50% formamide, 5xSSC (150 mM NaCI, 15 mM trisodium citrate), " 0.2% sodium dodecyl sulfate, 0.1% N-lauroylsarcosine, and 2% blocking reagent (Roche Diagnostics GmbH), followed by washing the filters in 0.
  • DIG digoxygenin
  • methods of determining whether reducing or blocking NOTCH 1 activation will be effective to treat, prevent, or ameliorate the effects of a cancer in a patient comprise determining if NOTCH 1 comprises a tetrapeptide sequence of SEQ ID NO:2 proximal to the transmembrane domain of NOTCH-1.
  • determining whether NOTCH 1 comprises the tetrapeptide sequence may comprise the steps of (a) extracting a protein sample from the patient, (b) contacting the protein sample with an antibody (or FAB fragment thereof) that selectively recognizes and binds to the tetrapeptide sequence or a portion thereof, and (c) determining whether the antibody (or FAB fragment thereof) recognized and bound to the tetrapeptide sequence within the protein sample.
  • an antibody or FAB fragment thereof
  • Such immunochemical methods of detecting the presence of the tetrapeptide sequence may be carried out using, e.g., an immunoblot, ELISA, RIA, flow cytometry, or a combination thereof.
  • Antibody includes an antibody of classes IgG, IgM, IgA,
  • the antibody may be a monoclonal antibody, polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.
  • the antibody may also be a chimeric antibody.
  • the antibody may be derivatized by the attachment of one or more chemical, peptide, or polypeptide moieties known in the art.
  • the antibody may be conjugated with a chemical moiety.
  • the antibody may be a human or humanized antibody.
  • Suitable antibody mimetics generally can be used as surrogates for the antibodies and antibody fragments described herein. Such antibody mimetics may be associated with advantageous properties (e.g., they may be water soluble, resistant to proteolysis, and/or be nonimmunogenic). For example, peptides comprising a synthetic beta-loop structure that mimics the second complementarity-determining region (CDR) of monoclonal antibodies have been proposed and generated. See, e.g., Saragovi et a/., Science. Aug. 16, 1991 ;253(5021):792-5.
  • CDR complementarity-determining region
  • Peptide antibody mimetics also have been generated by use of peptide mapping to determine "active" antigen recognition residues, molecular modeling, and a molecular dynamics trajectory analysis, so as to design a peptide mimic containing antigen contact residues from multiple CDRs. See, e.g., Cassett et a/., Biochem Biophys Res Commun. JuI. 18, 2003;307(1): 198-205. Additional discussion of related principles, methods, etc., which may be applicable in the context of this invention are provided in, e.g., Fassina, Immunomethods. October 1994;5(2):121-9.
  • methods of (a) determining whether reducing or blocking NOTCH 1 activation will be effective to treat, prevent, or ameliorate the effects of a cancer, (b) identifying whether a patient is sensitive to one or more ⁇ -secretase inhibitors, and (c) identifying a patient population for inclusion in a clinical trial of a drug candidate for treating cancer are provided. These methods comprise carrying out a screen for mutations in exon 26, exon 27, intron 27, exon 28, and exon 34 of a NOTCH 1 gene in a sample of DNA from a patient.
  • the invention provides that screens for such mutations may be performed using the PCR / sequencing methods described above or, e.g., hybridization-based assays (such as southern blots and/or microarray analyses).
  • the presence of a mutation in the patient's DNA sample is indicative that reducing or blocking NOTCH 1 activation will be effective to treat, prevent, or ameliorate the effects of the cancer,
  • (ii) is indicative that a patient will be sensitive to ⁇ -secretase inhibitors, and
  • (iii) should be considered in determining whether to include each patient in the clinical trial (in view of the mode of action of the drug candidate to be tested).
  • kits for determining whether reducing or blocking NOTCH 1 activation will be effective to treat, prevent, or ameliorate the effects of a cancer comprise a set of PCR primers, such as the primers used in the examples below, which may be used to generate an amplicon from patient DNA which comprises intron 27 and/or exon 28 of the NOTCH 1 gene.
  • the amplicon that is generated using such PCR primers may be subsequently sequenced in order to determine whether the amplicon contains a mutation in intron 27 and/or exon 28 of the NOTCH1 gene.
  • NOTCH 1 expression pi asm ids.
  • the pcDNA3 NOTCH1 expression plasmid is a full-length NOTCH1 expression construct containing codons 1-2555 of NOTCH 1 followed by a Flag tag sequence.
  • pcDNA3 NOTCH1 L1601 P encodes a HD (substitution of L to P at position 1601) mutant allele of NOTCH 1 tagged with a Flag tag epitope in the C-terminus.
  • pcDNA3 NOTCH1 L1601 P- ⁇ PEST encodes a double HD (substitution of L to P at position 1600) plus ⁇ PEST (truncation at position 2473) mutant allele of NOTCH 1 tagged with a Flag tag epitope in the C-terminus.
  • pcDNA3 NOTCH-1 pcDNA3 NOTCH1 L1601 P and pcDNA3 NOTCH1 L1601 P- ⁇ PEST constructs were provided by New York University.
  • the pcDNA3 NOTCH1 Jurkat JME17 mutant was generated by cloning a partial NOTCH 1 transcript (exons 19 to 29) amplified by PCR from Jurkat cells, which contain an internal tandem duplication of 51 bases within exon 28 of the NOTCH1 gene, in the unique BamH1 and Notl restriction sites of pcDNA3 NOTCH-1.
  • the pcDNA3 NOTCH1 P12 mutant was generated by cloning a partial NOTCH1 transcript (exons 19 to 29) amplified by PCR from P12-ICHIKAWA cells, which contain an internal tandem duplication of 51 bases within exon 28 of the NOTCH1 gene, in the unique BamH1 and Notl restriction sites of pcDNA3 NOTCH-1.
  • S2 cleavage site (AV 1720-1721 VH and AV 1720-1721 ED) were generated using the
  • Genomic DNA from each sample was extracted using a commercial kit (GENTRA,
  • Renilla luciferase Chemicon
  • tubulin SC-8035
  • NOTCH 1 mutation analysis in the Jurkat cell line was performed by direct sequencing of RT-PCR-amplified NOTCH 1 transcripts.
  • Primary T-ALL samples were analyzed by direct sequencing of PCR products expanding NOTCH 1 exons 26, 27, 28 and the distal part of exon 34 encompassing the sequences encoding the TAD and
  • Exon 27 FW CATGGGCCTCAGTGTCCT (SEQ ID NO: 5); [0059] Exon 27 RVTAGCAACTGGCACAAACAGC (SEQ ID NO: 6);
  • NOTCH1 expression plasmids (pcDNA3) were transiently transfected using FuGEne (Roche, Basel, Switzerland) transfection reagent together with the pGaLUC artificial luciferase reporter construct provided by Kyoto University, which contains six tandem CSL binding sites; and pRL, a plasmid expressing the Renilla luciferase gene under the control of the CMV promoter (which was used as an internal control). Total DNA was kept constant by adding empty vector as needed. All transfections were carried out in triplicate.
  • Jurkat cells were obtained from the ATCC. LOUCY, MOLT16, SUPT11 ,
  • T- ALL cell lines were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum, 100U/ml penicillin G, and 100 ⁇ g/ml streptomycin at 37°C in a humidified atmosphere under 5% CO2.
  • HeIa cells and immortalized murine fibroblast lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100U/ml penicillin G, and 100 ⁇ g/ml streptomycin at 37°C in a humidified atmosphere under 5% CO2.
  • DMEM Dulbecco's modified Eagle's medium
  • NOTCH1 mini-receptors corresponding to wild type NOTCH1 amino acids
  • HA immunoprecipitates were prepared as described above and following three washes with wash buffer, were incubated with 1 ml of wash buffer with 0 to 4M urea for 30 minutes at 24 0 C. Following two additional washes, immunoprecipitated proteins were eluted and analyzed by
  • Activating mutations in the HD and PEST domains of NOTCH 1 have been detected in over 70% of T-ALL cell lines and are associated with the expression of high levels of ICN1.
  • the levels of ICN1 protein were measured by Western blot using the VaI 1744 antibody, which specifically recognizes the ⁇ -secretase cleaved activated form of NOTCH-1 , in a panel of T-ALL lines lacking mutations in exons 26, 27 and 34 of the NOTCH 1 gene.
  • a first patient showed an in-frame insertion of the TGAAGGGG octanucleotide (SEQ ID NO: 14) followed by an internal tandem duplication encompassing the distal 49 bases of intron 27 and the 45 proximal bases of exon 28.
  • a second patient showed an in-frame internal tandem duplication encompassing the distal 54 bases of intron 27 and the proximal 54 bases of exon 28.
  • the first of these mutations is predicted to encode an insertion of 35 amino acids (LKGSRCLMSGHLPLAPVPAGETVEPPPPAQLHFMY) (SEQ ID NO: 15) at position 1738 in the extracellular juxtamembrane segment of NOTCH-1 , while the second one is predicted to encode an insertion of 36 amino acids (VRSRCLMSGHLPLAPVPAGETVEPPPPAQLHFMTWR) (SEQ ID NO: 16) at position 1741 , also extending the extracellular juxtamembrane segment of NOTCH1 ( Figure 6).
  • the other five internal tandem duplications, and the corresponding predicted amino acid insertions, are listed in Figure 6. Direct sequence analysis of exon 28 and flanking intronic sequences of T-ALL samples showing normal size PCR products failed to detect any additional NOTCH1 mutations in this region..
  • luciferase reporter assays were performed to compare the activity of wild type NOTCH 1 , the JME17 mutation isolated from Jurkat cells and a prototypical HD mutant allele of NOTCH 1 (L1600P).
  • Transfection of HeIa cells with the HD (L1600P), ⁇ PEST, and HD (L1600P) ⁇ PEST NOTCH 1 mutant alleles resulted in 12-, 2.5- and 105-fold activation of NOTCH 1 signaling over basal levels, respectively.
  • expression of the NOTCH1 Jurkat JME17 mutant allele induced over 2000-fold activation of the NOTCH1 reporter compared to controls ( Figure 2A).
  • NOTCH 1 JME mutations could be related to two NOTCH1 HD mutant alleles previously identified in the distal region of exon 27 in the P12-ICHIKAWA cell line and in a primary T-ALL patient sample, which consisted of internal tandem duplications resulting in the displacement of the NOTCH1 S2 cleavage site away from the inhibitory LNR-HD complex.
  • NOTCH 1 JME mutations might result in aberrant proteolytic processing of the extracellular juxtamembrane region of NOTCH 1 by creating an alternative S2-like site, possibly located within their common QLHF (SEQ ID NO: 2) sequence.
  • aberrant activation of NOTCH 1 might result from conformational changes in the HD-LNR complex upon its displacement away from the membrane.
  • NOTCH1 JEM mutations in NOTCH1 require y-secretase and S2 processing for activation.
  • Activating mutations in the HD domain of NOTCH 1 typically work by facilitating the S2 processing of the receptor and the subsequent S3 cleavage catalyzed by the ⁇ -secretase complex.
  • inhibition of S3 processing by small molecule inhibitors of the ⁇ -secretase complex effectively abrogate the activity of these mutants.
  • Treatment of Jurkat T-ALL cells with CompE resulted in almost complete clearance of ICN 1 at 24 hours, as determined by Western blot analysis with the NOTCH 1 VaI 1744 antibody; and in marked downregulation of NOTCH1 transcriptional activity - evidenced by a downregulation of HES1 (a direct target of NOTCH 1) (Figure 3A 1 B).
  • the extracellular HD and LNR repeat domains of NOTCH proteins play a critical role in the stabilization of the receptor in a resting state and are the target of activating mutations that result in aberrant NOTCH 1 signaling in T-ALL.
  • the resolution of the crystal structure of the NOTCH2 LNR-HD complex has provided mechanisms that mediate the inhibitory effects of these extracellular domains on the processing and activation of NOTCH receptors.
  • the third LNR domain buries the S2 cleavage site located in the C-terminal HD subunit, thereby protecting it from cleavage by metalloproteases.
  • physiologic NOTCH 1 signaling is initiated by the binding of a DSL ligand to the EGF repeats in the extracellular subunit of the receptor. This triggers a conformational change in the LNR-HD complex that exposes the S2 site to proteolytic cleavage by ADAM metalloproteases.
  • the short extracellular juxtamembrane stub of the transmembrane-intracelluar subunit binds to nicastrin, a membrane protein that works as a receptor for the ⁇ -secretase complex, which ultimately cleaves NOTCH 1 at the S3 site located in the transmembrane domain of the receptor.
  • This final proteolytic cleavage activates the receptor by releasing the intracellular domains of NOTCH 1 from the membrane, which then translocate to the nucleus and activate the expression of target genes.
  • HD mutations do not affect the stability of the LNR-HD complex, but introduce extra amino acids between the distal part of the HD domain and the S2 cleavage site displacing it away from the protective effects of the LNR-HD complex (class 2 mutations) ( Figure 5).
  • Common features of both class 1 and class 2 HD mutations are: (i) their strict dependence on the integrity of the S2 cleavage site for activation, (ii) having ADAM10 as the main metalloprotease involved in S2 processing, and (iii) the requirement of y ⁇ secretase activity for NOTCH 1 activation. However, they differ in their relative activity with class 2 mutants being significantly more active than class 1 NOTCH 1 alleles.
  • JME mutations could result in aberrant processing of NOTCH 1 at the typical S2 metalloprotease cleavage site, or alternatively, in proteolytic cleavage of the receptor at an alternative S2-like cleavage site located in the inserted peptide sequences encoded by these NOTCH1 alleles, followed by ⁇ -secretase cleavage.
  • NOTCH1 JME alleles and class 2 HD mutants have several features in common: (i) both are generated by relatively long insertions resulting from internal tandem duplications in the NOTCH 1 gene, (ii) they result in high levels of NOTCH 1 activity compared to class 1 HD alleles and (iii) their activity is dependent on the integrity of the S2 cleavage site and the function of the ⁇ -secretase complex.
  • class 2 HD NOTCH1 mutants aberrant S2 cleavage is induced by the insertion of extra amino acids immediately proximal to the classic S2 cleavage site, which is displaced closer to the membrane and out of reach from the structure of the LNR-HD complex ( Figure 5).
  • the S2 cleavage site is located 11 amino acids distant from the membrane in NOTCH2 and NOTCH3, while in NOTCH 1 and NOTCH4 the extracellular juxtamembrane region is 12 amino acids long.

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Abstract

La présente invention porte sur certains procédés et sur certaines compositions permettant de diagnostiquer, de prévenir, de traiter et/ou d'améliorer les effets de divers états chez un mammifère, tels qu'une leucémie lymphoblastique de lymphocytes T et un lymphome. La présente invention porte également, entre autres, sur des procédés permettant de déterminer si la réduction ou le blocage de l'activation de NOTCH-1 est, ou non, efficace pour traiter, prévenir ou améliorer les effets d'un cancer chez un patient. De tels procédés peuvent comprendre la détermination de savoir si le patient subit une ou plusieurs mutations de l'exon 28 ou de l'intron 27 du gène NOTCH-1.
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