EPC-TDNA as biomarkers and methods of measuring EPC-tdna

Abstract

The present disclosure relates to methods of determining a subject's suitability for treatment with an agent capable of disrupting the Fanconi anemia, complementation group M (FANCM) complex. In particular examples, the subject has a cancer that is not an alternative lengthening of telomeres positive (ALT+) cancer. In some examples, the subject has a degenerative disease caused by DNA damage. The present disclosure also relates to methods of treating subjects that have been determined to be suitable for treatment by the methods disclosed herein. The present disclosure also relates to methods of measuring extracellular primed single-stranded circular telomeric DNA.

Description

[0001] EPC-TDNA AS BIOMARKERS AND METHODS OF MEASURING EPC-TDNA

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to methods of determining a subject’s suitability for treatment with an agent capable of disrupting the Fanconi anemia, complementation group M (FANCM) complex. In particular examples, the subject has a cancer that is not an alternative lengthening of telomeres positive (ALT+) cancer. In some examples, the subject has a degenerative disease caused by DNA damage. The present disclosure also relates to methods of treating subjects that have been determined to be suitable for treatment by the methods disclosed herein.

[0004] BACKGROUND OF THE INVENTION FANCM may be targeted for the treatment of ALT cancers (W02020 / 242330 A2). There is a need to identify subjects who would benefit from treatment with agents capable of dismpting the FANCM complex.

[0005] SUMMARY OF THE INVENTION

[0006] In a first aspect, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the Fanconi anemia, complementation group M (FANCM) complex, optionally wherein the subject does not have an ALT+ cancer, the method comprising:

[0007] measuring the quantity of extracellular primed single-stranded circular telomeric DNA (epc-tDNA), extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles in a sample from the subject; and based on the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles, determining whether the subject is suitable for treatment with the agent.

[0008] The subject may not comprise detectable ALT cells. The subject may have a cancer. The subject may have a degenerative disease or a neurodegenerative disease, for instance Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, spinocerebellar ataxias, or amyotrophic lateral sclerosis (ALS).

[0009] The sample from the subject may be a liquid sample or liquid biopsy. The sample may be or may comprise cerebrospinal fluid, bronchoalveolar lavage, urine, saliva, blood, a blood fraction, serum, or plasma.

[0010] The measurement may be of epc-tDNA that comprises C-circles, G-circles, and / or TF-circles. The measured epc-tDNA, C-circles, G-circles, and / or TF-circles may be from extracellular vesicles and / or may have been cell-free. The method may comprise removing cells from the sample to obtain an extracellular fraction prior to measuring the quantity of epc-tDNA, C-circles, G-circles, and / or TF-circles. The method may comprise enriching the sample or extracellular fraction for extracellular vesicles. The extracellular vesicles may be exosomes. The method may comprise enriching the sample or extracellular fraction for extracellular nucleic acids. The extracellular nucleic acids may be free circulating DNA.

[0011] The sample, extracellular fraction, or enriched fraction may be split into a first fraction and a second fraction, and the first fraction may be contacted with a polymerase or amplified whereas the second fraction is not.

[0012] The measuring the quantity of epc-tDNA, C-circles, G-circles, and / or TF-circles may comprise amplification of telomeric DNA, epc-tDNA, C-circles, G-circles, and / or TF-circles. The amplification may be selective amplification, isothermal amplification, rolling circle amplification, and / or comprises the use of φ29 DNA polymerase.

[0013] The measuring the quantity of epc-tDNA, C-circles, G-circles, and / or TF-circles may comprise qPCR, for instance to detect telomeric DNA, epc-tDNA, C-circles, G-circles, and / or TF-circles. qPCR may be applied to the amplification product. The quantity of epc-tDNA in the sample may be expressed as the fold change of telomeric DNA between the first fraction and the second fraction.

[0014] The method of the first aspect may comprise treating the subject with the agent capable of disrupting the FANCM complex if the subject is determined to be suitable for treatment. The agent may be a small molecule drug, antibody, antigen-binding fragment of an antibody, aptamer, nucleic acid molecule, nucleic acid analogue, antisense molecule, siRNA, shRNA, peptide, protein, inactivated FANCM protein, the inactivated FANCM protein comprising a F1232A / F1236A double substitution, targeted nuclease that reduces expression of FANCM, ZFN, TALEN, meganuclease, or a CRISPR associated nuclease.

[0015] In a second aspect, there is provided an agent capable of disrupting the FANCM complex for use in treating a non-ALT cancer or a degenerative disease in a subject. The subject may have been determined to be suitable for treatment by a method of the first aspect.

[0016] In a third aspect, there is provided a kit comprising components for carrying out a method of the first aspect. In a fourth aspect, there is provided a method of determining the presence of epc-tDNA in a biological sample, the method comprising:

[0017] separating the sample into a first fraction and a second fraction;

[0018] contacting the first fraction but not the second fraction with a polymerase under conditions for amplification of telomeric DNA,

[0019] quantifying the telomeric DNA levels in the post-amplification first fraction and in the second fraction; and

[0020] determining the presence of epc-tDNA in the sample based on the fold change of telomeric DNA between the first fraction and the second fraction.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Fig 1. Thirty cell models have been evaluated for primed circular telomeric DNA (pc-tDNA) presence (y-axis) according to the presented methodology for both intracellular (top) and extracellular-exosomes enriched pc-tDNA content (bottom). The pc-tDNA presence correlates with FANCM sensitivity (x-axis) showed as TAE score (Tessellate Analysis Engine- derived from DepMap CRISPR knock out dependency score). Dotted lines ony- and x- axes indicates the threshold for pc-tDNA positivity (>1.2) and FANCM sensitivity (>25) respectively. Open circles are FANCM insensitive cell models; close circles are FANCM sensitive cell models also positive for pc-tDNA both intra- and extracellular; open triangles are FANCM sensitive cell models negative for intracellular pc-tDNA.

[0023] Fig 2. Brief description of the FANCM complex (adapted from Figure 7 of Deans AJ el a / ., FANCM Connects the Genome Instability Disorders Bloom's Syndrome and Fanconi Anemia, Molecular Cell, 2009).

[0024] Fig 3. ALT features are assessed in the cell models in comparison with U2OS ALT cells (A) Telomere restriction fragment analysis of the indicated cells. HeLa cells are well characterized telomerase-positive cells, U2OS are well-characterized ALT cells. Genomic DNA was restriction digested with Hinfl+Rsal and hybridized in-gel in native conditions to radiolabeled oligonucleotides comprising five CCCTAA repeats (to detect G-rich telomeric DNA; left panels) or five TTAGGG repeats (to detect C-rich telomeric DNA; right panels). After signal acquisition, gels were denatured and re-hybridized to the same probes. The molecular weights of a size marker are shown on the left of each gel in kb. Note that: i) c-rich single stranded telomeric DNA is only detected in U2OS cells; and ii) all cell lines, except for U2OS, have homogeneous telomeres of sizes similar to the ones in HeLa cells. (B) Northern blot analysis of TERRA expression in the indicated cell lines. Total RNA was hybridized to a radiolabeled telomeric probe specifically detecting TERRA UUAGGG repeats. Ethidium bromide-stained 28 rRNA serves as a loading control. Note that TERRA levels are elevated in U2OS cells only. (C) The indicated cell lines were grown in medium containing colchicine and metaphase spreads were prepared and subjected to DNA fluorescence in situ hybridization (FISH) using fluorescently labeled telomeric PNA probes. Fragile telomeres (FTs) were scored for 5 to 10 metaphases for each cell line. Note that all cells, except for U2OS, present relatively low levels of FTs. (D) The indicated cells were fixed on slides and subjected to indirect immunofluorescence using anti-PML antibodies, combined with telomeric DNA FISH. Co-localization events correspond to APBs and were scored for at least 50 nuclei for each cell line. In C and D, bars are means; ****: p<0.001 (one-way ANOVA). E) ipc-tDNA and epc-tDNA for the cell models described (expressed as Fold change).

[0025] DETAILED DESCRIPTION

[0026] The inventors have found that the presence of extracellular primed single-stranded circular telomeric DNA (epc-tDNA) structures, such as extracellular C-circles, extracellular G-circles and / or extracellular telomeric fusion circles (TF circles), positively correlate with sensitivity of cells or tissues for treatment with an agent capable of disrupting the FANCM complex. Hence, it is demonstrated herein that epc-tDNA may be used as a biomarker to establish the sensitivity of tissues to treatment with an agent capable of disrupting the FANCM complex.

[0027] Thus, in a first aspect, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, optionally wherein the subject does not have an ALT+ cancer, the method comprising:

[0028] measuring the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF -circles in a sample from the subject; and

[0029] based on the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF -circles determining whether the subject is suitable for treatment with the agent.

[0030] epc-tDNA is a partially single stranded circular DNA structure consisting of a C- or G-rich telomeric sequence with a nicked complementary sequence (primer). Thus, epc-tDNA can comprise C-circles, G-circles, and / or TF-circles. epc-tDNA does not naturally occur in healthy humans.

[0031] C-circles are structures where the intact circular single stranded DNA consists of the C-rich telomeric sequence and is primed by the G-rich telomeric sequence.

[0032] G-circles are structures where the intact circular single stranded DNA consists of the G-rich telomeric sequence and is primed by the C-rich telomeric sequence.

[0033] TF -circles are structures where the intact circular single stranded DNA consists of the both C-rich and G-rich telomeric sequences which are fused and is primed by a complementary telomeric sequence.

[0034] Measuring the quantity of epc-tDNA may be the determination of a relative quantity of epc-tDNA in the sample. For instance, in some embodiments the relative quantity is expressed as a fold change. In some instances, measuring the quantity of epc-tDNA may be the determination of whether epc-tDNA is present or absent.

[0035] Alternatively, measuring the quantity of epc-tDNA may be the determination of an absolute quantity of epc-tDNA in the sample. And so, the measurement may lead to a quantification of the actual amount of epc-tDNA in the sample, for instance by mass, copy number, or concentration.

[0036] Measuring the quantity of extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles may be the determination of a relative quantity of extracellular C-circles. extracellular G-circles, and / or extracellular TF-circles in the sample. For instance, in some embodiments the relative quantity is expressed as a fold change. In some instances, measuring the quantity of extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles may be the determination of whether extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles are present or absent. Alternatively, measuring the quantity of extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles may be the determination of an absolute quantity of extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles in the sample. And so, the measurement may lead to a quantification of the actual amount or number of extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles in the sample, for instance by mass, copy number, or concentration.

[0037] In an embodiment, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, optionally wherein the subject does not have an ALT+ cancer, the method comprising:

[0038] measuring the quantity of epc-tDNA in a sample from the subject; and

[0039] based on the quantity of epc-tDNA, determining whether the subject is suitable for treatment with the agent.

[0040] In another embodiment, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, optionally wherein the subject does not have an ALT+ cancer, the method comprising:

[0041] measuring the quantity of extracellular C-circles in a sample from the subject; and

[0042] based on the quantity of extracellular C-circles, determining whether the subject is suitable for treatment with the agent.

[0043] A subject suitable for treatment with an agent capable of disrupting the FANCM complex may be referred to as a FANCM-sensitive subject.

[0044] The subject may have a cancer, and so the methods of the first aspect may be for determining if a cancer patient can be treated with an agent capable of disrupting the FANCM complex. It is surprisingly shown herein that epc-tDNA, such as extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles, can be used as a marker to differentiate between cancer cells that are FANCM sensitive and cancer cells that are FANCM insensitive, even when the cancer cells are not ALT cancer cells. A “non-ALT cancer” is a cancer cell that is not an ALT cancer. Thus, the methods of the first aspect may be for determining if a subject with a non-ALT cancer can be treated with an agent capable of disrupting the FANCM complex. The methods of the first aspect may be for determining if a subject without detectable ALT cells can be treated with agent capable of disrupting the FANCM complex.

[0045] Further explanation of ALT cells and ALT cancers is provided in the section herein titled “ALT cells and ALT cancers”.

[0046] In some embodiments, the subject has a cancer and intracellular C-circles, intracellular G-circles, and / or intracellular TF-circles are not detectable in cancer cells from subject. For instance, intracellular C-circles, intracellular G-circles, and / or intracellular TF-circles may be measured in a biopsy of cancerous or suspected cancerous cells. In some embodiments, the subject has a cancer, and intracellular C-circles are not detectable in cancer cells from subject. For instance, intracellular C-circles may be measured in a biopsy of cancerous or suspected cancerous cells.

[0047] There is provided a method of determining a cancer patient’s suitability for treatment with an agent capable of dismpting the FANCM complex, the method comprising: identifying the subject as suitable for treatment when epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles are detected in a sample from the subject, wherein intracellular C-circles, intracellular G-circles, and / or intracellular TF-circles are not detectable in cancer cells from subject. In an embodiment, there is provided a method of determining a cancer patient’s suitability for treatment with an agent capable of disrupting the FANCM complex, the method comprising: identifying the subject as suitable for treatment when epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles are detected in a sample from the subject, wherein intracellular C-circles, intracellular G-circles, and / or intracellular TF-circles are not detectable in a biopsy of cancer cells from subject. There is provided a method of determining a cancer patient’s suitability for treatment with an agent capable of dismpting the FANCM complex, the method comprising: identifying the subject as suitable for treatment when epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles are detected in a sample from the subject, wherein intracellular C-circles are not detectable in cancer cells from subject. In an embodiment, there is provided a method of determining a cancer patient’s suitability for treatment with an agent capable of disrupting the FANCM complex, the method comprising: identifying the subject as suitable for treatment when epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles are detected in a sample from the subject, wherein intracellular C-circles are not detectable in a biopsy of cancer cells from subject.

[0048] There is provided a method of determining a cancer patient’s suitability for treatment with an agent capable of dismpting the FANCM complex, the method comprising: identifying the subject as suitable for treatment when extracellular C-circles are detected in a sample from the subject, wherein intracellular C-circles are not detectable in cancer cells from subject. In an embodiment, there is provided a method of determining a cancer patient’s suitability for treatment with an agent capable of disrupting the FANCM complex, the method comprising: identifying the subject as suitable for treatment when extracellular C-circles are detected in a sample from the subject, wherein intracellular C-circles are not detectable in a biopsy of cancer cells from subject.

[0049] Degenerative diseases, such as neurodegenerative diseases, can be associated with DNA damage that involves the FANCM complex and so the present methods can determine if a subject with a degenerative disease is FANCM-sensitive. Hence, in some embodiments the methods of the first aspect are for determining if a subject with a degenerative disease is suitable for treatment with an agent capable of disrupting the FANCM complex. The degenerative disease may be a disease associated with or caused by DNA damage. Examples of degenerative diseases to which the present methods may be applied are Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, spinocerebellar ataxias, and ALS.

[0050] In a particular embodiment, therefore, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, wherein the subject has a non- ALT cancer or a degenerative disease, the method comprising:

[0051] measuring the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles in a sample from the subject; and

[0052] based on the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles determining whether the subject is suitable for treatment with the agent.

[0053] The methods of the first aspect comprise measuring the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles. In some embodiments, only one of these species is measured. In some embodiments, a fraction comprising multiple of these species is measured. For instance, the methods may comprise measuring the quantity of extracellular TF-circles, C-circles, and G-circles. In examples described herein, the methods measure the quantity of epc-tDNA without differentiating between the species, such as TF-circles, C-circles and G-circles, within the epc-tDNA.

[0054] epc-tDNA, extracellular C-circles, extracellular G-circles, and extracellular TF-circles are measured in a sample from the subject. The measurement is performed in vitro. The sample may be a liquid sample. The sample may be a liquid biopsy. The sample may be blood, such as whole blood, or contain a blood fraction, such as serum or plasma. The sample may be serum or plasma. The sample may be cerebrospinal fluid, a bronchoalveolar lavage, urine, or saliva. The methods of the first aspect may comprise in vitro processing of a sample from a subject to be suitable for the detection methods disclosed herein. Cells may be removed from the sample. For instance, cells may be filtered out, centrifuged into a pellet, or excluded from the sample. In some embodiments, the cells are lysed and the epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles may be labelled prior to the lysing to differentiate from intracellular primed circular telomeric DNA. Thus, a sample may be processed to obtain an extracellular fraction. Methods of obtaining an extracellular fraction are known in the art. For instance, such techniques may comprise size exclusion, such as filtration. In an example, a 0.8-micron filter is used to obtain an extracellular fraction. In some embodiments, the methods of the first aspect may comprise the removal of cells from a liquid sample or liquid biopsy, such as a blood sample, to result in an extracellular fraction.

[0055] The sample may be processed to deplete linear single or double stranded telomeric DNA. Methods of processing samples to deplete linear single or double stranded telomeric DNA, such as treatment by exonuclease, are known in the art. The depletion of linear single or double stranded telomeric DNA may take place before, during, or after further enrichment steps.

[0056] In some embodiments, the methods of the first aspect comprise processing a sample from a subject to enrich a fraction potentially containing epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles. The sample may be processed to enrich nucleic acids or extracellular vesicles. The sample may be processed to enrich exosomes. Methods of processing samples to enrich for nucleic acids, such as DNA, are known in the art. Methods of processing samples to enrich for extracellular vesicles are known in the art. For instance, the methods may comprise size exclusion or centrifugation. Extracellular vesicles, such as exosomes, are associated with markers. For instance, they display markers on their surface (e.g. CD81, CD9, and / or CD63). Thus, extracellular vesicles may be enriched by labelling the makers and enriching for labelled entities or by binding molecules to the markers and enriching for bound entities. The sample may be processed as disclosed in Chen et al. (Cancers 2021, 13, 5369. https: / / doi.org / 10.3390 / cancersl3215369 - herein incorporated by reference). The exosomes may be isolated by differential centrifugation, for instance as disclosed in Chen et al. The exosomes may be isolated by density gradient purification, for instance iodixanol density gradient separation. The density gradient purification may be as disclosed in Chen et al. The exosomes may be isolated by immunoprecipitation, e.g. by using any one or a combination of CD81, CD9, and CD63. The immuno-precipitation may be as disclosed in Chen et al.

[0057] The extracellular fraction may be further processed to enrich for a fraction that potentially contains epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles. For instance, the extracellular fraction may be processed to enrich for nucleic acids, such as free circulating DNA, or extracellular vesicles, such as exosomes.

[0058] In an embodiment, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, optionally wherein the subject has a non-ALT cancer or a degenerative disease, the method comprising:

[0059] enriching extracellular vesicles (such as exosomes) from a sample from the subject;

[0060] measuring the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles from the extracellular vesicles; and

[0061] based on the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles, determining whether the subject is suitable for treatment with the agent.

[0062] In an embodiment, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, optionally wherein the subject has a non-ALT cancer or a degenerative disease, the method comprising:

[0063] removal of cells from a sample from the subject to obtain an extracellular fraction,

[0064] enriching extracellular vesicles (such as exosomes) from the extracellular fraction;

[0065] measuring the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles from the extracellular vesicles; and

[0066] based on the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles, determining whether the subject is suitable for treatment with the agent. In an embodiment, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, optionally wherein the subject has a non-ALT cancer or a degenerative disease, the method comprising:

[0067] enriching for extracellular nucleic acids (such as free circulating DNA) from a sample from the subject; measuring the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF -circles from the extracellular nucleic acids; and

[0068] based on the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF -circles, determining whether the subject is suitable for treatment with the agent.

[0069] In an embodiment, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, optionally wherein the subject has a non-ALT cancer or a degenerative disease, the method comprising:

[0070] removal of cells from a sample from the subject to obtain an extracellular fraction

[0071] enriching for extracellular nucleic acids (such as free circulating DNA) from the extracellular fraction; measuring the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF -circles from the extracellular nucleic acids; and

[0072] based on the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF -circles, determining whether the subject is suitable for treatment with the agent.

[0073] Exemplary methods for measuring the quantity of epc-tDNA, C-circles, G-circles, and TF-circles are known in the art. As a non-limiting example, Chen et al. discloses methods for detecting exosomal C-circles (a type of epc-tDNA). The methods may comprise amplification and detection or direct detection of epc-tDNA, C-circles, G-circles and / or TF-circles. The methods may comprise rolling circle amplification and / or qPCR.

[0074] In some embodiments, measuring the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or TF-circles comprises amplification. The amplification may be selective amplification. The amplification may be isothermal amplification. The amplification may be rolling circle amplification. The amplification may allow the direct or indirect detection of the epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles. The rolling circle amplification may use a circular strand of the epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles as a template. The amplification may comprise the use of a polymerase with stand-displacement and proofreading exonuclease activity. The amplification may comprise the use of a polymerase suitable for rolling circle amplification. The amplification may comprise the use of a polymerase suitable for rolling circle amplification with strand displacement activity. An exemplary polymerase is the φ29 DNA polymerase.

[0075] In some embodiments, the amplification is any that amplifies telomeric DNA. In some embodiments, the amplification is any that selectively amplifies circular DNA comprising the nucleotide sequence (CCCTAA)n, wherein n is 1 or any integer greater than 1, and / or the nucleotide sequence (TTAGGG)n, wherein n is 1 or any integer greater than 1, and / or the combination of the nucleotide sequences (CCCTAA)-(TTAGGG)n, wherein n is 1 or any integer greater than 1.

[0076] The amplification products may be detected by any suitable means. For example, hybridisation, sequencing, PCR, molecular beacons, nucleic acid enzymes such as DNA partzymes, and / or by incorporating suitably labelled nucleotides. In one example, the amplification products may be detected using a labelled nucleotide probe. The labelled probe may comprise the nucleotide sequence (CCCTAA)n, wherein n is 1 or any integer greater than 1. The labelled probe may comprise the nucleotide sequence (TTAGGG)n, wherein n is 1 or any integer greater than 1. The label may be any detectable label. For example, the label may be a fluorescent label. In some embodiments, a first fraction of the sample is contacted with a polymerase for amplification and a second fraction of the sample is not contacted with the polymerase.

[0077] In some embodiments, the epc-tDNA, C-circles, G-circles, TF-circles, or amplification products are quantified by the use of PCR. Techniques for quantitative PCR (qPCR) are known in the art. For instance, the qPCR may comprise the displacement of an intercalating dye by a polymerase. The qPCR may comprise amplification directed by primers specific for sequences associated with epc-tDNA, C-circles, G-circles, TF-circles. The qPCR may comprise the quantification of the amount of a housekeeping gene, to allow normalisation of data.

[0078] The quantity of the epc-tDNA may be expressed as a fold change. The quantity of the epc-tDNA may be expressed as copy number. The quantity of the epc-tDNA may be expressed as an absolute value. The quantity of the epc-tDNA may be expressed as mass. The quantity of the epc-tDNA may be expressed as concentration. The quantity of the epc-tDNA may be expressed as the presence or absence of epc-tDNA in the sample (i.e. whether epc-tDNA is detectable or not). In particular embodiments, the quantity of the epc-tDNA may be expressed as the fold change between a first fraction of the sample (a fraction contacted with the polymerase) and a second fraction of the sample.

[0079] The quantity of extracellular C-circles, G-circles, and / or TF-circles may be expressed as a fold change. The quantity of extracellular C-circles, G-circles, and / or TF-circles may be expressed as copy number. The quantity of extracellular C-circles, G-circles, and / or TF-circles may be expressed as an absolute amount. The quantity of extracellular C-circles, G-circles, and / or TF-circles may be expressed as mass. The quantity of extracellular C-circles, G-circles, and / or TF-circles may be expressed as concentration. The quantity of extracellular C-circles, G-circles, and / or TF-circles may be expressed as the presence or absence of extracellular C-circles, G-circles, and / or TF-circles in the sample (i.e. whether extracellular C-circles, G-circles, and / or TF-circles are detectable or not). In particular embodiments, the quantity of extracellular C-circles, G-circles, and / or TF-circles may be expressed may be expressed as the fold change of C-circles, G-circles, and / or TF-circles between a first fraction of the sample (a fraction contacted with the polymerase) and a second fraction of the sample.

[0080] In an embodiment, measuring the quantity of epc-tDNA comprises:

[0081] separating the sample into a first fraction and a second fraction;

[0082] contacting the first fraction with a polymerase under conditions for the selective amplification of telomeric DNA,

[0083] quantifying the telomeric DNA levels in the post-amplification first fraction and in the second fraction; and

[0084] expressing the quantity of epc-tDNA in the sample as the fold change of telomeric DNA between the first fraction and the second fraction.

[0085] In a particular embodiment, measuring the quantity of epc-tDNA comprises:

[0086] separating the sample into a first fraction and a second fraction;

[0087] contacting the first fraction with a rolling circle polymerase under conditions for the selective amplification of telomeric DNA,

[0088] quantifying the telomeric DNA levels in the post-amplification first fraction and in the second fraction by qPCR; and

[0089] expressing the quantity of epc-tDNA in the sample as the fold change of telomeric DNA between the first fraction and the second fraction.

[0090] In some embodiments, the methods comprise:

[0091] measuring the quantity of telomeric DNA in a sample from the subject;

[0092] determining the presence of epc-tDNA from the quantity of telomeric DNA; and based on the presence of epc-tDNA, determining whether the subject is suitable for treatment with the agent.

[0093] The methods of the first aspect determine if the subject is suitable for treatment with an agent capable of disrupting the FANCM complex. Such patients can be referred to as “FANCM sensitive”. Thus, any of the methods of the first aspect may be phrased as methods of determining if a subject has a FANCM-sensitive pathology or a FANCM-sensitive cancer. In particular embodiments, the methods are for determining if a subject has a FANCM-sensitive cancer that is not an ALT cancer. In other embodiments, the methods are for determining if a subject has a FANCM-sensitive degenerative disease or neurodegenerative disease.

[0094] A subject may be determined to be suitable for treatment with an agent capable of disrupting the FANCM complex if the quantity of epc-tDNA exceeds a predetermined threshold. The threshold may be determined based on control samples, such as negative and / or positive controls. For instance, samples from subjects that contain cells that are not FANCM-sensitive and / or samples from subjects that contain cells that are FANCM-sensitive. The control samples may be tissue samples that match the samples from the subject. The control samples may be samples from FANCM-insensitive cancer patients and / or FANCM-sensitive cancer patients. The control samples may be samples from FANCM-insensitive subjects and / or FANCM-sensitive subjects with degenerative diseases. A subject may be determined to be suitable for treatment with an agent capable of disrupting the FANCM complex if the quantity of extracellular or exosomal C-circles, G-circles, and / or TF -circles exceeds a predetermined threshold. The threshold may be determined based on control samples, such as negative and / or positive controls. For instance, samples from subjects that contain cells that are not FANCM-sensitive and / or samples from subjects that contain cells that are FANCM-sensitive. The control samples may be tissue samples that match the samples from the subject. The control samples may be samples from FANCM-insensitive cancer patients and / or FANCM-sensitive cancer patients. The control samples may be samples from FANCM-insensitive subjects and / or FANCM-sensitive subjects with degenerative diseases.

[0095] In illustrative embodiments, the subject is determined to be suitable for treatment with an agent capable of disrupting the FANCM complex if the fold change of telomeric DNA as measured in the aforementioned embodiments is >1.0. For instance, in Figure 1, 1.2 is used, but other fold changes >1.0 such as for instance at least 1.3, 1.4, 1.5, or 2 may also indicate FANCM sensitivity.

[0096] In other instances, the subject is determined to be suitable for treatment with agent capable of disrupting the FANCM complex if epc-tDNA is determined to be present. The subject may be determined to be suitable for treatment if extracellular or exosomal C-circles, G-circles, and / or TF-circles are determined to be present.

[0097] In an embodiment, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, optionally wherein the subject has a non-ALT cancer or a degenerative disease, the method comprising:

[0098] removal of cells from a sample (e.g. a liquid sample or liquid biopsy, such as a blood sample) from the subject to obtain an extracellular fraction;

[0099] enriching for extracellular vesicles (such as exosomes) or extracellular nucleic acids (such as free circulating DNA) from the extracellular fraction to form an enriched fraction;

[0100] measuring the quantity of epc-tDNA in the enriched fraction; and

[0101] based on the quantity or presence of epc-tDNA, determining whether the subject is suitable for treatment with the agent capable of disrupting the FANCM complex.

[0102] In an embodiment, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, wherein the subject has a non-ALT cancer or a degenerative disease, the method comprising: enriching for exosomes from a blood sample or fraction of a blood sample to form an enriched fraction; measuring the quantity of epc-tDNA levels in enriched fraction; and

[0103] based on the quantity or presence of epc-tDNA, determining whether the subject is suitable for treatment with the agent capable of disrupting the FANCM complex.

[0104] In an embodiment, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, optionally wherein the subject has a non-ALT cancer or a degenerative disease, the method comprising:

[0105] removal of cells from a sample (e.g. a liquid sample or liquid biopsy) from the subject to obtain an extracellular fraction;

[0106] enriching for extracellular vesicles (such as exosomes) or extracellular nucleic acids (such as free circulating DNA) from the extracellular fraction to form an enriched fraction;

[0107] separating the enriched fraction into a first fraction and a second fraction;

[0108] contacting the first fraction but not the second fraction with a polymerase under conditions for the amplification of telomeric DNA (e.g. rolling circle amplification);

[0109] quantifying the telomeric DNA levels in the post-amplification first fraction and in the second fraction (e.g. by qPCR);

[0110] expressing the quantity of epc-tDNA in the sample as the fold change of telomeric DNA between the first fraction and the second fraction; and

[0111] based on the quantity or presence of epc-tDNA, determining whether the subject is suitable for treatment with the agent capable of disrupting the FANCM complex.

[0112] In an embodiment, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, wherein the subject has a non-ALT cancer or a degenerative disease, the method comprising:

[0113] enriching for exosomes from a blood sample or fraction of a blood sample to form an enriched fraction; separating the enriched fraction into a first fraction and a second fraction;

[0114] contacting the first fraction but not the second fraction with a polymerase under conditions for the amplification of telomeric DNA (e.g. rolling circle amplification);

[0115] quantifying the telomeric DNA levels in the post-amplification first fraction and in the second fraction (e.g. by qPCR);

[0116] expressing the quantity of epc-tDNA in the sample as the fold change of telomeric DNA between the first fraction and the second fraction; and

[0117] based on the quantity or presence of epc-tDNA, determining whether the subject is suitable for treatment with the agent capable of disrupting the FANCM complex.

[0118] In an embodiment, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, wherein the subject has a non-ALT cancer or a degenerative disease, the method comprising:

[0119] enriching for exosomes from a blood sample or fraction of a blood sample to form an enriched fraction; separating the enriched fraction into a first fraction and a second fraction;

[0120] contacting the first fraction but not the second fraction with a rolling circle polymerase under conditions for the amplification of telomeric DNA (e.g. a φ29 DNA polymerase);

[0121] quantifying the telomeric DNA levels in the post-amplification first fraction and in the second fraction by qPCR;

[0122] expressing the quantity of epc-tDNA in the sample as the fold change of telomeric DNA between the first fraction and the second fraction; and

[0123] based on the quantity or presence of epc-tDNA, determining whether the subject is suitable for treatment with the agent capable of disrupting the FANCM complex. In an embodiment, there is provided a method of determining a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, wherein the subject has a non-ALT cancer or a degenerative disease, the method comprising:

[0124] removal of cells from a liquid biopsy or liquid sample from the subject to obtain an extracellular fraction, optionally wherein the liquid biopsy or liquid sample comprises a blood fraction;

[0125] enriching for exosomes or free circulating DNA from the extracellular fraction to form an enriched fraction;

[0126] separating the enriched fraction into a first fraction and a second fraction;

[0127] contacting the first fraction but not the second fraction with a rolling circle polymerase under conditions for the amplification of telomeric DNA (e.g. a φ29 DNA polymerase);

[0128] quantifying the telomeric DNA levels in the post-amplification first fraction and in the second fraction by qPCR;

[0129] expressing the quantity of epc-tDNA in the sample as the fold change of telomeric DNA between the first fraction and the second fraction; and

[0130] based on the quantity or presence of epc-tDNA, determining whether the subject is suitable for treatment with the agent capable of disrupting the FANCM complex.

[0131] The methods of the first aspect may comprise any method of the fourth aspect.

[0132] In some embodiments, when a subject is determined to be suitable for treatment, a further step of treating the subject with an agent capable of disrupting the FANCM complex is performed. Thus, in some embodiments, the methods of the first aspect further comprise treating the subject with the agent capable of disrupting the FANCM complex if the subject is determined to be suitable for treatment.

[0133] The agent may be any suitable for having a therapeutic effect on FANCM-sensitive subjects. The agent may be one that has a therapeutic effect on ALT cancers. This applies even when the methods of the first aspect are for subjects that do not have an ALT cancer. The agent may be any as described in the section herein titled “Agents for the disruption of the FANCM complex”.

[0134] In a second aspect, there is provided an agent capable of disrupting the FANCM complex for use in treating a non-ALT cancer or a degenerative disease in a subject. The subject may have been determined to be suitable for treatment with said agent by a method of the first aspect. The subject may have been determined to be suitable for treatment by a method of determining the presence of epc-tDNA according to the fourth aspect. The subject may be any as disclosed herein and, in particular, may be a human.

[0135] Thus, there is provided an agent capable of disrupting the FANCM complex for use in treating a non-ALT cancer or a degenerative disease in a subject, wherein the subject has been determined to be suitable for treatment by a method comprising:

[0136] measuring the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF -circles in a sample from the subject; and

[0137] based on the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF -circles, determining whether the subject is suitable for treatment with the agent.

[0138] All of the subject matter and embodiments disclosed for the first aspect and the fourth aspect are also applicable to the second aspect. The agent may be any suitable for having a therapeutic effect on FANCM-sensitive subjects. The agent may be one that has a therapeutic effect on ALT cancers. The agent may be any as described in the section herein titled “Agents for the disruption of the FANCM complex”.

[0139] For instance, the degenerative disease may be Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, spinocerebellar ataxias, or ALS. In a third aspect, there is provided a kit comprising components for carrying out a method of the first aspect. In a fourth aspect, there is provided a method of determining the presence of epc-tDNA in a biological sample, the method comprising:

[0140] separating the sample into a first fraction and a second fraction;

[0141] contacting the first fraction but not the second fraction with a polymerase under conditions for amplification of telomeric DNA,

[0142] quantifying the telomeric DNA levels in the post-amplification first fraction and in the second fraction; and

[0143] determining the presence of epc-tDNA in the sample based on the fold change of telomeric DNA between the first fraction and the second fraction.

[0144] Any subject matter and features disclosed for the first aspect may be applied to the fourth aspect.

[0145] Determining the presence of epc-tDNA may be the measurement of a relative quantity of epc-tDNA. For instance, the relative quantity may be expressed as a fold change. In some instances, determining the presence of epc-tDNA may be the determination of whether epc-tDNA is present or absent. Alternatively, determining the presence of epc-tDNA may be the measurement of the absolute quantity of epc-tDNA. And so, the measurement may lead to a quantification of the actual amount of epc-tDNA in the sample, for instance by mass, copy number, or concentration.

[0146] The biological sample in which the epc-tDNA is detected may be any sample as disclosed for the first aspect. The methods of the fourth aspect may comprise processing a sample from a subject to be suitable for the detection methods disclosed herein. The processing may as disclosed for the first aspect. For instance, the processing may be to obtain an extracellular fraction. The sample may be processed to deplete linear single or double stranded telomeric DNA, as disclosed for the first aspect.

[0147] In some embodiments, the sample may be enriched to enrich a fraction potentially containing epc-tDNA, as disclosed for the first aspect. The sample may be processed to enrich extracellular nucleic acids or extracellular vesicles, as disclosed for the first aspect.

[0148] The amplification of the first fraction may be any amplification as disclosed for the first aspect. For example, selective amplification, isothermal amplification, rolling circle amplification, and / or amplification with a φ29 DNA polymerase.

[0149] The quantification of the telomeric DNA may be by any suitable means, as discussed for the first aspect. For example, hybridisation, sequencing, PCR, molecular beacons, nucleic acid enzymes such as DNA partzymes, and / or by incorporating suitably labelled nucleotides. In one example, the amplification products may be detected using a labelled nucleotide probe. In some embodiments, the telomeric DNA is quantified by the use of PCR, as discussed for the first aspect.

[0150] The presence of epc-tDNA may be expressed as a fold change. The presence of epc-tDNA may be expressed as copy number. The presence of epc-tDNA may be expressed as an absolute value. The presence of epc-tDNA may be expressed as mass. The presence of epc-tDNA may be expressed as concentration. The presence of epc-tDNA may be expressed as the presence or absence of epc-tDNA (i.e. whether epc-tDNA is detectable or not). In particular embodiments, the presence of epc-tDNA may be expressed as the fold change between a first fraction of the sample (a fraction contacted with the polymerase) and a second fraction of the sample. ALT cells and ALT cancers

[0151] As discussed herein, the methods of the first aspect determine a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex, and the methods of the first aspect can be applied to subjects that do not have an ALT+ cancer. An ALT+ cancer is one that comprises ALT cells. The subject may be referred to as not comprising ALT cells or not comprising detectable ALT cells.

[0152] In order to achieve unlimited proliferation, cancer cells must maintain their telomeres. The majority of cancers (85-90%) achieve this by reactivating telomerase (telomerase- positive cells). Telomerase is a reverse transcriptase enzyme involved in synthesizing telomeric DNA from an RNA template. The sequence of telomerase is publicly available. The remaining 10-15% of tumour cells must stabilize their chromosome ends by alternative mechanisms to avert cessation of growth. These telomerase independent strategies are collectively known as ALT. Thus, an ALT cell as defined herein may be a cell exhibiting an active ALT mechanism. The ALT mechanism may 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 may operate in a cell to maintain telomeres. Thus, “ALT mechanism” does not necessarily refer to only one specific biochemical mechanism or pathway by which an ALT mechanism operates. There may be more than one specific pathway by which ALT operates. Thus, an ALT cell may be any cell which does not rely on telomerase activity to maintain telomere length.

[0153] Alternatively or in addition, an ALT cell may be considered to be any cell which does not rely on telomerase activity to maintain telomere stability. Thus, an ALT cell may be considered to be a cell which is not telomerase positive; or a telomerase negative cell.

[0154] Alternatively or in addition, an ALT cell may be considered to be a cell in which telomerase expression and / or activity is reduced compared to a telomerase positive cell. Expression and / or activity of telomerase can be determined by any means. For example, telomerase expression levels can be determined by quantifying the level of production of telomerase mRNA by any suitable method of mRNA detection (for example but without limitation: quantitative PCR, real time qPCR; next generation sequencing (NGS) methods; nanopore sequencing methods; northern blotting; and others). Alternatively or in addition, telomerase expression levels can be determined by quantifying the level of production of telomerase protein by any suitable protein detection methods. Telomerase protein levels can be detected, for example but without limitation, by western blotting; antibody detection methods (e.g., ELISA; or detection of a label such as a fluorescent label conjugated to an antibody capable of binding specifically to telomerase); and other methods. Alternatively or in addition, telomerase expression and / or activity levels can be determined through performance of a telomerase functional assay, wherein the level of telomerase activity is indicative of the level of expression and / or activity of telomerase in a 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 that described in Cohen and Reddel, 2008. It will be appreciated that any of the methods of identifying an ALT cell or identifying an ALT cancer or determining whether a subject is suffering from ALT cancer disclosed herein may comprise determining whether a cell is a telomerase positive cell by any of the methods disclosed herein, wherein the cell is identified as not being an ALT cell if that cell is determined to be a telomerase positive cell.

[0155] ALT cells may be characterized by elevated levels of DNA damage compared to mortal or telomerase-positive cells, indicative of heightened telomeric replication stress in ALT cells. This heightened telomeric replication stress is attributed to cumulative inadequacies in telomere structural integrity. Frequent or persistent replication fork stalling causes nicks and breaks in the DNA, and it has been hypothesized that the ALT mechanism emanates from stalled replication forks that deteriorate to form double stranded breaks (DSBs), which then provide the substrate for the engagement of homology -directed repair pathways, culminating in break induced telomere synthesis. ALT cells therefore achieve a fine balance between telomere protection and repair activities and telomere damage, and disruption of this balance can be applied as a means of dysregulating the ALT mechanism. An ALT cell as defined herein may be identified by detection of one or more phenotypic traits of ALT telomere repair or ALT telomeric replication stress, including any one or more of: replication fork stalling above a level that is typical of non-ALT cells (e.g., above a level that is typical of telomerase positive cells or mortal cells); DSB occurrence above a level that is typical of non-ALT cells (e.g., above a level that is typical of telomerase positive cells or mortal cells); and others. It will be appreciated that levels of telomeric replication fork stalling and / or DSBs that are typical of ALT cells can be established through identification and / or measurement of these traits in a sample of ALT cells and in a sample of non-ALT cells (e.g., telomerase positive cells or mortal cells). Suitable threshold levels can then be determined according to the particular methodology used to identify and / or measure these traits, such that a given cell can then be identified as an ALT cell or a non-ALT cell using the same or similar methodology. It will be appreciated that the precise thresholds will vary depending on the samples used to establish those threshold levels and according to the particular analytical methodology used in each instance. ALT involves recombination-dependent DNA replication (Dunham et al., 2000) and ALT may generate sudden, large increases in telomere length (Murnane et al., 1994), consistent with either a long, linear telomeric template or a rolling mechanism, such as rolling circle amplification (RCA). Cells with ALT activity also undergo rapid decreases in individual telomere lengths (Jiang et al., 2005 and Perrem et al., 2001) leading to 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, an ALT cell as defined herein may comprise any one or more of these phenotypic traits. For example, an ALT cell may exhibit recombination-dependent DNA replication, and / or may exhibit sudden, large increases in telomere length (e.g., compared to a non-ALT cell such as a telomerase positive cell or a mortal cell), and / or may exhibit heterogeneous telomere length distribution (e.g., compared to a non-ALT cell such as a telomerase positive cell or a mortal cell), and / or may comprise APBs (e.g., a level of APBs that is greater than in a non-ALT cell, such as a telomerase positive cell or a mortal cell). Alternatively, an ALT cell 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 appreciated that telomeres are repetitive DNA sequences present at or near the termini of linear chromosomes. Telomeres in humans typically comprise multiple repeats of the nucleotide sequence 5’-TTAGGG-3’. Thus, the identification of telomere length may comprise determining the number of repeats of this nucleotide sequence.

[0156] The ALT cell may be a cancer cell.

[0157] Agents for the disruption of the FANCM complex

[0158] The methods of the first aspect determine a subject’s suitability for treatment with an agent capable of disrupting the FANCM complex. In addition, the methods of the first aspect may comprise treating subjects deemed to be suitable with said agent. Exemplary agents are disclosed in W02020 / 242330 A2 (herein incorporated by reference).

[0159] The agent may be any that disrupts the FANCM complex in a manner that has a therapeutic effect on FANCM-sensitive patients. The agent may inhibit cell viability and / or cell growth of FANCM-sensitive cells. The agent may induce synthetic lethality of FANCM-sensitive cells. The agent may be referred to as a FANCM inhibitor. The agent may prevent the assembly of the FANCM complex as illustrated in Figure 2. The agent may reduce the expression, function, or binding capability of a member of the FANCM complex. The agent may target FANCM or may target RMI. The agent may disrupt the FANCM-RMI interaction.

[0160] FANCM is an ATP-dependent DNA helicase / translocase (EC 3.6.4.13) involved in homologous recombination, meiosis and DNA repair. The human gene (Gene ID: 57697) encoding the FANCM polypeptide has 25 exons and is located at 14q21.2. Reference human FANCM amino acid sequences have the database accession numbers NP 001295062.1, NP 1295063.1, and NP 065988.1. Reference humanFANCM coding sequences have the database accession numbers NM 001308133.1, NM 001308134.1, and NM 020937.4.

[0161] A non-limiting exemplary human FANCM amino acid sequence is:

[0162] MSGRQRTLFQTWGSSISRSSGTPGCSSGTERPQSPGSSKAPLPAAAEAQLESDDDVLLVAAYEAERQLCLENGGFC TSAGALWIYPTNCPVRDYQLHISRAALFCNTLVCLPTGLGKTFIAAWMYNFYRWFPSGKWFMAPTKPLVTQQIE ACYQVMGIPQSHMAEMTGSTQASTRKEIWCSKRVLFLTPQVMVNDLSRGACPAAEIKCLVIDEAHKALGNYAYCQA VQQVITNLLIGQIELRSEDSPDILTYSHERKVEKLIVPLGEELAAIQKTYIQILESFARSLIQRNVLMRRDIPNLT KYQIILARDQFRKNPSPNIVGIQQGIIEGEFAICISLYHGYELLQQMGMRSLYFFLCGIMDGTKGMTRSKNELGRN EDFMKLYNHLECMFARTRSTSANGISAIQQGDKNKKFVYSHPKLKKLEEWIEHFKSWNAENTTEKKRDETRVMIF SSFRDSVQEIAEMLSQHQPIIRVMTFVGHASGKSTKGFTQKEQLEWKQFRDGGYNTLVSTCVGEEGLDIGEVDLI ICFDSQKSPIRLVQRMGRTGRKRQGRIVIILSEGREERIYNQSQSNKRSIYKAISSNRQVLHFYQRSPRMVPDGIN PKLHKMFITHGVYEPEKPSRNLQRKSSIFSYRDGMRQSSLKKDWFLSEEEFKLWNRLYRLRDSDEIKEITLPQVQF SSLQNEENKPAQESTTGIHQLSLSEWRLWQDHPLPTHQVDHSDRCRHFIGLMQMIEGMRHEEGECSYELEVESYLQ MEDVTSTFIAPRNESNNLASDTFITHKKSSFIKNINQGSSSSVIESDEECAEIVKQTHIKPTKIVSLKKKVSKEIK KDQLKKENNHGIIDSVDNDRNSTVENIFQEDLPNDKRTSDTDEIAATCTINENVIKEPCVLLTECQFTNKSTSSLA GNVLDSGYNSFNDEKSVSSNLFLPFEEELYIVRTDDQFYNCHSLTKEVLANVERFLSYSPPPLSGLSDLEYEIAKG TALENLLFLPCAEHLRSDKCTCLLSHSAVNSQQNLELNSLKCINYPSEKSCLYDIPNDNISDEPSLCDCDVHKHNQ NENLVPNNRVQIHRSPAQNLVGENNHDVDNSDLPVLSTDQDESLLLFEDVNTEFDDVSLSPLNSKSESLPVSDKTA ISETPLVSQFLISDELLLDNNSELQDQITRDANSFKSRDQRGVQEEKVKNHEDIFDCSRDLFSVTFDLGFCSPDSD DEILEHTSDSNRPLDDLYGRYLEIKEISDANYVSNQALIPRDHSKNFTSGTVIIPSNEDMQNPNYVHLPLSAAKNE ELLSPGYSQFSLPVQKKVMSTPLSKSNTLNSFSKIRKEILKTPDSSKEKVNLQRFKEALNSTFDYSEFSLEKSKSS GPMYLHKSCHSVEDGQLLTSNESEDDEIFRRKVKRAKGNVLNSPEDQKNSEVDSPLHAVKKRRFPINRSELSSSDE SENFPKPCSQLEDFKVCNGNARRGIKVPKRQSHLKHVARKFLDDEAELSEEDAEYVSSDENDESENEQDSSLLDFL NDETQLSQAINDSEMRAIYMKSLRSPMMNNKYKMIHKTHKNINIFSQIPEQDETYLEDSFCVDEEESCKGQSSEEE VCVDFNLITDDCFANSKKYKTRRAVMLKEMMEQNCAHSKKKLSRIILPDDSSEEENNVNDKRESNIAVNPSTVKKN KQQDHCLNSVPSGSSAQSKVRSTPRVNPLAKQSKQTSLNLKDTISEVSDFKPQNHNEVQSTTPPFTTVDSQKDCRK FPVPQKDGSALEDSSTSGASCSKSRPHLAGTHTSLRLPQEGKGTCILVGGHEITSGLEVISSLRAIHGLQVEVCPL NGCDYIVSNRMWERRSQSEMLNSVNKNKFIEQIQHLQSMFERICVIVEKDREKTGDTSRMFRRTKSYDSLLTTLI GAGIRILFSSCQEETADLLKELSLVEQRKNVGIHVPTWNSNKSEALQFYLSIPNISYITALNMCHQFSSVKRMAN SSLQEISMYAQVTHQKAEEIYRYIHYVFDIQMLPNDLNQDRLKSDI ( SEQ ID NO: 1 )

[0163] A non-limiting exemplary human FANCM coding sequence is: tgtgcgaaggaaaccgatggggatcggaaccgtagcggttgagctgctgctgctacggatatctgacagaagcctt cggtggttgtcggcctaatgagcggacggcaaagaacgctttttcagacgtggggctcaagtatctcccgatcatc tgggactccgggttgcagctccggaactgagcgacctcagagccctggcagctccaaggcgcctttgccagcagca gcggaggctcagctggagtcggacgatgatgtgttgcttgtcgcggcgtacgaggctgagcggcagttgtgtctag agaatggcgggttctgcacctccgcgggcgccctgtggatttaccctaccaattgcccagtgcgggactaccagct gcacatttcccgggctgctctgttttgcaatacgctggtgtgtctgcctaccggactgggaaagacctttattgcc gccgtggtcatgtacaatttctaccgctggttcccttcaggaaaggtggtcttcatggccccaacgaaacccttgg tgacacagcagatcgaggcttgctaccaggtgatgggtatcccgcaatcccacatggccgaaatgacagggtctac acaagcttccaccaggaaggaaatatggtgcagtaagagagtgctttttcttacacctcaggtcatggtaaatgac ctttctagaggagcttgtcccgctgctgaaataaagtgtttagttattgatgaagctcataaagctctcggaaact atgcttattgccaggctgtgcaacaagttattactaacctgctaattgggcagatagagcttcgttctgaagattc tccagatattttgacatattctcatgaaagaaaagttgaaaagcttattgttccgcttggtgaagaacttgcagcc atccaaaagacctatatccagattttggaatcatttgctcgttctttgattcagaggaatgttttgatgagaaggg atatcccaaatctaacaaaatatcagataattctggcaagagatcagtttaggaaaaacccatctccgaatattgt gggaatacaacaaggcataatcgagggagagtttgctatttgtattagtttatatcatggttatgaattattgcag caaatgggaatgagatcattatatttcttcctttgtggaattatggatggaactaaagggatgacacggtcaaaaa atgaacttggccgaaatgaagacttcatgaaactctataatcatctagagtgtatgtttgcacgtacacgtagtac ttcagcaaatggtatttctgctatccaacaaggagataaaaataaaaaatttgtttatagtcatccaaagttaaag aaattagaagaagttgtaattgaacacttcaagtcatggaatgctgaaaacactactgaaaagaaacgtgatgaga cccgagttatgatcttctcttcatttcgagatagtgttcaagaaattgcagaaatgctttcacagcatcagccaat tattagagtaatgacttttgtcggccatgcctcagggaaaagcacgaagggttttacccagaaggagcaactggag gtagtgaaacagtttcgtgacggtggttacaacacgctggtttctacctgtgtgggtgaagaaggtttggatatag gagaagttgatcttataatatgttttgattcccagaagagcccaattcgtcttgtacaacgaatgggtagaactgg ccgtaaacgtcaaggcaggatagttattatcctttctgaaggacgagaggaacgtatttataatcagagtcagtcc aacaaaagaagtatatataaagctatttcaagtaacaggcaggtccttcatttttaccaaagaagtccacgaatgg ttcctgatggaatcaacccaaaattacacaaaatgttcatcacacatggtgtctatgaaccagagaagccttctcg gaacttgcagcgaaagtcatctatcttttcctatagggatggaatgaggcaaagtagcctaaagaaagattggttc ttatcagaagaagaatttaaattatggaacagactttatagattaagggacagtgatgaaattaaagagataacat tgcctcaagttcagttttcttctttacaaaatgaggaaaacaaaccagctcaagaatcaaccactggaattcatca actctctctctctgaatggagactgtggcaagatcatcctttgcctacacatcaagttgatcactcagatcgatgc cgccattttataggccttatgcaaatgatagagggaatgagacacgaagagggagaatgcagctatgaattggaag ttgaatcttatttacaaatggaagatgttacctcaacatttattgctcccaggaatgaatctaataatcttgccag tgacacctttatcactcacaagaaatcgtcatttataaagaacataaatcaaggcagttcatcctcagtgatagaa tctgatgaagaatgtgctgaaattgttaaacaaactcatatcaaacctactaaaattgtttctttaaagaaaaaag tgtctaaagaaataaaaaaagatcagcttaaaaaagaaaataatcacggtattatagattctgtagataatgacag aaattccactgttgaaaatatttttcaagaagacctaccaaatgataaaaggacatcagatacagatgaaattgct gccacatgtactattaatgaaaatgttattaaagaaccgtgtgtgttattaacagagtgtcagtttacaaataaat ccactagttcacttgctggaaatgttttagattctggttataacagtttcaatgatgaaaaatctgtttcatctaa cttatttcttccattcgaagaagagctttatattgttagaacagatgaccaattttataattgtcactcattgaca aaagaggtactagctaatgtagagagatttttatcttattctcctccgcctctcagtggactctcagacttggaat atgaaattgctaagggtactgcacttgagaatttgcttttcttaccctgtgcagagcatttacgaagtgataaatg cacctgtttgctgtcacattcagctgtgaattctcaacagaatttagaattgaattcacttaaatgtataaattat ccatctgaaaaaagttgcctttatgatatacctaatgataatatttctgatgagccaagtctctgtgactgtgatg tacataaacataatcaaaatgaaaatttagtacctaacaatcgtgttcaaatacacagaagccctgcacagaattt agttggagagaacaatcatgatgttgataacagtgacctcccagtattgtccactgatcaagatgaaagtttgctg ttatttgaagatgttaatacagagttcgacgatgtgagtctttcacccttgaacagtaaaagcgaatctttacctg tgtcagacaaaactgctattagtgaaacgcctctggtctctcagttcttaatttctgatgaacttttgttggacaa taattctgaactccaagatcaaatcacccgtgatgctaatagttttaaatctcgtgatcagagaggtgtacaggaa gaaaaagtgaagaatcatgaggatatttttgattgctctagggatttattttctgttacctttgatttaggattct gtagtccagattctgatgatgaaatattggaacatacatcagatagcaatagacctctagatgatctatatggaag gtatttggaaattaaggagataagtgatgcaaattatgtttcgaatcaagcactaataccaagagatcatagtaaa aattttactagtggaactgttattatcccatcaaatgaagatatgcagaatccaaattatgtacatttgccactga gtgcagcaaaaaatgaagaattgttatctcctggttattctcagttttctttaccagtgcaaaaaaaagttatgag tacaccactctctaaatcaaacacattgaactcattttctaagataagaaaggaaatacttaagacaccagattct agtaaggaaaaagtaaacctacaaagattcaaagaagcattgaattcaacttttgattattcagaattttctctag aaaagtctaaaagcagtggtccaatgtatctgcataaatcctgtcattctgttgaagatggacaattattaacaag taacgaaagtgaagatgacgagattttccgaagaaaagttaaaagagcaaaaggaaatgttttaaactctcctgag gatcagaaaaatagtgaagttgattctccacttcatgctgtcaaaaagcgcagatttcctataaacagatcagaat tatcatctagtgatgagagtgagaattttcccaaaccatgttcacaattagaagacttcaaggtttgtaacgggaa tgccagaagaggcatcaaagtcccaaagagacagagtcacttaaagcatgtagctaggaagtttttagatgatgaa gcagaactttctgaagaagatgcagaatatgtttcatcagatgaaaatgatgagtcagaaaatgaacaagattcct cattacttgactttttaaatgatgaaactcaactttcacaggctataaatgattctgaaatgagagctatttacat gaaatctttgcgtagtccaatgatgaacaataagtacaaaatgattcataagacacataaaaacataaacattttc tcgcagattcctgaacaagatgaaacctatttagaggatagtttttgtgttgatgaagaggagtcttgcaaaggcc aatcaagtgaagaagaagtttgtgttgattttaacttaataactgatgattgctttgcaaatagtaaaaagtataa aactcgacgtgcagtaatgctaaaagaaatgatggaacaaaattgtgcacattcaaaaaagaaattatccagaatt attttaccagatgattcaagtgaggaggagaacaatgtaaatgataaaagagaatctaatattgcggttaacccaa gcactgttaagaagaacaaacaacaggaccattgtttaaattcagtgccttctggatcttctgcgcagtccaaggt gcgttctactccaagagttaatccattagcaaagcagagcaaacagacatcgctgaatttaaaggatacaatttcc gaagtctcagacttcaaacctcagaatcataatgaagtccagtctaccacaccacccttcactactgttgattcac agaaagactgtagaaaatttccagttccacagaaggatggtagtgctttggaggattctagcacttcaggggcatc ctgttccaagtcaagaccacatttagctgggacacatacttctcttagacttccgcaggaaggaaaaggaacctgt attcttgtaggtggtcatgaaatcacttctggattagaagtaatttcttccctaagagcaattcatgggttgcaag tagaagtttgtcctcttaatggctgtgattacatcgtgagtaatcgcatggtggtggaaaggaggtctcaatctga gatgttaaatagtgtcaataagaacaagttcattgagcagatccagcacctgcagagtatgtttgaaagaatatgt gtgattgtggaaaaggacagagaaaaaacaggagacacatcaaggatgtttaggagaacaaagagctatgacagcc tgctgactaccttaattggcgctggaatccgaattcttttcagttcctgccaagaagaaaccgcagatttgctaaa ggaactgtctttagtggaacaaagaaagaatgttggtattcatgttccaacagtggtgaatagtaataaaagtgag gcactccagttttatttaagtattcccaatataagttatataactgcattaaatatgtgtcaccagttttcatctg t gaaaagga t ggct aacagctcacttcaagaaatctccatgt a tgcacaagtaactcatcagaaggct gaggaga t ctatagatatattcactatgtatttgacatacaaatgttaccaaatgatcttaaccaagatagactgaaatctgat atataatcaagctgctcaagatggggttttcaaagacctctcacaatattaaatgcacttcaataatcattgctgt tttatgtttatttgtaaataagagaatattttatttaaatattttatattgtatacatttttatttatagattata gaaattattaaaaaagaaaaatctgatgttcagtgatcattttgactagattataaaactaatttttcttattaaa taaaacaaggtttattaaaagtgttactaaggatagtttaagaaagtaaaagctaagctagagatatactttggaa tgtttcccaaaattaaagttgtactgttgtgataaatagtaaagttgacatgtctatgactacagccaacttgtcg attttccctatgtgtagatagtatacttttaagtgtactgattctaaatacatgtacttggtaaggtgtgggtgat gggtgggttgtgagataaatgacccagtaactaggaaagtagaaaacttaactgaatgtttatctgaccaaaggtg tgtcccagttaagtactgtcaaatctattaatatgaactctgatatggtttggctgtgtccccaaccaaaatctca tcttgacttgtaatctgaattataatcccaatatattggggagggacctcctggaacgtgattagctcatgggggc ggttcccccatgctgttctagtgatagttctcagaggatctgatggttttataagcttttcctctgttcactctgc agttctcttgcctactgccatgtggaaaaggaaacgtttgcttcccctccaccatgattgtaagttcccgaggcct ccccagccatgcaggactgtgagtcaattaaacatcttttccttataaattaaaaaaaaaaaaaaaaa ( SEQ ID NO: 2 )

[0164] The inhibitors may disrupt the FANCM-RMI interaction. In other embodiments, the inhibitors may reduce or abolish FANCM expression or activity. In certain embodiments, FANCM inhibitors inhibit or reduce FANCM ATPase activity.

[0165] It will be understood by a person skilled in the art that FANCM inhibitor may be a direct inhibitor or an indirect inhibitor of FANCM, i.e., it may exert its inhibitory effect by directly binding to FANCM, or a nucleic acid sequence encoding FANCM, or it may exert its inhibitory effect indirectly, e.g., by inhibiting another protein required for FANCM expression or activity. In particular embodiments, an inhibitor such as those disclosed herein may be capable of inhibiting FANCM such that one or more endogenous activity of FANCM is inhibited. In certain embodiments, a FANCM inhibitor inhibits or reduces DNA translocase activity or ATPase activity of FANCM. In particular embodiments, a FANCM inhibitor is capable of inhibiting or reducing ALT activity of an ALT cell, e.g., an ALT cancer cell, enough though it is for use in treating a subject with a non-ALT cancer as a part of the methods of the present invention. In particular embodiments, FANCM DNA translocase activity or ATPase 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, as compared to the activity present in an ALT cell not contacted with the FANCM inhibitor. In particular embodiments, 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, as compared to the activity present in an ALT cell not contacted with the FANCM inhibitor.

[0166] In certain embodiments, the FANCM-RMI interaction may be disrupted by upstream or downstream effectors 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 may bind to FANCM to inhibit its function by changing its conformation or by affecting its binding site such that it is no longer able to bind to RMI. In another example, the inhibitor may bind to RMI to inhibit its function by changing its conformation or by affecting its binding site such that it is no longer able to bind to FANCM. For example, the inhibitor may disrupt the RMI1-RMI2 subcomplex such that it is no longer able to bind to FANCM. In one example, the binding of FANCM to RMI is disrupted at 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. FANCM inhibitors may be any type of molecule with inhibitory activity, for example, small chemical molecules, polypeptides (which includes peptides and proteins), nucleic acids, or molecules comprising a combination of any of these classes of molecules. The terms “FANCM inhibitor” or “agent capable of disrupting the FANCM complex” as used herein, cover pharmaceutically acceptable salts and solvates of any inhibitors, agents, biological molecules, or compounds disclosed herein.

[0167] In particular embodiments, an inhibitor may cause a reduction in the expression of a target protein, e.g., FANCM, or a reduction in one or more biological activities of a target protein, e.g., FANCM, in each case, e.g., a reduction of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100%. Reducing the amount of active FANCM protein to 20% of the amount in control cells or lower is shown to be sufficient to induce cell death. For example, a cell may express up to 5%, up to 10%, up to 15% or up to 20%, of the active FANCM polypeptide that is expressed by control cells. In particular embodiments, FANCM ATPase activity is reduced, e.g., by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% as compared to a control cell not treated with the inhibitor.

[0168] FANCM inhibitors may, for example, include biological molecules that specifically bind to FANCM. The ATPase activity of FANCM is located within the amino-terminal DEAH helicase-like domain, responsible for translocase and branch migration activities. This ATPase activity is generally found to be dispensable for core complex targeting and FANCD2 ubiquitination but is required for replication fork stability and efficient checkpoint response. In some embodiments, a biological molecule may specifically bind to the region of FANCM associated with ATPase activity. In some embodiments, a biological molecule may specifically bind to the DEAH helicaselike domain corresponding to residues 83-591 of SEQ ID NO: 1. In some embodiments, a biological molecule may specifically bind to the MM2 domain of FANCM corresponding to EDIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO:3).

[0169] FANCM inhibitors may, for example, include small chemical molecules, for example non-polymeric organic compounds having a molecular weight of 900 Daltons or less. Suitable small molecule FANCM inhibitors may, for example, inhibit ATP binding to the ATPase domain of FANCM; DNA binding to the translocase domain of FANCM and / or FANCM binding to a binding partner, such as MHF, FAAP24, BLM, RMI, Topo Illa.

[0170] In one example, the FANCM inhibitor is a small molecule.

[0171] In certain embodiments, an inhibitor is a biological molecule, such as a polypeptide, e.g., a peptide or protein. Peptides may comprise or consist of from 5 to 40 amino acids, for example, from 6 to 10 amino acids and may be derived from FANCM or binding partners thereof as described herein. Polypeptide molecules may also include antibodies, antibody fragments and antibody derivatives and non-immunoglobulin binding molecules, such as aptamers, trinectins, anticalins, kunitz domains, transferrins, nurse shark antigen receptors and sea lamprey leucine-rich repeat proteins. Suitable techniques for the generation of biological molecules that specifically bind to FANCM are well known in the art. The peptide may be any peptide mimicking all or part of the MM2 domain of FANCM. For example, the peptide may 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:4). The peptide may 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: 4). For example, the peptide may 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: 5). The peptide may 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: 5). The peptide may comprise from 0 to 5, 0 to 4, 0 to 3, 0 to 2, 1, or 0 substitutions, insertions or deletions relative to SEQ ID NO: 4 or SEQ ID NO: 5. In particular, the peptide may comprise from zero to three substitutions relative to SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the peptide may be a peptide comprising or consisting of an amino acid sequence that is at least 90% identical to EDIFDCSRDLFSVTFDLGFCSPDSDDEILEHTSD (SEQ ID NO: 3). The peptide may 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: 3).

[0172] In some embodiments, the peptide or protein FANCM inhibitor may be any peptide capable of binding to FANCM and capable of occluding the MM2 binding domain. The occlusion may be such that the normal (endogenous) binding interaction between FANCM and its endogenous binding partner is disrupted. The peptide or protein may be capable of binding to the MM2 binding domain directly or indirectly. Alternatively, the peptide or protein may be any peptide of the RMI complex capable of binding to FANCM.

[0173] In some embodiments, a FANCM inhibitor may be a mutant FANCM protein that has reduced binding capability to the RMI protein or a mutant RMI protein that has reduced binding capability to the FANCM protein. Without wishing to be bound by theory, such proteins may act as decoys to the endogenous FANCM or RMI proteins, saturating the available binding sites on the endogenous proteins and thereby inhibiting their function. In one example, the mutant FANCM may be an inactivated FANCM protein comprising a F1232A / F1236A double substitution. The protein may comprise an immunoglobulin binding domain.

[0174] In some embodiments, the inhibitor may be a genetic inhibitor of FANCM or of RMI. Methods of 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 RNAs, antisense RNAs, mRNAs, tRNAs, rRNAs, small interfering RNAs (siRNAs), short hairpin RNAs (ShRNAs), micro RNAs (miRNAs), small nucleolar RNAs (SnoRNAs), small nuclear RNAs (snRNAs), ribozymes, aptamers, DNAzymes, antisense oliogonucleotides, vectors, plasmids, 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.

[0175] Examples of suitable genetic inhibitors include siRNA inhibitors comprising or consisting of GCUCGUUCUUUGAUUCAGA (SEQ ID NO: 6) or GAACAUACAUCAGAUAGCA (SEQ ID NO: 7).

[0176] In certain embodiments, inhibitors for reducing or suppressing FANCM expression include suppressor nucleic acids, targetable nucleases and nucleic acids encoding such agents. Nucleic acids encoding a suppressor nucleic acid or targetable nuclease may be contained in a vector. Suitable expression vectors are well-known in the art and include viral vectors, such as retroviral, adenoviral, adeno-associated viral, lentiviral, vaccinia or herpes vectors. The expression of active FANCM protein may be reduced by a suppressor nucleic acid or targetable nuclease compared to a control cell or may be absent, i.e. the transcription of the FANCM gene and / or translation of FANCM mRNA may be reduced or absent, such that the cell treated with the suppressor nucleic acids or targetable nuclease lacks or has a reduced amount of active FANCM protein compared to a control cell. Reducing the amount of active FANCM protein to 20% of the amount in control cells or lower is shown to be sufficient to induce cell death. For example, a cell may express up to 5%, up to 10%, up to 15% or up to 20%, of the active FANCM polypeptide that is expressed by control cells.

[0177] In some embodiments, nucleic acid suppression may be used to reduce the expression of active FANCM polypeptide. The use of nucleic acid suppression techniques such as anti-sense and RNAi suppression, to downregulate expression of target genes is well-established in the art.

[0178] Cells may be transfected with a suppressor nucleic acid (i.e. a nucleic acid molecule which suppresses FANCM expression), such as an siRNA or shRNA, or a heterologous nucleic acid encoding the suppressor 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 cells.

[0179] RNAi involves the expression or introduction into a cell of an RNA molecule which comprises a sequence which is identical or highly similar to the FANCM coding sequence. The RNA molecule interacts with mRNA which is transcribed from the FANCM gene, resulting in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of the mRNA. This reduces or suppresses expression of active FANCM polypeptide (Angell & Baulcombe (1997) The EMBO Journal 16, 12:3675-3684; Voinnet & Baulcombe (1997) Nature 389: Pg 553).

[0180] 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 expression of endogenous and heterologous genes in a wide range of mammalian cell lines (Elbashir SM. et al. Nature, 411, 494-498, (2001)).

[0181] Suitable RNA molecules for use in RNAi suppression include short interfering RNA (siRNA). siRNA are double stranded RNA molecules of 15 to 40 nucleotides in length, preferably 15 to 28 nucleotides or 19 to 25 nucleotides in length, for example 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, two unmodified 21 mer oligonucleotides may be annealed together to form a siRNA. A siRNA molecule may contain a 3' and / or 5' overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The overhang lengths of the strands are independent, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand.

[0182] Other suitable RNA molecules for use in RNAi include small hairpin RNAs (shRNAs). shRNA are single-chain RNA molecules which comprise or consist of a short (e.g. 19 to 25 nucleotides) antisense nucleotide sequence, followed by a nucleotide loop of 5 to 9 nucleotides, and the complementary sense nucleotide sequence (e.g. 19 to 25 nucleotides). Alternatively, the sense sequence may precede the nucleotide loop structure, and the antisense sequence may follow. The nucleotide loop forms a hairpin turn which allows the base pairing of the complementary sense and antisense sequences to form the shRNA.

[0183] A suppressor nucleic acid, such as a siRNA or shRNA, may comprise or consist of a sequence which is identical or substantially identical (i.e. at least 90%, at least 95% or at least 98% identical) to all or part (for example, 15 to 40 nucleotides) of a reference FANCM nucleotide coding sequence, such as SEQ ID NO: 2, or its complement. Suitable reference sequences coding FANCM that may be used for the design of suppressor nucleic acids are publicly available and include SEQ ID NO 2. FANCM activity is suppressed in the cancer cells by downregulation of the production of active FANCM polypeptide by the suppressor nucleic acid. For example, a siRNA to suppress the expression of human FANCM may comprise 18 to 22 contiguous nucleotides from SEQ ID NO: 2. Examples of preferred siRNA molecules for the suppression of human FANCM include GGAUGUUUAGGAGAACAAAGAGCUA (SEQ ID NO: 8 - siFa) and CCCAUCAAAUGAAGAUAUGCAGAAU (SEQ ID NO: 9 - siFb).

[0184] Suppressor nucleic acids, such as siRNAs and shRNAs, for reducing FANCM expression may be readily designed using reference FANCM coding sequences and software tools which are widely available in the art and may be produced using routine techniques. For example, a suppressor nucleic acid may be chemically synthesized; produced recombinantly in vitro or cells (Elbashir, S. M. etal., 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)).

[0185] In some embodiments, two or more suppressor nucleic acids may be used to suppress the expression of FANCM. For example, a pool of siRNAs may be employed. Suitable siRNAs and siRNA pools may be produced using standard techniques.

[0186] Nucleic acid suppression may also be carried out using anti-sense techniques. Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of the base excision repair pathway component so that its expression is reduced or completely or substantially completely prevented. In addition to targeting coding sequence, anti-sense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the anti-sense oligonucleotides can interfere with expression control sequences. The construction of anti-sense sequences and their use is well known in the art (Peyman and Ulman, Chemical Reviews, 90:543-584, (1990); Crooke, Ann. Rev. Pharmacol. Toxicol.

[0187] 32:329-376, (1992)).

[0188] Anti-sense oligonucleotides may be generated in vitro or ex vivo for administration or anti-sense RNA may be generated in vivo within the cancer cells in which down-regulation of FANCM is desired. Thus, double-stranded DNA may be placed under the control of a promoter in a "reverse orientation" such that transcription of the antisense strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the sense strand of the target gene. The complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein.

[0189] The complete sequence corresponding to the FANCM coding sequence in reverse orientation need not be used. For example, fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A suitable fragment may have about 14-23 nucleotides, e.g. about 15, 16 or 17.

[0190] In other embodiments, targeted mutagenesis may be used to reduce the expression of active FANCM polypeptide. The use of targeted mutagenesis techniques such as gene editing, to knock out or abolish expression of target genes is well established in the art (see for example Gaj et al (2013) Trends Biotechnol. 31(7) 397-405).

[0191] One or more mutations, such as insertions, substitutions or deletions, may be introduced into the FANCM gene in the cancer cells. Suitable mutations include deletions of all or part of the FANCM gene, for example, one, two or more exons, frameshift mutations, or nonsense mutations introducing premature stop codons. In some preferred embodiments, one or more premature stop codons may be introduced into the FANCM coding sequence.

[0192] Preferably, mutations, such as premature stop codons, are introduced into the first 400 codons of the coding sequence, to eliminate the ATPase domain of FANCM. The mutations may prevent the expression of active FANCM polypeptide, for example by impairing transcription or translation of the FANCM gene or causing an inactive polypeptide to be expressed.

[0193] Targeted mutagenesis to introduce one or more mutations may be performed by any convenient method. For example, the cancer cells may be transfected with a heterologous nucleic acid which encodes a targetable nuclease. The targetable nuclease may inactivate the FANCM gene encoding FANCM in one or more cells of the individual, for example, by introducing one or more mutations that prevent the expression of active FANCM polypeptide.

[0194] The targetable nuclease may inactivate the FANCM gene encoding FANCM selectively in cancer cells of the individual. The targetable nuclease may be targeted to specifically to cancer cells by conventional techniques, including cell targeted delivery vehicles, such as viral vectors that express a ligand for a specific cell type; direct administration of the targetable nuclease to a tumour e.g. by injection; or the expression of the targetable nuclease from heterologous nucleic acid selectively in cancer cells, for example using a tissue specific promoter.

[0195] The targetable nuclease may be site-specific (e.g. ZFN or TALEN) or may be expressed with one or more targeting sequences that target the nuclease to the FANCM gene (e.g. CRISPR / Cas).

[0196] The heterologous nucleic acid encoding the targetable nuclease may include an inducible promoter that promotes expression of the targetable nuclease and optional targeting sequence within a specific cell type, for example a tumour cell. For example, the inducible promoter could be a promoter-enhancer cassette that selectively favours expression of the targetable nuclease and the optional targeting sequence within the tumour cell over other types of host cells.

[0197] Suitable targeting 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.

[0198] Zinc-finger nucleases (ZFNs) comprise one or more Cys₂-His₂ zinc-finger DNA binding domains and a cleavage domain (i.e., nuclease). The DNA binding domain may be engineered to recognize and bind to any nucleic acid sequence using conventional techniques (see for example Qu et al. (2013) Nucl Ac Res 41(16):7771-7782). The use of ZFNs to introduce mutations into target genes is well-known in the art (see for example, Beerli et al Nat. Biotechnol.2002; 20:135-141; Maeder et al Mol. Cell. 2008; 31:294-301; Gupta et al Nat. Methods. 2012; 9:588-590) and engineered ZFNs are commercially available (Sigma-Aldrich (St. Louis, MO).

[0199] Transcription activator-like effector nucleases (TALENs) comprise a nonspecific DNA-cleaving nuclease fused to a DNA-binding domain comprising a series of modular TALE repeats linked together to recognise a contiguous nucleotide sequence. The use of TALEN targeting nucleases is well known in the art (e.g. Joung & Sander (2013) Nat Rev Mol Cell Bio 14:49-55; Kim et al Nat Biotechnol. (2013); 31:251-258. Miller JC, et al. Nat. Biotechnol. (2011) 29:143-148. ReyonD, et al. Nat. Biotechnol. (2012); 30:460-465).

[0200] Meganucleases are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs); as a result this site generally occurs only once in any given genome (see for example Silva et al. (2011) Curr Gene Ther 11(1): 11-27).

[0201] CRISPR targeting nucleases (e.g. Cas9) complex with a 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 may be combined into a single RNA to enable site-specific mammalian genome cutting within the FANCM gene or its regulatory elements. The use of CRISPR / Cas9 systems to introduce insertions or deletions into genes as a way of decreasing transcription is well known in the art (see for example 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; JinekM 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).

[0202] In some preferred embodiments, the targetable nuclease is a Cas endonuclease, preferably Cas9, which is expressed in the cancer cells in combination with a guide RNA targeting sequence that targets the Cas endonuclease to cleave genomic DNA within the FANCM gene and generate insertions or deletions that prevent expression of active FANCM polypeptide.

[0203] Nucleic acid sequences encoding a suppressor nucleic acid or targetable nuclease and optionally a guide RNA may be comprised within an expression vector. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the encoding nucleic acid in a host cell. Suitable regulatory sequences to drive the expression of heterologous nucleic acid coding sequences in a range of expression systems are well-known in the art and include constitutive promoters, for example viral promoters such as CMV or SV40. In some preferred embodiments, a tissue-specific or inducible promoter, such as a photoinducible promoter, may be employed to selectively express the suppressor nucleic acid or targetable nuclease and optionally guide RNA in cancer cells. A vector may also comprise sequences, such as origins of replication and selectable markers, which allow for its selection and replication and expression in bacterial hosts, such as E. coli and / or in eukaryotic cells, such as yeast, insect or mammalian cells. Vectors suitable for use in expressing a suppressor nucleic acid or targetable nuclease in mammalian cells include plasmids and viral vectors e.g. retroviruses, lentivimses, adenoviruses, and adeno-associated vimses. 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, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992; Recombinant Gene Expression Protocols Ed RS Tuan (Mar 1997) Humana Press Inc). Whilst an agent capable of disrupting the FANCM complex / FANCM inhibitor may be administered alone it will usually be administered in the form of a pharmaceutical composition, which may comprise at least one additonal component. The agent / inhibitor may be admixed with other reagents, such as buffers, carriers, diluents, preservatives and / or pharmaceutically acceptable excipients in order to produce a pharmaceutical composition. In some embodiments, a FANCM inhibitor is one that can inhibit ALT cell viability and / or growth, e.g., ALT cancer or tumour cell viability or growth. In some embodiments, a FANCM inhibitor can increase or induce death of ALT cells, e.g., ALT cancer or tumour cells. These definitions can apply even where, as in the methods of the invention, the FANCM inhibitor is for use in treating a non-ALT cancer.

[0204] With respect to “inhibiting” an activity of FANCM, such as binding to RMI, DNA translocase activity, ATPase activity and / or translocase activity, the inhibitor may be partial or complete. In certain embodiments, inhibition is a reduction of 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%, e.g., as compared to the amount in a comparable cell not contacted with the FANCM inhibitor, or as compared to a pre-determined value.

[0205] In certain contexts used herein, the term “inhibit ALT cell viability and / or growth” shall be taken to mean hinder, reduce, restrain or prevent ALT cell viability and / or growth relative to an ALT cell in which the FANCM -RMI interaction is intact. Likewise, in certain contexts used herein, the term “inhibit ALT cell viability and / or growth” shall be taken to mean hinder, reduce, restrain or prevent ALT cell viability and / or growth relative to an ALT cell in which FANCM is not inhibited. In certain contexts used herein, the term “inhibit ALT cell viability and / or growth” shall be taken to mean hinder, reduce, restrain or prevent ALT cell viability and / or growth relative to an ALT cell in which RMI is not inhibited. In certain contexts used herein, the term “inhibit ALT cell viability and / or growth” shall be taken to mean hinder, reduce, restrain or prevent ALT cell viability and / or growth relative to an ALT cell in which the FANCM-RMI interaction is not disrupted.

[0206] Cell viability and / or growth may be inhibited in any measurable amount. Inhibition of cell viability may be complete or may be partial. Thus, the methods disclosed herein may comprise at least partial inhibition of ALT cell viability and / or growth. For example, cell viability and / or growth may 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% following disruption of the FANCM-RMI interaction.

[0207] In certain contexts used herein, the term “induce ALT cell death” shall be taken to mean induce, increase, cause, or promote ALT cell death relative to an ALT cell in which the FANCM-RMI interaction is intact. Likewise, in certain contexts used herein, the term “induce ALT cell death” shall be taken to mean induce, increase, cause, or promote ALT cell death relative to an ALT cell in which FANCM is not inhibited. In certain contexts used herein, the term “induce ALT cell death” shall be taken to mean induce, increase, cause, or promote ALT cell death relative to an ALT cell in which RMI is not inhibited. In certain contexts used herein, the term “induce ALT cell death” shall be taken to mean induce, increase, cause, or promote ALT cell viability and / or growth relative to an ALT cell in which the FANCM-RMI interaction is not disrupted.

[0208] Cell death may be increased in any measurable amount. Increasing cell death may be complete or may be partial. For example, ALT cell death may 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% following disruption of the FANCM-RMI interaction. Cell death may be determined in a plurality of cells, e.g., as a percentage of non-viable or dead cells following contact with an inhibitor disclosed herein.

[0209] General The medicaments of the present disclosure may be administered to a subject by any suitable means and in any suitable form. Suitable means for administering the agents disclosed herein are known in the art. A suitable dosing regimen may be used depending on the organism to be treated. It will be appreciated that medicaments of the present disclosure may be used in a monotherapy. Alternatively, medicaments of the present disclosure may be used as an adjunct to, or in combination with, known therapies. The medicaments of the present disclosure may be for administration before, during or after onset of the pathological condition.

[0210] The methods of the first aspect and the medicaments for use of the second aspect may be applied to any subject in need thereof. The subject may be a vertebrate, mammal, or domestic animal. The subject may be a model organism. Most preferably, the subject is a human.

[0211] The terms “treat”, “treating”, “treatment” and the like, as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting the process of, or preventing the disease, disorder or condition to which such term applies, or one or more symptoms of such disease, disorder or condition and includes the administration of any of the medicaments, pharmaceutical compositions, or dosage forms described herein, to prevent the onset of the symptoms or the complications, or alleviating the symptoms or the complications, or eliminating the disease, condition, or disorder. For example, treatment is curative or ameliorating. As used herein, “preventing” means preventing in whole or in part, or ameliorating or controlling, or reducing or halting the production or occurrence of the thing or event, for example, the disease, disorder or condition, to be prevented.

[0212] The terms “administering”, “administer”, “administration” and the like, as used herein, refer to any mode of transferring, delivering, introducing, or transporting a therapeutic agent to a subject in need of treatment with such an agent.

[0213] Sequence comparisons can be conducted with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences.

[0214] The skilled technician will appreciate how to calculate the percentage identity between two nucleic sequences or two amino acid sequences. In order to calculate the percentage identity, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on: (i) the method used to align the sequences, for example, the Needleman-Wunsch algorithm (e.g. as applied by Needle(EMBOSS) or Stretcher(EMBOSS), the Smith-Waterman algorithm (e.g. as applied by Water(EMBOSS)), or the LALIGN application (e.g. as applied by Matcher(EMBOSS); and (ii) the parameters used by the alignment method, for example, local versus global alignment, the matrix used, and the parameters applied to gaps. In a particular embodiment, the sequence identities disclosed herein may be calculated based on a global alignment of the relevant feature.

[0215] Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length-dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

[0216] A calculation of percentage identities between two nucleic acid sequences may then be calculated from such an alignment as (N / T)* 100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs. The sequence alignment may be a pairwise sequence alignment. Suitable services include Needle (EMBOSS), Stretcher (EMBOSS), Water (EMBOSS), Matcher (EMBOSS), LALIGN, or GeneWise. In an example, the identity between two amino acid sequences may be calculated using the service Needle(EMBOSS) set to the default parameters, e.g. matrix (BLOSUM62), gap open (10), gap extend (0.5), end gap penalty (false), end gap open (10), and end gap extend (0.5). In another example, the identity between two amino acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g. matrix (BLOSUM62), gap open (14), gap extend (4), alternative matches (1). In an example, the identity between two nucleic acid sequences may be calculated using the service Needle(EMBOSS) set to the default parameters, e.g. matrix (DNAfull), gap open (10), gap extend (0.5), end gap penalty (false), end gap open (10), and end gap extend (0.5). In another example, the identity between two nucleic acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g. matrix (DNAfull), gap open (16), gap extend (4), alternative matches (1).

[0217] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Other aspects and embodiments of the invention provide the aspects and embodiments described herein with the term “comprising” replaced by the term “consisting of’ and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of’.

[0218] All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0219] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made to the Examples, which are not intended to limit the invention in any way. EXAMPLES

[0220] Example 1 - Outline

[0221] Principles. A liquid sample is 0.8-mcron filtered to remove cellular matter. The flow through is optionally further treated to obtain a particular fraction, e.g., free circulation DNA or exosomes. Subsequently, the nucleic acids are isolated and circular DNA is selectively amplified. In this isothermal amplification step, each sample has two reactions, with and without DNA polymerase. For both reactions, the circular telomeric is quantified by quantitative Polymerase Chain Reaction and content is expressed as fold change between samples with and without selective amplification.

[0222] Example 2 - Exemplary protocol

[0223] This methodology is suitable for any liquid sample, for example but not limited to serum, plasma, and culture media.

[0224] 1. Obtaining extracellular fraction - The extracellular fraction is obtained by size exclusion. The primary step is filtration of the liquid sample by a 0.8-micron filter. The flow through is used for further processing.

[0225] 2. The primary flow through (step 1) can be further enriched for a particular fraction, for example but not limited to free circulating DNA or extracellular vesicles by exploiting different unique characteristics.

[0226] a. Size exclusion utilization filtration.

[0227] b. Separation on mass utilization centrifugation.

[0228] c. Isolation by binding specific (surface) markers.

[0229] 3. Nucleic acid isolation and enrichment utilizing a kit for liquid sample based on the Boom protocol.

[0230] 4. Selective amplification of circular telomeric DNA by isothermal amplification utilizing a DNA polymerase with strand displacement and proofreading exonuclease activity, for example but not limited to Φ29 DNA polymerase. Each sample will have two reactions, with and without DNA polymerase.

[0231] 5. Polymerase chain reaction-based quantification of telomeric DNA in both selective amplification reactions of each sample by utilizing an intercalating dye and primers targeting telomeric DNA and a housekeeping.

[0232] 6. Quantity of circular telomeric DNA is expressed the fold change between sample reactions with and without selective amplification (step 4).

[0233] Example 3 - Exemplary protocol

[0234] The methodology is currently used for the determination of FANCM-sensitivity by quantification of extracellular circular telomeric DNA.

[0235] 1. Filtration of the liquid sample by a 0.8-micron filter.

[0236] 2. Size exclusion to obtain exosome fraction utilizing the exoEasy kit by QIAGEN.

[0237] 3. Nucleic acid isolation and enrichment utilizing the QiaAmp Blood mini kit by QIAGEN. 4. Isothermal selective amplification of circular telomeric DNA (tDNA) by <t>29 DNA polymerase by New England Biolabs. Two reactions for each sample, with and without Φ29 DNA polymerase DNA polymerase.

[0238] 5. For both selective amplification reactions per sample, the telomeric DNA content is quantified by quantitative polymerase chain reaction.

[0239] 6. Quantity is expressed as ΔCt-derived fold change between sample reactions with and without selective amplification.

Claims

CLAIMS1. A method of determining a subject’s suitability for treatment with an agent capable of disrupting the Fanconi anemia, complementation group M (FANCM) complex, optionally wherein the subject does not have an alternative lengthening of telomeres positive (ALT+) cancer, the method comprising:measuring the quantity of extracellular primed single-stranded circular telomeric DNA (epc-tDNA), extracellular C-circles, extracellular G-circles, and / or extracellular telomeric fusion circles (TF-circles) in a sample from the subject; andbased on the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles, determining whether the subject is suitable for treatment with the agent.

2. The method of claim 1, wherein the subject does not comprise detectable ALT cells.

3. The method of claim 1 or claim 2, wherein the subject has a cancer.

4. The method of claim 1, wherein the subject has a degenerative disease or a neurodegenerative disease; optionally wherein the disease is Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, spinocerebellar ataxias, or amyotrophic lateral sclerosis (ALS).

5. The method of any preceding claim, wherein the sample from the subject is a liquid sample or liquid biopsy.

6. The method of any preceding claim, wherein the sample from the subject is or comprises cerebrospinal fluid, bronchoalveolar lavage, urine, saliva, blood, a blood fraction, serum, or plasma.

7. The method of any preceding claim, wherein the measurement is of epc-tDNA that comprises:C-circles; orC-circles and / or G-circles; orC-circles, G-circles, and / or TF-circles.

8. The method of any preceding claim, wherein the measured epc-tDNA, C-circles, G-circles, and / or TF-circles was contained in extracellular vesicles and / or was in a cell-free form.

9. The method of any preceding claim, comprising removing cells from the sample to obtain an extracellular fraction prior to measuring the quantity of epc-tDNA, C-circles, G-circles, and / or TF-circles, and whereinthe measuring the quantity of epc-tDNA, C-circles, G-circles, and / or TF-circles is applied to the extracellular fraction.

10. The method of any preceding claim, comprising enriching the sample or extracellular fraction for extracellular vesicles to produce an enriched fraction, and whereinthe measuring the quantity of epc-tDNA, C-circles, G-circles, and / or TF-circles is applied to the enriched fraction.

11. The method of claim 10, wherein the extracellular vesicles are exosomes.

12. The method of any preceding claim, comprising enriching the sample or extracellular fraction for extracellular nucleic acids to produce an enriched fraction, and whereinthe measuring the quantity of epc-tDNA, C-circles, G-circles, and / or TF-circles is applied to the enriched fraction.

13. The method of claim 12, wherein the extracellular nucleic acids are free circulating DNA.

14. The method of any preceding claim, wherein the sample, extracellular fraction, or enriched fraction is split into a first fraction and a second fraction, and wherein the first fraction is contacted with a polymerase whereas the second fraction is not.

15. The method of any preceding claim, wherein the measuring the quantity of epc-tDNA, C-circles, G-circles, and / or TF-circles comprises amplification of telomeric DNA, epc-tDNA, C-circles, G-circles, and / or TF-circles.

16. The method of claim 15, wherein the amplification is selective amplification, isothermal amplification, rolling circle amplification, and / or comprises the use of φ29 DNA polymerase.

17. The method of claim 14, wherein the first fraction is amplified according to claim 15 or claim 16, whereas the second fraction is not.

18. The method of any preceding claim, wherein the measuring the quantity of epc-tDNA, C-circles, G-circles, and / or TF-circles comprises qPCR.

19. The method of any preceding claim, wherein the measuring the quantity of epc-tDNA, C-circles, G-circles, and / or TF-circles comprises qPCR to detect telomeric DNA, epc-tDNA, C-circles, G-circles, and / or TF-circles.

20. The method of claims 15 to 19, wherein the measuring the quantity of epc-tDNA, C-circles, G-circles, and / or TF-circles comprises qPCR applied to the amplification product.

21. The method of any preceding claim, comprisingseparating the sample, extracellular fraction, or enriched fraction into a first fraction and a second fraction;contacting the first fraction but not the second fraction with a polymerase under conditions for amplification of telomeric DNA,quantifying the telomeric DNA levels in the post-amplification first fraction and in the second fraction; andexpressing the quantity of epc-tDNA in the sample as the fold change of telomeric DNA between the first fraction and the second fraction.

22. The method of any preceding claim, wherein the quantity of epc-tDNA, extracellular C-circles, extracellular G-circles, and / or extracellular TF-circles positively correlates with suitability for treatment with the agent.

23. The method of any preceding claim, the method further comprising:iii) treating the subject with the agent capable of disrupting the FANCM complex if the subject is determined to be suitable for treatment.

24. The method of any preceding claim, wherein the agent capable of disrupting the FANCM complex is a small molecule drug, antibody, antigen-binding fragment of an antibody, aptamer, nucleic acid molecule, nucleic acid analogue, antisense molecule, siRNA, shRNA, peptide, protein, inactivated FANCM protein, the inactivated FANCM protein comprising a F1232A / F1236A double substitution, targeted nuclease that reduces expression of FANCM, ZFN, TALEN, meganuclease, or a CRISPR associated nuclease.

25. An agent capable of disrupting the FANCM complex for use in treating a non- ALT cancer or a degenerative disease in a subject.

26. The agent for use of claim 25, wherein the subject has been determined to be suitable for treatment by a method of any one of claims 1 to 22.

27. A method of determining the presence of epc-tDNA in a biological sample, the method comprising: separating the sample into a first fraction and a second fraction;contacting the first fraction but not the second fraction with a polymerase under conditions for amplification of telomeric DNA,quantifying the telomeric DNA levels in the post-amplification first fraction and in the second fraction; anddetermining the presence of epc-tDNA in the sample based on the fold change of telomeric DNA between the first fraction and the second fraction.

28. The method of claim 27, wherein the sample is processed to obtain an extracellular fraction, and the extracellular fraction is separated into the first fraction and the second fraction.

29. The method of claim 27 or claim 28, the method comprising enriching the sample or extracellular fraction for extracellular vesicles to produce an enriched fraction.

30. The method of claim 29, wherein the extracellular vesicles are exosomes.

31. The method of claim 27 or claim 28, the method comprising enriching the sample or extracellular fraction for extracellular nucleic acids to produce an enriched fraction.

32. The method of claim 31, wherein the extracellular nucleic acids are free circulating DNA.

33. The method of any one of claims 29 to 32, wherein the enriched fraction is split into the first fraction and the second fraction.

34. The method of any one of claims 27 to 33, wherein the epc-tDNA is or comprises:C-circles; orC-circles and / or G-circles; orC-circles, G-circles, and / or TF-circles.

35. The method of any one of claims 27 to 34, wherein the amplification is selective amplification, isothermal amplification, rolling circle amplification, and / or comprises the use of φ29 DNA polymerase.

36. The method of any one of claims 27 to 35, wherein quantifying the telomeric DNA levels comprises qPCR.

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