Anti-cancer drugs based on synthetic lethal interactions with TP53 or MLH1 mutations

Abstract

Methods of treating a cancer comprising a mutation in tumor protein p53 (TP53) or DNA mismatch repair protein Mlhl (MLH1) comprising: determining the cancer comprises the mutation and administering an agent that decreases abundance or function of a protein synthetically lethal with TP53 or MLH1 are provided. Agents that decrease abundance or function of a protein synthetical lethal with TP53 or MLH1 for use in treating a cancer comprising a mutation in TP53 or MLH1 are also provided.

Description

DEVELOPMENT OF ANTI-CANCER DRUGS BASED ON SYNTHETIC LETHAL INTERACTIONS UTILIZING HAPLOID HUMAN EMBRYONIC STEM CELLS (HHESCS)REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (HUJI-P-0136-PCT.xml; Size: 7,125 bytes; and Date of Creation: August 22, 2025) is herein incorporated by reference in its entirety.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 687,368, filed on August 27, 2024, the contents of which are all incorporated herein by reference in their entirety.FIELD OF INVENTION

[0003] The present invention is in the field of cancer therapeutics.BACKGROUND OF THE INVENTION

[0004] Haploid human embryonic stem cells (HhESCs) such as those produced in US patent 10,961,503, incorporated herein by reference in its entirety, are pluripotent and have a normal genome. However, unlike common hESCs that have two sets of chromosomes, or cancer cell lines that in most cases have several duplications of the chromosomes and numerous aberrations, HhESCs have a clean genetic background and only one set of chromosomes. These characteristics turn them into the perfect platform for genome-wide genetic screening.

[0005] TP53 is the most frequently mutated gene in cancer. MLH1 is a cancer-related gene that has a central role in DNA mismatch repair (MMR) and is the cause of MMR system defects in most tumors with MMR deficiency. MMR deficiency leads to high microsatellite instability (MSI-H) and to the accumulation of mutations across the genome. Deficiency inthe MMR pathway is common in colorectal cancer (-15%), which is the fourth most common cancer and the second most common cause of cancer-related deaths, as well as additional types of gastrointestinal cancer, uterus cancer and can also be found in additional cancer such as breast, prostate, bladder, and thyroid.

[0006] Synthetic lethality is defined as a type of genetic interaction where the combination of two genetic events results in cell death, whereas each of them separately does not. Drugs that can produce synthetic lethality in combination with MLH1 and TP53 mutations already present in various cancers are greatly needed.SUMMARY OF THE INVENTION

[0007] The present invention provides methods of treating a cancer comprising a mutation in tumor protein p53 (TP53) or DNA mismatch repair protein Mlhl (MLH1) comprising: determining the cancer comprises the mutation and administering an agent that decreases abundance or function of a protein synthetically lethal with TP53 or MLH1 are provided. Agents that decrease abundance or function of a protein synthetical lethal with TP53 or MLH1 for use in treating a cancer comprising a mutation in TP53 or MLH1 are also provided.

[0008] According to a first aspect, there is provided a method of treating a cancer in a subj ect in need thereof, wherein the cancer comprises a mutation in tumor protein p53 (TP53) or DNA mismatch repair protein Mlhl (MLH1), the method comprising: a. determining the cancer comprises a mutation in TP53 and administering to the subject an agent that decreases abundance or function of a protein synthetically lethal with TP53, wherein the protein synthetically lethal with TP53 is myosin heavy chain 10 (MYH10); or b. determining the cancer comprises a mutation in MLH1 and administering to the subject an agent that decreases abundance or function of a protein synthetically lethal with MLH1, wherein the protein synthetically lethal with MLH1 is selected from exonuclease 1 (EXO1), nuclear receptor subfamily 5, group A, member 2 (NR5A2) and Serine / threonine-protein kinase PLK2 (PLK2);thereby treating a cancer in a subject in need thereof.

[0009] According to some embodiments, the method, further comprises receiving a sample from the subject comprising cancer cells and measuring expression and / or function of MLH1 or TP53 in the cancer cells, wherein cancer cells with decreased expression and / or function as compared to control cells are cells with a mutation in MLH1 or TP53.

[0010] According to some embodiments, the control cells are selected from cancer cells without a mutation in MLH1 or TP53 or non-cancerous cells from the same tissue or cell type as the cancer cells.[Oi l] According to some embodiments, the method, further comprises selecting a subject that suffers from a cancer comprising a mutation in MLH1 or TP53.

[0012] According to some embodiments, a mutation is a loss of function (LOF) mutation.

[0013] According to some embodiments, the cancer with a mutation does not express MLH1 or TP53 and wherein the determining comprises determining the cancer does not express MLH1 or TP53.

[0014] According to some embodiments, express is express RNA of MLH1 or TP53 or express protein of MLH1 or TP53.

[0015] According to some embodiments, the agent is a nucleic acid molecule that hybridizes to an mRNA of the protein synthetically lethal with MLH1 or TP53 and inhibits expression of the protein synthetically lethal with MLH1 or TP53.

[0016] According to some embodiments, the agent is a genome editing complex that binds to a genomic region encoding the protein synthetically lethal with MLH1 or TP53 and modifies the genomic region to inhibit expression of the protein synthetically lethal with MLH1 or TP53.

[0017] According to some embodiments, the agent is a small molecule direct inhibitor of the protein synthetically lethal with MLH1 or TP53.

[0018] According to some embodiments, the cancer comprises a mutation in MLH1 and the small molecule direct inhibitor is selected from SRI 848 and derivatives thereof that inhibit NR5A2, ON1231320 and derivatives thereof that inhibit PLK2, and BI2536 and derivatives thereof that inhibit PLK2.

[0019] According to some embodiments, the cancer comprises a mutation in TP53 and the small molecule direct inhibitor is selected from Blebbistatin, para-amino-blebbistatin and derivative thereof that inhibit MYH10.

[0020] According to another aspect, there is provided an agent that decreases abundance or function of myosin heavy chain 10 (MYH10), for use in treating a cancer comprises a mutation in tumor protein p53 (TP53) in a subject in need thereof.

[0021] According to another aspect, there is provided an agent that decreases abundance or function of a protein synthetically lethal with MLH1, for use in treating a cancer comprises a mutation in DNA mismatch repair protein Mlhl (MLH1) in a subject in need thereof, wherein the protein synthetically lethal with MLH1 is selected from exonuclease 1 (EXO1), nuclear receptor subfamily 5, group A, member 2 (NR5A2) and Serine / threonine-protein kinase PLK2 (PLK2).

[0022] According to some embodiments, the cancer has been determined to comprise the mutation.

[0023] According to some embodiments, the cancer was determined to comprise the mutation by receiving a sample from the subject comprising cancer cells and measuring expression and / or function of MLH1 or TP53 in the cancer cells, wherein cancer cells with decreased expression and / or function as compared to control cells are cells with a mutation in MLHl or TP53.

[0024] According to some embodiments, the control cells are selected from cancer cells without a mutation in MLH1 or TP53 or non-cancerous cells from the same tissue or cell type as the cancer cells.

[0025] According to some embodiments, a mutation is a loss of function (LOF) mutation.

[0026] According to some embodiments, the cancer with a mutation does not express MLH1 or TP53.

[0027] According to some embodiments, express is express RNA of MLH1 or TP53 or express protein of MLH1 or TP53.

[0028] According to some embodiments, the agent is a nucleic acid molecule that hybridizes to a MLH1 or TP53 mRNA and inhibits expression of MLH1 or TP53.

[0029] According to some embodiments, the agent is a genome editing complex that binds to a MLH1 or TP53 genomic region and modifies the genomic region to inhibit MLH1 or TP53 expression.

[0030] According to some embodiments, the agent is a small molecule direct inhibitor of MLH1 or TP53.

[0031] According to some embodiments, the cancer comprises a mutation in TP53 and the small molecule direct inhibitor is selected from Blebbistatin, para-amino-blebbistatin and derivative thereof that inhibit MYH10.

[0032] According to some embodiments, the cancer comprises a mutation in MLH1 and the small molecule direct inhibitor is selected from SRI 848 and derivatives thereof that inhibit NR5A2, ON1231320 and derivatives thereof that inhibit PLK2, and BI2536 and derivatives thereof that inhibit PLK2.

[0033] According to another aspect, there is provided a method of treating a cancer in a subject in need thereof, wherein the cancer comprises a mutation in DNA mismatch repair protein Mlhl (MLH1) or tumor protein p53 (TP53), the method comprising: a. determining the cancer comprises a mutation in TP53 and administering to the subject an agent that decreases abundance or function of a protein synthetically lethal with TP53, wherein the protein synthetically lethal with TP53 is selected from the proteins provided in Table 4; or b. determining the cancer comprises a mutation in MLH1 and administering to the subject an agent that decreases abundance or function of a protein synthetically lethal with MLH1, wherein the protein synthetically lethal with MLH1 is selected from the proteins provided in Table 2; thereby treating a cancer in a subject in need thereof.

[0034] According to some embodiments, the protein synthetically lethal with MLH1 is selected from PTP4A1, QPCTL, LDB1, CBX3, EXO1, TTYH3, LMAN1, COL5A2, NR5A2, E2F3, MAP1LC3A, EXTL2, PLK2, SMPD2, PC, ATF5, ST3GAL4, and TYK2.

[0035] According to some embodiments, the protein synthetically lethal with TP53 is selected from FOXI3, SNAP23, TMED10, ELAVL1, TGFBRAP1, MYH10, SLC2A6,MANF, KMT2A, SENP6, MDH1, MAEA, VPS37A, 0R1I1, PRKAB2, CTPS2, PLBD1, and OR2B3.

[0036] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figures 1A-1F: Establishing and validating haploid models for synthetic lethality genome-wide screening. (1A) Schematic illustration of the experiment. (1B-1C) Sanger sequencing results of (IB) MLH1-K0 and (1C) TP53-KO demonstrating one nucleotide (IB) insertion or (1C) deletion that causes a frameshift. PAM sequence is highlighted in grey and mutation loci in red rectangles. (ID) The similarity between the SBS44 signature - associated with defective DNA mismatch repair (upper panel) to the MLH1-K0 (middle panel) and WT clones (lower panel). The colors describe the different types of base changes. The x-axis is all possible single-base-substitutions (SBS), and the y- axis is the frequency of each substitution in the different groups. (IE) Relative contribution of SBS44-like signature to WT or MLH1-K0 cells. Number of WT or MLH1-K0 clones that were analyzed utilizing whole-genome sequencing is n=3 and n=8, respectively. (IF) Gene set enrichment analysis (GSEA) was performed on the RNAseq results comparing TP53-KO cells to WT cells demonstrating enrichment of TP53 related pathways.

[0038] Figures 2A-2F: Genome-wide and custom-made genetic screening for synthetic lethality interaction identification. (2A) Volcano plot displaying the CRISPR score (CS = log2 fold change (MLH1-K0 vs. WT)) and adjusted p-values for all the nuclear genes in the genome-wide screen. Blue dots = depleted genes that were selected for further analysis. Red dots = three most promising candidate genes. Green dots = three genes previously suggested as synthetic lethal partners for mismatch repair (MMR) deficiency. (2B) STRING analysis results indicate the connection between MMR genes (grey) and the genes highlighted in red / green on the volcano plot (2A). (2C) Boxplot of NR5A2 (left), EXO1 (middle), and PLK2 (right) sgRNAs distribution in the different samples = WT (blue) / MLH1-K0 (red),on the last time point. Read counts were normalized by each sample (read count per sgRNA / total counts in a sample), and then the Z-score was calculated for each sgRNA across all samples. (2D) i: Volcano plot displaying the CRISPR score (CS = log2 fold change (TP53-KO vs. WT)) and adjusted p-values for all the genes in the genome-wide screen. Blue dots = significantly depleted genes that were selected for further analysis. Light red dots = genes related to signaling by Rho GTPases. Red dot = MYH10. ii: Gene set enrichment analysis (GSEA) was performed on the significantly depleted genes that were highlighted in 2D-i. (2E) Volcano plot displaying the CRISPR score (CS = log2 fold change (TP53-KO vs. WT)) and adjusted p-values for the genes in the costume screen. (2F) Boxplot of MYH10 sgRNAs distribution in the different samples - WT (blue) / TP53-KO (red), and time points (x-axis). Read counts were normalized by each sample (read count per sgRNA / total counts in a sample) and then the Z-score was calculated for each sgRNA across all samples.

[0039] Figures 3A-3D: Genetic and chemical validation of synthetic lethality candidate genes in haploid hESCs MLH1 deficient model. (3A) Haploid hESC MLH1-K0 cells were mixed with WT-GFP cells in a 50:50 ratio. The mixed cells were infected with empty CAS9 lentivector (blue), and EXO1 sgRNA CAS9 lentivector (red). Control uninfected cells (dark blue). Changes in WT vs. MLH1-K0 dynamics were monitored at various time points using FACS. The percentages of the MLH1-K0 cells are represented. (3B) A mixture of WT-GFP and MLH1-K0 cells, in a 50:50 ratio, was cultured both with (red lines) and without (blue lines) NR5A2 -inhibitor - SR1848 (left), PLK2 -inhibitor 1 - ON1231320 (middle), and PLK2 inhibitor 2 -= BI2536 (right). The dynamic of MLH1 mutant cell abundance in the culture was monitored over time using FACS and normalized to the initial time point. Without treatment, MLH1-K0 cells gradually dominate the culture, while their abundance significantly decreases under treatment. (3C) Haploid hESCs with (MLH1-K0) and without (WT) mutation in the MLH1 gene were treated with BI2536. Images depicting treated and untreated (DMSO) cells are displayed on the left, with quantitative analysis of viable cell count provided on the right (n=3, p-value < 0.05). (3D) Annexin-V-PI staining, utilized for apoptosis detection, is depicted in the FACS density-contour plot (left) of WT / MLH1-K0 cells treated with BI2536. Quantitative analysis of apoptotic cells provided on the right (n=3, p-value < 0.05, T-test).

[0040] Figures 4A-4D: Chemical validation of synthetic lethality candidate genes in MLH1 deficient cancer models. (4A) Cancer cell lines (CCLs) with (LS411N, LoVo, SW48) and without (Caco2) MMR deficiency were treated with SR1848 (top) ON1231320(middle), and BI2536 (bottom). Representative images of treated and untreated (DMSO) CCLs are displayed (left), with quantitative analysis (right) (n=3, *p-value < 0.05). (4B) Dose-response curves of DNAMMR deficient (red) and proficient (blue) CCLs treated with SR1848 (top) ON1231320 (middle) and BI2536 (bottom). Dots represent the mean of technical triplicates. Error bars represent the standard error of the mean (SEM) of technical triplicates. (4C) Bioinformatic analysis of CCLs to treatment with BI2536 - box plot and density plot of MMR-proficient CCLs (blue), and MMR-deficient CCLs (red). The left panel is the sensitivity data obtained from the Broad Repurposing Library and the PRISM multiplexed cell-line viability assay. The y-axis is log2 of the ratio of the abundance of cells in the treatment group vs. cells in the control DMSO group. The right panel is the sensitivity data obtained from the CTD2 database. * : p-value < 0.05, ** : p-value < 0.005. (4D) Upper panel: Schematic illustration of the experiment - 13 NOD-SCID I12rg- / - mice were subcutaneously injected with Caco2 (MMR proficient) and SW48 (MMR deficient) cells. 12 days after xenotransplantation, mice were subjected to a 6-day treatment with BI2536 (red, N=4 for each cell line) or vehicle (blue, N=3 for Caco2 and N=2 for SW48). Tumor mass was measured following the treatment and are illustrated in box plots. p-value= 0.45 for Caco2 (N.S) and 0.06 for SW48, KS test.

[0041] Figures 5A-5F: Genetic and chemical validation of the synthetic lethality candidate gene MYH10, in haploid hESCs TP53 deficient model. (5A) Comparison of LoF mutation distribution at different time points following the introduction of mutations to the MYH10 gene in both haploid WT (blue) and TP53-KO (red) cells. N=138 different LoF mutations. The Y-axis is the averaged normalized values according to the first time point (tp), that is, the first tp is equal to 1, and the other tps are the fold-change that is relative to tp 1. *** p < 0.0001. (5B) Visual depiction of the MYH10 amino acid sequence surrounding the CRISPR-Cas9 cut site (red arrow). The original amino acid sequence is presented at the top (WT), while variations in the sequence for non -frameshift mutations (middle, n=955) and frameshift mutations (bottom, n=l 551). (5C) A mixture of WT-GFP and TP53-KO cells, in a 1 :99 ratio, was cultured both with (red lines) and without (blue lines) para-amino- blebbistatin treatment. The progression of TP53 mutant cell abundance in the culture was continuously monitored over time using FACS and normalized to the initial time point. Without treatment, TP53-KO cells gradually dominate the culture, while their abundance significantly decreases with the application of treatment. (5D) Haploid hESCs with (TP53- KO) and without (WT) mutation in the TP53 gene were treated with para-amino-blebbistatinfor 4 days. Images depicting treated and untreated (DMSO) cells are displayed on the left, with quantitative analysis of viable cell count provided on the right (n=3, *p-value < 0.01, T-test). (5E) Principal Component Analysis (PCA) was performed on RNAseq results, comparing WT and TP53-KO haploid hESCs treated or untreated with para-amino- blebbistatin for 4 days. (5F) Heat map displaying False Discovery Rate (FDR) values of Gene Set Enrichment Analysis (GSEA), comparing wild-type (WT) and TP53-KO haploid hESCs treated or untreated with para-amino-blebbistatin for 4 days. Genes with increased expression were labelled as "enriched," while those with decreased expression were labelled as "depleted." The various comparison groups are specified beneath the heat map.

[0042] Figures 6A-6E: Genetic and chemical validation of the synthetic lethality candidate gene MYH10, in TP53 deficient cancer models. (6A) Dose-response curves of TP53-LoF (red) and TP53-WT (blue) CCLs treated with blebbistatin for 3-6 days. Dots represent the mean of technical triplicates. Error bars represent the SEM of triplicates. (6B) LoVo and HT29 cell lines, originally without TP53-LoF mutation, underwent TP53 knockout using CRISPR-Cas9 technology to generate isogenic cell lines. Dose-response curves of TP53-KO (red) and TP53-WT (blue) CCLs treated with blebbistatin for 3-6 days are depicted. The dots represent the mean of technical triplicates, with error bars indicating SEM for triplicates. (6C) CCLs with (LS41 IN) and without (LoVo) TP53-LoF were treated for 4 days with para-amino-blebbistatin. Left panel - representative images of treated and untreated (DMSO) CCLs. Right panel - cell count quantitative analysis (n=3, *p-value < 0.01, T.test). (6D) PCA was performed on RNAseq results to compare WT (LoVo) and TP53-LoF (LS411N) CCLs treated or untreated with para-amino-blebbistatin for 4 days. (6E) Left panel: Schematic illustration of the experiment - NOD-SCID I12rg- / - mice were subcutaneously injected with HCT116 (TP53-WT, n=8), LoVo (TP53-WT, n=8) and LS411N (TP53-LoF, n=8) CCLs. 8 days after xenotransplantation, mice were subjected to an 11 -day treatment with blebbistatin (red) or vehicle (blue). N=4 for all different conditions. Tumor analysis at the endpoint was conducted, and their masses are illustrated in box plots at the right. p-value= 0.63 for HCT116 , 0.71 for LoVo and 0.1 for LS411N, KS test.

[0043] Figures 7A-7D: Establishing and validating haploid models for synethetic lethality genome-wide screening. (7 A) Western blot analysis demonstrates loss of MLH1 protein. (7B) Western blot analysis demonstrates loss of p53 protein. (7C) The similarity between the Indel 2 (ID2) signature (upper panel) to the MLH1-K0 (middle panel) and WT cells (lower panel). The colors describe the length of the insertion / deletion, and the x-axis isa subclassification that describes the repeats of each insertion / deletion. The y-axis describes the frequency of each change. (7D) Relative contribution of ID2-like signature to WT or MLH1-K0 cells. Number of clones that were analyzed utilizing whole-genome sequencing is n=3 and n=8, respectively.

[0044] Figures 8A-8B: Genome-wide and custom-made genetic screening for synthetic lethality interaction identification. (8A) Volcano plot displaying the CRISPR score (CS = log2 fold change (MLH1-K0 last time point (tp) vs. WT last tp)) and adjusted p-values for DNA repair genes in the genome-wide MLH1-K0 screen. Blue dots = EXO1, POLB, and WRN. (8B) The most significant genes in the genome-wide TP53-KO screen, were analyzed for protein interactions STRING resource. Genes’ nodes color in STRING. The colored nodes categorize genes based on the pathways analyzed in gene set enrichment analysis (Fig. 2D-ii).

[0045] Figures 9A-9B: Chemical validation of synthetic lethality candidate genes in haploid hESCs MLH1 deficient model. (9 A) Annexin-V-PI staining, utilized for apoptosis detection, is depicted in the FACS density-contour plot (left) of WT / MLH1-K0 cells treated with SRI 848. quantitative analysis of apoptotic cells provided on the right (n=3, *p-value < 0.05, T-test). (9B) Images depicting SR1848 treated WT or MLH1-K0 cells.

[0046] Figure 10: Bioinformatic validation of synthetic lethality candidate genes in MLH1 deficient CCL models. Bioinformatic analysis of cancer cell lines (CCLs) response to 5-FU (left) or oxaliplatin (right) treatment is presented through box plots and density plots. MMR-proficient CCLs are represented in blue, while MMR-deficient CCLs are in red. The top panel displays sensitivity data obtained from the Broad Repurposing Library and the PRISM multiplexed cell-line viability assay. The y-axis indicates the log2 ratio of the abundance of cells in the treatment group versus cells in the control DMSO group. The bottom panel reflects sensitivity data from the CTD2 database. All comparisons yielded nonsignificant results (p-value > 0.1).

[0047] Figures 11A-11C: Genetic validation of the synthetic lethality candidate gene MYH10, in haploid hESCs TP53 deficient model. (11 A) Location of all mutations in MYH10 in the genetic validation. On the X-axis, we denote the mutation locations relative to the PAM sequence, where zero signifies the position of the CRISPR-Cas9 cut site, also indicated by the arrow. The Y-axis is the cumulative counts of all mutations that occurred in a specific location across all samples, for indels longer than 1, the location was defined asthe one nearest to the cut site locus. (11B) Top 5 LoF mutations in MYH10 in the genetic validation. Presented mutations were selected based on summed frequency across all samples, and their differences were compared in the trends over time between the WT and TP53-KO. The Y-axis is the normalized values according to the first tp, that is, the first tp is equal to 1, and the other tps are the fold-change that is relative to tp 1. (11C) Results of pathway enrichment analysis of WT and TP53-KO haploid hESCs treated or untreated with para-amino-blebbistatin. Significant pathways enriched or depleted in TP53-KO cells.

[0048] Figures 12A-12D: Genetic and chemical validation of the synthetic lethality candidate gene MYH10, in TP53 deficient CCLs. (12A) Effect of nutlin-3 on cancer cell lines (CCLs). HT29 and LoVo are colorectal CCLs without TP53 LoF mutation. Total number of cells was compared between treated (dark blue) and untreated (light blue) cells. X axis = sample, Y axis = total cell number. (12B) Effect of blebbistatin on colorectal CCLs is presented via area under the curve (AUC) comparison. CCLs with TP53 LoF (red) and CLLs without TP53 LoF (Blue) were treated. X axis = CCLs, Y axis = AUC values. (12C- D) Effect of nutlin-3 and blebbistatin on lung CCLs. (12C) NCH4460 and (12D) Calu-6 are lung CCLs originally without TP53 LoF mutation are sensitive to nutlin-3 treatment (light blue vs. Dark blue). The CCLs underwent TP53 knockout using CRISPR-Cas9 technology to generate isogenic cell lines. Dose-response curves of TP53-KO (red) and TP53-WT (blue) CCLs treated with blebbistatin for 3-6 days are depicted. The dots represent the mean of technical triplicates, with error bars indicating the standard error of the mean (SEM) for technical triplicates. X axis = concentration (uM), Y axis = cell viability. * = p-value < 0.05.DETAILED DESCRIPTION OF THE INVENTION

[0049] The present invention, in some embodiments, provides methods of treating a cancer comprising a mutation in tumor protein p53 (TP53) or DNA mismatch repair protein Mlhl (MLH1) comprising: determining the cancer comprises the mutation and administering an agent that decreases abundance or function of a protein synthetically lethal with TP53 or MLH1 are provided. Agents that decrease abundance or function of a protein synthetical lethal with TP53 or MLH1 for use in treating a cancer comprising a mutation in TP53 or MLH1 are also provided.

[0050] By a first aspect, there is provided a method of treating a cancer in a subject, the method comprising administering to a subject with a cancer comprising a mutation in tumorprotein p53 (TP53) an agent that inhibits a protein that is synthetically lethal with TP53; thereby treating a cancer in a subject.

[0051] By another aspect, there is provided a method of treating a cancer in a subject, the method comprising administering to a subject with a cancer comprising a mutation in DNA mismatch repair protein Mlhl (MLH1) an agent that inhibits a protein that is synthetically lethal with MLH1; thereby treating a cancer in a subject.

[0052] By another aspect, there is provided an agent that inhibits a protein that is synthetically lethal with TP53 for use in treating a cancer comprising a mutation in TP53 in a subject.

[0053] By another aspect, there is provided an agent that inhibits a protein that is synthetically lethal with MLH1 for use in treating a cancer comprising a mutation in MLH1 in a subject.

[0054] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is in need of a method of the invention. In some embodiments, the subject suffers from cancer. In some embodiments, the subject comprises a cancer. In some embodiments, the method is a therapeutic method. In some embodiments, the method is a diagnostic method. In some embodiments, the method is a diagnostic and therapeutic method.

[0055] As used herein "cancer" is a disease associated with abnormal or out of control cell proliferation. Non-limiting types of cancer include carcinoma, sarcoma, lymphoma, leukemia, blastoma and germ cells tumors.

[0056] In some embodiments, the cancer is solid cancer. In some embodiments, the cancer is a tumor. In some embodiments, the solid cancer is selected from hepato-biliary cancer, cervical cancer, urogenital cancer (e.g., urothelial cancer), testicular cancer, prostate cancer, thyroid cancer, ovarian cancer, nervous system cancer, ocular cancer, lung cancer, soft tissue cancer, bone cancer, pancreatic cancer, bladder cancer, skin cancer, intestinal cancer, hepatic cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, breast cancer (e.g., triple negative breast cancer), renal cancer (e.g., renal carcinoma), skin cancer, and head and neck cancer. In some embodiments, the cancer is a hematopoietic cancer. In some embodiments, a hematopoietic cancer is selected from leukemia and lymphoma. In some embodiments, a hematopoietic cancer is a hematological cancer.

[0057] In some embodiments, the cancer is a cancer comprising a TP53 mutation. In some embodiments, the cancer is a TP53-mutant cancer. TP53 is the most frequently mutated gene in human cancers and cancers with TP53 mutation can be found in virtually every type of cancer, from every tissue. TP53 mutations are very common in breast cancers, ovarian cancers, lung cancers, colorectal cancers, brain cancers, head and neck cancers, sarcomas and hematological cancers, though for at least 20 tumor types it has been shown that more than 50% of cancers bear a TP53 mutation. Additional tumor types that commonly bear TP53 mutations include but are not limited to esophageal cancer, gastric cancer, liver cancer, adrenocortical cancer, skin cancer, bladder cancer, pancreatic cancer and cervical cancer. In some embodiments, the TP53 mutant cancer is selected from breast cancers, ovarian cancers, lung cancers, colorectal cancers, brain cancers, head and neck cancers, sarcomas, hematological cancers, esophageal cancer, gastric cancer, liver cancer, adrenocortical cancer, skin cancer, bladder cancer, pancreatic cancer and cervical cancer bearing a TP53 mutation.

[0058] TP53 is also known as p53, cellular tumor antigen p53 and TRP53. The human TP53 gene can be found at Entrez gene ID number 7157 and the human TP53 protein can be found in UniProt ID P04637. Various human mRNA isoforms are provided in RefSeqs NM_001276761, NM_000546, NM_001126112, NM_001126113, and NM_001126114. Various human protein isoforms are provided in RefSeqs NP_000537, NP_001119584, NP_001119585, NP_001119586 and NP_001119587.

[0059] In some embodiments, the cancer is a cancer comprising a MLH1 mutation. In some embodiments, the cancer is a MLH1 -mutant cancer. MLH1 mutations have been reported in a wide variety of cancers including but not limited to colorectal cancers, endometrial cancers, ovarian cancers, gastric cancers, intestinal cancers, bladder cancers, kidney cancers, pancreatic cancers, biliary tract cancers and brain cancers. In some embodiments, the MLH1 mutant cancer is selected from colorectal cancer, endometrial cancer, ovarian cancer, gastric cancer, intestinal cancer, bladder cancer, kidney cancer, pancreatic cancer, biliary tract cancer and brain cancer bearing a MLH1 mutation.

[0060] MLH1 is also known as MutL protein homolog 1. The human MLH1 gene can be found at Entrez gene ID number 4292 and the human MLH1 protein can be found in UniProt ID P40692. Various human mRNA isoforms are provided in RefSeqs NM_000249, NM_001167617, NM_001167618, NM_001167619, and NM_001258271. Various humanprotein isoforms are provided in RefSeqs NP_000240, NP_001161089, NP_001162090, NP_001161091 and NP_001245200.

[0061] In some embodiments, the cancer is confirmed to have a mutation in TP53. In some embodiments, the method further comprises determining the cancer comprises a mutation in TP53. In some embodiments, the cancer is confirmed to have a mutation in MLH1. In some embodiments, the method further comprises determining the cancer comprises a mutation in MLH1. In some embodiments, determining comprises measuring expression of TP53. In some embodiments, determining comprises measuring expression of MLH1. In some embodiments, expression is protein expression in some embodiments, expression is RNA expression. In some embodiments, RNA is messenger RNA (mRNA). Methods of measuring protein and RNA expression are well known in the art, as are specific methods of measuring expression in cancer samples. These methods include, but are not limited to genome sequencing, whole genome analysis, Next-generation sequencing, whole exome analysis, PCR, qPCR, real-time PCR, western blotting, immunostaining, microarray analysis, proteinarray analysis, and FACS analysis.

[0062] In some embodiments, the method further comprises receiving a sample comprising cells from the cancer. In some embodiments, the method further comprises extracting from the subject a sample comprising cells from the cancer. In some embodiments, the determining is the received cells. In some embodiments, the sample is a tumor sample. In some embodiments, the sample is a biopsy. In some embodiments, the biopsy is a liquid biopsy. In some embodiments, the sample comprises cancer cell. In some embodiments, the cancer cell are circulating tumor cells.

[0063] In some embodiments, a mutation is a loss of function (LOF) mutation. In some embodiments, a mutation results in reduced expression of MLH1. In some embodiments, a mutation results in reduced expression of TP53. In some embodiments, reduced expression is no expression. In some embodiments, no expression is loss of expression. In some embodiments, a cancer with a mutation in MLH1 does not express MLH1. In some embodiments, a cancer with a mutation in TP53 does not express TP53. In some embodiments, determining is determining loss of expression. In some embodiments, determining is determining reduced expression. In some embodiments, determining is determining the cancer does not express MLH1. In some embodiments, determining is determining the cancer does not express TP53. In some embodiments, determining is determining the cancer cell has decreased TP53 expression as compared to control cells. Insome embodiments, determining is determining the cancer cell has decreased MLH1 expression as compared to control cells. In some embodiments, a cancer cell with decreased TP53 expression as compared to control cells is a cell with a mutation in TP53. In some embodiments, a cancer cell with decreased MLH1 expression as compared to control cells is a cell with a mutation in MLH1. In some embodiments, control cells are cancer cells without a mutation in MLH1. In some embodiments, control cells are cancer cells without a mutation in TP53. In some embodiments, control cells are from the same tissue or cell type as the cancer cells. In some embodiments, the control cells are non-cancerous cells from the same tissue or cell type as the cancer cells.

[0064] In some embodiments, the mutation is a frame shift mutation. In some embodiments, the mutation is a deletion. In some embodiments, the mutation is an insertion. In some embodiments, the mutation is a point mutation. In some embodiments, the mutation is a splice site mutation. In some embodiments, the mutation is a stop codon mutation. In some embodiments, the mutation generates a premature stop codon.

[0065] In some embodiments, the mutation is a mutation that alters protein function of TP53. In some embodiments, the mutation is a mutation that alters protein function of MLH1. In some embodiments, alters is reduces. In some embodiments, alters is abolishes. In some embodiments, alters is inhibits. In some embodiments, TP53 function is tumor suppressor function. In some embodiments, MLH1 function is DNA mismatch repair function. In some embodiments, the mutation is in an active site of TP53. In some embodiments, the mutation in an active site of MLH1. In some embodiments, determining is determining the cancer cell has decreased TP53 function as compared to control cells. In some embodiments, determining is determining the cancer cell has decreased MLH1 function as compared to control cells. In some embodiments, a cancer cell with decreased TP53 function as compared to control cells is a cell with a mutation in TP53. In some embodiments, a cancer cell with decreased MLH1 function as compared to control cells is a cell with a mutation in MLH1.

[0066] In some embodiments, a mutation is determined by genomic sequencing of a cancer cell. In some embodiments, a mutation is determined by absence of mRNA or protein expression of TP53. In some embodiments, a mutation is determined by absence of mRNA or protein expression of MLH1. It will be understood that genomic sequencing and thus determination of the actual mutation is not required. Rather, detecting the absence of TP53 / MLH1 expression is sufficient to indicate that a mutation is present and thus to determine that the subject is suitable to be treated with the agent.

[0067] In some embodiments, the method further comprises selecting a subject that suffers from a cancer comprising a mutation in TP53. In some embodiments, the method further comprises selecting a subject that suffers from a cancer comprising a mutation in MLH1. In some embodiments, the agent is for use in a subject selected based on a mutation in TP53. In some embodiments, the agent is for use in a subject selected based on a mutation in MLH1.

[0068] In some embodiments, the agent inhibits a gene that is synthetically lethal with TP53. In some embodiments, the agent inhibits a gene that is synthetically lethal with MLH1. It will be understood that by inhibiting the gene, the protein produced by the gene is also inhibited. In some embodiments, inhibits is decreases expression of. In some embodiments, inhibits is decreases abundance of a protein. In some embodiments, inhibits is decreases function of. In some embodiments, the agent is an inhibitor of the protein. In some embodiments, an inhibitor is a small molecule inhibitor. In some embodiments, the inhibitor is a direct inhibitor. In some embodiments, a direct inhibitor directly binds to the protein that is inhibited. As used herein, a “small molecule inhibitor” is a drug that is smaller than 900 Daltons or sometimes even smaller than 500 Daltons. Thus, these molecules are smaller than proteins and yet interact with proteins and produce inhibition. In some embodiments, the agent is a nucleic acid molecule that hybridizes to the gene and inhibits transcription of the gene. In some embodiments, the agent is a nucleic acid molecule that hybridizes to an mRNA of the gene and inhibits translation of the mRNA or degrades the mRNA. In some embodiments, the agent is an antisense oligonucleotide (ASO) against the gene. In some embodiments, the agent is a small interfering RNA (siRNA) against the gene. In some embodiments, the agent is a small hairpin RNA (shRNA) against the gene. In some embodiments, the agent is a genome editing complex that binds to a genomic region of the gene and modifies the genomic region to inhibit expression.

[0069] In some embodiments, the agent is a vector that encodes the nucleic acid molecule. In some embodiments, the agent is a composition comprising the nucleic acid molecule of the vector. In some embodiments, the vector is an expression vector. In some embodiments, the expression vector comprises a promoter operably linked to the nucleic acid molecule. In some embodiments, the promoter is active in the cancer cells. In some embodiments, the promoter is active in the tissue or cell type of the cancer. In some embodiments, the promoter is a constitutive promoter. It will be understood that since inhibiting the gene alone is not lethal and is only lethal in combination with silencing of TP53 / MLH1 it is not hazardous toexpress the agent in non-cancerous cells. These cells will have intact TP53 / MLH1 and so the agent will not have an effect. Thus, a benefit of the agent of the invention is that it is lethal only in the cancer cells and not in other cells, thereby reducing off-target effects.

[0070] In some embodiments, the cancer comprises a mutation in TP53 and the synthetically lethal gene is myosin heavy chain 10 (MYH10). MYH10 is also known as non-muscle myosin IIB or NM-IIB. The human MYH10 gene can be found at Entrez gene ID number 4628 and the human MYH10 protein can be found in UniProt ID P35580. Various human mRNA isoforms are provided in RefSeqs NM_001256012, NM_001256095, NM_005964, and NM 001375266. Various human protein isoforms are provided in RefSeqs NP_001242941, NP_001243024, NP_005955, and NP_001362195.

[0071] In some embodiments, the agent is a nucleic acid molecule that hybridizes to a MYH10 mRNA. In some embodiments, the nucleic acid molecule degrades the MYH10 mRNA. In some embodiments, the nucleic acid molecule inhibits translation of the MYH10 mRNA. In some embodiments, the agent is a genome editing complex that binds to a genomic region of the MYH10 gene. In some embodiments, the genome editing complex modifies the MYH10 gene. In some embodiments, the modifying inhibits expression of MYH10.

[0072] Genome editing complexes are well known in the art and any such complex that would allow for modification of the genomic locus of the synthetic lethal gene may be used. In some embodiments, the genome editing complex is CRISPR-CAS. In some embodiments, the CAS is CAS9. In some embodiments, the complex comprises a guide RNA (gRNA). In some embodiments, the gRNA hybridizes to the MYH10 genomic region. In some embodiments, the genome editing complex is selected from CRISPR, meganuclease, Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). In some embodiments, the genome editing complex is used to generate a mutation in the synthetically lethal gene. In some embodiments, the mutation is an LOF mutation.

[0073] In some embodiments, the agent is a small molecule inhibitor of MYH10. In some embodiments, the inhibitor is a direct inhibitor of MYH10. In some embodiments, the inhibitor binds to MYH10. In some embodiments, the MYH10 inhibitor is blebbistatin. In some embodiments, the MYH10 inhibitor is a derivative of blebbistatin. In some embodiments, a derivative is a derivative that inhibits MYH10. In some embodiments, the MYH10 inhibitor is para-amino-blebbistatin. Other examples of MYH10 small moleculeinhibitors include, but are not limited to MT -228 and MT-110 and derivatives thereof (disclosed in Radnai et al., “Development of clinically viable non-muscle myosin II small molecule inhibitors”, Cell, 2025 Jun 27:S0092-8674(25)00640-3, the contents of which are hereby incorporated by reference in their entirety).

[0074] In some embodiments, the cancer comprises a mutation in TP53 and the synthetically lethal protein / gene is selected from those provided in Table 4. In some embodiments, the cancer comprises a mutation in TP53 and the synthetically lethal protein / gene is selected from those provided in Table 5. In some embodiments, the cancer comprises a mutation in TP53 and the synthetically lethal protein / gene is selected from Angiomotin-like protein 2 (AMOTL2), kallikrein 5 (KLK5), diphthamide biosynthesis 2 (DPH2), spartin (SPART / SPG20), CCR4-NOT transcription complex subunit 9 (CNOT9 / RQCD1), TCF3 fusion partner (TFPT), forkhead box 13 (FOXI3), synaptosome associated protein 23 (SNAP23), transmembrane p24 trafficking protein 10 (TMED10), zinc finger protein 689 (ZNF689), casein kinase 2 alpha 2 (CSNK2A2), ZNF608, vesicle-trafficking protein SEC22a (SEC22A), ELAV-like protein 1 (ELAVL1), Transforming Growth Factor-beta Receptor-Associated Protein 1 (TGFBRAP1), MYH10, Trans-Golgi Network Vesicle Protein 23 Homolog B (TVP23B), transmembrane protein 106C (TMEM106C), ArfGAP With SH3 Domain, Ankyrin Repeat And PH Domain 1 (ASAP1), G protein-coupled receptor 158 (GPR158), Tubulin Beta 2A Class Ila (TUBB2A), PAX-interacting protein 1 (PAXIP1), Primase And DNA Directed Polymerase (PRIMPOL), Solute Carrier Family 2 Member 6 (SLC2A6), DPH1, zinc finger C3HC-type containing 1 (ZC3HC1), Erythroid membrane-associated protein (ERMAP), Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF), Kin of IRRE-like protein 1 (KIRREL / KIRREL1), TUBB2B, ZNF211, ZNF326, BAG Cochaperone 6 (B AG6), olfactory receptor 6A2 (OR6A2), Ubiquitin Binding Protein 2-Like (UBAP2L), Histone-lysine N-methyltransferase 2A (KMT2A), SUMO Specific Peptidase 6 (SENP6), GRBIO-Interacting GYF Protein 1 (GIGYF1), ZNF510, ZNF710, Fat mass and obesity-associated gene (FTO), SRSF Protein Kinase 2 (SRPK2), Brain Expressed X-Linked 5 (BEX5), Spectrin beta chain, brain 1 (SPTBN1), Apoptosis- associated speck-like protein containing a CARD (PYCARD), calcium / calmodulin- dependent protein kinase type II delta (CAMK2D), NAD-dependent methylenetetrahydrofolate dehydrogenase 2-like protein (MTHFD2L), Caspase 2 (CASP2), retinoblastoma protein (RBI), Lectin, Mannose Binding 1 (LMAN1), Cerebellar Degeneration-Related Protein 2 (CDR2), Histone H2A type 1-J (HIST1H2AJ),Phosphoinositide-3 -kinase-interacting protein 1 (PIK3IP1), transcription factor E2F3 (E2F3), Ribonucleoprotein, PTB Binding 2 (RAVER2), Dentin matrix acidic phosphoprotein 1 (DMP1), SH2 Domain Containing 6 (SH2D6), Ring Finger Protein 181 (RNF181), LLGL Scribble Cell Polarity Complex Component 1 (LLGL1), eukaryotic translation initiation factor 4E type 2 (EIF4E2), WD repeat domain 11 protein (WDR11), E2F Transcription Factor 7 (E2F7), (BNIPL) Bel -2 Interacting Protein Like, (ANKFY1) Ankyrin Repeat and FYVE Domain Containing 1, MYH9, (SCAF8) SCAF8 Scaffold Protein, (MLLT6) Myeloid / Lymphoid or Mixed-Lineage Leukemia Translocation Gene 6, (PCNP) Polycomb Neoplastic Transforming Gene, (MDH1) Malate Dehydrogenase 1, (YWHAH) Tyrosine 3-Monooxygenase / tryptophan 5 -Monooxygenase Activation Protein, Theta, (GLT6D1) Glucosaminyltransferase 6 Domain Containing 1, (MAEA) Myc- Associated Zinc Finger Protein, (PDCD10) Programmed Cell Death 10, (SAV1) Salvador Family WW Domain Containing 1, (RECQL) RecQ Like Helicase, (PPP ICC) Protein Phosphatase 1 Catalytic Subunit Gamma, (TBL2) TBL2, Transducer of ERBB2, (VPS37A) Vacuolar Protein Sorting 37A, (OR1I1) Olfactory Receptor Family 1 Subfamily I MemberI, ZNF704, (ZDHHC24) Zinc Finger DHHC-Type Palmitoyltransferase 24, (ROCK2) Rho Associated Coiled-Coil Containing Protein Kinase 2, (RBPJ) Recombining Binding ProteinJ, (AAGAB) Alpha- Actinin- Associated Protein, (CAV1) Caveolin 1, (CNTN2) Contactin 2, (TTC5) Tetratri copeptide Repeat Domain 5, (ADGRG6) Adhesion G Protein-Coupled Receptor G6, (DCHS1) Dachsous Cadherin-Related 1, (PRKAB2) Protein Kinase AMP- Activated Beta 2 Subunit, (M0RN2) MORN Domain Containing 2, (TRIM45) Tripartite Motif Containing 45, (SMIM24) Small Integral Membrane Protein 24, (MAPK7) Mitogen- Activated Protein Kinase 7, (CSTF2T) Cleavage Stimulation Factor 2, Tail Subunit, (CDV3) carnitine deficiency-associated gene expressed in ventricle 3, (F13A1) Coagulation Factor XIII A Chain, (SSR2) Signal Sequence Receptor 2, (F AMI 10C) Family With Sequence Similarity 110 Member C, (FLOT2) Flotillin 2, (TAOK1) TAO Kinase 1, (CTPS2) CTP Synthase 2, (ZBTB49) Zinc Finger and BTB Domain Containing 49, (MAVS) Mitochondrial Antiviral Signaling Protein, (STMN3) Stathmin 3, (PLBD1) Phospholipase B Domain Containing 1, (MOG) Myelin Oligodendrocyte Glycoprotein, (GPNMB) Glycoprotein Nmb, (OR2B3) Olfactory Receptor Family 2 Subfamily B Member 3, (KCTD17) Potassium Channel Tetramerization Domain Containing 17, (DNMT3A) DNA Methyltransferase 3 Alpha, ZNF764, (MCM4) Minichromosome Maintenance Complex Component 4, (CDKN2A) Cyclin-Dependent Kinase Inhibitor 2A, (TTC9C) Tetratri copeptide Repeat Domain 9C, (TSSK2) Testis-Specific Serine Kinase 2, (TTC28)Tetratri copeptide Repeat Domain 28, (WBSCR17) Williams Beuren Syndrome Chromosome Region 17, (KANSL1L) KAT8 Enhancer RNA Subunit 1-Like, (OR4M2) Olfactory Receptor Family 4 Subfamily M Member 2, ZNF615, (IRX1) Iroquois Homeobox 1, (DDR1) Discoidin Domain Receptor Tyrosine Kinase 1, (TIAM2) T-lymphoma Invasion and Metastasis, (AFF3) AF4 / FMR2 Family Member 3, (STS) Steroid Sulfatase, (IQGAP1) IQ Motif Containing GTPase Activating Protein 1, (C17orfl00) Chromosome 17 Open Reading Frame 100, (RANBP6) Ran Binding Protein 6, (MAP4K4) Mitogen- Activated Protein Kinase Kinase Kinase Kinase 4, (SBF2) Sucrose Nonfermenting 2, and (HPCAL4) Hippocalcin-Like 4. In some embodiments, the cancer comprises a mutation in TP53 and the synthetically lethal protein / gene is selected from DPH2, SPG20, TFPT, FOXI3, SNAP23, TMED10, SEC22A, ELAVL1, TGFBRAP1, MYH10, TMEM106C, ASAP1, SLC2A6, MANF, ZNF326, UBAP2L, KMT2A, SENP6, GIGYF1, RAVER2, RNF181, LLGL1, WDR11, BNIPL, MLLT6, MDH1, MAEA, TBL2, VPS37A, OR1I1, PRKAB2, TRIM45, CSTF2T, CTPS2, ZBTB49, PLBD1, OR2B3, TTC9C, TTC28, TAOK1, and CNTN2. In some embodiments, the cancer comprises a mutation in TP53 and the synthetically lethal protein / gene is selected from FOXI3, SNAP23, TMED10, ELAVL1, TGFBRAP1, MYH10, SLC2A6, MANF, KMT2A, SENP6, MDH1, MAEA, VPS37A, OR1I1, PRKAB2, CTPS2, PLBD1, and OR2B3.

[0075] In some embodiments, the cancer comprises a mutation in MLH1 and the synthetically lethal gene is exonuclease 1 (EXO1). EXO1 is also known as HEX1. The human EXO1 gene can be found at Entrez gene ID number 9156 and the human EXO1 protein can be found in UniProt ID Q9UQ84 or Q5T397. Various human mRNA isoforms are provided in RefSeqs NM_003686, NM_006027, NMJ30398, and NM_001319224. Various human protein isoforms are provided in RefSeqs NP_001306153, NP_003677, NP_006018, and NP_569082.

[0076] In some embodiments, the agent is a nucleic acid molecule that hybridizes to an EXO1 mRNA. In some embodiments, the nucleic acid molecule degrades the EXO1 mRNA. In some embodiments, the nucleic acid molecule inhibits translation of the EXO1 mRNA. In some embodiments, the agent is a genome editing complex that binds to a genomic region of the EXO1 gene. In some embodiments, the genome editing complex modifies the EXO1 gene. In some embodiments, the modifying inhibits expression of EXO1. In some embodiments, the genome editing complex is a CRISPR complex comprising a gRNA that hybridizes to the EXO1 genomic region.

[0077] In some embodiments, the agent is a small molecule inhibitor of EXO1. In some embodiments, the inhibitor is a direct inhibitor of EXO 1. In some embodiments, the inhibitor binds to EXO1. In some embodiments, the EXO1 inhibitor is EXO1-IN-1 (also known as Compound F684). In some embodiments, the EXO1 inhibitor is a derivative of EXO1-IN-1. In some embodiments, a derivative is a derivative that inhibits EXO1.

[0078] In some embodiments, the cancer comprises a mutation in MLH1 and the synthetically lethal gene is nuclear receptor subfamily 5, group A, member 2 (NR5A2). NR5A2 is also known as liver receptor homolog- 1, LRH-1, totipotency pioneer factor NR5A2 among other names. The human NR5A2 gene can be found at Entrez gene ID number 2494 and the human NR5A2 protein can be found in UniProt ID 000482. Various human mRNA isoforms are provided in RefSeqs NM_001276464, NM_003822, and NM_205860. Various human protein isoforms are provided in RefSeqs NP_001263393, NP_003813, and NP_995582.

[0079] In some embodiments, the agent is a nucleic acid molecule that hybridizes to a NR5A2 mRNA. In some embodiments, the nucleic acid molecule degrades the NR5A2 mRNA. In some embodiments, the nucleic acid molecule inhibits translation of the NR5A2 mRNA. In some embodiments, the agent is a genome editing complex that binds to a genomic region of the NR5A2 gene. In some embodiments, the genome editing complex modifies the NR5A2 gene. In some embodiments, the modifying inhibits expression of NR5A2. In some embodiments, the genome editing complex is a CRISPR complex comprising a gRNA that hybridizes to the NR5A2 genomic region.

[0080] In some embodiments, the agent is a small molecule inhibitor of NR5A2. In some embodiments, the inhibitor is a direct inhibitor of NR5A2. In some embodiments, the inhibitor binds to NR5A2. In some embodiments, the NR5A2 inhibitor is SRI 848. In some embodiments, the NR5A2 inhibitor is a derivative of SRI 848. SRI 848 is a well-known NR5A2 inhibitor that is commercially available. Its full chemical name is 6-[4-(3- Chlorophenyl)-l-piperazinyl]-3-cyclohexyl-2,4(lH,3H)-pyrimidinedione and it is also known as ML180 and CDI 3238389. Its CAS number is 863588-32-3. In some embodiments, a derivative is a derivative that inhibits NR5A2. Other examples of NR5A2 inhibitors include, but are not limited to Cpd3 and GSK2033.

[0081] In some embodiments, the cancer comprises a mutation in MLH1 and the synthetically lethal gene is Serine / threonine-protein kinase PLK2 (PLK2). PLK2 is alsoknown as SNK and polo like kinase 2. The human PLK2 gene can be found at Entrez gene ID number 10769 and the human PLK2 protein can be found in UniProt ID Q9NYY3. Various human mRNA isoforms are provided in RefSeqs NM 006622, and NM_001252226. Various human protein isoforms are provided in RefSeqs NP_001239155, and NP_006613.

[0082] In some embodiments, the agent is a nucleic acid molecule that hybridizes to a PLK2 mRNA. In some embodiments, the nucleic acid molecule degrades the PLK2 mRNA. In some embodiments, the nucleic acid molecule inhibits translation of the PLK2 mRNA. In some embodiments, the agent is a genome editing complex that binds to a genomic region of the PLK2 gene. In some embodiments, the genome editing complex modifies the PLK2 gene. In some embodiments, the modifying inhibits expression of PLK2. In some embodiments, the genome editing complex is a CRISPR complex comprising a gRNA that hybridizes to the PLK2 genomic region.

[0083] In some embodiments, the agent is a small molecule inhibitor of PLK2. In some embodiments, the inhibitor is a direct inhibitor of PLK2. In some embodiments, the inhibitor binds to PLK2. In some embodiments, the PLK2 inhibitor is ON1231320. In some embodiments, the PLK2 inhibitor is a derivative of ON1231320. ON1231320 is a well- known PLK2 inhibitor that is commercially available. It is also known as poloxin and its CAS number is 321688-88-4. In some embodiments, the PLK2 inhibitor is BI2536. In some embodiments, the PLK2 inhibitor is a derivative of BI2536. BI2536 is a well-known PLK2 inhibitor that is commercially available. Its CAS number is 755038-02-9. In some embodiments, a derivative is a derivative that inhibits PLK2. Other examples of PLK2 inhibitors include, but are not limited to Volasertib (BI 6727), TC-S 7005, Rigosertib, ELN 582175 and ELN 582646.

[0084] In some embodiments, the cancer comprises a mutation in MLH1 and the synthetically lethal protein / gene is selected from those provided in Table 2. In some embodiments, the cancer comprises a mutation in MLH1 and the synthetically lethal protein / gene is selected from those provided in Table 3. In some embodiments, the cancer comprises a mutation in MLH1 and the synthetically lethal protein / gene is selected from Ring Finger Protein 19A (RNF19A), Zinc Finger Protein 646 (ZNF646), EP300 Interacting Inhibitor of Differentiation 2 (EID2), MLX Interacting Protein (MLXIP), Transforming Growth Factor Beta Receptor Associated Protein 1 (TGFBRAP1), Protein Tyrosine Phosphatase Type IVA, Member 1 (PTP4A1), Coiled-Coil Domain Containing 127(CCDC127), Glutaminyl -Peptide Cyclotransferase Like (QPCTL), Zinc Finger CCCH-Type Containing 7B (ZC3H7B), LIM Domain Binding 1 (LDB1), Chromobox 3 (CBX3), LIM and Calponin Homology Domains 1 (LIMCH1), UBAP1-MVB12 Associated Domain Containing 1 (UMAD1), Exonuclease 1 (EXO1), Golgin A7 (GOLGA7), LIM Homeobox 4 (LHX4), Tweety Family Member 3 (TTYH3), Lectin, Mannose Binding 1 (LMAN1), Collagen Type V Alpha 2 Chain (COL5 A2), Ring Finger Protein 144B (RNF 144B), Nuclear Receptor Subfamily 5 Group A Member 2 (NR5A2), Trophinin Associated Protein (TROAP), Rho GDP Dissociation Inhibitor Gamma (ARHGDIG), Serine and Arginine Rich Splicing Factor 9 (SRSF9), LLGL Scribble Cell Polarity Complex Component 1 (LLGL1), E2F Transcription Factor 3 (E2F3), Microtubule Associated Protein 1 Light Chain 3 Alpha (MAP1LC3A), Chromosome 1 Open Reading Frame 159 (Clorfl59), Activator of HSP90 ATPase Activity 2 (AHSA2), AT -Rich Interaction Domain 4B (ARID4B), Exostosin Like Glycosyltransferase 2 (EXTL2), Zinc Finger Protein 783 (ZNF783), TCF3 Fusion Partner (TFPT), Polo Like Kinase 2 (PLK2), Tetraspanin 12 (TSPAN12), MAGE Family Member Hl (MAGEH1), Sphingomyelin Phosphodiesterase 2 (SMPD2), Pyruvate Carboxylase (PC), Transmembrane Protein 191C (TMEM191C), Activating Transcription Factor 5 (ATF5), ST3 Beta-Galactoside Alpha-2, 3-Sialyltransferase 4 (ST3GAL4), POU Class 4 Homeobox 1 (POU4F1), GRIP1 Associated Protein 1 (GRIP API), Coronin 2B (CORO2B), Cell Division Cycle Associated 7 (CDCA7), Sorting Nexin 4 (SNX4), KIAA0513 (KIAA0513), Tyrosine Kinase 2 (TYK2), and ZFP91 Zinc Finger Protein (ZFP91). In some embodiments, the cancer comprises a mutation in MLH1 and the synthetically lethal protein / gene is selected from RNF19A, PTP4A1, LHX4, LMAN1, COL5A2, RNF144B, NR5A2, E2F3, MAP1LC3A, EXTL2, PLK2, SMPD2, ATF5, and TYK2. In some embodiments, the cancer comprises a mutation in MLH1 and the synthetically lethal protein / gene is selected from PTP4A1, QPCTL, LDB1, CBX3, EX01, TTYH3, LMAN1, COL5A2, NR5A2, E2F3, MAP1LC3A, EXTL2, PLK2, SMPD2, PC, ATF5, ST3GAL4, and TYK2.

[0085] Agents for inhibiting the proteins / genes listed in Tables 2, 3, 4, and 5 are known in the art. These include nucleic acid molecules and small molecule inhibitors. A skilled artisan can selected the agent in order to be able to inhibit expression or function of the synthetically lethal gene / protein.

[0086] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.

[0087] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0088] In those instances where a convention analogous to "at least one of A, B, and C, etc. " is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0089] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0090] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0091] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0092] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are included.

[0093] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0094] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0095] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor LaboratoryPress, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I- III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Example 1: Establishing a model for MMR deficiency in HhESCs

[0096] To establish a model for MMR deficiency, HhESCs were transiently transfected with a vector containing GFP, Cas9 and a sgRNA for the MLH1 gene. Clones were established from transfected cells and the introduction of a loss-of-function mutation to the MLH1 gene was validated with Sanger sequencing (Fig. IB) and Western-Blot analysis (Fig. 7A).

[0097] Tumor cells are known to accumulate mutations that can be classified into different mutation signatures. These mutations signatures are the result of various biological perturbations and mutational processes that occur during the disease. To demonstrate the validity of the HhESCs MLH1 knock-out (KO) model, their mutational signatures were examined. Thus, MLH1 mutated HhESCs were grown for three months to allow mutation accumulation, subclones of MLH1 KO (n=8) and wild-type (WT) (n=4) cells were established and whole-genome sequencing analysis was performed. Using the Cosmic sigProfiler, which is a bioinformatic tool for performing mutational signature analysis, two signatures, SBS44 and ID2, that are correlated to MMR deficiency were identified. These signatures were identified in MLH1 KO and not in the WT cells (Fig. 1C and 7C) and are highly represented in MMR deficient tumors, such as uterus and colorectal cancers. This demonstrates that the HhESC MMR model recapitulates the mutational process observed in patients with MMR deficiency.Example 2: Synthetic lethal (SL) interaction screens

[0098] To identify novel SL interaction to the MLH1 gene, a genome wide genetic screen was performed on WT and MLH1 KO cells. A CRISPR knockout pooled library, targeting -19,000 genes (-76,400 sgRNAs), each by 4 different sgRNAs, was utilized.

[0099] To ensure that most of the cells are infected with one sgRNA virus amount was calibrated, to achieve a 0.3 MOI. 3xl0A8 WT / MLH1 KO cells were infected with the LentiCrispr pooled library. Two days post-infection Puromycin treatment was initiated to eliminate cells that were not properly infected with the virus. The cells were expanded for 27 days and samples containing 5xlOA7 cells were collected at 5 different time points- Day 1, Day 10, Day 15, Day 22, and Day 27. This allowed a ~x650 coverage, meaning that if the entire sgRNA library was equally distributed between the cells, each sgRNA would be found in 650 cells. Genomic DNA was isolated and libraries forNGS sequencing were established using unique primers that identified the pooled sgRNAs. The libraries were sequenced on MiSeq, Illumina, with 6x10A7 reads per sample.

[0100] Fastq files of the 3 different conditions (WT Dayl, WT_Day21 and MLHl_Day27) were analyzed using 3 different bioinformatic pipelines to identify depleted genes. The pipelines use different normalization methods and significance tests. A comparison of the pipelines can be found in Table 1. The output of each pipeline is: (1) Crispr Score (CS) which is calculated by the Avg(log2(cond. 1 / cond. 2)) for all different sgRNAs of a gene and (2) p-value which is based on the sgRNAs distribution. By the end of this process, each gene has a CS and p-value for 3 comparisons: (1) WT_day21 vs. WT dayl which we’ll refer to as “WT” (2) MLHl_day27 vs. WT day 1 which we’ll refer to as “MLH1” (3) MLHl_day27 vs. WT_day21 which we’ll refer to as “MLH1 To obtain the most promising genes, which are depleted in the MLH1 screen and enriched or do not significantly change in the WT screen, we applied several selection criteria.I. Removing genes that are not expressed in hESCs.II. Genes that show one of the following trends in at least two pipelines:1. Depleted in MLH1*, static in the MLH1 and enriched in the WT2. Depleted in MLH1*, depleted in MLH1 and enriched in the WT3. Depleted in MLH1*, depleted in MLH1 and static in the WTIII. Genes in which the third quartile of the A7 / .7 / 7_day27 sgRNAs is lower than the first quartile of WT dayl and WT_day21 sgRNAs.

[0101] This analysis led to the identification of 49 genes that can serve as novel therapeutic targets in MMR deficient tumors (Table 2). These genes were ranked according to their CS and p-value in MLH1*. Low values of CS and p-value got the high ranks and vice versa.The final ranking was calculated as an average of the ranks in pipelines in which the genes were depleted.

[0102] Literature overview revealed that the list of 49 targets is enriched with genes that are related to ER stress and to protein folding (Table 3, 14 genes). Suggesting an SL interaction between the DNA MMR pathway and ER stress response.

[0103] 18 out of 49 targets were found to be druggable- having the potential to be modulated by a small molecule drug, and for 12 an inhibitor was reported in the literature (Table 2). Targets’ druggability data was obtained from the DGIdb and DepMap databases, and from literature overview.

[0104] Table 1 : Comparison of the different parameters of normalization methods and significance tests in the 3 pipelines used in the analysis.

[0105] Table 2: Novel synthetic lethal interaction targets and drugs to MLH1.

[0106] Table 3: ER stress and protein folding related genes.Example 3: Genetic validation

[0107] Genetic assays were performed in HhESCs to validate SL interactions between the identified genes and the MLH1 gene. EX01 was targeted utilizing CRISPR-Cas9 in a mixed culture of MLH1 mutant and WT cells. The different cell populations were labeled and monitored utilizing FACS at different time points (Fig. 3A). If a synthetic lethal interaction exists between MLH1 and EXO1 a decrease in MLH1 mutant cells over time would be expected and indeed that is exactly what is observed.Example 4: Chemical validation

[0108] For some of the identified genes there are known small molecules that can inhibit the function of the relevant protein. It is predicted that MLH1 mutant cells will be more sensitive to such molecules. We treated HhESCs and CCLs and demonstrated that cells that lack functional MLH1 protein are indeed more sensitive to such molecules.

[0109] The molecules that were validated are: SRI 848 that inhibits NR5A2 (Fig. 3B, 4A- 4B, 9A-9B); BI2536 that inhibits PLK2 (This molecule also inhibits PLK1, PLK3, and BRD4) (Fig. 3B-3D, 4A-4D) and ON1231320 that inhibits PLK2 (Fig. 3B, 4A-4B).

[0110] Furthermore, we illustrated the distinct impact of the BI2536 molecule in a xenograft model, wherein both MMR-proficient and MMR-deficient CCLs were subcutaneously injected into immunocompromised mice (Fig. 4D). This experiment demonstrated the sensitivity of MMR-deficient CCL-derived tumors to BI2536, whereas MMR-proficient CCL-derived tumors did not exhibit any major effect. These molecules can be used to treat tumors deficient in MLH1 and DNA MMR.Example 5: The TP53 gene

[0111] TP53 is the most frequently mutated gene in cancer, and in response to DNA damage, initiates either cell cycle arrest to allow DNA repair or apoptosis. The inventors performed genome-wide genetic screening using HhESCs with and without loss-of-function (LoF) mutation in the TP53 gene. These experiments uncovered a list of putative synthetic lethal interaction partners that were further validated and can lead to tumor elimination upon inhibition.

[0112] To establish a model for TP53 deficiency, HhESCs were transiently transfected with a vector containing GFP, Cas9 and sgRNA for the TP53 gene. Nutlin-3 resistance clones were established from transfected cells and the introduction of a loss-of-function mutation to the TP53 gene was validated with Sanger sequencing and Western-Blot analysis (Fig. 1C, 7B).

[0113] To Identify novel SL interaction to the TP53 gene, a genome wide genetic screen was performed on WT and TP53 KO HhESCs. A CRISPR knockout pooled library, targeting -19,000 genes (-76,400 sgRNAs), each by 4 different sgRNAs, was utilized.

[0114] To assure that most of the cells are infected with one sgRNA virus amount was calibrated, to achieve a 0.3 MOI. 3x108 WT / TP53 KO cells were infected with the LentiCrispr pooled library. Two days post-infection Puromycin treatment was initiated to eliminate cells that were not properly infected with the virus. The cells were expanded for 23 days and samples containing 5x107 cells were collected at 4 different time points- Day 1, Day 9, Day 17, and Day 23. This allowed a ~x650 coverage, meaning that if the entire sgRNA library was equally distributed between the cells, each sgRNA would be found in650 cells. Genomic DNA was isolated and libraries for NGS sequencing were established using unique primers that identified the pooled sgRNAs. The libraries were sequenced on MiSeq, Illumina, with 6x107 reads per sample.

[0115] Fastq files of the 3 different conditions (WT Dayl, WT_Day21 and TP53_Day23) were analyzed using 3 different bioinformatic pipelines to identify depleted genes. The pipelines use different normalization methods and significance tests. A comparison of the pipelines can be found in Table 1. The output of each pipeline is: (1) Crispr Score (CS) which is calculated by the Avg(log2(cond. 1 / cond. 2)) for all different sgRNAs of a gene and (2) p-value which is based on the sgRNAs distribution. By the end of this process, each gene has a CS and p-value for 3 comparisons: (1) WT_day21 vs. WT dayl which we’ll refer to as “WT” (2) TP53_day23 vs. WT_dayl which we’ll refer to as “TP53” (3) TP53_day23 vs. WT_day21 which we’ll refer to as “TP53*”. To obtain the most promising genes, which are depleted in the TP53 screen and enriched or do not significantly change in the WT screen, we applied several selection criteria which resulted in a list of 132 genes (Table 4).

[0116] To refine this list, a secondary, customized "mini" screen targeting these 132 genes was designed. The “mini” screen has two main advantages over a genome wide genetic screen- 1) Unlike a genome wide genetic screen in which -76,400 sgRNAs are utilized, a customized screen contains only 2000 sgRNAs. Thus, enabling deeper sequencing and increasing screen sensitivity. 2) Each gene can be targeted with an increased number of sgRNAs to improve assay robustness. In this screen each gene was targeted by 8 different sgRNAs.

[0117] Virus amount was calibrated to achieve a 0.3 MOI. 6xlOA7 WT / TP53 KO cells were infected with the LentiCrispr pooled “mini” library. One day post-infection puromycin treatment was initiated to eliminate cells that were not properly infected with the virus. The cells were expanded for 24 days and samples containing 7xlOA6 cells were collected at 5 different time points- Day 1, Day 9, Day 14, Day 18, and Day 24. This allowed an x3,500 coverage. Genomic DNA was isolated and used to create a library for NGS sequencing. The libraries were sequenced on MiSeq, Illumina, with -6x106 reads per sample.

[0118] Sequencing results were analyzed as previously described to identify potential SL interaction targets.

[0119] A bioinformatic analysis of the 132 gene list reduced the number of potential targets to 41 genes. 18 out of 41 targets were found to be druggable - having the potential to bemodulated by a small molecule drug, and for 12 an inhibitor was reported in the literature (Table 5). Targets’ druggability data was obtained from the DGIdb and DepMap databases, and from literature overview.

[0120] The MYH10 gene demonstrated the most significant results (Fig. 2D-2F).

[0121] Genetic assays were performed in HhESCs to validate SL interactions between the identified genes and the TP53 gene. Thus, potential genes were targeted utilizing CRISPR- Cas9 in TP53 mutant and WT cells. Targeted loci were sequenced at different time points. Since haploid cells were used in this assay, each NGS read represents one cell and enables following mutation impact on cells. Using this method, SL interaction of the MYH10 gene and TP53 was validated (Fig. 5A-5B, 11A-11B), and additional validation experiments are performed for the other genes (Table 5).

[0122] For some of the identified genes there are known small molecules that can inhibit the function of the relevant protein. It is predicted that TP53 mutant cells will be more sensitive to such molecules. HhESCs and CCLs were treated and it was demonstrated that cells that lack functional p53 protein are indeed more sensitive to such molecules.

[0123] The molecules that were validated are: Blebbistatin or Para-aminoblebbistatin which inhibit MYH10 (Fig. 5C-5F, 6A-6D, 12A-12D). Further, the Iventors demonstrated the differential effect of the Blebbistatin molecule in a xenograft model where both p53 proficient and deficient CCLs were subcutaneously injected to immunocompromised mice (Fig. 6E). In this experiment it was shown that a p53 deficient CCL derived tumor is more sensitive to Blebbistatin than a p53 proficient CCL derived tumor. Thus, these molecules can be used to treat tumors deficient in the TP53 gene.

[0124] Table 4: Novel synthetic lethal interaction targets to TP53

[0125] Table 5: Novel synthetic lethal interaction targets and drugs to TP53

[0126] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS:

1. A method of treating a cancer in a subject in need thereof, wherein said cancer comprises a mutation in tumor protein p53 (TP53) or DNA mismatch repair protein Mlhl (MLH1), the method comprising: a. determining said cancer comprises a mutation in TP53 and administering to said subject an agent that decreases abundance or function of a protein synthetically lethal with TP53, wherein said protein synthetically lethal with TP53 is myosin heavy chain 10 (MYH10); or b. determining said cancer comprises a mutation in MLH1 and administering to said subject an agent that decreases abundance or function of a protein synthetically lethal with MLH1, wherein said protein synthetically lethal with MLH1 is selected from exonuclease 1 (EXO1), nuclear receptor subfamily 5, group A, member 2 (NR5A2) and Serine / threonine-protein kinase PLK2 (PLK2); thereby treating a cancer in a subject in need thereof.

2. The method of claim 1, further comprising receiving a sample from said subject comprising cancer cells and measuring expression and / or function of MLH1 or TP53 in said cancer cells, wherein cancer cells with decreased expression and / or function as compared to control cells are cells with a mutation in MLH1 or TP53.

3. The method of claim 2, wherein said control cells are selected from cancer cells without a mutation in MLH1 or TP53 or non-cancerous cells from the same tissue or cell type as said cancer cells.

4. The method of any one of claims 1 to 3, comprising selecting a subject that suffers from a cancer comprising a mutation in MLH1 or TP53.

5. The method of any one of claims 1 to 4, wherein a mutation is a loss of function (LOF) mutation.

6. The method of any one of claims 1 to 5, wherein said cancer with a mutation does not express MLH1 or TP53 and wherein said determining comprises determining said cancer does not express MLH1 or TP53.

7. The method of claim 6, wherein express is express RNA of MLH1 or TP53 or express protein of MLH1 or TP53.

8. The method of any one of claims 1 to 7, wherein said agent is a nucleic acid molecule that hybridizes to an mRNA of said protein synthetically lethal with MLH1 or TP53 and inhibits expression of said protein synthetically lethal with MLH1 or TP53.

9. The method of any one of claims 1 to 7, wherein said agent is a genome editing complex that binds to a genomic region encoding said protein synthetically lethal with MLH1 or TP53 and modifies said genomic region to inhibit expression of said protein synthetically lethal with MLH1 or TP53.

10. The method of any one of claims 1 to 7, wherein said agent is a small molecule direct inhibitor of said protein synthetically lethal with MLH1 or TP53.

11. The method of claim 10, wherein said cancer comprises a mutation in MLH1 and said small molecule direct inhibitor is selected from SRI 848 and derivatives thereof that inhibit NR5A2, ON1231320 and derivatives thereof that inhibit PLK2, and BI2536 and derivatives thereof that inhibit PLK2.

12. The method of claim 10, wherein said cancer comprises a mutation in TP53 and said small molecule direct inhibitor is selected from Blebbistatin, para-amino-blebbi statin and derivative thereof that inhibit MYH10.

13. An agent that decreases abundance or function of myosin heavy chain 10 (MYH10), for use in treating a cancer comprises a mutation in tumor protein p53 (TP53) in a subject in need thereof.

14. An agent that decreases abundance or function of a protein synthetically lethal with MLH1, for use in treating a cancer comprises a mutation in DNA mismatch repair protein Mlhl (MLH1) in a subject in need thereof, wherein said protein synthetically lethal with MLH1 is selected from exonuclease 1 (EXO1), nuclear receptor subfamily 5, group A, member 2 (NR5A2) and Serine / threonine-protein kinase PLK2 (PLK2).

15. The agent of claim 12 or 13, wherein said cancer has been determined to comprise said mutation.

16. The agent of claim 15, wherein said cancer was determined to comprise said mutation by receiving a sample from said subject comprising cancer cells and measuring expression and / or function of MLH1 or TP53 in said cancer cells, wherein cancer cells with decreased expression and / or function as compared to control cells are cells with a mutation in MLH1 or TP53.

17. The agent of claim 16, wherein said control cells are selected from cancer cells without a mutation in MLH1 or TP53 or non-cancerous cells from the same tissue or cell type as said cancer cells.

18. The agent of any one of claims 12 to 17, wherein a mutation is a loss of function (LOF) mutation.

19. The agent of nay one of claims 12 to 18, wherein said cancer with a mutation does not express MLH1 or TP53.

20. The agent of claim 19, wherein express is express RNA of MLH1 or TP53 or express protein of MLH1 or TP53.

21. The agent of any one of claims 12 to 20, wherein said agent is a nucleic acid molecule that hybridizes to a MLH1 or TP53 mRNA and inhibits expression of MLH1 or TP53.

22. The agent of any one of claims 12 to 20, wherein said agent is a genome editing complex that binds to a MLH1 or TP53 genomic region and modifies said genomic region to inhibit MLH1 or TP53 expression.

23. The agent of any one of claims 12 to 20, wherein said agent is a small molecule direct inhibitor of MLH1 or TP53.

24. The agent of claim 23, wherein said cancer comprises a mutation in TP53 and said small molecule direct inhibitor is selected from Blebbistatin, para-amino-blebbi statin and derivative thereof that inhibit MYH10.

25. The agent of claim 23, wherein said cancer comprises a mutation in MLH1 and said small molecule direct inhibitor is selected from SRI 848 and derivatives thereof thatinhibit NR5A2, ON1231320 and derivatives thereof that inhibit PLK2, and BI2536 and derivatives thereof that inhibit PLK2.

26. A method of treating a cancer in a subject in need thereof, wherein said cancer comprises a mutation in DNA mismatch repair protein Mlhl (MLH1) or tumor protein p53 (TP53), the method comprising: a. determining said cancer comprises a mutation in TP53 and administering to said subject an agent that decreases abundance or function of a protein synthetically lethal with TP53, wherein said protein synthetically lethal with TP53 is selected from the proteins provided in Table 4; or b. determining said cancer comprises a mutation in MLH1 and administering to said subject an agent that decreases abundance or function of a protein synthetically lethal with MLH1, wherein said protein synthetically lethal with MLH1 is selected from the proteins provided in Table 2; thereby treating a cancer in a subject in need thereof.

27. The method of claim 26, wherein said protein synthetically lethal with MLH1 is selected from PTP4A1, QPCTL, LDB1, CBX3, EX01, TTYH3, LMAN1, COL5A2, NR5A2, E2F3, MAP1LC3A, EXTL2, PLK2, SMPD2, PC, ATF5, ST3GAL4, and TYK2.

28. The method of claim 26, wherein said protein synthetically lethal with TP53 is selected from F0XI3, SNAP23, TMED10, ELAVL1, TGFBRAP1, MYH10, SLC2A6, MANF, KMT2A, SENP6, MDH1, MAEA, VPS37A, OR1I1, PRKAB2, CTPS2, PLBD1, and OR2B3.

Images