Treatment of ALT cancers

Abstract

To provide a technique that generally relates to a cancer and the treatment of the cancer, including the treatment of Alternative Lengthening of Telomeres (ALT) cancers.SOLUTION: The disclosure provides a method including a step of inhibiting FANCM activity or expression, for example, a step of inhibiting interaction between FANCM and RMI and / or FANCM's ATPase activity, to inhibit the growth and / or proliferation of ALT tumor cells and / or to induce death of ATL tumor cells. The method may be practiced on patients diagnosed with an ALT tumor.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to Australian Provisional Application No. 2019901766, filed on May 24, 2019; and UK Application No. 1907518.3, filed on May 28, 2019, both of which are hereby incorporated by reference in their entirety.

[0002] Statement regarding the sequence listing The sequence listing associated with this application is provided in text format instead of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is PCT Sequence Listing_TREATMENT OF ALT CANCERS.txt. The text file is approximately 305 KB, was created on May 20, 2020, and is electronically submitted as part of the description of this specification.

[0003] The disclosure of the present invention generally relates to the treatment of cancer, including the treatment of cancer and cancers with alternative lengthening of telomeres (ALT).

Background Art

[0004] Genomic integrity is partially maintained by telomeres in normal cells. Continuously shortening telomeres through successive cell divisions induces chromosomal instability. Immortalization by replication is often achieved by cancer cells by overcoming telomere shortening. Telomere shortening must be counteracted in immortal cells, including the majority of cancer cells, to avoid senescence or death. 1 In the majority of cancer cells, telomere length is maintained by telomerase. Approximately 90% of human cancers are in a state where telomerase, a reverse transcriptase, is reactivated, whereby newly synthesized telomere repeat sequences are added to the 3' ends of linear chromosomes. 2、3Approximately 10-15% of immortal cancer cells are telomerase-negative and replenish telomeres by a strategy independent of telomerase, which is collectively referred to as Alternative Lengthening of Telomeres (ALT) (Bryan et al., 1995 and Bryan et al., 1997) or the ALT pathway. 4 In humans, ALT has been reported in tumors of mesenchymal or epithelial origin such as osteosarcoma, liposarcoma, glioblastoma, astrocytoma, and bladder cancer, as well as in immortalized cell lines in vitro. 4~8 .

[0005] The characteristics of molecules considered as ALT markers are: i) telomeres with heterogeneous lengths at different chromosomal ends, such as telomeres that are even longer than the average telomeres in telomerase-positive cells, etc. 8 ; ii) an increase in the level of the long non-coding RNA (IncRNA) TERRA of telomeres 9~13 ; iii) clustering of multiple telomeres to ALT-associated PML bodies (APBs), nuclear structures containing the promyelocytic leukemia protein (PML), telomere factors such as TRF1, TRF2, and RAP1, TERRA, and DNA repair factors such as RAD51, RAD52, replication protein A (RPA), Brcal, and Bloom (BLM) as well as Werner helicase 11、14~19 ; iv) abundant extrachromosomal telomere repeat sequences (ECTRs) including double-stranded (ds), circular (t-circle), partially single-stranded (ss) circles (C and G circles), and linear dsDNA 20~23 ; v) including recurrent mutations in the X-linked alpha-thalassemia / mental retardation (ATRX) gene 12 .

[0006] ALT cells are characterized by elevated levels of DNA damage compared to lethal or telomerase-positive cells, indicating increased telomeric replication stress in ALT cells. This is due to cumulative defects in the structural integrity of telomeres. Frequent or persistent replication fork stalling causes nicks and breaks in DNA, leading to the hypothesis that the ALT mechanism arises from stalled replication forks that deteriorate to form double-strand breaks (DSBs), which then provide substrates for the participation of the homologous recombination repair pathway and end with break-induced telomere synthesis. Therefore, ALT telomeres achieve a delicate balance between telomere protection, telomere damage, and repair activity, and disruption of this balance has the potential to dysregulate the ALT mechanism.

[0007] In ALT cells, multiple DNA metabolic pathways cooperate to maintain telomeres. During the G2 phase of the cell cycle, break-induced replication (BIR) is active at ALT telomeres and is stimulated by experimentally induced DSBs using the DNA endonuclease TRF1-FokI tethered to telomeres. 24、25 ALT BIR requires POLD3 and POLD4, two regulatory subunits of DNA polymerase delta. 24、25 Conservative mitotic DNA synthesis (MiDAS) has also been demonstrated in human ALT cells. 26 ALT MiDAS is stimulated by replication stress and requires RAD52. 2S Finally, the clustering of ALT telomeres within APBs is promoted by RAD51-dependent long-range movements, which are also stimulated by DSBs induced by TRF1-FokI. 27 Telomere movement can promote efficient homology search and telomere synthesis, even though both ALT BIR and MiDAS are independent of RAD51. 25、26 。

[0008] The common concept emerging from all this research is that persistent physiological damage must be maintained at ALT telomeres to promote telomere elongation. This is consistent with the presence of markers of replication stress and DNA damage in APB 11,14~18 The cause of this damage remains unclear, but as candidates, RNA:DNA hybrids (R-loops), G-quadruplexes and oncogene expression have been proposed 11、26 This scenario necessarily involves that the telomere damage level is maintained within a specific threshold range that is high enough to initiate DNA synthesis-based repair but not high enough to induce cell death. Consistently, telomeric R-loops (telR-loops) formed by TERRA and telomeric DNA activate replication stress at ALT telomeres, and the levels are tightly regulated by the endoribonuclease RNase HI 11、28 Depletion of RNase HI rapidly causes increased replication stress, resulting in abundant telomere-free chromosome ends (TFEs) and C-circles. Conversely, overexpression of RNase HI causes progressive TFE accumulation, which may be due to inefficient de novo synthesis of telomeric DNA 11 Also, the DNA damage signaling kinases ATM and Rad3-related (ATR) and the annealing helicase SWI / SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein 1 (SMARCAL1) have also been reported to limit replication stress at ALT telomeres 29、30 。

[0009] The Fanconi anemia complementation group M (FANCM) ATPase / translocase is a component of the Fanconi anemia (FA) complex, which maintains efficient FANCD2 ubiquitination when replication forks stall due to physical obstacles such as DNA crosslinks 31 Independently of the FA complex, FANCM remodels replication forks, recruits DNA repair factors to damage sites, suppresses meiotic crossovers, and facilitates ATR checkpoint activation 32~35Furthermore, the ATPase / translocase activity of FANCM resolves RNA:DNA hybrids in vitro, and its R-loops accumulate genome-wide in FANCM-deficient cells. 36 。

[0010] FANCM is an essential factor in the stabilization of stalled replication forks. FANCM contains two DNA-binding domains at its N and C termini, with three highly conserved regions (MM1-MM3) in between. The MM1 domain recruits the FA core complex, a multi-subunit ubiquitin ligase essential for DNA interstrand crosslink (ICL) repair, while the MM2 domain directly binds to the RMI1-RMI2 subcomplex of BLM-TOP3A-RMI (BTR). The BTR complex contains BLM helicase activity, TOP3A decatenation activity, branch migration, and overall resolvase activity, and it has been suggested that FANCM and BTR cooperate to reverse stalled forks and thus stabilize them. FANCM retention at stalled replication forks depends on its interaction with a functional BTR complex but not on its interaction with the FA core complex.

[0011] In ALT cells, FANCM enables efficient progression of replication forks through the telomere pathway, and FANCM depletion induces telomere replication stress. 17 FANCM depletion causes the accumulation of BLM and Brca1 at ALT telomeres, and co-depletion of FANCM and Brca1 or BLM has been shown to be lethal. 17 。

[0012] Many cancer therapeutics act by indiscriminately damaging DNA, and cancer cells without robust DNA repair capabilities cannot survive from chemotherapeutic doses that are tolerated in healthy tissues. DNA-damaging chemotherapy is initially effective, but reactivation of tumor DNA repair pathways can lead to treatment failure and poor patient outcomes. The Fanconi anemia (FA) pathway is generally activated during tumorigenesis, and reactivation or upregulation of the FA pathway has been shown to be associated with chemotherapy resistance in many cancers. Proper execution of the FA pathway requires the interaction between the FANCM protein and the RMI complex, and mutations that disrupt the FANCM-RMI interaction can render cells sensitive to DNA crosslinking agents. It has been hypothesized that inhibitors that block the formation of the FANCM-RMI complex may be useful as therapeutics to resensitize tumors that have acquired chemotherapeutic agent resistance. In 2016, Voter et al. developed a screening to identify such inhibitors. However, the effect of these inhibitors on the ALT mechanism has not been investigated.

[0013] In WO2017 / 146947, depletion of at least one of FANCM and BLM or BRCA1 can induce replication stress in ALT cells, which occurs mainly at telomeres and has been found to dramatically reduce replication efficiency at ALT telomeres. However, the direct effect of FANCM depletion on ALT activity has not been fully determined.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0015] [Non-Patent Document 1] Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 4th Edition (2012), Volume I, Volume II, and Volume III [Non-Patent Document 2] DNA Cloning: A Practical Approach, Volume I and Volume II (D. N. Glover, 2nd Edition, 1995), IRL Press, Oxford [Non-Patent Document 3] Oligonucleotide Synthesis: A Practical Approach (edited by M. J. Gait, 1984) IRL Press, Oxford [Non-Patent Document 4] 4. Nucleic Acid Hybridization: A Practical Approach (edited by B. D. Hames & S. J. Higgins, 1985) IRL Press, Oxford [Non-Patent Document 5] Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford [Non-Patent Document 6] Perbal, B., A Practical Guide to Molecular Cloning (1984) [Non-Patent Document 7] Methods In Enzymology (edited by S. Colowick and N. Kaplan, Academic Press, Inc.) [Non-Patent Document 8] Altschul et al., (1990) J. Mol. Biol. 215: 405 - 410 [Non-Patent Document 9] Pearson and Lipman (1988) PNAS USA 85: 2444 - 2448 [Non - Patent Document 10] Smith and Waterman (1981) J. Mol Biol. 147: 195 - 197 [Non - Patent Document 11] Johnson LS et al., BMC Bioinformatics. August 18, 2010;11( ):431 [Non - Patent Document 12] Pearson Curr Prot Bioinformatics(2013) Chapter 3, Uniy 3.1 doi: 10.1002 / 0471250953.bi0301s42 [Non - Patent Document 13] Altschul et al., Nucl. Acids Res. (1997) 25 3389 - 3402 [Non - Patent Document 14] Angell and Baulcombe (1997) The EMBO Journal 16, 12:3675 - 3684 [Non - Patent Document 15] Voinnet and Baulcombe (1997) Nature 389: 553 [Non - Patent Document 16] Fire A. et al., Nature 391, (1998) [Non - Patent Document 17] Elbashir SM. et al., Nature, 411, 494 - 498, (2001) [Non - Patent Document 18] Elbashir, S. M. et al., Nature 411:494 - 498 (2001) [Non - Patent Document 19] Elbashir, S. M. et al., Genes & Development 15:188 - 200 (2001) [Non - Patent Document 20] Peyman and Ulman, Chemical Reviews, 90:543 - 584, (1990) [Non - Patent Document 21] Crooke, Ann. Rev. Pharmacol. Toxicol. 32: 329 - 376 (1992)

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Summary of the Invention

Problems to be Solved by the Invention

[0016] Obviously, there is a need for new drug targets and improved treatments for cancers including ALT cancer.

Means for Solving the Problems

[0017] The inventors have discovered that FANCM depletion in cells relying on the alternative lengthening of telomeres (ALT) pathway induces cell death. The inventors have demonstrated that inhibition of FANCM activity mediated by its interaction with RMI promotes ALT activity, and this inhibition can be applied to inhibit the growth and / or proliferation of ALT cells. In addition, the inventors have demonstrated that inhibition of the ATPase activity of FANCM can also be applied to inhibit the growth and / or proliferation of ALT cells. Based on these findings, the inventors have developed a new method for inhibiting the viability and / or growth of ALT cells, and a related method for treating ALT cancer, which includes the steps of inhibiting or depleting FANCM, inhibiting the ATPase activity of FANCM, and / or disrupting the FANCM-RMI interaction in ALT cells. Furthermore, the inventors have found that simultaneous inhibition of FANCM and BLM results in alleviation of telomere dysfunction associated with FANCM depletion alone. Thus, in any particular embodiment of any aspect of the invention disclosed herein, BLM and / or BRCA1 are not inhibited or depleted.

[0018] A first aspect of the invention provides a method for treating ALT cancer in an individual in need thereof, the method comprising the step of reducing the expression or activity of Fanconi anemia complementation group M (FANCM) in the individual.

[0019] A second aspect of the invention provides an agent for reducing the expression or activity of FANCM for use in the method of the first aspect.

[0020] A third aspect of the invention provides the use of an agent for reducing the expression or activity of FANCM in the manufacture of a medicament for use in the method of the first aspect.

[0021] A fourth aspect of the invention provides a method for screening for a compound that reduces viability or induces cell death in ALT cancer cells, the method comprising the step of determining the binding of a test compound to FANCM.

[0022] A fifth aspect of the present invention provides a method for screening a compound that reduces viability or induces cell death in ALT cancer cells, the method comprising determining the effect of a test compound on the expression or activity of FANCM.

[0023] A sixth aspect of the present invention provides a method for determining the responsiveness of cancer in an individual to an agent that reduces the expression or activity of FANCM, the method comprising determining the presence of one or more ALT cancer cells in a sample of cancer cells from the individual, wherein the presence of one or more ALT cancer cells in the sample indicates that the cancer responds to the agent.

[0024] An individual having cancer determined to respond to the agent may be treated by the method of the first aspect.

[0025] In a seventh aspect, the disclosure of the present invention provides a method for inhibiting the viability and / or growth of ALT cells, the method comprising disrupting the FANCM-RMI interaction. The method may be a method for treating ALT cancer in a subject.

[0026] Any of the methods disclosed herein may include disrupting the FANCM-RMI interaction by administering an inhibitor of the FANCM-RMI interaction.

[0027] The methods disclosed herein may include disrupting the binding between FANCM and RMI in the MM2 domain.

[0028] Any of the methods disclosed herein may include inhibiting the ATPase activity of FANCM by administering an inhibitor of the ATPase activity of FANCM.

[0029] Any of the methods disclosed herein may include the step of disrupting the FANCM-RMI interaction by administering an inhibitor of the FANCM-RMI interaction, and the step of inhibiting the ATPase activity of FANCM by administering an inhibitor of the ATPase activity of FANCM.

[0030] In certain embodiments, any of the methods disclosed herein may not include the step of inhibiting BLM and / or BRCA1.

[0031] The methods disclosed herein may further include the concurrent, sequential, or separate administration of chemotherapeutic agents.

[0032] Alternatively, the methods disclosed herein may not include the administration of chemotherapeutic agents. Thus, the methods disclosed herein may not include the concurrent, sequential, or separate administration of chemotherapeutic agents. For example, the methods disclosed herein may not include the concurrent administration of chemotherapeutic agents.

[0033] In an eighth aspect, the disclosure of the present invention is a method of selecting a subject for treatment with an inhibitor of the FANCM-RMI interaction, the method including the step of determining whether the subject has ALT cancer, and if the subject has ALT cancer, the subject is selected for treatment with an inhibitor of the FANCM-RMI interaction.

[0034] In a ninth aspect, the disclosure of the present invention is a method of identifying whether a subject having cancer is suitable for treatment with an inhibitor of the FANCM-RMI interaction, the method including the step of determining whether the cancer is ALT cancer, and if the subject has ALT cancer, the subject is identified as being suitable for treatment with an inhibitor of the FANCM-RMI interaction.

[0035] In a tenth aspect, the disclosure of the present invention is a method of determining whether a subject is responsive to treatment with an inhibitor of the FANCM-RMI interaction, Determining the presence and / or extent of genomic instability in one or more telomeres in cells harvested from a subject; and / or Determining the presence and / or level of ALT activity in cells harvested from a subject A method is provided that includes the above steps.

[0036] In another aspect, the disclosure of the present invention is a method for determining whether a subject responds to treatment with an inhibitor of the FANCM-RMI interaction, the method including determining the growth and / or viability of ALT cells in or derived from the subject, wherein a reduction in the growth and / or viability of ALT cells after the start of treatment compared to before the start of treatment indicates that the subject is positively responding to the treatment. In this regard, any of the methods for identifying or detecting ALT cells disclosed herein can be used.

[0037] In an eleventh aspect, the disclosure of the present invention provides a pharmaceutical composition comprising an inhibitor of the FANCM-RMI interaction for use in treating ALT cancer.

[0038] In a twelfth aspect, the disclosure of the present invention provides the use of an inhibitor of the FANCM-RMI interaction in the manufacture of a medicament for the treatment of ALT cancer.

[0039] The pharmaceutical composition may consist essentially of an inhibitor of the FANCM-RMI interaction. Thus, the medicament may consist essentially of an inhibitor of the FANCM-RMI interaction.

[0040] The inhibitor of the FANCM-RMI interaction can be any one or more of a genetic inhibitor, a small molecule, a peptide, and a protein.

[0041] In one embodiment, the inhibitor is a genetic inhibitor. For example, the genetic inhibitor can be siRNA.

[0042] In one embodiment, the inhibitor is a small molecule. For example, the small molecule can be ethyl 4-[(1-hydroxy-2-phenyl-1H-indol-3-yl)-pyridin-2-yl-methyl]-piperazine-1-carboxylate.

[0043] In one embodiment, the inhibitor is a peptide. For example, the peptide can be a peptide having an amino acid sequence that is at least 90% identical to a peptide selected from the group consisting of DLFSVTFDLGFC (SEQ ID NO: 49), DIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 50), and EDIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 5). In one example, the peptide is at least 90% identical to DLFSVTFDLGFC (SEQ ID NO: 49). In another example, the peptide is at least 90% identical to DIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 50). In another example, the peptide is at least 90% identical to EDIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 5).

[0044] In one embodiment, the inhibitor is a protein. For example, the protein can be an inactivated FANCM protein or an inactivated RMI complex. In one example, the inactivated FANCM protein may contain an F1232A / F1236A double replacement.

[0045] In another example, the protein may contain an immunoglobulin binding domain.

[0046] In a 13th aspect, the disclosure of the present invention provides a pharmaceutical composition comprising an inhibitor of the ATPase activity of FANCM for use in treating ALT cancer.

[0047] In a 14th aspect, the disclosure of the present invention provides the use of an inhibitor of the ATPase activity of FANCM in the manufacture of a medicament for the treatment of ALT cancer.

[0048] The pharmaceutical composition may essentially consist of an inhibitor of the ATPase activity of FANCM. Thus, the medicament may essentially consist of an inhibitor of the ATPase interaction of FANCM with FANCM.

[0049] The inhibitor of the ATPase activity of FANCM can be any one or more of a genetic inhibitor, a small molecule, a peptide, and a protein.

[0050] In one embodiment, the inhibitor is a genetic inhibitor. For example, the genetic inhibitor can be siRNA.

[0051] In a fifteenth aspect, the disclosure of the present invention provides a pharmaceutical composition comprising an inhibitor of the FANCM-RMI interaction and an inhibitor of the ATPase activity of FANCM for use in treating ALT cancer.

[0052] In a sixteenth aspect, the disclosure of the present invention provides the use of an inhibitor of the FANCM-RMI interaction and an inhibitor of the ATPase activity of FANCM in the manufacture of a medicament for the treatment of ALT cancer.

[0053] The pharmaceutical composition may essentially consist of an inhibitor of the FANCM-RMI interaction and an inhibitor of the ATPase activity of FANCM. Thus, the medicament may essentially consist of an inhibitor of the FANCM-RMI interaction and an inhibitor of the ATPase interaction of FANCM with FANCM.

[0054] The inhibitor of the FANCM-RMI interaction and the inhibitor of the ATPase activity of FANCM can be any one or more of a genetic inhibitor, a small molecule, a peptide, and a protein.

[0055] Other aspects and embodiments of the present invention are described in more detail below.

[0056] The following drawings form a part of this specification and are included to further demonstrate specific aspects of the disclosure of the present invention. The present disclosure can be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments set forth herein.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0058] Symbol Table of the Sequence Listing SEQ ID NO: 1 Amino acid sequence of the reference human telomerase protein (Uniprot accession number O14746). SEQ ID NO: 2 Amino acid sequence of the reference human FANCM protein (Uniprot accession number Q8IYD8). SEQ ID NO: 3 Amino acid sequence of the reference human RMI1 protein (Uniprot accession number Q9H9A7). SEQ ID NO: 4 Amino acid sequence of the reference human RMI2 protein (Uniprot accession number Q96E14). SEQ ID NO: 5 Amino acid sequence of the reference human MM2 protein domain. SEQ ID NO:6 Amino acid sequence of reference human BLM protein (Uniprot accession number P54132). SEQ ID NO:7 Amino acid sequence of reference human TOP3A protein (Uniprot accession number Q13472). SEQ ID NO:8 Amino acid sequence of reference human BRCA1 protein (Uniprot accession number P38398). SEQ ID NO:9 Nucleotide sequence of reference human FANCM sequence (Genbank accession number NM_020937.1). SEQ ID NO:10 Nucleotide sequence of reference human RMI1 sequence (Genbank accession number NM_001358291.1). SEQ ID NO:11 Nucleotide sequence of reference human RMI2 sequence (Genbank accession number NM_152308.3). SEQ ID NO:12 Nucleotide sequence of FANCM1 siRNA. SEQ ID NO:13 Nucleotide sequence of FANCM2 siRNA. SEQ ID NO:14 Nucleotide sequence of POLD3 siRNA. SEQ ID NO:15 Nucleotide sequence of BLM siRNA. SEQ ID NO:16 Nucleotide sequence of RAD51 siRNA. SEQ ID NO:17 Nucleotide sequence of RAD52 siRNA. SEQ ID NO:18 Nucleotide sequence of RMI1 siRNA. SEQ ID NO:26 Amino acid sequence of mutant human FANCM protein (PIP). SEQ ID NO:28 Amino acid sequence of mutant human FANCM protein (K117R). SEQ ID NO:30 Amino acid sequence of mutant human FANCM protein (MID). SEQ ID NO:32 Amino acid sequence of mutant human FANCM protein (S1045A). SEQ ID NO:34 Amino acid sequence of mutant human FANCM protein (ΔMM1). SEQ ID NO:36 Amino acid sequence of mutant human FANCM protein (ΔMM2). SEQ ID NO:38 Amino acid sequence of mutant human FANCM protein (FF>AA). Amino acid sequence of mutant human FANCM protein (ΔMM3) with SEQ ID NO: 26. Amino acid sequence of mutant human FANCM protein (ΔERCC4) with SEQ ID NO: 27. Amino acid sequence of mutant human FANCM protein (ΔHhH) with SEQ ID NO: 28. Amino acid sequence of wild-type human FANCM MM2 peptide with SEQ ID NO: 29. Amino acid sequence of mutant human FANCM MM2 peptide with SEQ ID NO: 30. Amino acid sequence of reference human FANCM protein with SEQ ID NO: 31. Nucleotide sequence of reference human FANCM coding sequence with SEQ ID NO: 32. Nucleotide sequence of siRNA molecule (siFa) related to the suppression of human FANCM with SEQ ID NO: 33. Nucleotide sequence of siRNA molecule (siFb) related to the suppression of human FANCM with SEQ ID NO: 34. Amino acid sequence of double mutant human FANCM protein (K117R and FF>AA) with SEQ ID NO: 35. Nucleotide sequence of siRNA molecule (siBl) related to the suppression of human FANCM with SEQ ID NO: 36. Nucleotide sequence of siRNA molecule (siATRXa) related to the suppression of human FANCM with SEQ ID NO: 37. Nucleotide sequence of siRNA molecule (siATRXb) related to the suppression of human FANCM with SEQ ID NO: 38. Nucleotide sequence of siRNA molecule (siTRF1) related to the suppression of human FANCM with SEQ ID NO: 39. Nucleotide sequence of siRNA molecule (siRNase H1) related to the suppression of human FANCM with SEQ ID NO: 40.

[0059] General Techniques and Definitions Unless otherwise specifically specified, all technical and scientific terms used in this specification shall be construed to have the same meaning as commonly understood by those skilled in the art in the relevant fields (e.g., genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0060] The disclosure of the present invention is carried out using conventional techniques in molecular biology, microbiology, recombinant DNA technology, and immunology, unless otherwise specified. Such procedures are described, for example, in their entirety in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 4th Edition (2012), Volumes I, II, and III; DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover, 2nd Edition, 1995), IRL Press, Oxford, the entire text; Oligonucleotide Synthesis: A Practical Approach (edited by M. J. Gait, 1984) IRL Press, Oxford, the entire text, and in particular the articles by Gait described therein, pages 1-22; Atkinson et al., pages 35-81; Sproat et al., pages 83-115; and Wu et al., pages 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (edited by B. D. Hames & S. J. Higgins, 1985) IRL Press, Oxford, the entire text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, the entire text; Perbal, B., A Practical Guide to Molecular Cloning (1984) and Methods In Enzymology (edited by S. Colowick and N. Kaplan, Academic Press, Inc.), the entire series.

[0061] Those skilled in the art will understand that the disclosure of the present invention also permits variations and modifications other than those specifically described. It will be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all of the steps, features, compositions and compounds individually or collectively mentioned or shown herein, as well as any and all combinations of any two or more of said steps or features.

[0062] The disclosure of the present invention is not limited to the scope by the specific embodiments described herein, which are merely intended for illustrative purposes. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure as described herein.

[0063] Each feature of any particular aspect or embodiment of the disclosure of the present invention can be applied to any other aspect or embodiment of the disclosure of the present invention with the necessary modifications.

[0064] Throughout this specification, unless otherwise specified or the context requires otherwise, references to a single step, composition of matter, group of steps or group of compositions of matter shall be construed to include one and more (i.e., one or more) of those steps, compositions of matter, group of steps or group of compositions of matter.

[0065] As used herein, the singular forms "a", "and" and "the" include the plural forms of these words unless the context clearly dictates otherwise.

[0066] The term "and / or", e.g., "X and / or Y", is understood to mean either "X and Y" or "X or Y", and is construed to provide explicit support for both meanings or either meaning.

[0067] As used herein, the term "and / or" shall be construed as a specific disclosure of whether each of two specified features or components is or is not accompanied by the other. For example, "A and / or B" shall be construed as a specific disclosure of (i) A, (ii) B, and (iii) A and B, respectively, as if each were individually recited herein.

[0068] Throughout this specification, variations such as the words "comprise", "comprises", or "comprising" shall be understood to imply the inclusion of the stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0069] ALT cells and ALT cancer The disclosure of the present invention provides a method for inhibiting the viability and / or growth of ALT cells. In this regard, the inventors have surprisingly shown that the step of disrupting the FANCM-RMI interaction is selectively toxic to ALT cells. This disclosure also relates to the discovery that reduction or elimination of the expression or activity of FANCM in ALT cancer cells induces G2 / M arrest and cell death. Accordingly, methods for disrupting the FANCM-RMI interaction and for reducing or inhibiting the expression or activity of FANCM can be used, for example, in the treatment of ALT cancer.

[0070] Cancer cells must maintain their telomeres in order to achieve unlimited proliferation. Most cancers (85 - 90%) achieve this by reactivating telomerase (telomerase-positive cells). Telomerase is a reverse transcriptase involved in synthesizing telomeric DNA from an RNA template. The sequence of telomerase is publicly available. An exemplary sequence is set forth in SEQ ID NO: 1. The remaining 10 - 15% of tumor cells must stabilize their chromosome ends by alternative mechanisms in order to avoid growth arrest. These telomerase-independent strategies are collectively known as alternative lengthening of telomeres (ALT). Thus, ALT cells as defined herein can be cells that exhibit an active ALT mechanism. The ALT mechanism can be any mechanism of telomere stabilization that does not rely on telomerase. The ALT mechanism is not necessarily limited to any one specific mechanism by which ALT might operate in a cell to maintain telomeres. Thus, the "ALT mechanism" does not necessarily refer only to one specific biochemical mechanism or pathway by which the ALT mechanism operates. There may be more than one specific pathway by which ALT operates.

[0071] Thus, ALT cells can be any cells that do not rely on telomerase activity to maintain the length of their telomeres. Alternatively, or in addition, ALT cells may be considered any cells that do not rely on telomerase activity to maintain the stability of their telomeres. Thus, ALT cells can be cells that are not telomerase-positive; or may be considered telomerase-negative cells.

[0072] Alternatively, or in addition, ALT cells may be considered cells in which the expression and / or activity of telomerase is reduced compared to telomerase-positive cells. The expression and / or activity of telomerase can be determined by any means known in the art. For example, the expression level of telomerase can be determined by quantifying the level of production of telomerase mRNA by any suitable mRNA detection method (e.g., but not limited to, quantitative PCR, real-time qPCR; next-generation sequencing (NGS) methods; nanopore sequencing methods; Northern blotting, etc.). Alternatively, or in addition, the expression level of telomerase may be determined by quantifying the level of production of telomerase protein by any suitable protein detection method. The telomerase protein level can be detected, for example, by, but not limited to, Western blotting; antibody detection methods (e.g., ELISA; or detection of labels such as fluorescent labels conjugated to antibodies capable of specifically binding to telomerase); and other methods. Alternatively, or in addition, the expression and / or activity level of telomerase may be determined via the performance of a telomerase function assay, in which case the level of telomerase activity is an indicator of the level of expression and / or activity of telomerase in the cell. Telomerase activity may be detected by the telomerase repeat amplification protocol (TRAP), quantitative TRAP (qTRAP), or by a direct telomerase activity assay, such as those described in Cohen and Reddel, 2008. Any method for identifying ALT cells or identifying ALT cancer, or for determining whether a subject has ALT cancer disclosed herein, may include determining whether a cell is a telomerase-positive cell by any of the methods disclosed herein, and it will be understood that if a cell is determined to be a telomerase-positive cell, that cell is identified as not being an ALT cell.

[0073] ALT cells can be characterized by an elevated level of DNA damage compared to dead or telomerase-positive cells, which indicates increased telomere replication stress in ALT cells. This increased telomere replication stress is due to the cumulative inadequacy of telomere structural integrity. Frequent or persistent replication fork stalling causes nicks and breaks in DNA, so the ALT mechanism is hypothesized to arise from stalled replication forks that deteriorate to form double-strand breaks (DSBs), which then provide substrates for the participation of homologous recombination repair pathways and end with break-induced telomere synthesis. Therefore, ALT cells achieve a delicate balance between telomere protection, repair activity, and telomere damage, and disruption of this balance may be applied as a means of disabling the ALT mechanism.

[0074] ALT cells, as defined herein, may be identified by detection of one or more phenotypic features of ALT telomere repair or ALT telomere replication stress, including, for example, replication fork stalling above levels typical of non-ALT cells (e.g., above levels typical of telomerase-positive cells or dead cells); DSB occurrence above levels typical of non-ALT cells (e.g., above levels typical of telomerase-positive cells or dead cells), and the like. It will be understood that the levels of telomere replication fork stalling and / or DSBs typical of ALT cells can be established through identification and / or measurement of these features in samples of ALT cells and samples of non-ALT cells (e.g., telomerase-positive cells or dead cells). Suitable threshold levels can then be determined according to the particular techniques used to identify and / or measure these features, and then in this way, a given cell can be identified as an ALT cell or a non-ALT cell using the same or similar techniques. It will be understood that the exact threshold will vary depending on the samples used to establish that threshold level and further according to the particular analytical techniques used in each instance.

[0075] ALT includes recombination-dependent DNA replication (Dunham et al., 2000), and ALT can result in a sudden substantial increase in telomere length (Murnane et al., 1994), which is consistent with either a long linear telomere template or a rolling mechanism, such as rolling circle amplification (RCA). Cells with ALT activity also experience a rapid decrease in the length of individual telomeres (Jiang et al., 2005 and Perrem et al., 2001), resulting in a highly heterogeneous telomere length distribution. ALT cells often contain telomeric chromatin within promyelocytic leukemia (PML) nuclear bodies (ALT-associated promyelocytic leukemia nuclear bodies; APBs) (Yeager et al., 1999). Thus, ALT cells as defined herein may include any one or more of these phenotypic characteristics. For example, ALT cells may exhibit recombination-dependent DNA replication and / or may exhibit a sudden substantial increase in telomere length (e.g., compared to non-ALT cells such as telomerase-positive cells or dead cells), and / or may exhibit a heterogeneous telomere length distribution (e.g., compared to non-ALT cells such as telomerase-positive cells or dead cells), and / or may contain APBs (e.g., a higher level of APBs in non-ALT cells such as telomerase-positive cells or dead cells). Alternatively, ALT cells may be identified by the maintenance of telomere length over one or more cell divisions in the absence of telomerase activity and / or expression. It will be understood that telomeres are repetitive DNA sequences that exist at or near the ends of linear chromosomes. Telomeres in humans typically contain multiple repeats of the nucleotide sequence 5'-TTAGGG-3'. Thus, identification of telomere length may include determining the number of repeats of this nucleotide sequence.

[0076] The ALT cells disclosed herein can be cancer cells. Thus, the ALT cells disclosed herein can be derived from a subject suffering from a disease or condition associated with abnormal cell proliferation, a subject suspected of suffering therefrom, or a subject predisposed thereto. The cancer can be of any physiological origin. ALT has been identified in a variety of cancers, such as, but not limited to, carcinomas arising from tissues such as those from the bladder, cervix, endometrium, esophagus, gallbladder, kidney, liver, lung, brain, bone, and connective tissue. ALT has also been found in medulloblastoma, oligodendroglioma, meningioma, schwannoma, and glioblastoma multiforme in children. In one example, the cancer can be any one of bladder cancer, cervical cancer, endometrial cancer, esophageal cancer, gallbladder cancer, kidney cancer, liver cancer, lung cancer, brain cancer, bone cancer, or connective tissue cancer. The ALT cancer or cell can be, for example, a sarcoma, blastoma, carcinoma, mesothelioma, or astrocytoma. The sarcoma can be osteosarcoma, malignant fibrous histiocytoma, liposarcoma, synovial sarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, or leiomyosarcoma. The blastoma can be neuroblastoma. The carcinoma can be non-small cell lung cancer, such as lung adenocarcinoma or breast cancer. The mesothelioma can be peritoneal mesothelioma. The astrocytoma can be low-grade astrocytoma, anaplastic astrocytoma, or glioblastoma multiforme. The ALT cancer or cell can be medulloblastoma, oligodendroglioma, meningioma, schwannoma, and / or glioblastoma multiforme in children. In certain embodiments, the ALT cancer is osteosarcoma, soft tissue sarcoma (such as liposarcoma, undifferentiated pleomorphic sarcoma, or leiomyosarcoma), glioblastoma, astrocytoma, neuroblastoma, or bladder cancer. The cancer can be primary cancer or metastatic cancer. The metastatic cancer can have a known or unknown origin. The methods disclosed herein can be used to treat any of these or other ALT cancers.

[0077] The ALT cells can be derived from any vertebrate, such as a mammal, and in particular, can be of human origin.

[0078] As shown herein, methods for determining whether a cell is an ALT cell or whether a cancer is an ALT cancer are known in the art. For example, C-circle biomarkers are ALT-specific molecules that can be detected using a C-circle assay (Henson et al., 2009 and WO / 2011 / 035375). The entire content of WO / 2011 / 035375 is incorporated herein by reference. Briefly, a C-circle assay involves extracting DNA from a sample and then quantifying it. For example, C-circles can be amplified by rolling circle amplification and the products can be detected.

[0079] Any of the methods disclosed in Henson et al., 2009 and / or WO / 2011 / 035375 can be used in combination with the disclosure of the present invention to identify a cell as an ALT cell. Thus, for example, the methods disclosed herein may include identifying a cell as an ALT cell by determining the presence and / or amount of partially double-stranded telomeric DNA circles in the cell, where the presence and / or amount of the partially double-stranded telomeric DNA circles identifies the cell as an ALT cell. The partially double-stranded telomeric DNA circles may include a closed circular strand and a linear strand. The circular strand may include a C-rich or G-rich telomeric sequence. The linear strand may include a G-rich or C-rich telomeric DNA sequence. The partially double-stranded telomeric circle may include repeats of the sequence (CCCTAA) n in the circular strand, and / or repeats of the sequence (TTAGGG) n in the linear strand (where n is any integer greater than 1). The partially double-stranded telomeric circle may include repeats of the sequence (TTAGGG) n in the circular strand, and / or repeats of the sequence (CCCTAA) n in the linear strand (where n is any integer greater than 1). In one example, the presence and / or amount of partially double-stranded telomeric DNA circles in a cell can be detected using rolling circle amplification.

[0080] The circular and / or linear strands may contain variant telomere repeat sequences, mutant telomere repeat sequences and / or non-telomere sequences.

[0081] The partially double-stranded telomere circle may be detected directly or indirectly. For example, the detection may be indirect according to rolling circle amplification. Rolling circle amplification can use the circular strand of the partially double-stranded circle as a template. In one example, the detection is (a) Optionally isolating DNA from cells; (b) incubating the DNA in the presence of a DNA polymerase and one or more dNTPs under suitable conditions such that polymerase-mediated extension from the incomplete (linear) strand generates a concatemer of single-stranded telomeric DNA; and (c) detecting the concatemer comprising.

[0082] The concatemer may be detected by any suitable means, such as hybridization, sequencing, PCR, molecular beacons, ribozymes, such as DNA partzymes, etc., or by incorporating a suitably labeled dNTP in the incubation step (b). In one example, the concatemer may be detected using a labeled nucleotide probe. The labeled probe may contain the nucleotide sequence (CCCTAA) n wherein n is any integer of 1 or greater than 1. The label can be any detectable label. For example, the label can be a fluorescent label.

[0083] The DNA polymerase can be, for example, φ29 DNA polymerase. Typically, when the partially double-stranded telomere circle contains repeats of the sequence (CCCTAA) n in the circular strand, the dNTPs consist of dATP, dGTP and dTTP, and optionally dCTP.

[0084] Detection of the partially double-stranded telomere circle may be detection of the circle present in cells, or alternatively may include detection of the circle in a biological sample, such as a sample from a subject. The biological sample may include, for example, blood, urine, sputum, pleural fluid, peritoneal fluid, bronchial and bronchoalveolar lavage fluid, or a tissue section. The sample may be obtained, for example, by fine needle aspiration biopsy. The blood may be whole blood, serum or plasma.

[0085] Rolling circle amplification may be performed with the provision of exogenous primers or without them. Advantageously, rolling circle amplification may be performed without exogenous primers. Thus, the method of identifying ALT cells by performing the C-circle assay disclosed herein may not include the use of exogenous primers.

[0086] Other suitable methods for determining ALT activity include, but are not limited to, telomere DNA and C-circle telomerase quantitative PCR (Lau et al., 2012); absence of telomerase activity; presence of very long and heterogeneous telomeres; presence of ALT-associated promyelocytic leukemia (PML) bodies (APBs) containing telomere DNA and telomere-binding proteins (Yeager et al., 1999); increased telomere sister chromatid exchange (T-SCE) events and presence of extrachromosomal telomere repeat (ECTR) DNA. Tumor samples can also be evaluated by a combination of telomere-specific fluorescence in situ hybridization and immunofluorescent labeling of the PML protein (Heaphy et al., 2011).

[0087] ALT cells contain a novel form of the promyelocytic leukemia (PML) bodies (ALT-associated PML bodies, APBs), in which the PML protein co-localizes with telomeric DNA and telomere-binding proteins hTRF1 and hTRF2. APBs are not found in dead cells, telomerase-positive cell lines or tumors (Yeager et al., 1999). Any method known in the art for detecting APBs can be used in conjunction with the disclosure of the present invention. For example, APBs may be detected visually. For example, APBs may be visualized by immunohistochemical methods (e.g., using anti-hTRF1 and / or anti-PML antibodies).

[0088] Significant differences in telomere variant repeat content have been found in tumors that use the ALT mechanism and those that do not (Lee et al., 2018). Accordingly, any method known in the art for determining telomere variant repeat content can be used in conjunction with the disclosure of the present invention. For example, whole genome sequencing can be used to determine telomere variant repeat content.

[0089] FANCM 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 can be human FANCM. The human gene (gene number 57697) encoding the FANCM polypeptide has 25 exons and is located at 14q21.2. The amino acid sequence of reference human FANCM is shown in SEQ ID NO: 31. Other reference human FANCM amino acid sequences have database accession numbers NP_001295062.1, NP_1295063.1, and NP_065988.1. The FANCM polypeptides described herein may include an amino acid sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% identity, or at least 98% identity with a reference amino acid sequence, such as SEQ ID NO: 31, or a reference amino acid sequence, such as SEQ ID NO: 31.

[0090] The nucleotide sequence encoding reference human FANCM is shown in SEQ ID NO: 32. Other reference human FANCM coding sequences have GenBank accession numbers NM_001308133.1, NM_001308134.1, and NM_020937.4. The FANCM nucleotide sequences described herein may include a reference human FANCM coding sequence, such as the nucleotide sequence of SEQ ID NO: 32, or a reference human FANCM coding sequence, such as SEQ ID NO: 32, and a nucleotide sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% identity, or at least 98% identity.

[0091] Sequence identity is generally defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximize the number of matches and minimize the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and a GAP extension penalty = 4. Although the use of GAP may be preferred, other algorithms can also be used, such as BLAST (which uses the method of Altschul et al., (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses 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. August 18, 2010; 11(): 431), or the TBLASTN program of Altschul et al. (1990) above which generally employs default parameters (see, for example, Pearson Curr Prot Bioinformatics (2013), Chapter 3, Uniy 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 comparisons are preferably made over the full length of the relevant sequences described herein.

[0092] In other embodiments, for example, if the individual to be treated is a non-human mammal, FANCM can be a non-human mammalian FANCM.

[0093] In cells that utilize the alternative lengthening of telomeres (ALT) pathway to maintain telomeres (ALT cells), reduction in the expression or activity of FANCM is shown herein to reduce viability and increase cell cycle arrest and death. In contrast, reduction in the expression or activity of FANCM has no effect on telomerase-positive cells (which do not maintain telomeres via the ALT pathway) or primary cells. Therefore, an agent that reduces the expression or activity of FANCM can be used to induce cell death in ALT cancer cells (i.e., cancers that rely on the ALT pathway to maintain telomeres), for example, in the treatment of ALT cancers.

[0094] FANCM activity can be reduced in an individual by administering an agent that reduces the expression or activity of FANCM, such as a FANCM antagonist. A FANCM antagonist can inhibit FANCM activity, for example, be a FANCM inhibitor, or can reduce or suppress the expression of FANCM, for example, be a suppressor nucleic acid or a targeted nuclease.

[0095] FANCM-RMI complex FANCM is an essential factor in the stabilization of stalled replication forks. It contains two DNA-binding domains at its N and C termini, with three highly conserved regions (MM1-MM3) in between. The sequence of FANCM is publicly available. An exemplary sequence is set forth in SEQ ID NO: 2. The MM1 domain replenishes the FA core complex, a multi-subunit ubiquitin ligase essential for DNA interstrand crosslink (ICL) repair, while the MM2 domain is described as a 34-amino acid motif that directly binds to the RMI (RecQ-mediated genomic instability) subcomplex (RMI1 and RMI2) of the BLM-TOP3A-RMI (BTR) subcomplex. The MM2 domain is also described in (Deans et al., 2009). The sequences of RMI1 and RMI2 are publicly available, and exemplary sequences are set forth in SEQ ID NO: 3 and 4. An exemplary sequence containing the MM2 domain is set forth in SEQ ID NO: 5. Thus, the MM2 domain may contain the amino acid sequence set forth in SEQ ID NO: 5. Alternatively, the MM2 domain described herein may contain an amino acid sequence that is at least 90% identical to the amino acid sequence DLFSVTFDLGFC (SEQ ID NO: 49). The MM2 domain may contain an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence DLFSVTFDLGFC (SEQ ID NO: 49). This sequence has been identified as the core sequence within the MM2 domain (Hoadley et al., 2012).

[0096] The BTR complex encompasses BLM helicase activity, TOP3A decatenation activity, branch migration, and overall resolvase activity, and it is suggested that FANCM and BTR cooperate to be able to reverse stalled forks and thus stabilize them. The sequences of BLM and TOP3A are publicly available, and exemplary sequences are set forth in SEQ ID NO: 6 and 7.

[0097] Retention of FANCM at stalled replication forks depends on its interaction with the functional BTR complex, but not on its interaction with the FA core complex.

[0098] Inhibitor In certain embodiments, the FANCM inhibitor and the RMI inhibitor inhibit or reduce the expression of FANCM or RMI, respectively, in cells, such as ALT cancer cells. In certain embodiments, the FANCM inhibitor and the RMI inhibitor inhibit or reduce one or more biological activities of FANCM or RMI, respectively, in cells, such as ALT cancer cells. In certain embodiments, the FANCM inhibitor and the RMI inhibitor inhibit or reduce the binding of FANCM or RMI to another protein, respectively, in cells, such as ALT cancer cells. In certain embodiments, the FANCM inhibitor inhibits or reduces the ATPase activity of FANCM in cells, such as ALT cancer cells.

[0099] The FANCM inhibitor or RMI inhibitor may be a direct or indirect inhibitor of FANCM or RMI, respectively. That is, they can exert their inhibitory effects by directly binding to FANCM or RMI, or to the nucleic acid sequences encoding FANCM or RMI, respectively. Or they can indirectly exert their inhibitory effects, for example, by inhibiting another protein required for either the expression or activity of FANCM or RMI. It will be understood by those skilled in the art that this is possible. In certain embodiments, an inhibitor such as those disclosed herein may be capable of inhibiting FANCM such that one or more endogenous activities of FANCM are inhibited. In certain embodiments, an inhibitor such as those disclosed herein may be capable of inhibiting RMI such that one or more endogenous activities of RMI are inhibited. In certain embodiments, the FANCM antagonist or inhibitor inhibits or reduces the DNA translocase activity or the ATPase activity of FANCM. In certain embodiments, the FANCM antagonist or inhibitor inhibits or reduces the ALT activity in ALT cells, such as ALT cancer cells. In certain embodiments, the DNA translocase activity or the ATPase activity of FANCM is reduced by at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or completely reduced compared to the activity present in ALT cells not contacted with the FANCM antagonist or inhibitor. In certain embodiments, the ALT activity is reduced by at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or completely reduced compared to the activity present in ALT cells not contacted with the FANCM antagonist or inhibitor.

[0100] In certain embodiments, the FANCM-RMI interaction can be disrupted by effectors upstream or downstream of FANCM or RMI. The inhibitor may be a direct inhibitor of the FANCM-RMI interaction or an indirect inhibitor of the FANCM-RMI interaction. For example, the inhibitor can inhibit the function of FANCM by binding to FANCM and changing its conformation or affecting its binding site such that it cannot bind to RMI. In another example, the inhibitor can inhibit the function of RMI by binding to RMI and changing its conformation or affecting its binding site such that it cannot bind to FANCM. For example, the inhibitor can disrupt the RMI1-RMI2 partial complex such that the RMI1-RMI2 partial complex cannot bind to FANCM. In one example, the binding of FANCM and RMI is disrupted in the MM2 domain. Any inhibitor, such as those disclosed herein, may be capable of disrupting the FANCM-RMI interaction such that the endogenous function of the FANCM-RMI complex is inhibited.

[0101] FANCM activity can be measured via a C-circle assay or by any suitable means known in the art or by any of the methods disclosed herein.

[0102] FANCM inhibitors and RMI inhibitors (and other inhibitors) can be any type of molecule having inhibitory activity, e.g., small molecule chemical molecules, polypeptides (including peptides and proteins as examples of this), nucleic acids, or molecules comprising any combination of these classes of molecules. The terms "FANCM inhibitor" and "RMI inhibitor" as used herein encompass pharmaceutically acceptable salts and solvates of any biological molecule or compound disclosed herein.

[0103] In certain embodiments, the inhibitor can result in a reduction in the expression of a target protein, such as FANCM, or a reduction in one or more biological activities of a target protein, such as FANCM, and the reduction is, in each case, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% reduction. Reducing the amount of active FANCM protein to 20% or less of the amount in control cells has been shown to be sufficient to induce cell death. For example, the cells can express up to 5%, up to 10%, up to 15% or up to 20% of the active FANCM polypeptide expressed by the control cells. In certain embodiments, the ATPase activity of FANCM is reduced by, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% compared to control cells not treated with the inhibitor.

[0104] Examples of FANCM inhibitors can include biological molecules that specifically bind to FANCM. The ATPase activity of FANCM is located within the amino-terminal DEAH helicase-like domain involved in translocase and branch point migration activities. This ATPase activity has generally been found not to be important for targeting of the core complex and FANCD2 ubiquitination, but is required for replication fork stability and efficient checkpoint response. In some embodiments, the biological molecule can specifically bind to the region of FANCM associated with the ATPase activity. In some embodiments, the biological molecule can specifically bind to the DEAH helicase-like domain corresponding to residues 83 - 591 of SEQ ID NO: 31. In some embodiments, the biological molecule can specifically bind to the MM2 domain of FANCM corresponding to SEQ ID NO: 5.

[0105] Examples of FANCM antagonists and FANCM inhibitors include, for example, low molecular weight chemical molecules, such as non-polymeric organic compounds having a molecular weight of 900 daltons or less. Suitable small molecule FANCM inhibitors can, for example, inhibit the binding of ATP to the ATPase domain of FANCM; the binding of DNA to the translocase domain of FANCM, and / or the binding of FANCM to binding partners such as MHF, FAAP24, BLM, RMI, Topo IIIα. Suitable techniques for the rational design of small molecule inhibitors through structural analysis of FANCM are well known in the art.

[0106] In one example, the FANCM inhibitor is a small molecule. According to one example where the FANCM inhibitor is a small molecule, the small molecule is ethyl 4-[(1-hydroxy-2-phenyl-1H-indol-3-yl)-pyridin-2-yl-methyl]-piperazine-1-carboxylate. Thus, the small molecule inhibitor can be an inhibitor defined by the following formula I: Formula I:

Chemical formula

[0107] In certain embodiments, the inhibitor is a biological molecule, such as a polypeptide, such as a peptide or a protein. The peptide may contain or consist of 5 to 40 amino acids, such as 6 to 10 amino acids, and may be derived from FANCM or its binding partner described herein. Polypeptide molecules also include antibodies, antibody fragments and antibody derivatives, as well as non-immunoglobulin binding molecules such as aptamers, trinectins, anticalins, knotted domains, transferrins, tenecteplase antigen receptors, and sea lamprey leucine-rich repeat proteins. Suitable techniques for generating biological molecules that specifically bind to FANCM are well known in the art.

[0108] In some embodiments, the FANCM inhibitor is a peptide. The peptide can be any peptide that mimics all or part of the MM2 domain of FANCM. For example, the peptide can be a peptide comprising or consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence DLFSVTFDLGFC (SEQ ID NO: 49). The peptide can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence DLFSVTFDLGFC (SEQ ID NO: 49). For example, the peptide can be a peptide comprising or consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence DIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 50). The peptide can be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence DIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 50).

[0109] In some embodiments, the peptide can be a peptide comprising or consisting of an amino acid sequence that is at least 90% identical to EDIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 5). The peptide can be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence EDIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 5).

[0110] In some embodiments, the FANCM inhibitor, which is a peptide or protein, can be any peptide capable of binding to FANCM and further capable of shielding the MM2 binding domain. The shielding can be such that the normal (endogenous) binding interaction between FANCM and its endogenous binding partner is disrupted. The peptide or protein can be capable of binding directly or indirectly to the MM2 binding domain. Alternatively, the peptide or protein can be any peptide of the RMI complex capable of binding to FANCM.

[0111] In some embodiments, the FANCM inhibitor can be a mutant FANCM protein with reduced binding ability to the RMI protein, or a mutant RMI protein with reduced binding ability to the FANCM protein. Without wishing to be bound by theory, such proteins can act as decoys for endogenous FANCM or RMI proteins that saturate the available binding sites on the endogenous proteins and thereby inhibit their function. In one example, the mutant FANCM can be an inactivated FANCM protein containing the F1232A / F1236A double replacement. This protein may contain an immunoglobulin binding domain.

[0112] In some embodiments, the inhibitor can be a genetic inhibitor of FANCM or RMI. Methods for designing suitable genetic inhibitors are known in the art. Suitable examples of genetic inhibitors include, but are not limited to, DNA (gDNA, cDNA), RNA (sense RNA, antisense RNA, mRNA, tRNA, rRNA, short interfering RNA (siRNA), short hairpin RNA (ShRNA), microRNA (miRNA), small nucleolar RNA (SnoRNA), small nuclear RNA (snRNA), ribozyme, aptamer, DNAzyme, antisense oligonucleotide, vector, plasmid, other ribonuclease-type complexes, and mixtures thereof. The gene sequences of FANCM and RMI1 and RMI2 are publicly available and can be used to design suitable genetic inhibitors by methods known in the art. The reference nucleotide sequences of FANCM, RMI1, and RMI2 are provided in SEQ ID NOs: 9, 10, and 11, respectively.

[0113] Examples of suitable genetic inhibitors are described in the experimental examples herein. Thus, the genetic inhibitor may comprise or consist of an siRNA inhibitor comprising the nucleotide sequence disclosed in SEQ ID NO: 12 or SEQ ID NO: 13.

[0114] In certain embodiments, inhibitors for reducing or suppressing the expression of FANCM include suppressor nucleic acids, targetable nucleases, and nucleic acids encoding such agents. The nucleic acid encoding the suppressor nucleic acid or targetable nuclease may be contained in a vector. Suitable expression vectors are well known in the art and examples thereof include viral vectors such as retrovirus, adenovirus, adeno-associated virus, lentivirus, vaccinia or herpes vectors.

[0115] The expression of active FANCM protein may be reduced or eliminated by a suppressor nucleic acid or a targetable nuclease, i.e., the transcription of the FANCM gene and / or the translation of FANCM mRNA can be reduced or eliminated such that cells treated with the suppressor nucleic acid or targetable nuclease lack active FANCM protein or have a reduced amount of active FANCM protein compared to control cells. Reducing the amount of active FANCM 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 FANCM polypeptide expressed by control cells.

[0116] In some embodiments, nucleic acid inhibition can be used to reduce the expression of active FANCM polypeptide. The use of nucleic acid inhibition techniques such as antisense and RNAi inhibition to downregulate the expression of a target gene is well established in the art.

[0117] Cells can be transfected with a suppressor nucleic acid (i.e., a nucleic acid molecule that suppresses the expression of FANCM), such as an siRNA or shRNA encoding a suppressor nucleic acid, or a heterologous nucleic acid. The suppressor nucleic acid reduces the expression of active FANCM polypeptide by interfering with transcription and / or translation, thereby reducing FANCM activity in the cell.

[0118] RNAi involves the expression or introduction into cells of an RNA molecule comprising a sequence identical or highly similar to the FANCM coding sequence. The RNA molecule interacts with the 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 the active FANCM polypeptide (Angell and Baulcombe (1997) The EMBO Journal 16, 12: 3675-3684; Voinnet and Baulcombe (1997) Nature 389: 553).

[0119] 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 variety of mammalian cell lines (Elbashir SM. et al., Nature, 411, 494-498, (2001)).

[0120] Suitable RNA molecules for use in RNAi inhibition include short interfering RNA (siRNA). The siRNA is a double-stranded RNA molecule having a length of 15 to 40 nucleotides, preferably a length of 15 to 28 nucleotides or 19 to 25 nucleotides, for example, a length of 19, 20, 21, 22, 23, 24 or 25 nucleotides. For example, two unmodified 21mer oligonucleotides can anneal together to form an siRNA. The siRNA molecule may contain 3' and / or 5' overhangs having a length of about 0, 1, 2, 3, 4, or 5 nucleotides in each strand. The lengths of the overhangs of the strands are independent, i.e., the length of the overhang in one strand does not depend on the length of the overhang in the second strand.

[0121] Other suitable RNA molecules 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 nucleotides) antisense nucleotide sequence, followed by a 5 - 9 nucleotide nucleotide loop, and a complementary sense nucleotide sequence (e.g., 19 - 25 nucleotides).

[0122] Alternatively, the sense sequence may be present before the nucleotide loop structure and the antisense sequence may be present after it. The nucleotide loop forms a hairpin turn, thereby enabling base pairing between the complementary sense and antisense sequences and the formation of the shRNA.

[0123] Suppressor nucleic acids, such as siRNAs or shRNAs, may comprise or consist of a sequence that is identical or substantially identical (i.e., at least 90%, at least 95% or at least 98% identical) to the coding sequence of a reference FANCM nucleotide, such as all or part (e.g., 15 - 40 nucleotides) of SEQ ID NO: 32, or its complement. Suitable reference sequences encoding FANCM that can be used in the design of suppressor nucleic acids are publicly available and an example thereof is SEQ ID NO: 32. FANCM activity is suppressed in cancer cells by down - regulation of the production of active FANCM polypeptide by the suppressor nucleic acid. For example, an siRNA for suppressing the expression of human FANCM may comprise 18 - 22 consecutive nucleotides from SEQ ID NO: 32.

[0124] Examples of preferred siRNA molecules for the suppression of human FANCM include SEQ ID NO: 33 (siFa) and SEQ ID NO: 34 (siFb).

[0125] Suppressor nucleic acids for reducing the expression of FANCM, such as siRNA and shRNA, can be easily designed using reference FANCM coding sequences and software tools widely available in the art, and can be produced using conventional techniques. For example, the suppressor nucleic acids may be chemically synthesized, recombinantly produced in vitro or in cells (Elbashir, S. M. et al., Nature 411:494-498 (2001); Elbashir, S. M. et al., Genes & Development 15:188-200 (2001)), or obtained from commercial sources (e.g., Cruachem (Glasgow, UK), Dharmacon Research (Lafayette, Colo., USA)).

[0126] In some embodiments, two or more suppressor nucleic acids can be used to suppress the expression of FANCM. For example, a pool of siRNAs can be employed. Suitable siRNAs and siRNA pools can be produced using standard techniques.

[0127] Nucleic acid suppression can also be performed using antisense technology. Antisense oligonucleotides hybridize to complementary sequences of nucleic acids, pre-mRNA or mature mRNA, and are designed to interfere with the production of base excision repair pathway components so that their expression is reduced or completely or substantially completely prevented. In addition to targeting coding sequences, antisense technology can be used to target regulatory sequences of genes, such as regulatory sequences in the 5' flanking sequences, whereby the antisense oligonucleotide can interfere with expression control sequences. The construction and use of antisense sequences are well known in the art (Peyman and Ulman, Chemical Reviews, 90:543-584, (1990); Crooke, Ann. Rev. Pharmacol. Toxicol. 32:329-376, (1992)).

[0128] Antisense oligonucleotides may be generated in vitro or ex vivo for administration, or antisense RNA may be generated in vivo in cancer cells where downregulation of FANCM is desired. Thus, the double-stranded DNA may be placed under the control of a promoter "in the reverse direction" such that transcription of the antisense strand of the DNA results in an RNA that is complementary to the normal mRNA transcribed from the sense strand of the target gene. The complementary antisense RNA sequence is then thought to bind to the mRNA to form a duplex and inhibit the translation of the endogenous mRNA from the target gene into protein.

[0129] It is not necessary to necessarily use the complete sequence corresponding to the reverse FANCM coding sequence. For example, a fragment of sufficient length may be used. Screening fragments derived from various portions of the coding or flanking sequences of genes having various sizes and attempting to optimize the level of antisense inhibition is routine for those skilled in the art. It may be advantageous to include the starting methionine ATG codon and, in some cases, also one or more nucleotides upstream of the start codon. Suitable fragments may have about 14 to 23 nucleotides, such as about 15, 16 or 17 nucleotides.

[0130] In other embodiments, targeted mutagenesis can be used to reduce the expression of the active FANCM polypeptide. The use of targeted mutagenesis techniques such as gene editing to knock out or eliminate the expression of a target gene is well established in the art (see, for example, Gaj et al., (2013) Trends Biotechnol. 31(7) 397-405).

[0131] One or more mutations, such as insertions, substitutions or deletions, may be introduced into the FANCM gene in cancer cells. Suitable mutations include deletion of all or part of the FANCM gene, for example, deletion of 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 may be introduced into the FANCM coding sequence. Preferably, in order to exclude the ATPase domain of FANCM, a mutation, such as a premature stop codon, is introduced into the first 400 codons of the coding sequence. The mutation can prevent the expression of an active FANCM polypeptide, for example, by reducing the transcription or translation of the FANCM gene or by expressing an inactive polypeptide.

[0132] Targeted mutagenesis to introduce one or more mutations can be performed by any convenient method. For example, cancer cells can be transfected with a heterologous nucleic acid encoding a targetable nuclease. The targetable nuclease can inactivate the FANCM gene encoding FANCM in one or more cells of an individual, for example, by introducing one or more mutations that prevent the expression of an active FANCM polypeptide.

[0133] The targetable nuclease can selectively inactivate the FANCM gene encoding FANCM in the cancer cells of an individual. The targetable nuclease can be specifically targeted to cancer cells by conventional techniques, such as delivery means that target cells, such as viral vectors expressing ligands for specific cell types; direct administration of the targetable nuclease to the tumor, such as by injection; or selectively expressing the targetable nuclease from a heterologous nucleic acid in cancer cells, such as expression using a tissue-specific promoter.

[0134] The targetable nuclease may be site-specific (e.g., ZFN or TALEN), or may be expressed together with one or more target sequences (e.g., CRISPR / Cas) that target the nuclease to the FANCM gene.

[0135] The heterologous nucleic acid encoding the targetable nuclease may include an inducible promoter that promotes the expression of the targetable nuclease and optional target sequences in a specific cell type, e.g., a tumor cell. For example, the inducible promoter can be a promoter-enhancer cassette in which the expression of the targetable nuclease and optional target sequences is selectively supported in tumor cells as compared to other types of host cells.

[0136] Suitable targetable 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.

[0137] Zinc finger nucleases (ZFNs) contain one or more Cys2-His2 zinc finger DNA binding domains and a cleavage domain (i.e., a nuclease). The DNA binding domains can be engineered to recognize and bind to any nucleic acid sequence using conventional techniques (see, e.g., Qu et al., (2013) Nucl Ac Res 41(16): 7771-7782). The use of ZFNs to introduce mutations into target genes is well known in the art (see, e.g., 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 engineered ZFNs are commercially available (Sigma-Aldrich (St. Louis, MO).

[0138] Transcription activator-like effector nucleases (TALENs) contain a non-specific DNA cleavage nuclease fused to a DNA binding domain that includes a series of modular TALE repeats linked together to recognize contiguous nucleotide sequences. The use of TALEN-targeted nucleases is well known in the art (see, e.g., Joung and 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).

[0139] Meganucleases are endodeoxyribonucleases characterized by large recognition sites (12-40 base pair double-stranded DNA sequences), such that as a result this site generally occurs only once in any given genome (see, e.g., Silva et al., (2011) Curr Gene Ther 11(1): 11-27).

[0140] CRISPR-targeted nucleases (e.g., Cas9) complex with guide RNA (gRNA) to cleave genomic DNA in a sequence-specific manner. The crRNA and tracrRNA of the guide RNA may be used separately or combined into a single RNA to enable site-specific cleavage of the mammalian genome within the FANCM gene or its regulatory elements. The use of the CRISPR / Cas9 system to introduce insertions or deletions into genes as a method of reducing transcription is well known in the art (see, 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).

[0141] In some preferred embodiments, the targetable nuclease is a Cas endonuclease, preferably Cas9, which is combined with a guide RNA target sequence that targets the Cas endonuclease to cleave genomic DNA within the FANCM gene and generate insertions or deletions that prevent the expression of an active FANCM polypeptide, and is expressed in cancer cells.

[0142] A nucleic acid sequence encoding a suppressor nucleic acid or a targetable nuclease, and optionally a guide RNA, may be included within an expression vector. Suitable vectors can be selected or constructed that contain appropriate regulatory sequences such as promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as necessary. Preferably, the vector contains regulatory sequences appropriate for promoting the expression of the encoded nucleic acid in the host cell. Regulatory sequences suitable for promoting the expression of heterologous nucleic acid coding sequences in various expression systems are well known in the art, examples of which 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 employed 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 selectable marker that enable its selection, replication, and expression in a bacterial host such as Escherichia coli (E. coll), and / or in a eukaryotic cell such as yeast, insect, or mammalian cells. Suitable vectors for use in the expression of a suppressor nucleic acid or targetable nuclease in mammalian cells include plasmids and viral vectors such as retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Suitable techniques for expressing a suppressor nucleic acid or targetable nuclease in mammalian cells are well 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, 2nd Edition, Ausubel et al. eds., John Wiley & Sons, 1992; Recombinant Gene Expression Protocols Ed RS Tuan (March 1997) Humana Press Inc).

[0143] Identification of FANCM inhibitors Another aspect of the invention relates to the use of FANCM for screening compounds that may increase cell death or reduce the viability of ALT cells and may be useful in the treatment of ALT cancer.

[0144] Using a screening method, test compounds that bind to the isolated FANCM protein can be identified. A method for screening compounds that reduce viability or increase cell death in ALT cells may include determining the binding of a test compound to FANCM. For example, a test compound may be contacted with FANCM to determine the binding of the test compound to FANCM. Binding of the test compound to FANCM may indicate that the test compound reduces viability or increases cell death in ALT cells.

[0145] Binding of the test compound to FANCM may be determined by standard techniques such as surface plasmon resonance (SPR).

[0146] In some embodiments, the ability of a test compound to inhibit the interaction of FANCM with a binding partner can be determined. A method for screening compounds that reduce viability or increase cell death in ALT cells may include determining the effect of a test compound on the binding of FANCM to a binding partner. For example, FANCM may be contacted with a binding partner in the presence and absence of a test compound. A reduction in the binding of FANCM to a binding partner in the presence of a test compound compared to its absence may indicate that the test compound reduces viability or increases cell death in ALT cells.

[0147] The binding partner is, for example, a protein that naturally binds to intracellular FANCM during homologous recombination, meiosis, and DNA repair. Examples of FANCM binding partners include MHF, FAAP24, HCLK2, BLM, RMI, Topo IIIα.

[0148] A screening method can be used to identify test compounds that inhibit FANCM activity. A method of screening for compounds that reduce viability or increase cell death in ALT cells may include the step of determining the effect of the test compound on FANCM activity. For example, FANCM activity can be determined in the presence and absence of the test compound. A decrease in FANCM activity in the presence of the test compound compared to its absence indicates that the compound reduces viability or increases cell death in ALT cells.

[0149] The ATP-dependent DNA helicase / translocase activity of FANCM can be determined in the presence of the test compound compared to its absence. A decrease or reduction in the ATP-dependent DNA helicase / translocase activity in the presence of the test compound may indicate that the test compound inhibits the activity of the FANCM protein. For example, the test compound can be a FANCM inhibitor. Suitable methods for determining activities such as ATPase and translocase assays are well known in the art.

[0150] The exact manner of any of the screening or assay methods of the present invention can be modified by those skilled in the art using conventional techniques and knowledge. Those skilled in the art are well aware of the need to employ appropriate control experiments.

[0151] FANCM for use in a screening method can be an isolated polypeptide comprising a full-length FANCM sequence, such as a FANCM reference sequence, such as SEQ ID NO:1 described herein, or a fragment thereof. Suitable fragments can include at least 50, at least 100, or at least 150 contiguous amino acids from the FANCM reference sequence. In some embodiments, a FANCM fragment comprising ATPase or translocase activity can employ a fragment comprising or consisting of the N-terminal DEAH helicase-like domain corresponding to residues 83-591 of SEQ ID NO:1. The isolated FANCM polypeptide can be produced using standard recombinant techniques.

[0152] A test compound can be an isolated molecule or can be contained in a sample, mixture or extract, such as a biological sample. Compounds that can be screened using the methods described herein can be natural or synthetic chemical substances used in drug screening programs, examples of which include small organic molecules, polypeptides and nucleic acids, such as aptamers. Extracts of plants, microorganisms or other organisms containing several characterized or uncharacterized components can also be used.

[0153] Suitable test compounds for screening include compounds that inhibit an activity similar to the ATP-dependent DNA helicase / translocase activity of FANCM. For example, a suitable test compound can be an ATP analog. Suitable test compounds can be produced using rational drug design to provide test candidate compounds having specific molecular shape, size and charge characteristics suitable for modulating FANCM activity.

[0154] Combinatorial library technology provides an efficient way to test a vast number of different compounds for their ability to modulate FANCM activity. Such libraries and their use are known in the art with respect to all kinds of natural products, especially small molecules and peptides. The use of peptide libraries may be preferred in certain circumstances. In some embodiments, libraries of biological molecules, such as aptamers or antibody molecules.

[0155] The amount of test compound that can be added to the assay of the present invention is generally expected to be determined by trial and error depending on the type of compound used. Typically, putative inhibitor compounds at a concentration of about 0.001 nM to 1 mM or higher can be used, for example 0.01 nM to 100 μM, for example 0.1 to 50 μM, for example about 10 μM of inhibitor compound can be used. Compounds with weak effects may still be useful lead compounds for further investigation and development.

[0156] Test compounds can include peptides from FANCM or its binding partners as described above. Membrane-permeable peptide fragments of 5 to 40 amino acids, for example 6 to 10 amino acids, can be tested for their ability to bind to FANCM or inhibit its activity. The regulatory properties of the above peptides can be increased by the addition of one of the following groups: chloromethyl ketone, aldehyde and boronic acid to the C-terminus. These groups are transition state analogs of serine, cysteine and threonine proteases. The N-terminus of the peptide fragment can be blocked with carbobenzyl to inhibit aminopeptidase and improve stability (Proteolytic Enzymes, 2nd Edition, edited by R. Beynon and J. Bond, Oxford University Press, 2001).

[0157] Test compounds can include antibodies, antibody fragments and antibody derivatives, as well as non-immunoglobulin binding molecules such as aptamers, trinectins, anticalins, knotted domains, transferrins, tenecteplase antigen receptors, and sea lamprey leucine-rich repeat proteins. Suitable molecules may be directed against the DEAH helicase-like domain corresponding to residues 83-591 of SEQ ID NO: 1, or another part of the FANCM protein. Candidate regulatory antibody molecules can be characterized and their binding regions determined by providing single-chain antibodies and fragments thereof that are involved in inhibiting activity or blocking interaction with binding partners. Suitable antibodies can be produced using standard techniques in the art, for example, immunizing a mammal with a suitable peptide, such as a fragment of a pro-inflammatory polypeptide, or isolating specific antibodies from a recombinantly produced library of expressed immunoglobulin variable domains, for example, using lambda bacteriophage or filamentous bacteriophage that display functional immunoglobulin binding domains on their surface; see, for example, W092 / 01047.

[0158] An aptamer directed against FANCM is also a putative agent for modulating FANCM. An aptamer is a nucleic acid that specifically binds to a target molecule. Typically, an aptamer is a small nucleic acid in the range of 15-50 bases in length that folds into a defined secondary and tertiary structure, such as a stem-loop or G-quadruplex. An aptamer can bind very tightly to the target molecule with a Kd of less than 10 -12 M. An aptamer can bind to FANCM with a very high degree of specificity. For example, aptamers have been isolated where the difference in binding affinity between the target molecule and another molecule that differs only at a single position on the molecule is greater than 10,000-fold. The aptamer has a Kd with FANCM that is at least 1 / 10, 1 / 100, 1 / 1000, 1 / 10,000, or 1 / 100,000 of the Kd with a control polypeptide d d d ​​It may have. The production and use of aptamers are well known in the art (see, for example, Bunka et al., Curr Opin Pharmacol 2010 10 (5) 557-562).

[0159] Test compounds identified as inhibiting FANCM activity may be further investigated using one or more secondary screenings. Secondary screenings may include testing for biological functions or activities in vitro and / or in vivo, for example, in animal models. For example, the ability of a test compound to reduce the viability of ALT cells or increase cell death can be determined. In some embodiments, secondary screening may include determining the selectivity of the compound for FANCM by screening against a panel of isolated enzymes.

[0160] The effect of a test compound identified as a FANCM inhibitor can be determined in mammalian cells in vitro. For example, the effect of a test compound on an ALT cell line can be determined. Increased ALT cell death in the presence of the compound compared to in the absence of the compound may indicate that the compound exhibits useful activity in the treatment of ALT cancer.

[0161] After a FANCM inhibitor that may be useful in the treatment of ALT cancer as described herein has been identified, the method may further include modifying the compound so that its pharmaceutical properties are optimized. Suitable optimization methods, for example, by structural modeling, are well known in the art. Further optimization or modification may then be performed to reach one or more final compounds during in vivo testing or clinical trials.

[0162] Test compounds identified as FANCM inhibitors may be isolated and / or purified, or alternatively, synthesized using conventional techniques of recombinant expression or chemical synthesis. Furthermore, such test compounds can be manufactured and / or used in the preparation, i.e., manufacture or formulation, of compositions such as pharmaceuticals, pharmaceutical compositions or drugs. Accordingly, the methods described herein may include formulating a test compound into a pharmaceutical composition using pharmaceutically acceptable excipients, vehicles or carriers for therapeutic use.

[0163] Pharmaceutical composition Therapeutic agents described herein, such as FANCM antagonists, inhibitors, suppressor nucleic acids, targetable nucleases, suppressor nucleic acids or nucleic acids encoding targetable nucleases, may be administered alone, but the therapeutic agents are typically expected to be administered in the form of a pharmaceutical composition, which may contain at least one component in addition to the active agent. The therapeutic agent may be mixed with other reagents, such as buffers, carriers, diluents, preservatives and / or pharmaceutically acceptable excipients, to produce a pharmaceutical composition for use in cancer immunotherapy. Suitable reagents are described in more detail below.

[0164] Aspects of the invention provide (i) a pharmaceutical composition comprising a therapeutic agent selected from (a) an FANCM inhibitor, (b) an FANCM suppressor nucleic acid, (c) an FANCM targetable nuclease, or (d) a nucleic acid encoding an FANCM suppressor nucleic acid or targetable nuclease, and a pharmaceutically acceptable excipient; and (ii) a method of producing a pharmaceutical composition for use in cancer immunotherapy, the method comprising the step of mixing the above-described therapeutic agent with a pharmaceutically acceptable excipient.

[0165] Aspects of the present invention provide a pharmaceutical composition comprising: (i) a therapeutic agent selected from (a) an RMI inhibitor, (b) an RMI-suppressing nucleic acid, (c) an RMI-targetable nuclease, or (d) a nucleic acid encoding an RMI-suppressing nucleic acid or a targetable nuclease, and a pharmaceutically acceptable excipient; and (ii) a method of producing a pharmaceutical composition for use in cancer immunotherapy, the method comprising the step of mixing the above-described therapeutic agent with a pharmaceutically acceptable excipient.

[0166] Aspects of the present invention provide a pharmaceutical composition comprising: (i) a therapeutic agent that inhibits the binding of FANCM and RMI, selected from (a) a small molecule inhibitor, (b) a polypeptide inhibitor, or (c) a nucleic acid inhibitor, and a pharmaceutically acceptable excipient; and (ii) a method of producing a pharmaceutical composition for use in cancer immunotherapy, the method comprising the step of mixing the above-described therapeutic agent with a pharmaceutically acceptable excipient.

[0167] Aspects of the present invention provide a pharmaceutical composition comprising a therapeutic agent that inhibits the binding of FANCM and RMI and a therapeutic agent that inhibits the ATPase activity of FANCM. In certain embodiments, the therapeutic agent is selected from (a) a small molecule inhibitor, (b) a polypeptide inhibitor, or (c) a nucleic acid inhibitor, and a pharmaceutically acceptable excipient. In certain embodiments, the present disclosure provides a method of producing a pharmaceutical composition for use in cancer immunotherapy, the method comprising the step of mixing the above-described therapeutic agent with a pharmaceutically acceptable excipient.

[0168] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.

[0169] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment, have a reasonable benefit / risk ratio, are suitable for use in contact with the tissues of a subject (e.g., a human) without causing excessive toxicity, irritation, allergic response, or other problems or complications, and are "acceptable" in the sense of being compatible with the other ingredients of the formulation. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0170] The disclosure of the present invention also provides a pharmaceutical composition comprising an inhibitor of the FANCM-RMI interaction for use in treating ALT cancer. The disclosure of the present invention also provides the use of an inhibitor of the FANCM-RMI interaction in the manufacture of a medicament for the treatment of ALT cancer. In one example, the pharmaceutical composition or medicament consists essentially of an inhibitor of the FANCM-RMI interaction. The disclosure of the present invention also provides a pharmaceutical composition comprising an inhibitor of the ATPase activity of FANCM for use in treating ALT cancer. The disclosure of the present invention also provides the use of an inhibitor of the ATPase activity of FANCM in the manufacture of a medicament for the treatment of ALT cancer. The disclosure of the present invention also provides a pharmaceutical composition comprising an inhibitor of the FANCM-RMI interaction and an inhibitor of the ATPase activity of FANCM for use in treating ALT cancer. The disclosure of the present invention also provides the use of an inhibitor of the FANCM-RMI interaction and an inhibitor of the ATPase activity of FANCM in the manufacture of a medicament for the treatment of ALT cancer. In one example, the pharmaceutical composition or medicament consists essentially of an inhibitor of the FANCM-RMI interaction. In one example, the pharmaceutical composition or medicament consists essentially of an inhibitor of the ATPase activity of FANCM. In one example, the pharmaceutical composition or medicament consists essentially of an inhibitor of the ATPase activity of FANCM and an inhibitor of the ATPase activity of FANCM. However, the medicament or composition may also contain excipients that are physiologically compatible and not harmful for the inhibitors or their use described herein, or agents such as solvents, diluents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents. The use of such carriers and agents for preparing compositions of pharmaceutical active substances is well known in the art (see, for example, Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, PA, 2005).

[0171] Suitable pharmaceutical compositions for administration (e.g., by infusion) include aqueous and non-aqueous isotonic pyrogen-free sterile injectable solutions, and such injectable solutions may contain antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, and such sterile suspensions may contain 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 injection. Suitable vehicles can be found in standard pharmaceutical textbooks, e.g., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Easton, Pa., 1990.

[0172] The pharmaceutical composition may be diluted before use. Suitable diluents may be selected, for example, from Ringer's solution, Hartmann's solution, dextrose solution, physiological saline, and sterile water for injection.

[0173] Pharmaceutical compositions include those for oral, rectal, nasal, topical (including buccal and sublingual), parenteral administration (including intramuscular, intraperitoneal, subcutaneous, and intravenous), or in a form suitable for administration by inhalation or insufflation. Inhibitors of the FANCM-RMI interaction may be incorporated into pharmaceutical compositions and unit dosage forms thereof together with conventional adjuvants, carriers, or diluents, and in such forms may be employed, all for oral use, as solids, e.g., tablets or filled capsules, or as liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with them, or in the form of sterile injectable solutions for parenteral (e.g., subcutaneous) use.

[0174] The pharmaceutical composition for administration of the antagonist or inhibitor of this disclosure may be supplied in a convenient dosage unit form and can be prepared by any of the methods well known in the pharmaceutical art.

[0175] The pharmaceutical compositions and methods disclosed herein may also generally further comprise a compound having other therapeutic activity that is applicable to the treatment of the disclosed disorder or condition. The selection of suitable agents for use in combination therapy can be made by one of ordinary skill in the art according to conventional pharmaceutical principles. Combinations of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders or conditions disclosed herein. Using this approach, the dosage of each agent can be lowered to achieve therapeutic efficacy and thus the potential for harmful side effects can be reduced.

[0176] When other therapeutic agents are employed in combination with those disclosed herein, they may be used, for example, in amounts as described in the Physician Desk Reference (PDR) or otherwise as determined by one of ordinary skill in the art.

[0177] Method In certain embodiments, the present disclosure provides, for example, methods for inhibiting FANCM, methods for inhibiting RMI, methods for inhibiting or disrupting the FANCM-RMI interaction, and methods for inhibiting the ATPase and / or translocase activity of FANCM in ALT cells, such as ALT cancer or tumor cells. In certain embodiments of any such method, the method inhibits the viability and / or growth of ALT cells, such as the viability or growth of ALT cancer or tumor cells. In certain embodiments, the method increases or induces the death of ALT cells, such as ALT cancer or tumor cells. The method can be carried out in vitro or in vivo, for example, to treat a subject having an ALT cancer or tumor. In certain embodiments, the subject is a mammal, such as a human diagnosed with ALT cancer.

[0178] "Inhibiting" FANCM activity, for example, binding to RMI, DNA translocase activity, ATPase activity and / or translocase activity, the inhibitor may be partial or complete. In certain embodiments, the inhibition is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% reduction compared to the amount in equivalent cells not contacted with a FANCM antagonist or inhibitor, or compared to a predetermined value.

[0179] Accordingly, in one embodiment, the present disclosure provides a method of inhibiting the viability and / or growth of ALT cells, the method comprising contacting ALT cells (e.g., ALT cancer cells) with a FANCM antagonist or inhibitor. In certain embodiments, the antagonist or inhibitor inhibits the expression of FANCM in ALT cells. In certain embodiments, the antagonist or inhibitor inhibits the biological activity of one or more FANCMs in ALT cells. In certain embodiments, the biological activity is the DNA translocase activity of FANCM. In certain embodiments, the biological activity is the ATPase and / or translocase activity of FANCM. In certain embodiments, the biological activity is mediated by the binding of FANCM to the BLM-TOP3-RMI (BTR) complex. In certain embodiments, the biological activity is mediated by the ATP-dependent translocase activity (DEAH domain) of FANCM. In certain embodiments, the method comprises contacting ALT cells (e.g., ALT cancer cells) with one or more FANCM antagonists or inhibitors, wherein the one or more FANCM antagonists or inhibitors collectively inhibit both the DNA translocase activity of FANCM and the ATPase and / or translocase activity of FANCM. In certain embodiments, the method comprises contacting ALT cells (e.g., ALT cancer cells) with one or more FANCM antagonists or inhibitors, wherein the one or more FANCM antagonists or inhibitors collectively disrupt the FANCM-RMI interaction and inhibit the ATPase / translocase activity of FANCM. The inhibitor that disrupts the FANCM-RMI interaction and the inhibitor that inhibits the ATPase and / or translocase activity of FANCM may be the same inhibitor or different inhibitors.

[0180] As used in the specific context of this specification, the term "inhibiting the viability and / or growth of ALT cells" is to be construed as meaning interfering with, reducing, limiting, or preventing the viability and / or growth of ALT cells as compared to ALT cells in which the FANCM-RMI interaction is intact. Similarly, in the specific context used herein, the term "inhibiting the viability and / or growth of ALT cells" is to be construed as meaning interfering with, reducing, limiting, or preventing the viability and / or growth of ALT cells as compared to ALT cells in which FANCM is not inhibited. In the specific context used in this specification, the term "inhibiting the viability and / or growth of ALT cells" is to be construed as meaning interfering with, reducing, limiting, or preventing the viability and / or growth of ALT cells as compared to ALT cells in which RMI is not inhibited. In the specific context used in this specification, the term "inhibiting the viability and / or growth of ALT cells" is to be construed as meaning interfering with, reducing, limiting, or preventing the viability and / or growth of ALT cells as compared to ALT cells in which the FANCM-RMI interaction has not decayed.

[0181] The viability and / or growth of the cells may be inhibited in any measurable amount. The inhibition of cell viability may be complete or partial. Thus, the methods disclosed herein may include at least partial inhibition of the viability and / or growth of ALT cells. For example, the viability and / or growth of the cells can be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% after disruption of the FANCM-RMI interaction.

[0182] Accordingly, in one embodiment, the present disclosure provides a method of inducing ALT cell death, the method comprising contacting an ALT cell (e.g., an ALT cancer cell) with a FANCM antagonist or inhibitor. In certain embodiments, the antagonist or inhibitor inhibits the expression of FANCM in the ALT cell. In certain embodiments, the antagonist or inhibitor inhibits the biological activity of one or more FANCMs in the ALT cell. In certain embodiments, the biological activity is the DNA translocase activity of FANCM. In certain embodiments, the biological activity is the ATPase and / or translocase activity of FANCM. In certain embodiments, the biological activity is mediated by the binding of FANCM to the BLM-TOP3-RMI (BTR) complex. In certain embodiments, the biological activity is mediated by the ATP-dependent translocase activity (DEAH domain) of FANCM. In certain embodiments, a method comprising contacting an ALT cell (e.g., an ALT cancer cell) with one or more FANCM antagonists or inhibitors, wherein the one or more FANCM antagonists or inhibitors collectively inhibit both the DNA translocase activity of FANCM and the ATPase and / or translocase activity of FANCM. In certain embodiments, a method comprising contacting an ALT cell (e.g., an ALT cancer cell) with one or more FANCM antagonists or inhibitors, wherein the one or more FANCM antagonists or inhibitors collectively disrupt the FANCM-RMI interaction and inhibit the ATPase and / or translocase activity of FANCM. The inhibitor that disrupts the FANCM-RMI interaction and the inhibitor that inhibits the ATPase and / or translocase activity of FANCM may be the same inhibitor or different inhibitors.

[0183] As used in the specific context of this specification, the term "inducing ALT cell death" is to be construed to mean inducing, increasing, causing, or promoting ALT cell death as compared to ALT cells in which the FANCM-RMI interaction is intact. Similarly, in the specific context used herein, the term "inducing ALT cell death" is to be construed to mean inducing, increasing, causing, or promoting ALT cell death as compared to ALT cells in which FANCM is not inhibited. In the specific context used in this specification, the term "inducing ALT cell death" is to be construed to mean inducing, increasing, causing, or promoting ALT cell death as compared to ALT cells in which RMI is not inhibited. In the specific context used in this specification, the term "inducing ALT cell death" is to be construed to mean inducing, increasing, causing, or promoting the viability and / or growth of ALT cells as compared to ALT cells in which the FANCM-RMI interaction has not decayed.

[0184] Cell death can be increased in any measurable amount. Increasing cell death can be complete or partial. Thus, the methods disclosed herein may include at least partial induction of ALT cell death. For example, ALT cell death can be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% after disruption of the FANCM-RMI interaction. Cell death can be determined in a plurality of cells, for example, as the percentage of non-viable or dead cells after contact with an antagonist or inhibitor disclosed herein. In certain embodiments, the plurality of cells are ALT tumor cells.

[0185] Thus, in one embodiment, the present disclosure provides a method of inhibiting ALT cancer in a subject, the method comprising contacting the subject with a FANCM antagonist or inhibitor. In certain embodiments, the antagonist or inhibitor inhibits the expression of FANCM in ALT cells. In certain embodiments, the antagonist or inhibitor inhibits the biological activity of one or more FANCMs in ALT cells. In certain embodiments, the biological activity is the DNA translocase activity of FANCM. In certain embodiments, the biological activity is the ATPase and / or translocase activity of FNCM. In certain embodiments, the biological activity is mediated by the binding of FANCM to the BLM-TOP3-RMI (BTR) complex. In certain embodiments, the biological activity is mediated by the ATP-dependent translocase activity (DEAH domain) of FANCM. In certain embodiments, a method comprising contacting a subject with one or more FANCM antagonists or inhibitors, wherein the one or more FANCM antagonists or inhibitors collectively inhibit both the DNA translocase activity of FANCM and the ATPase and / or translocase activity of FANCM. In certain embodiments, a method comprising contacting ALT cells (e.g., ALT cancer cells) with one or more FANCM antagonists or inhibitors, wherein the one or more FANCM antagonists or inhibitors collectively disrupt the FANCM-RMI interaction and inhibit the ATPase and / or translocase activity of FANCM. The inhibitor that disrupts the FANCM-RMI interaction and the inhibitor that inhibits the ATPase and / or translocase of FANCM may be the same inhibitor or different inhibitors. In certain embodiments, the ALT cancer is osteosarcoma, soft tissue sarcoma (e.g., liposarcoma, undifferentiated pleomorphic sarcoma, or leiomyosarcoma), glioblastoma, astrocytoma, neuroblastoma, or bladder cancer.

[0186] As used in the specific context of this specification, the term "inhibiting ALT cancer" is to be construed to mean inhibiting the growth or metastasis of an ALT cancer or tumor, reducing the size of an ALT cancer or tumor, or reducing the growth rate of an ALT cancer or tumor, as compared to ALT cells in which the FANCM-RMI interaction is intact. Similarly, in the specific context used herein, the term "inhibiting ALT cancer" is to be construed to mean interfering with, reducing, restricting, or preventing the viability and / or growth of ALT cells, as compared to ALT cells in which FANCM is not inhibited. As used in the specific context of this specification, the term "inhibiting ALT cancer" is to be construed to mean interfering with, reducing, restricting, or preventing the viability and / or growth of ALT cells, as compared to ALT cells in which RMI is not inhibited. As used in the specific context of this specification, the term "inhibiting ALT cancer" is to be construed to mean interfering with, reducing, restricting, or preventing the viability and / or growth of ALT cells, as compared to ALT cells in which the FANCM-RMI interaction has not collapsed.

[0187] Inhibition of ALT cancer can occur in any measurable amount. Inhibition of ALT cancer may be complete or partial. Accordingly, the methods disclosed herein may include at least partial inhibition of the growth or metastasis of an ALT cancer or tumor, at least partial reduction in the size of an ALT cancer or tumor, or at least partial reduction in the growth rate of an ALT cancer or tumor.

[0188] Inhibition of FANCM or RMI can be achieved by any suitable means in the art, including the use of any of the FANCM and RMI inhibitors disclosed herein. The interaction between FANCM and RMI can be disrupted by any suitable means known in the art, including the use of an inhibitor of FANCM-RMI or a substance that disrupts it disclosed herein. Inhibition of FANCM, RMI, or disruption of the FANCM-RMI interaction may be partial or complete. FANCM activity can be measured via a C-circle assay or by any suitable means known in the art or by any of the methods disclosed herein.

[0189] In certain embodiments of any of the methods disclosed herein, the method may also not include inhibiting one of BLM and / or breast cancer type 1 susceptibility protein (BRCA1). In certain embodiments of any of the methods disclosed herein, the method may also not include inhibiting BLM. In certain embodiments of any of the methods disclosed herein, the method may also not include inhibiting BRCA1. The sequence of BRCA1 is publicly available. An exemplary sequence is set forth in SEQ ID NO: 8.

[0190] Agents that reduce the expression or activity of FANCM are shown herein to induce G2 / M arrest and cell death in ALT cancer cells and may therefore be useful in the treatment of ALT cancer.

[0191] As used herein, the terms "treating," "treat," or "treatment" and variations thereof refer to a clinical intervention designed to alter the natural course of a treated individual or cell during the course of clinical pathology. When used in the context of treating a condition herein, the term "treatment" generally refers to treatment and therapy with respect to which some desirable therapeutic effect, such as inhibition of progression of the condition, is achieved, including reduction in the rate of progression, halting of the rate of progression, as well as alleviation of the condition, and cure of the condition. Desirable effects of treatment include reducing the rate of disease progression, reducing the size of a cancer, inhibiting tumor growth, inhibiting the progression or metastasis of cancer, alleviating or reducing the condition of a disease, and improving remission or prognosis.

[0192] Treatment can be any treatment and therapy in which some desirable therapeutic effect, such as inhibition or delay of progression of a condition, is achieved, regardless of whether it is treatment and therapy of a human or an animal (e.g., in veterinary applications), including reduction in the rate of progression, halting of the rate of progression, alleviation of the condition, cure or remission (partial or complete) of the condition, preventing, delaying, alleviating, or halting one or more symptoms and / or signs of the condition, or increasing the survival period of the subject or patient beyond that expected in the absence of treatment.

[0193] Treatment as a prophylactic measure (i.e., prevention) is also included. For example, an individual who is prone to or at risk of developing or recurring cancer can be treated as described herein. Such treatment can prevent or delay the occurrence or recurrence of cancer in the individual.

[0194] In particular, the treatment may include inhibition of cancer growth, including complete remission of cancer, and / or inhibition of cancer metastasis. Cancer growth generally refers to any one of various indicators that show a change to a more developed form within the cancer. Thus, indicators for measuring inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumor volume or morphology (e.g., as determined using computed tomography (CT), ultrasonography, or other imaging methods), a delay in tumor growth, destruction of tumor vasculature, improvement in performance in a delayed hypersensitivity skin test, an increase in the activity of cytolytic cancer cells, and a decrease in the level of tumor-specific antigens.

[0195] As used herein, the term "subject" refers to any animal, such as a mammal, including but not limited to, for example, humans, non-human primates, livestock (such as sheep, horses, cows, pigs, donkeys), companion animals (such as pets, for example dogs and cats), laboratory test animals (such as mice, rabbits, rats, guinea pigs), performance animals (such as racehorses, camels, greyhounds) or captive wild animals, etc. In one embodiment, the "subject" is a human. In certain embodiments, a suitable subject or individual for treatment with a therapeutic agent, such as (a) a FANCM antagonist or inhibitor, (b) a FANCM suppressor nucleic acid, (c) a FANCM-targetable nuclease, or (d) a nucleic acid encoding a FANCM suppressor nucleic acid or a targetable nuclease, can be a mammal, such as a rodent (such as guinea pigs, hamsters, rats, mice), murine animals (such as mice), canine animals (such as dogs), feline animals (such as cats), equine animals (such as horses), primates, anthropoid apes (such as monkeys or apes), monkeys (such as marmosets, baboons), apes (such as gorillas, chimpanzees, orangutans, langurs), or a human, as described herein. Typically, the terms "subject" and "patient" are used interchangeably, especially when referring to a human subject. A subject may be receiving administration of a chemotherapeutic agent simultaneously, sequentially, or separately. A subject can be a subject with cancer, a subject suspected of having cancer, or a subject at risk of developing cancer. The cancer can be any cancer disclosed herein.

[0196] The inventors have surprisingly shown for the first time that the disruption of the FANCM-RMI complex is selectively toxic to ALT cancer cells. Based on this discovery, the inventors herein provide (i) a method of inhibiting the viability and / or growth of ALT cells, (ii) a method of treating ALT cancer, (iii) a method of selecting a subject for treatment, or a method of identifying whether a subject suffering from cancer is suitable for treatment with an inhibitor of the FANCM-RMI interaction, and (iv) a method of determining whether a subject is responsive to treatment with an inhibitor of the FANCM-RMI interaction.

[0197] In certain embodiments, the disclosure of the present invention provides a method of treating ALT cancer in a subject, the method comprising the step of disrupting the FANCM-RMI interaction. In certain embodiments, the method comprises providing to a subject having ALT cancer an effective amount of an inhibitor that disrupts the FANCM-RMI interaction. In certain embodiments, the ALT cancer is any of those disclosed herein. In certain embodiments, the ALT cancer is osteosarcoma, soft tissue sarcoma (such as liposarcoma, undifferentiated pleomorphic sarcoma, or leiomyosarcoma), glioblastoma, astrocytoma, neuroblastoma, or bladder cancer. In certain embodiments, the method comprises providing to the subject one or more FANCM antagonists or inhibitors, wherein the one or more FANCM antagonists or inhibitors collectively inhibit both the DNA translocase activity of FANCM and the ATPase and / or translocase activity of FANCM.

[0198] In certain embodiments, the disclosure of the present invention provides a method of treating ALT cancer in a subject, the method comprising inhibiting the ATPase activity of FANCM. In certain embodiments, the method comprises providing to a subject having ALT cancer an effective amount of an ATPase inhibitor of FANCM. In certain embodiments, the ALT cancer is any of those disclosed herein. In certain embodiments, the ALT cancer is osteosarcoma, soft tissue sarcoma (such as liposarcoma, undifferentiated pleomorphic sarcoma, or leiomyosarcoma), glioblastoma, astrocytoma, neuroblastoma, or bladder cancer.

[0199] In one example, the disclosure of the present invention provides a method of treating ALT cancer, comprising the step of disrupting the FANCM-RMI interaction and / or the step of inhibiting the ATPase activity of FANCM, wherein the subject is receiving concurrent administration of a chemotherapeutic agent. In another example, the disclosure of the present invention provides a method of treating ALT cancer, comprising the step of disrupting the FANCM-RMI interaction, wherein the subject is receiving sequential administration of a chemotherapeutic agent. In another example, the disclosure of the present invention provides a method of treating ALT cancer, comprising the step of disrupting the FANCM-RMI interaction and / or the step of inhibiting the ATPase activity of FANCM, wherein the subject is receiving separate administration of a chemotherapeutic agent. In certain embodiments, the ALT cancer is any of those disclosed herein. In certain embodiments, the ALT cancer is osteosarcoma, soft tissue sarcoma (such as liposarcoma, undifferentiated pleomorphic sarcoma, or leiomyosarcoma), glioblastoma, astrocytoma, neuroblastoma, or bladder cancer.

[0200] In one example, the disclosure of the present invention provides a method of treating ALT cancer, comprising the step of disrupting the FANCM-RMI interaction and / or the step of inhibiting the ATPase activity of FANCM, wherein the subject is not receiving concurrent, sequential, or separate administration of a chemotherapeutic agent. Thus, the methods disclosed herein may include the step of disrupting the FANCM-RMI interaction and / or the step of inhibiting the ATPase activity of FANCM as a single treatment modality or as a single anti-cancer treatment modality. In certain embodiments, the ALT cancer is any of those disclosed herein. In certain embodiments, the ALT cancer is osteosarcoma, soft tissue sarcoma (such as liposarcoma, undifferentiated pleomorphic sarcoma, or leiomyosarcoma), glioblastoma, astrocytoma, neuroblastoma, or bladder cancer.

[0201] Any chemotherapeutic agent approved for the treatment of cancer is suitable for optional use in combination with any of the inhibitors disclosed herein, or in combination with the step of disrupting the FANCM-RMI interaction disclosed herein. Examples of suitable chemotherapeutic agents include, but are not limited to, paclitaxel, doxorubicin, carboplatin, cyclophosphamide, daunorubicin, doxorubicin, epirubicin, fluorouracil, gemcitabine, eribulin, ixabepilone, methotrexate, mutamycin, mitoxantrone, virorelbine, docetaxel, thiotepa, vincristine, and capecitabine.

[0202] In certain embodiments of any of the methods disclosed herein, the method includes treating a subject having ALT cancer. Any of the methods described herein may also include identifying or diagnosing cancer in an individual as ALT cancer. This can be accomplished, for example, by obtaining such a diagnosis from a physician, hospital, or diagnostic laboratory. This may also be accomplished by performing an assay on a biological sample obtained from the subject, such as a tumor sample, or may include any of the various assays disclosed herein. For example, the presence of C circles or ALT-associated PML bodies in cancer cells from an individual can be determined. Suitable methods for the identification of ALT cancer are well known in the art 75 。

[0203] As discussed herein, methods for determining whether a subject has ALT cancer are known in the art. Suitable assays include, but are not limited to, measurement of C circles (e.g., as disclosed in Henson et al., 2009 and WO2011035375), quantitative PCR of telomeric DNA and C circles (Lau et al., 2012), absence of telomerase activity, presence of extremely long and heterogeneous telomeres, presence of ALT-associated PML bodies (APBs), elevated telomere sister chromatid exchange (T-SCE) (e.g., elevated in relation to non-ALT cells, e.g., telomerase-positive cells or dead cells), and presence of extrachromosomal telomere repeat (ECTR) DNA. Any of the methods disclosed herein for identifying ALT cells can be used to determine whether a subject has ALT cancer. These methods can be performed on a subject or a sample taken from the subject.

[0204] In one example, the disclosure of the invention is a method of selecting a subject for treatment with an inhibitor of the FANCM-RMI interaction, comprising the step of determining whether the subject has ALT cancer, and if the subject has ALT cancer, the subject is selected for treatment with an inhibitor of the FANCM-RMI interaction.

[0205] In one example, the disclosure of the invention is a method of identifying whether a subject with cancer is suitable for treatment with an inhibitor of the FANCM-RMI interaction, comprising the step of determining whether the cancer is ALT cancer, and if the subject has ALT cancer, the subject is identified as being suitable for treatment with an inhibitor of the FANCM-RMI interaction.

[0206] In one example, the disclosure of the present invention is a method for determining whether a subject is responsive to treatment with an inhibitor of the FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM, the method comprising determining the presence and / or extent of genomic instability at one or more telomeres in cells taken from the subject. Alternatively, the disclosure of the present invention is a method for determining whether a subject is responsive to treatment with an inhibitor of the FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM, the method comprising determining the presence and / or level of ALT activity in a cell or tissue sample taken from the subject. The presence and / or level of ALT activity can be determined using any suitable method known in the art or using any of the methods disclosed herein for identifying ALT cells. In certain embodiments, for example, if the ALT activity is reduced in the cell or tissue sample as compared to the amount pre-detected in a cell or tissue sample taken from the subject prior to treatment or as compared to a pre-determined value, the subject is responsive.

[0207] Additionally, the methods disclosed herein may include assaying a sample that can be obtained from a subject prior to treatment with an inhibitor of the FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM to determine the presence and / or extent of genomic instability in one or more telomeres and / or the presence and / or level of ALT activity in cells taken from the subject. This can be compared to a sample taken from the subject after treatment with an inhibitor of the FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM. Thus, an increase in the level of genomic instability or ALT activity after treatment with an inhibitor of the FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM compared to the amount / presence of the level of genomic instability or ALT activity prior to treatment with an inhibitor of the FANCM interaction and / or an inhibitor of the ATPase activity of FANCM indicates that the subject is responsive to treatment with an inhibitor of the FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM. Conversely, no increase or decrease in the level of genomic instability or ALT activity after treatment with an inhibitor of the FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM compared to the amount / presence of the level of genomic instability or ALT activity prior to treatment with an inhibitor of the FANCM interaction and / or an inhibitor of the ATPase activity of FANCM indicates that the subject is not responsive to treatment with an inhibitor of the FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM.

[0208] It will be understood by those skilled in the art that the assays used to determine whether a cell is an ALT cell or whether a cancer is an ALT cancer can also be used to determine whether a subject is responsive to treatment with an inhibitor of the FANCM-RMI interaction.

[0209] For example, using any of the methods disclosed in Henson et al., 2009 and / or WO / 2011 / 035375, it is possible to determine whether a subject is responding to the treatment methods disclosed in the present invention. Thus, for example, the methods disclosed herein may include assaying a sample obtained from a subject treated with an inhibitor of the FANCM-RMI interaction for the presence and / or amount of a partially double-stranded telomeric circle, where the partially double-stranded telomeric circle is detected according to rolling circle amplification using a partially double-stranded circular telomeric DNA as a template, and where the presence and / or amount of said circle indicates whether the subject is responding to the treatment. The method may be carried out with or without an exogenous primer. For example, the method may be carried out without an exogenous primer. Samples can be obtained from the subject before starting treatment with an inhibitor of the FANCM-RMI interaction and after treatment with an inhibitor of the FANCM-RMI interaction. By comparing the presence and / or amount of C circles in the samples, it is possible to determine whether the subject is responding to the treatment. For example, an increase in the presence and / or amount of C circles in a sample from the subject indicates that the subject is responding to treatment with an inhibitor of the FANCM-RMI interaction.

[0210] In certain embodiments, the disclosure of the present invention is a method for treating ALT cancer in a subject in need thereof, comprising providing to the subject an effective amount of a therapeutic agent, such as (a) a FANCM inhibitor, (b) a FANCM-suppressing nucleic acid, (c) a FANCM-targetable nuclease, or (d) a nucleic acid encoding a FANCM-suppressing nucleic acid or a targetable nuclease, each of which may be useful in therapy as described herein. For example, a therapeutic agent that reduces the expression or activity of FANCM may be administered to an individual for the treatment of ALT cancer. In certain embodiments, the ALT cancer is any of those disclosed herein. In certain embodiments, the ALT cancer is osteosarcoma, soft tissue sarcoma (such as liposarcoma, undifferentiated pleomorphic sarcoma, or leiomyosarcoma), glioblastoma, astrocytoma, neuroblastoma, or bladder cancer.

[0211] Reduction of the expression or activity of FANCM is shown herein to have a strongly specific effect on ALT cancer cells. Thus, in some embodiments, the therapeutic agents described herein, such as (a) a FANCM inhibitor, (b) a FANCM-suppressing nucleic acid, (c) a FANCM-targetable nuclease, or (d) a nucleic acid encoding a FANCM-suppressing nucleic acid or a targetable nuclease, may therefore be administered to an individual without other co-existing cancer therapies, such as cytotoxic chemotherapy or radiation therapy, i.e., the therapeutic agent may be administered alone.

[0212] In any other embodiment of the methods disclosed herein, the therapeutic agents described herein, such as (a) a FANCM inhibitor, (b) a FANCM-suppressing nucleic acid, (c) a FANCM-targetable nuclease, or (d) a nucleic acid encoding a FANCM-suppressing nucleic acid or a targetable nuclease, may be administered in combination with one or more other therapies, such as cytotoxic chemotherapy or radiation therapy. This may be useful, for example, in treating cancers that include ALT cancer cells and telomerase-positive cancer cells or cancers where the ALT / telomerase status has not been determined. In some embodiments, the cytotoxic chemotherapy suitable for use in combination with an ATM antagonist may not be a DNA-damaging agent.

[0213] When an inhibitor or therapeutic agent is used in combination with an additional therapeutic agent, the compounds can be administered either sequentially or simultaneously by any convenient route. When a therapeutic agent is used in combination with an additional therapeutic agent that is active against the same disease, the dosage of each agent in the combination may differ from the dosage when the therapeutic agent is used alone. Appropriate dosages are expected to be readily recognizable by those of ordinary skill in the art.

[0214] The data described herein indicate that FANCM depletion alone increases ALT cell death and reduces the viability of ALT cells, whereas co - depletion of both BLM and FANCM does not have this effect. In some preferred embodiments, a FANCM antagonist, such as a FANCM inhibitor, a FANCM suppressor nucleic acid, a FANCM - targetable nuclease, a nucleic acid encoding a FANCM suppressor nucleic acid or a targetable nuclease, or an agent that disrupts the FANCM - RMI interaction and / or an inhibitor of the ATPase activity of FANCM can be administered without reducing the expression or activity of BLM and / or BRCA1. For example, a FANCM antagonist and / or an inhibitor of the ATPase activity of FANCM may be administered to an individual without co - administering a BRCA1 antagonist and / or a BLM antagonist to the individual. Thus, any embodiment of the methods disclosed herein also does not include providing or administering a BRCA1 antagonist or a BLM antagonist to a subject.

[0215] Administration of an inhibitor or therapeutic agent described herein, such as (a) a FANCM inhibitor, (b) a FANCM suppressor nucleic acid, (c) a FANCM - targetable nuclease, (d) a nucleic acid encoding a FANCM suppressor nucleic acid or a targetable nuclease, (e) an agent that disrupts the FANCM - RMI interaction, or (f) an inhibitor of the ATPase activity of FANCM, can be carried out in a single dose, continuously or intermittently (e.g., in several divided doses at appropriate intervals) over the course of treatment. Methods for determining the most effective means of administration and dosage are well known to those of skill in the art and are expected to vary depending on the formulation used in the therapy, the purpose of the therapy, the target cells being treated, and the subject being treated. Single or multiple administrations may be performed, and the dosage levels and patterns are selected by the treating physician.

[0216] In some preferred embodiments, the subject or individual is a human. In other preferred embodiments, non-human mammals can be employed, particularly mammals that are customarily used as models to demonstrate therapeutic efficacy in humans (e.g., mice, primates, pigs, dogs, or rabbit animals).

[0217] In some embodiments, the individual may have minimal residual disease (MRD) after a first cancer treatment.

[0218] Individuals with ALT cancer may exhibit at least one identifiable sign, symptom, or laboratory finding sufficient to make a diagnosis of ALT cancer according to clinical criteria known in the art. Examples of such clinical criteria can be found in medical textbooks, such as Harrison's Principles of Internal Medicine, 15th Edition, edited by Fauci AS et al., McGraw-Hill, New York, 2001. In some cases, the diagnosis of ALT cancer in an individual may include the identification of a specific cell type (e.g., ALT cancer cells) in a sample of body fluid or tissue obtained from the individual. The methods disclosed herein can be used for the diagnosis of ALT cancer.

[0219] Another aspect of the invention relates to the identification of an individual having cancer who is suitable for treatment with an FANCM antagonist described herein, such as (a) an FANCM antagonist or inhibitor, (b) an FANCM-suppressing nucleic acid, (c) an FANCM-targetable nuclease, or (d) a nucleic acid encoding an FANCM-suppressing nucleic acid or a targetable nuclease. For example, an individual having cancer can be evaluated using the methods described herein to determine whether treatment with an FANCM antagonist is likely to be beneficial to the individual, i.e., whether the individual is suitable for treatment by the method of the first aspect of the invention. A method for predicting, determining, or evaluating the responsiveness of cancer in an individual to an agent that reduces the expression or activity of FANCM may include determining the presence of one or more ALT cancer cells in a sample of cancer cells from the individual, wherein the presence of one or more ALT cancer cells in the sample indicates that the cancer responds to the agent.

[0220] A sample of cancer cells can be obtained from an individual using conventional techniques. The presence of ALT cancer cells in the sample can be determined by determining the presence of cancer cells having one or more characteristic features of ALT cancer cells, such as one or more of the features (i)-(v) detailed above. Suitable methods for identifying the presence of characteristic features of ALT cancer cells, such as the presence of C-circles or ALT-associated PML bodies, can be determined using standard techniques.

[0221] An individual identified as having cancer that responds to an FANCM antagonist can be treated as described herein, for example, using the method of the first aspect of the invention.

[0222] The therapeutic agent or pharmaceutical composition containing the therapeutic agent described in this specification can be administered to a subject by any convenient route of administration, whether systemic / peripheral or at the desired site of action. For example, but not limited to, it can be administered parenterally, for example, by infusion such as intravenous infusion, especially intravenous bolus infusion. Suitable infusion techniques are known in the art and are commonly used in therapy (see, for example, Rosenberg et al., New Eng. J. of Med., 319:1676, 1988).

[0223] It will be understood that the appropriate dosage of the therapeutic agent and the composition containing the therapeutic agent can vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic utility against any risks or adverse side effects of the treatment of the present invention. The selected dosage level is expected to depend on, among other things, the activity of specific cells, the route of administration, the time of administration, the rate of cell loss or inactivation, the treatment period, other drugs, compounds, and / or materials used in combination, and various factors such as the age, sex, weight, condition, general health, and previous medical history of the patient. Thus, in certain embodiments, the specific dosage levels and frequencies of dosing for any particular subject can vary and will depend on various factors such as the activity of the specific compound employed, the metabolic stability and duration of action of that compound, age, weight, general health, sex, diet, mode and time of administration, frequency of excretion, drug combinations, severity of the particular condition, and the host being treated. The amount of cells and the route of administration are ultimately expected to be at the discretion of the physician, but generally the dosage is expected to be an amount that achieves a local concentration at the site of action that achieves the desired effect without causing substantial toxic or harmful side effects.

[0224] In some embodiments, the typical oral dosage of the small molecule inhibitor is in the range of about 0.05 to about 1000 mg, preferably about 0.1 to about 500 mg, more preferably about 1.0 mg to about 200 mg, and is administered in one or multiple doses, for example, 1 to 3 doses. The exact dosage is expected to depend on the frequency and mode of administration, the gender, age, weight and general condition of the subject being treated, the nature and severity of the condition being treated, as well as any co-existing diseases to be treated, and other factors apparent to those skilled in the art. In the case of parenteral routes, such as intravenous, intrathecal, intramuscular and similar administrations, typically the dosage is about half of the dosage employed in oral administration.

[0225] Other aspects and embodiments of the present invention provide embodiments and aspects described herein with the term "comprising" replaced by the term "consisting of", and embodiments and aspects described above with the term "comprising" replaced by the term "consisting essentially of".

[0226] It will be understood that this application discloses all combinations of any of the above-described aspects and embodiments, as long as the context does not require otherwise. Similarly, this application discloses all combinations of preferred and / or optional features, either alone or in combination with any of the other aspects, as long as the context does not require otherwise.

[0227] Modifications, further embodiments and modifications of the above embodiments are expected to be apparent to those skilled in the art upon reading this disclosure, and as such they are within the scope of the present invention.

[0228] All documents and sequence database registrations mentioned in this specification are hereby incorporated by reference in their entirety for all purposes.

Examples

[0229] (Example 1) Cell culture and cell lines Cell lines U-2 OS (ALT), IIICF / c (ALT), HeLa (telomerase positive), HeLa 1.2.11 (telomerase positive), HCT116 (telomerase positive), GM847 (ALT), Saos-2 (ALT) and HEK-293 (telomerase positive) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v / v) fetal bovine serum (FBS) at 37 °C and 10% CO2 in a humidified incubator. The cell lines were authenticated by short tandem repeat profiling at 16 loci and tested for mycoplasma contamination by CellBank Australia (Children's Medical Research Institute).

[0230] (Example 2) RNA interference The following Silencer Select siRNAs were designed and synthesized by Life Technologies: FANCM1 (s33619) (SEQ ID NO: 12) and FANCM2 (s33621) (SEQ ID NO: 13), POLD3 (s21045) (SEQ ID NO: 14), BLM (sl998) (SEQ ID NO: 15), RAD51 (sll735) (SEQ ID NO: 16), RAD52 (sll747) (SEQ ID NO: 17) and siRNA negative control #2 (#4390847) of Silencer Select RNAi. Cell suspensions were transfected at a density of 20 - 50% with Lipofectamine RNAiMAX (Life Technologies) at a final concentration of 30 nM siRNA. The culture medium was changed after 48 hours and the cells were harvested for analysis 72 hours after transfection. The knockdown efficiency was verified by Western blot analysis.

[0231] (Example 3) Vector Empty vectors and wild-type FANCM (pCMV6-Myc-DDK) constructs were obtained from Origene Technologies. Wild-type FANCM (SEQ ID NO: 2) and FANCM mutants (SEQ ID NOs: 19 - 28) were cloned into the pLenti-C-Myc-DDK-IRES-Neo backbone (Origene Technologies) by restriction enzyme subcloning of gene blocks synthesized by Integrated DNA Technologies (IDT). Lentiviruses were produced by the vector and the Genome Engineering Facility (Children's Medical Research Institute). For stable overexpression, cells were transduced with lentiviruses, allowed to recover for 24 hours, and subjected to G418 selection. Cells were maintained with G418 to ensure persistent overexpression.

[0232] (Example 4) Extraction and purification of genomic DNA Cells were harvested by trypsinization, washed in PBS, and lysed in DNA extraction buffer (100 mM Tris-HCl pH 7.6, 100 mM NaCl, 10 mM EDTA, 1% (w / v) N-lauroyl sarcosine). The lysate was digested with 50 μg / ml RNase A at room temperature for 20 minutes, followed by digestion with 100 μg / ml proteinase K at 55 °C overnight. DNA was extracted using a phenol / chloroform / isoamyl alcohol (25:24:1) solution (Sigma Aldrich) three times in MaXtract High Density tubes (Qiagen). DNA from the aqueous phase was precipitated using 0.1 volume of 3 M sodium acetate pH 5.2 and 2.5 volumes of cold 100% ethanol. The DNA was washed with 70% ethanol, dried, and dissolved in 10 mM Tris-HCl pH 8.0, 1 mM EDTA.

[0233] (Example 5) C-circle assay The C circle was amplified overnight with Phi29 polymerase using dATP, dTTP, and dGTP. The product was dot blotted onto a BioDyne B membrane (Pall) and pre-hybridized in PerfectHyb Plus (Sigma) for at least 30 minutes. Then, a radiolabeled telomere C-probe was added and the blot was hybridized overnight at 37°C (Henson et al., 2009). The blot was washed three times with 0.5× SSC, 0.1% SDS for 5 minutes each and then exposed to a phosphor screen. Imaging was performed with a Typhoon FLA 7000 system (GE Healthcare) using a 750V PMT.

[0234] (Example 6) Terminal restriction fragment (TRF) analysis Genomic DNA was digested overnight at 37°C with 4 U / μg of HinfI and RsaI. The digested DNA was precipitated using 0.1 volume of 3 M sodium acetate pH 5.2 and 2.5 volumes of 100% ethanol. The DNA was washed with 70% ethanol, dried, and dissolved in 10 mM Tris-HCl pH 7.6, 1 mM EDTA. For one-dimensional gel electrophoresis, digested DNA (2 μg) was loaded onto a 1% (w / v) pulse field certified agarose (Bio-Rad) gel and separated at 6 V / cm for 12 hours using an initial switch time of 1 second and a final switch time of 6 seconds. For two-dimensional gel electrophoresis, digested DNA (20 μg) was separated in the first direction by standard gel electrophoresis in a 0.6% (w / v) agarose gel in 0.5×TBE at 1 V / cm for 13.5 hours. The lane was excised and run in the second direction in a 1.1% (w / v) agarose gel containing 300 ng / ml ethidium bromide at 6 V / cm for 4 hours. The gel was dried at 50°C for 150 minutes and rehydrated in 2×SSC for 30 minutes. The gel was pre-hybridized in Church buffer (250 mM sodium phosphate buffer pH 7.2, 7% (w / v) SDS, 1% (w / v) BSA fraction V grade (Roche), 1 mM EDTA) at 50°C for 2 hours. The native gel was hybridized overnight with a γ-[32P]-ATP labeled (GGGTTA)4 or (CCCTAA)4 oligonucleotide probe. The gel was washed 3 times in 0.2×SSC at room temperature for 15 minutes and exposed to a PhosphorImager screen for 3 days. The gel was then denatured in 0.5 M NaOH, 1.5 M NaCl at 65°C for 40 minutes, followed by washing 2 times in 2×SSC. The gel was pre-hybridized for 1 hour and then hybridized overnight in Church buffer at 50°C with a γ-[32P]-ATP labeled (GGGTTA)4 or (CCCTAA)4 oligonucleotide probe. The denatured gel was washed and exposed to a PhosphorImager screen overnight. Imaging was performed using a Typhoon FLA7000 system (GE Healthcare) with a 750 V PMT.

[0235] (Example 7) Immunoblotting Cells were collected and lysed in RIPA buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 4 mM EDTA) supplemented with a complete mini EDTA-free protease inhibitor cocktail (Roche). Proteins were separated on either 3–8% Tris acetate or 4–12% Bis-Tris gels (Life Technologies). Proteins were transferred to Immobilon P PVDF membranes (Merck). The membranes were optionally stained with Ponceau S (Sigma Aldrich) and then destained with PBST. The membranes were then blocked with either 5% skim milk or bovine serum albumin (BSA) in PBST. The blots were incubated with primary antibodies (see Table 1 for a list of antibodies) either overnight at 4 °C or for 2 h at room temperature. The membranes were then incubated with the corresponding HRP-conjugated secondary antibodies (Dako) for 1 h at room temperature, and bands were visualized using PICO, PICO PLUS, or FEMTO enhanced chemiluminescence reagents (Thermo Scientific).

[0236]

Table 1

[0237] (Example 8) Co-Immunoprecipitation (co-IP) For co-IP experiments involving an ER-inducible system, cells were lysed in IP buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 10% (v / v) glycerol, 0.5 mM EDTA, 1 mM DTT) supplemented with either 1× mammalian protease inhibitor cocktail (Sigma Aldrich) with or without the MM2 peptide variant (mimotope) and 50 U / ml benzonase (Novagen) for 2 hours at 4°C. After lysis, the NaCl concentration was increased to 250 mM and then the lysate was clarified by centrifugation at 16,000×g for 15 minutes. The supernatant was then mixed with 1 μg of α-FANCM (Abcam) or α-ER (Santa Cruz Biotechnology) and 20 μl of protein G sepharose (GE Healthcare), or using α-Flag M2 agarose (Sigma Aldrich). After mixing for 3 hours at 4°C, the beads were washed 4 times with IP buffer and once with 50 mM NH4(CO3)2, 0.5 mM EDTA, and then eluted with 500 mM NH4OH (pH 11.0), 0.5 mM EDTA. The samples were then lyophilized and resuspended in 1× LDS loading buffer (Life Technologies) prior to immunoblotting.

[0238] For the co-IP experiment containing PIP-199, HeLa cells treated with PIP-199 or DMSO for 72 hours were lysed at 4°C for 1 hour in 20 mM HEPES-KOH pH 7.9 supplemented with 1 mM PMSF, 1 mM DTT and 1× complete protease inhibitor (Roche), 200 mM NaCl, 2 mM MgCl2, 10% glycerol, 0.1% Triton X-100. The lysate was clarified at 13,000 rpm at 4°C for 40 minutes. Then the supernatant was incubated at 4°C overnight with 30 μl of protein G Dynabeads, and 2.5 μg of anti-BLM (Bethyl: #A300-110A), 0.5 μg of anti-RMI1 (Proteintech: 14630-1-AP) or 2.5 μg of normal rabbit IgG control (Cell Signalling: 2729S). The proteins were eluted at 70°C in 30 μl of LDS buffer (Life Technologies) before analysis by immunoblotting.

[0239] (Example 9) Immunofluorescence (IF) and Fluorescence in situ Hybridization (FISH) Indirect IF and telomere FISH were performed on both interphase nuclei and metaphase spreads. For the interphase IF experiment, cells were grown on cover glasses or LabTek chamber slides (Thermo Scientific). Slides were prepared as described by Sobinoff et al., 2017. Cells on cover glasses were washed twice with PBS, permeabilized with KCM buffer (120 mM KCl, 20 mM NaCl, 10 mM Tris pH 7.5, 0.1% Triton), washed again with PBST and PBS, and then fixed with ice-cold 4% paraformaldehyde in PBS solution for 10 minutes at room temperature. Cover glasses were blocked with antibody dilution buffer (20 mM Tris-HCl, pH 7.5, 2% (w / v) BSA, 0.2% (v / v) fish gelatin, 150 mM NaCl, 0.1% (v / v) Triton X-100, and 0.1% (w / v) sodium azide) and 0.1 mg / ml RNase A for 30 minutes at 37°C. Cells were incubated with primary antibody (Supplementary Table 1) for 1 hour at 37°C or 2 hours at room temperature, and then incubated with a 1:1,000 dilution of the appropriate Alexa Fluor-conjugated secondary antibody (Thermo Scientific). Cover glasses were rinsed with PBS and then fixed with 4% (v / v) formaldehyde at room temperature prior to telomere FISH. Cover glasses were subjected to a stepwise series of ethanol (75% for 2 minutes, 85% for 2 minutes, and 100% for 2 minutes) and air-dried. The dehydrated cover glasses were overlaid with 0.3 μg / ml FAM-OO-(CCCTAA)3 telomere PNA probe (Panagene) in PNA hybridization solution (70% deionized formamide, 0.25% (v / v) NEN blocking reagent (PerkinElmer), 10 mM Tris-HCl, pH 7.5, 4 mM Na2HPO4, 0.5 mM citric acid, and 1.25 mM MgCl2), denatured at 80°C for 5 minutes, and hybridized overnight at room temperature. Cover glasses were washed twice with PNA wash solution A (70% formamide, 10 mM Tris pH 7.5), and then with PNA wash solution B (50 mM Tris pH 7.5, 150 mM NaCl, 0.8% Tween-20) for 5 minutes each.DAPI was added to the second PNA washing solution B at 50 ng / ml. Finally, the cover glass was rinsed briefly in deionized water and air-dried, and then fixed in DABCO (2.3% 1,4-diazabicyclo(2.2.2)octane, 90% glycerol, 50 mM Tris pH 8.0). Microscopic images were acquired using an appropriate filter set on a Zeiss Axio Imager microscope.

[0240] (Example 10) EdU Detection Cells were pulsed with 10 μM EdU for 2 hours. Cells were permeabilized and then fixed with 4% formaldehyde in PBS solution. Next, the Click-iT® Alexa Fluor 647 azide reaction was performed according to the manufacturer's instructions before blocking with antibody dilution buffer and RNase A. Telomeres were visualized by FISH using a TAMRA-OO-(CCCTAA)3 telomere PNA probe (Panagene), and PML was visualized by IF.

[0241] (Example 11) Single Molecule Analysis of Telomeric DNA (SMAT) Cells were labeled with 100 μM CldU for 5 hours and then recovered by trypsinization. Cells were embedded in low melting point agarose plugs and then subjected to proteinase K digestion overnight. The plugs were lysed using agarase (Thermo Scientific) according to the manufacturer's instructions. Molecular combing was performed using a molecular combing system (Genomic Vision S.A.) with a constant stretching factor of 2 kb / μm and using vinyl silane cover glasses (20 × 20 mm; Genomic Vision S.A.) according to the manufacturer's instructions.

[0242] After coming, the cover glass was dried at 60 °C for 4 hours. The quality and integrity of the coming DNA fibers were checked using YoYo-1 counterstaining (Molecular Probes). The cover glass was denatured in alkaline denaturation buffer (0.2 M NaOH, 0.1% b-mercaptoethanol in 70% ethanol) for 25 minutes and fixed by adding 0.5% glutaraldehyde for 5 minutes. Telomeric DNA was visualized by hybridization using a TAMRA-OO-KKK(TTAGGG)3 PNA probe (Panagene). Halogenated nucleotides were detected with a rat anti-CldU monoclonal antibody (Accurate) and an Alexa Fluor 488-conjugated goat anti-rat antibody (Molecular Probes). Telomeric fibers were detected with a Zeiss Axio Imager microscope equipped with an ApoTome module and analyzed using Zen software (Zeiss).

[0243] (Example 12) Neonatal Telomere Analysis The BrdU pull-down was performed with partial modifications as described by Verma et al., 2018. Cells were harvested after a 5-hour pulse with 100 μM BrdU (Sigma). Genomic DNA (gDNA) was extracted and resuspended in 100 μl of EB buffer (Qiagen). The gDNA was sheared into fragments of 100 - 1,000 bp using a Covaris M220 sonicator. 4 μg of sheared gDNA was denatured at 95 °C for 10 minutes and then immediately cooled. The denatured gDNA was incubated with 2 μg of control mouse IgG (Millipore) or anti-BrdU antibody (BD Biosciences) in 250 μl of immunoprecipitation buffer (0.0625% (v / v) Triton X-100 in PBS) and rotated overnight at 4 °C. The samples were then incubated overnight at 4 °C with 60 μl of BSA-blocked (nuclease-free) protein G agarose beads (Roche). The next day, the beads were collected by centrifugation at 13,000 rpm for 60 seconds and washed with 1 ml of buffer A (20 mM HEPES-KOH, pH 8.0, 2 mM MgCl 2、300 mM KCl, 1 mM EDTA, 10% (v / v) glycerol, and 48 0.1% (v / v) Triton X-100), and then washed twice with 1 ml of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The immunoprecipitated DNA was eluted from the protein G agarose beads into 100 μl of elution buffer (50 mM NaHCO3 and 1% (v / v) SDS), then purified with the QIAquick PCR Purification Kit (Qiagen) and eluted into 140 μl of TE. Samples and inputs were diluted with 200 μl of 0.46 M NaOH, denatured at 95°C for 5 minutes, cooled on ice, and then dot-blotted onto Hybond XL (GE Healthcare Life Sciences). The membrane was cross-linked in a Stratalinker (Stratagene) at 254 nm with 2 × 240 mJ, pre-hybridized in PerfectHyb Plus hybridization buffer (Sigma) at 37°C for 54 minutes, and hybridized overnight with a γ-[32P]-ATP-labeled (TTAGGG)4 telomere probe to detect the C strand. To detect the G strand, the blot was stripped by washing 5 times for 20 minutes with 0.1% SDS that was almost boiling, and then probed again with a γ-[32P]-ATP-labeled (CCCTAA)4 telomere probe. The membrane was washed 3 times in 2× SSC at room temperature for 15 minutes and then exposed to a PhosphorImager screen. Imaging was performed using the array analysis function of ImageQuant software (GE Healthcare Life Sciences).

[0244] Alternatively, gDNA was extracted and digested with HinfI and RsaI before BrdU immunoprecipitation. The immunoprecipitated DNA was then eluted from the protein G agarose beads into 100 μl of elution buffer (50 mM NaHCO3 and 1% 61 (v / v) SDS), purified with the QIAquick PCR Purification Kit (Qiagen), and eluted into 25 μL of TE. Amplification and detection of C circles were performed as described by Henson et al., 2009.

[0245] (Example 13) Automated image analysis ZEN microscope images (.czi) were processed into enlarged projection views of z-stacks using ZEN desk 2011 software (Zeiss) and imported into Cellprofiler v2.1.1 (Carpenter et al., 2006) for analysis. Using the DAPI channel, individual nuclei were masked as the main objects. Lesions within each segmented nucleus were identified using an intensity threshold-based mask. Any given object was considered to overlap with another object if at least 20% of the area of the first object was within the area of the second object.

[0246] (Example 14) Sister chromatid exchange (SCE) assay The SCE assay was performed as described by Bayani, 2005. Cells were cultured in 100 μM BrdU (Sigma Aldrich) for two cell cycles, with or without 200 nM 4-hydroxytamoxifen (Sigma Aldrich), and subsequently cultured in 0.2 μg / ml colcemid (Life Technologies) for 1 hour. Images of mitotic spreads were captured using a Zeiss Axioplan 2 microscope, and SCEs were scored manually and verified in a blinded manner. Exchanges due to obvious "flipping" at the centrosome were excluded from quantification. Mitotic abnormalities were scored as described by Caldon et al., 2013 and Bayani, 2005.

[0247] (Example 15) Colony formation assay The clonogenic assay was performed as described by Franken et al., 2006. Cells were plated at 1,000 cells / 10 cm culture dish with or without 200 nM 4-hydroxytamoxifen (Sigma Aldrich), and 4-hydroxytamoxifen was replenished on days 4 and 7. For cells treated with PIP-199 (Aobious #AOB33732, CAS number 622795-76-0), cells were plated at 300 cells / well in 6-well plates and treated with 0 - 10 μM drug in dimethyl sulfoxide (DMSO). Cells were grown for 11 days without change. Plates were washed with 1×PBS, then fixed and stained with 0.5% (w / v) crystal violet in 1 part acetic acid, 7 parts methanol for 2 hours at room temperature. The plates were then washed with tap water and air dried. Colonies on each plate were counted, and plating efficiency and survival fraction were calculated.

[0248] (Example 16) Imaging of living cells Twenty-four hours prior to imaging, cells were seeded into 12-well 1.5 mm glass bottom wells (MatTek) coated with alcian blue. 120 cells and subsequent daughter cells were monitored for 48 hours at 6-minute intervals. The number of nuclei, the duration of interphase, the duration of mitosis (defined as the period from the first cell aggregation to the completion of cytokinesis), and the result of mitosis (normal, lethal during mitosis, arrested in the middle, multipolar division, and cell fusion resulting in multinucleated cells) were recorded.

[0249] (Example 17) Flow cytometry Ethanol-fixed single cell suspensions (approximately 1×10 cells 6Cells (number) were stained with 2 mg / ml RNase A and 0.1 mg / ml propidium iodide (PI) in 0.25 ml PBS for DNA analysis. The cells were incubated at 37 °C for 30 minutes and equilibrated at least for 10 minutes in the dark at room temperature. Cells were analyzed by BD FACSCanto flow cytometry (BD Biosciences) using an air-cooled 488 nm argon laser to excite PI. A total of 9,800 - 10,000 stopping gate events were collected at a flow rate of approximately 200 events / second. Forward scatter (FSC, size) and side scatter (SSC, internal granularity) of each cell were recorded. Pulse area (PI-A) was plotted against pulse width (PI-W) to identify and exclude cell debris and doublets. Doublets identified as cells with an increased pulse width and a DNA content of 4N were excluded. Cell cycle population analysis was performed using FlowJo v5 software (FlowJo). Gating of cell division phases (G0 / G1 and G2 / M) was performed using the Dean-Jett algorithm. The percentage of S-phase cells was calculated as the remaining percentage after G0 / G1 and G2 / M gating.

[0250] (Example 18) Statistical analysis Details regarding quantification and statistical analysis are provided in the figure legends. For data assumed to be normally distributed, a two-sided Student's t-test was performed, while for data assumed not to be normally distributed, a two-sided Mann-Whitney test was performed. No statistical analysis was performed on the length of telomere elongation due to a substantial difference in events between treatments. Data analysis was performed using Microsoft Excel and GraphPad Prism.

[0251] (Example 19) FANCM depletion induces telomere dysfunction and ALT markers ALT telomeres are characterized by an elevated DNA damage response (DDR) compared to lethal and telomerase-positive cells (Cesare et al., 2009). Such damage is observed as foci (TIFs) induced by telomere dysfunction, which are characterized by the co-localization of the DNA damage marker γ-H2AX and telomeric DNA. Knockdown of FANCM using siFANCM1 and siFANCM2 (Figure 1a) resulted in a significant increase in metaphase TIFs (meta-TIFs) in U-2 OS ALT cells compared to scrambled controls (Figure 2a). In contrast to the ALT-specific induction of telomere dysfunction, FANCM depletion induced comparable levels of overall DDR signaling in both U-2 OS (ALT) and HeLa (telomerase-positive) cell lines (Figure 1b).

[0252] ALT cells characteristically have long and heterogeneous telomere lengths, as well as abundant extrachromosomal telomeric repeat (ECTR) DNA containing t-circles and C-circles (Henson et al., 2009). One-dimensional gel electrophoresis of isolated terminal restriction fragments (TRFs), followed by hybridization under native conditions, revealed both a decrease in G-rich overhangs and a marked increase in low molecular weight single-stranded (ss) C-rich telomeric DNA associated with FANCM depletion (Figure 2b; black and red arrows, respectively). Since this low molecular weight species of DNA was detected even after separation of undigested genomic DNA (Figure 1c), it was shown to be inherently extrachromosomal. Despite these observations, no change in average telomere length was observed (Figure 2b). These effects were observed after FANCM depletion using both siRNAs, with the most pronounced effects seen with siFANCM2. As a result, siFANCM2 was used in subsequent experiments. Separation of TRFs by two-dimensional gel electrophoresis identified an increase in extrachromosomal t-circles, which characteristically resolved as an arc above the arc of linear telomeric DNA after FANCM depletion (Figure 2c; blue arrow). Low molecular weight ss C-rich telomeric DNA migrated as a distinct separated arc below both the t-circle and the arc of linear telomeric DNA in FANCM-depleted cells (Figure 2c; red arrow).

[0253] FANCM depletion led to a marked increase in C circles detected by rolling circle amplification (Fig. 1d, Fig. 2d). The increase in the amplified C circles corresponded to low molecular weight ss ECTR C-rich telomeric DNA identified by one- and two-dimensional gel electrophoresis (Fig. 2b and Fig. 2c), consistent with this type of DNA being C circles. This is the first visualization of C circles reported by gel electrophoresis, demonstrating that t circles and C circles are distinct DNA species. Quantification of telomeric DNA and telomere-related proteins co-localized with the PML protein in ALT-associated PML bodies (APBs) revealed that the number of APBs increased significantly after FANCM depletion in U-2 OS and IIICF / c cells, but not in GM847 and Saos-2 cells (Fig. 1e, Fig. 2e and Fig. 2f).

[0254] Interestingly, a marked increase in the intensity of telomeric signals within APBs was also observed in all ALT cell lines analyzed (Fig. 1f, Fig. 2e and Fig. 2g). This indicates increased telomeric DNA accumulation or telomere clustering in APBs in response to FANCM depletion. Collectively, these data demonstrate that the ALT phenotype is significantly induced after transient loss of FANCM, but no overall telomere elongation is observed. These observations are specific to ALT cell lines (U-2 OS, IIICF / c, GM847 and Saos-2) and independent of the p53 status, while induction of ALT features was not observed in telomerase-positive cell lines (HeLa, HeLa 1.2.11 and HCT116) in response to FANCM depletion (Fig. 1). This indicates that induction of the ALT phenotype may be due to ALT-specific telomere dysfunction.

[0255] (Example 20) FANCM depletion promotes telomere synthesis induced by breakage ALT involves a mechanism of telomere synthesis induced by a conservative break similar to break-induced replication (BIR) in yeast and depends on the POLD3 subunit of Polδ. A significant increase in POLD3 replenishment at telomeres after FANCM depletion (Figure 3a) was identified. This was consistent with an increase in the generation of total nascent telomere repeats (Figure 3b). Nascent telomere DNA was localized with APB (Figure 3c) and contributed to the dramatic induction of C-circles generated in response to FANCM depletion (Figure 3d).

[0256] Previously, it has been shown that telomere synthesis induced by breakage depends on BLM, and that both RAD51-dependent and -independent pathways of telomere elongation can exist (Sobinoff et al., 2017; Dilley et al., 2016; Cho et al., 2014; and Verma et al., 2016). These pathways are reminiscent of the survival of Saccharomyces cerevisiae lacking type I (RAD51-dependent) and type II (RAD51-independent) telomerases, which require Rad52 and the BLM homolog Sgs1. To characterize the involvement of these proteins in the context of FANCM knockdown, we co-depleted FANCM with either POLD3, BLM, RAD51, or RAD52 (Supplementary Figure 4a). Interestingly, FANCM depletion resulted in a concomitant decrease in the protein levels of POLD3, BLM, and RAD51 (Figures 4a and 4b), and was most prominent for RAD51, suggesting that FANCM co-regulates these proteins. This co-regulation is unlikely to contribute to the observed exacerbated ALT phenotype, as independent depletion of POLD3, BLM, or RAD51 caused only a minor or antagonistic effect on ALT activity compared to that seen with FANCM depletion. Co-depletion experiments showed that the elevated levels of C-circles detected by both C-circle assay and TRF analysis after FANCM depletion were dependent on POLD3 and BLM, and partially dependent on RAD51 and RAD52 (Figures 4c and 5a). Similarly, the number and intensity of APBs increased in response to FANCM depletion were dependent on POLD3 and BLM, and partially dependent on RAD51 and RAD52 (Figures 5b and 5c).

[0257] To directly measure the frequency and length of telomere synthesis events, single molecule analysis of telomeres (SMAT) in DNA fibers incorporating CldU was used. FANCM depletion led to a significant increase in the number of telomere elongation events, while the length of the elongation products remained unchanged (Figure 5d). This increase was predominantly dependent on POLD3, BLM, and RAD52 (Figure 5d). Overall, these data demonstrate that FANCM depletion results in an increase in telomere synthesis induced by breaks mediated by POLD3 and BLM at ALT telomeres, which is consistent with the rapid induction of ss and C-rich predominant nascent ECTRs. These data show a stronger dependence on RAD52 than RAD51, supporting the role of both proteins in break-induced telomere synthesis.

[0258] (Example 21) The MM2 domain of FANCM is required to attenuate ALT activity Multiple functional domains of FANCM are well characterized. In particular, the PIP domain interacts with proliferating cell nuclear antigen (PCNA) via a conserved PIP-box sequence (Rohleder et al., 2016). The conserved DEAH domain has ATP-dependent DNA remodeling translocase activity and binds to replication forks and DNA repair intermediates to promote replacement and annealing of nascent and parental DNA strands (Xue et al., 2008 and Gari et al., 2008). The major histone fold 1 and 2 (MHF1 / 2) heterotetramer is an essential cofactor that binds to the domain that interacts with MHF1 / 2 (MID), targets FANCM to DNA branch points, and facilitates replication fork traversal (Yan et al., 2010, Huang et al., 2013, Fox et al., 2014 and Zhao et al., 2014). The MM1 domain replenishes the FA core complex to the ICL site; the MM2 domain interacts with the BTR complex to regulate replication fork stabilization; the MM3 domain has no known function (Singh et al., 2013). The major site at S1045 is phosphorylated upon genotoxic stress, which is required for efficient ATR-CHK1 checkpoint activation. The ERCC4 endonuclease domain and the helix-hairpin-helix (HhH) motif recognize ssDNA gaps and lesions in DNA present at the ICL site and facilitate heterodimerization with FAAP24, another essential FANCM cofactor (Xue et al., 2008, Blackford et al., 2012, Kim et al., 2008 and Huang et al., 2010).

[0259] FANCM has been shown elsewhere to directly associate with telomeric DNA (Pan et al., 2017 and Pentz et al., 2019).

[0260] To determine the specific functional FANCM activities required to suppress the ALT phenotype, a panel (Figure 6a) of 10 mutant lentiviral constructs (SEQ ID NOs: 19 - 28) with each identified domain disrupted was established. The PIP mutant (SEQ ID NO: 19) is substituted with L8R and W12S. The K117R mutant (SEQ ID NO: 20) is substituted with K117R. The MID domain mutant (SEQ ID NO: 21) is substituted with V749G / H751G to disrupt the interaction with the boundary of the MHF1 / 2 heterotetramer. The S1045A mutant (SEQ ID NO: 22) is substituted with S1045A. The MM1 domain mutant (SEQ ID NO: 23) has a deletion in amino acid residues 943 - 1004. The MM2 domain mutant (SEQ ID NO: 24) has a deletion in amino acid residues 1219 - 1251. The FF>AA mutant (SEQ ID NO: 25) has a double substitution of phenylalanine with alanine (FANCM F1232A / F1236A) within the MM2 domain and has previously been characterized as substantially disrupting BTR binding (Deans et al., 2009). The MM3 domain mutant (SEQ ID NO: 26) has a deletion in amino acid residues 1337 - 1707. The ERCC4 domain mutant (SEQ ID NO: 27) has a deletion in amino acid residues 1815 - 1922. The HhH domain mutant (SEQ ID NO: 28) has a deletion in amino acid residues 1971 - 2048. These constructs, including wild - type FANCM, were stably transduced into U - 2 OS cells, and the exogenous expression of FANCM mutants was confirmed by Western blot analysis (Figure 7a). Stable overexpression of FANCM resulted in a significant decrease in both telomere dysfunction (Figure 6b) and the number of fragile telomeres (Figure 6c). Most of the mutants also suppressed telomere DDR and telomere fragility (Figure 6b and Figure 6c). Exceptions were the MM2 (SEQ ID NO: 24), FF>AA, and MID domain mutants, in which both telomere dysfunction and telomere fragility were significantly increased, and the K117R mutant (SEQ ID NO: 20) with a disrupted DEAH domain that could not suppress the phenotype (Figure 6b and Figure 6c). The effects of FANCM mutant overexpression on ECTR generation and ALT activity were examined.Consistent with the stable overexpression of wild-type FANCM, overexpression of most FANCM mutants resulted in a significant decrease in C-circle generation (Figure 6d). Exceptions were the K117R (SEQ ID NO: 20), ERCC4 (SEQ ID NO: 27), and HhH domain (SEQ ID NO: 28) mutants that did not cause changes in C-circle levels, and the MM2 (SEQ ID NO: 24) and FF>AA (SEQ ID NO: 25) domain mutants that caused approximately a two-fold increase in C-circles compared to the vector control (Figure 6d). No change in average telomere length was observed after overexpression of wild-type or mutant FANCM, but the smear of low molecular weight ss C-rich telomeric DNA that had been previously identified was observed in cells overexpressing the K117R, MM2, and FF>AA domain mutants (Figure 7b). These changes were consistent with a significant increase in both the number of APBs and the intensity of the telomere signal within the APBs after overexpression of the K117R, MM2, and FF>AA mutants (Figure 7c). A relatively minor difference in the number and intensity of APBs was observed after overexpression of some of the other mutants, indicating the complexity underlying APB formation and function.

[0261] The number and length of telomere elongation events were measured to determine whether overexpression of FANCM mutants has a direct effect on telomere synthesis. A significant increase in the number of elongation events was observed in response to overexpression of the MM2 and FF>AA domain mutants (Figure 6e), but the length of the elongation events remained relatively stable after overexpression of wild-type FANCM and the other mutants. These data indicate that wild-type FANCM suppresses telomere replication stress but does not directly affect the length of telomere elongation events. Overall, the functional significance of replication fork remodeling and FANCM restart activity provided by its ATP-dependent translocase activity (DEAH domain) and its BTR-binding ability (MM2 domain) in regulating ALT activity was demonstrated. This data also indicates a dominant negative effect after overexpression of the MM2 domain mutant.

[0262] Further studies were performed using a double mutant (DM) lentiviral construct (SEQ ID NO: 35) containing both the K117R substitution and the FF>AA double substitution. The effects of the DM on telomere dysfunction, APB frequency, and C-circle levels were examined compared to the K117R mutant and the FF>AA mutant. As shown in FIGS. 23A - 23C, the DM caused a significantly greater increase in C-circles compared to either the K117R mutant or the FF>AA mutant. The DM mutant also showed enhanced telomere dysfunction compared to either the empty vector or the wild type. These changes were associated with an increase in the number of APBs that was greater compared to either the K117R mutant or the FF>AA mutant. Thus, the inventors have two independent identified targets for FANCM (the MM2 interaction domain and the ATPase domain). Considering data indicating that the ATPase domain of FANCM is required to remodel telomeric R-loops, a major source of replication stress at telomeres, these studies suggest that the ATPase activity and RMI interaction of FANCM independently support telomere stability in ALT cells. These studies suggest that ALT cancers may also be treated using inhibitors of the FANCM - RMI interaction alone or in combination with inhibitors of the ATPase and / or translocase activity of FANCM.

[0263] Defects in ALT phenotype suppression are exacerbated by overexpression of a double mutant (DM) FANCM in which both the MM2 domain (FF>AA) and the translocase domain (K117R) of FANCM are mutated.

[0264] (a) Representative dot blot and quantification of the C-circle assay in U - 2 OS cells stably overexpressing wild type (WT) or FANCM domain mutants (K117R, FF>AA, DM). C-circles were normalized to the mean of the empty vector control (EV). Error bars represent mean ± SEM from n = 3 experiments. * p<0.05, **p < 0.005, Student's t-test. (b) Quantification of APB frequency in U-2 OS cells overexpressing wild-type (WT) or FANCM mutants. Bars in the scatter plots represent mean ± SEM. From three experiments, n = 150 cells were scored for each mutant, ** p < 0.005, Mann-Whitney test. (c) Quantification of metaphase TIFs in U-2 OS cells stably overexpressing wild-type (WT) or FANCM mutants. Bars in the scatter plots represent mean ± SEM. From three experiments, n = 120 metaphases were scored for each mutant, * p < 0.05, ** p < 0.005, Mann-Whitney test. All statistical comparisons were made against the EV control.

[0265] (Example 22) Disruption of the FANCM-BTR complex inhibits the viability of ALT cells Previously, it has been shown that FANCM depletion induced replication stress at ALT telomeres but did not affect cell viability (Pan et al., 2017). In contrast, throughout the course of this study, a consistent deficiency in mitotic cells following FANCM depletion was observed. To determine the effect of FANCM depletion on cell cycle progression, cell cycle analysis and live cell imaging were used. An accumulation of cells in G2 / M was observed in FANCM-depleted U-2 OS cells compared to FANCM-depleted HeLa cells (Figures 8a and 8b). Both U-2 OS and HeLa cells showed a delay in cycling speed after FANCM depletion, as evidenced by an increase in the duration of the interphase period (Figures 8c and 8d). FANCM-depleted U-2 OS cells exhibited cell cycle decay in less than one-third of the cells that did not enter mitosis over a 48-hour observation time frame, while the effect of FANCM depletion on mitotic entry in HeLa cells was significantly less (Figures 8c and 8d). After FANCM depletion, changes in the outcome of mitosis (aberrant mitosis or mitotic death) were not apparent, which is consistent with cells arrested prior to mitosis (Figures 8e and 8f). Cells that were able to progress to mitosis may have avoided a dangerous level of FANCM knockdown. These data demonstrate that ALT cells are hypersensitive to replication stress induced by FANCM depletion.

[0266] To further evaluate the effect of FANCM perturbation on cell survival, Project Achilles was utilized, which is the first step to identify genes and enumerate their requirements across cancer cell lines (Meyers et al., 2017). Gene dependency scores for FANCM after CRISPR-Cas9-mediated knockout were compared. When the gene dependency scores were calculated using CERES43, it was shown that FANCM might be essential in that cell line. Lower gene dependency scores indicate a higher likelihood that the gene is essential, and -1 represents the median score for all pan-cancer essential genes. Overall, across the panel of cell lines, FANCM did not appear to be essential for cell viability (Figure 9). However, for the subset of identified ALT cell lines, most (4 / 5) clustered around a gene dependency score of -1 (Figure 9). These data support an essential role for FANCM in the viability of ALT cancer cells.

[0267] To investigate the possibility of specifically targeting the viability of ALT cells via FANCM, two approaches were employed to disrupt the FANCM-BTR complex. First, a chemically synthesized peptide (SEQ ID NO: 29) corresponding to 28 highly conserved amino acids of the MM2 domain, which was shown to have binding affinity to the BTR complex similar to full-length FANCM, was used (Deans et al., 2009). Addition of the MM2 peptide to cell lysates resulted in dose-dependent inhibition of FANCM-BTR complex formation, whereas addition of an MM2 peptide containing an FF>AA mutant that does not bind to BTR (SEQ ID NO: 30) was unable to inhibit complex formation (FIGS. 10a and 10b). To investigate the effect of FANCM-BTR complex disruption in ALT cells, the MM2 peptide was made functionally dependent on tamoxifen by fusion to the C-terminus of the estrogen receptor (MM2-ER) (FIG. 11a). The ability of the MM2-ER fusion protein to inhibit FANCM-BTR complex formation was tested by immunoprecipitation with either FANCM or ER. In the absence of tamoxifen, the TOP3A and RMI1 components of the BTR complex were immunoprecipitated by FANCM but not by ER (FIG. 11b). Addition of tamoxifen resulted in activation of the MM2-ER fusion protein, which was detected by immunoprecipitation of TOP3A and RMI1 with ER but not with FANCM. Activation of the control FF>AA mutant MM2-ER fusion protein was unable to sequester the BTR complex from FANCM (FIG. 11b).

[0268] To confirm the genomic efficacy of FANCM-BTR complex disruption, sister chromatid exchange (SCE) was quantified as previously shown to result in increased SCE formation upon FANCM depletion (Deans et al., 2009). An increase in the frequency of SCE in response to activation of the MM2-ER fusion protein was identified, but not in the FF>AA mutant (Figures 10c and 10d), which is consistent with efficient disruption of the FANCM-BTR complex in both ALT and telomerase-positive cell lines. Activation of the MM2-ER fusion protein resulted in a marked increase in TIF in U-2 OS cells, but a small but significant decrease in TIF was observed after activation of the FF>AA mutant fusion protein (Figure 11c). Since activation of the MM2-ER fusion protein, but not the FF>AA mutant, also induced C-circles (Figure 11d), it is shown that enhanced replication stress at ALT telomeres leads to increased ALT activity. From the clonogenic survival assay, a 10- to 20-fold decrease in the survival of U-2 OS, GM847, and Saos-2 ALT cells after activation of the MM2-ER fusion protein was elucidated, but activation of the FF>AA mutant MM2-ER fusion protein had no effect on cell survival (Figure 11e). In the telomerase-positive cell lines HeLa and HCT116, no effect on cell survival was observed upon activation of either wild-type MM2-ER or FF>AA mutant MM2-ER fusion protein (Figure 11e).

[0269] The second approach involved treating cells by increasing the concentration of PIP-199, a small molecule inhibitor of the MM2-RMI interaction within FANCM-BTR. An increase in C circles (Figure 11f) was observed in U-2 OS, GM847, and Saos-2 ALT cell lines. This is consistent with the disruption of the FANCM-BTR complex mediated by PIP-199. Clonogenic assays identified the hypersensitivity of ALT cells to PIP-199 compared to telomerase-positive cells (Figures 11g and 12a). Co-immunoprecipitation experiments of BLM and RMI1 in U-2 OS cells confirmed the disruption of FANCM-BLM and FANCM-RMI1 interactions by increasing the concentration of PIP-199 after 72 hours (Figure 12b).

[0270] These experiments employed two independent approaches to disrupt the important binding interaction between FANCM and the BTR complex, demonstrating that inhibition of the FANCM-BTR complex results in replication stress and elevated C circles.

[0271] In summary, the studies of the present inventors defined the mechanism by which FANCM regulates ALT activity. Specifically, FANCM functions in a BTR-dependent manner, independent of the FA-core complex, to resolve replication stress that spontaneously occurs within telomeres. ALT telomeres are hypersensitive to FANCM depletion because they have unique structural abnormalities that render them replication-defective. In the absence of FANCM or through inhibition of the synthesis of the FANCM-BTR complex, stalled forks deteriorate to form DSBs. This leads to the induction of telomere synthesis events induced by breaks to repair dysfunctional telomeres, which coincides with the production of nascent ECTR DNA species. ECTR accumulation may further exacerbate DDR by depleting the cellular RPA reserve. The consequences of DSB formation and subsequent DDR include excessive ALT activity and loss of cell viability via G2 / M arrest. Importantly, the present inventors found that the use of FANCM-BTR complex inhibition can selectively suppress the growth and viability of ALT cancer cells, and targeting the FANCM-RMI interaction is a potent strategy for ALT cancer cell lethality.

[0272] (Example 23) FANCM regulates BML in ALT cells Method Cell lines and culture conditions HeLa cervical cancer, HT1080 fibrosarcoma, and HEK293 fetal kidney cells were purchased from ATCC. U20S osteosarcoma cells were a kind gift from Dr. M. Lopes (IMCR, Zurich, Switzerland). Hu09, Saos2, and HOS osteosarcoma cells were a kind gift from Dr. B. Fuchs (Balgrist University Hospital, Zurich, Switzerland). WI-38 VA13 in vitro SV40-transformed lung fibroblasts were a kind gift from Dr. A. Londoño-Vallejo (CNRS, Paris, France). SKNAS neuroblastoma cells were a kind gift from Dr. O. Shakhova (University Hospital Zurich, Switzerland). HeLa, HT1080, HEK293, U20S, and WI-38 VA13 cells were cultured in high-glucose DMEM, GlutaMAX (Thermo Fisher Scientific) supplemented with 10% tetracycline-free fetal bovine serum (Pan BioTech) and 100 U / ml penicillin-streptomycin (Thermo Fisher Scientific). Hu09, Saos2, HOS, and SKNAS cells were cultured in high-glucose DMEM / F12, GlutaMAX (Thermo Fisher Scientific) supplemented with 10% tetracycline-free fetal bovine serum (Pan BioTech), 100 U / ml penicillin-streptomycin (Thermo Fisher Scientific), and non-essential amino acids (Thermo Fisher Scientific). Mycoplasma contamination was tested using the VenorGeM Mycoplasma PCR Detection Kit (Minerva Biolabs) according to the manufacturer's instructions. Where indicated, cells were incubated with 1 μM camptothecin (Sigma-Aldrich) for 3 h, 0.2 mM hydroxyurea (Sigma-Aldrich) for 16 h, 20 μM BIBR1532 (Merck Millipore) for 7 days, and 10 μM RO-3306 (Selleckchem) for 18 h.

[0273] Ectopic expression of protein For FANCM complementation experiments, siFa- and siFb-resistant cDNAs encoding FANCM variants with a V5 tag at the N-terminus were synthesized by GenScript and cloned into the lentiviral vector pLVX-TetOne-Puro (Clontech). The resulting plasmids, pLVX-V5FANCM and pLVX-V5FANCMK117R, were used to produce lentiviruses, which were then used to infect U20S cells and subsequently selected in medium containing 1 μg / ml puromycin (Merck Millipore). Experiments were performed in medium containing 1 μg / ml doxycycline (Sigma-Aldrich). For overexpression of RNase H1, U20S cells were transfected with the pLHCX-MYC-RHIWT and pLHCX-MYC-RHID145A plasmids 11Infected with retroviruses produced using [relevant method], and subsequently selected in medium containing 200 μg / ml hygromycin B (VWR). For TRF1 overexpression, U20S cells were infected with retroviruses produced using pLPC-NFLAG-TRF1 (kind gift from T. de Lange, Addgene plasmid #16058), and subsequently selected with puromycin. Transgene expression was verified by Western blotting. For telomerase overexpression, U20S and HeLa cells were infected with retroviruses produced using the pBABEpuroUThTERT+U3-hTR-500 plasmid (Addgene plasmid #27665), and subsequently selected with puromycin. Viruses were produced in HEK293 cells according to standard procedures. Ectopic expression of FANCM, RNase H1 and TRF1 was verified by Western blotting (see below). hTERT and hTR expression was verified by quantitative RT-PCR on total RNA using the following oligonucleotides: hTERT forward, 5'-agagtgtctggagcaagttgc-3' (SEQ ID NO: 41); hTERT reverse, 5'-cgtagtccatgttcacaatcg-3' (SEQ ID NO: 42); hTR forward, 5'-gtggtggccattttttgtctaac-3' (SEQ ID NO: 43); hTR reverse, 5'-tgctctagaatgaacggtggaa-3' (SEQ ID NO: 44); actin B1 forward, 5'-tccctggagaagagctacga-3' (SEQ ID NO: 45); actin B1 reverse, 5'-agcactgtgttggcgtacag-3' (SEQ ID NO: 46). Actin B1 was used as a standard.

[0274] Protein depletion mediated by siRNA DsiRNA (Integrated DNA Technologies) was transfected using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer's instructions. DsiRNA was used at a final concentration of 20 nM unless otherwise specified. The medium was changed 5 hours after transfection, and samples were collected 48 hours after transfection unless otherwise specified. The following mRNA target sequences were used: siFa: 5'-GGATGTTTAGGAGAACAAAGAGCTA-3' (SEQ ID NO: 33); siFb: 5'-CCCATCAAATGAAGATATGCAGAAT-3' (SEQ ID NO: 34); siBl: 5'-GCTAGGAGTCTGCGTGCGAGGATTA-3' (SEQ ID NO: 36); siATRXa: 5'-GAGGAAACCUUCAAUUGUAACAAAGUA-3' (SEQ ID NO: 37); siATRXb: 5'-UGCAAGCUCUAUCAGUACUACUUAGAU-3' (SEQ ID NO: 38); siTRFl: 5'-CUUUCUUUCUUAUUAAGGUCUUGUUGC-3' (SEQ ID NO: 39); siRNase H1: 5'-UUGUCUAAUGCCUACAUUUAAAGGAUG-3' (SEQ ID NO: 40). The NC1 negative control (51-01-14-03) was used as siCt.

[0275] Cell proliferation and viability assays For the colony formation assay, cells were transfected with siRNA, and 24 hours later, 300 - 500 cells were plated in 3-cm culture dishes and grown until visible colonies were formed. Cells were stained with 1% crystal violet, 1% formaldehyde, 1% MeOH (Sigma-Aldrich) for 20 minutes at room temperature, followed by washing with tap water. The plates were air-dried and photographed using a FluorChem HD2 imaging device (Alpha Innotech).

[0276] Colonies were counted using ImageJ software. For growth curves, cells were transfected with siRNA and after 24 hours, 1×10 5 cells were seeded into 6 cm culture dishes and passaged every 10 days. Cells were transfected again with siRNA and counted every 3 days. For fluorescence-activated cell sorting, cells were trypsinized and pelleted by centrifugation at 500 g for 5 minutes at 4 °C. The cell pellet was either left untreated for viability assays or fixed in 70% ethanol at -20 °C for 30 minutes and treated in 25 μg / ml RNase A (Sigma-Aldrich) in 1×PBS at 37 °C for 20 minutes. Cells were then washed in 1×PBS and stained with 20 μg / ml propidium iodide (Sigma-Aldrich) in 1×PBS for 10 minutes at 4 °C. Flow cytometry was performed on a BD FACSCalibur or BD Accuri C6 (BD Biosciences). Data were analyzed using FlowJo software.

[0277] Western blotting Cells were trypsinized and pelleted by centrifugation at 500 g for 5 minutes at 4 °C. The pellet was resuspended in 2× lysis buffer (4% SDS, 20% glycerol, 120 mM Tris-HCl pH 6.8), boiled at 95 °C for 5 minutes, and centrifuged at 1600 g for 10 minutes at 4 °C. The supernatant was collected and the protein concentration was determined by the Lowry assay using bovine serum albumin (BSA; Sigma-Aldrich) as a standard. 0.004% bromophenol blue and 1% β-mercaptoethanol (Sigma-Aldrich) were added to 20 - 40 μg of protein, incubated at 95 °C for 5 minutes, separated on a 6 or 10% polyacrylamide gel, and transferred to a nitrocellulose membrane (Maine Manufacturing, LLC) using a Trans-Blot SD semi-dry cell transfer apparatus (Bio-Rad). The following primary antibodies were also used: mouse monoclonal anti-FANCM (CV5.1 72, 1:1000 dilution); mouse monoclonal anti-Golgin 97 (Molecular Probes, A-21270, 1:5000 dilution); rabbit polyclonal anti-KAPl (Bethyl Laboratories, A300-274A, 1:2000 dilution); rabbit polyclonal anti-pKAPl(Ser824) (Bethyl Laboratories, A300-767A, 1:2000 dilution); mouse monoclonal anti-CHK1 (Santa Cruz Biotechnology, sc-8408, 1:1000 dilution); rabbit monoclonal anti-pCHK1 Ser345 (Cell Signaling, 2348, 1:500 dilution); rabbit polyclonal anti-RPA32 (Bethyl Laboratories, A300-244A, 1:3000 dilution); rabbit polyclonal anti-pRPA32 Ser33 (Bethyl Laboratories, A300-246A, 1:1000 dilution); rabbit polyclonal anti-Lamin B1 (GeneTex, GTX103292, 1:1000 dilution); rabbit polyclonal anti-RNase H1 (GeneTex, GTX117624, 1:500 dilution); mouse monoclonal anti-beta-actin (Abcam, ab8224, 1:5000 dilution); rabbit polyclonal anti-BLM (Bethyl Laboratories, A300-110A, 1:3000 dilution); rabbit polyclonal anti-PML (1:1000 dilution); rabbit polyclonal anti-PARPl (Cell Signaling, 9542, 1:1000 dilution); mouse monoclonal anti-POLD3 (Novus Biologicals, H00010714-M01, 1:500 dilution); rabbit polyclonal anti-ATRX (Bethyl Laboratories, A301-045A-T, 1:1000 dilution); sheep polyclonal anti-TRFl (R&D Systems, AF5300, 1:1000 dilution). Secondary antibodies were HRP-conjugated goat anti-mouse and goat anti-rabbit IgG (Bethyl Laboratories, A90-116P and A120-101P, 1:2000 dilution) and HRP-conjugated donkey anti-sheep IgG (Novus Bio, NBP1-75437, 1:3000 dilution).Signal detection was performed using an ECL detection reagent (GE Healthcare) and a FluorChem HD2 imaging device (Alpha Innotech).

[0278] Fluorescence in situ hybridization (FISH) Metaphase spreads were prepared by incubating cells with 200 ng / ml colchicine (Sigma-Aldrich) for 2 - 6 hours, harvesting mitotic cells by shaking them off, and incubating them in 0.075 M KCl at 37 °C for 10 minutes. Chromosomes were fixed in ice-cold methanol / acetic acid (3:1) and spread onto glass slides. The slides were treated with 20 μg / ml RNase A (Sigma-Aldrich) in 1× PBS at 37 °C for 1 hour, fixed in 4% formaldehyde (Sigma-Aldrich) in 1× PBS for 2 minutes, and then treated with 70 μg / ml pepsin (Sigma-Aldrich) in 2 mM glycine, pH 2 (Sigma-Aldrich) at 37 °C for 5 minutes. The slides were fixed again in 4% formaldehyde in 1× PBS for 2 minutes, then incubated in 70%, 90%, and 100% ethanol for 5 minutes each and air-dried.

[0279] A hybridization solution (Cy3-labeled C-rich telomere PNA probe (TelC-Cy3; 5'-Cy3-OO-CCCTAACCCTAACCCTAA-3' [SEQ ID NO: 47]; Panagene) diluted with 10 mM Tris-HCl pH 7.2, 70% formamide, 0.5% blocking solution (Roche)) was applied onto the slides, followed by incubation at 80°C for 5 minutes once and at room temperature for 2 hours once. The slides were washed twice in 10 mM Tris-HCl pH 7.2, 70% formamide, 0.1% BSA and three times in 0.1 M Tris-HCl pH 7.2, 0.15 M NaCl, 0.08% Tween-20, each for 10 minutes at room temperature. For natural FISH experiments in interphase nuclei, cells grown on coverslips were incubated in CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES pH 6.8, 0.5% Triton-X) on ice for 7 minutes. The cells were then fixed with 4% formaldehyde in 1×PBS for 10 minutes and permeabilized with CSK buffer for 5 minutes at room temperature.

[0280] RNase H treatment was performed by incubating the slides with 30 U of RNase H (Takara) in 1×RNase H buffer or with buffer only at 37°C for 2 hours. Hybridization and washing were performed as described above, except that the TYE563-labeled G-rich telomere LNA probe (TelG-TYE563; 5'-TYE563-T * TAGGGT * TAGGGT *TAGGG-3', the asterisk indicates LNA nucleotides; performed using Exiqon). DNA was counterstained with 100 ng / ml DAPI (Sigma-Aldrich) in 1×PBS, and the slides were mounted with Vectashield (Vectorlabs). Images were acquired with an Olympus IX81 microscope equipped with a Hamamatsu ORCA-ER camera and a 60X / 1.42NA oil-immersion PlanApo N objective, or with a Zeiss Cell Observer equipped with a cooled AxioCam 506m camera and a 63X / 1.4NA oil-immersion DIC M27 PlanApo N objective. Image analysis was performed using ImageJ and Photoshop software.

[0281] Combination of FISH and EdU incorporation / detection Twenty-four hours after siRNA transfection, the cells were incubated in 10 μM RO-3306 (Selleckchem). After 21.5 hours, 10 μM EdU (Thermo Fisher Scientific) was added to the culture medium, followed by incubation for 2.5 hours. The cells were first stained using the TelC-Cy3 probe as in DNA FISH, then washed twice with 1×PBS, and subsequently EdU was detected using the Click-iT EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. DNA was counterstained with 100 ng / ml DAPI in 1×PBS, and the coverslips were mounted on the slides in Vectashield. Image acquisition and analysis were performed as in DNA FISH.

[0282] Indirect immunofluorescence (IF) Cells were grown on coverslips and incubated on ice for 7 minutes in CSK buffer. All subsequent procedures were carried out at room temperature. Cells were fixed with 4% formaldehyde (Sigma-Aldrich) in 1×PBS for 10 minutes, permeabilized with CSK buffer for 5 minutes, and incubated in blocking solution (0.5% BSA, 0.1% Tween-20 in 1×PBS) for 1 hour. Coverslips were incubated in blocking solution containing primary antibody for 1 hour, washed three times with 0.1% Tween-20 in 1×PBS for 10 minutes each, and incubated for 50 minutes with secondary antibody diluted in blocking solution. DNA was counterstained with 100 ng / ml DAPI in 1×PBS. For the combination of IF and DNA FISH, cells were fixed again with 4% formaldehyde in 1×PBS for 10 minutes, washed three times with 1×PBS, incubated in 10 mM Tris-HCl pH 7.2 for 5 minutes, then denatured and hybridized with TelC-Cy3 probe as described above. DNA was counterstained with 100 ng / ml DAPI in 0.1 M Tris-HCl pH 7.2, 0.15 M NaCl, 0.08% Tween-20, and coverslips were mounted on slides in Vectashield. The following primary antibodies were used: rabbit polyclonal anti-pRPA32 pSer33 (Bethyl Laboratories, A300-246A, 1:1000 dilution); rabbit polyclonal anti-53BP1 (Abcam, ab21083, 1:1000 dilution); mouse monoclonal anti-TRF2 (Millipore, 05-521, 1:500 dilution); rabbit polyclonal anti-BLM (Bethyl, A300-110A, 1:5000 dilution); rabbit polyclonal anti-PML (kind gift from M. Carmo-Fonseca, iMM, Lisbon, Portugal, 1:500 dilution); mouse monoclonal anti-RAD51 (Abcam, ab213, 1:100 dilution); mouse monoclonal anti-POLD3 (Novus Biologicals, H00010714-M01, 1:100 dilution); rabbit polyclonal anti-RAP1 (Bethyl, A300-306A, 1:500 dilution).The secondary antibodies were Alexa Fluor 568-conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific, A10042) and Alexa Fluor 488-conjugated donkey anti-mouse IgG (Thermo Fisher Scientific, A21202). Image acquisition and analysis were performed as in DNA FISH.

[0283] Genomic DNA analysis Genomic DNA was isolated by phenol:chloroform extraction and treatment with 40 μg / ml RNase A, followed by ethanol precipitation. The reconstructed DNA was digested with HinfI and RsaI (New England Biolabs) and purified again by phenol:chloroform extraction. For TRF analysis, 2 μg of digested DNA was separated on a 0.6% agarose gel, which was vacuum-dried at 50 °C for 50 minutes. The gel was labeled at the 5'-end with T4 polynucleotide kinase (New England Biolabs) and a telomere oligonucleotide probe (5'-(TTAGGG)5-3' or 5'-(CCCTAA)5-3') labeled with [α- 32 P]ATP and hybridized overnight at 50 °C. Post-hybridization washes were 2×SSC, 0.2% SDS for 20 minutes twice, and 0.5×SSC, 0.2% SDS, 50 °C for 30 minutes once.

[0284] After acquisition of the radioactive signal, the gel was incubated in denaturing solution (1.5 M NaCl, 0.5 M NaOH) at room temperature for 20 minutes, then hybridized overnight at 55 °C with a double-stranded telomere probe (Telo2 probe), and Klenow fragment (New England Biolabs) and [α- 32It was radiolabeled using [P]dCTP. The washes after hybridization were twice for 20 minutes in 2×SSC, 0.2% SDS and once for 30 minutes at 55°C in 0.2×SSC, 0.2% SDS. For dot blot hybridization, 1 μg of genomic DNA was digested as described above, denatured at 98°C for 5 minutes or left untreated, and dot blotted onto a nylon membrane. The membrane was first hybridized with the telomere oligonucleotide probe as described above. After acquisition of the radioactive signal, the gel was incubated in the denaturing solution as described above and then re-hybridized overnight at 50°C with the radiolabeled Alu repeat oligonucleotide (5'-GTGATCCGCCCGCCTCGGCCTCCCAAAGTG-3' [SEQ ID NO: 48]). The washes after hybridization were twice for 20 minutes in 2×SSC, 0.2% SDS and once for 30 minutes at 50°C in 0.5×SSC, 0.2% SDS. For the C circle assay, 150 - 500 ng of digested DNA was incubated with 7.5 U of phi29 DNA polymerase (New England Biolabs) in the presence of dATP, dTTP and dGTP (1 mM each) at 30°C for 8 hours, followed by heat inactivation at 65°C for 20 minutes. The amplification products were dot blotted onto a nylon membrane (GE Healthcare) and hybridized with the radiolabeled Telo2 probe as described above. For two-dimensional gel electrophoresis, 10 μg of digested DNA was separated on a 0.6% agarose gel (pulsed field certified agarose; Biorad) at 30 V for 7 hours, followed by excision of the lane and separation of the DNA in the second direction on a 1.1% agarose gel (UltraPure agarose; Life Technologies) at 100 V for 3 hours. The DNA was then transferred onto a nylon membrane, denatured and hybridized with the radiolabeled Telo2 probe as described above. The radioactive signal was detected using a Typhoon FLA9000 imager (GE Healthcare) and quantified using ImageJ software.

[0285] Northern blotting Total RNA was isolated using TRIzol reagent (Invitrogen) and treated three times with DNase I (New England Biolabs). 15 μg of RNA was separated on a 1.2% agarose gel containing 0.7% formaldehyde. The RNA was then transferred onto a nylon membrane and hybridized to the radiolabeled Telo2 probe as described above. Loading was controlled using tRNA stained with ethidium bromide (Sigma-Aldrich). Radioactive signals were detected using a Typhoon FLA9000 imager (GE Healthcare) and quantified using ImageJ software.

[0286] Chromatin immunoprecipitation (ChIP) 10 6 10 individual cells were collected by scraping and resuspended in 1 ml of 1% formaldehyde at room temperature for 15 minutes. After quenching with 125 mM glycine, the cells were washed three times in 1× PBS by centrifugation at 800 g for 5 minutes. The cell pellet was resuspended in 500 μl of lysis buffer (1% SDS, 50 mM Tris-HCl pH 8, 10 mM EDTA pH 8) supplemented with a complete protease inhibitor cocktail (Roche) and sonicated twice at 4°C using a Bioruptor device (Diagenode) (settings: 30 seconds "on" / 30 seconds "off"; output: "high"; time: 15 minutes).

[0287] Cell debris was pelleted by centrifugation at 1600 g for 10 minutes at 4°C, and 100 μl of the supernatant was mixed with 1.1 ml of IP buffer (1% Triton X-100, 20 mM Tris-HCl pH 8, 2 mM EDTA pH 8, 150 mM NaCl). The diluted extract was incubated with 50 μl of protein A / G-Sepharose beads (GE Healthcare) blocked with sheared Escherichia coli genomic DNA and BSA on a rotating wheel at 4°C for 30 minutes, followed by pre-clearing by centrifugation at 800 g for 5 minutes at 4°C. The pre-cleared extract was incubated with 1 μg of anti-FANCM mouse monoclonal antibody (CE56.172 ) together with the rotated wheel at 4 °C for 4 hours and incubated. The immune complex was isolated by incubating overnight at 4 °C with the blocked protein A / G beads on a rotated wheel. The beads were washed 4 times with wash buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8, 150 mM NaCl, 20 mM Tris-HCl pH 8) and once with wash buffer 2 (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8, 500 mM NaCl, 20 mM Tris-HCl pH 8) by centrifugation at 800 g for 5 minutes at 4 °C. The beads were then incubated in 100 μl of elution buffer (1% Triton X-100, 20 mM Tris-HCl pH 8, 2 mM EDTA pH 8, 150 mM NaCl) containing 40 μg / ml RNase A at 37 °C for 1 hour, followed by incubation overnight at 65 °C to reverse crosslinking. DNA was purified using the Wizard SV Gel and PCR Clean-Up Kit (Promega), dot blotted onto a nylon membrane, and hybridized overnight to a radiolabeled Telo2 probe as in the case of TRF analysis. After signal detection, the membrane was stripped and hybridized again overnight to the radiolabeled Alu repetitive oligonucleotide as described above. The radioactive signal was detected using a Typhoon FLA9000 imager (GE Healthcare) and quantified using ImageJ software.

[0288] DNA:RNA Immunoprecipitation (DRIP) Cells were harvested by scraping and dissolved in 1 ml of RA1 buffer (Macherey-Nagel) containing 1% v / v β-mercaptoethanol and 100 mM NaCl. Nucleic acids were extracted with phenol / chloroform / isoamyl alcohol (25:24:1 saturated with 10 mM Tris-Cl pH 7.0, 1 mM EDTA), precipitated with isopropanol, and then centrifuged at 15,000 g for 10 minutes at 4°C. The pellet was washed in 70% ethanol, resuspended in 200 μl of Tris-EDTA, 100 mM NaCl, and sonicated using a Bioruptor device (Diagenode) at 4°C (settings: 30 seconds “on” / 30 seconds “off”; output: “high”; time: 5 minutes). 5 μg of nucleic acids were incubated with 1 μg of S9.6 antibody (kind gift from B. Luke, IMB, Mainz, Germany) in IP buffer (0.1% SDS, 1% Triton X-100, 10 mM HEPES pH 7.2, 0.1% sodium deoxycholate, 275 mM NaCl) on a rotating wheel at 4°C for 5 hours. For RNase H control experiments, nucleic acids were incubated with 60 U of RNase H in 1× RNase H buffer or with buffer alone for 3 hours at 37°C, and then incubated with S9.6 antibody. Immune complexes were isolated by incubation with protein G Sepharose beads (GE Healthcare) blocked with sheared E. coli DNA and BSA. The beads were washed 4 times by centrifugation at 800 g in IP buffer and incubated in elution buffer (50 mM Tris-Cl pH 8, 10 mM EDTA, 0.5% SDS) containing 10 μg / ml proteinase K (Sigma-Aldrich) and 40 μg / ml RNase A for 30 minutes at 50°C. The beads were centrifuged as above and the supernatant was recovered. Isopropanol-precipitated DNA was dot-blotted onto a nylon membrane and hybridized to a radiolabeled 5'-(TTAGGG)5-3' oligonucleotide as in the case of TRF analysis. After signal detection, the membrane was stripped and re-hybridized to a radiolabeled Alu repeat oligonucleotide as in the case of ChIP analysis.The radioactive signal was detected using a Typhoon FLA9000 imager (GE Healthcare) and quantified using ImageJ software.

[0289] In vitro R-loop resolution assay The Flag-FANCM-8HIS:FAAP24 complex was purified using a baculovirus expression system in Sf9 cells. Cells were pelleted at 500×g and resuspended in 0.5 M NaCl, 0.02 M triethanolamine pH 7.5, 1 mM DTT, 10% glycerol, and mammalian protease inhibitors (Sigma-Aldrich) on ice and sonicated in 5×10 s bursts on ice. The clarified lysate was incubated with equilibrated Flag M2 resin (Sigma-Aldrich) for 1 h at 4°C. The Flag resin was subjected to 5 batch washes and eluted with 100 μg / ml Flag peptide. The eluate containing pooled FANCM-FAAP24 was diluted to a final concentration of 100 mM NaCl, 20 mM TEA pH 7.5, 10% glycerol, 1 mM DTT (buffer B) and bound to 400 μl of ssDNA affinity resin (Sigma-Aldrich). The resin was washed with 10 CV of buffer B. The FANCM-FAAP24 complex was eluted with buffer B containing 0.5 M NaCl. 2 μg of pcDNA6-Telo or pcDNA6-TeloR plasmid containing an approximately 1 kb fragment of the human telomere repeat sequence cloned downstream of the T7 promoter was used in vitro with T7 polymerase (New England Biolabs) and CTP, GTP, ATP (2.25 mM each), 825 nM of [α 32Transcription was carried out in the presence of [[P]]UTP (3000 Ci / mmol; Perkin Elmer). The reaction was stopped by heating to 65 °C for 20 minutes followed by treatment with RNase A (Epicentre) in 330 mM NaCl. Plasmids containing R-loops were purified by two phenol:chloroform extractions. Unincorporated nucleotides were removed by passing the nucleic acid through an S-400 column (GE Healthcare) twice. The R-loop unwind reaction (10 μl final volume) contained 1 nM R-loop plasmid, 1 mM ATP, 2.5 nM FANCM-FAAP24 in R-loop buffer (6.6 mM Tris pH 7.5, 3% glycerol, 0.1 mM EDTA, 1 mM DTT, 0.5 mM MgCl2). The reaction was run at 37 °C for 10 minutes and then stopped by adding 2 μl of stop buffer (10 mg / ml proteinase K (New England Biolabs), 1% SDS) and incubated at 37 °C for 15 minutes. Samples were run on a 0.8% agarose TAE gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) at 100 V for 60 - 90 minutes, followed by drying the gel and performing autoradiography.

[0290] Statistical analysis For direct comparison of two groups, we employed either a paired two-sided Student's t-test using Microsoft Excel or a non-parametric two-sided Mann-Whitney U-test using GraphPad Prism. For comparison of two or more factors and their interactions within each group, we used two-way analysis of variance (ANOVA) followed by Tukey's HSD for pairwise comparisons. Analyses were performed using the aov and Tukey HSD functions of R version 3.3.2. The significance level was from the P-value adjusted by Tukey's HSD. The P-values are * P < 0.05, ** P < 0.005, *** P < 0.001, **** shown as P < 0.0001.

[0291] Results FANCM supports the viability of ALT cells We used short interfering RNAs (siRNAs) against two sequences (siFa and siFb) from the FANCM coding region to deplete FANCM in several ALT (U20S, Hu09, Saos2, and WI-38VA13) and telomerase-positive (Tel+; HeLa, HOS, HT1080, and SKNAS) cells. A non-targeted siRNA was used as a control (siCt). Two days after transfection, near-complete depletion of FANCM protein was detected by Western blot in cells transfected with Fa and Fb, except for SKNAS cells transfected with siFb, in which ~10% of the protein remained (Figure 15A). Fluorescence-activated cell sorting (FACS) of ethanol-fixed propidium iodide (PI)-stained cells revealed that FANCM-depleted ALT cells accumulated in G2 / M phase, except for Tel+ (Figures 15B and 15C). The clonogenic ability of ALT cells was mostly abrogated by transfection with FANCM siRNA, whereas one of the Tel+ cells remained essentially unaffected (Figures 15D and 15E). For the colony formation assay, cells were transfected only once with siRNA and then plated, and colonies were counted at least 8 days later. Thus, the antiproliferative effect exerted by FANCM depletion in ALT cells is rapid and irreversible. Analysis of cell growth with long-term siRNA treatment showed that FANCM-depleted U20S cells were rapidly excluded from the population, whereas HeLa cells continued to grow at a lower rate (Figure 15F). Finally, FANCM-depleted U20S cells, but not HeLa cells, became permeable to PI as early as 3 days after siRNA treatment. This indicates cell death. No major changes in PARP1 cleavage were detected in the same cells. Thus, FANCM depletion causes abnormal accumulation of ALT cells in G2 / M phase, followed by PARPl-independent cell death.

[0292] Our data show that in ALT cells, FANCM is essential for cell cycle progression and viability, which is different from what has been observed so far. 17 Less efficient protein depletion or retained expression of important FANCM splice variants obtained by Pan and co-workers (including cell type-specific ones that may not have been reported in public databases in some cases) may have left sufficient residual amounts of FANCM protein to maintain cell proliferation. Survival assays of less sensitive cells may also have underestimated the effect of FANCM depletion in previous studies.

[0293] Telomere replication stress sensitizes ALT cells to FANCM depletion Several features of ALT cells, namely, the absence of telomerase activity, very long telomeres, ATRX inactivation, and persistent telomere replication stress, can explain their sensitivity to FANCM depletion. We generated supertelomerase cells by ectopically expressing the catalytic (hTERT) and RNA (hTR) subunits of telomerase in U20S and HeLa cells. 37 Overexpression of hTERT and hTR was confirmed by quantitative RT-PCR. As expected, HeLa supertelomerase cells had longer telomeres than HeLa control cells, and U20S supertelomerase cells had reduced TFE frequency, but the incidence of fragile telomeres (TF) with insufficient replication remained unchanged. 11、37 FANCM depletion inhibited cell proliferation and caused G2 / M accumulation in U20S supertelomerase cells but not in HeLa supertelomerase cells (Figures 15B - 15E). Furthermore, the telomerase inhibitor BIBR1532 38HeLa cells treated with [the method] did not accumulate at G2 / M when FANCM was depleted. The inventors then simultaneously depleted FANCM and ATRX in HeLa cells, and no accumulation of G2 / M cells was observed. Therefore, the presence of extremely long telomeres or the absence of active telomerase or ATRX alone does not account for the sensitivity of ALT cells to FANCM depletion.

[0294] The inventors then overexpressed the shelterin factor TRF1 by retroviral infection in U20S cells, because this treatment halves the incidence of FT. 39 . FANCM depletion in cells overexpressing TRF1 still resulted in G2 / M accumulation, but was significantly less than the accumulation in cells infected with an empty vector (ev) retrovirus (Figures 15G and 15H). However, when FANCM was simultaneously depleted, HeLa cells were depleted of TRF1 using siRNA, which has been shown to induce telomere fragility 39 , and did not accumulate at G2 / M. Similarly, HeLa cells depleted of FANCM did not show an altered cell cycle distribution when treated with the replication stress inducer hydroxyurea (HU) followed by release of the block. Therefore, telomere replication stress contributes to the sensitivity of ALT cells to FANCM depletion, but nevertheless, telomeres or generalized replication stress alone are insufficient to render non-ALT cells sensitive to FANCM depletion.

[0295] FANCM suppresses telomere replication stress in ALT cells To test the involvement of FANCM in telomere stability, the inventors performed indirect immunofluorescence (IF) using a combination of an antibody against TRF2 and an antibody against RPA32 phosphorylated at serine 33 (pS33) or p53 binding protein 1 (53BP1). RPA32 is phosphorylated at serine 33 by ATR during S phase when replication forks stall. 40;53BP1 forms foci in dysfunctional telomeres that have activated either ATR or another DNA damage signaling kinase, ataxia telangiectasia mutated (ATM), or both 41、42 . Within 48 hours of transfection, pS33 and 53BP1 accumulated at telomeres in ALT cells depleted of FANCM (Figure 16A) and formed so-called telomere dysfunction-induced foci (TIF) 41 . FANCM depletion did not induce TIF formation in Tel+ cells (Figure 16A). Accumulation of pSer33 and 53BP1 outside telomeres was minimal in all cell lines depleted of FANCM (Figure 16A). Since the protein associates with telomeric DNA in chromatin immunoprecipitation (ChIP) experiments, the FANCM-mediated suppression of telomere instability is likely to be direct (Figure 16B and Figure 16C). FANCM also immunoprecipitated with abundant, genome-wide Alu repetitive DNA (Figure 16B and Figure 16C), indicating that the protein does not associate exclusively with telomeres. This is consistent with the reported localization of FANCM to the cellular chromatin fraction 43 .

[0296] Western blot analysis confirmed that FANCM depletion caused pS33 accumulation and elucidated the phosphorylation of another ATR target checkpoint kinase 1 (CHK1) in U20S but not in HeLa cells (Figure 16D). The ATM target KRAB domain-associated protein 1 (KAP1) was not phosphorylated in any of the cell lines tested (Figure 16D). Furthermore, pS33 accumulation was attenuated in FANCM-depleted U20S cells overexpressing TRF1 (Figure 15G), but telomerase inhibition, ATRX or TRF1 depletion, and HU treatment did not promote pS33 accumulation in FANCM-depleted HeLa cells. Indeed, pS33 could not accumulate efficiently in FANCM-depleted HeLa cells treated with HU, which is consistent with the role of FANCM in supporting the activation of the normal ATR-dependent intra-S phase checkpoint 32The inventors propose that FANCM deficiency in ALT cells activates a specific ATR-dependent signaling cascade, which is not completely identical to that caused by generalized replication stress and is at least partially due to excessive telomere replication stress. Such an ATR response may cause the observed G2 / M arrest and cell death.

[0297] FANCM suppresses the characteristics of ALT In the inventors' IF images, the TRF2 foci in FANCM-depleted ALT cells are both larger and brighter than those in control cells. To confirm that this is not simply due to increased TRF2 at telomeres, the inventors subjected U20S interphase cells transfected with siRNA to DNA fluorescence in situ hybridization (FISH) using a telomere probe and measured the number and area of telomere foci. The inventors controlled for possible secondary effects related to the cell cycle stage by arresting cells transfected with siCt at the G2 / M boundary with RO-3306, a cyclin-dependent kinase 1 (CDK1) inhibitor 44 (FIGS. 17A and 17B). The overall number of telomere foci decreased when FANCM was depleted (FIGS. 17C and 17D), but their area distribution became broader, the frequency of very small foci increased slightly (S in FIGS. 17C and 17E), and the frequency of very large foci increased substantially (L in FIGS. 17C and 17E). Also, the number of telomere foci in cells treated with RO-3306 was also less than that in control cells (FIG. 17D), which may be due to clustering of ALT telomeres in G2 19、45However, the increases in very small and very large lesions were more prominent in FANCM depletion than in RO-3306 treatment (FIGS. 17C and 17E). Approximately 60% of the cells depleted of FANCM had at least 5 large lesions, whereas for the untreated or RO-3306-treated cells transfected with siCt, they were approximately 10% and 15%, respectively (FIG. 17F).

[0298] The inventors then analyzed the localization of PML, RAD51, and POLD3 at telomeres by combining IF for PML and RAD51 with telomere FISH and, in the case of POLD3 and RAP1, by double IF. The inventors observed an increase in the telomeric localization of all three factors in FANCM-depleted U20S cells, but no obvious increase in the total protein levels of PML, POLD3, and RAD51 (FIG. 17A; FIG. 18). RO-3306 treatment did not substantially affect the number of PML and RAD51 foci at telomeres but increased one of the POLD3 foci at telomeres, but was not as significant as FANCM depletion (FIG. 18). Furthermore, the inventors incubated the cells treated with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) as described above for 2.5 hours and performed telomere FISH combined with EdU detection to visualize newly synthesized telomeric DNA. To exclude S-phase cells, the inventors scored only the cells showing punctuate EdU staining and having 25 or fewer EdU foci. FANCM depletion increased the incidence of EdU foci at telomeres, as did RO-3306 treatment, but to a lower extent (FIG. 18).

[0299] The inventors have concluded that FANCM depletion worsens ALT activity, as shown by robust telomere clustering within large APBs containing PML, RAD51, and POLD3, and increased telomere DNA synthesis outside of S phase. FANCM depletion also generates, in some cases, short telomere species that represent ECTR (see below). G2 / M arrest alone cannot account for the abnormally elevated ALT features observed in FANCM-depleted cells.

[0300] FANCM suppresses telomeric ssDNA and ECTR in ALT cells The inventors performed telomere restriction fragment (TRF) analysis in gels of genomic DNA from ALT (U20S and WI-38VA13) and Tel+ (HOS and HeLa) cells harvested 48 hours after siRNA transfection. Blots were hybridized with oligonucleotides of telomeres with either 5'-TTAGGG-3' or 5'-CCCTAA-3' repeats. When hybridization was performed under native (non-denaturing) conditions, the inventors observed an increase in C-rich telomeric ssDNA of very diverse lengths in FANCM-depleted ALT cells (Figure 19A, upper panel). Conversely, a decrease in G-rich ssDNA was observed for most counterparts of telomeres. This could be due to shortening of the G-overhang (Figure 19A, lower panel). For both probes, the fraction of the signal was in the gel well and corresponded to exposed ssDNA from molecules with potentially important secondary structures (Figure 19A). The inventors did not observe alterations in telomeric ssDNA in Tel+ cells (Figure 19A). From hybridization of the same gels under denaturing conditions using a long telomere probe (Telo2 probe), it was elucidated that there were no obvious alterations in telomere length in FANCM-depleted cells (Figure 19A).

[0301] The inventors then dot-blotted genomic DNA from the above-described cells, hybridized it to telomere oligonucleotides under native conditions, subsequently denatured it, and hybridized it to Alu repeats as a control for the total DNA loaded. This experiment confirmed that ALT cells depleted of FANCM contain more telomeric C-rich ssDNA than cells transfected with siCt (Figure 19B). As previously reported 11 , depletion of RNase H1 in U20S cells also increased telomeric C-rich ssDNA, although at a lower level than FANCM depletion (Figure 19B). Using denatured DNA dot-blot hybridization for U20S, HOS, and HeLa cells, no significant difference was detected in total telomeric DNA (Figure 19B). An increase in total C-rich telomeric DNA was observed in WI-38VA13 cells depleted of FANCM (Figure 19B).

[0302] The inventors then performed a phi-29-mediated C-circle assay using DNA from ALT and Tel+ cells 46 and found a surprising increase in C-circles in ALT cells depleted of FANCM (Figure 19C). The accumulation of ECTR likely corresponds, at least in part but not exclusively, to C-circles and was also detected in U20S cells depleted of FANCM using two-dimensional gel electrophoresis (Figure 19D). Metaphase chromosome FISH of U20S cells depleted of FANCM showed abundant extrachromosomal telomeric signals, probably corresponding to ECTR, and DNA threads extending from the ends of a single chromosome or bridging the ends of two independent chromosomes. C-rich ssDNA-containing ECTR and DNA threads could account for the well-retained DNA molecules observed in our TRF analysis and the increased telomeric ssDNA observed in our dot-blot analysis (Figures 19A and 19B). The inventors did not observe an increase in the incidence of TFE in U20S cells depleted of FANCM.

[0303] FANCM regulates BLM in ALT cells FANCM and BLM have been reported to cooperate in maintaining ALT telomeres 17 . We depleted FANCM in U20S and HeLa cells and performed indirect IF using anti-BLM and anti-TRF2 antibodies. Since FANCM is required for BLM recruitment to stalled replication-induced damage sites 34 , we included cells treated with the topoisomerase I inhibitor camptothecin (CPT). CPT induced robust formation of nuclear (non-telomeric) BLM foci in U20S and HeLa cells transfected with siCt but not in cells transfected with siFa. On the other hand, BLM TIFs were already abundant in U20S transfected with siCt and were only very rarely observed in HeLa cells transfected with siCt. In U20S cells, FANCM depletion increased the number of BLM TIFs. In all samples, CPT treatment had a minor effect on TIF frequency. The relocalization of BLM to the nucleus occurred without major changes in total protein levels. Thus, we conclude that FANCM depletion causes BLM accumulation at ALT telomeres 17 , but confirm that it prevents this at non-telomeric sites of damage in both ALT and Tel+ cells 34 . The accumulation of BLM at telomeres upon FANCM depletion may involve the reported interactions with TRF1 and TRF2 47 .

[0304] The inventors then simultaneously depleted FANCM and BLM in U20S cells (Figure 20A). BLM depletion alone did not alter cell cycle distribution or the number of colonies formed, but decreased the growth rate and, to a very minimal extent, increased the proportion of PI-permeable cells (Figures 20B–20D). Unexpectedly, simultaneous depletion of FANCM and BLM resulted in an abnormal cell cycle distribution due to FANCM depletion and a partial rescue of cell growth and viability (Figures 20B–20D). Furthermore, BLM depletion halved the incidence of pS33 TIF in cells depleted of FANCM (Figure 20E). These results establish that BLM depletion alleviates the deleterious effects exerted by FANCM deficiency in ALT cells.

[0305] FANCM suppresses TERRA and telR loops in ALT cells To test whether FANCM suppresses telomere replication stress in ALT cells by regulating TERRA and / or telR loops, the inventors first performed TERRA Northern blots and found that the levels of this IncRNA were 3.5- and 2.5-fold higher, respectively, in cells transfected with siFa and siFb than in those transfected with siCt. TERRA species up to approximately 2 kb in length were most affected (Figure 21A). The inventors then performed in vitro R-loop resolution assays using plasmids containing telR loops generated by T7 transcription of approximately 1 kb of the telomere tract 11 . The inventors used two plasmids with different insert orientations for producing transcripts containing TERRA-like G-rich RNA repeats or transcripts complementary C-rich transcripts (Figure 21B). As expected 11, G-rich transcripts were produced with lower efficiency than C-rich ones (Figure 21B). TelR-loop plasmids were incubated with or without ATP, together with recombinant FANCM in a heterodimer with its stabilizing partner FAAP24, and then digested with agarose gel. FANCM promoted the complete release of both G-rich and C-rich transcripts from the R-loop - plasmid, without RNA degradation and, furthermore, in an ATP-dependent manner (Figure 21B). Thus, FANCM efficiently unwinds the RNA portion of the telR loop in vitro. To examine telR loops in FANCM-depleted U20S cells, the inventors performed DNA:RNA immunoprecipitation (DRIP) using the monoclonal antibody S9.6 48 . Dot blot hybridization detected telomeric DNA in the immunoprecipitated material from all samples, with an approximately 3-fold increase in the siFa and siFb samples (Figure 21C). Treatment of the nucleic acids with recombinant RNase H prior to antibody incubation abolished most of the hybridization signal, confirming that they originated from DNA:RNA hybrids (Figure 21C). The inventors also performed native DNA FISH using a G-rich telomeric probe on interphase nuclei treated or not treated with RNase H 11。The punctate staining corresponding to C-rich telomeric DNA was already visible by eye in cells transfected with untreated siCt, and its intensity was higher in RNase H-treated cells. This may be due to the degradation of TERRA transcripts within the telR loop and the resulting increased binding sites to its probe (Figures 21D and 21E). In cells transfected with untreated siFa, the signal of C-rich ssDNA was more prominent than in control cells and was further increased by RNase H treatment (Figures 21D and 21E). The total number of foci per cell was higher in cells depleted of FANCM than in the total number of cells, but was not affected by RNase H treatment (Figures 21D and 21E). The inventors have concluded that FANCM suppresses TERRA and telR loops containing TERRA in ALT cells. Considering the ability of FANCM to disassemble telR loops in vitro (Figure 21B) and the localization of FANCM to telomeres (Figures 16C and 16D), the inventors propose that FANCM directly disassembles telR loops on telomeric chromatin. The more prominent C-rich ssDNA signal present in cells depleted of FANCM that have not already been treated with RNase H (Figures 21D and 21E) may be due to gaps in DNA replication or the degradation by cells of the RNA portion of the telR loop. The inventors have also mentioned the RNA:DNA hybrid structure of telomeres that occurs upon depletion of FANCM as a telR loop, but their experiments do not distinguish between conventional R loops, triple-stranded nucleic acids containing ssDNA replaced with an RNA:DNA hybrid, and ds RNA:DNA hybrids lacking displaced loops.

[0306] FANCM avoids telomeric replication stress induced by telR loops The inventors hypothesized that FANCM suppresses telomere replication stress by resolving the telR loop. The inventors depleted FANCM in U20S cells infected with retroviruses expressing siRNA-resistant, V5 epitope-tagged FANCM variant (V5-FANCM WT), or an ATPase / translocase-inactive counterpart (V5-FANCM K117R 36 ) that is unable to resolve R loops. Both variants were expressed at higher levels than endogenous FANCM (Figure 22A). When the specificity of the inventors' siRNA was confirmed, in cells transfected with siFa, most of the V5-FANCM WT avoided G2 / M arrest and the accumulation of pS33 TIF and APB (Figures 22B and 22C). In contrast, the cell cycle distribution and the incidence of pS33 TIF and APB were similar between V5-FANCM K117R and control cells transfected with siFa (Figures 22B and 22C). The inventors then exploited the ability of overexpressed RNase H1 to suppress telR loops in cells 11、39、49The inventors depleted FANCM alone or in combination with BLM in U20S cells infected with a retrovirus driving overexpression of RNase H1 with a MYC epitope tag (MYC-RH1 WT), or a catalytically inactive counterpart (MYC-RH1 D145A), or infected with an empty vector control retrovirus (Figure 22D). MYC-RH1 WT further enhanced the rescue of the G2 / M arrest defect in cells co-depleted of FANCM and BLM (Figure 22E). The decrease in the frequency of pS33 TIFs induced by FANCM depletion in cells expressing MYC-RH1 WT was measured compared to empty vector-infected cells (Figure 22F). In cells overexpressing MYC-RH1 WT co-depleted of FANCM and BLM, pS33 TIFs recovered to levels similar to those of empty vector control cells (Figure 22F). In all experiments, MYC-RH1 D145A was unable to function as its catalytically active counterpart (Figure 22E and Figure 22F). These results indicate that telomeric replication stress that occurs upon FANCM depletion is suppressed by FANCM enzyme activity and is due to undissolved telR loops and uncontrolled BLM.

[0307] Here, the inventors demonstrate that in the absence of FANCM, ALT cells experience severe telomeric replication stress, activating DNA damage signaling mediated by ATR, which may be responsible for the observed G2 / M arrest and cell death. 50 The fact that overexpression of TRF1 makes ALT cells less sensitive to FANCM depletion (Figures 15G and 15H) and the lack of accumulation of non-telomeric pSer33 and 53BP1 foci in FANCM-depleted ALT cells (Figure 16A) demonstrate the centrality of the signal released from damaged telomeres in promoting G2 / M arrest. However, as suggested by its physical interaction with Alu repetitive DNA, FANCM may also have an essential function outside of telomeres in ALT cells.

[0308] Furthermore, the inventors have also confirmed that since the proliferation and viability of Tel+ cells are not significantly affected by FANCM depletion, FANCM is not essential in all cells. Consistent with this, adult humans with loss-of-function biallelic FANCM mutations have been reported. 51、52 Furthermore, Tel+ human colorectal cancer cells, mouse embryonic fibroblasts, and chicken lymphoblasts with FANCM knocked out developed and proliferated normally as long as they were not attacked by DNA damage. 53~55 As such, FANCM is an attractive target in ALT cancer therapy. Although FANCM deficiency is indeed associated with a higher risk of breast and liver cancers, 51、55 the irreversible lesions that occurred rapidly due to FANCM depletion in ALT cells indicate that short-term inhibition of FANCM can efficiently eradicate ALT tumors without causing secondary effects. From another perspective, therapies based on the co-inhibition of FANCM and BLM proposed so far should be avoided. 17 .

[0309] The inventors also show that FANCM suppresses ALT-related features such as telomere clustering in APBs containing PML, POLD3, and RAD51, and the production of ECTRs, including C-circles, and possibly other forms in some cases (Figure 18A; Figures 19C and 19D). When the inventors depleted FANCM in Tel+ cells, the same features were not obvious, so FANCM deficiency alone is insufficient to initiate ALT anew. Consistent with FANCM restricting ALT, its depletion increased telomeric DNA synthesis outside of S phase (Figures 18A and 18B) and led to the appearance of DNA threads that may represent intermediates of intermolecular recombination events. The inventors propose that FANCM suppresses POLD3-dependent telomeric BIR in G2 and possibly MiDAS. 24~26 Consistent with this, deletion of the yeast helicase Mph1, a Saccharomyces cerevisiae FANCM ortholog, 56 directs the repair of DSBs induced by the HO endonuclease to BIR.57 Furthermore, overexpression of Mph1 inhibited BIR at chromosomal DSBs, but 58 did not prevent the survival rebellion of telomerase-deficient type II ALT yeast that maintains its telomeres via BIR. 58~62 Finally, Mph1 localizes to short telomeres in an R-loop-dependent manner. 63 The FANCM protein appears to play distinct specific roles at ALT and uncapped telomeres, different from those it plays at chromosomal damage sites and those mediated by R-loops. The telR loop can directly promote the recruitment and / or stabilization of FANCM at telomeres in human ALT cells, and in turn activate and thus regulate POLD3-dependent telomeric BIR. The relevance of RAD51 accumulation at APBs in the case of FANCM depletion remains unclear (Figure 18A). RAD51 can mediate the clustering of telomeres observed in cells depleted of FANCM or other molecular events not investigated by the inventors, such as sister telomere exchange.

[0310] Despite increasing ALT activity, FANCM depletion did not induce a large gain in total (C-rich in addition to G-rich) telomeric DNA (Figure 19B). In the inventors' experiments, the amount of newly produced telomeric DNA may have been below the detection limit. In addition, de novo synthesis of telomeric DNA in FANCM-depleted cells may be offset by incomplete semi-conservative replication of telomeric DNA in S phase, 17 and furthermore, by the exclusion of ECTR from the cells. The exact mechanism by which circular ECTR is generated remains unclear, but such a mechanism is associated with features that increase in FANCM-depleted ALT cells, such as replication stress, activated ATR, C-rich telomeric ssDNA, and telR loops. 11、64~66 One observation from the inventors' study is that FANCM depletion does not alter the TFE frequency. This suggests that the observed ECTR is not obtained by excising the entire telomeric pathway.

[0311] The replication stress that occurs at ALT telomeres upon FANCM depletion arises mainly from two sources, namely deregulated BLM and telR loops. Considering the increased BLM recruitment to ALT telomeres when FANCM is depleted, FANCM can directly replace BLM from telomeres. Alternatively, FANCM can suppress the triggers that cause BLM recruitment, such as in arrested telomeric replication forks or R- and D-loop intermediates. BLM activity promotes ALT by degrading recombination intermediates formed during strand invasion associated with BIR, which is sometimes part of the BLM-TOP3A-RMI (BTR) resolvase complex, thereby supporting telomere recombination and BIR-based telomeric DNA synthesis. 15、67、68 . Consistently, FANCM depletion increases telomere synthesis outside of S-phase (Figure 18A). Furthermore, because BLM mediates long-range resection of DNA ends 69、70 , overly active BLM is likely to directly contribute to the production of telomeric ssDNA when FANCM is depleted. This is consistent with the low levels of pS33 TIF detected in cells doubly depleted for FANCM and BLM (Figure 22F). Since FANCD2 has also been shown to suppress BLM toxicity in ALT cells 68 , it can be hypothesized that this may be a general role for the FA pathway. Nevertheless, the ATPase / helicase activity of FANCM is not important for the monoubiquitination of FANCD2 31 , and overexpression of a variant of FANCM that cannot recruit the FA complex to chromatin in U20S cells did not suppress any of the ALT-related features (see above). Therefore, it seems unlikely that the entire FA complex functions to maintain ALT.

[0312] Similar to the case of the nature of telR loops in FANCM-depleted cells, our data suggest that they accumulate because they are not properly resolved by the ATPase / translocase activity of FANCM at telomeric chromatin (Figures 21B - 21E). Since telomeric DNA is a difficult substrate for RNA polymerase, telR loops can be generated by co-transcription 11、49 。The increased short TERRA species observed in FANCM-depleted ALT cells (Figure 21A) may actually be due to premature termination of telomeric transcription resulting from inappropriate telR loop resolution. The accumulation of pS33 also indicates that FANCM is most likely to resolve telR loops during S phase. Thus, inappropriate telR loop resolution can at least partially explain the decrease in the efficiency of replication fork progression through the telomere pathway in FANCM-depleted cells 17 。Furthermore, the accumulation of C-rich telomeric ssDNA and C circles, which are features of the replication stress associated with some FANCM depletion (Figures 19A, 19B, and 19E), is more apparent in U20S cells than in other ALT cells. This can be explained by the fact that TERRA and telR loops are particularly abundant in U20S cells 11 。

[0313] Due to the rapid and dramatic response of ALT cells to FANCM inactivation (Figures 15B - 15F), our study had to be performed using siRNA rather than CRISPR / Cas9-based gene inactivation, which confounded the analysis of genetic interactions. Nevertheless, the synergistic effect of BLM depletion and RNase H1 overexpression in suppressing replication stress in FANCM-depleted cells suggests that BLM activity and telR loops may be functionally related. Although BLM suppresses R loops on a genomic scale 71, BLM can specifically promote telR loop formation in ALT cells, for example, by generating C-rich ssDNA followed by TERRA annealing. Conversely, telR loops can recruit BLM to telomeres by stalling the telomeric replication fork or by forming a structure that mimics a D loop.

[0314] Future studies may utilize conditional knockout cells for FANCM and BLM, which are expected to refine this interesting cellular scenario and pave the way for new approaches to treat ALT cancers.

[0315] (References) TIFF2025108697000003.tif215152TIFF2025108697000004.tif215152TIFF2025108697000005.tif224152TIFF2025108697000006.tif225153TIFF2025108697000007.tif115152

Claims

Claim 1 A method for inhibiting the viability of telomere alternative lengthening (ALT) cells and / or the growth of ALT cells, the method comprising the step of reducing the expression or activity of Fanconi anemia complementation group M (FANCM) in the ALT cells. Claim 2 The method according to claim 1, wherein the expression or activity of FANCM is reduced by administering a FANCM antagonist to an individual. Claim 3 The method according to claim 2, wherein the FANCM antagonist inhibits one or more activities of FANCM. Claim 4 The method according to claim 3, wherein the FANCM antagonist is an organic compound having a molecular weight of 900 Da or less. Claim 5 The method according to claim 3, wherein the FANCM antagonist is an antibody molecule or aptamer that specifically binds to FANCM. Claim 6 The method according to claim 2, wherein the FANCM antagonist reduces the expression of FANCM. Claim 7 The method according to claim 6, wherein the FANCM antagonist is a suppressor nucleic acid. Claim 8 The method according to claim 7, wherein the suppressor nucleic acid is siRNA or shRNA. Claim 9 The method according to claim 8, wherein the suppressor nucleic acid comprises a nucleotide sequence that is at least 95% identical to a continuous sequence of nucleotides 15 to 40 of SEQ ID NO:

32. Claim 10 The method according to claim 9, wherein the suppressor nucleic acid comprises the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO:

4. Claim 11 The method according to claim 7, wherein the suppressor nucleic acid is an antisense oligonucleotide. Claim 12 The method according to claim 6, wherein the FANCM antagonist is a targeted nuclease that reduces the expression of FANCM. Claim 13 The method according to claim 12, wherein the targeted nuclease is a ZFN, TALEN or meganuclease that recognizes a target sequence within the FANCM gene. Claim 14 The method according to claim 12, 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. Claim 15 The method according to claim 12 or 13, wherein the targeted nuclease cleaves genomic DNA at a target sequence of the FANCM gene, thereby resulting in a deletion or insertion that reduces or prevents the expression of an active FANCM polypeptide.

16. The method according to any one of claims 1 to 15, wherein the activity or expression of BLM and / or BRCA1 is not reduced in the ALT cells.

17. The method according to any one of claims 1 to 16, wherein the ALT cells are mesenchymal or epithelial cancer cells.

18. The method according to claim 17, wherein the ALT cells are osteosarcoma, liposarcoma, glioblastoma, astrocytoma, or bladder cancer cells.

19. The method according to claim 1, wherein the method comprises a step of disrupting the FANCM-RMI interaction and / or a step of inhibiting the ATPase activity of FANCM.

20. The method according to claim 19, wherein the step of disrupting the FANCM-RMI interaction comprises a step of administering an inhibitor of the FANCM-RMI interaction, and the step of inhibiting the ATPase activity of FANCM comprises a step of administering an inhibitor of the ATPase activity of FANCM.

21. The method according to claim 19 or 20, comprising a step of disrupting the binding between FANCM and RMI in the MM2 domain.

22. The method according to any one of claims 19 to 21, wherein the inhibitor is any one or more of a genetic inhibitor, a small molecule, a peptide, and a protein.

23. The method according to claim 22, wherein the genetic inhibitor is siRNA.

24. The method according to claim 22, wherein the small molecule is ethyl 4-[(1-hydroxy-2-phenyl-1H-indol-3-yl)-pyridin-2-yl-methyl]-piperazine-1-carboxylate.

25. The peptide is DLFSVTFDLGFC (SEQ ID NO: 49), DIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 50), and IFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 5) The method according to claim 22, which is a peptide comprising an amino acid sequence that is at least 90% identical to a peptide selected from the group consisting of.

26. The method according to claim 22, wherein the protein is an inactivated FANCM protein.

27. The method according to claim 26, wherein the inactivated FANCM protein comprises an F1232A / F1236A double replacement. **Claim 28** The method according to claim 22, wherein the protein comprises an immunoglobulin binding domain. **Claim 29** The method according to claim 19, which is a method for treating ALT cancer in a subject. **Claim 30** A method for treating telomere alternative lengthening (ALT) cancer in an individual in need thereof, the method comprising the step of reducing the expression or activity of Fanconi anemia complementation group M (FANCM) in the individual. **Claim 31** The method according to claim 30, wherein the expression or activity of FANCM is reduced by administering an FANCM antagonist to the individual. **Claim 32** The method according to claim 31, wherein the FANCM antagonist inhibits the activity of FANCM. **Claim 33** The method according to claim 32, wherein the FANCM antagonist is an organic compound having a molecular weight of 900 Da or less. **Claim 34** The method according to claim 32, wherein the FANCM antagonist is an antibody molecule or aptamer that specifically binds to FANCM. **Claim 35** The method according to claim 31, wherein the FANCM antagonist reduces the expression of FANCM. **Claim 36** The method according to claim 35, wherein the FANCM antagonist is a suppressor nucleic acid. **Claim 37** The method according to claim 36, wherein the suppressor nucleic acid is siRNA or shRNA. **Claim 38** The method according to claim 37, wherein the suppressor nucleic acid comprises a nucleotide sequence that is at least 95% identical to a continuous sequence of nucleotides 15 to 40 of SEQ ID NO:

32. **Claim 39** The method according to claim 38, wherein the suppressor nucleic acid comprises the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO:

4. **Claim 40** The method according to claim 36, wherein the suppressor nucleic acid is an antisense oligonucleotide. **Claim 41** The method according to claim 35, wherein the FANCM antagonist is a targeted nuclease that reduces the expression of FANCM. **Claim 42** The method according to claim 41, wherein the targeted nuclease is a ZFN, TALEN or meganuclease that recognizes a target sequence within the FANCM gene. **Claim 43** The method according to claim 41, wherein the targeted nuclease is a CRISPR-related nuclease, and the CRISPR-related nuclease is administered in combination with a guide RNA that recognizes a target sequence within the FANCM gene.

44. The method according to claim 41 or 42, wherein the targeted nuclease cleaves genomic DNA at a target sequence of the FANCM gene, thereby resulting in a deletion or insertion that reduces or prevents the expression of an active FANCM polypeptide.

45. The method according to any one of claims 30 to 44, wherein the activity or expression of BLM and / or BRCA1 is not reduced in ALT cells.

46. The method according to any one of claims 30 to 45, wherein the ALT cancer is a mesenchymal or epithelial cancer.

47. The method according to claim 46, wherein the ALT cancer is osteosarcoma, soft tissue sarcoma (e.g., liposarcoma, undifferentiated pleomorphic sarcoma, or leiomyosarcoma), glioblastoma, astrocytoma, neuroblastoma, or bladder cancer.

48. An agent for reducing the expression or activity of FANCM for use in the method of treatment according to any one of claims 30 to 47.

49. Use of a FANCM antagonist in the manufacture of a medicament for use in the method of treatment according to any one of claims 30 to 47.

50. The method according to claim 30, wherein the method comprises a step of disrupting the FANCM-RMI interaction and / or a step of inhibiting the ATPase activity of FANCM.

51. The method according to claim 50, wherein the step of disrupting the FANCM-RMI interaction comprises a step of administering an inhibitor of the FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM, and / or a step of administering an agent that inhibits the ATPase activity of FANCM.

52. The method according to claim 50 or 51, wherein the method comprises a step of disrupting the binding between FANCM and RMI in the MM2 domain.

53. The method according to any one of claims 50 to 52, wherein the inhibitor is any one or more of a genetic inhibitor, a small molecule, a peptide, and a protein.

54. The method according to claim 53, wherein the genetic inhibitor is siRNA.

55. The method according to claim 53, wherein the small molecule is ethyl 4-[(1-hydroxy-2-phenyl-1H-indol-3-yl)-pyridin-2-yl-methyl]-piperazine-1-carboxylate.

56. The peptide is DLFSVTFDLGFC (SEQ ID NO: 49), DIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 50), and EDIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 5), The method according to claim 53, wherein the peptide is a peptide comprising an amino acid sequence that is at least 90% identical to a peptide selected from the group consisting of.

57. The method according to claim 53, wherein the protein is an inactivated FANCM protein.

58. The method according to claim 57, wherein the inactivated FANCM protein comprises F1232A / F1236A double replacement and / or K117R replacement.

59. The method according to claim 53, wherein the protein comprises an immunoglobulin binding domain.

60. The method according to claim 51 or 52, further comprising simultaneous, sequential, or separate administration of a chemotherapeutic agent.

61. The method according to claim 51 or 52, wherein the method does not comprise simultaneous, sequential, or separate administration of a chemotherapeutic agent.

62. A pharmaceutical composition comprising an inhibitor of FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM for use in treating telomere alternative lengthening (ALT) cancer.

63. Use of an inhibitor of FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM in the manufacture of a medicament for the treatment of telomere alternative lengthening (ALT) cancer.

64. The pharmaceutical composition according to claim 62, wherein the pharmaceutical composition consists essentially of an inhibitor of FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM; or the use according to claim 19, wherein the medicament consists essentially of an inhibitor of FANCM-RMI interaction and / or an inhibitor of the ATPase activity of FANCM.

65. The pharmaceutical composition according to claim 62 or 64, or the use according to claim 19 or 20, wherein the inhibitor of FANCM-RMI interaction and / or the inhibitor of the ATPase activity of FANCM is any one or more of a genetic inhibitor, a small molecule, a peptide, and a protein.

66. The pharmaceutical composition or use according to claim 65, wherein the genetic inhibitor is siRNA.

67. The pharmaceutical composition or use according to claim 65, wherein the small molecule is ethyl 4-[(1-hydroxy-2-phenyl-1H-indol-3-yl)-pyridin-2-yl-methyl]-piperazine-1-carboxylate.

68. The peptide is DLFSVTFDLGFC (SEQ ID NO: 49), DIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 50) and EDIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 5), The pharmaceutical composition or use according to claim 65, comprising an amino acid sequence that is at least 90% identical to a peptide selected from the group consisting of.

69. The pharmaceutical composition or use according to claim 65, wherein the protein is an inactivated FANCM protein.

70. The pharmaceutical composition or use according to claim 69, wherein the inactivated FANCM protein comprises F1232A / F1236A double replacement and / or K117R replacement.

71. The pharmaceutical composition or use according to claim 65, wherein the protein comprises an immunoglobulin binding domain.

72. A method for determining the responsiveness of cancer in an individual to treatment with a FANCM antagonist, comprising determining the presence of one or more ALT cancer cells in a sample of cancer cells derived from the individual, wherein the presence of one or more ALT cancer cells in the sample indicates that the cancer responds to treatment with the FANCM antagonist.

73. The method according to claim 72, wherein the presence of ALT cancer cells is determined by evaluating the presence of C circles or ALT-related PML bodies in one or more cancer cells in the sample, and the presence of C circles or ALT-related PML bodies in the one or more cancer cells indicates that the cancer cells are ALT cancer cells.

74. The method according to claim 72 or 73, comprising identifying the cancer in the individual as responsive to a FANCM antagonist.

75. A method of selecting a subject for treatment with an inhibitor of the FANCM-RMI interaction, said method comprising the step of determining whether said subject has an ALT cancer, and if said subject has an ALT cancer, said subject is selected for treatment with said inhibitor of the FANCM-RMI interaction.

76. A method of identifying whether a subject having cancer is suitable for treatment with an inhibitor of the FANCM-RMI interaction, said method comprising the step of determining whether said cancer is an ALT cancer, and if said subject has an ALT cancer, said subject is identified as being suitable for treatment with said inhibitor of the FANCM-RMI interaction.

77. A method of determining whether a subject is responsive to treatment with an inhibitor of the FANCM-RMI interaction, determining the presence and / or degree of genomic instability at one or more telomeres in cells taken from the subject; and / or determining the presence and / or level of ALT activity in cells taken from the subject comprising the method.

78. The method according to claim 77, wherein said cells are ALT cancer cells.

79. The method according to any one of claims 75 to 78, wherein said inhibitor is any one or more of a genetic inhibitor, a small molecule, a peptide and a protein.

80. The method according to claim 79, wherein said genetic inhibitor is siRNA.

81. The method according to claim 79, wherein said small molecule is ethyl 4-[(1-hydroxy-2-phenyl-1H-indol-3-yl)-pyridin-2-yl-methyl]-piperazine-1-carboxylate.

82. Said peptide is DLFSVTFDLGFC (SEQ ID NO: 49), DIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 50), EDIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 5) The method according to claim 79, which is a peptide comprising an amino acid sequence that is at least 90% identical to a peptide selected from the group consisting of.

83. The method according to claim 79, wherein said protein is an inactivated FANCM protein.

84. The method according to claim 83, wherein the inactivated FANCM protein comprises F1232A / F1236A double replacement and / or K117R replacement.

85. The method according to claim 79, wherein the protein comprises an immunoglobulin binding domain.

86. A method for screening a compound that induces death of ALT cancer cells, comprising: determining the binding between a test compound and FANCM and the binding to FANCM indicates that the compound induces cell death in ALT cells.

87. A method for screening a compound that induces death of ALT cancer cells, comprising determining the effect of a test compound on the expression or activity of FANCM, and a reduction in the expression or activity of FANCM indicates that the compound induces cell death in ALT cells.

88. The method according to claim 87 or 88, comprising identifying the test compound as a compound that reduces the expression or activity of FANCM.

89. The method according to claim 88, comprising isolating or purifying the identified compound.

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