Composition for preventing or treating alternative lengthening of telomeres-positive cancer or aging

Abstract

The present invention relates to a method for preventing or treating alternative lengthening of telomeres-positive cancer by identifying factors involved in alternative lengthening of telomeres and correlations therebetween, and by regulating expression of the factors. The present invention provides an effective therapeutic target for alternative lengthening of telomeres-positive cancer, for which an effective treatment method has not yet been developed due to the previously unclear mechanism. Accordingly, the present invention can be effectively used for efficient prevention or treatment of alternative lengthening of telomeres-positive cancer by regulating expression of the target.

Description

Composition for preventing or treating cancer or aging with an alternative telomere maintenance mechanism

[0001] The present invention relates to a method for specifically inhibiting cancers that maintain telomere length by an alternative telomere maintenance mechanism (Alternative Lengthening of Telomeres) and a method for treating aging diseases caused by structural and functional damage to telomeres.

[0002]

[0003] The dynamic interaction between BRCA2 and the telomeric G-quadruplex is a mechanism for maintaining replicative homeostasis, and cells lacking BRCA2 exhibit telomere damage. However, certain cells overcome telomere damage-induced cellular senescence by maintaining telomere length through the break-induced replication (BIR) mechanism even in the presence of BRCA2 deficiency. Meanwhile, BRCA2-deficient ALT (Alternative Lengthening of Telomeres)-like cells characteristically contain telomeric PML bodies that exhibit liquid-liquid phase separation (LLPS) characteristics. “Alternative telomere maintenance-positive” cancer cells, which are BRCA2 deficient and maintain telomere length through the ALT mechanism, account for only a small portion of all cancers, but the mechanism of telomere length maintenance through the ALT mechanism has not been elucidated in detail, so there is no effective treatment method for this cancer.

[0004] Accordingly, the present inventors aimed to develop a new treatment method that can be specifically used for alternative telomere maintenance mechanism-positive cancers by elucidating in detail the factors involved in the telomere length maintenance mechanism used in BRCA2-deficient cancer cells and their interrelationships.

[0005] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0006]

[0007] The present inventors have diligently researched and developed novel treatment methods for "alternative telomere maintenance mechanism-positive cancers," which account for only a small portion of all cancers but for which the mechanism of telomere length maintenance through the ALT mechanism has not been fully elucidated. Therefore, there is a high demand for effective treatment methods. As a result, the present invention was completed by elucidating various factors involved in the liquid-liquid phase separation phenomenon involved in the alternative telomere maintenance mechanism and their interrelationships.

[0008] Accordingly, the purpose of the present invention is to provide a composition for preventing or treating alternative telomere maintenance mechanism-positive cancer.

[0009] Another object of the present invention is to provide a method for screening a composition for preventing or treating alternative telomere maintenance mechanism-positive cancer.

[0010] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0011]

[0012] According to one aspect of the present invention, the present invention provides a composition for preventing or treating alternative telomere lengthening (ALT) mechanism-positive cancer or aging, comprising as an active ingredient at least one selected from the group consisting of inhibitors of liquid-liquid phase separation (LLPS) phenomenon and abnormal stabilizers of telomere G-quadruplexes.

[0013] According to another aspect of the present invention, the present invention provides a method for preventing or treating alternative telomere maintenance mechanism-positive cancer or aging, comprising administering to a subject at least one active ingredient selected from the group consisting of an inhibitor of liquid-liquid phase separation phenomenon and an abnormal stabilizer of telomere G-quadruplex.

[0014] The present inventors have diligently researched and developed novel treatment methods for "alternative telomere maintenance mechanism-positive cancers," which account for only a small portion of all cancers but for which the mechanism of telomere length maintenance through the ALT mechanism has not been fully elucidated. Therefore, there is a high demand for effective treatment methods. As a result, the present invention was completed by elucidating various factors involved in the liquid-liquid phase separation phenomenon involved in the alternative telomere maintenance mechanism and their interrelationships.

[0015] The term “Liquid-Liquid Phase Separation (LLPS)” used herein refers to a type of membrane-less organelle, a structure in which specific biomolecules are locally gathered despite the absence of a cell membrane. Specifically, when a solution containing macromolecules such as proteins or nucleic acids undergoes LLPS, it condenses into a dense phase similar to a liquid droplet. LLPS is a process in which macromolecule / water interactions change to macromolecule / macromolecule and water / water interactions, and the driving force behind this process is that the total free energy can be minimized more by interactions between the same molecules than by the entropy that increases when different molecules are mixed, making it thermodynamically more stable. According to what has been discovered by the present invention, essential substances in the alternative telomere maintenance mechanism (ALT) function by forming a condensed structure through liquid-liquid phase separation, and thus, when the liquid-liquid phase separation phenomenon is inhibited in telomeres, cells that maintain telomere length through the ALT mechanism, specifically, alternative telomere maintenance (ALT) mechanism-positive cancers, can be prevented or treated.

[0016] The term “alternative telomere maintenance mechanism” as used herein refers to a telomere length maintenance mechanism that extends telomeres through homologous recombination, specifically, the break-induced telomere synthesis (BITS) mechanism, in the absence of telomerase in certain cells, including cancer cells, as a type of telomerase-independent mechanism for maintaining telomere length.

[0017] As used herein, the term “alternative telomere maintenance mechanism-positive cancer” refers to a cancer that maintains telomere length through an alternative telomere maintenance mechanism.

[0018] As used herein, the term "aging" or "senescence" refers to the gradual loss of the original biological functions and characteristics of living cells or organs, and more specifically, refers to pathological aging in which telomere length is not maintained normally and thus progresses abnormally compared to normal controls. Aging includes, but is not limited to, ischemic necrosis diseases such as avascular necrosis of the femoral head, and may include all aging diseases caused by structural and functional damage to telomeres.

[0019] The term “inhibitor” as used herein means a substance that causes a decrease in the activity or expression of a target substance, or inhibits the initiation or progression of a desired phenomenon, and thereby reduces the activity or expression of the target substance and the occurrence of the desired phenomenon to an undetectable or insignificant level, as well as a substance that reduces the activity or expression to such an extent that the biological function of the target substance and the biological effect of the desired phenomenon can be significantly reduced.

[0020] The term “prevention” as used herein means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to such disease or condition.

[0021] As used herein, the term “treatment” means (a) suppressing the development of a disease, condition, or symptom; (b) alleviating the disease, condition, or symptom; or (c) eliminating the disease, condition, or symptom. When the composition of the present invention is administered to a subject, it inhibits or suppresses the liquid-liquid phase separation phenomenon, thereby preventing telomere length maintenance by the Alternative Lengthening of Telomeres (ALT) mechanism, thereby suppressing, eliminating, or alleviating the development of symptoms caused by cancer, specifically, Alternative Lengthening of Telomeres (ALT) mechanism-positive cancer. Therefore, the composition of the present invention may be a composition for treating these diseases on its own, or may be administered together with other pharmacological ingredients and applied as an adjuvant treatment for the diseases. Accordingly, the terms “treatment” or “therapeutic agent” as used herein include the meaning of “adjuvant treatment” or “adjuvant treatment agent.”

[0022] The term “Telomere G-quadruplexes” as used herein refers to structures formed in nucleic acids by guanine-rich sequences, which have a helical shape and a structure comprising guanine tetrads that can be formed into one, two, or four strands.

[0023] As used herein, the term “excessive stabilizer of telomere G-quadruplexes” refers to a substance that induces the formation of a G-quadruplex structure or binds to a G-quadruplex and abnormally (or excessively) stabilizes the G-quadruplex structure. Here, excessive stabilization means that after the telomere G-quadruplexes are formed in cancer cells, excessive telomere damage occurs due to this, or the cancer cells are stabilized to the point where it is difficult to repair the damage to the telomeres, leading to the death of the cancer cells. Specifically, the excessive stabilizer of telomere G-quadruplexes is pyridostatin (PDS).

[0024]

[0025] According to a specific embodiment of the present invention, the inhibitor of the liquid-liquid phase separation (LLPS) phenomenon of the present invention is an inhibitor of APB (ALT-associated promyelocytic leukemia bodies) formation.

[0026] The liquid-liquid phase separation phenomenon can be inhibited by reducing the expression or activity of a substance related to the liquid-liquid phase separation phenomenon identified by the present invention, and the inhibitor of the liquid-liquid phase separation phenomenon can be an inhibitor that inhibits any one of the subcomponents or phenomena of APB (ALT-associated promyelocytic leukemia bodies) (e.g., formation of TERRA-R loops, disruption of G4 dynamics, trimethylation by PRC2, EZH2, SUZ12 or EED, which are subcomponents constituting PRC2), and the means for inhibiting them can be a substance that inhibits the activity of these substances or inhibits the expression of these substances at the protein or gene level, as described below.

[0027] Inhibitors that inhibit the expression of the target substance at the genetic level include, but are not limited to, shRNA, siRNA, miRNA, ribozyme, PNA (peptide nucleic acids) antisense oligonucleotide that inhibit the expression of the gene at the genetic level, the sequence of which is already known in the art, or a CRISPR system that includes a guide RNA that encodes the target substance or recognizes a gene involved in the expression of the target substance, antibodies or aptamers that inhibit at the protein level, as well as compounds, peptides and natural products that inhibit the activity thereof, and all gene and protein level inhibitory means known in the art can be used.

[0028] The term “shRNA (small hairpin RNA)” as used herein refers to an RNA sequence that forms a tight hairpin structure to suppress the expression of a target gene through RNA interference, which is a single-stranded structure consisting of 50-70 nucleotides that forms a stem-loop structure in vivo. Typically, a long RNA of 19-29 nucleotides complementarily forms a double-stranded stem by base pairing on both sides of a loop region of 5-10 nucleotides, and is transduced into cells through a vector containing a U6 promoter to ensure constant expression, and is usually passed on to daughter cells to ensure heritable suppression of the expression of the target gene.

[0029] The term “siRNA” in this specification refers to a short double-stranded RNA that can induce RNAi (RNA interference) by cleavage of a specific mRNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases, and either blunt-ended or cohesive-ended is possible as long as it can suppress the expression of the target gene through the RNAi effect. The cohesive-ended structure can be either a 3-terminal protruding structure or a 5-terminal protruding structure.

[0030] In this specification, the term “miRNA (microRNA)” refers to a single-stranded RNA molecule that is an oligonucleotide that is not expressed in cells and has a short stem-loop structure and suppresses target gene expression through complementary binding to the mRNA of the target gene.

[0031] As used herein, the term "ribozyme" refers to an RNA molecule that functions like an enzyme, recognizing a specific base sequence in RNA and cleaving it. A ribozyme consists of a region that specifically binds to a complementary base sequence of a target mRNA strand and a region that cleaves the target RNA.

[0032] As used herein, the term "PNA (Peptide Nucleic Acid)" refers to a molecule that possesses properties of both nucleic acids and proteins and can complementarily bind to DNA or RNA. PNA is not found in nature and is artificially synthesized through chemical methods. It forms a double strand through hybridization with a natural nucleic acid of complementary base sequence, thereby regulating the expression of target genes.

[0033] As used herein, the term “antisense oligonucleotide” refers to a nucleic acid molecule that is a nucleotide sequence complementary to a sequence of a specific mRNA and binds to the complementary sequence in the target mRNA, thereby inhibiting its translation into protein, translocation into the cytoplasm, maturation, or any other essential activity for its overall biological function. Antisense oligonucleotides can be modified at one or more base, sugar, or backbone positions to enhance their potency (De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55, 1995). The oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatoms, heterocyclic sugar sulphonates, etc.

[0034] As used herein, the term "gRNA (guideRNA)" refers to an RNA molecule used in a gene editing system that recognizes a target gene and induces a nuclease to specifically cleave the recognized region. A representative example of such gene editing systems is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system.

[0035] According to the present invention, the inhibitor of the present invention may be a specific antibody that inhibits the activity of a protein (or target substance) encoded by the aforementioned genes. The antibody that specifically recognizes the target protein is a polyclonal or monoclonal antibody, and is preferably a monoclonal antibody.

[0036] The antibodies of the present invention can be produced by methods commonly practiced in the art, for example, the fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), the recombinant DNA method (U.S. Patent No. 4,816,567), or the phage antibody library method (Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:58, 1-597 (1991)). General procedures for antibody production are described in detail in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; and Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984.

[0037] As used herein, the term “antigen binding fragment” means a portion of a polypeptide in the overall structure of an immunoglobulin capable of binding an antigen, including, but not limited to, F(ab')2, Fab', Fab, Fv, and scFv.

[0038] As used herein, the term “specifically binding” is synonymous with “specifically recognizing” and means that an antigen and an antibody (or fragment thereof) specifically interact through an immunological reaction.

[0039] The present invention can also utilize aptamers that specifically bind to a target protein instead of antibodies. As used herein, the term "aptamer" refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity. General information on aptamers is described in detail in Hoppe-Seyler F, Butz K, "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):426-30 (2000); Cohen BA, Colas P, Brent R, "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24):14272-7 (1998).

[0040] The term “APB (ALT-associated promyelocytic leukemia bodies)” as used herein refers to a type of PML body, a structure that exists specifically in telomerase-negative tumors (cancer). “PML bodies”, which may also be referred to as “PML bodies” or “promyelocytic leukemia bodies,” are intracellular structures that are spherical structures present within the nucleus with a diameter of up to 1 μm, and are composed of various substances including the PML protein, and are substances related to cellular mechanisms such as telomere elongation and DNA damage response.

[0041]

[0042] According to a specific embodiment of the present invention, the APB formation inhibitor of the present invention is at least one selected from the group consisting of a TERRA-R-loop inhibitor, a histone H3K9 trimethylation (H3K9me3) inhibitor, and a histone H3K27 trimethylation (H3K27me3) inhibitor.

[0043] In this specification, the term “TERRA” refers to a type of long noncoding RNA (lncRNA) transcribed from chromosome ends that regulates telomeric chromatin structure and telomere maintenance through telomerase and homology-directed repair. TERRA-R-loop promotes homologous recombination at telomeres through the formation of DNA:RNA (R-loop) structures.

[0044] The term “histone H3K9 trimethylation (H3K9me3)” as used herein refers to an epigenetic modification of histone H3, a DNA packaging protein, which refers to a phenomenon in which the 9th lysine residue of the histone H3 protein is trimethylated or a histone H3 protein in which the phenomenon has occurred.

[0045] The term “histone H3K27 trimethylation (H3K27me3)” as used herein refers to an epigenetic modification of histone H3, a DNA packaging protein, which refers to a phenomenon in which the 27th lysine residue of the histone H3 protein is trimethylated or the histone H3 protein in which the phenomenon has occurred.

[0046]

[0047] According to a specific embodiment of the present invention, the histone H3K27 trimethylation (H3K27me3) inhibitor is a polycomb repressive complex (PRC2) inhibitor.

[0048] The term "polycomb repressive complex (PRC2)" used herein refers to a family of protein complexes, first discovered in Drosophila, and the only enzyme in mammals that methylates histone H3K27. PRC2 is a key epigenetic factor responsible for transcriptional repression, and defects in this enzyme can lead to problems during fetal development as well as various human diseases, including cancer.

[0049]

[0050] According to a specific embodiment of the present invention, the polycomb repressive complex (PRC2) of the present invention is composed of EZH2, SUZ12 and EED, and the polycomb repressive complex inhibitor is at least one selected from the group consisting of an EZH2 inhibitor, a SUZ12 inhibitor and an EED inhibitor.

[0051] The term “EZH2” in this specification is an abbreviation for “Enhancer of zeste homolog 2” and refers to a functional catalytic subunit of polycomb repressive complex 2 (PRC2), a highly conserved histone methyltransferase that methylates lysine 27 of histone H3, and is encoded by the EZH2 gene in humans. Accordingly, all inhibitors of EZH2, including the aforementioned proteins or nucleic acids encoding them, may be applied.

[0052] The term “SUZ12” in this specification is an abbreviation for “suppressor of zeste 12” and refers to a catalytic subunit of polycomb repressive complex 2 (PRC2), a highly conserved histone methyltransferase that methylates lysine 27 of histone H3, and is encoded by the SUZ12 gene in humans. Accordingly, all inhibitors of SUZ12, including the aforementioned proteins or nucleic acids encoding them, may be applied.

[0053] The term “EED” in this specification is an abbreviation for “embryonic ectoderm development” and refers to the catalytic subunit of polycomb repressive complex 2 (PRC2), a highly conserved histone methyltransferase that methylates lysine 27 of histone H3, and is encoded by the EED gene in humans. Accordingly, any inhibitor of EED, including the aforementioned proteins or nucleic acids encoding them, may be used.

[0054]

[0055] According to a specific embodiment of the present invention, the EZH2 inhibitor of the present invention is EPZ-6348.

[0056] In this specification, the term “EPZ-6348” refers to a low-molecular-weight compound having selective inhibitory activity against EZH2, and its CAS number corresponds to 1403254-99-8.

[0057] According to a specific embodiment of the present invention, the alternative telomere maintenance (ALT) mechanism positive cancer of the present invention is a telomerase-negative cancer or a BRCA-2 deficient cancer.

[0058] The term “telomerase-negative cancer” as used herein refers to a cancer in which the expression of telomerase is inhibited or suppressed, and the cancer can extend telomeres through alternative telomere maintenance mechanisms instead of telomerase.

[0059] The term “BRCA-2 deficient cancer” in this specification refers to a cancer in which the human gene BRCA2 is knocked out.

[0060] According to a specific embodiment of the present invention, the BRCA-2 deficient cancer is BRCA-2 deficient breast cancer.

[0061] According to one aspect of the present invention, the present invention provides a composition for preventing or treating cancer or aging with an alternative lengthening of telomeres (ALT) mechanism, which comprises an abnormal stabilizer of telomere G-quadruplexes as an active ingredient, in combination with a histone H3K27 trimethylation (H3K27me3) inhibitor.

[0062] According to another aspect of the present invention, the present invention provides a method for preventing or treating alternative telomere maintenance mechanism-positive cancer or aging, comprising the step of co-administering to a subject an abnormal stabilizer of telomere G-quadruplex and an inhibitor of histone H3K27 trimethylation (H3K27me3).

[0063] In the present invention, the meanings of the terms “G4 (telomere G-quadruplex)”, “histone H3K27 trimethylation (H3K27me3)”, “alternative lengthening of telomeres (ALT) mechanism-positive cancer”, and “aging” have already been described above, and therefore, their description is omitted to avoid excessive duplication.

[0064] According to a specific embodiment of the present invention, the abnormal stabilizer of the telomere G-quadruplexes is pyridostatin (PDS).

[0065] According to a specific embodiment of the present invention, the trimethylation (H3K27me3) inhibitor is a polycomb repressive complex (PRC2) inhibitor.

[0066] According to a specific embodiment of the present invention, the polycomb repressive complex (PRC2) comprises EZH2, SUZ12 and EED, and the inhibitor of the polycomb repressive complex is at least one selected from the group consisting of an EZH2 inhibitor, a SUZ12 inhibitor and an EED inhibitor.

[0067] According to a specific embodiment of the present invention, the alternative telomere maintenance (Alternative Lengthening of Telomeres, ALT) mechanism-positive cancer is a telomerase-deficient cancer or a BRCA-2-deficient cancer.

[0068] According to a specific embodiment of the present invention, the BRCA-2 deficient cancer is BRCA-2 deficient breast cancer.

[0069] In this specification, the meanings of the terms “pyridostatin (PDS)”, “trimethylation (H3K27me3)”, “polycomb repressive complex (PRC2)”, “EZH2”, “SUZ12”, “EED”, “alternative lengthening of telomeres (ALT) mechanism-positive cancer”, “telomerase-deficient cancer”, and “BRCA-2-deficient cancer” have already been described above, and therefore, their description is omitted to avoid excessive duplication.

[0070]

[0071] According to another aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating an alternative telomere lengthening (ALT) mechanism-positive cancer, comprising the following steps:

[0072] (a) administering a test substance to a biological sample containing cells exhibiting liquid-liquid phase separation (LLPS) phenomenon; and

[0073] (b) a step of measuring the LLPS level of cells contained in the biological sample;

[0074] If the above LLPS level decreases, the test substance is determined to be a composition for preventing or treating alternative lengthening of telomeres (ALT) mechanism-positive cancer.

[0075] The term "biological sample" as used herein refers to any sample obtained from a mammal, including a human, that contains cells exhibiting the LLPS phenomenon, including, but not limited to, tissues, organs, cells, or cell cultures. More specifically, the biological sample may be cancer tissue, cancer cells, or a culture thereof. Most specifically, the cancer cells may be ALT-positive cancer cells.

[0076] The term "test substance" used in referring to the screening method of the present invention refers to an unknown substance used in screening to determine whether it affects a sample containing cells exhibiting the aforementioned phenomenon by adding the unknown substance. The test substance includes, but is not limited to, compounds, nucleotides, peptides, and natural extracts. The step of measuring the level of liquid-liquid phase separation within cells in a biological sample treated with the test substance can be performed using various measurement methods known in the art.

[0077]

[0078] According to a specific embodiment of the present invention, the liquid-liquid phase separation (LLPS) phenomenon is due to the formation of ALT-associated promyelocytic leukemia bodies (APB).

[0079] According to a specific embodiment of the present invention, the APB formation is by at least one selected from the group consisting of TERRA-R-loop formation, histone H3K9 trimethylation (H3K9me3), and histone H3K27 trimethylation (H3K27me3).

[0080] According to a specific embodiment of the present invention, the H3K27 trimethylation (H3K27me3) is mediated by the polycomb repressive complex (PRC2).

[0081] According to a specific embodiment of the present invention, the polycomb repressive complex (PRC2) is composed of EZH2, SUZ12, and EED.

[0082] According to a specific embodiment of the present invention, the step (b) is performed by measuring the level of one or more selected from the group consisting of formation of APB (ALT-associated promyelocytic leukemia bodies), formation of TERRA-R loop, trimethylation of histone H3K9 (H3K9me3), and trimethylation of histone H3K27 (H3K27me3).

[0083] The meanings of alternative telomere maintenance mechanisms, benign cancer, liquid-liquid phase separation phenomenon, prevention, treatment, liquid-liquid phase separation, APB, TERRA-R-loop, telomeric G-quadruplex, histone H3K9 trimethylation (H3K9me3), histone H3K27 trimethylation (H3K27me3), polycomb repressive complex, EZH2, SUZ12, and EED have been described above, so their description is omitted to avoid excessive duplication.

[0084]

[0085] According to another aspect of the present invention, the present invention provides a composition for diagnosing cancer or aging of an alternative telomere maintenance (ALT) mechanism, which comprises as active ingredients an agent for detecting telomeres in a biological sample isolated from an individual and an agent for measuring the degree of telomere damage.

[0086] According to a specific embodiment of the present invention, the agent for measuring the degree of damage to the telomere is selected from the group consisting of an agent for detecting telomere G-quadruplexes, an agent for detecting rH2AX, an agent for detecting APB (ALT-associated promyelocytic leukemia bodies), an agent for detecting TERRA (Telomeric Repeat-containing RNA), and an agent for detecting telomere R-loop.

[0087] Since the ALT mechanism-positive cancer and aging that can be diagnosed with the composition of the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0088] The term “diagnosis” as used herein includes determining an individual’s susceptibility to a particular disease, determining whether an individual currently has a particular disease, and determining a prognosis for an individual with a particular disease.

[0089] The term “diagnostic composition” in this specification means an integrated mixture or device including a means for measuring the expression level of the marker proteins or nucleic acid molecules listed above to determine whether a subject has developed ALT mechanism-positive cancer or aging or to predict the possibility of developing the same, and may also be expressed as a “diagnostic kit.”

[0090] In this specification, the “agent for detecting” a target protein or nucleic acid molecule means an agent that can provide visual, quantitative information about the presence, expression level, or expression location (localization) of a protein or nucleic acid molecule that is a diagnostic marker for the ALT mechanism-positive cancer or aging listed above in a biological sample, for example, in the case of a protein, it may be an antibody or an antigen-binding fragment thereof that specifically binds thereto, and in the case of a nucleic acid molecule, it may be a primer, probe, antibody, or an antigen-binding fragment thereof that specifically binds to the nucleic acid molecule.

[0091] According to a specific embodiment of the present invention, the agent for detecting the telomere is an antibody or an antigen-binding fragment thereof that specifically binds to TRF1 (Telomeric Repeat Binding Factor 1).

[0092] According to the present invention, the TRF1 protein of the present invention can be detected using an immunoassay method utilizing an antigen-antibody reaction, and used to analyze the presence and location of telomeres in a biological sample, and based on this, the presence or absence of telomere damage and the subsequent development of ALT-mediated cancer. This immunoassay can be performed according to various immunoassay or immunostaining protocols developed in the past.

[0093] When TRF1 protein is used as a diagnostic marker, markers reflecting the degree of telomere damage (e.g., G4, rH2AX, APB, TERRA) can be measured together as a composite marker, and when their expression locations are the same (co-localization), the risk of ALT mechanism-positive cancer can be determined to be increased.

[0094] According to a specific embodiment of the present invention, the agent for detecting APB is an antibody or an antigen-binding fragment thereof that specifically binds to PML (Promyelocytic leukemia protein).

[0095] According to a specific embodiment of the present invention, the agent for detecting the telomere R-loop is an antibody or an antigen-binding fragment thereof that specifically recognizes DNA-RNA hybridization.

[0096] According to a specific embodiment of the present invention, the alternative telomere maintenance mechanism positive cancer to be diagnosed with the composition of the present invention is triple-negative breast cancer or ovarian cancer.

[0097] According to a specific embodiment of the present invention, the aging disease to be diagnosed with the composition of the present invention is ischemic necrosis disease, and more specifically, avascular necrosis of the femoral head.

[0098]

[0099] The features and advantages of the present invention are summarized as follows:

[0100] (a) The present invention identifies factors involved in the alternative telomere maintenance mechanism and their correlations, and provides a method for preventing or treating alternative telomere maintenance mechanism-positive cancer by regulating the expression of the factors.

[0101] (b) The present invention provides an effective treatment target for alternative telomere maintenance mechanism-positive cancer, the mechanism of which has not been fully elucidated and thus no effective treatment method has been available, and thus can be usefully used for the efficient prevention or treatment of alternative telomere maintenance mechanism-positive cancer by regulating the expression of the target.

[0102]

[0103] Figure 1 illustrates the results of experiments confirming that assembly of telomere PML bodies is essential for telomere synthesis in BRCA2-deficient ALT-like cells. Figure 1a illustrates the relative levels of PML mRNA (left) and protein (right) in telomerase-positive (+mTR) and telomerase-negative (-mTR) MEFs with or without Brca2. BI-derived fibroblasts (telomerase-positive (mTR + / + );Brca2 F11 / F11 ;Cre-ER TM ) or telomerase-null TBI fibroblasts (telomerase-null (mTR - / - );Brca2 F11 / F11 ;Cre-ER TM) were conditionally Brca2-deficient under 4-OHT treatment conditions. The bar graphs are representative of the results of four or more independent experiments. Figure 1b is a graph depicting the relative mRNA levels of interferon-α, -β, and -γ in telomerase-positive (+mTR) and telomerase-negative (-mTR) MEFs in the presence or absence of Brca2, respectively. The bar graphs are representative of the results of four or more independent experiments. Figure 1c is a graph depicting the results of analyzing telomere synthesis in the G2 phase after transfecting fibroblasts with two different siRNAs targeting PML (siPML#3, siPML#4) in the presence or absence of Brca2. The left image depicts the extent of EdU incorporation at telomeres in TBI fibroblasts double knocked out of Brca2 and telomerase. Fibroblasts were arrested in the G2 phase by treatment with 18 μM RO-3306, a CDK1 inhibitor. Magnified images of EdU-positive telomeres are indicated by white squares, and the scale bar is 5 μm. The upper right corner shows the percentage of cells with three or more EdU-positive telomeres. The bar graph represents the results of three independent experiments. >100 BI cells (+mTR) and >200 TBI cells (-mTR) fibroblasts were scored for each experiment. The lower right corner shows the results of Western blot analysis after siRNA transfection to evaluate the efficiency of PML knockdown (Student's t-test (mean ± SEM)).

[0104] Figure 2 illustrates the results of experiments confirming that liquid-liquid phase separation properties play an essential role in telomere synthesis in ALT cells. Figure 2a illustrates examples of TRF1-IDR expression constructs. IDR domains derived from the N-terminus of FUS (FUSN) and the N-terminus of DDX4 (DDX4N), two different IDR-containing proteins, were fused to the TRF1-expressing construct, respectively, and the cDNA expressing mCherry (mCh) fluorescent protein was inserted downstream of TRF1. When these constructs were introduced into cells, LLPS-induced telomere clustering was induced. Figure 2b illustrates representative time-lapse capture images of TRF1-FUS movement in WT fibroblasts. Telomere foci formed in droplets are indicated by arrows, and enlarged images are shown below (scale bar, 10 μm). Figure 2c is a plot depicting the integrated fluorescence intensity of TRF1-IDR clusters within the nucleus per cell, with the y-axis expressed in arbitrary units. >400 cells were scored, and x / y is the integrated intensity of TRF1 foci / average intranuclear mCherry intensity (black, TRF1-mch; red, TRF1-FUS; pink, TRF1-DDX). In Figures 2d and 2e, TBI fibroblasts were transfected with siPML in the presence (+) or absence (-) of Brca2, and then fibroblasts were infected with the indicated construct-expressing lentiviruses (M, TRF1-mCh; F, TRF1-FUS; D, TRF1-DDX). Figure 2d is a plot depicting the percentage of cells with ≥3 EdU-positive telomeres, with the bar graph representing the results of three independent experiments, with >120 cells scored for each condition. Figure 2e depicts the results of telomere length comparisons. Telomere length was measured using interphase T-FISH and expressed as arbitrary units of fluorescence intensity. For each condition, >1000 telomere foci derived from >50 cells were scored.In Figures 2F and 2G, U2OS cells were transfected with siLuc (control, +) or siPML (-) and lentivirally infected with the indicated constructs (M; F; D). Figure 2F is a representative image depicting colocalization of EdU (pink) and telomeres (green) in G2 phase cells (scale bar, 5 μm). On the right is the quantification of EdU-positive telomeres in U2OS cells, with >100 cells counted for each condition. Figure 2G is a representative image of colocalization of BLM (red) and telomeres (green) in cells (scale bar, 5 μm). The figure on the right shows the results of quantifying BLM-positive telomeres in U2OS cells, with >120 cells counted for each condition (* p<0.0001, Student's t-test (mean ± SEM)).

[0105] Figure 3 illustrates the results of an experiment confirming that the increase in telomere G4 increases the TERRA-R loop, which in turn causes the assembly of PML bodies and telomere synthesis. The experiments in Figures 3a to 3f were performed in BI and TBI fibroblasts. For conditional depletion of Brca2, fibroblasts were treated with tamoxifen (4-OHT), and the molecular characteristics of ALT were measured daily. Figure 3a illustrates the results of Western blot analysis for Brca2 deletion and Brca2F11 allele generation after 4-OHT treatment. Figure 3b illustrates the results of the evaluation of G4-positive telomeres. The top image shows representative images of colocalization of G4 (green) and telomeres (red). Fibroblasts were fixed and immunofluorescent with BG4 (anti-DNA G4 monoclonal antibody, green), followed by T-FISH using the TelC-PNA probe (red). An enlarged image of the white square is shown at the bottom. The bottom shows the percentage of cells with three or more G4-positive telomeres, scored for each of 100 or more cells (scale bar, 5 μm). Figure 3c shows the results of evaluating TERRA RNA levels. The upper panel shows the results of TERRA RNA detection by TelC-PNA hybridization (green) at 37°C following immunofluorescence with anti-TRF1 antibody (red) (scale bar, 5 μm). The lower panel shows the results of RNA-FISH for TERRA RNA, quantified as fluorescence intensity in arbitrary units, with >500 TERRA foci counted in >40 cells. Figure 3d shows the results of immunofluorescence coupled with T-FISH for the evaluation of R-loops at telomeres. R-loops were detected by immunofluorescence with S9.6 (anti-DNA:RNA hybrid antibody, green). A magnified image of the white square area is shown at the bottom.The lower part is a bar graph showing the percentage of cells with ≥4 R-loop positive telomeres, scored for >120 cells each. Figure 3e is a graph showing the results of quantification of APBs per cell, scored for >100 cells each. Figure 3f is a graph showing telomere length measured by interphase T-FISH as arbitrary units of fluorescence intensity, measured for >500 telomere foci in >30 cells. Figure 3g is a graph showing the results of measuring TERRA RNA abundance levels in HeLa LT TERC KO cells in the presence or absence of BRCA2, transfected with siLuc(+) or siBRCA2(-) 3 days prior to cell fixation. Representative images of TRF1 (green) and TERRA (red) are shown on the left (scale bar, 2.5 μm), and the intensity of TERRA RNA measured in RNA-FISH images is shown on the right. Fluorescence intensity is expressed in arbitrary units, and >500 TERRA foci in >30 cells each were scored. All results are from three independent experiments (p<0.0001, Student's t-test (mean ± SEM)).

[0106] Figure 4 illustrates the results of an experiment confirming that telomere R-loops induce LLPS formation. Telomerase-positive BI and telomerase-negative TBI fibroblasts in the experiments of Figures 4a to 4e were infected with a lentivirus encoding RNase H1-GFP. The upper part of Figure 4a is a graph showing the number of APBs per cell, and more than 100 cells were measured for each condition. The lower part is a figure showing the results of Western blot analysis to evaluate deletion and generation of the Brca2F11 allele after 4-OHT treatment, evaluating the effects of Brca2 deletion and / or RNH1-GFP overexpression on PML and POLD. Figure 4b shows representative fluorescence images showing APBs in TBI fibroblasts transduced with or without RNH1-GFP in the presence (+) or absence (-) of Brca2 (PML, anti-PML immunofluorescence (green); telomeres, T-FISH (red). Scale bar, 5 μm). Figure 4c shows the results of an experiment to determine the effect of R loop and LLPS formation on G2 telomere synthesis. TBI fibroblasts expressing RNH1-GFP were transduced with lentiviruses expressing TRF1-mCh (M), -FUS (F), and -DDX (D), respectively, and then subjected to experiments to assess G2 telomere synthesis. The bar graph represents the percentage of TBI cells with ≥3 EdU-positive telomeres, scored for >100 cells included in each condition. Figure 4d shows the results of an experiment to determine the effect of stabilized G4 on R-loop accumulation at telomeres. BI or TBI fibroblasts were depleted of Brca2, treated (-) or not (+) with 4-OHT, and exposed to 5 μM pyridostatin, a G-quadruplex stabilizer, for 24 h. Cells not treated with PDS were included as a control. The percentage of cells with ≥5 R-loop-positive telomeres is shown, with >120 cells measured under each condition.Figure 4e shows the results of experiments examining the effects of PDS and / or RNH1 on APB formation, with >100 cells measured in each. Figure 4f shows the results of experiments using HeLa LT TERC KO cells transfected with siLuc (control) or siBRCA2 and co-transfected with mCherry- or RNH1-mCherry expression constructs. The top row is a representative image of co-localization of R-loops (green) and telomeres (red), with white arrows indicating R-loop-positive telomeres, and the middle row is a representative image of co-localization of PML (green) and telomeres (red), with white arrows indicating APBs (scale bar, 2.5 μm). The lower panel shows the quantification of R-loop-positive telomeres and APBs in HeLa LT TERC KO cells. Each condition contained >120 cells, and all data were derived from three independent experiments. Figure 4g shows the experimental results confirming a model of the mechanism by which BRCA2 deletion promotes ALT-like telomere synthesis. Brca2 deletion induced G4 stabilization, followed by an increase in TERRA-R loops. R-loops triggered the formation of LLPS, which contains a protein complex required for BIR at telomeres (*p<0.0001, Student's t-test (mean ± SEM)).

[0107] Figure 5 shows the results of an experiment confirming that trimethylation of lysine 27 of histone H3 at telomeres within LLPS is mediated by the telomeric R-loop. The left side shows the results of hybridization with telomere and Alu probes after applying the chromatin IP (ChIP) method for H3K27me3, H3K9me3, and histone H3 to Brca2-deleted (-) or non-deleted (+) BI (+mTR) and TBI (-mTR) fibroblasts. The right side is a bar graph showing the results of quantifying the ChIP experiment results in the blot on the left. The results for H3K27me3 and H3K9me3 at telomeres were normalized to the level of H3, and the experiment was repeated four times independently. Figure 5b shows the results of ChIP using anti-H3K27me3 and -H3 antibodies, respectively, on RNH1-expressing BI and TBI fibroblasts with or without Brca2 deletion, followed by hybridization with a telomere probe. On the right is the result of measuring H3K27me3 levels normalized to H3, and the results are from three independent experiments. Figures 5c and 5e show the results of experiments using BI and TBI fibroblasts depleted of the PRC2 core components EZH2, SUZ12, and EED proteins using siRNA transfection in the presence (+) or absence (-) of Brca2. Figure 5c shows depletion of the PRC2 complex by removing the telomeric LLPS, and >100 cells were counted in each. Figure 5d illustrates the results of an experiment to remove APB by administering EZH2 inhibitors. BI and TBI fibroblasts, treated (-) or not (+) with 4-OHT to remove Brca2, were exposed to 5 μM PDS for 24 h and / or 5 μM EPZ-6438 for 48 h prior to cell fixation, and analyzed for APB formation. Scores were assigned to >100 cells under each condition.Figure 5e shows the results of an experiment confirming that PRC2 depletion significantly reduces telomere R-loop formation, with >100 cells counted for each. Figure 5f shows the results of an experiment confirming that telomere LLPS decreases upon depletion of BIR factors POLD3 or BLM, with >140 cells scored. Figure 5g shows the results of an oligonucleotide pull-down assay analyzing the region to which PRC2 protein binds, with cell lysates incubated with the indicated biotinylated oligonucleotides and Western blot analysis using the indicated antibodies. RNA oligonucleotides (CCCUAA)8 and (UUAGGG)8 represent TERRA antisense and TERRA, respectively, (TTAGGG)8+(CCCTAA)8 represents a hybrid double-stranded telomere, (UUAGGG)8+(CCCTAA)8 represents a DNA:RNA hybrid (R-loop), and (UUAGGG)8+(CCCTAA)3 represents a DNA:RNA hybrid with five exposed TERRA repeats. Figure 5h is a diagram illustrating the telomeric chromatin remodeling model essential for telomere synthesis in ALT-like cells within LLPS, where the PRC2 complex catalyzing H3K27me3 is recruited at TERRA RNA telomeres protruding from the R-loop. All results are representative of three independent experiments (* p<0.0001, Student's t-test (mean ± SEM)).

[0108] Figure 6 illustrates the abnormal G4 stabilization of BRCA2-deficient ALT-like cells and their selective sensitivity to EZH2 inhibitors. Figure 6a compares the relative growth rates of Brca2-deficient or -deleted BI and TBI fibroblasts following treatment with PDS (left) or EPZ-6438 (right, EZH2i), and the calculated cell numbers were normalized to the cell growth rates in the untreated group (WT control: circles and black lines; BI and -Brca2 squares and blue lines; TBI: open circles and black dashed lines; open squares and red dashed lines: TBI and -Brca2). Figure 6b is a graph comparing the relative growth of TBI fibroblasts with or without Brca2 deletion when co-treated with 2.5 μM PDS and 1 μM EZH2i. Cells were counted every 2 days, and the growth rate was normalized compared to the untreated group (circles and black line: TBI+EZH2i; squares and blue line: TBI+EZH2i+PDS; black dotted line and open circles: TBI & -Brca2+EZH2i; red dotted line and open squares: TBI & -Brca2+EZHi+PDS). Figures 6c and 6d are pictures showing the results of analyzing the cell viability of BRCA2-deleted or -non-deleted HeLa LT TERC KO cell lines according to PDS and / or EPZ-6438 (EZH2i) treatment. HeLa LT TERC KO cells were transfected with siLuc (control) or siBRCA2 for 24 hours, seeded in 96-well plates, and then treated with the indicated concentrations of drugs. Cell viability was measured using an MTT assay after 3 days. Figure 6c is a graph showing the results according to PDS treatment at concentrations of 0 to 2.5 μM for 3 days (circles and black line: siLuc; squares and red dotted line: siBRCA2).Figure 6d is a graph showing the response of BRCA2 deletion and non-deletion HeLa LT TERC KO cells to EZH2i and co-treatment with PDS and EZH2i. Cells treated with 1 μM PDS were co-treated with EPZ-6438 at concentrations ranging from 0 to 10 μM (circles and black lines: siLuc+EZH2i; squares and black dashed lines: siLuc+EZH2i+PDS; open circles and red lines: siBRCA2+EZH2i; open squares and red dashed lines: siBRCA2+EZH2i+PDS). Experiments were repeated three times independently (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Student's t-test (mean ± SEM)).

[0109] Figure 7 illustrates the results confirming that approximately half of the mutant human breast cancers expressing APB have BRCA2 deletions. Figure 7a shows a representative fluorescent image of APB-positive human breast cancer tissue, which was performed by combining immunofluorescence using an anti-PML antibody and T-FISH on paraffin-embedded tissue sections (white arrow: APB). The bar graph represents the frequency of APB-positivity in breast cancer specimens, comparing wild-type BRCA2 (WT) breast cancer and mutant BRCA2 breast cancer. 19 samples were used for each (x / y: number of APB-positive sections / total number of samples; scale bar, 2 μm). Figure 7b shows the heterogeneity of telomere length (coefficient of variation), which was calculated as the standard deviation of telomere length divided by the mean telomere length. Figure 7c is a model illustrating how BIR functions for telomere synthesis after BRCA2 deletion. BRCA2 deletion stabilizes telomeric G4s, accumulates TERRA-containing R-loops at telomeres, induces telomere phase separation by the increased R-loops, the interior of the resulting telomeric LLPS is characterized by H3K27me3, and DNA breaks recruit BIR DNA repair factors. The abnormally stabilized telomeric G4s induce TERRA-R-loop accumulation, which induces phase separation with protein aggregation. BIR is induced by telomere damage due to replication problems in S phase, forming liquid condensates (Student's t-test (mean ± SEM)).

[0110] Figure 8 illustrates the experimental results confirming the induced phase separation and its effect on break-induced replication (BIR). The left side of Figure 8a is a representative image of fluorescence recovery after photobleaching (FRAP) analysis of TRF1-IDR (scale bar, 0.5 μm), and the right side shows the FRAP recovery curves measured by the average intensity of mCherry, expressed in arbitrary units (black: TRF1-mCh; red: TRF1-FUS; pink: TRF1-DDX). The left side of Figure 8b shows a control construct lacking TRF1, in which two nuclear localization signals (NLSs) from SV40 were attached to the N-terminus, and the mCherry (mCh) fluorescent protein-expressing cDNA was linked downstream of the NLSs. When these constructs were introduced into cells, LLPS-induced aggregation was induced. The right side shows representative images of mCherry fluorescence of each NLS-IDR expression within the nucleus (scale bar, 5 μm). Figures 8c-8f show G2 telomere synthesis (Figures 8c and 8f) and telomere length (Figures 8d and 8f) measured in telomerase-positive BI and telomerase-negative TBI fibroblasts after PML depletion and / or M, F, D expression using lentiviral transduction in the presence (+) or absence (-) of Brca2. Transfection with siLuc(+) served as a control for PML depletion using siPML(-). Figures 8c and 8e are graphs depicting the percentage of cells with three or more EdU-positive telomeres, scored for >100 cells each. Figures 8d and 8f show the results of T-FISH measurements, with >800 telomere foci measured in >50 cells each. Figure 8g shows the results of Western blot analysis in U2OS cells, showing mCherry protein levels after transfection with the TRF1-IDR construct; and PML knockdown efficiency after siPML transfection. All results are representative of three independent experiments (* p<0.05).0001, Student's t-test (mean ± SEM)).

[0111] Figure 9 illustrates the results of RNA-FISH validation for detecting G4 and R-loop-specific antibodies used in immunofluorescence and TERRA. Figure 9a is a representative image of G4 (green) and telomeres (red). G4 was detected by immunofluorescence with a monoclonal antibody against FLAG, and the G4-specific antibody BG4 tagged FLAG (Merck Millipore). Telomeres were labeled by T-FISH in the presence or absence of BG4 in Brca2-depleted TBI fibroblasts (white arrows: G4-positive telomeres). Figure 9b is a representative image of TERRA RNA (green) in Brca2-depleted TBI fibroblasts in the presence or absence of RNase A. Figure 9c Left shows R-loops (green) immunofluorescently labeled with S9.6 antibody in Brca2-depleted TBI fibroblasts and telomeres (red) in the presence or absence of RNH fused to overexpressed GFP (RNH1-GFP) (white arrows: R-loop-positive telomeres). Right shows the results of counting the number of R-loop-positive telomeres per cell, with 100 cells counted each. All experiments consisted of at least three independent experiments (scale bar, 5 μm. Student's t-test (mean ± SEM)).

[0112] Figure 10 illustrates the results of experiments confirming that R-loops are essential for Brca2-deficiency-induced ALT-like telomere synthesis. The experiments in Figures 10a to 10d were performed in RNH1-GFP-expressing TBI fibroblasts, which were infected with the indicated lentiviruses M, F, and D, respectively, in the presence (+) or absence (-) of Brca2. Figure 10a shows the results of Western blot analysis using anti-RNH1 antibodies; the same blot was reprobed with anti-actin antibodies for normalization. Figures 10b and 10d show that telomere length was scored as arbitrary fluorescence units by T-FISH in interphase, with more than 700 telomere foci measured in more than 40 cells under each condition. Figure 10c shows the percentage of cells with three or more EdU-positive telomeres. The results were derived from three independent experiments, and scores were calculated for each of >110 cells. Figure 10e shows Western blot analysis results showing mCherry levels after siBRCA2 knockdown and transfection of a construct expressing RNH1-mCherry in HeLa LTTERCKO cells. Figures 10f and 10g show BJ cells transfected with siLuc (control) or siBRCA2, followed by transduction using a RNH1-GFP expressing lentivirus. Figure 10f shows the results of Western blot analysis using the indicated antibodies. Figure 10g Left: Representative image of co-localization of R-loop (green) and telomeres (red) (white arrows: R-loop-positive telomeres), right: Representative image of co-localization of PML (green) and telomeres (red) (white arrows: APBs; scale bar, 2.5 μm), and bottom: Quantitative results of R-loop-positive telomeres and APBs in BJ cells, each counted for >100 cells (* p<0.0001, Student's t-test (mean ± SEM)).

[0113] Figure 11 illustrates the results of an experiment confirming the effect of PRC2 inhibition on phase separation and telomere synthesis. Figure 11a shows the results of an experiment in which wild-type MEFs were transfected with siRNA targeting EZH2, and Western blot analysis confirmed that EZH2 and H3K27me3 were depleted by siEZH2 transfection. The levels of H3K9me3 and histone H3 were also shown on the same blot. Figure 11b, left, shows the results of chromatin IP showing the levels of H3K27me3 and H3K9me3 before and after siEZH2 transfection. The abundance of H3K27me3, H3K9me3, and H3 at telomeres was normalized to the input value, and the experiment was repeated three times independently. Figure 11c shows the results of Western blot analysis using the indicated antibodies on BI (left) and TBI (right) fibroblasts after siRNA transfection of EZH2, SUZ12, and EED, respectively. Figure 11d shows the results of an experiment to determine the effect of PRC2 complex depletion on telomere synthesis. The graph shows the percentage of cells with three or more EdU-positive telomeres, and >100 cells were counted for each. Figure 11e shows the results of an experiment in which BI and TBI fibroblasts were exposed to 5 μM of the EZH2 inhibitor EPZ-6438 for 48 hours, followed by Western blot analysis using the indicated antibodies to assess the levels. The same blots were reprobed with anti-actin antibody for normalization. Figure 11f shows the results of measuring the abundance of TERRA RNA in PRC2-deficient fibroblasts. The measured intensity of TERRA RNA is expressed in arbitrary units, and >500 TERRA foci from >30 cells in each condition were scored. Figure 11g shows the results of Western blots confirming the efficiency of POLD3 or BLM knockdown after shRNA transduction and selection in BI and TBI fibroblasts.Figure 11h illustrates the results of an experiment confirming that ALT-like telomere synthesis function is reduced by depletion of POLD3 or BLM. Mouse fibroblasts were transduced with shRNA targeting POLD3 or BLM, and the level of EdU incorporation at telomeres was then assessed. Three or more EdU-positive telomeres are represented as a bar graph, and data were derived from three independent experiments and scored for >150 cells. Figure 11i is a diagram illustrating the results of oligonucleotide pull-down assays to analyze the PRC2 binding domain, where cell lysates were incubated with the indicated biotinylated substances, and oligonucleotides were subjected to Western blot analysis using the indicated antibodies (TERRA antisense: (CCCUAA)8; single-stranded telomere: (TTAGGG)8; TERRA sense: (UUAGGG)8; double-stranded telomere: (TTAGGG)8+(CCCTAA)8; DNA:RNA hybrid (R-loop): (UUAGGG)8+(CCCTAA)8; DNA:RNA hybrid with three repeats of exposed TERRA: (UUAGGG)8+(CCCTAA)5; DNA:RNA hybrid with five repeats of exposed TERRA: (UUAGGG)8+(CCCTAA)3, * p<0.0001, Student's t-test (mean ± SEM)).

[0114] Figure 12 is a drawing showing the results of confirming the degree of structural damage of telomeres, a characteristic of ALT cancer, in surgical tissues of triple-negative breast cancer (TNBC) patients and ovarian cancer patients, and shows the results of immunofluorescence staining for G4, rH2AX, and TRF1 in paraffin surgical tissue samples of TNBC patients (Figure 12a) and ovarian cancer patients (Figure 12b), respectively.

[0115] Figure 13 is a drawing showing the results of immunofluorescence staining for rH2AX and telomeres and telomere-FISH performed to determine the degree of telomere damage, a characteristic of ALT cancer, in paraffin surgical tissue samples from patients with avascular necrosis of the femoral head (Figure 13a), patients with ovarian cancer (Figure 13b), and patients with triple-negative breast cancer (Figure 13c).

[0116] Figure 14 is a diagram showing the results of immunofluorescence staining for PML and telomeres and telomere-FISH performed to determine the presence of APB, a characteristic of ALT cancer, in tissue paraffin samples from surgical ovarian cancer patients.

[0117] Figure 15 is a drawing showing the results of immunofluorescence staining for TERRA according to the presence or absence of BRCA2 mutations in order to confirm the degree of TERRA, a characteristic of ALT cancer, in paraffin samples of surgical tissues of ovarian cancer patients.

[0118] Figure 16 is a drawing showing the results of immunofluorescence staining that confirmed the telomere R-loop, a characteristic of ALT cancer, in paraffin samples of surgical tissue from ovarian cancer patients.

[0119]

[0120] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0121]

[0122] Example

[0123] Liquid-liquid phase separation is crucial for break-induced replication (BIR)-mediated telomere synthesis upon Brca2 loss.

[0124] Brca2 depletion in telomerase-negative cells induces break-induced replication (BIR) at telomeres and induces ALT-like activity. The appearance of PML bodies, namely ALT-associated promyelocytic leukemia bodies (APBs), at telomeres is one of the characteristics of these Brca2 depletion-induced ALT-like cells. We focused on APB formation and sought to elucidate the factors that induce APB formation upon Brca2 depletion. To elucidate this, we used mouse embryonic fibroblasts in which Brca2 can be conditionally deleted. Brca2 F11 / F11 ;ER-Cre mice to mTR + / - Crossbreeding with mice to produce Brca2 F11 / F11 ;ER-Cre; mTR + / + (BI,Brca2-inducible) orBrca2 F11 / F11 ;ER-Cre;mTR - / - (TBI, Telomerase-null and Brca2-inducible) progeny were generated. Subsequently, BI and TBI MEFs were isolated and immortalized by expressing the large T antigen of SV40. The present inventors have generated telomerase-positive BI (mTR + / + ) or negative TBI (mTR - / -) Cre expression was induced using tamoxifen (4-OHT) treatment in the background. We then first assessed whether Brca2 depletion affected the levels of promyelocytic leukemia protein (PML), which is important for recruiting multivalent protein interactions in APB assembly. Quantitative RT-PCR confirmed that PML RNA was increased twofold following Brca2 deletion (Fig. 1a, -Brca2; +mTR) or telomerase loss (Fig. 1a, +Brca2; -mTR). PML was further increased up to threefold when both Brca2 and telomerase were depleted (Fig. 1a, -Brca2; -mTR). Western analysis (WB) revealed that PML protein was increased up to fourfold in Brca2-depleted cells and telomerase-negative ALT cells (Fig. 1a, right and Fig. 1c, -Brca2 in TBI).

[0125]

[0126] PML expression is elevated in inflamed and tumor tissues, suggesting its involvement in inflammation, cell growth regulation, and other potential aspects of tumorigenesis. The increase in PMLRNA upon Brca2 loss or telomerase deficiency suggests that DNA damage may directly or indirectly increase PML expression. We sought to determine whether proinflammatory cytokines influence PML elevation. Quantitative RT-PCR analysis revealed that loss of either Brca2 or telomerase slightly increased interferon-α (IFNα), but no additive effect was observed when both were lost (Fig. 1b). In contrast, interferon-β (IFNβ) levels increased more than fourfold upon Brca2 loss but were unaffected by telomerase loss (Fig. 1b). Interferon-γ (IFNγ) levels increased ~2-fold upon Brca2 or telomerase loss. Deletion of Brca2 and telomerase resulted in an additive effect of more than a threefold increase in interferon-γ (Fig. 1b).

[0127] Next, we tested whether PML protein is essential for ALT activity in Brca2-deficient cells. To this end, we depleted PML in TBI fibroblasts, which exhibit BIR and ALT-like activity. BRCA2 loss causes telomere damage during replication, resulting in a break in the late S / G2 phase. Thus, G2 or mitotic telomere synthesis is responsible for ALT in the presence of BRCA2 loss. Therefore, we synchronized cells at the G2 / M boundary by treating them with the CDK1 inhibitor RO-3306. Telomere synthesis activity was then measured using the EdU (5-ethynyl-2'-deoxyuridine) Click Assay combined with T-FISH (telomere in situ hybridization). When PML was depleted in Brca2-deficient TBI fibroblasts using siRNA transfection, no EdU incorporation was observed at any telomeres (Fig. 1c). In conclusion, we were able to confirm that Brca2 loss increases PML, which is required for telomere synthesis, similar to ALT.

[0128] In a subset of ALT cells, telomeres cluster at APBs, promoting telomere synthesis, and APBs exhibit liquid-liquid phase separation properties. It remains unclear whether elevated levels of DNA repair factors at APBs and the induction of liquid condensates, thereby promoting telomere clustering, are unrelated. Therefore, we sought to determine whether BRCA2 deficiency, which increases PML and induces APBs, confers the essential phase separation properties of telomeres for ALT activity.

[0129] Intrinsically disordered regions (IDRs) contribute to complex promiscuous interactions that synergize with specific protein-protein and RNA-protein interactions to form LLPS. In particular, FUS and DDX4, which harbor IDRs, induce LLPS. Considering this, we generated lentiviral constructs expressing IDRs of FUS or DDX4 fused to the telomere repeat binding protein (TRF1) (Fig. 2a). To eliminate functions that could interfere with ALT, the expression constructs contained only the N-terminus of FUS or DDX (FUSN and DDXN, respectively). Furthermore, the red fluorescent mCherry expression gene was fused between TRF1 and the IDRs, allowing for microscopic visualization (Fig. 2a). These lentiviruses were transduced into cells in the presence or absence of PML and their LLPS formation ability and telomere synthesis activity were analyzed.

[0130] When TRF1-IDR was expressed in cells, telomere clustering was observed, similar to previous observations of APB in ALT telomere rearrangement (Fig. 2b). Clustered telomere foci showed rapid recovery after photobleaching, indicating that they had liquid droplet characteristics (Fig. 8a). To measure the degree of telomere clustering, various TRF1-IDR constructs were expressed in wild-type fibroblasts, and the integrated intensity of each focus was measured. The results were then normalized to the mean intensity of nuclear mCherry fluorescence. According to the normalized results, TRF1-FUS expression showed the highest fluorescence intensity at these foci, followed by TRF1-DDX. Control TRF1-mch expression showed the weakest fluorescence (Fig. 2c). These results suggest that telomere-induced phase separation using TRF1-IDR induces telomere clustering.

[0131] Next, we sought to determine whether artificially induced phase separation could replace PML-induced APB in telomere synthesis. To this end, we transduced PML-depleted TBI fibroblasts with TRF1-IDR-expressing lentiviral particles. The results showed that expression of TRF1-IDR, -FUS(F), or -DDX(D) completely restored telomere synthesis activity to levels comparable to those in PML-proficient cells (Fig. 2d). Consistent with this, telomere shortening induced by PML depletion was restored by TRF1-IDR expression (Fig. 2e), but not by expression of the control TRF1-mCh(M). In contrast, introduction of NLS-IDR, in which TRF1 is replaced by two nuclear localization signal sequences (NLS) from SV40 (SV40-NLS) (Fig. 8b), resulted in large condensations in the nucleus (Fig. 8b). However, it was unable to reactivate telomere synthesis by inducing phase separation in the non-telomeric region (Fig. 8b-d). Next, we tested whether a human ALT cancer cell line behaved similarly. We used U2OS, a well-defined ALT cancer cell line that utilizes BIR for telomere maintenance. We found that PML depletion significantly reduced telomere synthesis in human U2OS ALT cells (Fig. 2f). The BLM helicase localizes to the APB of human ALT cell lines and is essential for BIR. PML depletion in U2OS cells significantly reduced the number of BLM-positive telomeres (Fig. 2g). In contrast, lentiviral transduction of TRF1-IDR into PML-depleted cells completely restored ALT activity (Fig. 2f). Furthermore, BLM localization at telomeres was also restored (Fig. 2g), indicating that phase separation induction is crucial for the assembly of BIR proteins for ALT activity in human cancer cells.These results suggest that the pivotal role of phase separation in telomere synthesis is not limited to BRCA2-deficient cells but extends to BRCA2-proficient ALT cancer cells.

[0132]

[0133] Telomere G4 stabilization due to BRCA2 deficiency stimulates telomere separation.

[0134] Despite the formation of telomere LLPS (Fig. 2b), introduction of TRF1-IDR did not initiate telomere synthesis in the presence of Brca2 (Figs. 2d and 2e, +Brca2). This suggests that, in addition to the assembly of telomere LLPS, Brca2 depletion induces the recruitment of appropriate recombination factors to telomere LLPS. BRCA2 ensures telomere replication homeostasis through dynamic interactions with telomeric G-quadruplexes (G4). BRCA2 interacts with a G-quadruplex (G3) intermediate, which is formed during the interconversion between two different G-quadruplex forms. Indeed, BRCA2 loss likely stabilizes telomeric G4s. Considering the above, we sought to determine whether abnormally increased telomeric G4 is linked to telomere phase separation upon BRCA2 loss. First, we analyzed the effect of Brca2 loss on telomeric G4 formation using the G4-specific monoclonal antibody BG4 by immunofluorescence (Fig. 9a). As expected, Brca2 depletion (Fig. 3a, +4-OHT) significantly increased telomeric G4 in both BI and TBI fibroblasts 1 day after Brca2 depletion (Fig. 3b). The effect of Brca2 depletion on G4 stabilization was stronger in telomerase-negative TBI cells compared to telomerase-positive BI cells 1 day after Brca2 depletion (Fig. 3b, bottom).

[0135]

[0136] Loss of the dynamics of interconversion between two different G4 forms leads to G4 stabilization, which in turn leads to replication fork stalling. If the stalled replication fork is not restored, transcription-replication collisions occur, leading to increased transcription of unscheduled telomeric RNA (TERRA) and the formation of DNA:RNA hybrids, i.e., R-loops. Therefore, we analyzed whether TERRA and R-loop levels increase after telomeric G4 insertion. TERRA abundance was measured using immunofluorescence coupled with RNA telomere-FISH (T-FISH) hybridization in the presence or absence of Brca2 (Fig. 9b). Regardless of Brca2 or telomerase status, TERRA mostly colocalized with TRF1 (Fig. 3c). TERRA abundance was measured based on intensity, and its abundance varied depending on the presence or absence of telomerase. TERRA levels increased from day 1 in TBI fibroblasts (+4-OHT, day 1, TBI) due to Brca2 depletion, whereas this increase was delayed by one day in the presence of telomerase (+4-OHT, day 2, BI). From day 2 after Brca2 depletion (+4-OHT), Brca2-depleted telomerase-deficient double null TBI fibroblasts showed a greater increase in TERRA levels (Fig. 3C, bottom, +4-OHT, TBI) compared to telomerase-positive BI fibroblasts (Fig. 3C, bottom, +4-OHT, TBI). Immunofluorescence using the R-loop-specific monoclonal antibody S9.6 (Fig. 9C) showed that Brca2 depletion increased telomeric R-loops. Telomere R-loops were significantly increased 1 day after Brca2 depletion in telomerase-deficient TBI fibroblasts, and increased further from day 2 post-depletion (Fig. 3d, TBI). The number of APBs significantly increased from day 3 after Brca2 depletion in TBI fibroblasts, but the effect of Brca2 depletion was significantly less in the presence of telomerase (Fig. 3e).Meanwhile, in telomerase-positive fibroblasts (Fig. 3e, +4-OHT, day 4, BI), APBs slightly increased on day 4 after Brca2 depletion. Consistent with APB assembly, shortened telomeres due to telomerase deficiency lengthened from day 3 after Brca2 depletion in TBI fibroblasts (Fig. 3f, +4-OHT, day 3, TBI). On the other hand, telomere elongation was not observed in telomerase-positive cases (Fig. 3f, +4-OHT, BI). In summary, in the absence of Brca2, telomere LLPS is formed and sequential telomere synthesis occurs. The sequential steps are as follows: increase in telomeric G4 due to abnormal stabilization; transcription of TERRA and subsequent increase in TERRA-R-loops, especially in telomerase-deficient cases; Phase-separated APB assembly and telomere elongation. By demonstrating that telomerase-deficient mice are significantly affected by Brca2 depletion, we demonstrate that telomerase activity plays a central role in inducing ALT or ALT-like activity.

[0137] It is particularly significant that more R-loops are generated at shorter telomeres. Meanwhile, because mouse telomeres are much longer than human telomeres, we sought to determine whether this effect also holds true in human cells. We measured and compared TERRA levels in HeLa cells lacking TERC (HeLa LT TERC KO) in the presence or absence of BRCA2. Consistent with the data from mouse fibroblasts, BRCA2 depletion in human cells significantly increased TERRA levels (Fig. 3g).

[0138]

[0139] Telomere R-loops are essential for telomere LLPS formation.

[0140] Based on the sequence of events revealed above, we sought to confirm the possibility that telomeric R-loops may be essential for the assembly of phase-separated APBs. We sought to determine whether APB assembly was affected when telomeric R-loops were deleted. To this end, we exploited the function of RNase H1, an R-loop-specific nuclease that specifically degrades RNA in DNA:RNA hybrids. The effect of expression of GFP-tagged RNase H1 (RNH1-GFP) was confirmed in Brca2-depleted TBI fibroblasts (Fig. 9c). When RNH1-GFP was ectopically expressed in Brca2-depleted cells (Fig. 10a), this resulted in a significant reduction in phase-separated telomeric PML bodies (Figs. 4a and 4b). These results indicate that R-loops are involved in telomere LLPS formation. In WB, RNH1-GFP expression did not interfere with PML or POLD3 protein levels. The elevation of PML due to Brca2 depletion was not affected by RNH1 expression, despite a decrease in telomeric PML bodies (Fig. 4a, PML, TBI; -Brca2). Next, we sought to determine whether telomere synthesis was affected when R-loops were disrupted. Furthermore, we sought to determine whether artificially engineered phase separation at telomeres could restore APB and telomere synthesis activity in the absence of telomeric R-loops. To test this, RNH1 was ectopically expressed in TBI fibroblasts in the presence (+) or absence (-) of Brca2. We then transduced TRF1-IDR-expressing lentivirus to induce phase separation at telomeres. Fifty hours after transduction, EdU incorporation assay was performed on G2 / M arrested cells.As a result, we were able to confirm that telomere synthesis activity was significantly reduced by expressing RNH1-GFP in Brca2-deficient TBI fibroblasts. In addition, expression of TRF1-FUS (F) or TRF1-DDX (D) restored G2 telomere synthesis that was lost due to RNH1-GFP expression in Brca2-depleted ALT cells, but not in the control group (M) (Fig. 4c). In contrast, expression of nuclear IDR (NLS-IDR) had no effect on telomere synthesis or telomere elongation (Fig. S3D) (Fig. 10c). These results confirmed that R-loops are required for phase separation induction and that telomere LLPS formation is essential for BIR-mediated telomere synthesis.

[0141]

[0142] Pyridostatin (PDS) is a G4-stabilizing ligand that induces DNA damage. PDS-induced DNA damage sites appear to correlate with the proliferation of R-loops, suggesting that unplanned G4 stabilization and increased R-loops are associated with DNA damage and repair. We scored telomeric R-loops in cells with and without Brca2 and compared these scores to PDS-treated cells. Brca2 depletion or PDS treatment resulted in an increase in telomeric R-loops. The effects of Brca2 depletion or PDS treatment on R-loop formation were exacerbated in the absence of telomerase (Fig. 4d). Interestingly, combining Brca2 loss and PDS treatment further exacerbated the effects of R-loop formation (Fig. 4d, TBI; -Brca2; +PDS). The number of APBs increased upon PDS treatment or Brca2 depletion in both telomerase-positive and -negative cells. However, the magnitude of the increase was greater in telomerase-negative cells (Fig. 4e). Expression of RNH1-GFP abolished all telomere LLPS in both PDS-induced and Brca2-depleted cells. LLPS was also abolished when RNH1 was expressed, and the degree of LLPS loss was further increased when Brca2 depletion and PDS were combined in TBI fibroblasts (Fig. 4e, -Brca2; +PDS; +RNH1). These results suggest that stabilized G4s generate telomere R-loops, which in turn trigger phase separation at telomeres. Next, we sought to determine whether the same phenomenon occurred in human cells. HeLa LT TERC KO cell line (Fig. 10e) and wild-type BJ cell line lacking telomerase activity (Fig. 10f) were made into BRCA2-depleted cells using siRNA (siBRCA2). RNH1-GFP was then ectopically expressed, and the level of telomeric R-loops was determined.Simultaneously, APB formation was analyzed (Figs. 4f and 10g). BRCA2 depletion in telomerase-deficient human cells increased the level of telomeric R-loops (Figs. 4f and 10g). The increased telomeric PML bodies induced by BRCA2 depletion were significantly reduced when RNH1 was expressed (Figs. 4f and 10g). These results confirm that telomeric R-loops induce phase separation in human ALT-like cells, and that BRCA2 abrogation results in loss of telomeric G4 dynamics, leading to G4 stabilization. Abnormally stabilized telomeric G4s induce transcription-replication collisions, resulting in the accumulation of TERRA-containing R-loops. Telomeric R-loops induce liquid-liquid phase separation and BIR at telomeres. In summary, loss of telomeric G4 dynamics leads to phase separation and BIR at telomeres (Fig. 4g).

[0143]

[0144] Histone H3 trimethylated lysine 27 indicates telomere activity in BIR.

[0145] The state of telomeric chromatin (euchromatin or heterochromatin) during ALT telomere synthesis remains controversial. Previously, we confirmed that BIR, a conventional replication mechanism, underlies ALT telomere synthesis in cases of BRCA2 loss. Furthermore, considering that MiDAS, a mitotic DNA synthesis process, underlies BRCA2 deficiency-induced ALT, we hypothesized that telomere synthesis induced by BRCA2 deficiency occurs in a heterochromatin state.

[0146] To elucidate the molecular structure of telomeric chromatin in BRCA2 deficiency-induced ALT, we examined the level of chromatin condensation by measuring the abundance of histone H3 trimethylation at lysine 9 (H3K9me3) and lysine 27 (H3K27me3). Telomere ChIP was performed using antibodies against H3K9me3 and H3K27me3 in BI and TBI fibroblasts treated with or without tamoxifen. The ratio of H3K9me3 or H3K27me3 to H3 was compared to normalize for differences in telomere length. H3K9me3 was detected at all telomeres (Fig. 5a, H3K9me3 / H3). In contrast, H3K27me3 levels were increased up to 2-fold (~2-fold) by Brca2-depletion in the absence of telomerase (Fig. 5a, -Brca2; -mTR, H3K27me3 / H3).

[0147] H3K27 trimethylation is mediated by the polycomb repressive complex (PRC2), which consists of three core proteins (EZH2, SUZ12, and EED). Telomere ChIP revealed that depletion of EZH2, the methyltransferase component of PRC2 (Fig. 11a), significantly reduced H3K27me3 (Fig. 11b). Meanwhile, H3K9me3 decreased by ~15%, while H3 was unaffected (Fig. 11b). Taken together, these results suggest that telomeres in Brca2-deficient cells are in a heterochromatin state enriched in H3K27me3.

[0148] Next, we sought to determine whether H3K27me3 marks were affected when the R-loop was removed. We observed that Brca2 deficiency led to a dramatic increase in telomeres marked with H3K27me3 in the absence of telomerase (Fig. 5B, -Brca2; -mTR). When RNH1-GFP was expressed, H3K27me3-marked telomeres were significantly reduced (Fig. 5B, -Brca2; -mTR; +RNH1-GFP). RNH1 expression was barely affected in telomerase-high-expressing cells depleted of Brca2 or in telomerase-deficient cells with Brca2 present (Fig. 5B). These results indicate that the R-loop is required for H3K27me3 condensation at telomeres. Next, we sought to determine whether PRC2-catalyzed H3K27me3 marking is required for BIR and telomere synthesis. Each of the three core components of PRC2 was depleted in immortalized fibroblasts, followed by G2 telomere synthesis assays. Depletion of EZH2, SUZ12, or EED (Fig. 11c) significantly reduced telomere synthesis (Fig. S4D) as well as APB number (Fig. 5c) in Brca2-deficient TBI fibroblasts. To confirm that H3K27 trimethylation is important for BIR in ALT-like activity, EZH2 was inhibited using EPZ-6438, a S-adenosyl-methionine (SAM) competitor that specifically inhibits EZH2 activity (Fig. 11e). The effect of EPZ-6438 on APB formation in telomerase-positive BI fibroblasts was minimal (Fig. 5d, BI; + / - Brca2). In contrast, EZH2 inhibition caused a significant decrease in APB number in Brca2-deficient and telomerase-deficient TBI fibroblasts (Fig. 5d, TBI; -Brca2).PDS treatment of Brca2-deficient TBI cells significantly increased the number of APBs (Fig. 5d, TBI; -Brca2; -EZH2i; +PDS), whereas EZH2 inhibition significantly reduced the level (Fig. 5d, TBI; -Brca2; +EZH2i; +PDS). These results suggest that abnormal stabilization of telomeric G4 leads to telomere LLPS formation, and that increased telomere H3K27me3 levels are associated with phase-separated APBs.

[0149] Since inhibition of PRC2 reduces APBs, we examined whether R-loops, which trigger APB assembly, were affected by PRC2 complex depletion. Depletion of the PRC2 core complex significantly reduced R-loops at telomeres, consistent with the decrease in APBs (Fig. 5e). Correspondingly, TERRA transcription was also downregulated upon PRC2 depletion (Fig. 11f). However, because the PRC2 complex appeared to be recruited to APBs only after TERRA-R-loop formation was induced, the reduction of TERRA transcription or R-loop formation by PRC2 depletion was unexpected. These results suggest that disruption of any single component of the LLPS disrupts droplet collapse and function, and that the density of the LLPS is crucial.

[0150] To verify the above results, we depleted POLD3 or BLM, essential components of BIR and telomere synthesis thought to exist within the telomere LLPS (Fig. 11g). Depletion of POLD3 or BLM inhibited APB formation (Fig. 5f) and telomere synthesis (Fig. 11h). Taken together, these results allowed us to conclude that all components of the telomere LLPS are interdependent, and that disruption of any one of these components impairs the maintenance of LLPS structure and its function, thereby inhibiting BIR-mediated telomere synthesis.

[0151]

[0152] TERRA-R-loop recruits PRC2 to enrich H3K27me3 at telomeres within LLPS.

[0153] We sought to determine whether the telomeric R-loop plays a direct role in H3K27me3 marking of telomeres. We were able to confirm that PRC2 can bind through the single-stranded non-translated RNA TERRA, catalyzing the trimethylation of histone lysine 27 and thereby establishing H3K9me3 for telomere heterochromatin formation. Since we have shown that the TERRA-containing R-loop is essential for telomere LLPS formation and BIR, we additionally sought to determine whether the TERRA-R-loop can bind to PRC2 and whether this interaction is essential for the maintenance of LLPS. DNA and / or RNA oligonucleotides representing various regions of the telomeric R-loop were biotinylated, and pull-down experiments were performed with EZH2-, SUZ12-, or EED-immune complexes derived from Brca2-depleted TBI fibroblasts. EZH2, SUZ12, and EED bound to TERRA RNA (Fig. 5g, (UUAGGG)8) but not to the complementary sequence (Fig. 5g, (CCCUAA)8). The telomeric DNA:RNA hybrid (UUAGGG)8 + (CCCTAA)8, which mimics a triple R-loop without single-stranded TERRA, did not interact with any PRC2 protein (Fig. 5g, (UUAGGG)8 + (CCCTAA)8). In contrast, (UUAGGG)8 + (CCCTAA)3, which contains five repeats of single-stranded RNA protruding from the DNA:RNA hybrid, bound to all three PRC2 core components (Fig. 5g, (UUAGGG)8 + (CCCTAA)3).

[0154] Next, we confirmed whether a certain length of protruding TERRA was required for binding to PRC2. In a DNA:RNA triplet, (UUAGGG)8 + (CCCTAA)5, which protruded three TERRA repeats, could not bind to the PRC2 complex, whereas in a DNA:RNA hybrid, (UUAGGG)8 + (CCCTAA)3, which protruded five TERRA repeats, could bind to the PRC2 complex (Fig. 11i). These results indicate that single-stranded TERRA must be repeated three or more times and protrude from the R-loop to bind to PRC2. According to Fig. 5, depletion of the PRC2 complex causes a decrease in APB. These results strongly support that the PRC2 complex is part of the telomere LLPS. Thus, the TERRA-R loop recruits the PRC2 complex to the telomeric LLPS, which requires three or more TERRA repeats protruding from the DNA:RNA hybrid. PRC2 within the telomeric LLPS plays a pivotal role in telomeric heterochromatin formation, which is essential for BIR (Fig. 5h).

[0155]

[0156] Disrupting phase separation impairs the growth and viability of BRCA2-deficient ALT-like cells.

[0157] We sought to determine whether disruption of phase-separation properties could have clinical potential. BRCA2-deficient cells are sensitive to PDS treatment, a G4-stabilizing agent. We sought to determine whether BRCA-2 deficiency results in abnormal G4 growth or stabilization by loss of dynamics. The stabilization and growth of telomere G4 were further enhanced by PDS administration, and R-loop and APB accumulation were also significantly increased after treatment. These results suggest that BRCA2-deficient cells, particularly those exhibiting ALT-like activity, are highly sensitive to PDS treatment. EZH2 inhibition sensitizes BRCA2-deficient breast cancer to PARP inhibitors. Because inhibition of EZH2 methyltransferase disrupts telomere LLPS maintenance in Brca2-deficient cells (Fig. 5d) and PDS treatment enhances telomere G4 stabilization and R-loop formation, we investigated whether co-administration of PDS with an EZH2 inhibitor could selectively inhibit the growth and / or viability of Brca2-deficient and telomerase-deficient ALT-like cells. BI and TBI fibroblasts, with or without Brca2, were treated with 5 μM PDS or EPZ-6438, and relative cell growth was assessed by scoring cell number. Data were normalized to untreated cells (NT). A value greater than 1.0 on the Y-axis indicates no response to the drug, while a value less than 1.0 indicates growth inhibition. Wild-type fibroblasts were unaffected by PDS or EPZ-6438 (Fig. 6a, black circles). In comparison, Brca2- and telomerase-negative TBI cells showed significant growth inhibition in response to PDS treatment (Fig. 6a, PDS, red squares).

[0158] EPZ-6438 treatment did not show any growth inhibition in telomerase single-null cells (Fig. 6A, EZH2i, open black circles). However, EZH2 inhibition moderately reduced cell number in Brca2-deficient and telomerase-null double mutant fibroblasts (Fig. 6A, EZH2i, red squares). We then evaluated the combination treatment effect in TBI fibroblasts. The growth rate was measured when 1 μM EPZ-6438 was treated alone or together with 2.5 μM PDS. EZH2 inhibition moderately inhibited the growth of Brca2-deficient TBI cells (Fig. 6B, open black circles), whereas Brca2-present telomerase-null cells were unaffected by EZH2 inhibition (Fig. 6B, open black circles). Surprisingly, combined treatment with PDS and EPZ-6438 significantly inhibited the growth of Brca2-deficient TBI fibroblasts, with almost complete growth inhibition achieved by day 4 (Fig. 6b, red squares). Next, we investigated whether human cells also responded to this inhibitor combination. HeLa cells were depleted of telomerase (HeLa LT TERC KO) and then transfected with siLuc (control) or siBRCA2.

[0159] Then, MTT (3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to measure cell viability 3 days after administration of PDS alone, EPZ-6438 alone, or EPZ-6438 and PDS together. PDS treatment inhibited cell viability of BRCA2-deficient HeLa LT TERC KO cells (Fig. 6c). Similar to mouse fibroblasts, EZH2 inhibition alone in human cells showed only minimal effects (Fig. 6d). However, when EPZ-6438 was combined with PDS, selective cytotoxicity was observed in BRCA2-deficient ALT cells (Fig. 6d, empty squares). These results demonstrate that disrupting the dynamics of telomere G4 and LLPS maintenance not only impairs BIR-mediated telomere synthesis but also cell survival in Brca2-deficient ALT-like cells.

[0160] Finally, we investigated whether BRCA2-mutant human cancers express ALT. The presence of APB was assessed in tissue sections from 38 paraffin-embedded breast cancer specimens, using a combination of immunohistochemistry on paraffin sections with anti-PML antibodies and telomere-FISH. Approximately half (47%, n = 19) of BRCA2-mutant cancer cells expressed APB. None of the breast cancer specimens harboring wild-type BRCA2 expressed APB (Fig. 7a). Telomere length heterogeneity, a hallmark of ALT cancer, was increased in BRCA2-mutant cancers expressing APB (Fig. 7b). In summary, disruption of BRCA2 expression can lead to ALT-like cancers. Telomerase positivity and ALT activity can be present in both cancer cell lines and human tumors. However, since BRCA2 deficiency-induced accumulation of TERRA-R loops, APB assembly, and BIR was more severe in telomerase-deficient cells, ALT cells may be more dependent on the BIR mechanism for telomere maintenance and much less dependent on telomerase. These results suggest that BRCA2 deficiency is associated with the induction of ALT or ALT-like cancers through abnormal stabilization of telomeric G4 and subsequent formation of phase-separated APBs (Fig. 7c).

[0161]

[0162] Determining the degree of structural damage to telomeres in TNBC and ovarian cancer patient tissues.

[0163] In order to determine whether the markers that are characteristic of ALT-positive cancer, such as APB, telomere damage, telomere structural damage, TERRA, and R-loop, appear in tissues isolated from various cancer patients and patients with aging diseases, we sought to determine whether these markers could function as markers of ALT-positive cancer and aging. To this end, immunofluorescence staining for G4, rH2AX, and telomere binding factor (TRF1) was performed on paraffin samples of surgical tissues derived from TNBC patients and ovarian cancer patients. Paraffin tissue slides isolated from patients were baked at 55°C for 2 hours to deparaffinize and rehydrate, then washed three times with xylene for 15 minutes each, twice with 100% ethanol for 10 minutes each, twice with 95% ethanol for 10 minutes each, twice with 70% ethanol for 10 minutes each, and twice with distilled water for 5 minutes each, and then rehydrated with PBS for 5 minutes. Antigen retrieval was performed by treating the sections twice with 1x citrate buffer at 95°C for 10 minutes each, washing them twice with PBS for 5 minutes each, and permeabilization with 0.5% Triton X-100 in PBS for 1 hour and 30 minutes. The sections were then blocked with 5% goat serum (PBS-T) for 1 hour and incubated overnight with the following primary antibodies diluted in 5% goat serum and 0.1% Triton X-100 in PBS:

[0164] G4: anti-DNA G-quadruplex structure, BG4, Sigma-aldrich MABE 917

[0165] rH2Ax: anti-histone H2A.X(ser139), Cell Signaling Technology, #2577L

[0166] TRF1:anti-TRF1 (TRF-78), Abcam, ab10579.

[0167] Afterwards, they were incubated with secondary antibodies (Alexa488-goat-anti-mouse, Alexa568-goat-anti-rabbit, Alexa647-goat-anti-mouse) at room temperature for 2 hours. After washing four times with 0.1% Triton X-100 in PBS for 15 minutes each, they were fixed with 4% formaldehyde for 10 minutes, washed four times with PBS, mounted using a mounting solution, sealed with nail polish, and stored at -20℃ under light protection. As a result of the analysis, as shown in Fig. 12, significant telomere structural damage was observed in both tissues of triple-negative breast cancer (Fig. 12a) and ovarian cancer patients (Fig. 12b), confirming that G4, rH2AX, and TRF1 can be reliable markers for ALT-positive cancer.

[0168]

[0169] Determination of the degree of telomere damage in tissues from patients with avascular necrosis of the femoral head, ovarian cancer, and TNBC.

[0170] Immunofluorescence staining for rH2AX and telomeres and telomere-FISH were performed on paraffin surgical tissue samples from patients with avascular necrosis of the femoral head, ovarian cancer, and triple-negative breast cancer. Paraffin tissue slides isolated from patients were baked at 55°C for 2 h for deparaffinization and rehydration, washed three times with xylene for 15 min each, washed twice with 100% ethanol for 10 min each, washed twice with 95% ethanol for 10 min each, washed twice with 70% ethanol for 10 min each, and washed twice with distilled water for 5 min each, and then rehydrated with PBS for 5 min. Antigen retrieval was performed by treating the sections twice with 1x citrate buffer at 95°C for 10 minutes each, washing them twice with PBS for 5 minutes each, and permeabilizing them with PBS 0.5% Triton X-100 for 1 hour and 30 minutes. The sections were then blocked with 5% goat serum (PBS-T) for 1 hour and incubated overnight with the primary antibody (rH2Ax: anti-histone H2A.X (ser139), Cell Signaling Technology, #2577L) diluted in 5% goat serum and PBS 0.1% Triton X-100. This was followed by incubation with the secondary antibody (Alexa488-goat-anti-rabbit) for 2 hours at room temperature. The sections were washed four times with PBS 0.1% Triton X-100 for 15 minutes each, fixed with 4% formaldehyde for 10 minutes, and washed four times with PBS.

[0171] Telomere FISH (fluorescent in situ hybridization) was performed as follows: Slides were dehydrated by sequentially treating them with 70%, 95%, and 100% ethanol for 5 minutes each at room temperature, then air-dried. 200 μl of hybridization buffer (Telomere-cy3 probe 500:1) was applied onto coverslips and denatured on an 85°C hotplate for 20 minutes. Hybridization was then performed with a wet paper towel in the dark at room temperature for 2 hours or at 4°C overnight. The slides were then removed and washed twice in Hybridization Wash Solution #1 for 15 minutes each on a vortex. They were then washed three times in Hybridization Wash Solution #2 for 5 minutes each on a vortex. In the second wash step, 0.5 mg / ml DAPI stock was added at a 1:300 ratio. After treating the slides with 70%, 95%, and 100% ethanol for 5 minutes each at room temperature, the slides were dehydrated by air-drying, mounted using a mounting solution, sealed with nail polish, and stored at -20℃ under light protection.

[0172] As a result of the analysis, significant telomere damage was observed in all tissues of patients with avascular necrosis of the femoral head (Fig. 13a), patients with ovarian cancer (Fig. 13b), and patients with TNBC (Fig. 13c), as shown in Fig. 13.

[0173]

[0174] Determination of APB levels in ovarian cancer patient tissues

[0175] Immunofluorescence staining and telomere-FISH for PML and telomeres were performed on surgical tissues (paraffin samples) from ovarian cancer patients. Paraffin tissue slides isolated from patients were deparaffinized and rehydrated by baking at 55°C for 2 h, washing with xylene three times for 15 min each, washing with 100% ethanol twice for 10 min each, washing with 95% ethanol twice for 10 min each, washing with 70% ethanol twice for 10 min each, and washing with distilled water twice for 5 min each, and rehydrating with PBS for 5 min. Antigen retrieval was performed by treating with 1x citrate buffer twice for 10 min each at 95°C, washing with PBS twice for 5 min each, and permeabilization with PBS 0.5% Triton X-100 for 1 h 30 min. Afterwards, the sections were blocked with 5% goat serum (PBS-T) for 1 hour and incubated overnight with the primary antibody (anti-PML (PG-M3), Santa Cruz Biotechnology, sc966) diluted in 5% goat serum and 0.1% Triton X-100 in PBS. Afterwards, they were incubated with the secondary antibody (Alexa488-goat-anti-mouse) for 2 hours at room temperature. After washing four times with 0.1% Triton X-100 in PBS for 15 minutes each, they were fixed with 4% formaldehyde for 10 minutes and washed four times with PBS.

[0176] Telomere FISH was performed as follows: Slides were dehydrated by sequentially treating them with 70%, 95%, and 100% ethanol for 5 minutes each at room temperature, then air-dried. 200 μl of hybridization buffer (Telomere-cy3 probe 500:1) was applied onto coverslips and denatured on an 85°C hotplate for 20 minutes. Subsequently, the slides were blotted with a damp paper towel and hybridized for 2 hours at room temperature in the dark or overnight at 4°C. The slides were then removed and washed twice in Hybridization Wash Solution #1 for 15 minutes each on a vortex. Subsequently, the slides were washed three times in Hybridization Wash Solution #2 for 5 minutes each on a vortex. In the second wash step, 0.5 mg / ml DAPI stock was added at a 1:300 ratio. After treating the slides with 70%, 95%, and 100% ethanol for 5 minutes each at room temperature, the slides were dehydrated by air-drying, mounted using a mounting solution, sealed with nail polish, and stored at -20°C in a light-protected environment. As a result of the analysis, as shown in Fig. 14, PML (APB) was specifically observed in ovarian cancer patient tissues, confirming that APB can be used as a marker for the detection of ALT-positive cancer.

[0177]

[0178] Identification of TERRA in ovarian cancer patient tissues

[0179] Immunofluorescence staining for TERRA was performed on surgical tissues (paraffin samples) from ovarian cancer patients according to the presence or absence of BRCA2 mutations. Paraffin tissue slides isolated from patients were baked at 55°C for 2 hours for deparaffinization and rehydration, washed three times with xylene for 15 minutes each, washed twice with 100% ethanol for 10 minutes each, washed twice with 95% ethanol for 10 minutes each, washed twice with 70% ethanol for 10 minutes each, and washed twice with distilled water for 5 minutes each, and then rehydrated with phosphate-buffered saline (PBS) for 5 minutes. After antigen retrieval, the slides were washed twice with PBS for 5 minutes each, and then permeabilized with 0.5% Triton X-100 in PBS for 1 hour and 30 minutes. Control samples were treated with 1 mg / ml RNaseA in PBS, and experimental samples were treated with 10 mM Ribonucleoside Vanadyl Complex in PBS, and incubated at 37°C for 1 hour. After blocking with 0.5% BSA PBS-T for 2 hours, the primary antibody was diluted in 3% BSA and 0.1% Triton X-100 in PBS and incubated overnight. After washing four times for 15 minutes each with 0.1% Triton X-100 in PBS, the sections were incubated with the secondary antibody for 2 hours, and then washed four times for 15 minutes each with 0.1% Triton X-100 in PBS. After fixation with 4% formaldehyde for 10 minutes, the sections were washed eight times with PBS.

[0180] TERRA FISH was performed as follows: Slides were dehydrated by sequentially treating them with 70%, 95%, and 100% ethanol for 5 minutes each at room temperature, then air-dried. 200 μl of hybridization buffer (Telomere-cy3 probe 500:1) was applied onto coverslips and denatured on a 37°C hotplate for 1.5 hours. Subsequently, the slides were hybridized for 2 hours at room temperature in the dark or overnight at 4°C with a damp paper towel. The slides were then removed and washed twice in Hybridization Wash Solution #1 for 15 minutes each on a vortex. Subsequently, the slides were washed three times in Hybridization Wash Solution #2 for 5 minutes each on a vortex. In the second wash step, 0.5 mg / ml DAPI stock was added at a 1:300 ratio. After treating the slides with 70%, 95%, and 100% ethanol for 5 minutes each at room temperature, the slides were dehydrated by air-drying, mounted using a mounting solution, sealed with nail polish, and stored at -20℃ in a light-protected environment. As shown in Fig. 15, TERRA was not detected when the BRCA2 gene was normal, but TERRA was detected when there was a mutation (bottom right of Fig. 15). The disappearance of the TERRA signal when treated with RNaseA confirmed that the signal was RNA, and this confirmed that ALT-positive cancer can be diagnosed by detecting TERRA in patient tissue samples.

[0181]

[0182] Identification of telomeric R-loops in ovarian cancer patient tissues

[0183] Immunofluorescence staining for R-loop and telomeres according to the presence or absence of BRCA2 mutations was performed on surgical tissues (paraffin samples) of ovarian cancer patients. Paraffin tissue slides isolated from patients were baked at 55℃ for 2 hours for deparaffinization and rehydration, washed three times with xylene for 15 minutes each / twice with 100% ethanol for 10 minutes each / twice with 95% ethanol for 10 minutes each / twice with 70% ethanol for 10 minutes each / twice with distilled water for 5 minutes each, and rehydrated with phosphate-buffered saline (PBS) for 5 minutes. After antigen retrieval, the slides were washed twice with PBS for 5 minutes each and permeabilized with 0.5% Triton X-100 in PBS for 1 hour and 30 minutes. Afterwards, the sections were blocked with 5% BSA PBS-T for 2 hours and incubated with the primary antibody (anti-DNA-RNA hybrid (S9.6), Merck Millipore, MABE 1095) in PBS 0.1% Triton X-100 containing 3% BSA overnight. After washing four times for 15 minutes each with PBS 0.1% Triton X-100, the sections were incubated with the secondary antibody for 2 hours at room temperature. After washing four times for 15 minutes each with PBS 0.1% Triton X-100, the sections were fixed with 4% formaldehyde for 10 minutes and washed eight times with PBS.

[0184] Telomere FISH was performed as follows: Slides were dehydrated by sequentially treating them with 70%, 95%, and 100% ethanol for 5 minutes each at room temperature, then air-dried. 200 μl of hybridization buffer (Telomere-cy3 probe 500:1) was applied onto coverslips and denatured on an 85°C hotplate for 20 minutes. Subsequently, the slides were blotted with a damp paper towel and hybridized for 2 hours at room temperature in the dark or overnight at 4°C. The slides were then removed and washed twice in Hybridization Wash Solution #1 for 15 minutes each on a vortex. Subsequently, the slides were washed three times in Hybridization Wash Solution #2 for 5 minutes each on a vortex. In the second wash step, 0.5 mg / ml DAPI stock was added at a 1:300 ratio. After treating the slides with 70%, 95%, and 100% ethanol for 5 minutes each at room temperature, the slides were dehydrated by air-drying, mounted using a mounting solution, sealed with nail polish, and stored at -20℃ in a light-protected environment. As a result of the analysis, as shown in Fig. 16, it was confirmed that the telomere R-loop did not appear when the BRCA2 gene was normal, but appeared when a mutation occurred (bottom right of Fig. 16). This confirmed that the detection of the telomere R-loop in patient tissue samples can function as a marker for the diagnosis of ALT-positive cancer (BRCA-2-deficient cancer) and aging.

[0185]

[0186] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for preventing or treating alternative lengthening of telomeres (ALT) mechanism-positive cancer or aging, comprising as an active ingredient at least one selected from the group consisting of inhibitors of liquid-liquid phase separation (LLPS) and stabilizers of abnormal telomere G-quadruplexes.

2. A composition according to claim 1, characterized in that the inhibitor of liquid-liquid phase separation (LLPS) is an inhibitor of APB (ALT-associated promyelocytic leukemia bodies) formation.

3. A composition according to claim 2, characterized in that the APB formation inhibitor is at least one selected from the group consisting of a TERRA-R-loop inhibitor, a histone H3K9 trimethylation (H3K9me3) inhibitor, and a histone H3K27 trimethylation (H3K27me3) inhibitor.

4. A composition according to claim 1, characterized in that the abnormal stabilizer of the telomere G-quadruplexes is pyridostatin (PDS).

5. A composition according to claim 3, characterized in that the histone H3K27 trimethylation (H3K27me3) inhibitor is a polycomb repressive complex (PRC2) inhibitor.

6. A composition according to claim 5, wherein the polycomb inhibitory complex (PRC2) comprises EZH2, SUZ12 and EED, and the inhibitor of the polycomb inhibitory complex is at least one selected from the group consisting of an EZH2 inhibitor, a SUZ12 inhibitor and an EED inhibitor.

7. A composition according to claim 6, characterized in that the EZH2 inhibitor is EPZ-6348.

8. A composition according to claim 1, wherein the alternative lengthening of telomeres (ALT) mechanism-positive cancer is a telomerase-deficient cancer or a BRCA-2-deficient cancer.

9. A composition according to claim 8, wherein the BRCA-2 deficient cancer is BRCA-2 deficient breast cancer.

10. A composition for preventing or treating alternative lengthening of telomeres (ALT) mechanism-positive cancer or aging, comprising as an active ingredient an abnormal stabilizer of telomere G-quadruplexes, in combination with a histone H3K27 trimethylation (H3K27me3) inhibitor.

11. A composition according to claim 10, characterized in that the abnormal stabilizer of the telomere G-quadruplexes is pyridostatin (PDS).

12. A composition according to claim 10, wherein the triple methylation (H3K27me3) inhibitor is a polycomb repressive complex (PRC2) inhibitor.

13. A composition according to claim 12, wherein the polycomb inhibitory complex (PRC2) comprises EZH2, SUZ12 and EED, and the inhibitor of the polycomb inhibitory complex is at least one selected from the group consisting of an EZH2 inhibitor, a SUZ12 inhibitor and an EED inhibitor.

14. A composition according to claim 13, wherein the EZH2 inhibitor is EPZ-6348.

15. A composition according to claim 10, wherein the alternative lengthening of telomeres (ALT) mechanism-positive cancer is a telomerase-deficient cancer or a BRCA-2-deficient cancer.

16. A composition according to claim 15, wherein the BRCA-2 deficient cancer is BRCA-2 deficient breast cancer.

17. A method for screening a composition for preventing or treating an alternative lengthening of telomeres (ALT) mechanism-positive cancer comprising the following steps: (a) administering a test substance to a biological sample containing cells exhibiting liquid-liquid phase separation (LLPS) phenomenon; and (b) a step of measuring the level of LLPS within cells contained in the biological sample; If the above LLPS level decreases, the test substance is determined to be a composition for preventing or treating alternative lengthening of telomeres (ALT) mechanism-positive cancer.

18. A composition for diagnosing an alternative lengthening of telomeres (ALT) mechanism-positive cancer, comprising as active ingredients an agent for detecting telomeres in a biological sample isolated from a subject and an agent for measuring the degree of telomere damage.

19. A composition according to claim 18, wherein the agent for measuring the degree of telomere damage is selected from the group consisting of an agent for detecting telomere G-quadruplexes, an agent for detecting rH2AX, an agent for detecting APB (ALT-associated promyelocytic leukemia bodies), an agent for detecting TERRA (Telomeric Repeat-containing RNA), and an agent for detecting telomere R-loop.

20. A composition according to claim 19, characterized in that the agent for detecting the telomere is an antibody or an antigen-binding fragment thereof that specifically binds to TRF1 (Telomeric Repeat Binding Factor 1).

21. A composition according to claim 19, characterized in that the agent detecting APB is an antibody or an antigen-binding fragment thereof that specifically binds to PML (Promyelocytic leukemia protein).

22. A composition according to claim 19, characterized in that the agent for detecting the telomere R-loop is an antibody or an antigen-binding fragment thereof that specifically recognizes DNA-RNA hybridization.

23. A composition according to claim 18, wherein the alternative telomere maintenance mechanism positive cancer is triple-negative breast cancer or ovarian cancer.

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