WO2014160364A1 - Methods of treating cancer - Google Patents

Abstract

The present invention provides methods of treating cancer, particularly cancers that are null or have decreased expression or activity of the FBXW7 gene or the STAG2 gene. Also included are methods of identifying therapeutic targets for the treatment of cancer.

Description

METHODS OF TREATING CANCER

RELATED APPLICATIONS

[0001] This application claims benefit of and priority to U. S. S.N. 61/778,992 filed on March 13, 2013; the contents of which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to treating cancer. Also included are methods of identifying therapeutic targets for the treatment of cancer.

BACKGROUND OF THE INVENTION

[0003] A large proportion of human cancers exhibit chromosomal instability (CIN), an increased rate of chromosome gain, loss, and rearrangement. This is a feature of tumor cells that distinguishes cancer cells from normal cells and thus potentially provides a therapeutic index for drug therapy.

[0004] 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.

[0005] A need exists for the identification of ploidy specific lethal gene knockdowns in in diploid and tertrapolod cancer cells. More specifically, a need exists to identify therapies that target genetically unstable cancer cells, in particular genetic instabilities that can be readily identified by biomarkers such as mutations.

SUMMARY OF THE INVENTION

[0006] 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. For example, 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.

[0007] The compound is is a nucleic acid, an antibody or a small molecule. In some aspects the method further includes administering a chemotherapeutic agent to the subject. For example the the chemotherapeutic agent is a serine-threonine kinase inhibitor. In another aspect, 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).

[0008] Also included in the invention are methods of screening for therapeutic targets for treating cancer by providing a cell having a chromosome bridge, micronuclei and/or extra centrosomes; contacting the cell with a library of RNAi or small molecule; and identifying an RNAi or small molecule which is lethal to said cell.

[0009] The invention further provide a method of treating cancer comprising administering to the subject a compound identified by the screening methods disclosed herein.

[00010] In one aspect, 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.

[00011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In cases of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[00012] Other features and advantages of the invention will be apparent from and encompassed by the following detailed description and claims.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[00013] Figure 1. Tetraploid-selective lethality of VRK2 inhibition. (A) Isolation of tetraploid HCTl 16 cells. FACS profile (left) and karyotypes (right), comparing the parental

HCTl 16 diploid line to a representative HCTl 16 tetraploid line. 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. (D and E) Frequency of chromosome bridges and micronuclei in diploid and tetraploid cells, n > 500. Data represent average + s.d. of three individual experiments. (F) Tetraploid-selective lethality from VRK2 knockdown. The indicated VRK2 shRNAs or controls were delivered by lentiviral infection to two diploid (blue) and two tetraploid (red) HCT116 clones. Cell number was determined at the indicated intervals after infection. The data are an average from three replicates + s.d. (G and H) Complementation of VRK2 shRNA effect by an shRNA- resistant construct and requirement for the VRK2 kinase activity. An 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-Glo™ reagent. The data represent averages from 3 replicates + s.d. (H) Levels of the indicated proteins from "H" by western blotting. (I-K) Non-phosphorylatable BAF phenocopies VRK2 inhibition. (I) Cartoon of BAF, the 3A nonphosphorylatable mutant and the 3D phosphomimetic mutant (17) (18). These constructs were expressed in the indicated cells after lentiviral-mediated

delivery. Shown are crystal violet- stained plates (J) or CellTiter-Glo™ viability assay (K) 21 -days after infection. Average + s.d.

[00014] Figure 2. 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. (C) Average GFP-BAF intensity (normalized to mRFP-H2B) on chromosome bridges relative to the main chromosome mass. The data are averages of movies from 18 cells. Error bars are s.e.m. Control cells had ~3- fold accumulation of GFP-BAF on chromosome bridges as compared to ~18-fold after VRK2 knockdown. [00015] Figure 3. The fate of cells after VRK2 inhibition. (A and B) Cytokinesis failure after VRK2 inhibition. (A) The indicated shRNA treatments were performed in HeLa Kyoto cells expressing H2B-GFP and mRFP-LAP2 . Cells were treated with 100 ng/ml nocodazole and 1.5 μg/ml bleomycin for 8hrs. After drug washout, spinning disc confocal images were acquired at, 15 min intervals. Scale bar, 10 μιη. (B) Cumulative distribution of the time of chromosome bridge resolution for control (blue) and VRK2- inhibited (red) samples (p < 0.01, Mann- Whitney test). Shown is the fraction of cells that had bridge resolution at the indicated time intervals. The last column shows the fraction of cells that failed cytokinesis during the 15 hr period of imaging. (C) Synthetic lethality of VRK2 inhibition with bleomycin treatment. Cells were treated with the indicated shRNAs, exposed to bleomycin 1 μg/ml or the vehicle for 8 hr, and viability was measured after 14 days of culture. Data are averages from 3 replicates + s.d. (D) Fate of HCT116 cells, with or without chromosome bridges, after VRK2 inhibition. Wide-field imaging was performed for 96 hrs at 15 min intervals for cells expressing GFP-BAF and mCherry-Cdtl. Cells were scored for the indicated outcomes after completion of mitosis. Cells were scored as having chromosome bridges (BR) if a bridge was detected during mitosis or after mitotic exit. "No BR" indicates cells without detectable bridges. Gl cell cycle arrest was defined by a 40 hr or longer time interval in Gl phase. (E) Representative images of binucleation in HCT116 tetraploid cells after VRK2 inhibition. Scale bar, 10 μιη.

[00016] Figure 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 Z¾W7-mutant cells. Cells of the indicated genotypes were infected with lentiviruses expressing GFP, GFP-BAF, or GFP-BAF-3A and viability was scored 21-days post infection. All samples were normalized to the GFP control + s.d. (D) Nuclear structure abnormalities of colon cancer cell lines predict sensitivity to VRK2 inhibition. Top left: shRNA (VRK2-207) was delivered to 2 non- transformed colonic epithelial cell lines and 21 colon cancer cell lines. Viability was assayed 21-days after VRK2 inhibition. Blue letters indicate nontransformed cell lines, red letters indicate colon cancer cells with FBW7 mutations, and black letters indicate other colon cancer cell lines. The FBXW7 mutations are listed in Table S3. Shown are the average + s.d. Bottom left: Fraction of interphase cells in each cell line with the indicated nuclear morphology defects. Right: Representative images of a cell line that is resistant to VRK2 inhibition (RKO) and one that is sensitive (NCI-H747). Nuclear membranes were detected with an anti-LAP2 antibody (white) and the plasma membrane was detected with an anti- -catenin antibody (red). Scale bars, 10 μιη.

[00017] Figure 5. 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. Cells were fixed and immunostained with anti-centrin2 (green), anti-oc-tubulin antibodies (red), and Hoechst 33342 (blue). (C) Representative metaphase images of diploid and tetraploid cells. Scale bars, 5μιη. (D) Centriole number in mitotic cells and (E) the frequency of bipolar or pseudo-bipolar metaphase figures, n > 200 for each sample. (F and G) Frequency of chromosome missegregation in diploid and tetraploid cells. Paired daughter cells were examined for chromosome missegregation by anaphase FISH using a chromosome 8 oc-satellite probe as in Ganem et al. (F) Representative images of normal chromosome segregation and missegregation in diploid and tetraploid cells. The chromosome 8 signal is green, DNA is blue. Scale bar, 10 μιη. (G) Rate of chromosome mis-segregation in diploid and tetraploid cells. Shown are the average + s.e.m. Note that diploid cells missegregated one chromosome in every 34.5 mitoses, whereas tetraploid cells had a chromosome missegregation every 5.5 mitoses.

[00018] Figure 6. 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 Glo™ reagent at 72 hrs after the reverse transfection procedure. (B) Scatter plot of the primary screening data. Each blue dot corresponds to an siRNA pool targeting an individual gene. Y-axis, viability in diploid cells; X-axis, viability in tetraploid cells. Knockdowns with equivalent viability in diploids and tetraploids fall on the black line (R = 0.90). The area in pink contains siRNAs that gave 50% or greater viability of diploids relative to tetraploids, which was one criteria for candidate gene selection. (C) The result of the primary screen is displayed as the degree of selective lethality in tetraploid cells relative to diploid cells using TTEST metric (32). TTEST metric uses the difference of means scaled by the standard deviation and number of samples (see more in the Supplementary materials). Each dot represents a pool of siRNA, and the red arrow show the position of siRNA targeting VRK2. (D) Each of the selected 633 genes from the primary screen was re-tested by four individual siRNAs. The results were analyzed using RNAi gene enrichment ranking (RIGER) method (33) and are presented as a normalized enrichment score. (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.

[00019] Figure 7. 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 (β-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

[00020] Figure 8. VRK2 inhibition increases the frequency of cells with large pathological chromosome bridges, but not those with ultrafine bridges. (A) Schematic of the experiment. Images were acquired with a spinning disk confocal microscope.

The frequency of cells with PICH-positive ultrafine bridges in late anaphase (B) and LAP2- positive chromosome bridges in telophase cells (C). n>200 cells for each sample.

Representative images of cells with PICH-labeled ultrafine bridges (D) and LAP2-labeled large chromosome bridges (E) are shown. Scale bars, 5um.

[00021] Figure 9. Synthetic lethality between VRK2 inhibition and topoisomerase

II inhibition. (A) Schematic of the experiments to test synthetic lethality between topoisomerase II inhibitors and VRK2-inhibition. (B) Results of the experiment in "A" at varying concentrations of ICRF-159 or Doxorubicin. Viability was assayed with CellTiter Glo™ reagent at 14 days post VRK2-inhibition.

[00022] Figure 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).

[00023] Figure 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 μιη. (B) Fluorescence intensity measurements showing the oscillation of mCherry- Cdtl from (A).

[00024] Figure 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 γ-Η2ΑΧ, p53, and p21 fourteen days after infection with the indicated lentiviruses. (A) Representative images of cells. Scale bar, 20 μιη. (B) Quantification of the experiment from (A). The amount of DNA damage was examined by using an anti- γΗ2ΑΧ antibody. Cells were scored as y-H2AX-positive if they contained at least 3 large foci. The data are the average + s.d., n > 300. [00025] Figure 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.

[00026] 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.

[00027] Figure 15. VRK2 is highly expressed in colorectal adenoma and

adenocarcinoma. (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.

DETAILED DESCRIPTION OF THE INVENTION

[00028] 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. These gentetic insatbilities can be identified by morphologically by routine methods such as immunohistochemistry or by identifying specific genetic mutations for example by PCR.

[00029] Human cells contain three proteins in the VRK family: 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; and VRK3 is an enzymatically inactive pseudo- kinase. During normal cell division, VRKs prevent premature nuclear envelope assembly by phosphorylating an abundant (> 100,000 molecules per cell) small DNA binding protein called BAF (Barrier to Autointegration Factor). BAF binds nonspecifically to DNA along the rim of decondensing chromosomes. 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.

[00030] 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. In the absence of additional VRK activity, 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. BAF binding to newly replicated retroviral DNA prevents suicidal autointegration and therefore favors viral integration into the host genome. Altogether, outside of its role in normal cell division, 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.

[00031] These results indicate that genetically unstable cancer cells containing chromosome bridges, micronuclei and/or extra centrosomes can be selectively targeted by VRK inhibition (VRK1 or VRK2). As a consequence of VRK inhibition, BAF mistakes chromosome bridges, micronuclei as foreign DNA binds them preventing the cells form solving these problematic chromosome structures and eventually leads cell death.

Chromosome bridges and micronuclei are highly prevelant in cells containing extra centrosomes. [00032] The nucleic acid sequence of VRK1 (GenBank Accession No. NM_003384.2) is provided below:

ACTGCAGGGTGCGAAGGGGCCGGCGCCGCTGCCGAGTTACGAGTCGGCGAAAGCGGCGGGAAGTTCGTAC TGGGCAGAACGCGACGGGTCTGCGGCTTAGGTGAAAATGCCTCGTGTAAAAGCAGCTCAAGCTGGAAGAC AGAGCTCTGCAAAGAGACATCTTGCAGAACAATTTGCAGTTGGAGAGATAATAACTGACATGGCAAAAAA GGAATGGAAAGTAGGATTACCCATTGGCCAAGGAGGCTTTGGCTGTATATATCTTGCTGATATGAATTCT TCAGAGTCAGTTGGCAGTGATGCACCTTGTGTTGTAAAAGTGGAACCCAGTGACAATGGACCTCTTTTTA CTGAATTAAAGTTCTACCAACGAGCTGCAAAACCAGAGCAAATTCAGAAATGGATTCGTACCCGTAAGCT GAAGTACCTGGGTGTTCCTAAGTATTGGGGGTCTGGTCTACATGACAAAAATGGAAAAAGTTACAGGTTT ATGATAATGGATCGCTTTGGGAGTGACCTTCAGAAAATATATGAAGCAAATGCCAAAAGGTTTTCTCGGA AAACTGTCTTGCAGCTAAGCTTAAGAATTCTGGATATTCTGGAATATATTCACGAGCATGAGTATGTGCA TGGAGATATCAAGGCCTCAAATCTTCTTCTGAACTACAAGAATCCTGACCAGGTGTACTTGGTAGATTAT GGCCTTGCTTATCGGTACTGCCCAGAAGGAGTTCATAAAGAATACAAAGAAGACCCCAAAAGATGTCACG ATGGCACTATTGAATTCACGAGCATCGATGCACACAATGGCGTGGCCCCATCAAGACGTGGTGATTTGGA AATACTTGGTTATTGCATGATCCAATGGCTTACTGGCCATCTTCCTTGGGAGGATAATTTGAAAGATCCT AAATATGTTAGAGATTCCAAAATTAGATACAGAGAAAATATTGCAAGTTTGATGGACAAATGTTTTCCTG AGAAAAACAAACCAGGTGAAATTGCCAAATACATGGAAACAGTGAAATTACTAGACTACACTGAAAAACC TCTTTATGAAAATTTACGTGACATTCTTTTGCAAGGACTAAAAGCTATAGGAAGTAAGGATGATGGCAAA TTGGACCTCAGTGTTGTGGAGAATGGAGGTTTGAAAGCAAAAACAATAACAAAGAAGCGAAAGAAAGAAA TTGAAGAAAGCAAGGAACCTGGTGTTGAAGATACGGAATGGTCAAACACACAGACAGAGGAGGCCATACA GACCCGTTCAAGAACCAGAAAGAGAGTCCAGAAGTAATTCAGATGCTGTGAACCAGATTTCCTTTTCTTT GTTTTCTTTTGACTTTTTTCTCCTTTTCTATTTGAACTGTTTTATTTTCCTGTGAGTCTTGCGAGGTGGA AGTAATGATTAAATACTCATGTGTTCAGAAAACATAAACTTTTTTTATAAAAATATTTTGTACAATTCAT TAAAGGCTAATTTATGAAATTTGAAAATCTTCAGGTTATACTCCTTAAGTTATCCCAAAGCCGTGTGTTT GTGATGTTTTGGAGTACATATATATGAAAATTATTATGACACGCACTTTTCTAATCATTGTACATTTCTC AGAGTGGATAAAAATGTTTGACAAAGTCCTCACTTTTAAGGAAATGCAAAGCTTAAAATAAAACTCTCTT TTGTTTGATGCAAACACACAGTAAAAAAAAAAAAAAAAAA (SEQ ID NO: 8)

[00033] The amino acid sequence of VRK1 (GenBank Accession No. NP_003375.1) is provided below. 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.

MPRVKAAQAGRQSSAKRHLAEQFAVGE I ITDMAKKEWKVGLPIGQGGFGCIYLADMNSSESVGSDAPCVV KVEPSDNGPLFTELKFYQRAAKPEQIQKWIRTRKLKYLGVPKYWGSGLHDKNGKSYRFMIMDRFGSDLQK IYEANAKRFSRKTVLQLSLRILDILEYIHEHEYVHGDIKASNLLLNYKNPDQVYLVDYGLAYRYCPEGVH KEYKEDPKRCHDGTIEFTSIDAHNGVAPSRRGDLE ILGYCMIQWLTGHLPWEDNLKDPKYVRDSKIRYRE NIASLMDKCFPEKNKPGE IAKYMETVKLLDYTEKPLYENLRDILLQGLKAIGSKDDGKLDLSWENGGLK AKTITKKRKKEIEESKEPGVEDTEWSNTQTEEAIQTRSRTRKRVQK (SEQ ID NO: 9)

[00034] 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

(GenBank Accession No. NM_006296.6), VRK2 mRNA transcript 2 (GenBank Accession

No. NM_001130480.2), VRK2 mRNA transcript 3 (GenBank Accession No.

NM_001130481.2), VRK2 mRNA transcript 4 (GenBank Accession No.

NM_001130482.2), VRK2 mRNA transcript 5 (GenBank Accession No.

NM_001130483.2), VRK2 mRNA transcript 6 (GenBank Accession No.

NM_001288836.1), VRK2 mRNA transcript 7 (GenBank Accession No.

NM_001288839.1), VRK2 mRNA transcript 8 (GenBank Accession No. NM_001288837.1), and VRK2 mRNA transcript 9 (GenBank Accession No.

NM_001288838.1).

[00035] The nucleic acid sequence of the complete mRNA for VRK2 (GenBank

Accession No. AB000450.1) is provided below:

CTGCACTGCGAGGCCGACGCAGCTGGAGAGAAGTTAGGCAGGTCCTAGGGAGGGCAGGCTCGAGTGCTGG GCCCGCCTCCCCGCGGGACTGTAGGCCCGGGGGCTCCGCCTCGTCGCAGCGGCAGAAGTGATGCCACCAA AAAGAAATGAAAAATACAAACTTCCTATTCCATTTCCAGAAGGCAAGGTTCTGGATGATATGGAAGGCAA TCAGTGGGTACTGGGCAAGAAGATTGGCTCTGGAGGATTTGGATTGATATATTTAGCTTTCCCCACAAAT AAACCAGAGAAAGATGCAAGACATGTAGTAAAAGTGGAATATCAAGAAAATGGCCCGTTATTTTCAGAAC TTAAATTTTATCAGAGAGTTGCAAAAAAAGACTGTATCAAAAAGTGGATAGAACGCAAACAACTTGATTA TTTAGGAATTCCTCTGTTTTATGGATCTGGTCTGACTGAATTCAAGGGAAGAAGTTACAGATTTATGGTA ATGGAAAGACTAGGAATAGATTTACAGAAGATCTCAGGCCAGAATGGTACCTTTAAAAAGTCAACTGTCC TGCAATTAGGTATCCGAATGTTGGATGTACTGGAATATATACATGAAAATGAATATGTTCATGGTGATGT AAAAGCAGCAAATCTACTTTTGGGTTACAAAAATCCAGACCAGGTTTATCTTGCAGATTATGGACTTTCC TACAGATATTGTCCCAATGGGAACCACAAACAGTATCAGGAAAATCCTAGAAAAGGCCATAATGGGACAA TAGAGTTTACCAGTTTGGATGCCCACAAGGGAGTAGCCTTGTCCAGACGAAGTGACGTTGAGATCCTCGG CTACTGCATGCTGCGGTGGTTGTGTGGGAAACTTCCCTGGGAACAGAACCTGAAGGACCCTGTGGCTGTG CAGACTGCTAAAACAAATCTGTTGGACGAGCTCCCCCAGTCAGTGCTTAAATGGGCTCCTTCTGGAAGCA GTTGCTGTGAAATAGCCCAATTTTTGGTATGTGCTCATAGTTTAGCATATGATGAAAAGCCAAACTATCA AGCCCTCAAGAAAATTTTGAACCCTCATGGAATACCTTTAGGACCACTGGACTTTTCCACAAAAGGACAG AGTATAAATGTCCATACTCCAAACAGTCAAAAAGTTGATTCACAAAAGGCTGCAACAAAGCAAGTCAACA AGGCACACAATAGGTTAATCGAAAAAAAAGTCCACAGTGAGAGAAGCGCTGAGTCCTGTGCAACATGGAA AGTGCAGAAAGAGGAGAAACTGATTGGATTGATGAACAATGAAGCAGCTCAGGAAAGCACAAGGAGAAGA CAGAAATATCAAGAGTCTCAAGAACCTTTGAATGAAGTAAACAGTTTCCCACAAAAAATCAGCTATACAC AATTCCCAAACTCATTTTATGAGCCTCATCAAGATTTTACCAGTCCAGATATATTCAAGAAGTCAAGATC TCCATCTTGGTATAAATACACTTCCACAGTCAGCACGGGGATCACAGACTTAGAAAGTTCAACTGGACTT TGGCCTACAATTTCCCAGTTTACTCTTAGTGAAGAGACAAACGCAGATGTTTATTATTATCGCATCATCA TACCTGTCCTTTTGATGTTAGTATTTCTTGCTTTATTTTTTCTCTGAAGATGATACCAAAATTCCTTTTG ATAATTTTTTAAGTTTCCAGCTCTTCACCGAAATGTTGTATTCTTATTTCAGTGTTTCCTTCCAGACATT TTTAAGGTAATTGGCTTTAAAAAGAGAACATATTTTAACAAAGTTTGTGGACACTCTAAAAAATAAAATT GCTTTGTACTAGT (SEQ ID NO: 10)

[00036] The amino acid sequence of VRK1 (GenBank Accession No. BAA19109.1) is provided below. 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.

MPPKRNEKYKLPIPFPEGKVLDDMEGNQWVLGKKIGSGGFGLIYLAFPTNKPEKDARHWKVEYQENGPL FSELKFYQRVAKKDCIKKWIERKQLDYLGIPLFYGSGLTEFKGRSYRFMVMERLGIDLQKISGQNGTFKK STVLQLGIRMLDVLEYIHENEYVHGDVKAANLLLGYKNPDQVYLADYGLSYRYCPNGNHKQYQENPRKGH NGTIEFTSLDAHKGVALSRRSDVEILGYCMLRWLCGKLPWEQNLKDPVAVQTAKTNLLDELPQSVLKWAP SGSSCCEIAQFLVCAHSLAYDEKPNYQALKKILNPHGIPLGPLDFSTKGQSINVHTPNSQKVDSQKAATK QVNKAHNRLIEKKVHSERSAESCATWKVQKEEKLIGLMNNEAAQESTRRRQKYQESQEPLNEVNSFPQKI SYTQFPNSFYEPHQDFTSPDIFKKSRSPSWYKYTSTVSTGITDLESSTGLWPTISQFTLSEETNADVYYY RI I IPVLLMLVFLALFFL (SEQ ID NO: 11)

[00037] VRK Inhibitors

[00038] Accordingly, the invention provides methods of treating or alleviating a symptom of cancer by administering to a subject in need thereof a VRK inhibitor. [00039] A Vaccinia-related kinase (VRK) inhibitor is a compound that decreases expression or activity of VRK1 or VRK2. VRKs are ATP-dependent serine-threonine kinases that are widely expressed in human tissues and has increased expression in actively dividing cells.

[00040] 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. For example, 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. For example, the control is the activity of the VRK kinase before treatment or in a subject that has not received any treatment.

[00041] For example, the VRK inhibitor is a kinase inhibitor. Preferably, the kinase inhibitor is a serine/threonine kinase inhibitor.

[00042] 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. For example, 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.

[00043] 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).

[00044] 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,

carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.

[00045] The VRK inhibitor is an antibody or fragment thereof specific to VRK. For example, 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). Methods for designing and producing specific antibodies are well-known in the art.

[00046] Alternatively, the VRK inhibitor is for example an antisense VRK2 nucleic acid, a VRK-specific short-interfering RNA, or a VRK -specific ribozyme. By the term "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. Optionally, 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.

[00047] 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. Preferably, the

oligonucleotide is 19-25 nucleotides in length. Most preferably, the oligonucleotide is less than 75, 50, 25 nucleotides in length.

[00048] 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. Alternatively, 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 μΜ 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.

[00049] Therapeutic Methods

[00050] 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). By 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.

[00051] Cells are directly contacted with the compound. Alternatively, the compound is administered systemically. [00052] The tumor cell exhibits chromosomal instability (CIN). For example the tumor cell has chromosome bridges, and or micronuclei. Cells containing extra centrosomes are highly susceptible to chromosome bridges and micronuclei.

[00053] 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.

[00054] The methods described herein are useful to alleviate the symptoms of a variety of cancers. Any cancer exhibiting chromosomal instability such as chromosome bridges, micronuclei and/or extra centrosomes is amenable to treatment by the methods of the invention

[00055] 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. When treatment is applied prophylactically, "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.

[00056] Therapeutic Administration

[00057] The invention includes administering to a subject composition comprising a VRK inhibitor.

[00058] 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.

[00059] 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.

[00060] The pharmaceutical compound is administered to such an individual using methods known in the art. Preferably, 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. Examples of 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.

[00061] The therapeutic compounds described herein are formulated into compositions for other routes of administration utilizing conventional methods. For example, the therapeutic compounds are formulated in a capsule or a tablet for oral administration.

Capsules may contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets may be formulated in accordance with conventional procedures by compressing mixtures of a therapeutic compound with a solid carrier and a lubricant.

Examples of solid carriers 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. 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.

[00062] 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.

[00063] Additionally, 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. [00064] In some embodiments, it is preferred that 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. In some aspects, 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. Examples of anti-mitotic agents include taxanes (i.e., paclitaxel, docetaxel), and vinca alkaloids (i.e., vinblastine, vincristine, vindesine, vinorelbine).

[00065] Screening Assays

[00066] The invention also provides a method of screening for therapeutic targets for treating cancers. In particular, 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.

[00067] Definitions

[00068] As used herein, the term "null" refers to the presence, expression or activity status of a particular gene or genes. For example, 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. In some embodiments, 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.

[00069] The term "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. In one embodiment, 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).

[00070] As used interchangeably herein, the terms "oligonucleotides", "polynucleotides", and "nucleic acids" include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term "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. The term "nucleotide" is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Although the term "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.

[00071] The term "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.

[00072] As used herein, the term "substantial sequence identity" or "substantial homology" is used to indicate that a sequence exhibits substantial structural or functional equivalence with another sequence. Any structural or functional differences between sequences having substantial sequence identity or substantial homology will be de minimus; that is, they will not affect the ability of the sequence to function as indicated in the desired application. Differences may be due to inherent variations in codon usage among different species, for example. Structural differences are considered de minimus if there is a significant amount of sequence overlap or similarity between two or more different sequences or if the different sequences exhibit similar physical characteristics even if the sequences differ in length or structure. Such characteristics include, for example, the ability to hybridize under defined conditions, or in the case of proteins, immunological crossreactivity, similar enzymatic activity, etc. The skilled practitioner can readily determine each of these characteristics by art known methods.

[00073] Additionally, 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.

[00074] To determine the percent identity of two sequences, 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). In a preferred embodiment, 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. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein 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.

[00075] The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).

[00076] "Treatment" is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy. As used herein, "ameliorated" or "treatment" 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.

[00077] Thus, 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. "Suppressing" or "inhibiting", refers inter alia to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof. The symptoms are primary, while in another embodiment, symptoms are secondary. "Primary" refers to a symptom that is a direct result of the proliferative disorder, while, secondary refers to a symptom that is derived from or consequent to a primary cause. Symptoms may be any manifestation of a disease or pathological condition. [00078] 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 associated with the disorder.

[00079] As used herein, "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.

[00080] As used herein, 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.

[00081] As used herein, 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. By "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. The specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. [00082] As used herein, "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. Examples of 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.

[00083] 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.

[00084] The terms "patient" or "individual" are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the 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.

[00085] By the term "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.

[00086] As used herein, the term "administering to a cell" (e.g., an expression vector, nucleic acid, a delivery vehicle, agent, and the like) refers to transducing, transfecting, microinjecting, electroporating, or shooting, the cell with the molecule. In some aspects, 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). [00087] As used herein, "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. For example, multiple different types of 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.

EXAMPLES

[00088] EXAMPLE 1: GENERAL METHODS

[00089] The examples as described herein were performed using the reagents and methods generally described below.

[00090] Cell lines

[00091] The following cell lines were utilized: HCT116 FBXWT¹'^' and FBXWT^1' cells; HCT116 FBXWT'^' and FBXW7a'^~ cells; HCT116 STAG2-null cells; and 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.

[00092] Cell culture

[00093] 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. For experiments in 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. For live-cell imaging, C02-independent medium (Life technologies) supplemented with 10% FBS, Glutamate, and Penicillin/Streptomycin was filtrated with 0.45 μιη PVDF membrane and then used during the imaging. [00094] Antibodies, shRNA and siRNA

[00095] Antibodies used in this study were summarized in Tables 1 and 2.

[00096] Table 1. Antibodies for Western Blotting

[00097] Table 2. Antibodies for Immunofluorescence

[00098] 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.

[00099] Table 3. shRNA

[000100] Table 4. siRNA

[000101] Plasmids

[000102] 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 Gateway™ LR recombinase enzyme mix.

[000103] pLenti-CMV-Hyg-mRFP-LAP2 : cDNA for mRFP-LAP2 β (encoding rat LAP2 β 244-452 a.a. tagged with mRFP) was obtained by digesting pmRFP-LAP2 β - 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.

[000104] pMSCV-IRES-GFP-VRK2R: VRK2R (RNAi-resistant VRK2 cDNA) was partially synthesized as follows (silent mutations are capitalized): atgccaccaaaaagaaatgaaaaatacaaacttcctattccatttccagaGggGaaAgtCctAgaCgaCatggaaggcaatca gtgggtactgggcaagaagattggctctggaggatttggattgatatatttagctttccccacaaataaaccagagaaagatgcaaga catgtagtaaaagtggaatatcaagaaaatggcccgttattttcagaacttaaattttatcagagagttgcaaaaaaagactgtatcaaa aagtggatagaacgcaaacaacttgattatttaggaattcctctgttttaCggTAGCggCTtAacAgaGttTaaAggGCg

GTCAtaTCgCttCatggtaatggaaagactaggaatagatttacaAaaAatTAGCggTcaAaaCggtacctttaaaaa gtcaactgtcctAcaGCtCggAatTAgGatgCtAgaCgtCTtAgaGtaCatTcaCgaaaatgaatatgttcatggtgat gtaaaagcagcaaatctacttttgggttacaaaaatccagaccaggtttatcttgcagattaCggTctGAGTtaTCgCtaCtgC ccTaaCggTaaTcaTaaGcaAtaCcaAgaGaaCccAagaaaaggccataatgggacaatagagtttaccagcttggatg cccacaagggagtagccttgtccagacgaagtgacgttgagatcctcggctactgcatgc (SEQ ID NO: 7). This fragment was cloned into pDONR221 vector together with the other half of VRK2 cDNA.

The resultant vector was recombined with pMSCV-DEST-IRES-GFP (gift from Drs.

Akinori Yoda and David M. Weinstock, Dana-Farber Cancer Institute, Boston, MA, USA) using Gateway LR recombinase. VRK2R K168E expression vector was produced by the same method except that the synthesized VRK2R sequence carried alterations corresponding to K168E.

[000105] 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).

[000106] Production of tetraploid HCTl 16 clones

[000107] The induction of genome doubling was modified form (6). HCTl 16 cells were treated with 4 μΜ Dehydro-cytochalacin B for 20 hrs, and incubated with 5 μg/ml of Hoechst 33342 at 37°C for 30 min. Cells were then trypsinized and applied to flow cytometry (BD FACS Aria II SORP UV). The 8C peak containing G2/M population of newly generated tetraploid cells was gated for single cell sorting into 96-well plates. The rate of successful single cell proliferation was about 1%. The clones were expanded and examined for karyotypes by Giemsa staining.

[000108] Numerical and structural chromosome abnormalities

[000109] 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.

[000110] Chromosome missegregation rate (anaphase FISH)

[000111] Exponentially growing HCTl 16 diploid and tetraploid clones were analyzed by FISH for chromosome 6, 7 and 8 (Cytocell) as described previously (35). Cells either in anaphase or telophase were examined for chromosome missegregation. Diploid, n = 3107; tetraploid, n = 2594. Data represent missegregation per chromosome per division +/- s.e.m.

[000112] Centrosome number and the polarity of mitosis

[000113] Two diploid and four tetraploid clones were fixed in cold Methanol at -20°C and immunostained with anti-Centrin2 rabbit antibody (sc-27793-R, Santa Cruz) together with anti-a- Tubulin mouse antibody (DMla, Sigma-Aldrich) and Hoechst 33342 (5 μg/ml). Cells in anaphase were analyzed for the number of centrosomes as well as the polarity of the mitotic spindle.

[000114] Growth rate analysis

[000115] 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).

[000116] Tetraploidy-specific lethality screen

[000117] Genome- wide siRNA screen was performed at ICCB-L (Harvard Medical School). siGENOME siRNA libraries (50 nM final concentration, Dharmacon) targeting 21,176 genes in human whole genome were aliquoted to 384-well plates in sextuplicate using an automated liquid-handling robot (Velocity 11 Bravo) and mixted with Lipofectamine RNAiMAX (75 nL per well, Invitrogen) diluted in 9 of McCoy's 5 A medium. The triplicates of the plates were added with 1500 HCT116 diploid cells and another triplicates were added with 750 HCT116 tetraploid cells for reverse transfection in the final volume of 30 lL using WellMate (Matrix). Six hours after transfection, the cells were supplemented with 5 lL of medium containing Penicillin/Streptomycin, resulting in 35 μΙ_ 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. Each assay plate contained 20 non-targeting controls (siGLO Red transfection indicator, siGLO RISC-Free Control siRNA and siGENOME Non-Targeting siRNA, Dharmacon) and 6 positive controls for successful transfection (Plkl SMARTpool siRNA, Dharmacon).

[000118] Each pool of siRNAs was scored by the viability ratio between diploid and tetraploid as well as false discovery rate (FDR) of Benjamini-Hockberg method. Those with strong toxicity (viability of less than 0.3) and high variability (FDR of more than 0.1) were excluded from the list. TTEST metric from the GenePattern (32) was used to rank order the primary screen results:

where μ is the mean, n is the number of samples, and σ is the standard deviation.

[000119] As a secondary screen, 633 top-ranked pools of 4 siRNA were retested as individual siRNAs. These results were analyzed by RIGER method (33).

[000120] Production of lentiviral and retroviral particles

[000121] HEK 293FT cells (8 x 10⁶ in 10 cm dish) were seeded 24 hrs before transfection. The following amount of plasmids was mixed with 60 μΐ of Lipofectamine 2000 and added to the cells according to manufacturere' s protocol: 3μg of psPAX2, 3μg of pMD2.G, and 6 μg transfer vector for lentiviruses; 3μg of pCMV-VSV-G, 3μg of pMD.MLVopg, and 6μg of transfer vector for retroviruses. The supernatant containing viral particles was collected at 48 hrs and 72 hrs after transfection, filtered with 0.45 μιη PVDF membrane, and concentrated using PEG-it solution (System Biosciences). The viruses were stored at -80°C in multiple aliquots to avoid freezing and thawing.

[000122] Lentivirus and retrovirus infection

[000123] Following number of cells were seeded in a 12-well plate 24 hrs before viral infection: HCT116 diploid cells, 8xl0⁵; HCT116 tetraploid cells, 4xl0⁵ cells; HeLa-Kyoto cells and all the cells in Fig. 4D, 4xl0⁵ cells. Cells were added with lentiviral particles and 8 μg/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 μg/ml Puromycin for the elimination of non-infected cells.

[000124] Viability assay after shRNA-mediated VRK2 knockdown

[000125] In Fig. IF, 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 2xl0⁵ (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)

[000126] Rescue experiment and kinase dependency of VRK2 knockdown phenotype [000127] 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 xlO⁵ diploid cells and 2 xlO⁵ tetraploid cells) followed by lentivirus-mediated knockdown of endogenous VRK2. Cell viability was measured by using CellTiter Glo™ reagent (Promega) at 7 days post infection. Protein samples were prepared for western blotting to examine the expression level of VRK2.

[000128] Viability effect of BAF expression

[000129] GFP-tagged BAF WT and mutants were expressed through lentiviral delivery. The infected cells were cultured in the presence of 100 μg/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.

[000130] Immunofluorescence

[000131] Cells plated on cover glasses were fixed in 4% paraformaldehyde for 15 min on ice, washed with PBS twice, and permeabilized in 0.3% TritonX-100 in PBS for 2 min, followed by blocking in 1% BSA, 0.1% TritonX-lOO/PBS for 1 hr. Antibodies (listed in Table SX) diluted in 1% BSA, 0.1% TritonX-lOO/PBS were incubated for 1 hr, washed with PBS twice, followed by Alexa- labeled secondary antibodies together with 5 μg/ml Hoechst 33342 for 1 hr. Prolong gold Antifade Reagent (Invitrogen) was used for mounting. Images for most experiments were collected with a Yokogawa CSU-22 spinning disk confocal mounted on a Zeiss Axiovert microscope using 488, 561 and 640 nm laser light. A series of 0.5 μιη optical sections were acquired using a 40x NA 1.x or 63X NA 1.x Plan Apo objective with an Orca ER CCD camera (Hamamatsu Photonics).

[000132] Immunofluorescence for PICH⁺ or LAP2⁺ bridges

[000133] The knockdown of VRK2 and synchronization was performed as fig. S4A. Cells before the start of nuclear envelope re-formation was examined to quantify PICH-positive ultra-fine bridges, whereas cells after the onset of nuclear envelope re-formation and before chromatin decondensation were examined to quantify LAP2 -positive chromosome bridges. n>200 cells for each sample.

[000134] Quantification of GFP-BAF signal on chromosome bridges

[000135] 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 Ιμιη 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. To avoid photo-bleaching, laser output and exposure time was minimized to 5% and -100 ms, respectively. Signal intensities of GFP-BAF and mRFP-H2B were quantified by using line scan (Nikon NIS Elements Ar). The boundaries between nucleus and bridge were determined based on the mRFP-H2B peaks. After background subtraction, signal intensities were averaged for nucleus and bridges. Each data represent the average + s.e.m. of >18 cells.

[000136] Sensitivity to VRK2 knockdown in FBXW7 or STAG2 mutant cells

[000137] HCT116 cells of the indicated genotypes were plated in 24-well plates (4 xlO⁵ 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).

[000138] Induction of chromosome bridges in HeLa Kyoto cells

[000139] 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 xlO⁵ cells/dish). On the following day cells were treated with 100 ng/ml nocodazole and 1.5 μg/ml bleomycin (Bleocin, EMD Millipore) for 8 hrs. Cells were then gently washed three times and subjected to time-lapse imaging for 15 hrs with the interval of 1 μιη Z-step and 15 min using a spinning disk confocal microscope equipped with 60x objective (Plan-Apo VC DIC NA 1.4 oil, Nikon) at room temperature. The persistence of chromosome bridges was analyzed by using NIS elements Ar software; the cells that did not resolve bridges nor underwent binucleation by the end of experiments were excluded from the analysis.

[000140] Fate tracking of cells with or without chromosome bridges

[000141] HCT116 diploid, tetraploid cells or STAG2-null cells were stably expressed with

GFP-BAF-WT and mCherry-hCdtl(30/120) using lentiviral vectors. After introducing VRK2 shRNA, diploid and tetraploid cells were plated in 12- well glass bottom plate (MatTek) at 1 xlO⁵ and 5xl0⁴ cells/well, respectively. Twenty four hrs after plating, live- cell imaging was performed for 96 hrs using a 20x objective at 15 min and 2μιη Z-step interval as described previously (36). Determination of the fate was performed when the cell spent at least 40 hrs after mitosis.

[000142] Gene expression analysis of VRK2

[000143] Expression data of VRK2 in colon cancer samples and cell lines were retrieved from the Oncomine Database. Fold change and p value for most datasets were listed in the oncomine database. For datasets without fold change and p value listed, raw data were retrieved from NCBI GE02R website and analyzed with 2-sample equal variance t-test.

[000144] EXAMPLE 2: GENERATION OF TETRAPLOID CELLS

[000145] Starting with a chromosomally stable cancer cell line (HCT116), 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). The majority of the tetraploid cells contained extra centrosomes (75-83%), which were usually clustered during mitosis, enabling the assembly of pseudo-bipolar spindles (92-98%) (Fig. 5C-E). Despite the instability of the tetraploid lines, they maintained a near tetraploid chromosome content even after 70 days of continuous culture (Fig. 5B), presumably because of selection to maintain gene expression balance (8).

[000146] EXAMPLE 3: IDENTIFICATION OF GENES SELECTIVELY LETHAL TO

TETRAPLOID CELLS

[000147] An siRNA screen was used to identify gene knockdowns that were selectively lethal to the tetraploid cells (Fig. 6A-D). Validating the overall approach, the number one gene identified in the secondary screen was 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. Here 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. 2E-G and in Table 3). The selective lethality of VRK2 inhibition in tetraploid cells was suppressed by reexpression of an shRNA-resistant variant of VRK2 (VRK2R-WT) and this suppression was abrogated by a mutation that abolishes VRK2's kinase activity (K168E, Fig. 1H and I) (10).

[000148] Interestingly, 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. Indeed, 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).

[000149] EXAMPLE 3: CHARACTERIZATION OF VRK2-MEDIATED LETHALITY

[000150] The data presented herein suggests that the tetraploid-selective lethality of VRK2 inhibition is mediated by hyperactivation of BAF. Expression of non- phosphorylatable BAF (BAF-3A) produced tetraploid-selective lethality, whereas overexpression of wild-type BAF or a phosphomimetic variant had no effect (Fig. II and J).

[000151] These data indicated that hyperactive BAF recognizes cancer cell-specific aberrant DNA structures, such as micronuclei or chromosome bridges, in a similar way that it recognizes viral or other foreign DNA. Live-cell imaging in cells containing mRFP-H2B and GFP-BAF was used to confirm the mechanism. In control diploid or tetraploid cells, GFP-BAF exhibited the expected dynamic pattern of localization during mitosis. GFP-BAF was not detected on metaphase chromosomes, was subsequently recruited to the microtubule-proximal "core" region of anaphase chromosomes, and eventually concentrated homogeneously at the nuclear periphery during interphase (Fig. 2A, Control) (21). Like its transient accumulation on the core region of anaphase chromosomes, GFP-BAF also transiently accumulated on chromosome bridges. By contrast, after VRK2 knockdown, GFP-BAF massively and persistently accumulated on chromosome bridges (~ 18-fold enrichment) as well as on micronuclei derived from these bridges (Fig. 2A, VRK2-207). This was observed in all of 18 cells with the fold accumulation ranging from 81-fold to 7.4- fold. Although VRK2 inhibition affected the duration and degree of BAF accumulation, it did not obviously alter the initial time of its recruitment to chromosomes during anaphase.

[000152] Because BAF promotes the interaction of chromatin with NE envelope proteins, we considered the possibility that VRK2 inhibition might delay chromosome bridge resolution, thereby compromising cell viability. Indeed, VRK2 inhibition produced a striking increase in the numbers of large DNA-containing chromosome bridges in synchronized cells released from a Gl cell cycle block (Fig. 9C and E). A similar tetraploid- specific effect was observed in cells overexpressing nonphosphorylatable BAF-3A (Fig. 8B).

[000153] The primary effect of VRK2 inhibition occurs late in the cell division cycle when chromosome bridges and micronuclei form. First, VRK2 knockdown does not appear to induce DNA replication abnormalities because there was no detectable intra-S or G2 DNA damage checkpoint activation within four days of VRK2 inhibition (Fig. 10A and B). Second, VRK2 inhibition did not activate the spindle checkpoint, as evidenced by measurement of the interval between NE breakdown and anaphase onset. Finally, no increase in so-called ultrafine bridges after VRK2 knockdown was detected (Fig. 10B and D). Ultrafine bridges, which are not visualized by DNA dyes, are generated from late replicating regions of chromosomes and late separating catenenes at the centromere (22) (23).

[000154] Live cell imaging was utilized to determine the lifetime of chromosome bridges after VRK2 inhibition and to track the fate of VRK2-inhibited cells. To monitor chromosome bridges HeLa-Kyoto cells expressing mRFP-LAP2 were imaged (24). Cells were synchronized by release from a nocodazole block and concurrently treated with bleomycin to increase the frequency of chromosome bridges. Consistent with the fixed cell experiments, chromosome bridges persisted significantly longer in VRK2-inhibited cells (Fig. 3A and B, p < 0.01 by Mann-Whitney test). Moreover, this effect is likely to be underestimated because persistent chromosome bridges induce cytokinesis failure (Fig. 3A and B), which then obscures the presence of chromosome bridges due to the close proximity of the juxtaposed nuclei. VRK2 inhibition caused a 4.4-fold increase in the frequency of binucleated cells within 15 hr of release from nocodazole and bleomycin. Consistent with these findings, VRK2 inhibition and bleomycin treatment had a synergistic effect on the viability of cells (Fig. 3C). A similar synergistic effect was observed when VRK2 inhibition was combined topoisomerase II inhibitor treatment, which also induces chromosome bridges (Fig. 9B).

[000155] Next, it was determined that delayed chromosome bridge resolution after VRK2 inhibition lead to cell cycle arrest or cell death. Live cell imaging was utilized to follow chromosome bridge formation by GFP-BAF and the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) for the Gl phase, mCherry-Cdtl, was used to identify cells that were viable, but arrested in the Gl phase of the cell cycle (25). Imaging was performed in diploid and tetraploid HCT116 cells with or without VRK2 inhibition. Lethality or growth arrest was common after VRK2 inhibition (Fig. 3D). The most frequent outcome was cell cycle arrest in Gl in binucleated cells that underwent cytokinesis failure (Fig. 3E). Cells that arrested in Gl phase, cells that died in Gl, and cells that underwent endoreduplication, DNA replication without an intervening mitosis were observed through visualization with the FUCCI fluorescence reporter (26). The presence of chromosome bridges created a strong bias for these outcomes, with effects ranging from a 1.7-fold to 11 -fold increased frequency in bridge-containing cells. Almost all the tetraploid cells that underwent endoreduplication after VRK2 inhibition had a chromosome bridge formed in the previous mitosis (Fig. 3D, Fig. 11). Consistent with endoreduplication being error prone, cells with giant polyploidy nuclei accumulated after VRK2 inhibition and these displayed extensive DNA damage, p53 activation, and increased expression of the cyclin-dependent kinase inhibitor, p21 (Fig. 12). Together our data suggest that VRK2 inhibition produces lethality or growth arrest in cells containing chromosome bridges.

[000156] EXAMPLE 4: VRK2 CAUSES LETHALITY IN CANCER CELLS

[000157] Next, it was determined if VRK2 inhibition would cause lethality in cancer cells with mutations known to cause chromosome bridges and micronuclei. As a first test, 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). 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). Finally, 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. [000158] Live cell imaging demonstrated common mechanisms of cell death between STAG2 null cells and tetraploid cells after VRK2 inhibition. As previously described, in HCT116 cells, loss of STAG2 increased the frequency of chromosome bridges (29). After VRK2 inhibition, about half of the cells with chromosome bridges failed cytokinesis and arrested as binucleated cells (Fig. 4B). Moreover, expression of nonphosphorylatable BAF caused selective lethality in both STAG2-null and FBXW7-mi\\ cells (Fig. 4C).

[000159] Finally, we examined a panel of colon cancer cell lines to determine whether nuclear structure abnormalities- chromosome bridges, micronuclei, and multinucleation- would generally predict sensitivity to VRK2 inhibition. Two nontransformed colonic epithelial cell lines were resistant to VRK2 inhibition whereas there was marked variability in the sensitivity of 21 colon cancer cell lines (Fig. 4D, top). There was a striking correlation between the frequency of nuclear structure abnormalities and growth inhibition after VRK2 knockdown (Fig. 4D, bottom, Pearson's r = 0.84). Moreover, included in this collection were five FBW7 heterozygous mutant lines, which were all strongly sensitive to VRK2 inhibition (Fig. 4D red, and Fig. 14).

[000160] The data and results presented herein demonstrate that the VRK-BAF system can be manipulated such that cytoplasmic DNA structures in cancer cells heavily accumulate BAF, in a manner that resembles the BAF-mediated response to Vaccinia virus. One major consequence of BAF hyperactivation is the stabilization of chromosome bridges, with accompanying cytokinesis failure, p53 activation, and cell cycle arrest. BAF hyperactivation also promotes the generation of micronuclei derived from chromosome bridges (Fig. 2A) (30). The nuclear envelopes of micronuclei formed by other mechanisms are fragile and prone to spontaneous rupture (31). BAF also massively accumulates on cytoplasmic DNA from ruptured micronuclei. In the future, it will be interesting to determine the degree to which BAF accumulation, or the activation of other innate antiviral responses, might contribute to the efficacy of cancer treatments, such as radiation therapy or Taxol, that significantly induce chromosome bridges and micronuclei. References

. C. Lengauer, K. W. Kinzler, B. Vogelstein, Genetic instabilities in human cancers.

Nature 396, 643-649 (1998); published online EpubDec 17 (10.1038/25292).

. S. J. Pfau, A. Amon, in EMBO Rep. (2012), vol. 13, pp. 515-527.

. J. M. Schvartzman, R. Sotillo, R. Benezra, Mitotic chromosomal instability and cancer: mouse modelling of the human disease. Nature reviews. Cancer 10, 102-115 (2010); published online EpubFeb (10.1038/nrc2781).

. D. J. Gordon, B. Resio, D. Pellman, in Nature Reviews Genetics. (2012), vol. 13, pp.

189-203.

. Z. Storchova, A. Breneman, J. Cande, J. Dunn, K. Burbank, E. O'toole, D. Pellman, in Nature. (2006), vol. 443, pp. 541-547.

. T. Fujiwara, M. Bandi, M. Nitta, E. V. Ivanova, R. T. Branson, D. Pellman, in

Nature. (2005), vol. 437, pp. 1043-1047.

. T. I. Zack, S. E. Schumacher, S. L. Carter, A. D. Cherniack, G. Saksena, B. Tabak, M. S. Lawrence, C.-Z. Zhang, J. Wala, C. H. Mermel, C. Sougnez, S. B. Gabriel, B. Hernandez, H. Shen, P. W. Laird, G. Getz, M. Meyerson, R. Beroukhim, in Nat Genet. (2013), vol. 45, pp. 1134-1140.

. Y. C. Tang, A. Amon, Gene copy-number alterations: a cost-benefit analysis. Cell 152, 394-405 (2013); published online EpubJan 31 (10.1016/j.cell.2012.11.043).. M. Castedo, A. Coquelle, S. Vivet, I. Vitale, A. Kauffmann, P. Dessen, M. O.

Pequignot, N. Casares, A. Valent, S. Mouhamad, E. Schmitt, N. Modjtahedi, W. Vainchenker, L. Zitvogel, V. Lazar, C. Garrido, G. Kroemer, Apoptosis regulation in tetraploid cancer cells. EMBO J 25, 2584-2595 (2006); published online EpubJun 7 (10.1038/sj.emboj.7601127).

0. S. Blanco, L. Klimcakova, F. M. Vega, P. A. Lazo, The subcellular localization of vaccinia-related kinase-2 (VRK2) isoforms determines their different effect on p53 stability in tumour cell lines. FEBS J 273, 2487-2504 (2006); published online EpubJun.

1. E. Klerkx, P. Lazo, P. Askjaer, in Histology and histopathology. (2009), vol. 24, pp.

749.

2. A. I. Agoulnik, B. Lu, Q. Zhu, C. Truong, M. T. Ty, N. Arango, K. K. Chada, C. E.

Bishop, in Hum Mol Genet. (2002), vol. 11, pp. 3047-3053.

3. M. Gorjanacz, E. P. F. Klerkx, V. Galy, R. Santarella, C. Lopez-Iglesias, P. Askjaer, I. W. Mattaj, in EMBO J. (2007), vol. 26, pp. 132-143.

4. O. M. Lancaster, C. F. Cullen, H. Ohkura, in The Journal of Cell Biology. (2007), vol. 179, pp. 817-824.

5. D. Skoko, M. Li, Y. Huang, M. Mizuuchi, M. Cai, C. M. Bradley, P. J. Pease, B.

Xiao, J. F. Marko, R. Craigie, K. Mizuuchi, in Proc Natl Acad Sci USA. (2009), vol. 106, pp. 16610-16615.

6. T. Haraguchi, T. Koujin, M. Segura-Totten, K. K. Lee, Y. Matsuoka, Y. Yoneda, K.

L. Wilson, Y. Hiraoka, in Journal of Cell Science. (2001), vol. 114, pp. 4575-4585.7. L. Bengtsson, K. L. Wilson, in Mol Biol Cell. (2006), vol. 17, pp. 1154-1163.

8. R. Nichols, M. Wiebe, P. Traktman, in Molecular biology of the cell. (2006), vol.

17, pp. 2451.

9. M. S. Wiebe, P. Traktman, in Cell Host Microbe. (2007), vol. 1, pp. 187-197.

0. J. Nezu, A. Oku, M. Jones, M. Shimane, in Genomics. (1997), vol. 45, pp. 327-331.1. T. Haraguchi, T. Kojidani, T. Koujin, T. Shimi, H. Osakada, C. Mori, A.

Yamamoto, Y. Hiraoka, Live cell imaging and electron microscopy reveal dynamic processes of BAF-directed nuclear envelope assembly. J Cell Sci 121, 2540-2554 (2008); published online EpubAug 1 (jcs.033597 [pii]

/jcs.033597).

H. W. Mankouri, D. Huttner, I. D. Hickson, in The EMBO Journal. (2013), vol. 32, pp. 2661-2671.

L. H.-C. Wang, B. Mayer, O. Stemmann, E. A. Nigg, in Journal of cell science. (2010), vol. 123, pp. 806-813.

P. Steigemann, C. Wurzenberger, M. H. A. Schmitz, M. Held, J. Guizetti, S. Maar, D. W. Gerlich, in Cell. (2009), vol. 136, pp. 473-484.

A. Sakaue-Sawano, H. Kurokawa, T. Morimura, A. Hanyu, H. Hama, H. Osawa, S. Kashiwagi, K. Fukami, T. Miyata, H. Miyoshi, T. Imamura, M. Ogawa, H. Masai, A. Miyawaki, in Cell. (2008), vol. 132, pp. 487-498.

T. Davoli, E. L. Denchi, T. de Lange, in Cell. (2010), vol. 141, pp. 81-93.

J. E. Grim, M. P. Gustafson, R. K. Hirata, A. C. Hagar, J. Swanger, M. Welcker, H. C. Hwang, J. Ericsson, D. W. Russell, B. E. Clurman, in The Journal of cell biology. (2008), vol. 181, pp. 913-920.

S. Sur, R. Pagliarini, F. Bunz, C. Rago, L. A. Diaz, K. W. Kinzler, B. Vogelstein, N. Papadopoulos, in Proceedings of the National Academy of Sciences of the United States of America. (2009), vol. 106, pp. 3964-3969.

D. A. Solomon, T. Kim, L. A. Diaz-Martinez, J. Fair, A. G. Elkahloun, B. T. Harris, J. A. Toretsky, S. A. Rosenberg, N. Shukla, M. Ladanyi, Y. Samuels, C. D. James, H. Yu, J.-S. Kim, T. Waldman, in Science. (2011), vol. 333, pp. 1039-1043.

D. R. Hoffelder, L. Luo, N. A. Burke, S. C. Watkins, S. M. Gollin, W. S. Saunders, in Chromosoma. (2004), vol. 112, pp. 389-397.

E. M. Hatch, A. H. Fischer, T. J. Deerinck, M. W. Hetzer, in Cell. (2013), vol. 154, pp. 47-60.

M. Reich, T. Liefeld, J. Gould, J. Lerner, P. Tamayo, J. P. Mesirov, in Nature Genetics. (2006), vol. 38, pp. 500-501.

B. Luo, H. W. Cheung, A. Subramanian, T. Sharifnia, M. Okamoto, X. Yang, G. Hinkle, J. S. Boehm, R. Beroukhim, B. A. Weir, C. Mermel, D. A. Barbie, T. Awad, X. Zhou, T. Nguyen, B. Piqani, C. Li, T. R. Golub, M. Meyerson, N. Hacohen, W. C. Hahn, E. S. Lander, D. M. Sabatini, D. E. Root, Highly parallel identification of essential genes in cancer cells. Proc Natl Acad Sci U S A 105, 20380-20385 (2008); published online EpubDec 23 (10.1073/pnas.0810485105).

S. Finkin, Y. Aylon, S. Anzi, M. Oren, E. Shaulian, in Oncogene. (2008), vol. 27, pp. 4411-4421.

N. J. Ganem, S. A. Godinho, D. Pellman, A mechanism linking extra centrosomes to chromosomal instability. Nature 460, 278-282 (2009); published online EpubJul 9 N. J. Ganem, S. A. Godinho, D. Pellman, in Nature. (2009), vol. 460, pp. 278-282.

Claims

We Claim:

1. A method of treating a cancer in a subject comprising administering to said subject a compound that inhibits the expression or activity of Vaccina-reiated kinase (VRK).

2. The method of claim 1, wherein the VRK is VRK1 or VRK2.

3. The method of claim 1, wherein the cancer is a STAG2 or FBXW7 mutant cancer.

4. The method of claim any one of the preceding claims, wherein the cell has a FBXW7 mutation a BRCA1 mutation, a BRCA2 mutation or a Rb mutation.

5. The method of claim 1, wherein said cancer is a leukemia, a lymphoma, a

melanoma, a carcinoma or a sarcoma.

6. The method of claim any one of the preceding claims, wherein the compound is a nucleic acid, an antibody or a small molecule.

7. The method of any one of the preceding claims claims, further comprising

administering a chemotherapeutic agent.

8. The method of claim 7, wherein the chemotherapeutic agent is a serine-threonine kinase inhibitor.

9. A method of screening for therapeutic targets for treating cancer comprising:

a. providing a cell having a chromosome bridge, micronuclei and/or extra centrosomes;

b. contacting the cell with a library of RNAi or small molecule; and c. identifying an RNAi or small molecule which is lethal to said cell; thereby identifying a therapeutic target for treating cancer.

10. A method of treating cancer comprising administering to said subject a compound that inhibits the expression of activity of the therapeutic target identified in claim 9.

11. Use of a composition that inhibits the expression or activity of Vaccina -related kinase (VRK) for treating a cancer in a subject.

12. The use of claim 11, wherein the VRK is VRK1 or VRK2.

13. The use of any one of claims 11 or 12, wherein the cancer is a STAG2 or FBXW7 mutant cancer.

14. The use of any one of claims 11-13, wherein the cell has a FBXW7 mutation a

BRCA1 mutation, a BRCA2 mutation or a Rb mutation.

15. The use of any one of claims 11-12, wherein said cancer is a leukemia, a lymphoma, a melanoma, a carcinoma or a sarcoma.

16. The use of claim any one of claims 11-15, wherein the compound is a nucleic acid, an antibody or a small molecule.

17. The use of any one of claims 11-16, further comprising administering a

chemotherapeutic agent.

18. The use of claim 18, wherein the chemotherapeutic agent is a serine-threonine

kinase inhibitor.