Induction of cell death in cancer

Abstract

The present invention relates to the discovery that depletion of FANC proteins or genes, such as FANCM, FANCD2, or FANCI, causes synthetic lethality in SETD2-deficient cells. Methods and compounds for use in treating SETD2-deficient cancers are provided.

Description

[Technical Field]

[0001] The present invention relates to the induction of cell lethality in cancer cells. [Background technology]

[0002] Many conventional anti-cancer therapies, such as radiation therapy or chemotherapy, target dividing cells and are highly toxic to non-cancerous cells, causing severe side effects.

[0003] A therapeutic strategy based on synthetic lethal interactions, i.e., inducing cell death by disrupting two genes simultaneously rather than separately, allows for highly efficient and selective targeting of tumor cells while protecting non-cancerous cells.

[0004] There is a need for new and improved cancer therapeutics based on synthetic lethal interactions. Summary of the Invention

[0005] We found that depletion of Fanconi anemia complementation group (FANC) proteins, for example by siRNA, is selectively lethal to SETD2-deficient cancer cells, and therefore FANC antagonists may be useful in treating SETD2-deficient cancers.

[0006] A first aspect of the present invention provides a method of treating cancer in an individual in need thereof, the method comprising reducing the expression or activity of FANC in the individual, wherein the cancer is a SETD2-deficient cancer.

[0007] A second aspect of the present invention provides a FANC antagonist for use in a method of treating SETD2-deficient cancer.

[0008] A third aspect of the invention provides the use of a FANC antagonist in the manufacture of a medicament for treating a SETD2-deficient cancer.

[0009] A fourth aspect provides a method for screening for a compound that induces cell death in SETD2-deficient cancer cells, comprising determining binding of a test compound to FANC, wherein binding to FANC indicates that the compound induces cell death in SETD2-deficient cells.

[0010] A fifth aspect provides a method for screening for a compound that induces cell death in SETD2-deficient cancer cells, comprising determining the effect of a test compound on the expression or activity of FANC, wherein a decrease in the expression or activity of FANC suggests that the compound induces cell death in SETD2-deficient cancer cells.

[0011] A sixth aspect provides a method for determining the responsiveness of a cancer in an individual to treatment with a FANC antagonist, the method comprising determining the presence of one or more SET2D-deficient cancer cells in a sample obtained from the individual, wherein the presence of one or more SETD2-deficient cancer cells in the sample indicates that the cancer will be responsive to treatment with a FANC antagonist.

[0012] A seventh aspect provides a method for selecting an individual having a SETD2-deficient cancer for treatment with a FANC antagonist, the method comprising identifying cancer cells obtained from the individual as being deficient in SETD2 relative to normal cells.

[0013] Preferred Fanconi anemia complementation group (FANC) proteins according to the first to seventh aspects include FANCM, FANCD2, and FANCI.

[0014] Preferred FANC antagonists according to the first to seventh aspects include FANCM antagonists, FANCD2 antagonists, and FANCI antagonists.

[0015] Other aspects and embodiments of the invention are described in more detail below. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 shows the decrease in cell culture density of SETD2-deficient clear cell carcinoma cells transfected with siRNA to deplete FANCM. Wild-type (WT) or CRISPR / Cas9 SETD2 knockout (KO) clear cell renal cell carcinoma cells (Caki2 cell line) were transfected with control (siCtrl) or FANCM siRNA. (A) Figure shows the decrease in cell culture density 3 days after FANCM knockdown. (B) Figure shows the decrease in cell culture density in SETD2-KO Caki cells 6 days after knockdown. [Figure 2] This figure shows increased cell death in SETD2-deficient clear cell carcinoma cells 3 days after transfection with siRNA depleting FANCM. WT or SETD2-KO clear cell carcinoma cells (786-O cell line) were transfected with control or FANCM siRNA. (A) After 3 days of culture, a viability assay was performed by measuring the level of propidium iodide staining in SETD2-KO 786-O cells by flow cytometry. (B) This figure shows the percentage of PI+ cells in WT or SETD2-KO clear cell carcinoma cells (786-O cell line) transfected with siCtrl or siFANM. P values ​​were calculated using a two-tailed Student's t-test. *P<0.05, **P<0.005, ***P<0.001. [Figure 3]This figure shows increased cell death in SETD2-deficient clear cell carcinoma cells 6 days after transfection with siRNA depleting FANCM. WT or SETD2-KO clear cell carcinoma cells (786-O cell line) were transfected with control or FANCM siRNA. (A) After 6 days of culture, a viability assay was performed by measuring the level of propidium iodide staining in SETD2-KO 786-O cells by flow cytometry. (B) This figure shows the percentage of PI+ cells in WT or SETD2-KO clear cell carcinoma cells (786-O cell line) transfected with siCtrl or siFANCM. P values ​​were calculated using a two-tailed Student's t-test. *P<0.05, **P<0.005, ***P<0.001. [Figure 4] Figure 1 shows increased cell death in SETD2-deficient cells transfected with siRNA depleting FANCD2. WT (ACHN and Caki-1) or SETD2-KO cells (RCC-AB, RCC-ER, RCC-MF) were transfected with control or FANCM siRNA. Figure 2 shows the percentage of PI+ cells in WT or SETD2-KO cells transfected with siCtrl or siFANCD3 after 3 days (A) and 6 days (B). P values ​​were calculated using Student's two-tailed t-test. *P<0.05, **P<0.005, ***P<0.001. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention relates to the discovery that reduced or absent expression or activity of FANC protein in SETD2-deficient cancer cells induces cell death, which may be useful in the treatment of cancerous conditions, such as cancerous conditions characterized by a SETD2 deficiency.

[0018] Su(var), Enhancer of zeste, Trithorax (SET)-domain containing protein 2 (SETD2; EC:2.1.1.359; Gene ID:29072) is a histone methyltransferase responsible for the trimethylation of histone H3 at lysine 36 (i.e., H3K36me3). SETD2-mediated H3K36me3 is introduced sequentially during transcription elongation and is important for maintaining nucleosome occupancy, DNA double-strand break repair, RNA splicing, and transcription termination (Li J, et al. Oncotarget. 2016; 7(31):50719-50734; de Almeida SF, Carmo-Fonseca M. Trends Biochem Sci. 2012; 37(6):248-253; Carvalho S, et al. Nucleic Acids Res. 2013; 41(5):2881-2893; Carvalho S, et al. Elife. 2014; 3:e02482; Grosso AR, et al. Elife. 2015; 4:e09214). Thus, loss of SETD2 causes genomic instability, and SETD2 is mutated in a variety of human cancers (Fahey CC, et al. Cold Spring Harb Perspect Med. 2017; 7(5):a026468).

[0019] The human gene encoding SETD2 has 25 exons and is located at 3p21.31. The reference coding sequence of human SETD2 is set forth in SEQ ID NO: 9. Other reference human SETD2 coding sequences have database accession numbers NM_001349370.3 and NM_014159.7. The SETD2 coding sequences described herein may comprise a reference nucleotide sequence such as SEQ ID NO: 9 or a variant thereof.

[0020] A SETD2-deficient cancer may comprise or consist of one or more cancer cells in which SETD2 expression and / or activity is reduced or absent. SETD2 expression and / or activity may be reduced or absent in cancer cells due to one or more alterations in one or both alleles of the SETD2 gene, such as one or more mutations, translocations, or deletions, or due to epigenetic modifications that impair SETD2 expression or activity, such as changes in DNA or histone methylation, or due to mutation(s) in genes encoding regulators, such as HOX transcript antisense RNA.

[0021] Mutations may include nonsynonymous mutations, including frameshift, missense, nonsense, splice site, nonstop, and translation start site changes; truncating mutations, including nonsense, nonstop, frameshift deletions, frameshift insertions, and splice site changes; and in-frame mutations, including in-frame deletions and in-frame insertions. Mutations in the gene encoding SETD2 can result in the production of mutant polypeptides with altered activity, such as reduced or lost activity. Genomic translocations or chromosomal rearrangements (e.g., inversions) involving the SETD2 gene and / or deletion of part or all of the SETD2 gene can also result in loss of functional SETD2 protein. Epigenetic changes, such as DNA methylation or histone (de)acetylation and (de)methylation, can also result in reduced or lost SETD2 activity.

[0022] The presence of one or more mutations in one or both alleles of the SETD2 gene can be determined by detecting the presence of a nucleic acid sequence from the SETD2 gene containing one or more mutations, or by detecting a mutant polypeptide encoded by the gene, in one or more cells of a test sample obtained from the individual.

[0023] A variety of methods are available for determining the expression and / or activity of SETD2 in a sample obtained from an individual.

[0024] Genetic testing such as DNA sequencing, exome sequencing, comparative genomic hybridization, in situ hybridization (e.g., fluorescent in situ hybridization (FISH)), PCR, RT-PCR, karyotyping, classical and molecular cytogenetics, or molecular testing such as enzyme activity assays, complementation assays, Western blot or immunofluorescence of SETD2 or H3K36me3, bisulfite sequencing, can reveal one or more mutations (including one or more nucleotide mutations, deletions, translocations, inversions, chromosomal rearrangements, or epigenetic changes) in the SETD2 gene or insufficient production or activity of the SETD2 protein.

[0025] The nucleic acid, which may be genomic DNA, RNA, or cDNA, or an amplified region thereof, can be sequenced to identify or determine the presence of one or more mutations in the nucleic acid. Mutations can be identified by comparing the obtained sequence with database sequences of the component, as defined above. In particular, the presence of one or more mutations that cause abrogation or loss of function of SETD2 can be determined. The presence of one or more of the mutation types described above indicates reduced or lost activity.

[0026] Sequencing can be performed using any one of a variety of standard techniques. Sequencing of the amplified products can involve, for example, precipitation with isopropanol, resuspension, and sequencing using a TaqFS+ dye terminator sequencing kit. Extension products can be electrophoresed on an ABI 377 DNA sequencer, and data analysis can be performed using Sequence Navigator software.

[0027] A specific amplification reaction, such as polymerase chain reaction (PCR), using one or more pairs of primers can be conveniently used to amplify a region of interest within a nucleic acid sequence, e.g., a portion of the sequence suspected of containing a mutation or polymorphism. As described above, the amplified nucleic acid can then be sequenced and / or tested in any other manner to determine the presence or absence of a mutation or polymorphism that reduces or eliminates the expression and / or activity of SETD2.

[0028] Furthermore, once the nucleic acid of an individual or sample has been sequenced, the sequence information is preserved and can then be retrieved without reliance on the original nucleic acid itself, allowing sequence variations or mutations to be identified, for example, by scanning databases of sequence information using sequence analysis software.

[0029] In some embodiments, a cancer can be identified as being deficient in SETD2 by assessing the level of SET2D expression or activity, which can be determined, for example, by Western blot, ELISA, RT-PCR, nucleic acid hybridization, or karyotype analysis.

[0030] Mutations and polymorphisms associated with SETD2-deficient cancers can also be detected at the protein level, for example, by detecting the presence of mutant polypeptides by immunohistochemistry.

[0031] Furthermore, because SETD2 is the only enzyme responsible for H3K36me3 (Edmunds et al., (2008) (The EMBO journal 27(2):406-420)), determining the level of H3K36me3 in a sample obtained from an individual can be used to identify cancer as a SETD2-deficient cancer. The level of H3K36me3 can be measured using immunofluorescence or Western blot. A decrease in the level of the trimethylation mark H3K36me3 suggests a decrease or loss of SETD2 activity and / or expression.

[0032] The sample may be a sample of cancer cells, which may be obtained from an individual using conventional techniques.

[0033] SETD2-deficient cancers can be treated by reducing or eliminating the expression or activity of Fanconi anemia complementation group (FANC) proteins, as described herein. FANC proteins are components of the FANC / BRCA pathway, which is involved in DNA replication and DNA damage response. FANC proteins may include FANCA, FANCC, FANCG (XRCC9), FANCJ, FANCE, FANCF, FANCP (SLX4), FANCD1 (BRCA2), FANCI, FANCL, FANCM, FANCN (PALB2), FANCO (RAD51C), FANCQ (ERCC4), FANCS (BRCA1), FANCT (UBE2T), FANCU (XRCC2), FANCV (REV7), FANCW (RFWD3), FANCB, FANCR, and (RAD51).

[0034] Preferred FANC proteins can include components of the FA core ubiquitin ligase complex, such as FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, and FANCL. Other preferred FANC proteins can include components of the heterodimeric complex (ID complex) that is monoubiquitinated by the FA core complex, such as FANCD2 and FANCI.

[0035] In some preferred embodiments, the FANC protein is FANCM.

[0036] In other preferred embodiments, the FANC protein is FANCD2 or FANCI.

[0037] Fanconi anemia group M protein (FANCM) is an ATP-dependent DNA helicase / translocase (EC 3.6.4.13) involved in homologous recombination, meiosis, and DNA repair. FANCM may be human FANCM. The human gene encoding the FANCM polypeptide (Gene ID: 57697) has 25 exons and is located at 14q21.2. A reference human FANCM amino acid sequence is shown in SEQ ID NO: 1. Other reference human FANCM amino acid sequences have database accession numbers NP_001295062.1, NP_1295063.1, and NP_065988.1. The FANCM polypeptides or proteins described herein may comprise a reference amino acid sequence such as SEQ ID NO: 1 or a variant thereof.

[0038] The Fanconi anemia group D2 protein (FANCD2) is a member of the heterodimeric ID (or FANCD2 / FANCI) complex, which is monoubiquitinated by the FA core complex and loads onto chromatin in response to DNA damage. FANCD2 may be human FANCD2. The human gene encoding the FANCD2 polypeptide (Gene ID: 2177) has 45 exons and is located at 3p25.3. A reference human FANCD2 amino acid sequence is set forth in SEQ ID NO: 5. Other reference human FANCD2 amino acid sequences have database accession numbers NP_001018125.1, NP_001306913.1, NP_001351182.1, NP_001361183.1, NP_001361184.1, and NP_149075.2. The FANCD2 polypeptides or proteins described herein may comprise a reference amino acid sequence such as SEQ ID NO: 5 or a variant thereof.

[0039] The Fanconi anemia group I protein (FANCI) is a member of the heterodimeric ID (or FANCD2 / FANCI) complex, which is monoubiquitinated by the FA core complex and localized to chromatin in response to DNA damage, such as interstrand crosslinks. FANCI may be human FANCI. The human gene encoding the FANCI polypeptide (Gene ID: 55215) has 41 exons and is located at 15q26.1. A reference human FANCI amino acid sequence is set forth in SEQ ID NO: 7. Other reference human FANCI amino acid sequences have database accession numbers NP_001363839.1, NP_001363840.1, and NP_060663.2. The FANCI polypeptides or proteins described herein may comprise a reference amino acid sequence, such as SEQ ID NO: 7, or a variant thereof.

[0040] As used herein, a variant of a reference amino acid sequence is an amino acid sequence that has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% identity, or at least 98% identity to a reference amino acid sequence such as SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:7.

[0041] A reference human FANCM-encoding nucleotide sequence is set forth in SEQ ID NO: 2. Other reference human FANCM-encoding sequences have database accession numbers NM_001308133.1, NM_001308134.1, and NM_020937.4. The FANCM nucleotide sequences described herein can include the nucleotide sequence of a reference human FANCM-encoding sequence, such as SEQ ID NO: 2, or a variant thereof.

[0042] A reference human FANCD2-encoding nucleotide sequence is set forth in SEQ ID NO: 6. Other reference human FANCD2-encoding sequences have database accession numbers NM_001319984.2, NM_001374253.1, NM_001374254.1, NM_001374255.1, and NM_0033084.6. The FANCD2 nucleotide sequences described herein may include the nucleotide sequence of a reference human FANCD2-encoding sequence, such as SEQ ID NO: 6, or a variant thereof.

[0043] A reference human FANCI-encoding nucleotide sequence is set forth in SEQ ID NO: 8. Other reference human FANCI-encoding sequences have database accession numbers NM_001376910.1, NM_001376911.1, and NM_018193.3. The FANCI nucleotide sequences described herein can include the nucleotide sequence of a reference human FANCI-encoding sequence, such as SEQ ID NO: 8, or a variant thereof.

[0044] Reference amino acid sequences and reference coding sequences for other FANC proteins are available in public databases.

[0045] Sequence identity is generally defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego, USA). GAP aligns two complete sequences using the Needleman and Wunsch algorithm, which maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters are used: gap creation penalty = 12 and gap extension penalty = 4. Although the use of GAP may be preferred, other algorithms may also be used, such as BLAST (using the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (using the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), SSEARCH (Smith and Waterman (1981) J. Mol. Biol. 147: 195-197;), HMMER3 (Johnson LS et al BMC Bioinformatics. 2010 Aug 18; 11():431), or the TBLASTN program of Altschul et al. (1990) (cited above), generally using default parameters (e.g., Pearson Curr Prot Bioinformatics (2013) Chapter 3 Univ. 3.1). doi:10.1002 / 0471250953.bi0301s42). In particular, the psi-Blast algorithm can be used (Altschul et al. Nucl. Acids Res. (1997) 25 3389-3402). Sequence identity and similarity can also be determined using Genomequest™ software (Gene-IT, Worcester MA USA). Sequence comparison is preferably performed over the entire length of the relevant sequences described herein.

[0046] In other embodiments, for example, when the individual to be treated is a non-human mammal, the FANC can be a non-human mammalian FANC. Reference non-human FANC amino acid sequences and reference non-human FANC coding sequences are available in public databases.

[0047] It is shown herein that reduced expression or activity of FANC, for example, reduced expression or activity of FANCM, FANCD2, and / or FANCI, reduces the viability of SETD2-deficient cancer cells. Reduced or absent expression or activity of the FANC protein described herein can reduce or inhibit the activity of the FANC / BRCA pathway, which repairs DNA interstrand crosslinks (ICLs) (see, e.g., Renaudin et al. Genes 202011(5) 585).

[0048] FANC activity in an individual can be reduced by administering a FANC antagonist that reduces the expression or activity of the FANC protein. The FANC antagonist can be one that can inhibit the activity of the FANC protein, such as a FANC inhibitor, or one that can reduce the activity of the FANC protein by degrading the FANC protein, such as a FANC degradation inducer such as a PROTAC or molecular glue, or one that can reduce or inhibit the expression of the FANC protein, such as a suppressor nucleic acid or targeted nuclease.

[0049] In some embodiments, the FANC antagonist may be a FANCM antagonist that reduces the expression or activity of a FANCM protein.

[0050] In other embodiments, the FANC antagonist may be a FANCD2 antagonist that reduces the expression or activity of a FANCD2 protein.

[0051] In other embodiments, the FANC antagonist may be a FANCI antagonist that reduces the expression or activity of a FANCDI protein.

[0052] FANC inhibitors may include, for example, small chemical molecules, e.g., non-polymeric organic compounds having a molecular weight of 900 daltons or less. For example, a suitable small molecule FANCM inhibitor can inhibit ATP binding to the ATPase domain of FANCM, DNA binding to the translocase domain of FANCM, and / or FANCM binding to binding partners such as MHF, FAAP24, BLM, RMI, and Topo IIIα. A suitable small molecule FANCD2 or FANCI inhibitor can inhibit monoubiquitination of FANCD2 or FANCI, binding of FANCI to FANCD2, or phosphorylation of FANCI or FANCD2 by ATR / ATM. Techniques suitable for rational design of small molecule inhibitors based on structural analysis of FANC proteins such as FANCM, FANCD2, and FANCI are known in the art.

[0053] Examples of FANC inhibitors include biomolecules that specifically bind to FANC proteins. Examples of FANCM inhibitors include biomolecules that specifically bind to FANCM. In some embodiments, the biomolecules can specifically bind to the DEAH helicase-like domain corresponding to residues 83 to 591 of SEQ ID NO: 1.

[0054] Biomolecules may include peptides. Peptides may comprise or consist of 5 to 40 amino acids, e.g., 6 to 10 amino acids, and may be derived from the above-described FANC proteins, e.g., FANCM, FANCD2, or FANCI, or their binding partners. Biomolecules may also include antibodies, antibody fragments, and antibody derivatives, as well as non-immunoglobulin binding molecules, such as aptamers, trinectins, anticalins, Kunitz domains, transferrin, nurse shark antigen receptor, and sea lamprey leucine-rich repeat proteins. Techniques suitable for generating biomolecules that specifically bind to FANC proteins are known in the art.

[0055] FANC degraders include small molecule ligands that regulate protein interactions in the ubiquitin-proteasome system. FANC degraders may include proteolysis-targeting chimeric molecules (PROTACs). PROTACs are heterobifunctional molecules containing a ligand for a protein of interest (POI) conjugated via a linker to a ligand that recruits an E3 ubiquitin ligase (E3). The PROTAC promotes the formation of a ternary complex between the POI and the E3, initiating degradation, followed by ubiquitination and degradation of the POI by the ubiquitin-proteasome system. The PROTAC can then be recycled to target another POI. This catalytic process reduces target levels (Sun, X., et al. Sig Transduct Target Ther 4, 64 (2019); Schneider, M., et al. Nat Rev Drug Discov 20, 789-797 (2021)). Thus, FANC degradation inducers include PROTACs that include a ligand for a FANC protein, such as FANCM, FANCD2, or FANCI, linked to a ligand that recruits an E3 ubiquitin ligase (E3). Methods for designing PROTACs are known in the art (see, e.g., Paiva SL, Curr Opin Chem Biol. 2019; 50:111-119).

[0056] FANC degradation inducers include molecular glues, which are linkerless scaffolds that induce proximity through direct ligase-target protein-protein interactions. The molecular glues insert into the natural protein-protein interaction (PPI) interface of the POI, inducing interaction between E3 ubiquitin ligase and the target protein, leading to target protein degradation (Kozicka Z, et al., Cell Chem Biol. 2021; 28(7):1032-1047; Zhao L, et al., Signal Transduct Target Ther. 2022; 7(1):113; Simonetta KR, et al. Nat Commun. 2019; 10(1):1402). Therefore, FANC degradation inducers include molecular glues that induce interaction between FANCM and E3 ubiquitin ligase. Methods for designing molecular glues are known in the art (see, for example, Kozicka Z, et al., Cell Chem Biol. 2021; 28(7):1032-1047).

[0057] Suitable E3 ligase ligands and methods for discovering E3 ligase ligands are known in the art (see, for example, Ishida T, Ciulli A. SLAS Discov. 2021; 26(4):484-502).

[0058] The terms "FANC antagonist" and "FANC inhibitor," as used herein, refer to compounds that antagonize or inhibit components of the FANC / BRCA pathway (FANC proteins), and may include "FANCM antagonists," "FANCM inhibitors," "FANCD2 antagonists," "FANCD2 inhibitors," as well as "FANCI antagonists" and "FANCI inhibitors."

[0059] The terms "FANC antagonist" and "FANC inhibitor" as used herein include FANCM antagonists, including pharmaceutically acceptable salts and solvates of these compounds.

[0060] Suitable antagonists for reducing or suppressing expression of a FANC protein, such as FANCM, FANCD2, or FANCI, include suppressor nucleic acids, targetable nucleases, and nucleic acids encoding such agents. The suppressor nucleic acid or nucleic acid encoding a targetable nuclease may be contained in a vector.

[0061] Suitable expression vectors are known in the art and include viral vectors such as retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, vaccinia vectors or herpes vectors.

[0062] Expression of active FANC protein can be reduced or abolished by a suppressor nucleic acid or targetable nuclease compared to control cells. That is, transcription of the FANC gene and / or translation of FANC mRNA can be reduced or abolished, such that cells treated with the suppressor nucleic acid or targetable nuclease lack or have reduced amounts of active FANC protein compared to control cells. Reducing the amount of active FANC protein to 20% or less of the amount in control cells has been shown to be sufficient to induce cell death. For example, cells can express up to 5%, up to 10%, up to 15%, or up to 20% of the active FANC polypeptide expressed by control cells.

[0063] In some embodiments, nucleic acid suppression can be used to reduce expression of active FANC polypeptides. The use of nucleic acid suppression methods, such as antisense suppression and RNAi suppression, to downregulate expression of target genes is well established in the art.

[0064] Cells may be transfected with a suppressor nucleic acid (i.e., a nucleic acid molecule that suppresses expression of a FANC protein), such as an siRNA or shRNA, or a heterologous nucleic acid encoding the suppressor nucleic acid. The suppressor nucleic acid reduces expression of active FANC polypeptide by interfering with transcription and / or translation, thereby reducing the activity of the FANC polypeptide in the cell.

[0065] RNAi involves expressing or introducing into cells an RNA molecule containing a sequence identical to or highly similar to the FANCM coding sequence. The RNA molecule interacts with mRNA transcribed from the FANCM gene, resulting in sequence-specific degradation of the mRNA or specific post-transcriptional gene silencing (PTGS). This reduces or suppresses the expression of active FANCM polypeptide (Angell & Baulcombe (1997) The EMBO Journal 16, 12:3675-3684; Voinnet & Baulcombe (1997) Nature 389: pg 553).

[0066] The RNA molecule is preferably double-stranded RNA (dsRNA) (Fire A. et al. Nature 391, (1998)). Synthetic siRNA duplexes have been shown to specifically suppress the expression of endogenous and heterologous genes in a wide range of mammalian cell lines (Elbashir SM. et al. Nature, 411, 494-498, (2001)).

[0067] RNA molecules suitable for use in RNAi suppression include short interfering RNAs (siRNAs). siRNAs are double-stranded RNA molecules 15 to 40 nucleotides in length, preferably 15 to 28 nucleotides or 19 to 25 nucleotides in length, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. For example, two unmodified 21-mer oligonucleotides may be annealed together to form siRNA. The siRNA molecule may include a 3' overhang and / or a 5' overhang on each strand, each having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The lengths of the overhangs on these strands are independent; i.e., the length of the overhang on one strand is independent of the length of the overhang on the second strand.

[0068] Other RNA molecules suitable for use in RNAi include small hairpin RNAs (shRNAs). shRNAs are single-stranded RNA molecules that contain or consist of a short (e.g., 19-25 nucleotide) antisense nucleotide sequence, followed by a 5-9 nucleotide loop and a complementary sense nucleotide sequence (e.g., 19-25 nucleotides). Alternatively, the sense sequence may precede the nucleotide loop structure and be followed by the antisense sequence. The nucleotide loop forms a hairpin turn, allowing base pairing between the complementary sense and antisense sequences to form the shRNA.

[0069] A suppressor nucleic acid, e.g., an siRNA or shRNA, can comprise or consist of a sequence identical or substantially identical (i.e., at least 90%, at least 95%, or at least 98% identical) to all or a portion (e.g., 15 to 40 nucleotides) of a reference FANC nucleotide coding sequence, e.g., SEQ ID NO:2, SEQ ID NO:6, or SEQ ID NO:8, or a complementary sequence thereof. Suitable reference sequences encoding FANCM that can be used to design a suppressor nucleic acid have been published and include SEQ ID NO:2. Suitable reference sequences encoding FANCD2 that can be used to design a suppressor nucleic acid have been published and include SEQ ID NO:6. Suitable reference sequences encoding FANCI that can be used to design a suppressor nucleic acid have been published and include SEQ ID NO:8.

[0070] The activity of the FANC protein is suppressed in cancer cells by downregulating the production of active FANCM polypeptide using a suppressor nucleic acid. For example, an siRNA that suppresses the expression of human FANCM may contain 18 to 22 consecutive nucleotides of SEQ ID NO: 2, an siRNA that suppresses the expression of human FANCD2 may contain 18 to 22 consecutive nucleotides of SEQ ID NO: 6, and an siRNA that suppresses the expression of human FANCi may contain 18 to 22 consecutive nucleotides of SEQ ID NO: 8.

[0071] Examples of preferred siRNA molecules for suppressing human FANCM include SEQ ID NO: 3 (siFa) and SEQ ID NO: 4 (siFb). Suitable siRNA molecules are also described in Silva B, et al., Nat Commun. 2019; 10(1):2253. siRNA molecules for suppressing human FANCM are also commercially available (siRNA IDs s33621 and s33619 (ThermoFisher)) and are also described in Lu R, et al., Nat Commun. 2019; 10(1):2252 (a corrected version is published in Nat Commun. 2019 Nov 20; 10(1):5345).

[0072] Suppressor nucleic acids, such as siRNAs and shRNAs, that reduce the expression of FANC proteins can be readily designed using reference FANC coding sequences and software tools widely available in the art and can be produced using conventional techniques. For example, suppressor nucleic acids can be chemically synthesized, recombinantly produced in vitro or intracellularly (Elbashir, SM et al., Nature 411:494-498 (2001); Elbashir, SM, et al., Genes & Development 15:188-200 (2001)), or obtained from commercial sources (e.g., Cruachem (Glasgow, UK), Dharmacon Research (Lafayette, Colo., USA)).

[0073] In some embodiments, two or more suppressor nucleic acids may be used to suppress the expression of the FANC protein. For example, a pool of siRNAs may be used. Suitable siRNAs and pools of siRNAs can be generated using standard techniques.

[0074] Nucleic acid suppression can also be achieved using antisense technology. Antisense oligonucleotides can be designed to hybridize to the complementary sequence of a nucleic acid, pre-mRNA, or mature mRNA, and reduce or completely or substantially completely block its expression by interfering with the production of base excision repair pathway components. In addition to targeting coding sequences, antisense technology can also be used to target gene regulatory sequences, such as regulatory sequences within the 5' flanking sequence, so that antisense oligonucleotides can interfere with expression control sequences. The construction of antisense sequences and their use are known in the art (Peyman and Ulman, Chemical Reviews, 90:543-584, (1990); Crooke, Ann. Rev. Pharmacol. Toxicol. 32:329-376, (1992)).

[0075] Antisense oligonucleotides can be produced in vitro or ex vivo for administration, or antisense RNA can be produced in cancer cells in vivo when desired to downregulate FANC protein.For this reason, double-stranded DNA can be placed under the control of promoter in " reverse orientation", so that the transcription of antisense strand of DNA generates RNA complementary to the normal mRNA transcribed from the sense strand of target gene.In this case, complementary antisense RNA sequence is thought to bind with mRNA to form duplex, and inhibit the translation of endogenous mRNA of target gene into protein.

[0076] It is not necessary to use the entire sequence corresponding to the FANC coding sequence in reverse orientation. For example, any fragment of sufficient length can be used. It is routine for one skilled in the art to screen fragments of various sizes from various portions of the coding sequence or flanking sequence of a gene to optimize the level of antisense inhibition. It may be advantageous to include the initiating methionine ATG codon and perhaps one or more nucleotides upstream of the initiating codon. Suitable fragments may have about 14 to 23 nucleotides, e.g., about 15, 16, or 17 nucleotides.

[0077] In other embodiments, targeted mutagenesis can be used to reduce expression of active FANC polypeptides. The use of targeted mutagenesis methods, such as gene editing, to knock out or eliminate expression of targeted genes is well established in the art (see, e.g., Gaj et al (2013) Trends Biotechnol. 31(7) 397-405).

[0078] One or more mutations, such as insertions, substitutions, or deletions, can be introduced into the FANC gene in cancer cells. Suitable mutations include deletions of all or part of the FANC gene (e.g., one, two, or more exons), frameshift mutations, or nonsense mutations that introduce premature stop codons. In some preferred embodiments, one or more premature stop codons can be introduced into the FANC coding sequence, preferably within the first 400 codons of the coding sequence. For example, a mutation, such as a premature stop codon, can be introduced into the first 400 codons of the FANCM coding sequence to remove the ATPase domain. These mutations can prevent expression of an active FANC polypeptide, for example, by inhibiting transcription or translation of the FANC gene or by causing expression of an inactive polypeptide.

[0079] Targeted mutagenesis for introducing one or more mutations can be carried out by simple method.For example, cancer cell can be transfected with heterologous nucleic acid that encodes targetable nuclease.Targetable nuclease can inactivate the FANC gene that encodes FANC protein in one or more cells of individual, for example, by introducing one or more mutations that prevent the expression of active FANC polypeptide.

[0080] A targetable nuclease can selectively inactivate the FANC gene encoding a FANC protein, such as FANCM, FANCD2, or FANCI, in an individual's cancer cells. The targetable nuclease can be specifically targeted to cancer cells by conventional techniques, including cell-targeting delivery vehicles, such as viral vectors expressing ligands for specific cell types; direct administration of the targetable nuclease to the tumor, for example, by injection; or selective expression of the targetable nuclease from a heterologous nucleic acid in the cancer cells, for example, using a tissue-specific promoter.

[0081] The targetable nuclease may be site-specific (e.g., ZFN or TALEN) or may be expressed using one or more targeting sequences (e.g., CRISPR / Cas) that target the nuclease to the FANCM gene.

[0082] The heterologous nucleic acid encoding the targetable nuclease may comprise an inducible promoter that promotes expression of the targetable nuclease and any targeting sequences in a particular cell type, such as a tumor cell. For example, the inducible promoter can be a promoter-enhancer cassette that preferentially supports expression of the targetable nuclease and any targeting sequences in tumor cells over other types of host cells.

[0083] Suitable targeted nucleases include, for example, site-specific nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases or RNA-guided nucleases, such as clustered regularly interspaced short palindromic repeat (CRISPR) nucleases.

[0084] Zinc finger nucleases (ZFNs) contain one or more Cys2-His2 zinc finger DNA binding domains and a cleavage domain (i.e., nuclease). The DNA binding domain can be modified to recognize and bind to any nucleic acid sequence using conventional techniques (see, for example, Qu et al. (2013) Nucl Ac Res 41(16):7771-7782). The use of ZFNs to introduce mutations into target genes is known in the art (see, for example, Beerli et al. Nat. Biotechnol. 2002; 20:135-141, Maeder et al. Mol. Cell. 2008; 31:294-301, Gupta et al. Nat. Methods. 2012; 9:588-590), and modified ZFNs are commercially available (Sigma-Aldrich (St. Louis, MO)).

[0085] Transcription activator-like effector nuclease (TALEN) comprises a non-specific DNA cleavage nuclease fused to a DNA binding domain, which comprises a series of modular TALE repeats linked to recognize consecutive nucleotide sequences.The use of TALEN targeting nuclease is known in the art (for example, Joung & Sander (2013) Nat Rev Mol Cell Bio 14:49-55, Kim et al. Nat Biotechnol. (2013); 31:251-258, Miller JC, et al. Nat. Biotechnol. (2011) 29:143-148, Reyon D, et al. Nat. Biotechnol. (2012); 30:460-465).

[0086] Meganucleases are endodeoxyribonucleases characterized by large recognition sites (double-stranded DNA sequences of 12 to 40 base pairs), and as a result, this site is generally found only once in any given genome (see, e.g., Silva et al. (2011) Curr Gene Ther 11(1):11-27).

[0087] CRISPR-targeting nucleases (e.g., Cas9) complex with guide RNAs (gRNAs) and cleave genomic DNA in a sequence-specific manner. The guide RNAs, crRNA and tracrRNA, can be used separately or combined into a single RNA to enable site-specific mammalian genome cleavage in the FANCM gene or its regulatory elements. The use of CRISPR / Cas9 systems to introduce insertions or deletions into genes as a method of reducing transcription is known in the art (e.g., Cader et al Nat Immunol 2016 17 (9) 1046-1056, Hwang et al. (2013) Nat. Biotechnol 31:227-229, Xiao et al., (2013) Nucl Acids Res 1-11, Horvath et al., Science (2010) 327:167-170, Jinek M et al. Science (2012) 337:816-821, Cong L et al. Science (2013) 339:819-823, Jinek M et al. (2013) eLife 2:e00471, Mali P et al. (2013) Science 339:823-826, Qi LS et al. (2013) Cell 152:1173-1183, Gilbert LA et al. (2013) Cell 154:442-451, Yang H et al. (2013) Cell 154:1370-1379 and Wang H et al. (2013) Cell 153:910-918).

[0088] In some embodiments, the targetable nuclease is a Cas endonuclease, preferably Cas9, that is expressed in cancer cells in combination with a guide RNA targeting sequence that targets the Cas endonuclease to cleave genomic DNA within the FANC gene, generating an insertion or deletion that prevents expression of an active FANC polypeptide.

[0089] The nucleic acid sequence encoding the suppressor nucleic acid or targetable nuclease, and optionally, the guide RNA, may be included in an expression vector. Suitable vectors can be selected or constructed containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as needed. Preferably, the vector contains appropriate regulatory sequences for driving expression of the encoding nucleic acid in host cells. Regulatory sequences suitable for driving expression of heterologous nucleic acid coding sequences in various expression systems are known in the art and include constitutive promoters, such as viral promoters such as CMV or SV40. In some preferred embodiments, tissue-specific or inducible promoters, such as light-inducible promoters, can be used to selectively express the suppressor nucleic acid or targetable nuclease, and optionally, the guide RNA, in cancer cells. The vector may also contain sequences such as an origin of replication and a selectable marker that allow its selection, replication, and expression in bacterial hosts, such as E. coli, and / or eukaryotic cells, such as yeast, insect, or mammalian cells. Suitable vectors for use in expressing suppressor nucleic acids or targetable nucleases in mammalian cells include plasmids and viral vectors, such as retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Techniques suitable for expressing suppressor nucleic acids or targetable nucleases in mammalian cells are known in the art (see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press, or Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992, Recombinant Gene Expression Protocols Ed. R.S. Tuan (Mar. 1997) Humana Press Inc.).

[0090] Another aspect of the present invention relates to the use of a FANC protein, such as FANCM, FANCD2, or FANCI, to screen for compounds that increase cell death or decrease viability of SETD2-deficient cells and are potentially useful in the treatment of SETD2-deficient cancers.

[0091] The screening method can be used to identify a test compound that binds to an isolated FANC protein. A method for screening a compound that reduces the viability or increases cell death of SETD2-deficient cells can include determining the binding of the test compound to a FANC protein, such as FANCM, FANCD2, or FANCI. For example, the test compound can be contacted with a FANC protein to determine the binding of the test compound to the FANC protein. The binding of the test compound to the FANC protein can indicate that the test compound reduces the viability or increases cell death of SETD2-deficient cells.

[0092] Binding of the test compound to the FANC protein can be determined by standard techniques, such as surface plasmon resonance (SPR).

[0093] In some embodiments, the ability of a test compound to inhibit the interaction of a FANC protein, such as FANCM, FANCD2, or FANCI, with a binding partner can be determined. A method for screening for a compound that reduces the viability or increases cell death of SETD2-deficient cells can include determining the effect of a test compound on the binding of a FANC protein with a binding partner. For example, a FANC protein can be contacted with a binding partner in the presence and absence of a test compound. A reduction in the binding of a FANC protein with a binding partner in the presence of the test compound compared to the absence of the test compound can indicate that the test compound reduces the viability or increases cell death of SETD2-deficient cells.

[0094] Binding partners are proteins that naturally bind to FANC proteins in cells, for example, during homologous recombination, meiosis, and DNA repair. FANCM binding partners can include MHF, FAAP24, HCLK2, BTR complex, and individual members of the BTR complex, such as BLM, RMI1, RMI2, and Topo IIIα. FANCD2 binding partners can include DNA, BRCA2, USP1, and FANCI. FANCI binding partners can include DNA and FANCD2.

[0095] The screening method can be used to identify test compounds that inhibit the activity of FANC protein. The method for screening compounds that reduce the viability of SETD2 cells or increase cell death can include determining the effect of the test compound on the activity of FANC protein. For example, the activity of FANC protein, such as FANCM, FANCD2 or FANCI, can be determined in the presence and absence of the test compound. A decrease in the activity of FANC protein in the presence of the test compound compared to the absence of the test compound indicates that the compound reduces the viability of SETD2-deficient cells or increases cell death.

[0096] In some embodiments, the ATP-dependent DNA helicase / translocase activity of FANCM may be determined in the presence of a test compound compared to its absence. A decrease or reduction in ATP-dependent DNA helicase / translocase activity in the presence of a test compound may indicate that the test compound inhibits the activity of the FANCM protein. For example, the test compound may be a FANCM inhibitor. Suitable methods for determining activity, including ATPase and translocase assays, are known in the art. For example, ATP hydrolysis can be measured in the presence of forked DNA, as described in Coulthard R, et al., Structure. 2013; 21(9):1648-1658. A reduction in ATP hydrolysis may indicate that the test compound reduces or impairs FANCM DNA helicase / translocase activity. Translocase activity can be determined using a D-loop dissociation assay, which tests the dissociation of the D-loop catalyzed by FANCM, as described in Gari K, Decaillet C, Delannoy M, Wu L, Constantinou A. Proc Natl Acad Sci U S A. 2008; 105(42):16107-16112. Reduction or elimination of D-loop dissociation in the presence of the test compound indicates that the test compound can reduce or inhibit the translocase activity of FANCM.

[0097] The exact format of any of the screening or assay methods of the present invention can be varied by those skilled in the art using routine skill and knowledge, and those skilled in the art will be well aware of the need to use appropriate control experiments.

[0098] The FANC protein used in the screening method may be an isolated polypeptide comprising a full-length FANC sequence, e.g., a FANCM reference sequence such as SEQ ID NO: 1 as defined herein, a FANCD2 reference sequence such as SEQ ID NO: 5 as defined herein, a FANCI reference sequence such as SEQ ID NO: 7 as defined herein, or a variant or fragment of any of these sequences. Suitable fragments may comprise at least 50, at least 100, or at least 150 consecutive amino acids of the FANC reference sequence. In some embodiments, a FANCM fragment containing ATPase or translocase activity may be used, e.g., a fragment comprising or consisting of the N-terminal DEAH helicase-like domain corresponding to residues 83-591 of SEQ ID NO: 1. Isolated FANC polypeptides can be produced using standard recombinant techniques.

[0099] The test compound may be an isolated molecule or may be contained in a sample, mixture, or extract, such as a biological sample. Compounds that can be screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programs, and may include, for example, small organic molecules, polypeptides, and nucleic acids, such as aptamers. Extracts of plants, microorganisms, or other organisms containing several characterized or uncharacterized components may also be used.

[0100] Suitable test compounds for screening include compounds that inhibit the ATP-dependent DNA helicase / translocase activity of FANCM. For example, a suitable test compound may be an ATP analog. Suitable test compounds may also be compounds created using rational drug design to obtain test candidate compounds with specific molecular shapes, sizes, and charge characteristics suitable for regulating FANCM activity.

[0101] Combinatorial library technology provides an efficient method for testing a potentially vast number of different compounds for their ability to modulate the activity of FANC proteins. Such libraries and their uses are known in the art, particularly for all kinds of natural products, small molecules, and peptides. The use of peptide libraries may be preferred in certain situations. In some embodiments, libraries of biomolecules, such as aptamer or antibody molecules, may be preferred.

[0102] The amount of test compound that can be added to the assay of the present invention will usually be determined by trial and error depending on the type of compound used. Typically, a putative inhibitor compound can be used at a concentration of about 0.001 nM to 1 mM or more, for example, 0.01 nM to 100 μM, for example, 0.1 μM to 50 μM, for example, about 10 μM. Even compounds with weak effects may be useful lead compounds for further investigation and development.

[0103] Test compounds may include peptides derived from the FANC protein or its binding partners described above. Membrane-permeable peptide fragments of 5 to 40 amino acids, e.g., 6 to 10 amino acids, can be tested for their ability to bind to or inhibit the activity of FANC protein. The regulatory properties of the peptides can be enhanced by adding one of the following groups to the C-terminus: chloromethyl ketone, aldehyde, and boronic acid. These groups are transition-state analogs of serine, cysteine, and threonine proteases. Blocking the N-terminus of the peptide fragment with carbobenzyl can inhibit aminopeptidases and improve stability (Proteolytic Enzymes, 2nd Ed., Edited by R. Beynon and J. Bond, Oxford University Press, 2001).

[0104] Test compounds may include antibodies, antibody fragments and antibody derivatives, as well as non-immunoglobulin binding molecules such as aptamers, trinectins, anticalins, Kunitz domains, transferrin, nurse shark antigen receptor, and sea lamprey leucine-rich repeat proteins.

[0105] In some embodiments, suitable molecules may be directed to the DEAH helicase-like domain corresponding to residues 83-591 of SEQ ID NO:1, or another portion of the FANCM protein.

[0106] Candidate regulatory antibody molecules can be characterized to determine their binding regions, thereby obtaining single-chain antibodies and fragments thereof that inhibit activity or block interaction with a binding partner. Suitable antibodies can be obtained using techniques standard in the art, including, for example, immunizing a mammal with a suitable peptide, such as a fragment of a proinflammatory polypeptide, or isolating specific antibodies from a recombinantly generated library of expressed immunoglobulin variable domains, for example, using lambda or filamentous bacteriophage that display functional immunoglobulin-binding domains on their surface. See, e.g., WO 92 / 01047.

[0107] Aptamers directed against the FANC protein are also putative agents that modulate FANC activity. Aptamers are nucleic acids that specifically bind to target molecules. Typically, aptamers are small nucleic acids ranging from 15 to 50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers are small nucleic acids that bind to target molecules. d is 10 -12 Aptamers can bind very tightly when the kM is less than M. Aptamers can bind to FANC proteins, such as FANCM, FANCI, or FANCD2, with very high specificity. For example, aptamers have been isolated that have binding affinities that differ by more than 10,000 times between a target molecule and another molecule that differs at only one position on the molecule. Aptamers can bind to FANC proteins, such as FANCM, with kM. dis the k with the control polypeptide d The affinity of an aptamer to a target molecule may be at least 10, 100, 1000, 10,000, or 100,000 times lower than the affinity of an aptamer to a target molecule. The generation and use of aptamers is known in the art (see, e.g., Bunka et al Curr Opin Pharmacol 2010 10 (5) 557-562).

[0108] Test compounds identified as inhibiting the activity of a FANC protein, such as FANCM, FANCI, or FANCD2, may be further investigated using one or more secondary screens. Secondary screens may involve testing biological function or activity in vitro and / or in vivo (e.g., in animal models). For example, the ability of a test compound to reduce SETD2 cell viability or increase cell death may be determined. In some embodiments, secondary screens may involve determining the selectivity of a compound for a FANC protein, such as FANCM, by screening against a panel of isolated enzymes or other protein factors.

[0109] The effect of the test compound identified as FANC inhibitor can be determined in vitro on mammalian cells.For example, the effect of the test compound on SETD2-deficient cell line can be determined.Compared with the absence of compound, the increase in cell death in its presence can suggest that the compound shows useful activity in treating SETD2-deficient cancer.

[0110] As described herein, after identifying a FANC inhibitor potentially useful in treating SETD2-deficient cancers, the method can further include modifying the compound to optimize its pharmaceutical properties. Suitable methods of optimization, for example, by structural modeling, are known in the art. Further optimization or modification can then be performed to arrive at one or more final compounds for in vivo or clinical trials.

[0111] Test compounds identified as FANC inhibitors may be isolated and / or purified, or alternatively, synthesized using conventional techniques of recombinant expression or chemical synthesis. They may then be manufactured and / or used in the preparation, i.e., manufacturing or formulating, of compositions such as drugs, pharmaceutical compositions, or medicaments. Thus, the methods described herein may include formulating the test compound with a pharmaceutically acceptable excipient, vehicle, or carrier into a pharmaceutical composition for therapeutic use.

[0112] Although the above-described FANC antagonists, such as FANC inhibitors, degradation inducers, suppressor nucleic acids, targetable nucleases, and nucleic acids encoding suppressor nucleic acids or targetable nucleases, may be administered alone, the FANC antagonists will typically be administered in the form of a pharmaceutical composition that may contain at least one component in addition to the active agent. The FANC antagonists can be mixed with other reagents, such as buffers, carriers, diluents, preservatives, and / or pharmaceutically acceptable excipients, to produce pharmaceutical compositions for use in cancer immunotherapy. Suitable reagents are described in more detail below.

[0113] Aspects of the present invention provide: (i) a pharmaceutical composition comprising a FANC antagonist selected from (a) a FANC inhibitor, (b) a FANC degradation inducer, (c) a FANC suppressor nucleic acid, (d) a FANC targetable nuclease, or (e) a nucleic acid encoding a FANC suppressor nucleic acid or a FANC targetable nuclease, and a pharmaceutically acceptable excipient; and (ii) a method for producing a pharmaceutical composition for use in cancer immunotherapy, e.g., a pharmaceutical composition for treating SETD2-deficient cancer, comprising mixing the FANC antagonist described above with a pharmaceutically acceptable excipient.

[0114] In some embodiments, the FANC antagonist is a FANCM antagonist selected from (a) a FANCM inhibitor, (b) a FANCM degradation inducer, (c) a FANCM suppressor nucleic acid, (d) a FANCM targetable nuclease, (e) a FANCM suppressor nucleic acid or a nucleic acid encoding a FANCM targetable nuclease; (a) a FANCD2 inhibitor, (b) a FANCD2 degradation inducer, (c) a FANCD2 suppressor nucleic acid, (d) a FANCD2 targetable nuclease. a FANCD2 antagonist selected from (a) a FANCI inhibitor, (b) a FANCI degradation inducer, (c) a FANCI suppressor nucleic acid, (d) a FANCI targetable nuclease, (e) a nucleic acid encoding a FANCI suppressor nucleic acid or a FANCI targetable nuclease; or a FANCI antagonist selected from (a) a FANCI inhibitor, (b) a FANCI degradation inducer, (c) a FANCI suppressor nucleic acid, (d) a FANCI targetable nuclease, (e) a nucleic acid encoding a FANCI suppressor nucleic acid or a FANCI targetable nuclease.

[0115] The term "pharmaceutically acceptable," as used herein, refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0116] Pharmaceutical compositions suitable for administration (e.g., by injection) include aqueous and non-aqueous isotonic, pyrogen-free, sterile injectable solutions which may contain antioxidants, buffers, preservatives, stabilizers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Examples of isotonic vehicles suitable for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Suitable vehicles can be found in standard pharmaceutical textbooks, e.g., Remington's Pharmaceutical Sciences, 18 thedition, Mack Publishing Company, Easton, Pa., 1990.

[0117] The FANC antagonists described herein, or pharmaceutical compositions comprising the FANC antagonists, can be administered to a subject by any convenient route of administration, including, but not limited to, parenteral administration, such as by injection (including intravenous injection, particularly intravenous bolus injection), whether systemically / peripherally or to the desired site of action. Suitable injection methods are known in the art and commonly used in therapy (see, e.g., Rosenberg et al., New Eng. J. of Med., 319:1676, 1988).

[0118] Reducing the activity or expression of FANC in SETD2-expressing cells does not affect cell death. Thus, systemic administration, e.g., oral administration or injection, of a FANC antagonist described herein can induce a selective cell death effect on SETD2-deficient cancer cells in an individual relative to other non-SETD2-deficient cells in the individual.

[0119] It is understood that appropriate dosages of FANC antagonists and compositions containing FANC antagonists may vary from patient to patient. Determining the optimal dosage generally requires balancing the level of therapeutic benefit against any risk or adverse side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors, including, but not limited to, the activity of the particular cells, the route of administration, the time of administration, the rate of cell loss or inactivation, the duration of treatment, other drugs, compounds, and / or materials used in combination, and the patient's age, sex, weight, condition, general health, and previous medical history. The amount of cells and the route of administration are ultimately at the discretion of the physician, but generally, the dosage will be that amount required to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harm or adverse side effects.

[0120] Typical oral dosages of small molecule inhibitors range from about 0.05 mg to about 1000 mg, preferably about 0.1 mg to about 500 mg, and more preferably about 1.0 mg to about 200 mg, administered in one or more doses, such as one to three doses. The exact dosage will depend on the frequency and mode of administration, the sex, age, weight, and general condition of the subject being treated, the nature and severity of the condition being treated and any complications being treated, and other factors apparent to those skilled in the art. For parenteral routes, such as intravenous, intrathecal, intramuscular, and similar administration, dosages are typically about half of those used for oral administration.

[0121] FANC antagonists described herein, such as (a) FANC inhibitors, (b) FANC degradation inducers, (c) FANC suppressor nucleic acids, (d) FANC targetable nucleases, or (e) nucleic acids encoding FANC suppressor nucleic acids or FANC targetable nucleases, can be useful in therapy. For example, a FANC antagonist that reduces the activity of FANC protein can be administered to an individual to treat SETD2-deficient cancers.

[0122] Methods according to some aspects of the invention can include determining binding of an oligonucleotide probe to nucleic acid, e.g., genomic DNA, RNA, or cDNA, obtained from a sample. The probe can comprise a nucleotide sequence that specifically binds to nucleic acid sequences that contain one or more mutations or polymorphisms and does not specifically bind to nucleic acid sequences that do not contain the one or more mutations or polymorphisms, or vice versa.

[0123] The oligonucleotide probe may contain a label, and binding of the probe may be determined by detecting the presence of the label.

[0124] The method may include hybridizing one or more (e.g., two) oligonucleotide probes or primers to the target nucleic acid. If the nucleic acid is double-stranded DNA, hybridization will generally be preceded by denaturation to produce single-stranded DNA. Hybridization may be performed as part of a PCR method or as part of a probing method without PCR. An illustrative approach would be a combination of PCR and low stringency hybridization.

[0125] Binding of a probe to a target nucleic acid (e.g., DNA) can be measured using any of a variety of techniques freely available to those skilled in the art. For example, probes may be radioactively, fluorescently, or enzyme-labeled. Other methods that do not involve labeling the probe include restriction fragment length polymorphism testing, PCR amplification, RNase cleavage, and allele-specific oligonucleotide probing. Probing can also be performed using standard Southern blotting techniques. For example, DNA can be extracted from cells and digested with various restriction enzymes. Restriction fragments can then be separated by agarose gel electrophoresis, followed by denaturation and transfer to a nitrocellulose filter. Labeled probes can be hybridized to the DNA fragments on the filter, and binding can be determined.

[0126] Those skilled in the art can apply appropriate conditions of stringency desired for selective hybridization, taking into account factors such as length and base composition of the oligonucleotide, temperature, and the like.

[0127] Selective hybridization conditions suitable for oligonucleotides of 17 to 30 bases include overnight hybridization in 6xSSC at 42°C, followed by washing in 6xSSC at temperatures gradually increasing from 42°C to 65°C.

[0128] Other suitable conditions and protocols are described in Molecular Cloning: a Laboratory Manual: 3 rd edition, Sambrook & Russell (2001) Cold Spring Harbor Laboratory Press, NY, and Current Protocols in Molecular Biology, Ausubel et al. eds. John Wiley & Sons (1992).

[0129] Cancer is characterized by the abnormal proliferation of malignant cancer cells compared to normal cells, and may include leukemias such as AML, CML, ALL, and CLL, lymphomas such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma, as well as solid cancers such as sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, e.g., glioblastoma multiforme, such as pediatric glioblastoma multiforme, breast cancer, uterine cancer, oral cancer, ovarian cancer, prostate cancer, lung cancer, e.g., lung adenocarcinoma and lung squamous cell carcinoma, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, renal cancer, e.g., clear cell renal carcinoma, gastric cancer, testicular cancer, gallbladder and biliary tract cancer, thyroid cancer, thymic cancer, bone cancer, and cerebral cancer. Cancers may be familial or sporadic. SET2D-deficient cancers suitable for treatment as described herein may be any of these types of cancer in which SET2D expression or activity is deficient.

[0130] Another aspect of the present invention relates to identifying individuals with cancer who are suitable for treatment with a FANC antagonist described herein, such as (a) a FANC inhibitor, (b) a FANC degradation-inducing agent, (c) a FANC suppressor nucleic acid, (d) a FANC-targetable nuclease, or (e) a nucleic acid encoding a FANC suppressor nucleic acid or a FANC-targetable nuclease. For example, an individual with cancer can be evaluated using the methods described herein to determine whether treatment with a FANC antagonist is likely to benefit the individual, i.e., whether the individual is suitable for treatment according to the first aspect of the present invention. A method for predicting, determining, or assessing the responsiveness of a cancer in an individual to a FANC antagonist that reduces the expression or activity of a FANC protein can include determining the presence of one or more SET2D-deficient cancer cells in a sample of cancer cells from the individual, where the presence of one or more SET2D-deficient cancer cells in the sample indicates that the cancer will be responsive to the FANC antagonist.

[0131] Individuals with cancer who are identified as being responsive to a FANC antagonist can be treated as described herein, for example, using the method of the first aspect of the invention.

[0132] An individual suitable for treatment with a FANC antagonist described herein, such as (a) a FANC inhibitor, (b) a FANC degradation inducer, (c) a FANC suppressor nucleic acid, (d) a FANC targetable nuclease, or (e) a nucleic acid encoding a FANC suppressor nucleic acid or a FANC targetable nuclease, can be a mammal, such as a rodent (e.g., a guinea pig, hamster, rat, mouse), murine (e.g., a mouse), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), primate, simian (e.g., a monkey or ape), monkey (e.g., a marmoset, baboon), ape (e.g., a gorilla, chimpanzee, orangutan, gibbon), or human.

[0133] In some preferred embodiments, the individual is a human. In other preferred embodiments, a non-human mammal, particularly a mammal traditionally used as a model for demonstrating therapeutic efficacy in humans (e.g., a murine, primate, porcine, canine, or lagomorph) can be used.

[0134] In some embodiments, the individual may have minimal residual disease (MRD) after initial cancer treatment.

[0135] The term "treatment", when used herein in relation to the treatment of a condition, generally relates to treatments and therapies that achieve some desired therapeutic effect, such as inhibiting the progression of the condition, including slowing the rate of progression, halting the rate of progression and ameliorating the condition, and curing the condition.

[0136] Treatment can be any treatment or therapy, whether in humans or animals (e.g., for veterinary use), that achieves some desired therapeutic effect, such as inhibiting or slowing the progression of a condition, including slowing the rate of progression, halting the rate of progression, ameliorating the condition, curing or remission (whether partial or complete) of the condition, preventing, delaying, alleviating or arresting one or more symptoms and / or signs of the condition, or prolonging the survival of a subject or patient beyond that expected in the absence of treatment.

[0137] Treatment as a preventative measure (i.e., prophylaxis) is also included. For example, an individual susceptible to or at risk of developing or recurring cancer can be treated as described herein. Such treatment may prevent or delay the development or recurrence of cancer in the individual.

[0138] In particular, treatment can include inhibiting cancer growth, including complete remission of cancer, and / or inhibiting cancer metastasis. Cancer growth generally refers to any one of a number of indicators that indicate a change in cancer to a more advanced form. Thus, indicators for measuring inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumor volume or morphology (e.g., determined using computed tomography (CT), ultrasound, or other imaging methods), slowing tumor growth, destruction of tumor vasculature, improved performance in delayed-type skin hypersensitivity tests, increased activity of cytolytic cancer cells, and a decrease in the level of tumor-specific antigens.

[0139] It is shown herein that reducing the expression or activity of FANC protein has a potent and specific effect on SETD2-deficient cancer cells. Thus, in some preferred embodiments, a FANC antagonist described herein, such as (a) a FANC inhibitor, (b) a FANC degradation-inducing agent, (c) a FANC suppressor nucleic acid, (d) a FANC targetable nuclease, or (e) a nucleic acid encoding a FANC suppressor nucleic acid or a FANC targetable nuclease, can be administered to an individual without concomitant other cancer therapies, such as cytotoxic chemotherapy or radiation therapy, i.e., the FANC antagonist can be administered alone.

[0140] In other embodiments, a FANC antagonist described herein, such as (a) a FANC inhibitor, (b) a FANC degradation inducer, (c) a FANC suppressor nucleic acid, (d) a FANC targetable nuclease, or (e) a nucleic acid encoding a FANC suppressor nucleic acid or a FANC targetable nuclease, may be administered in combination with one or more other therapies, such as cytotoxic chemotherapy or radiation therapy. This can be useful, for example, in treating cancers that contain both SETD2-deficient and SETD2-positive cancer cells, or cancers where SETD2 status has not been determined.

[0141] When FANC antagonist is used in combination with additional therapeutic agent, the compounds can be administered sequentially or simultaneously by any convenient route.When FANC antagonist is used in combination with additional therapeutic agent that is active against the same disease, the dosage of each active substance in the combination may be different from that when FANC antagonist is used alone.Appropriate dosage is easily understood by those skilled in the art.

[0142] Administration of the FANC antagonists described herein, such as (a) FANC inhibitors, (b) FANC degradation inducers, (c) FANC suppressor nucleic acids, (d) FANC targetable nucleases, or (e) nucleic acids encoding FANC suppressor nucleic acids or FANC targetable nucleases, can be achieved in a single administration, continuously, or intermittently (e.g., in divided administrations at appropriate intervals) throughout the course of treatment. Methods for determining the most effective means of administration and dosage are known to those skilled in the art and vary depending on the formulation used for therapy, the purpose of therapy, the target cells being treated, and the subject being treated. Single or multiple administrations can be used, with the dosage level and pattern being selected by the treating physician.

[0143] Other aspects and embodiments of the present invention provide aspects and embodiments described above where the term "comprising" is replaced with the term "consisting of," as well as aspects and embodiments described above where the term "comprising" is replaced with the term "consisting essentially of."

[0144] It is understood that the present application discloses all combinations of any of the above aspects and embodiments described above with each other unless the context requires otherwise. Likewise, the present application discloses all combinations of preferred and / or optional features alone or with any other aspect unless the context requires otherwise.

[0145] Variations of the above embodiments, further embodiments and variations thereof will be apparent to those skilled in the art upon reading this disclosure and as such are within the scope of the present invention.

[0146] All literature and sequence database entries referred to herein are incorporated by reference in their entirety for all purposes.

[0147] "And / or," as used herein, is to be understood as a specific disclosure of two specified features or components, each with or without the other. For example, "A and / or B" is to be understood as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0148] Other aspects and embodiments of the present invention provide aspects and embodiments described above where the term "comprising" is replaced with the term "consisting of," as well as aspects and embodiments described above where the term "comprising" is replaced with the term "consisting essentially of."

[0149] The term "downstream," as used herein, refers to the 5' to 3' direction in the nucleic acids described herein, and the term "upstream," as used herein, refers to the 3' to 5' direction in the nucleic acids described herein.

[0150] Reference to a nucleotide sequence defined herein, unless the context requires otherwise, encompasses DNA molecules having the specified sequence and also encompasses RNA molecules in which T in the specified sequence is replaced by U.

[0151] It is understood that the present application discloses all combinations of any of the above aspects and embodiments described above with each other unless the context requires otherwise. Likewise, the present application discloses all combinations of preferred and / or optional features alone or with any other aspect unless the context requires otherwise.

[0152] Variations of the above embodiments, further embodiments and variations thereof will be apparent to those skilled in the art upon reading this disclosure and as such are within the scope of the present invention.

[0153] All literature and sequence database entries referred to herein are incorporated by reference in their entirety for all purposes.

[0154] "And / or," as used herein, is to be understood as a specific disclosure of two specified features or components, each with or without the other. For example, "A and / or B" is to be understood as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein. [Example]

[0155] experiment Simultaneous loss of FANCM and SET2D leads to cancer cell death SET domain-containing protein 2 (SETD2) is a histone methyltransferase that is frequently mutated in several types of cancer. We aimed to test the effect of genetic ablation of FANCM on the survival of SETD2-deficient cancer cells.

[0156] Wild-type (WT) and CRISPR / Cas9 SETD2 knockout (KO) clear cell renal cell carcinoma (Caki2) cells were transfected with control (Ctrl) or FANCM siRNA. After 6 days of culture, SETD2-KO Caki2 cells transfected with FANCM-depleting siRNA showed a significant decrease in cell density, suggesting increased cell death in these cells (Figure 1).

[0157] To test whether simultaneous deletion of FANCM and SET2D results in increased cell death, we transfected WT and CRISPR / Cas9 SETD2 knockout (KO) clear cell renal cell carcinoma cells (786-O cell line) with control (Ctrl) or FANCM siRNA. Only 3 days after knockdown, SETD2-KO 786-O cells transfected with siRNA specifically depleting FANCM showed a significant loss of viability, as evidenced by increased PI staining levels detected in flow cytometry experiments (Figure 2). A further increase in specific cell death was observed 6 days after knockdown in SETD2-KO 786-O cells transfected with siRNA specifically depleting FANCM (Figure 3).

[0158] Simultaneous loss of FANCD2 and SET2D leads to cancer cell death To test whether co-deletion of FANCD2 and SET2D leads to increased cell death, we transfected WT (ACHN and Caki-1 cell lines) and SETD2-deficient (RCC-AB, RCC-ER, and RCC-MF cell lines) cells with control (Ctrl) or FANCD2 siRNA. Only 3 days after knockdown, SETD2-mutant RCC-AB, RCC-ER, and RCC-MF cell lines transfected with siRNA specifically depleting FANCD2 showed a significant loss of viability, as evidenced by increased PI staining levels detected by flow cytometry (Figure 4A). A further increase in specific cell death was observed 6 days after knockdown in SETD2-mutant RCC-AB, RCC-ER, and RCC-MF cell lines transfected with siRNA specifically depleting FANCD2 (Figure 4B).

[0159] These results suggest that the simultaneous loss of (i) FANCM or FANCD2 and (ii) SETD2 results in a synthetic lethal phenotype leading to cancer cell death. This synthetic lethal phenotype may be utilized as a monotherapy for cancers with somatic loss of SETD2 or may be administered in addition to chemotherapy and radiation therapy for the treatment of aggressive or recurrent cancers. This also represents a promising approach for the treatment of metastatic cancers, where treatment options are limited.

[0160] array 1 msgrqrtlfq twgssisrss gtpgcssgte rpqspgsska plpaaaeaql esdddvllva 61 ayeaerqlcl enggfctsag alwiyptncp vrdyqlhisr aalfcntlvc lptglgktfi 121 aavvmynfyr wfpsgkvvfm aptkplvtqq ieacyqvmgi pqshmaemtg stqastrkei 181 wcskrvlflt pqvmvndlsr gacpaaeikc lvideahkal gnyaycqavq qvitnlligq 241 ielrsedspd iltysherkv eklivplgee laaiqktyiq ilesfarsli qrnvlmrrdi 301 pnltkyqiil ardqfrknps pnivgiqqgi iegefaicis lyhgyellqq mgmrslyffl 361 cgimdgtkgm trsknelgrn edfmklynhl ecmfartrst sangisaiqq gdknkkfvys 421 hpklkkleev viehfkswna enttekkrde trvmifssfr dsvqeiaeml sqhqpiirvm 481 tfvghasgks tkgftqkeql evvkqfrdgg yntlvstcvg eegldigevd liicfdsqks 541 pirlvqrmgr tgrkrqgriv iilsegreer iynqsqsnkr siykaissnr qvlhfyqrsp 601 rmvpdginpk lhkmfithgv yepekpsrnl qrkssifsyr dgmrqsslkk dwflseeefk 661 lwnrlyrlrd sdeikeitlp qvqfsslqne enkpaqestt gihqlslsew rlwqdhplpt 721 hqvdhsdrcr hfiglmqmie gmrheegecs yelevesylq medvtstfia prnesnnlas 781 dtfithkkss fikninqgss ssviesdeec aeivkqthik ptkivslkkk vskeikkdql 841 kkennhgiid svdndrnstv enifqedlpn dkrtsdtdei aatctinenv ikepcvllte 901 cqftnkstss lagnvldsgy nsfndeksvs snlflpfeee lyivrtddqf ynchsltkev 961 lanverflsy sppplsglsd leyeiakgta lenllflpca ehlrsdkctc llshsavnsq 1021 qnlelnslkc inypsekscl ydipndnisd epslcdcdvh khnqnenlvp nnrvqihrsp 1081 aqnlvgennh dvdnsdlpvl stdqdeslll fedvntefdd vslsplnsks eslpvsdkta 1141 isetplvsqf lisdellldn nselqdqitr dansfksrdq rgvqeekvkn hedifdcsrd 1201 lfsvtfdlgf cspdsddeil ehtsdsnrpl ddlygrylei keisdanyvs nqaliprdhs 1261 knftsgtvii psnedmqnpn yvhlplsaak neellspgys qfslpvqkkv mstplsksnt 1321 lnsfskirke ilktpdsske kvnlqrfkea lnstfdysef slekskssgp mylhkschsv 1381 edgqlltsne seddeifrrk vkrakgnvln spedqknsev dsplhavkkr rfpinrsels 1441 ssdesenfpk pcsqledfkv cngnarrgik vpkrqshlkh varkflddea elseedaeyv 1501 ssdendesen eqdsslldfl ndetqlsqai ndsemraiym kslrspmmnn kykmihkthk 1561 ninifsqipe qdetyledsf cvdeeesckg qsseeevcvd fnlitddcfa nskkyktrra 1621 vmlkemmeqn cahskkklsr iilpddssee ennvndkres niavnpstvk knkqqdhcln 1681 svpsgssaqs kvrstprvnp lakqskqtsl nlkdtisevs dfkpqnhnev qsttppfttv 1741 dsqkdcrkfp vpqkdgsale dsstsgascs ksrphlagth tslrlpqegk gtcilvgghe 1801 itsgleviss lraihglqve vcplngcdyi vsnrmvverr sqsemlnsvn knkfieqiqh 1861 lqsmfericv ivekdrektg dtsrmfrrtk sydsllttli gagirilfss cqeetadllk 1921 elslveqrkn vgihvptvvn snksealqfy lsipnisyit alnmchqfss vkrmansslq 1981 eismyaqvth qkaeeiyryi hyvfdiqmlp ndlnqdrlks di Sequence number 1 1 tgtgcgaagg aaaccgatgg ggatcggaac cgtagcggtt gagctgctgc tgctacggat 61 atctgacaga agccttcggt ggttgtcggc ctaatgagcg gacggcaaag aacgcttttt 121 cagacgtggg gctcaagtat ctcccgatca tctgggactc cgggttgcag ctccggaact 181 gagcgacctc agagccctgg cagctccaag gcgcctttgc cagcagcagc ggaggctcag 241 ctggagtcgg acgatgatgt gttgcttgtc gcggcgtacg aggctgagcg gcagttgtgt 301 ctagagaatg gcgggttctg cacctccgcg ggcgccctgt ggatttaccc taccaattgc 361 ccagtgcggg actaccagct gcacatttcc cgggctgctc tgttttgcaa tacgctggtg 421 tgtctgccta ccggactggg aaagaccttt attgccgccg tggtcatgta caatttctac 481 cgctggttcc cttcaggaaa ggtggtcttc atggccccaa cgaaaccctt ggtgacacag 541 cagatcgagg cttgctacca ggtgatgggt atcccgcaat cccacatggc cgaaatgaca 601 gggtctacac aagcttccac caggaaggaa atatggtgca gtaagagagt gctttttctt 661 acacctcagg tcatggtaaa tgacctttct agaggagctt gtcccgctgc tgaaataaag 721 tgtttagtta ttgatgaagc tcataaagct ctcggaaact atgcttattg ccaggctgtg 781 caacaagtta ttactaacct gctaattggg cagatagagc ttcgttctga agattctcca 841 gatattttga catattctca tgaaagaaaa gttgaaaagc ttattgttcc gcttggtgaa 901 gaacttgcag ccatccaaaa gacctatatc cagatttgg aatcatttgc tcgttctttg 961 attcagagga atgttttgat gagaagggat atcccaaatc taacaaaata tcagataatt 1021 ctggcaag atcagttg gaaaaaccca tctccgaata tgtgggaat acaaaggc 1081 ataatcgagg gagagtttgc tatttgttatt agtttatc atggttatga attattgcag 1141 CAATGGAA TGAGATTATTATTCTTC CTTTGTGAA TTATTGATG ACTAAGgg 1201 atgacacggt siaaaatga acttggccga atgagact tcatgaact ctataatcat 1261 ctagagtgta tgtttgcacg tacacgtagt acttcagcaa atggtatttc tgctatccaa 1321 caggaata aaaaaaaa atttgtttat agtcatcha agttaagaa attagagaa 1381 gttgtattg aacactca gtcatggaat gctgaaaca ctactgaaaa gaacgtgat 1441 gagacccgag ttgatctt ctctcattt cgagatagtg ttcaagaat tgcagaatg 1501 ctttcacagc atcagccaat tattagagta atgactttg tcggcatgc ctcagggaa 1561 spy gttttaccca gaaggagcaa ctggaggtag tgaacagtt tcgtgacggt 1621 ggttacaca cgctggtttc tactgtgtg ggtgaagaag gtttggatat aggagaagtt 1681 gatcttataa tatgttttga ttcccagaag agcccaatc gtcttgtaca acgaatgggt 1741 agaactggcc gtaaacgtca agggaggata gttattacc tttctgagg acgagaggaa 1801 cgttattatta atcagtca gtccacaaa agagtatat ataagctat ttcagtaac 1861 Aggcaggtcc Tcatttta ccaagaagt ccacgaatgg ttccgatgg aatcaaccca 1921 aattacaca aaatgttcat cacacatgt gtctatgac cagagaagcc tctcggaac 1981 ttgcagcgaa agtcatctat cttttcctat agggatgga tgaggcaag tagcctaag 2041 aagattggt tcttacaga agaagaattt aaatttagga acagacttta tagattagg 2101 gaagtgatg aaattaaga gataacattg cctcaagttc agttttctc tttacaaat 2161 gaggaaaaaccagctca agaatcacc actggaattc atcaactc tctctctgaa 2221 tggactgt ggcagatca tcctttgct accacatcag tgatcactc agatcgatgc 2281 cgccatttta taggccttat gcaatgata gagggaatga cgacgaga gggagaatgc 2341 agctatgaat tggagttga atctttatta caatggaag atgttaccctc aacatttatt 2401 gctcccagga atgaatctaa taatcttgcc agtgacacct attcactca caagaatcg 2461 tcatttataa agaacataaa tcaaggcagt tcatcctcag tgatagaatc tgatgaagaa 2521 tgtgctgaaa ttgttaaaca aactcatatc aaacctacta aaattgtttc tttaaagaaa 2581 aaagtgtcta aagaaataaa aaaagatcag cttaaaaaag aaaataatca cggtattata 2641 gattctgtag ataatgacag aaattccact gttgaaaata tttttcaaga agacctacca 2701 aatgataaaa ggacatcaga tacagatgaa attgctgcca catgtactat taatgaaaat 2761 gttattaaag aaccgtgtgt gttattaaca gagtgtcagt ttacaaataa atccactagt 2821 tcacttgctg gaaatgtttt agattctggt tataacagtt tcaatgatga aaaatctgtt 2881 tcatctaact tatttcttcc attcgaagaa gagctttata ttgttagaac agatgaccaa 2941 ttttataatt gtcactcatt gacaaaagag gtactagcta atgtagagag atttttatct 3001 tattctcctc cgcctctcag tggactctca gacttggaat atgaaattgc taagggtact 3061 gcacttgaga atttgctttt cttaccctgt gcagagcatt tacgaagtga taaatgcacc 3121 tgtttgctgt cacattcagc tgtgaattct caacagaatt tagaattgaa ttcacttaaa 3181 tgtataaatt atccatctga aaaaagttgc ctttatgata tacctaatga taatatttct 3241 3301 cctaacaatc gtgttcaaat acacagaagc cctgcacaga atttagttgg agagaacaat 3361 catgatgttg ataagatga cctcccagta ttgtccactg ataagatga aagtttgctg 3421 ttatttgaag atgttaatac agagttcgac gatgtgagtc tttcaccctt gaagaca 3481 agcgaatctt tacctgtgtc agaaaact gctattagtg aaacgcctct ggtctctcag 3541 ttcttaattt ctgatgaact tttgttggac aataattctg aactccaaga tcaaatcacc 3601 cgtgatgcta atagttttaa atctcgtgat cagagaggtg tacagaga aaaagtgaag 3661 aatcatgagg atatttttga ttgctctagg gatttttt ctgttacctt tgatttagga 3721 3781 3841 tcgaatcaag cactaatacc aagagatcat agtaaaaatt ttactagtgg aactgttat 3901 atcccatcaa atgaagatat gcagaatcca aattatgtac atttgccact gagtgcagca 3961 aaaaatgaag aattgttatc tcctggttat tctcagttt ctttaccagt gcaaaaaaaa 4021 4081 gaatactta agacaccaga ttctagtaag gaaaagtaa acctacaaag attcaaaga 4141 gcattgaatt caacttttga ttattcagaa ttttctctag aaaagtctaa aagcagtggt 4201 4261 4321 aactctcctg aggatcagaa aaatagtgaa gttgattctc cacttcatgc tgtcaaaaag 4381 cgcagatttc ctataaacag atcagaatta tcatctagtg atgagagtga gaatttttcc 4441 aaaccatgtt cacaattaga agacttcaag gtttgtaacg ggaatgccag aagaggcatc 4501 4561 4621 atgaacaag attcctcatt acttgactt ttaatgatg aaaccact ttcacaggct 4681 aaatgatt ctgaatgag agctatttac atgaatctt tgcgtagtcc atgatgaac 4741 ataagtaca aaatgatca taagacaca aaaaactaa acatttctc gcagattcct 4801 gaacagatg aaacctattt agagatagt tttgtgttg atgagagga gtcttgcaaa 4861 ggccaatcaa gtgaagaga agtttgtgtt gatttttact taatactga tgattgcttt 4921 gcaatagta aaagtataa aactcgacgt gcagtaatgc tAAagaat gatgaacaa 4981 aattgtgcac attcaaaaa gaaattatcc agaattattt taccagatga ttcaagtgag 5041 gaggaaca atgtaatga taaaagagaa tctaatattg cggttaaccc aagcactgtt 5101 aagagaaaaaacagga ccattgttta attcagtgc cttctggatc tctcgcag 5161 tccaggtgc gttctactcc aagagttaat ccattagcaa agcagagcaa acagacatcg 5221 ctgaatttaa aggatacaat ttccgaagtc tcagactca aacctcagaa tcataatgaa 5281 gtccagtcta ccaccacc cttcactact gttgatcac agaagactg tagaaattt 5341 ccagttccac agaaggatgg tagtgctttg gaggattcta gcacttcagg ggcatcctgt 5401 tccaagtcaa gaccacattt agctgggaca catactttc ttagacttcc gcaggaagga 5461 aaaggaacct gtattcttgt aggtggtcat gaaatcactt ctggattaga agtaatttct 5521 tccctaagag caattcatgg gttgcaagta gaagtttgtc ctcttaatgg ctgtgattac 5581 atcgtgagta atcgcatggt ggtggaaagg aggtctcaat ctgagatgtt aaatagtgtc 5641 aataagaaca agttcattga gcagatccag cacctgcaga gtatgtttga aagaatatgt 5701 gtgattgtgg aaaaggacag agaaaaaaca ggagacacat caaggatgtt taggagaaca 5761 aagagctatg acagcctgct gactacctta attggcgctg gaatccgaat tcttttcagt 5821 tcctgccaag aagaaaccgc agatttgcta aaggaactgt ctttagtgga acaaagaaag 5881 aatgttggta ttcatgttcc aacagtggtg aatagtaata aaagtgaggc actccagttt 5941 tatttaagta ttcccaatat aagttatata actgcattaa atatgtgtca ccagttttca 6001 tctgtgaaaa ggatggctaa cagctcactt caagaaatct ccatgtatgc acaagtaact 6061 catcagaagg ctgaggagat ctatagatat attcactatg tatttgacat acaaatgtta 6121 ccaaatgatc ttaaccaaga tagactgaaa tctgatatat aatcaagctg ctcaagatgg 6181 ggttttcaaa gacctctcac aatattaaat gcacttcaat aatcattgct gttttatgtt 6241 tatttgtaaa taagagaata ttttatttaa atattttata ttgtatacat ttttatttat 6301 agattaga aattattaaa aaagaaaaat ctgatgttca gtgatcattt tgactagatt 6361 ataaaactaa ttttcttat taataaaac aaggtttatt aaaagtgtta ctaaggatag 6421 tttaagaaag taaaagctaa gctagagata tactttggaa tgtttcccaa aattaaagtt 6481 gtactgttgt gataaatagt aaagttgaca tgtctatgac tacagccaac ttgtcgattt 6541 tccctatgtg tagatagtat acttttaagt gtactgattc taaatacatg tacttggtaa 6601 ggtgtgggtg atgggtgggt tgtgagataa atgacccagt aactaggaaa gtagaaaact 6661 taactgaatg tttatctgac caaaggtgtg tcccagttaa gtactgtcaa atctattaat 6721 atgaactctg atatggtttg gctgtgtccc caaccaaaat ctcatcttga cttgtaatct 6781 gaattataat cccaatatat tggggaggga cctcctggaa cgtgattagc tcatgggggc 6841 ggttccccca tgctgttcta gtgatagttc tcagaggatc tgatggtttt ataagctttt 6901 cctctgttca ctctgcagtt ctcttgccta ctgccatgtg gaaaaggaaa cgtttgcttc 6961 ccctccacca tgattgtaag ttcccgaggc ctccccagcc atgcaggact gtgagtcaat 7021 taaacatctt ttccttataa attaaaaaaa aaaaaaaaaa SEQ ID NO: 2 GGATGTTTAGGAGAACAAAGAGCTA SEQ ID NO: 3 (siFa) CCCATCAAATGAAGATATGCAGAAT SEQ ID NO: 4 (siFb) 1 mvskrrlsks edkeslteda sktrkqplsk ktkkshiane veendsifvk llkisgiilk 61 tgesqnqlav dqiafqkklf qtlrrhpsyp kiieefvsgl esyiededsf rncllscerl 121 qdeeasmgas yskslikllll gidilqpaii ktlfeklpey ffenknsdei niprlivsql 181 kwldrvvdgk dlttkimqli siapenlqhd iitslpeilg dsqhadvgke lsdllients 241 ltvpildvls slrldpnfll kvrqlvmdkl ssirledlpv iikfilhsvt amdtlevise 301 lrekldlqhc vlpsrlqasq vklkskgras ssgnqessgq sciillfdvi ksaiyekti 361 seawikaien tasvsehkvf dlvmlfiiys tntqtkkyid rvlrnkirsg ciqeqllqst 421 fsvhylvlkd mcssilslaq sllhsldqsi isfgsllyky afkffdtycq qevvgalvth 481 icsgneaevd taldvllelv vlnpsammmn avfvkgildy ldnispqqir klfyvlstla 541 fskqneassh iqddmhlvir kqlsstvfky kligiigavt magimaadrs espsltqera 601 nlsdeqctqv tsllqlvhsc seqspqasal yydefanliq hekldpkale wvghticndf 661 qdafvvdscv vpegdfpfpv kalygleeyd tqdgiainll pllfsqdfak dggpvtsqes 721 gqklvsplcl apyfrllrlc verqhngnle eidglldcpi fltdlepgek lesmsakers 781 fmcslifltl nwfreivnaf cqetspemkg kvltrlkhiv elqiilekyl avtpdyvppl 841 gnfdvetldi tphtvtaisa kirkkgkier kqktdgskts ssdtlseekn secdptpshr 901 gqlnkeftgk eektslllhn shaffreldi evfsilhcgl vtkfildtem hteatevvql 961 gppellflle dlsqklesml tppiarrvpf lknkgsrnig fshlqqrsaq eivhcvfqll 1021 tpmcnhleni hnyfqclaae nhgvvdgpgv kvqeyhimss cyqrllqifh glfawsgfsq 1081 penqnllysa lhvlssrlkq gehsqpleel lsqsvhylqn fhqsipsfqc alylirllmv 1141 ilekstasaq nkekiaslar qflcrvwpsg dkeksnisnd qlhallciyl ehtesilkai 1201 eeiagvgvpe linspkdass stfptltrht fvvffrvmma elektvkkie pgtaadsqqi 1261 heekllywnm avrdfsilin likvfdshpv lhvclkygrl fveaflkqcm plldfsfrkh 1321 redvlsllet fqldtrllhh lcghskihqd trltqhvpll kktlellvcr vkamltlnnc 1381 reafwlgnlk nrdlqgeeik sqnsqestad eseddmssqa skskatedge edevsageke 1441 qdsdesydds d sequence number 5 1 gggaaagtcg aaaactacgg gcggcgacgg cttctcggaa gtaattttaag tgcacaagac 61 attggtcaaa atggtttcca aaagaagact gtcaaaatct gaggataaag agagcctgac 121 agaagatgcc tccaaaacca ggaagcaacc actttccaaa aagacaaaga aatctcatat 181 tgctaatgaa gttgaagaaa atgacagcat ctttgtaaag cttcttaaga tatcaggaat 241 tattcttaaa acgggagaga gtcagaatca actagctgtg gatcaaatag ctttccaaaa 301 gaagctcttt cagaccctga ggagacaccc ttcctatccc aaaataatag aagaatttgt 361 tagtggcctg gagtcttaca ttgaggatga agacagtttc aggaactgcc ttttgtcttg 421 tgagcgtctg caggatgagg aagccagtat gggtgcatct tattctaaga gtctcatcaa 481 actgcttctg gggattgaca tactgcagcc tgccattatc aaaaccttat ttgagaagtt 541 gccagaatat ttttttgaaa acaagaacag tgatgaaatc aacatacctc gactcattgt 601 cagtcaacta aaatggcttg acagagttgt ggatggcaag gacctcacca ccaagatcat 661 gcagctgatc agtattgctc cagagaacct gcagcatgac atcatcacca gcctacctga 721 gatcctaggg gattcccagc acgctgatgt ggggaaagaa ctcagtgacc tactgataga 781 gaatacttca ctcactgtcc caatcctgga tgtcctttca agcctccgac ttgacccaaa 841 cttcctattg aaggttcgcc agttggtgat ggataagttg tcgtctatta gattggagga 901. 961 aatttctgag cttcggggaga agttggatct gcagcattgt gttttgccat cacggttaca 1021 ggcttcccaa gtaaagttga aaagtaagg acgagcaagt tcctcagga atcaagaaag 1081 cagcggtcag agctgtatta ttctcctctt tgatgtaata aagtcagcta ttagatatga 1141 gaaaaccatt tcagaagcct ggattaaggc aattgaaaac actgcctcag tatctgaaca 1201 caaggtgttt gacctggtga tgcttttcat catctatagc accatactc agacaaagaa 1261 gtacattgac agggtgctaa gaaataagat tcgatcaggc tgcattcaag aacagctgct 1321 ccagagtaca ttctctgttc attacttagt tcttaaggat atgtgttcat ccattctgtc 1381 gctggctcag agtttgcttc actctctaga ccagagtata atttcatttg gcagtctcct 1441 attack gcatttaagt tttttgacac gtactgccag caggaagtgg ttggtgcctt 1501 agtgacccat atctgcagtg ggaatgaagc tgaagttgat actgccttag atgtccttct 1561 agagttggta gtgttaaacc catctgctat gatgatgaat gctgtctttg taaagggcat 1621 tttagattat ctggataaca tatcccctca gcaatacga aaactcttct atgttctcag 1681 cacactggca tttagcaaac agaatgaagc cagcagccac atccaggatg acatgcactt 1741 ggtgataaga aagcagctct ctagcaccgt attcaagtac aagctcattg ggattattgg 1801 tgctgtgacc atggctggca tcatggcggc agacagaagt gaatcaccta gtttgaccca 1861 agagagagcc aacctgagcg atgagcagtg cacacaggtg acctccttgt tgcagttggt 1921 tcattcctgc agtgagcagt ctcctcaggc ctctgcactt tactatgatg aatttgccaa 1981 cctgatccaa catgaaaagc tggatccaaa agccctggaa tgggttgggc ataccatctg 2041 taatgatttc caggatgcct tcgtagtgga ctcctgtgtt gttccggaag gtgactttcc 2101 atttcctgtg aaagcactgt acggactgga agaatacgac actcaggatg ggattgccat 2161 aaacctcctg ccgctgctgt tttctcagga ctttgcaaaa gatgggggtc cggtgacctc 2221 acaggaatca ggccaaaaat tggtgtctcc gctgtgcctg gctccgtatt tccggttact 2281 gagactttgt gtggagagac agcataacgg aaacttggag gagattgatg gtctactaga 2341 ttgtcctata ttcctaactg acctggagcc tggagagaag ttggagtcca tgtctgctaa 2401 agagcgttca ttcatgtgtt ctctcatatt tcttactctc aactggttcc gagagattgt 2461 aaatgccttc tgccaggaaa catcacctga gatgaagggg aaggtgctca ctcggttaaa 2521 2581 ccctcctctt ggaaactttg atgtggaaac tttagatata acacctcata ctgttactgc 2641 tatttcagca aaaatcagaa agaaaggaaa aatagaaagg aaacaaaaaa cagatggcag 2701 caagacatcc tcctctgaca cactttcaga agaaaaat tcagaatgtg accctacgcc 2761 atctcataga ggccagctaa acaaggagtt cacagggaag gaaaaaga catcattgtt 2821 actacataat tcccatgctt ttttccgaga gctggacatt gaggtcttct ctattctaca 2881 2941 tgtgcaactt gggccccctg agctgctttt cttgctggaa gatctctccc agaagctgga 3001 gagtatgctg acacctccta ttgccaggag agtccccttt ctcaagaaca aaagccg 3061 gaatattgga ttctcacatc tccaacagag atctgcccaa gaaattgttc attgtgtttt 3121 cacaacctg acccaatgt gcacactt ggagacatt cacaactatt tcagtgtt 3181 agctgctgag atcacggtg tagttgatg accaggagtg aaagttcagg agtaccacat 3241 aatgtctcc tgctatcaga ggctgctgca gattttcat gggctttg cttggagtgg 3301 atttctca cctgaaaatc agaatttact gtattcagcc ctccatgtcc ttagtagccg 3361 actgaacag ggagacaca gccagcctttt ggaggaacta ctcagccaga gcgtccatta 3421 cttgcagaat ttccatcaa gcattcccag tttccagt gctctttatc tcatcagact 3481 tttgatggtt atttggaga atcacagc ttctgctcag aaaaagaaaaattgctc 3541 ccttgccaga caatcctct gtcgggtgtg gccaagtgggg gataagaga agagcacac 3601 ctctaatgac cagctccatg ctctgctctg tattactg gagcacacag agagcattct 3661 gaaggccata gaggattg ctggtgttgg tgtcccagaa ctgatcaact ctcctaaga 3721 tgcatctcc tccacattcc ctacactgac caggcatact ttgttgtt tcttccgtgt 3781 gatgatggct gaactagaga agacggtgaa aaaattgag cctggcacag cagcagactc 3841 gcagcagatt catgaagaga aactcctcta ctggaacatg gctgttcgag acttcagtat 3901 cctcatcaac ttgataaagg tatttgatag tcatcctgtt ctgcatgtat gtttgaagta 3961 tgggcgtctc tttgtggaag catttctgaa gcaatgtatg ccgctcctag acttcagttt 4021 tagaaaacac cgggaagatg ttctgagctt actggaaacc ttccagttgg acacaaggct 4081 gcttcatcac ctgtgtgggc attccaagat tcaccaggac acgagactca cccaacatgt 4141 gcctctgctc aaaaagaccc tggaactttt agtttgcaga gtcaaagcta tgctcactct 4201 caacaattgt agagaggctt tctggctggg caatctaaaa aaccgggact tgcagggtga 4261 agagattaag tcccaaaatt cccaggagag cacagcagat gagagtgagg atgacatgtc 4321 atcccaggcc tccaagagca aagccactga ggatggtgaa gaagacgaag taagtgctgg 4381 agaaaaggag caagatagtg atgagagtta tgatgactct gattagaccc cagataaatt 4441 gttgcctgct tctgtgtctc tgccagcctg tgatcatttt gtgttagagt ttgaaatccg 4501 ctgtttgcct ttcttactgg taggatcctt ttttgttcct cttttttttt tttttttttt 4561 ttttttaaag acggggactc gctgtgtttc ccaggctgga gtgcagtgct gcaatcttgg 4621 ctcactgcaa cctccatctc ctaggttcaa gcgattctcc tgcctcagcc tcctgagtag 4681 ctgggacgac aggcacatgc caccatgccc agctaatttt tgtattttta gtagatacgg 4741 ggttttacca tgtcggccag atggtctcaa tctcctgaac tcatgatcca cctgcctcag 4801 cctcccaaag tgctgggatt acaggcatga gccaccgctc ccagccatat tttgttctta 4861 aagtggggtc tttattaact tgtggacatc atggattgtc taacaccatc acagtccctg 4921 gctcaggatt ctaatgtagc attatttatt ggtttggata aacccagctg tgctacactg 4981 cagagtaaaa tctctgagtc atgattctgg actttgggag ctagttttga aactctgatt 5041 tattgtagaa cttaggcttg taccaatttt acaaataaat tctgttctaa gttctg SEQ ID NO: 6 1 mdqkilslaa ektadklqef lqtlregdlt nllqnqavkg kvagallrai fkgspcseea 61 gtlrrrkiyt cciqlvesgd lqkeiaseii gllmleahhf pgpllvelan efisavregs 121 lvngkslell piiltalatk kenlaygkgv lsgeeckkql intlcsgrwd qqyviqltsm 181 fkdvpltaee vefvvekals mfskmnlqei pplvyqllvl sskgsrksvl egiiaffsal 241 dkqhneeqsg delldvvtvp sgelrhvegt iilhivfaik ldyelgrelv khlkvgqqgd 301 snnnlspfsi alllsvtriq rfqdqvldll ktsvvksfkd lqllqgskfl qnlvphrsyv 361 stmilevvkn svhswdhvtq glvelgfilm dsygpkkvld gktietspsl srmpnqhack 421 lganilletf kihemirqei leqvlnrvvt rasspishfl dllsnivmya plvlqscssk 481 vteafdylsf lplqtvqrll kavqpllkvs msmrdclilv lrkamfanql darksavagf 541 llllknfkvl gslsssqcsq slsvsqvhvd vhshynsvan etfcleimds lrrclsqqad 601 vrlmlyegfy dvlrrnsqla nsvmqtllsq lkqfyepkpd llpplkleac iltqgdkisl 661 qepldyllcc iqhclawykn tviplqqgee eeeeeeafye dlddilesit nrmikseled 721 feldksadfs qstsigiknn icaflvmgvc evlieynfsi ssfsknrfed ilslfmcykk 781 lsdilnekag kaktkmankt sdsllsmkfv sslltalfrd siqshqesls vlrssnefmr 841 yavnvalqkv qqlketghvs gpdgqnpeki fqnlcditrv llwrytsipt sveesgkkek 901 gksisllcle glqkifsavq qfyqpkiqqf lraldvtdke geeredadvs vtqrtafqir 961 qfqrsllnll ssqeedfnsk ealllvtvlt slskllepss pqfvqmlswt skickensre 1021 dalfckslmn llfslhvsyk spvillrdls qdihghlgdi dqdvevektn hfaivnlrta 1081 aptvcllvls qaekvleevd wlitklkgqv sqetlseas sqatlpnqpv ekaiimqlgt 1141 lltffhelvq talpsgscvd tllkdlckmy ttltalvryy lqvcqssggi pknmeklvkl 1201 sgshltplcy sfisyvqnks kslnytgekk ekpaavatam arvlretkpi pnlifaieqy 1261 ekflihlskk skvnlmqhmk lstsrdfkik gnildmvlre dgedeneegt asehggqnke 1321 packkkrkkk sequence number 7 1 aacggaagtg tggcggcgtt gggttgagcg ggctttttgg aagtttgtgg cggagttctg 61 tgatatgagc aacaatggac cagaagattt tatctctagc agcagaaaaa acagcagaca 121 aactgcaaga atttcttcaa accctgagag aaggtgattt gactaatctc cttcagaatc 181 aagcagtgaa aggaaaagtt gctggagcac tcctgagagc catcttcaaa ggttccccct 241 gctctgagga agctgggaaca cttaggagac gtaagatata cacttgttgt atccagttgg 301 tggaatcggg ggatttgcag aaagaaatag cgtctgagat cataggatta ctgatgctgg 361 aggctcacca ttttccagga ccattattgg ttgaattagc caatgagttt attagtgctg 421 tcagagaagg cagcctagtg aatggaaaat ctttggagtt actacctatc attctcactg 481 ccctggctac gaaaaggaa aatctggctt atggaaaagg tgtactgagt ggggaagaat 541 gtaagaaaca gttgattaac accctgtgtt ctggcaggtg ggatcagcaa tatgtaatcc 601 aactcacctc catgttcaag gatgtccctc tgactgcaga agaggtggaa tttgtggtgg 661 aaaaagcatt gagcatgttc tccaagatga atcttcaaga aataccacct ttggtctatc 721 agcttctggt tctctctccc aagggaagca gaaagagtgt tttggaagga atcatagcct 781 tcttcagtgc actagataag cagcacaatg aggaacagag tggtgacgag ctattggatg 841 ttgtcactgt gccatcaggt gaacttcgtc atgtggaagg caccattatt ctacacattg 901 tgtttgccat caaattggac tatgaactag gcagagaact cgtgaaacac ttaaaggtag 961 gacagcaagg agattccaat aataacttaa gtcccttcag cattgctctt cttctgtctg 1021 taacaagaat acaaagattt caggaccagg tgcttgatct tttaaagact tcggttgtaa 1081 agagctttaa ggatcttcaa ctcctccaag gctcaaaatt tcttcagaat ctagttcctc 1141 atagatctta tgtttcaacc atgatcttgg aagtagtgaa gaatagcgtt catagctggg 1201 accatgttac tcagggcctc gtagaacttg gtttcatttt gatggattca tatgggccaa 1261 agaaggttct tgatggaaaa actattgaaa ccagcccaag tctttctaga atgccaaacc 1321 agcatgcatg tagactcgga gctaatatcc tgttggaaac ttttaagatc catgagatga 1381 tcagacaaga aattttggag caggtcctca acagggttgt taccagagca tcttctccca 1441 tcagtcattt cttagacctg ctttcaaata tcgtcatgta tgcaccctta gttcttcaaa 1501 gttgttcttc taaagtcaca gaagcttttg actatttgtc cttctgccc cttcagactg 1561 tacaaggct gcttaaggca gtgcagcccc ttctcaaagt cagcatgtca atgagagact 1621 gcttgatact tgtccttcgg aaagctatgt ttgccaacca gcttgatgcc cgaaaatctg 1681 cagttgctgg gttttgctg ctcctgaaga actttaaagt tttaggcagc ctgtcatcct 1741 ctcagtgcag tcagtctctc agtgtcagtc aggttcatgt ggatgttcac agccattaca 1801 attctgtcgc caatgaaact ttttgccttg agatcatgga tagtttgagg agatgcttaa 1861 gccagcaagc tgatgttcga ctcatgcttt atgaggggtt tttgatgtt cttcgaagga 1921 actctcagct ggctaattca gtcatgcaaa ctctgctctc acagttaaaa cagttctatg 1981 agccaaaacc tgatctgctg cctcctctga aattagaagc ttgtattctg acccaaggag 2041 ataagatctc tctacaagaa ccactggatt atctgctgtg ttgtattcag cattgtttgg 2101 cctggtataa gaatacagtc atacccttac agcagggaga ggaggaagag gaggaggaag 2161 aggcattcta cgaagaccta gatgatatat tggagtccat tactaataga atgattaaga 2221 gtgagctgga agactttgaa ctggataaat cagcagattt ttctcagagc accagtattg 2281 gcataaaaaa tatatctgt gcttttcttg tgatgggagt ttgtgaggtt ttaatagaat 2341 acaatttctc cataagtagt ttcagtaaga ataggtttga ggacattctg agcttattta 2401 tgtgttacaa aaaactctct gacattctta atgaaaaagc gggtaaagcc aaaactaaaa 2461 tggccaacaa gacaagtgat agtcttttgt ccatgaaatt tgtgtccagt cttctcactg 2521 ctctttttcag ggatagtatc caaagccacc aagaaagcct ttctgttctc aggtccagca 2581 2641 cagggcatgt gagtggccct gatggccaaa acccagaaaa gatctttcag aacctctgtg 2701 acataactcg agtcttgcta tggagataca cttcaattcc tacttcagtg gaagagtcgg 2761 gaagaaaga gaaggaaag agcatctcac tgctgtgctt ggagggttta cagaaaatat 2821 tcagtgctgt gcaacagttc tatcagccca agattcagca gtttctcaga gctctggatg 2881 2941 cattccagat ccggcaattt cagaggtcct tgttgaattt acttagcagt caagaggaag 3001 attttaatag caaagaagcc ctcctgctag tcacggttct taccagtttg tccaagttac 3061 tggagccctc ctctcctcag tttgtgcaga tgttatcctg gacatcaaag atttgcaagg 3121 aaaacagccg ggaggatgcc ttgttttgca agagcttgat gaacttgctc ttcagcctgc 3181 atgtttcgta taagagtcct gtcattctgc tgcgtgactt gtcccaggat atccacgggc 3241 atctgggaga tatagaccag gatgtagagg tggagaaaac aaaccacttt gcaatagtga 3301 atttgagaac ggctgcccccc actgtctgtt tacttgttct gagtcaggcc gagaaggttc 3361 tagagaagt ggactggcta atcaccaagc ttaagggaca agtgagccaa gaaaccttat 3421 cagaagaggc ctcttctcag gcaacctac caaatcagcc tgttgagaaa gctatcatca 3481 tgcaactggg aactctgctt acatttttcc acgagctggt gcagacagct ctgccatcag 3541 gcagctgtgt ggacaccttg ttaaaggact tgtgcaaaat gtacaccaca cttacagccc 3601 ttgtcagata ttatctccag gtgtgtcaga gctccggagg aattccaaaa aatatggaaa 3661 agctggtgaa gctgtctggt tctcatctga cccccctgtg ttatctttc atttcttacg 3721 tacagaataa gagtaagagc ctgaactata cgggagaaa aaaggagaaa cctgctgccg 3781 ttgccacagc catggccaga gttcttcggg aaaccaagcc aatccctaac ctcatctttg 3841 ccatagaaca gtatgaaaaa tttctcatcc acctttctaa gaagtccaag gtgaacctga 3901 tgcagcacat gaagctcagc acctcacgag acttcaagat caaaggaaac atcctagaca 3961 tggttcttcg agaggatggt gaagatgaaa atgaagaggg cactgcatca gagcatgggg 4021 gacagaacaa agaaccagcc aagaagaaaa ggaaaaata aatgaaatgc ctgagttaat 4081 gtgaactttg gggcttctgc ttcattttta cccaacaagc aacaatgccc cttgtcctgt 4141 agtccacacc gatgttggca tcttggttct gaacccactg aattcaactg caccttcagt 4201 tagaaggaat cttcttggca ggtcctgcta ctgaaaaatg gctggcctta ggcaagccct 4261 ttgcaaaaa gcacagctga aagcctgagt ttgggagcct gcaccacccc gatgaagctc 4321 cacgggagca aatacagagc ctccaggcag tgctatggtc caggctggct tcgtttttcc 4381 aaggagcctt tggtgagttc aattatctgg taaatatcca gcgcttcacc tgaaagatag 4441 tgcaaattgg ttaggatgcc acctcaagaa ctgtaactga gagctcagaa gtgagcaaag 4501 gagcttaatg ctaaggtcaa aaggagagtg aaaggttgag aacaattgcc acgaacggta 4561 atgttacatg ttaggagggt ctgttttctt tttatataag tgtgtcttag atatatttta 4621 aatagaaaat aagctttctg atttacttgt ttggtattta aagcacagtt tgttttctg 4681 tcacctatag agtgcaagaa tgcactctat agaataaatt atctttaaac att sequence number 8 1 agagagcggc tggcgggcgt ccgagggagg gagggagcga cgagcgaggt agcgacgcgg 61 gccgcccctg ccgccgccgc cgccgccgcc gccgccgccg cggtcggacc tgcggcctcc 121 tcccctcccc tcccccgcgt cgccctgctg cgggagggg gctcgcgtcg ccgtctccag 181 ccgctcccga tgaagcagct gcagccgcag ccgcctccga agatggggga tttctacgac 241 ccggagcacc cgacccctga gcaaagattg aaaatgtgca gaaaacaggt ttcatcaaag 301 gaccaatgtt caaaggtgtt gcttctagtc gatttttgcc caaaggcacc aaaacaaaag 361 ttaatttgga agaacaggga cgacagaagg tgtcattcag cttcagcctt acaaagaaaa 421 ctttgcagaa taggtttctc actgcacttg gcaatgaaaa gcaaagtgat actccaaacc 481 ctccagctgt acctcttcag gtagactcga ctcctaaaat gaaaatggaa attggtgata 541 ccttatctac tgcagagaa tcttccccac caagtcaag ggtggaattg ggcaaattc 601 attttagaa acatctgctt catgtaacat ccaggccact gctgctact accacagcag 661 tagcatctcc acctactcat gcagcaccat taccagcagt gatagcagaa tcaacactg 721 tagactcacc gccctcatct cgcctccac cgcctccacc tgcccagcc acacactct 781 catcaccagc accagtaaca gagccagtgg ccttgccaca tacaccaata acagttctaa 841 tggcagcacc agtaccctta ccagtagatg tagcagttag atctctgaaa gaaccaccaa 901 ttatattgt accagaatct ttagaagcag atactaagca ggacactata tctaatagtt 961 tagagaca cgtactca atttgaatg agcaagcaga tattccctca aaaaagaag 1021 attcccatat tgggaggt gagaaatttc cagatagttc tagattagt ctgagctgta 1081 aaaaaacagg ttctagaag aaatcccac aatctgagg catctctt ggttcagaat 1141 ctgatgaaga ttctgtacgg acttctca gtcaagatc acatgattta aaatttcag 1201 caagcattga aaggaaaga gattttaaa agagctcagc acctttaaa agtgaggaatc 1261 tagggaaacc ttcacgatct aaaacagaca gagatgataa atattttagc tattcaaaac 1321 ttgaaagaga tactcggtat gtatcttccc gatgtagatc agaaagagag cgacggcgga 1381 gcagatctca ctctaggtct gagagaggct ctagaactaa tttatcctat tccaggtcag 1441 aacgatctca ttattatgac tctgatcgtc gctaccatag gagctcccct tatcgagaga 1501 ggacgcgcta ttctcggcca tacacagata acagagcacg agagagttct gactcagaag 1561 aagagtataa gaagacatac tcaaggcgta cctcatctca ttcctcttct tacagagacc 1621 taaggacatc atcctattct aaatctgatc gggactgtaa aactgagacc tcttacttag 1681 agatggaaag aagaggcaag tattcttcaa aactagaaag agaatctaaa aggacttcag 1741 aaaatgaagc attaaaaga tgttgttctc cccctaatga actgggattc cgacgagggt 1801 catcatattc taagcatgac agtagtgctt cccgttataa atctaccctt tcaaaaccta 1861 tacccaagtc tgataaattt aaaaattctt tctgttgtac agaattaaat gaagaaatca 1921 aacagtctca ttcttttagt ttacagacac cttgttcaaa aggtagtgaa ttaagaatga 1981 tttaaaaa tcctgaaaga gaaaaggctg ggtctccagc tccatcaat cgattaatg 2041 attcacctac ttaaaaag ctagatgaat tgcctatttt taagtccgaa tttatacac 2101 atgatagcca tgatagtatt aaggaattag actctttatc taagtgaag atgatcaat 2161 taagagttt ttgcccata gattaata taatggatc tcctggggca gatctgatt 2221 tgcacatt tgcactct aaaactgatg ctgtttat tgtctgat gatagtgtga 2281 ctggatcgga attatcccct ttggtcaag catgcatgct ttcatcaat ggatttcaga 2341 atattag gtgcaagaa aaagacttgg atgatacctg catgctgcat aagaagtcag 2401 aaagcccatt tagagaaca gaacctctgg tgtcaccaca ccagataa ctcatgtcta 2461 tgccagttat gactgtggat tattccaaa cagtagttaa agaaccagtt gatacgaggg 2521 tttctgctg siaaaccaaa gattcagaca tatactgtac tttgaacgat agcaaccctt 2581 ctttgtgtaa ctctgaagct gaaatattg agccttcagt tatgagat tctcaata 2641 gctttatgaa tgtgcatttg gatcaaac cagttatatg tgattag aatttgacag 2701 atcactcaa atttgcatgt gagaata agcagagcat cggtagcact agttcagctt 2761 ctgttaatca tttgatgat ttatatcac ctattggg ttcaggtatt gcttcatctc 2821 twenty-two twenty-two twenty-two twenty-two twenty-two 2881 acacatctcc agttctggat gcagtgctaa agagtaaaa aagttcagag ttttaagc 2941 atgcaggga agaacaata gtagaagtag gtagtgacct tcctgattca ggaagggat 3001 ttgcttccag ggagaacagg cgtaataatg ggttatctgg gaatgtttg caagaggctc 3061 aagagaagg gattccata ttgcctgaaa gagaggaag accagaatc tctttagatg 3121 aagaggaga aggacat gtgcatactt ctgatgactc agaagttgta tttctctt 3181 gtgatttgaa ttataccatg gagacagtg atggtgtaac ttatgcatta aagtgtgaca 3241 gtagtggtca tgccccagaa attgtgtcta cagttcatga agattattct ggctctctg 3301 aaagttcaa tgatgaagt gattcagaag atacagattc ggatgatagc agtattccaa 3361 gaaaccgtct ccagtctgtt gtggttgtgc caagaattc tactttgccc atggaagaaa 3421 caagtccttg ttctctcgg agcagtcaa gttatagaca ctattctgac cattgggaag 3481 attgagatt ggagtcaag agacattgt atgagaaaa atttgaagt atagcaagta 3541 aagcctgtcc tcaactgat aagtttcc ttcaataagg aacagagaag aatccggaaa 3601 tttctttac acagtccagt agaaaacaa tagataatcg cctgcctgaa ctttctcatc 3661 ctcagagtga tggttgat agtacaagtc atacagatgt gaaatctgac cctctgggtc 3721 acccaattc agaggaacc gtgaaagcca aaataccttc taggcagca gagagctgc 3781 caatttattc ttctgatt gaagagatgtcc caattagtc ttggcacag accactttcc 3841 aaaacaggcc agatagtaga ctgggaaaa cagaattgag tttctcc tcttgtgaga 3901 taccacatgt ggatgcttg cactcatcag agagctcag aaacttaggt tgggacttct 3961 ctcaagaaa gcctttacc acgtatcagc aacctgacag tagctatgga gcttgtggtg 4021 gandacagta tcagcaaat gcagacagt atggtgggac acgtgattac tggcaggca 4081 atggttactg ggatccaaga tcaggtagac ctcctggac tggttgtg tatgatcgaa 4141 ctcaggaca agtaccagat tccctacag atgatcgtga agagaggag aattgggatc 4201 aacaggatgg atcccatttt tcagaccagt ccgataaatt tcttattcc cttcagaaag 4261 acaagggtc agtgcaagca cctgaaataa gcagcaatc cattaaggac acttagctg 4321 tgaatgaaaa gaagatttt tcaaaaact tagaaaaaa tgaatcaaa gagaggggc 4381 ctcttaaaa aaggaggcag gaatagaga gtgattctga aagtgatgt gagcttcagg 4441 acagaaagaa agttagagtg gaggtagagc agggagac atcagtgccc ccaggttcag 4501 cactggttgg gccctgt gtcatggatg acttcaggga cccacagcga tggaggat 4561 gtgccaagca agggaaatg ccatgttact ttgatcttat tgaagaaat gttttatta 4621 Cagaagaaa gagaataa tctcatcgag atttaagcg aatgcagtgt gagtgtacac 4681 ctctttctaa agatgaaga gctcaggtg aaatagcatg tgggagat tgtcttaatc 4741 gtctctcat gattgaatgt tcttctcggt gtccaatgg ggattattgt tccatagac 4801 gtttcagag aaaaacagcat gcagatgtgg aagtcatact cacagaaag aaaggctggg 4861 gcttgagagc tgccaagac cttccttcga acaccttgt cctagaatat tgtggagagg 4921 tactcgatca taagagttt aaagctcgag tgaggagta tgcacgaac ac aaaaacatcc 4981 attackattt catggccctg aagaatgatg agatataga tgccactca aaggaaatt 5041 gctctcgttt catgaatcac agctgtgaac caattgtga aacccaaaa tggactgtga 5101 acggacact gagggttggg ttttacca acggactggt tccttcaggc tcagagttaa 5161 cgtttgacta tcagttccag agatatggaa aagaagccca gaatgttttc tgcggatcag 5221 ccaattgccg gggttacctg ggaggagaaa acagagtcag catchcagagca gcaggaggga 5281 aaatgagaa ggaacgatct cgtaagagg attcagtga tggagcta gaagctctga 5341 tggaaatgg tgaggtctc tctgataaaa accaggtgct cagcttatcc cggctatgg 5401 ttagaattga aacttggag cagaaactta cctgtctgga actcatacag aacacacact 5461 cacagtcctg cctgaagtcc tttctggac gtcatgggct gtcttgttg tggatctga 5521 tggcagct aggtgacggc cgggaagta accagaagct tcaggaagct attataaga 5581 ctttggaaca ctgcccatt cctactaaaa atatgttgga ggaagcaaa gtacttccaa 5641 ttattcacg ctggtctcag actaagactg ctgtccctcc gttgagtgaa ggagatgggt 5701 attctagtga gatacatcg cgtgctcata caccactca cacacctgat ccttccacca 5761 agctgagcac agaagctgac acagacactc ccagaaact aatgtttcgc agactgaaaa 5821 ttataagtga aaatagcatg vakagtgcaa tctctgatgc aaccagtgag ctagaaggca 5881 aggatggcaa agaggatctt gatcattag aaatgtccc tgtagagaa gaggagaat 5941 tgcagtcaca acagctactc ccacacagc tgctgaatg CAagttgat agtgaaacca 6001 acatagaagc tagtaagcta cctacatctg aaccagaagc tgacgctgaa atagagcccca 6061 aagagagcaa cggcacaaaa ctagagaac ctattaatga agaacacca tcccagatg 6121 aagaggagg tgtgtctgat gtggagagtg aaaggagcca agaacagcca gataaaacag 6181 tggatataag tgatttggcc accaactcc tggacagttg gaagaccta aaggaggtat 6241 atcgaatttcc aaagaaagt aaactgaaa aggaacac aaaactgaa cgaggaaggg 6301 atgctgttgg cttcagagat caaacacctg ccccgaagac tcctaatagg tcaagagaga 6361 gagacccaga caagcaaact caaaataaag agaaaaggaa acgaagaagc tccctctcac 6421 caccctcttc tgcctatgag cggggaacaa aaaggccaga tgacagatat gatacaccaa 6481 cttctaaaaa gaaagtacga attaaagacc gcaataaact ttctacagag gaacgccgga 6541 agttgtttga gcaagaggtg gctcaacggg aggctcagaa acaacagcaa cagatgcaga 6601 acctgggaat gacatcacca ctgccctatg actctcttgg ttataatgcc ccgcatcatc 6661 cctttgctgg ttacccacca ggttatccca tgcaggccta tgtggatccc agcaacccta 6721 atgctggaaa ggtgctcctg cccacaccca gcatggaccc agtgtgttct cctgctcctt 6781 atgatcatgc tcagcccttg gtgggacatt ctacagaacc cctttctgcc cctccaccag 6841 taccagtggt gccacatgtg gcagctcctg tggaagtttc cagttcccag tatgtggccc 6901 agagtgatgg tgtagtacac caagactcca gcgttgctgt cttgccagtg ccggcccccg 6961 gcccagttca gggacagaat tatagtgttt gggattcaaa ccaacagtct gtcagtgtac 7021 agcagcagta ctctcctgca cagtctcaag caaccatata ttatcaagga cagacatgtc 7081 caacagtcta tggtgtgaca tcaccttatt cacagacaac tccaccaatt gtacagagtt 7141 atgcccagcc aagtcttcag tatatccagg ggcaacagat tttcacagct catccacaag 7201 gagtggtggt acagccagcc gcagcagtga ctacaatagt tgcaccaggg cagcctcagc 7261 ccttgcagcc atctgaaatg gttgtgacaa ataatctctt ggatctgccg cccccctctc 7321 ctcccaaacc aaaaaccatt gtcttacctc ccaactggaa gacagctcga gatccagaag 7381 ggaagattta ttactaccat gtgatcacaa ggcagactca gtgggatcct cctacttggg 7441 aaagcccagg agatgatgcc agccttgagc atgaagctga gatggacctg ggaactccaa 7501 catatgatga aaaccccatg aaggcctcga aaaagcccaa gacagcagaa gcagacacct 7561 ccagtgaact agcaaagaaa agcaaagaag tattcagaaa agagatgtcc cagttcatcg 7621 tccagtgcct gaacccttac cggaaacctg actgcaaagt gggaagaatt accacaactg 7681 aagactttaa acatctggct cgcaagctga ctcacggtgt tatgaataag gagctgaagt 7741 actgtaagaa tcctgaggac ctggagtgca atgagaatgt gaaacacaaa accaaggagt 7801 acattaagaa gtacatgcag aagtttgggg ctgtttacaa acccaaagag gacactgaat 7861 tagagtgact gttgggccag ggtgggagga tgggtggtca ggtaagacag actctaggga 7921 gaggaaatcc tgtgggcctt tctgtcccac ccctgtcagc actgtgctac tgatgataca 7981 tcaccctggg gaattcaacc ctgcagatgt caactgaagg ccacaaaaat gaactccatc 8041 tacaagtgat tacctagttg tgagctgttg gcatgtggtt agaagccatc agaggtgcaa 8101 gggcttagaa aagaccctgg ccagacctga ctccactctt aaacctgggt cttctccttg 8161 gcggtgctgt cagcgcacag acccatgcgc atccccaccc acaacccttt accctgatga 8221 tctgtattat attttaatgt atatgtgaat atattgaaaa taatttgttt tttcctggtt 8281 tttgtttggt tttcgttttg cttttagcct ctacatgcta ggatcacagg aagactttgt 8341 aaggacagtt taagttctcc tgcaaggttt aatttgttat catgtaaata ttctaaagca 8401 ggctgccttg tggttttggc cagccttgtg ctatgttgat aagattgatt tactgcttaa 8461 aatcacttta ctttatccaa tttttactga actttttatg taaaaaaata aaatcaatta 8521 aaga sequence number 9

Claims

1. 1. A method of treating cancer in an individual in need thereof, comprising reducing the expression or activity of a Fanconi Anemia Complementation Group (FANC) protein in said individual, wherein said cancer is a SETD2-deficient cancer.

2. The method of claim 1, wherein the FANC protein is a FANCM protein, a FANCD2 protein, or a FANCI protein.

3. 3. The method of claim 1 or 2, wherein the expression or activity of FANC is reduced by administering a FANC antagonist to the individual.

4. 4. The method of claim 3, wherein the FANC antagonist is a FANCM antagonist.

5. The method of claim 4, wherein the FANCM antagonist reduces expression of FANCM.

6. The method of claim 5 , wherein the FANCM antagonist is a suppressor nucleic acid.

7. The method of claim 6 , wherein the suppressor nucleic acid is an siRNA or an shRNA.

8. The method of claim 6 , wherein the suppressor nucleic acid is an antisense oligonucleotide.

9. The method of claim 4, wherein the FANCM antagonist is a targeted nuclease that reduces expression of FANCM.

10. 10. The method of claim 9, wherein the targeted nuclease is a CRISPR-associated nuclease, and the CRISPR-associated nuclease is administered in combination with a guide RNA that recognizes a target sequence within the FANCM gene.

11. The method of claim 4, wherein the FANCM antagonist reduces the activity of FANCM.

12. 12. The method of claim 11, wherein the FANCM antagonist is a FANCM inhibitor.

13. 13. The method of claim 12, wherein the FANCM inhibitor is a small chemical molecule, and optionally the small chemical molecule is a non-polymeric organic compound.

14. The method of claim 11 or 12, wherein the FANCM antagonist is an antibody, antibody fragment, antibody derivative, or non-immunoglobulin binding molecule that specifically binds to FANCM.

15. 15. The method of any one of claims 12 to 14, wherein the antagonist reduces the ATP-dependent DNA helicase / translocase activity of FANCM, and optionally, the antagonist binds to the DEAH helicase domain.

16. 4. The method of claim 3, wherein the FANC antagonist is a FANCD2 antagonist.

17. 17. The method of claim 16, wherein the FANCD2 antagonist reduces expression of FANCD2.

18. 18. The method of claim 17, wherein the FANCD2 antagonist is a suppressor nucleic acid.

19. 19. The method of claim 18, wherein the suppressor nucleic acid is an siRNA or shRNA.

20. 19. The method of claim 18, wherein the suppressor nucleic acid is an antisense oligonucleotide.

21. 17. The method of claim 16, wherein the FANCD2 antagonist is a targeted nuclease that reduces expression of FANCM.

22. 22. The method of claim 21, wherein the targeted nuclease is a CRISPR-associated nuclease, and the CRISPR-associated nuclease is administered in combination with a guide RNA that recognizes a target sequence within the FANCD2 gene.

23. 17. The method of claim 16, wherein the FANCD2 antagonist reduces the activity of FANCD2.

24. 24. The method of claim 23, wherein the FANCD2 antagonist is a FANCD2 inhibitor.

25. 25. The method of claim 24, wherein the FANCD2 inhibitor is a small chemical molecule, and optionally the small chemical molecule is a non-polymeric organic compound.

26. 25. The method of claim 23 or 24, wherein the FANCD2 antagonist is an antibody, antibody fragment, antibody derivative, or non-immunoglobulin binding molecule that specifically binds to FANCD2.

27. 4. The method of claim 3, wherein the FANC antagonist is a FANCI antagonist.

28. 28. The method of claim 27, wherein the FANCI antagonist reduces expression of FANCI.

29. 29. The method of claim 28, wherein the FANCI antagonist is a suppressor nucleic acid.

30. 30. The method of claim 29, wherein the suppressor nucleic acid is an siRNA or shRNA.

31. 30. The method of claim 29, wherein the suppressor nucleic acid is an antisense oligonucleotide.

32. 28. The method of claim 27, wherein the FANCI antagonist is a targeted nuclease that reduces expression of FANCI.

33. 33. The method of claim 32, wherein the targeted nuclease is a CRISPR-associated nuclease, and the CRISPR-associated nuclease is administered in combination with a guide RNA that recognizes a target sequence within the FANCI gene.

34. 34. The method of claim 33, wherein the FANCI antagonist reduces the activity of FANCI.

35. 35. The method of claim 34, wherein the FANCI antagonist is a FANCI inhibitor.

36. 36. The method of claim 35, wherein the FANCI inhibitor is a small chemical molecule, and optionally the small chemical molecule is a non-polymeric organic compound.

37. 36. The method of claim 34 or 35, wherein the FANCI antagonist is an antibody, antibody fragment, antibody derivative, or non-immunoglobulin binding molecule that specifically binds to FANCI.

38. The method of any one of claims 1 to 37, wherein the SETD2-deficient cancer is selected from the group consisting of leukemia, such as AML, CML, ALL, and CLL, lymphoma, such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma, and solid cancer, such as sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, such as glioblastoma multiforme, including pediatric glioblastoma multiforme, breast cancer, uterine cancer, oral cancer, ovarian cancer, prostate cancer, lung cancer, such as lung adenocarcinoma and lung squamous cell carcinoma, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, renal cancer, such as clear cell renal carcinoma, gastric cancer, testicular cancer, gallbladder and biliary tract cancer, thyroid cancer, thymic cancer, bone cancer, and cerebral cancer.

39. 39. The method of any one of claims 1 to 38, wherein the SETD2-deficient cancer comprises one or more cancer cells with a mutation in or loss of SETD2.

40. 40. The method of any one of claims 1 to 39, further comprising identifying an individual with a cancer that is deficient in SETD2.

41. 41. The method of any one of claims 1 to 40, further comprising administering a chemotherapeutic agent to the individual.

42. FANC antagonists for use in methods of treating SETD2-deficient cancers.

43. 43. The FANC antagonist for use according to claim 42, wherein the FANC antagonist is a FANCM antagonist, a FANCD2 antagonist or a FANCI antagonist.

44. 44. A FANC antagonist for use according to claim 42 or 43, wherein the method is a method of treatment according to any one of claims 1 to 41.

45. Use of a FANC antagonist in the manufacture of a medicament for treating a SETD2-deficient cancer.

46. 46. ​​The use of claim 45, wherein the FANC antagonist is a FANCM antagonist, a FANCD2 antagonist, or a FANCI antagonist.

47. The use according to claim 46, wherein the treatment is a method of treatment according to any one of claims 1 to 41.

48. A method for screening for a compound that induces cell death in SETD2-deficient cancer cells, the method comprising determining binding of a test compound to a FANC protein, wherein binding to the FANC protein indicates that the compound induces cell death in SETD2-deficient cells.

49. A method for screening for a compound that induces cell death in SET2D-deficient cancer cells, comprising determining the effect of a test compound on the expression or activity of FANC protein, wherein a decrease in the expression or activity of FANC protein indicates that the compound induces cell death in SET2D-deficient cancer cells.

50. 50. The method of claim 48 or 49, wherein the FANC protein is a FANCM protein, a FANCD2 protein, or a FANCI protein.

51. 51. The method of any one of claims 48 to 50, comprising identifying the test compound as a compound that reduces the expression or activity of a FANC protein.

52. 52. The method of claim 51, further comprising isolating or purifying the identified compound.

53. 1. A method for determining the responsiveness of a cancer in an individual to treatment with a FANC antagonist, comprising determining the presence of one or more SETD2-deficient cells in a sample obtained from the individual, wherein the presence of one or more SETD2-deficient cancer cells in the sample indicates that the cancer will be responsive to treatment with the FANC antagonist.

54. 1. A method of selecting an individual having a SETD2-deficient cancer for treatment with a FANC antagonist, the method comprising identifying cancer cells obtained from the individual as being deficient in SETD2 relative to normal cells.

55. 55. The method of claim 53 or 54, wherein the FANC antagonist is a FANCM antagonist, a FANCD2 antagonist, or a FANCI antagonist.

56. 56. The method of any one of claims 53 to 55, further comprising providing an inhibitor of FANC suitable for administration to the individual.

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