WO2014160364A1 - Méthodes de traitement du cancer - Google Patents

Méthodes de traitement du cancer Download PDF

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WO2014160364A1
WO2014160364A1 PCT/US2014/026396 US2014026396W WO2014160364A1 WO 2014160364 A1 WO2014160364 A1 WO 2014160364A1 US 2014026396 W US2014026396 W US 2014026396W WO 2014160364 A1 WO2014160364 A1 WO 2014160364A1
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cells
vrk2
cancer
cell
chromosome
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David Pellman
Taruho KURODA
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Dana Farber Cancer Institute Inc
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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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/158—Expression markers
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates generally to treating cancer. Also included are methods of identifying therapeutic targets for the treatment of cancer.
  • CIN chromosomal instability
  • Tetraploid or subtetraploid DNA content is common in human cancers. Recent bioinformatic analysis suggests that tumor genomes often pass through an unstable tetraploid intermediate at some stage of tumor development. Known defects in cancer cells such as telomere attrition induce tetraploidy.
  • the present invention provides methods of treating cancer in a subject by administering to the subject a compound that inhibits the expression of activity of Vaccina- related kinase (VRK).
  • the VRK is VRK1 or VRK2.
  • the cancer is a cancer that exhibits chromosomal instability, such as chromosome bridges, micronuclei, and/or extra centrosomes.
  • the cancer is a STAG2 or FBXW7 mutant cancer.
  • the cancer cell has a BRCA1 mutation, a BRCA2 mutation or a Rb mutation.
  • the cancer is for example a leukemia, a lymphoma, a melanoma, a carcinoma or a sarcoma.
  • the compound is is a nucleic acid, an antibody or a small molecule.
  • the method further includes administering a chemotherapeutic agent to the subject.
  • the chemotherapeutic agent is a serine-threonine kinase inhibitor.
  • the agent is administered after treatment with a chromosome instability- causing therapeutic agent.
  • Therapeutic agents that cause chromosome instability include, for example, radiation therapy or cell-cycle-arresting agents, such as paclitaxel (Taxol).
  • the invention further provide a method of treating cancer comprising administering to the subject a compound identified by the screening methods disclosed herein.
  • the invention provides methods of treating a subject having a cancer with chromosomal instability by administering a compound, such as a VRK inhibitor, preferably a VRK2 inhibitor, to a subject in need thereof.
  • a compound such as a VRK inhibitor, preferably a VRK2 inhibitor
  • FIG. 1 Tetraploid-selective lethality of VRK2 inhibition.
  • A Isolation of tetraploid HCTl 16 cells. FACS profile (left) and karyotypes (right), comparing the parental
  • Blue boxes show chromosome rearrangements common between the diploid and tetraploid; orange boxes indicate new chromosome rearrangements in the tetraploid; red boxes indicate chromosome gain or loss specific to the tetraploid.
  • B and C Summary of the cytological analysis.
  • B The percentage of cells that display deviation from the modal chromosome number.
  • C Number of chromosome structural abnormalities per cell (translocations, rings, dicentric chromosomes, and arm-level gains or losses). Data represent averages of > 20 chromosome spreads, 2 diploid clones and 5 tetraploid clones + s.d.
  • RNAi-resistant VRK2 construct (VRK2R-WT) or a kinase-dead variant (VRK2R-K168E) were expressed in cells from a retroviral vectorand then infected with the indicated shRNAs.
  • G Viability was determined 7-days post shRNA delivery using CellTiter-GloTM reagent. The data represent averages from 3 replicates + s.d.
  • H Levels of the indicated proteins from "H” by western blotting.
  • II-K Non-phosphorylatable BAF phenocopies VRK2 inhibition.
  • II Cartoon of BAF, the 3A nonphosphorylatable mutant and the 3D phosphomimetic mutant (17) (18).
  • FIG. 1 Marked BAF accumulation on chromosome bridges and micronuclei after VRK2 inhibition.
  • A VRK2 knockdown or control tetraploid HCT116 cells expressing GFP-BAF and mRFP-H2B. Spinning disk confocal images were acquired at 1 min intervals. Arrowheads indicate chromosome bridges; arrow shows a micronucleus derived from a chromosome bridge.
  • B Extent of GFP-BAF accumulation on chromosome bridges. Line scans (broken arrows) showing the fluorescence intensity of GFP-BAF (green) and mRFP-H2B (red) after VRK2 knockdown.
  • FIG. 4 Mutations and nuclear morphology predict cancer cell sensitivity to VRK2 inhibition.
  • A Selective lethality of VRK2 inhibition in STAG2- or FBXW7- mutant cells. Cells of the indicated genotypes were infected with shRNA-expressing lentiviruses and viability was scored 14-days post infection. The viability of VRK2 knockdown cells was expressed as the percentage of the scramble control + s.d..
  • B The fate of STAG2 mutant cells after VRK2 inhibition. Outcomes were scored as in Figure 3D.
  • C Selective lethality of nonphosphorylatable BAF in STAG2- or Z3 ⁇ 4W7-mutant cells.
  • FIG. 1 Characterization of tetraploid HCT116 cells.
  • A Comparable growth of diploid and tetraploid HCT116 cells. Cells were seeded in 384-well plates and were stained with Hoechst 33342 every 24 hrs. The number of cells was examined with ImageXpress Micro (Molecular Devices) using an automated nuclear counting module.
  • B Tetraploid HCT116 cells maintain a near tetraploid chromosome content after 70 days in continuous culture. Representative FACS profiles are shown of the indicated cell lines after 40 days or 70 days in culture.
  • C-E Centrosome number and spindle morphology of tetraploid HCT116 lines.
  • the chromosome 8 signal is green, DNA is blue. Scale bar, 10 ⁇ .
  • FIG. 1 RNAi screen for ploidy-specific lethality.
  • A Schematic of the siRNA screen. A whole-genome Dharmacon siRNA library was aliquoted in six 384-well plates, mixed with Lipofectamine RNAiMAX, and then diploid or tetraploid HCT116 cells were added to the plates. Cell viability was measured with CellTiter GloTM reagent at 72 hrs after the reverse transfection procedure.
  • RNAi gene enrichment ranking 33
  • E and F Analysis of individual siRNAs from the pool targeting VRK2.
  • E Diploid and tetraploid cell lines were transfected with the indicated siRNAs on day 0 and day 3; viability was then assayed on day 6. Shown are averages normalized to the control (siGLORed) siRNA + s.d.
  • F Western blot showing VRK2 steady state levels after the indicated siRNA treatment.
  • G 3 independent shRNAs targeting VRK2 were introduced in diploid and tetraploid HCTl 16 cells. After the selection with Puromycin, these cells were harvested at 6-days post infection and assayed by western blotting for VRK2 expression.
  • Nonphosphorylatable BAF generates interphase cells with chromosome bridges.
  • A-C HCTl 16 diploid and tetraploid cells were infected with lentiviruses expressing GFP-BAF, GFP-BAF -3 A, GFP-BAF -3D or GFP, as a control.
  • A Expression levels of the indicated proteins 4-days post-infection.
  • B The indicated cells were processed for immunofluorescence 21 -days post infection to detect the nuclear envelope (anti-LAP2 antibody) and the plasma membrane ( ⁇ -Catenin antibody). The frequency of LAP2-positive chromosome bridge formation in interphase cells was examined (n > 500).
  • C Representative images of tetraploid cells expressing the indicated constructs are shown. Arrowheads and insets show chromosome bridges. Scale bars, 10
  • FIG. 10 Absence of S phase or metaphase-anaphase delay after short- term VRK2-inhibition.
  • a and B Progression from DNA replication to mitosis was monitored by pulse-labeling with BrdU, followed by 10 hr nocodozole treatment and labeling for histone H3 ser 10 phosphorylation, as described. Note that VRK2 inhibition does not diminish the fraction of BrdU + phospho-H3 + cells, either in the FBW7 +/+ or FBW7 /_ lines. Also note that FBW7 _/" cells display the expected delay in S phase progression relative to the FBW7 +/+ controls.
  • B Quantification of (A).
  • FIG. 11 Example of endoreduplication in a tetraploid HCT116 cell with a chromosome bridge after VRK2 inhibition.
  • A Images from a timelapse series of a cell expressing the indicted fluorescent proteins. Images were acquired at 15 min intervals as in Figure 3D. Endoreduplication is evidenced by oscillation of mCherry-Cdtl without intervening mitosis that is accompanied by increased nuclear size. Insets show the chromosome bridge. Dotted line outlines the nucleus undergoing endoreduplication. Scale bar, 20 ⁇ .
  • B Fluorescence intensity measurements showing the oscillation of mCherry- Cdtl from (A).
  • FIG. 12 Accumulation of DNA damage and activation of p53 after long- term VRK2 inhibition. Although we saw little or no evidence of DNA damage early after VRK2 inhibition ( ⁇ 5 days), at a late timepoint, during the period of maximal cell death, a significant population of cells with large nuclei and DNA damage accumulated. Cells were labeled for ⁇ - ⁇ 2 ⁇ , p53, and p21 fourteen days after infection with the indicated lentiviruses. (A) Representative images of cells. Scale bar, 20 ⁇ . (B) Quantification of the experiment from (A). The amount of DNA damage was examined by using an anti- ⁇ 2 ⁇ antibody. Cells were scored as y-H2AX-positive if they contained at least 3 large foci.
  • FIG. 13 Selective lethality of VRK2 inhibition in STAG2- and FBXW7- deficient cells.
  • A Knockdown efficiency of VRK2 in the indicated cell lines. The absence of FBW7 was inferred from the increased expression of Cyclin E and p21 as described (34).
  • B STAG2- and FBXW7 -deficient cells accumulate large cells with DNA damage late after VRK2 inhibition (14 days), like tetraploid HCT116 cells. DNA damage was scored after VRK2 knockdown as in Fig. 12.
  • C Independently derived ZW7-deficient HCT116 clones exhibit sensitivity to VRK2 inhibition. Independnetly derived FBXW7-null and FBXW7oc-null HCT116 clones were described in (27), and examined for sensitivity to VRK2 inhibition as in Fig. 4A.
  • Figure 14 Genomic alterations associated with the sensitivity to VRK2 inhibition. From the colon cancer cells in Fig. 4D, those in Cancer Cell Line Encyclopedia (CCLE, REF) were selected and the viability of these cell lines were cross-matched for cancer-associated genomic alterations.
  • CCLE Cancer Cell Line Encyclopedia
  • A Gene expression data from biopsy of colorectal adenoma and matched normal mucosa in 32 patients (Sabates-Bellver et al., GSE8671) were analyzed to compare the expression level of VRK2. p-value was calculated by Student's t-test (two tailed, paired).
  • B Gene expression data from Kaiser et al. (GSE5206) were analyzed to compare the expression level of VRK2 in normal colorectal tissue and colorectal adenocarcinoma, p-value was calculated by Student's t-test (two tailed, two sample equal distribution). Note that similar results were obtained in multiple different datasets through Oncomine database search.
  • the invention is based in part upon the surprising discovery that Vaccinia- related kinase 2 (VRK2) is preferentially required for the survival of genetically unstable cancer cells containing chromosome bridges, micronuclei and/or extra centrosomes.
  • VRK2 Vaccinia- related kinase 2
  • These gentetic insatbilities can be identified by morphologically by routine methods such as immunohistochemistry or by identifying specific genetic mutations for example by PCR.
  • VRK1 is mainly nuclear but also found in the cytoplasm
  • VRK2 has two splice isoforms, a short form that is similar in localization to VRK1 and a long form that is a tail-anchored ER membrane protein with its kinase domain facing the cytoplasm
  • VRK3 is an enzymatically inactive pseudo- kinase.
  • BAF Barrier to Autointegration Factor
  • BAF promotes nuclear envelope assembly at the end of mitosis by recruiting inner nuclear envelope proteins that contain LEM domains, which are BAF-binding protein interaction module. Structural studies show that BAF forms a bowtie-shaped dimer with DNA binding domains on each lateral surface. The LEM domain-binding region is located at the "knot" of the bowtie BAF can therefore crosslink DNA. Genetic experiments demonstrate that BAF is the functionally significant VRK substrate. However, other substrates, including p53 have been reported, although the physiological significance of these substrates is not definitively established.
  • the VRK-BAF system also plays a role in the defense against certain viral infections.
  • Vaccinia virus is a double- stranded DNA virus that replicates in the cytoplasm of infected cells.
  • BAF massively binds to viral DNA and prevents viral replication.
  • Vaccinia circumvents this barrier to infectivity by bringing into host cells its own VRK kinase (vvBlR)— hence the moniker "Vaccinia Related Kinases”.
  • BAF also has a function in the retroviral life cycle. After reverse- transcription of the viral genome, a preintegration complex is assembled that contains BAF.
  • VRK-BAF binding to newly replicated retroviral DNA prevents suicidal autointegration and therefore favors viral integration into the host genome.
  • the VRK-BAF system can be viewed as a detector of "stray" double stranded DNA, such as viral DNA. Different viruses manipulate the system in different ways.
  • VRK1 GenBank Accession No. NP_003375.1
  • Structural and domain analysis predicts a serine/threonine kinase domain at amino acids positions 37-293, and a PKC domain at amino acid positions 43-253.
  • VRK2 has multiple mRNA transcripts and slice forms.
  • the present invention provides for inhibitors of any of the transcripts, such as VRK2 mRNA transcript 1
  • VRK1 GenBank Accession No. BAA19109.1
  • Structural and domain analysis predicts a serine/threonine kinase domain at amino acids positions 29-421, and a PKC domain at amino acid positions 35-241.
  • a VRK inhibitor decreases expression or activity of VRK.
  • a decrease in VRK activity is defined by a reduction of a biological function of the VRK.
  • a biological function of VRK includes phosphorylation of BAF1 which disrupts its ability to bind DNA and reduces its binding to LEM domain-containing proteins. Phosphorylation of BAF1 can be detected by various standard methods known to the skilled person in the art, such as immunoblotting with phosphor-specific antibodies. BAF1 binding to LEM domain- containing proteins can be determined for example by immunoprecipitation studies or activity assy of downstream signaling.
  • a decrease or reduction in VRK biological activity refers to at least a 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% decrease in VRK activity compared to a control.
  • the control is the activity of the VRK kinase before treatment or in a subject that has not received any treatment.
  • the VRK inhibitor is a kinase inhibitor.
  • the kinase inhibitor is a serine/threonine kinase inhibitor.
  • VRK expression is measured by detecting a VRK1 or VRK2 transcript or protein using standard methods known in the art, such as RT-PCR, microarray, and immunoblotting or immunohistochemistry with VRK-specific antibodies.
  • a decrease in VRK expression refers to at least a 1 %, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% decrease in the level of VRK mRNA or VRK protein.
  • VRK2 has several characteristics that make it an attractive cancer therapeutic target. First, VRK2 is not essential for viability in the mouse (12). Second, the catalytic domains of VRK2 and VRK1 differ such that VRK2-selective inhibitors can be developed (11). Finally, as described in detail herein, cells with chromosomal instability, such as cancer cells, are sensitive to VRK2 inhibition.
  • VRK2 inhibitors are known in the art or are identified using methods described herein. For example, a VRK2 inhibitor is identified by detecting the phosphorylation status of downstream phosphorylation substrates (i.e. BAF).
  • the VRK inhibitor can be a small molecule.
  • a "small molecule” as used herein, is meant to refer to a composition that has a molecular weight in the range of less than about 5 kD to 50 daltons, for example less than about 4 kD, less than about 3.5 kD, less than about 3 kD, less than about 2.5 kD, less than about 2 kD, less than about 1.5 kD, less than about 1 kD, less than 750 daltons, less than 500 daltons, less than about 450 daltons, less than about 400 daltons, less than about 350 daltons, less than 300 daltons, less than 250 daltons, less than about 200 daltons, less than about 150 daltons, less than about 100 daltons.
  • Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics,
  • the VRK inhibitor is an antibody or fragment thereof specific to VRK.
  • the antibody specifically binds to the kinase domain of the VRK protein, and therefore, decreases, reduces, or inhibits kinase activity through either steric hindrance or competitive inhibition with the substrate (i.e., BAFl).
  • BAFl steric hindrance or competitive inhibition with the substrate
  • the VRK inhibitor is for example an antisense VRK2 nucleic acid, a VRK-specific short-interfering RNA, or a VRK -specific ribozyme.
  • siRNA is meant a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into a cell are used, including those in which DNA is a template from which an siRNA is transcribed.
  • the siRNA includes a sense VRK nucleic acid sequence, an anti-sense VRK nucleic acid sequence or both.
  • the siRNA is constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin (shRNA). Examples of siRNAs and shRNAs are disclosed in the examples herein.
  • binding of the siRNA to a VRK transcript in the target cell results in a reduction in VRK production by the cell.
  • the length of the oligonucleotide is at least 10 nucleotides and may be as long as the naturally-occurring VRK transcript.
  • the oligonucleotide is at least 10 nucleotides and may be as long as the naturally-occurring VRK transcript.
  • oligonucleotide is 19-25 nucleotides in length. Most preferably, the oligonucleotide is less than 75, 50, 25 nucleotides in length.
  • VRK kinases form their own branch of the kinase tree and are most closely related to casein kinases. Structures for VRKl (NMR) and VRK2 (crystal structure) are available. The kinase domains of VRKl and VRK2 have well-defined, typical kinase folds. The unique C-terminal tail of VRKl makes contacts with the catalytic center that is required for its structural stability and catalysis; these unique features could be a structural basis for selective inhibitors. Although not completely defined, VRK1 and VRK2 seem to functionally overlap to a significant degree such that pan- VRK inhibitors or isoform selective inhibitors would likely behave similarly in assays. Effects of several broad-acting kinase inhibitors on VRKs have been described, but these effects are non-specific.
  • Identification of novel VRK2 inhibitors can be performed by nonradioactive (Z'-LYTETM (SEQ ID NO: 12)) kinase assay for VRK1 and VRK2.
  • cell-based screens using available libraries of kinase inhibitors can also be performed. Screening will be performed in paired cell lines— either the diploid and tetraploid HCT116 cells or in paired mutant (e.g., STAG2 or FBXW7) and control cell lines. Cells will be screened at a compound concentration of 1 ⁇ for the ability to selectively inhibit proliferation of the cell lines with chromosome bridges, micronuclei and/or extra centrosomes. Any compound that causes greater than a 3-fold inhibition in cell proliferation at the screening
  • concentration will be re-tested in dose-response format.
  • Compounds that possess EC50s in the single-digit micromolar range and that exhibit greater than 3 -fold more potent inhibition of relevant cell lines will be characterized for effects on BAF localization, i.e. , by immunofluorescence or immunoblotting. Selectivity will be assessed using Ambit's KinomeScan and ActivX's KiNativ. Inhibitors exhibiting promising cell potency and selectivity, pathway engagement, and kinase selectivity will be further considered and optimized by medicinal chemistry.
  • the growth of tumor cells is inhibited, e.g. reduced, by contacting a tumor cell with a composition containing a compound that decreases the expression or activity of VRK (i.e. VRK1 or VRK2).
  • a composition containing a compound that decreases the expression or activity of VRK i.e. VRK1 or VRK2
  • inhibition of cell growth is meant the cell proliferates at a lower rate or has decreased viability compared to a cell not exposed to the composition.
  • Cell growth is measured by methods know in the art such as, the MTT cell proliferation assay, cell counting, measurement of ATP content, crystal violet staining, or measurement of total GFP from GFP expressing cell lines.
  • Cells are directly contacted with the compound. Alternatively, the compound is administered systemically.
  • the tumor cell exhibits chromosomal instability (CIN).
  • CIN chromosomal instability
  • the tumor cell has chromosome bridges, and or micronuclei.
  • Cells containing extra centrosomes are highly susceptible to chromosome bridges and micronuclei.
  • STAG2 and/or FBXW7 mutations in either in the gene, polypeptide or both are known to cause CIN. Accordingly, in various aspects the tumor cell has a STAG2 and/or a FBXW7 mutation.
  • STAG2 and or FBXW7 mutations or null mutations can be identified by methods known in the art. The mutation may be in the nucleic acid sequence encoding STAG2 or FBXW7 polypeptide or in the STAG2 or FBXW7 polypeptide, or both.
  • Treatment is efficacious if the treatment leads to clinical benefit such as, a decrease in size, prevalence, or metastatic potential of the tumor in the subject.
  • "efficacious” means that the treatment retards or prevents tumors from forming or prevents or alleviates a symptom of clinical symptom of the tumor. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type.
  • the invention includes administering to a subject composition comprising a VRK inhibitor.
  • An effective amount of a therapeutic compound is preferably from about 0.1 mg/kg to about 150 mg/kg.
  • Effective doses vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and coadministration with other therapeutic treatments including use of other anti-proliferative agents or therapeutic agents for treating, preventing or alleviating a symptom of a cancer.
  • a therapeutic regimen is carried out by identifying a mammal, e.g., a human patient suffering from a cancer using standard methods.
  • Doses may be administered once, or more than once. In some embodiments, it is preferred that the therapeutic compound is administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week for a predetermined duration of time.
  • the predetermined duration of time may be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or up to 1 year.
  • the pharmaceutical compound is administered to such an individual using methods known in the art.
  • the compound is administered orally, rectally, nasally, topically or parenterally, e.g., subcutaneously, intraperitoneally, intramuscularly, and intravenously.
  • the inhibitors are optionally formulated as a component of a cocktail of therapeutic drugs to treat cancers.
  • formulations suitable for parenteral administration include aqueous solutions of the active agent in an isotonic saline solution, a 5% glucose solution, or another standard pharmaceutically acceptable excipient.
  • Standard solubilizing agents such as PVP or cyclodextrins are also utilized as pharmaceutical excipients for delivery of the therapeutic compounds.
  • the therapeutic compounds described herein are formulated into compositions for other routes of administration utilizing conventional methods.
  • the therapeutic compounds are formulated in a capsule or a tablet for oral administration.
  • solid carriers examples include starch and sugar bentonite.
  • the compound is administered in the form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, conventional filler, and a tableting agent.
  • a binder e.g., lactose or mannitol
  • Other formulations include an ointment, suppository, paste, spray, patch, cream, gel, resorbable sponge, or foam. Such formulations are produced using methods well known in the art.
  • Therapeutic compounds are effective upon direct contact of the compound with the affected tissue. Accordingly, the compound is administered topically. Alternatively, the therapeutic compounds are administered systemically. For example, the compounds are administered by inhalation.
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • compounds are administered by implanting (either directly into an organ or subcutaneously) a solid or resorbable matrix which slowly releases the compound into adjacent and surrounding tissues of the subject.
  • the therapeutic compounds described herein are administered in combination with another therapeutic agent, such as a chemotherapeutic agent, radiation therapy, or an anti-mitotic agent.
  • the anti-mitotic agent is administered prior to administration of the present therapeutic compound, in order to induce additional chromosomal instability to increase the efficacy of the present invention to targeting cancer cells.
  • anti-mitotic agents include taxanes (i.e., paclitaxel, docetaxel), and vinca alkaloids (i.e., vinblastine, vincristine, vindesine, vinorelbine).
  • the invention also provides a method of screening for therapeutic targets for treating cancers.
  • the invention provides a method for identifying therapeutic targets for treating cancer by providing a contacting the cell with a library of RNAi or small moclecules.
  • Potential therapeutic targets are identified by determining what RNAi or small molecule is lethal to the cell, decreases cell viability or inhibits cell growth.
  • Assays for identification of potential therapeutic targets are known in the art, for example, MTT proliferation assay, cell growth curves, and analysis by staining and flow cytometry.
  • an STAG2 null cancer refers to those cancers that display a disruption in the STAG2 gene, such that the levels of the STAG2 gene, mRNA or protein or STAG2 protein activity is decreased.
  • the disruption in the gene can be caused by a mutation.
  • Disruption of the gene can be detected by sequencing or genotyping methods known in the art. Detection of decreased mRNA or protein levels and protein activity can be detected by standard methods known in the art, for example qRT-PCR, microarray, immunoassays, Western blots or various activity assays.
  • polypeptide refers, in one embodiment, to a protein or, in another embodiment, to protein fragment or fragments or, in another embodiment, a string of amino acids.
  • reference to "peptide” or “polypeptide” when in reference to any polypeptide of this invention is meant to include native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
  • Such modifications include, but are not limited to N terminal, C terminal or peptide bond modification, including, but not limited to, backbone modifications, and residue modification, each of which represents an additional embodiment of the invention.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).
  • oligonucleotides include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.
  • nucleotide as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form.
  • nucleotide is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, all as described herein.
  • homology when in reference to any nucleic acid sequence indicates a percentage of nucleotides in a candidate sequence that are identical with the nucleotides of a corresponding native nucleic acid sequence. Homology may be determined by computer algorithm for sequence alignment, by methods well described in the art. For example, computer algorithm analysis of nucleic acid or amino acid sequence homology may include the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.
  • two nucleotide sequences are "substantially complementary” if the sequences have at least about 70 percent or greater, more preferably 80 percent or greater, even more preferably about 90 percent or greater, and most preferably about 95 percent or greater sequence similarity between them.
  • Two amino acid sequences are substantially homologous if they have at least 50%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% similarity between the active, or functionally relevant, portions of the polypeptides.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • ameliorated refers to a symptom which is approaches a normalized value (for example a value obtained in a healthy patient or individual), e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests.
  • a normalized value for example a value obtained in a healthy patient or individual
  • treating may include suppressing, inhibiting, preventing, treating, or a combination thereof.
  • Treating refers inter alia to increasing time to sustained progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof.
  • Symptoms may be any manifestation of a disease or pathological condition.
  • the "treatment of cancer or tumor cells” refers to an amount of peptide or nucleic acid, described throughout the specification , capable of invoking one or more of the following effects: (1) inhibition of tumor growth, including, (i) slowing down and (ii) complete growth arrest; (2) reduction in the number of tumor cells; (3) maintaining tumor size; (4) reduction in tumor size; (5) inhibition, including (i) reduction, (ii) slowing down or (iii) complete prevention, of tumor cell infiltration into peripheral organs; (6) inhibition, including (i) reduction, (ii) slowing down or (iii) complete prevention, of metastasis; (7) enhancement of anti-tumor immune response, which may result in (i) maintaining tumor size, (ii) reducing tumor size, (iii) slowing the growth of a tumor, (iv) reducing, slowing or preventing invasion and/or (8) relief, to some extent, of the severity or number of one or more symptoms
  • an ameliorated symptom or “treated symptom” refers to a symptom which approaches a normalized value, e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests.
  • a "pharmaceutically acceptable” component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
  • the term "safe and effective amount” or “therapeutic amount” refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.
  • therapeutically effective amount is meant an amount of a compound of the present invention effective to yield the desired therapeutic response. For example, an amount effective to delay the growth of or to cause a cancer to shrink rr or prevent metastasis.
  • cancer refers to all types of cancer or neoplasm or malignant tumors found in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas.
  • cancers are cancer of the brain, breast, pancreas, cervix, colon, head and neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma.
  • Additional cancers include, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer.
  • a "proliferative disorder” is a disease or condition caused by cells which grow more quickly than normal cells, i.e., tumor cells.
  • Proliferative disorders include benign tumors and malignant tumors. When classified by structure of the tumor, proliferative disorders include solid tumors and hematopoietic tumors.
  • patient or “individual” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred.
  • methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
  • modulate it is meant that any of the mentioned activities, are, e.g., increased, enhanced, increased, augmented, agonized (acts as an agonist), promoted, decreased, reduced, suppressed blocked, or antagonized (acts as an antagonist). Modulation can increase activity more than 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., over baseline values. Modulation can also decrease its activity below baseline values.
  • administering to a cell refers to transducing, transfecting, microinjecting, electroporating, or shooting, the cell with the molecule.
  • molecules are introduced into a target cell by contacting the target cell with a delivery cell (e.g., by cell fusion or by lysing the delivery cell when it is in proximity to the target cell).
  • delivery cell e.g., by cell fusion or by lysing the delivery cell when it is in proximity to the target cell.
  • molecule is used generically to encompass any vector, antibody, protein, drug and the like which are used in therapy and can be detected in a patient by the methods of the invention.
  • nucleic acid delivery vectors encoding different types of genes which may act together to promote a therapeutic effect, or to increase the efficacy or selectivity of gene transfer and/or gene expression in a cell.
  • the nucleic acid delivery vector may be provided as naked nucleic acids or in a delivery vehicle associated with one or more molecules for facilitating entry of a nucleic acid into a cell.
  • Suitable delivery vehicles include, but are not limited to:
  • liposomal formulations polypeptides; polysaccharides; lipopolysaccharides, viral formulations (e.g., including viruses, viral particles, artificial viral envelopes and the like), cell delivery vehicles, and the like.
  • HCT116 FBXWT 1 ' ' and FBXWT 1' cells HCT116 FBXWT' ' and FBXW7a' ⁇ cells
  • HCT116 STAG2-null cells HCT116 STAG2-null cells
  • HeLa- Kyoto cell line Colorectal cancer cell lines HT55 and GP2d were obtained from Sigma- Aldrich. Other colorectal cancer and nontransformed colorectal cell lines were obtained from ATCC.
  • HCT116 colorectal cancer cells and their derivatives are cultured in McCoy's 5A medium supplemented with 10% FBS and Penicillin/Streptomycin unless noted otherwise.
  • HEK 293FT (Life technologies) and HeLa-Kyoto cells were cultured in DMEM supplemented with 10% FBS and Penicillin/Streptomycin.
  • Fig. 4D cells were cultured in RPMI (Life technologies) supplemented with 10% FBS and Penicillin/Streptomycin. All cell lines were maintained at 37 °C with 5% C02 atmosphere.
  • shRNA and siRNA were obtained from RNAi screening facility at Dana-Farber Cancer Institute and Dharmacon, respectively, and are summarized in Tables 3 and 4 respectively.
  • pLenti- CM V-Hyg- GFP-B AF WT, 3A, 3D: addition of restriction enzyme sites (BspEI and BamHI in the 5' and 3', respectively) and mutations in BAF cDNA was achieved by a standard PCR-based method.
  • the PCR fragment was inserted into pENTR- EGFP2 (addgene #22450).
  • the resultant vectors were recombined with pLenti-CMV- Hygro-DEST (addgene #17454) using GatewayTM LR recombinase enzyme mix.
  • pLenti-CMV-Hyg-mRFP-LAP2 cDNA for mRFP-LAP2 ⁇ (encoding rat LAP2 ⁇ 244-452 a.a. tagged with mRFP) was obtained by digesting pmRFP-LAP2 ⁇ - IRES-Puro2b (24) with Agel and BamHI , and the fragment was cloned into pENTR- EGFP2. The resultant vector was recombined with pLenti-CMV-Hygro-DEST (addgene.org #17454) using Gateway LR recombinase.
  • pMSCV-IRES-GFP-VRK2R RNAi-resistant VRK2 cDNA was partially synthesized as follows (silent mutations are capitalized): atgccaccaaaaagaaatgaaaatacaaacttcctattccatttccagaGggGaaAgtCctAgaCgaCatggaaggcaatca gtgggtactgggcaagaagattggctctggaggatttggattgatatatttagctttccccacaaataaccagagaaagatgcaaga catgtagtaaaagtggaatatcaagaaaatggcccgttattttcagaacttaaattttatcagagagttgcaaaaaaagactgtatcaaaaagactgtatcaaaaagactgtatcaaaaagactgtatcaaaaagactgt
  • the resultant vector was recombined with pMSCV-DEST-IRES-GFP (gift from Drs.
  • VRK2R K168E expression vector was produced by the same method except that the synthesized VRK2R sequence carried alterations corresponding to K168E.
  • CSII-EF-mCherry-hCdtl (30/120) is a gift from Dr. Atsushi Miyawaki (BSI, RIKEN, Wako, Saitama, Japan).
  • pBABE-Puro-mRFP-H2B is a gift from Dr. Randall King (Harvard medical School, Boston, MA, USA).
  • pLenti6-H2B-GFP is kindly provided by Dr. Masayuki Nitta (Tokyo women's medical university, Tokyo, Japan).
  • the karyotype of HCTl 16 diploid and tetraploid clones was examined by G- banding. The gains or losses of a whole chromosome from original HCTl 16 karyotype were counted as numerical abnormalities. The rate of structural chromosomal abnormalities, such as translocations, double minute chromosomes, marker chromosomes and isochromosomes was also examined. The number of numerical and structural abnormalities per cell was counted in 4 diploid and 5 tetraploid clones (25 cells/cell line) and presented as average + s.d.
  • Chromosome missegregation rate (anaphase FISH)
  • HCT116 diploid and tetraploid cells were seeded in 384-well plates. Cells were fixed and stained with Hoechst 33342 every 24 hrs and the images were acquired by ImageXpressMicro cellular imaging system (Molecular Devices). The cell number was counted using cell counting module of MetaXpress software (Molecular Devices).
  • the cells were supplemented with 5 lL of medium containing Penicillin/Streptomycin, resulting in 35 ⁇ _ of complete growth medium. After 3 days of incubation at 37°C in 5% C02, the plates were taken out of the incubator for lhr and then added with 20 lL of CellTiter Glo viability assay reagent (Promega) using Multidrop Combi nL (Thermo scientific). Luminescence was measured by using Envision microplate reader (PerkinElmer) in a high-throughput format.
  • ⁇ is the mean
  • n is the number of samples
  • ⁇ is the standard deviation
  • HCT116 diploid cells 8xl0 5
  • HCT116 tetraploid cells 4xl0 5 cells
  • HeLa-Kyoto cells all the cells in Fig. 4D, 4xl0 5 cells.
  • Cells were added with lentiviral particles and 8 ⁇ g/ml Polybrene in 1ml of growth medium, and centrifuged at 1,178 x g for 30 min at room temperature. After 24 hrs of incubation, virus-containing medium was replaced with fresh medium containing 2 ⁇ g/ml Puromycin for the elimination of non-infected cells.
  • HCT116 diploid and tetraploid cells were infected with lentiviral particles expressing VRK2 shRNA. At each time point, cells were trypsinized, collected, and the cell number was counted. Cells were then re-plated at the density of 2xl0 5 (diploid) and 1x10 s (tetraploid) in 6-well dishes for the next counting. To validate the knockdown, cells corresponding to 6-days post-infection were harvested and subjected to 10% SDS- PAGE and immunobloting with anti-VRK2 antibody (data not shown)
  • Retroviral particles expressing VRK2R WT or VRK2R K168E were produced and concentrated 10 fold with PEG-it solution (System Biosciences), and were infected in HCT116 diploid and tetraploid cells. After propagating for 7 days, the cells were subjected to FACS sorting to obtain top 5% of GFP-positive population. After expanding the cells, they were re-plated in 24-well plates (4 xlO 5 diploid cells and 2 xlO 5 tetraploid cells) followed by lentivirus-mediated knockdown of endogenous VRK2. Cell viability was measured by using CellTiter GloTM reagent (Promega) at 7 days post infection. Protein samples were prepared for western blotting to examine the expression level of VRK2.
  • GFP-tagged BAF WT and mutants were expressed through lentiviral delivery.
  • the infected cells were cultured in the presence of 100 ⁇ g/ml Hygromycin, passaged every ⁇ 7 days, and examined for viability on 21 days post infection. For crystal violet staining, cells were first fixed in 4% paraformaldehyde and then stained with 0.5% Crystal violet.
  • VRK2 knockdown was performed in HCT116 tetraploid cells stably expressing GFP-BAF and mRFP-H2B.
  • Cells were re-plated in 35-mm glass bottom dishes (MatTek) 2-days post infection and were visualized 4-days post infection.
  • Time-lapse images of mitotic cells were acquired at lmin interval and ⁇ Z-step for 90 min by using Nikon inverted microscope equipped with 40x objective (Plan-Apo DIC NA 1.3 oil, Nikon), spinning-disk head CSU-X1 (Yokogawa), EM-CCD camera iXon DU-897 (Andor), piezo z-stepper stage (Prior scientific) and environmental chamber (in vivo scientific) maintained at 37°C.
  • HCT116 cells of the indicated genotypes were plated in 24-well plates (4 xlO 5 cells/well) for 24 hr. Cells were infected with shRNA targeting VRK2 or scramble control. Puromycin (1 or 2 g/ml) was added 24 hrs after the infection to maintain selection for the infected cells. These cells were then propagated in the presence of Puromycin for 14 days. The viability was measured using CellTiter Glo reagent (Promega).
  • HeLa-Kyoto cells expressing GFP-H2B and mRFP-LAP2 were infected with shRNA targeting VRK2 or Scramble control. Three to four days after infection, cells were plated on 35-mm glass bottom dish (2 xlO 5 cells/dish). On the following day cells were treated with 100 ng/ml nocodazole and 1.5 ⁇ g/ml bleomycin (Bleocin, EMD Millipore) for 8 hrs.
  • HCT116 diploid, tetraploid cells or STAG2-null cells were stably expressed with
  • EXAMPLE 2 GENERATION OF TETRAPLOID CELLS
  • tetraploid derivatives were generated that acquired all the hallmarks of CIN (Fig. 1A). Tetraploid clones exhibited a 4.6-fold increase in aneuploidy (Fig. IB), 6.3-fold increased rates of whole chromosome missegregation (Fig. 5G), 5.2-fold increased frequency of nonreciprocal translocations (Fig. 1C), and an increase in a variety of nuclear structural abnormalities including micronuclei (2.8-fold) and chromosome bridges (6.5-fold) (Fig. ID and E).
  • siRNA screen was used to identify gene knockdowns that were selectively lethal to the tetraploid cells (Fig. 6A-D).
  • MDM2 which encodes an ubiquitin ligase that targets the tumor suppressor p53 for degradation.
  • Tetraploid cells are known to exhibit a small-scale increase in p53 activation (9), which is expected to render them more sensitive to further p53 activation by MDM2 inhibition.
  • the focus is on one gene selectively required in tetraploid cells, VRK2.
  • the selective effect was observed with 4/4 siRNAs from the originally screened pool, as well as with 5 different shRNAs targeting different VRK2 sequences (Fig. IF, Fig.
  • BAF is also required for an innate antiviral response (19): it binds heavily to Vaccinia virus DNA in the cytoplasm, and is thought to inhibit viral DNA replication.
  • Vaccinia has acquired its own VRK homologue as a virulence factor to circumvent BAF inhibition, which is why the family is known as "Vaccinia-related kinases" (20).
  • VRK2 inhibition would cause lethality in cancer cells with mutations known to cause chromosome bridges and micronuclei.
  • VRK2 was inhibited in isogenic HCT116-derived cell lines with or without mutations in STAG2 or FBW7/hCDC4.
  • STAG2 is a component of the cohesion complex that is required for normal chromosome segregation and for transcriptional regulation.
  • STAG2 and other cohesins are mutated in a growing list of human cancers including colorectal cancer, glioblastoma, melanoma, and acute myelogenous leukemia.
  • FBW7 encodes a component of an E3 ubiquitin ligase complex and is mutated in -8% of all human tumors.
  • VRK2 knockdown selectively inhibited the growth of STAG2 null cells, FBW7 null cells, and FBW7 + heterozygotes (Fig. 4A).
  • Fig. 4A A similar selective effect was obtained with independently generated FBW7 mutant cells lacking either all three or only the major (a) isoform of FBW7 (Fig. 13C) (27).
  • selective lethality from VRK2 inhibition was also seen in FBW7 ' derivatives of DLD1 colon cancer cells (Fig. 4A), which lack functional p53 (28), which is commonly mutated in ZW7-mutated cancers.

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Abstract

La présente invention concerne des méthodes de traitement du cancer, en particulier des cancers qui sont nuls ou ont une expression ou une activité réduite du gène FBXW7 ou du gène STAG2. L'invention concerne également des procédés d'identification de cibles thérapeutiques pour le traitement du cancer.
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WO2017127282A1 (fr) * 2016-01-19 2017-07-27 The General Hospital Corporation Traitements du cancer et méthodes de sélection de ceux-ci
US20220370408A1 (en) * 2021-04-21 2022-11-24 The Trustees Of Columbia University In The City Of New York Methods, compositions, kits and uses thereof targeting and/or treating vrk2 to enhance effectiveness of immune checkpoint inhibitor(s)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017127282A1 (fr) * 2016-01-19 2017-07-27 The General Hospital Corporation Traitements du cancer et méthodes de sélection de ceux-ci
US20220370408A1 (en) * 2021-04-21 2022-11-24 The Trustees Of Columbia University In The City Of New York Methods, compositions, kits and uses thereof targeting and/or treating vrk2 to enhance effectiveness of immune checkpoint inhibitor(s)
US12491176B2 (en) * 2021-04-21 2025-12-09 The Trustees Of Columbia University In The City Of New York Methods, compositions, kits and uses thereof targeting and/or treating VRK2 to enhance effectiveness of immune checkpoint inhibitor(s)

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