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WO2025150911A1 - Composition pour la prévention ou le traitement d'un cancer positif à l'allongement alternatif des télomères ou du vieillissement - Google Patents

Composition pour la prévention ou le traitement d'un cancer positif à l'allongement alternatif des télomères ou du vieillissement

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Publication number
WO2025150911A1
WO2025150911A1 PCT/KR2025/000480 KR2025000480W WO2025150911A1 WO 2025150911 A1 WO2025150911 A1 WO 2025150911A1 KR 2025000480 W KR2025000480 W KR 2025000480W WO 2025150911 A1 WO2025150911 A1 WO 2025150911A1
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WIPO (PCT)
Prior art keywords
telomere
inhibitor
telomeres
brca2
cancer
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Pending
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PCT/KR2025/000480
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English (en)
Korean (ko)
Inventor
이현숙
이재은
김형민
전사라
주소영
이수현
최시영
정성원
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SNU R&DB Foundation
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Seoul National University R&DB Foundation
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Publication of WO2025150911A1 publication Critical patent/WO2025150911A1/fr
Pending legal-status Critical Current
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds

Definitions

  • the present invention relates to a method for specifically inhibiting cancer that maintains 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.
  • the oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatomics, heterocyclic sugar sulphonates and the like.
  • 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.
  • ALT-associated promyelocytic leukemia bodies 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 in the nucleus with a diameter of up to 1 ⁇ m, and are composed of various substances including PML proteins, and are substances related to cellular mechanisms such as telomere elongation and DNA damage response.
  • 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.
  • TERRA in this specification refers to a type of long noncoding RNA (lncRNA) transcribed at chromosome ends that regulates telomeric chromatin structure and telomere maintenance through homology-directed repair with telomerase.
  • TERRA-R-loop promotes homologous recombination at telomeres through the formation of DNA:RNA (R-loop) structures.
  • histone H3K9 trimethylation 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.
  • histone H3K27 trimethylation 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 a histone H3 protein in which the phenomenon has occurred.
  • the histone H3K27 trimethylation (H3K27me3) inhibitor is a polycomb repressive complex (PRC2) inhibitor.
  • polycomb repressive complex 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 when the enzyme is defective, it causes problems during fetal development as well as various human diseases, including cancer.
  • 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.
  • EZH2 in this specification is an abbreviation for “Enhancer of zeste homolog 2” and means 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 of the inhibitory means of the above-mentioned proteins or nucleic acids encoding them can be applied as inhibitors of EZH2.
  • PRC2 polycomb repressive complex 2
  • 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 of the inhibitory means of the above-mentioned protein or the nucleic acid encoding it can be applied as an inhibitor of SUZ12.
  • PRC2 polycomb repressive complex 2
  • EED in this specification is an abbreviation for “embryonic ectoderm development” 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 EED gene in humans. Accordingly, all of the inhibitors of EED, including the above-mentioned proteins or the inhibitory means of the nucleic acid encoding them, can be applied.
  • PRC2 polycomb repressive complex 2
  • the EZH2 inhibitor of the present invention is EPZ-6348.
  • EPZ-6348 in this specification refers to a small molecule compound having selective inhibitory activity against EZH2 and has the CAS number 1403254-99-8.
  • the Alternative Lengthening of Telomeres (ALT) mechanism-positive cancer of the present invention is a telomerase-negative cancer or a BRCA-2 deficient cancer.
  • telomerase-negative cancer refers to a cancer in which the expression of telomerase is inhibited or suppressed, and which can extend telomeres through alternative telomere maintenance mechanisms instead of telomerase.
  • the BRCA-2 deficient cancer is BRCA-2 deficient breast cancer.
  • the present invention provides 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.
  • ALT telomeres
  • H3K27me3 histone H3K27 trimethylation
  • 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 telomere G-quadruplex stabilizer and a histone H3K27 trimethylation (H3K27me3) inhibitor.
  • the abnormal stabilizer of the telomere G-quadruplexes is pyridostatin (PDS).
  • the polycomb repressive complex comprises EZH2, SUZ12 and EED
  • 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.
  • test substance is determined to be a composition for preventing or treating alternative lengthening of telomeres (ALT) mechanism-positive cancer.
  • biological sample in the present invention refers to any sample containing cells exhibiting the LLPS phenomenon obtained from a mammal including a human, 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 mechanism-positive cancer cells.
  • the liquid-liquid phase separation (LLPS) phenomenon is due to the formation of APB (ALT-associated promyelocytic leukemia bodies).
  • the APB formation is caused by at least one selected from the group consisting of TERRA-R-loop formation, histone H3K9 trimethylation (H3K9me3), and histone H3K27 trimethylation (H3K27me3).
  • H3K27 trimethylation is mediated by the polycomb repressive complex (PRC2).
  • the polycomb repressive complex is composed of EZH2, SUZ12 and EED.
  • the step (b) is performed by measuring the level of at least one 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).
  • APB ALT-associated promyelocytic leukemia bodies
  • TERRA-R loop trimethylation of histone H3K9
  • H3K27me3 trimethylation of histone H3K27
  • 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, and therefore, their description is omitted to avoid excessive duplication.
  • the present invention provides a diagnostic composition for alternative lengthening of telomeres (ALT) mechanism-positive cancer or aging, comprising 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.
  • a diagnostic composition for alternative lengthening of telomeres (ALT) mechanism-positive cancer or aging comprising 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.
  • 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.
  • diagnosis includes determining the susceptibility of an individual to a particular disease, determining whether an individual currently has a particular disease, and determining the prognosis of an individual with a particular disease.
  • 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).
  • the agent for detecting APB is an antibody or an antigen-binding fragment thereof that specifically binds to PML (Promyelocytic leukemia protein).
  • the agent for detecting the telomere R-loop is an antibody or an antigen-binding fragment thereof that specifically recognizes DNA-RNA hybridization.
  • 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.
  • 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.
  • the present invention provides an effective therapeutic target for alternative telomere maintenance mechanism-positive cancer, the mechanism of which has not been fully elucidated and thus there has been no effective treatment method, and thus can be usefully used for the effective prevention or treatment of alternative telomere maintenance mechanism-positive cancer by regulating the expression of the target.
  • Figure 1 is a diagram showing the results of experiments that confirmed that assembly of telomeric PML bodies is essential for telomere synthesis in BRCA2-deficient ALT-like cells.
  • Figure 1a is a diagram showing 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 depleted of Brca2 under 4-OHT treatment conditions.
  • the bar graphs are representative of the results from four or more independent experiments.
  • Figure 1b is a graphical representation of the relative mRNA levels of interferon- ⁇ , - ⁇ , and - ⁇ in telomerase-positive (+mTR) and telomerase-negative (-mTR) MEFs in the presence or absence of Brca2, respectively.
  • Figure 1c is a diagram showing the results of analyzing telomere synthesis in the G2 phase after transfection of fibroblasts with two different siRNAs targeting PML (siPML#3, siPML#4) in the presence or absence of Brca2.
  • the left image shows the extent of EdU incorporation at telomeres in TBI fibroblasts double knocked out of Brca2 and telomerase.
  • Fibroblasts were treated with 18 ⁇ M RO-3306, a CDK1 inhibitor, to arrest cells in the G2 phase.
  • Magnified images of EdU-positive telomeres are indicated by white squares, and the scale bar is 5 ⁇ m.
  • the upper right is the percentage of cells with three or more EdU-positive telomeres.
  • the bar graphs are the results of three independent experiments. >100 BI cells (+mTR) and >200 TBI cells (-mTR) fibroblasts were scored in each experiment.
  • the lower right shows the results of evaluating the efficiency of PML knockdown through Western blot analysis after siRNA transfection (Student's t-test (mean ⁇ SEM)).
  • FIG. 2 illustrates the results of experiments that confirmed that liquid-liquid phase separation properties play an essential role in telomere synthesis in ALT cells.
  • Figure 2a is an illustration of an example of a TRF1-IDR expression construct. IDR domains derived from the N-terminus of FUS (FUSN) and the N-terminus of DDX4 (DDX4N), two different IDR-containing proteins, were each fused to the TRF1-expression construct, and the cDNA expressing mCherry (mCh) fluorescent protein was inserted behind TRF1. When these constructs were introduced into cells, LLPS-induced telomere clustering was induced.
  • Figure 2b is a representative time-lapse capture image of TRF1-FUS movement in WT fibroblasts.
  • Telomere foci formed in droplets are indicated by arrows, and an enlarged image is illustrated below (scale bar, 10 ⁇ m).
  • Figure 2c is a plot of the integrated fluorescence intensity of TRF1-IDR clusters within the nucleus per cell, with the y-axis 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).
  • 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 of 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 is a graphical representation of the results of telomere length comparison, where telomere length was measured using interphase T-FISH and expressed as arbitrary units of fluorescence intensity.
  • 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 of Figures 3a to 3f were performed in BI and TBI fibroblasts, and the fibroblasts were treated with tamoxifen (4-OHT) for conditional depletion of Brca2, 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 evaluating G4-positive telomeres.
  • the upper image is a representative image for co-localization of G4 (green) and telomeres (red), after fixing fibroblasts and performing immunofluorescence with BG4 (anti-DNA G4 monoclonal antibody, green), and T-FISH using the TelC-PNA probe (red).
  • the enlarged image of the white square is shown at the bottom.
  • the lower image shows the percentage of cells with ⁇ 3 G4-positive telomeres, and each score was assigned to ⁇ 100 cells (scale bar, 5 ⁇ m).
  • Figure 3c is a picture showing the results of evaluating TERRA RNA levels.
  • the lower frame is a bar graph showing the percentage of cells with ⁇ 4 R-loop-positive telomeres, each scored for >120 cells.
  • Figure 3e is a graph showing the results of quantification of APB per cell, with scores assigned to >100 cells each.
  • Figure 3f is a graph showing telomere length measured by interphase T-FISH as an arbitrary unit of fluorescence intensity, with >500 telomere foci measured 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.
  • HeLa LT TERC KO cells were transfected with siLuc(+) or siBRCA2(-) 3 days prior to cell fixation.
  • TRF1 green
  • TERRA red
  • RNA-FISH images Representative images of TRF1 (green) and TERRA (red) (scale bar, 2.5 ⁇ m) are shown on the left, 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, and all results are from three independent experiments (p ⁇ 0.0001, Student's t-test (mean ⁇ SEM)).
  • Figure 4 illustrates the results of experiments confirming that telomere R-loops induce LLPS formation.
  • Telomerase-positive BI and telomerase-negative TBI fibroblasts of the experiments of Figures 4a to 4e were infected with 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.
  • the bar graph represents the percentage of TBI cells with ⁇ 3 EdU-positive telomeres, and >100 cells were scored for each condition.
  • Figure 4d is a diagram illustrating the results of experiments examining 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 ⁇ M 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, and >120 cells were measured for each condition.
  • Figure 4e is a diagram illustrating the results of experiments examining the effect of PDS and/or RNH1 on APB formation, and >100 cells were measured for 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.
  • Top is a representative image of co-localization of R-loops (green) and telomeres (red), where white arrows indicate R-loop-positive telomeres; middle is a representative image of co-localization of PML (green) and telomeres (red), where white arrows indicate APBs (scale bar, 2.5 ⁇ m).
  • FIG. 4g illustrates the experimental results confirming the model of the mechanism by which BRCA2 deletion promotes ALT-like telomere synthesis.
  • Brca2 deletion induced stabilization of G4, which then induced an increase in TERRA-R loops.
  • R-loops triggered the formation of LLPS containing protein complexes required for BIR at telomeres (*p ⁇ 0.0001, Student's t-test (mean ⁇ SEM)).
  • Figures 5c and 5e are diagrams showing the results of experiments using BI and TBI fibroblasts in the presence (+) or absence (-) of Brca2, depleting the core components of PRC2, EZH2, SUZ12, and EED proteins using siRNA transfection.
  • Figure 5c is depleted of the PRC2 complex by removing the telomeric LLPS. >100 cells were counted for each.
  • Figure 5d shows the results of an experiment to remove APB by administering EZH2 inhibitor.
  • BI and TBI fibroblasts which were treated (-) or not (+) with 4-OHT to remove Brca2, were exposed to 5 ⁇ M PDS treatment for 24 h and/or 5 ⁇ M EPZ-6438 for 48 h before cell fixation, and analyzed for APB formation. >100 cells were scored for each condition.
  • Figure 5e shows the results of an experiment to confirm that telomere R-loop formation was significantly reduced through PRC2 depletion. >100 cells were counted for each.
  • Figure 5f shows the results of an experiment to confirm that telomere LLPS is reduced when BIR factors POLD3 or BLM are depleted. >140 cells were scored.
  • Figure 5g is a drawing showing the results of analyzing the region to which the PRC2 protein binds by oligonucleotide pull-down analysis, where cell lysates were incubated with the indicated biotinylated oligonucleotides and Western blot analysis was performed 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 repeats of TERRA.
  • Figure 5h is a schematic representation of the telomere chromatin remodeling model essential for telomere synthesis in ALT-like cells inside LLPS, in which the PRC2 complex catalyzing H3K27me3 is recruited at TERRA RNA telomeres protruding from the R loop. All results are from three independent experiments (* p ⁇ 0.0001, Student's t-test (mean ⁇ SEM)).
  • Figure 6 illustrates the selective sensitivity of BRCA2-deficient ALT-like cells to abnormal G4 stabilizers and 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 ⁇ M PDS and 1 ⁇ M EZH2i. Cells were counted every 2 days, and the growth rate was normalized 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 drawings 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, and 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 ⁇ M 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, where cells treated with 1 ⁇ M PDS were co-treated with EPZ-6438 at concentrations of 0 to 10 ⁇ M (circles and black line: siLuc+EZH2i; squares and black dashed line: siLuc+EZH2i+PDS; open circles and red line: siBRCA2+EZH2i; open squares and red dashed line: siBRCA2+EZH2i+PDS).
  • the experiments were repeated three times independently (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001, Student's t-test (mean ⁇ SEM)).
  • Figure 7 illustrates the results confirming that approximately half of the mutant human breast cancers expressing APB have BRCA2 deletion.
  • Figure 7a shows a representative fluorescence image of APB-positive human breast cancer tissue, which was performed by combining immunofluorescence using 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. Each sample was used (x/y: number of APB-positive sections/total number of samples; scale bar, 2 ⁇ m).
  • 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 showing how BIR functions for telomere synthesis after BRCA2 deletion, whereby telomeric G4 is stabilized upon BRCA2 deletion, TERRA-containing R-loops accumulate at telomeres, telomere phase separation is induced by the increased R-loops, the interior of the generated telomeric LLPS is characterized by H3K27me3, and DNA breaks recruit BIR DNA repair factors.
  • the abnormally stabilized telomeric G4 induces TERRA-R-loop accumulation, which induces phase separation along 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)).
  • Figure 8 is a drawing showing the experimental results confirming the effect of induced phase separation and its break-induced replication (BIR).
  • the left side of Fig. 8a is a representative image (scale bar, 0.5 ⁇ m) of fluorescence recovery after photobleaching (FRAP) analysis of TRF1-IDR, and the right side is a drawing of the FRAP recovery curves measured by the average intensity of mCherry expressed in arbitrary units (black: TRF1-mCh; red: TRF1-FUS; pink: TRF1-DDX).
  • FRAP fluorescence recovery after photobleaching
  • 8b is a drawing showing a control construct without TRF1 , in which two NLSs (nuclear localization signals) from SV40 were attached to the N-terminus, and mCherry (mCh) fluorescent protein-expressing cDNA was linked downstream of the NLS .
  • NLSs nuclear localization signals
  • mCh mCh
  • LLPS-induced aggregation was induced.
  • Right shows representative images of mCherry fluorescence for each NLS-IDR expression within the nucleus (scale bar, 5 ⁇ m).
  • 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, with siLuc (+) transfection used as a control for PML depletion using siPML (-).
  • Figures 8c and 8e are graphs depicting the percentage of cells with ⁇ 3 EdU-positive telomeres, scored for >100 cells each.
  • Figures 8d and 8f show the results of T-FISH measurements, in which >800 telomere foci were measured in >50 cells, respectively.
  • Figure 8g shows the results of Western blot analysis in U2OS cells, showing mCherry protein levels after TRF1-IDR construct transfection; and PML knockdown efficiency after siPML transfection. All results are from three independent experiments (* p ⁇ 0.0001, Student's t-test (mean ⁇ SEM)).
  • Figure 9 is a diagram showing the results of RNA-FISH validation for detecting G4 and R-loop-specific antibodies and TERRA used in immunofluorescence.
  • Figure 9a is a diagram showing a representative image of G4 (green) and telomeres (red), where G4 was detected by immunofluorescence with a monoclonal antibody against FLAG, and the G4-specific antibody BG4 designated a FLAG (Merck Millipore) tag, and 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 diagram showing a representative image of TERRA RNA (green) in Brca2-depleted TBI fibroblasts in the presence or absence of RNase A.
  • Figure 9c shows R-loops (green) labeled by immunofluorescence using the 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), while the right shows the results of counting the number of R-loop-positive telomeres per cell, with 100 cells counted in each experiment. All experiments consisted of at least three independent experiments (scale bar, 5 ⁇ m; Student's t-test (mean ⁇ SEM)).
  • Figure 10 illustrates the results of experiments confirming that R-loops are essential for Brca2-deficiency-induced ALT-like telomere synthesis.
  • the experiments of Figures 10a to 10d were performed in RNH1-GFP- expressing TBI fibroblasts, which were infected with the indicated lentiviruses M, F, D, respectively, in the presence (+) or absence (-) of Brca2.
  • Figure 10a illustrates the results of Western blot analysis using anti-RNH1 antibody, and the same blot was reprobed with anti-actin antibody for normalization.
  • Figures 10b and 10d show that telomere length was scored via arbitrary fluorescence units of T-FISH in interphase, and >700 telomere foci in >40 cells were measured for each condition.
  • Figure 10c shows the percentage of cells with ⁇ 3 EdU-positive telomeres, the results are from three independent experiments and scored for >110 cells each.
  • Figure 10e shows Western blot analysis results showing mCherry levels after transfection of a construct expressing RNH1-mcherry in BRCA2 knockdown and HeLa LT TERC KO cells after siBRCA2 transfection.
  • Figures 10f and 10g show BJ cells transduced 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-loops (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 ⁇ m), 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)).
  • 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 confirmed that EZH2 and H3K27me3 were depleted by siEZH2 transfection. The levels of H3K9me3 and histone H3 are also shown on the same blot.
  • Liquid-liquid phase separation is critical for BIR (Break-Induced Replication)-mediated telomere synthesis upon Brca2 loss
  • telomeres Brca2 depletion in telomerase-negative cells induces Break-Induced Replica (BIR) at telomeres and induces ALT-like activity.
  • BIR Break-Induced Replica
  • APB promyelocytic leukemia bodies
  • Brca2 F11/F11 ; ER-Cre mice were crossed with mTR +/- mice to generate Brca2 F11/F11 ; ER-Cre ; We generated mTR +/+ ( BI , Brca2 -inducible ) or Brca2 F11/F11 ; ER-Cre ; mTR -/- ( TBI , T telomerase-null and Brca2 -inducible ) progeny.
  • BI and TBI MEFs were then isolated and immortalized by expressing the large T antigen of SV40.
  • telomere-deficient mice are significantly affected by Brca2 depletion, we could imply that telomerase activity plays a pivotal role in inducing ALT or ALT-like activity.
  • telomere R-loops trigger phase separation at telomeres.
  • HeLa LT TERC KO cell line Fig. 10e
  • wild-type BJ cell line lacking telomerase activity Fig. 10f
  • siRNA siRNA
  • RNH1-GFP was then ectopically expressed, and the level of telomeric R-loops was determined.
  • APB formation was analyzed (Figs. 4f and 10g). BRCA2 depletion in human cells lacking telomerase increased the level of telomeric R-loops (Figs.
  • telomeric PML bodies due to BRCA2 depletion were markedly reduced when RNH1 was expressed (Figs. 4f and 10g).
  • telomeric R-loops induce phase separation in human ALT-like cells, and that abolishment of BRCA2 results in loss of telomeric G4 dynamics and stabilization of G4.
  • Abnormally stabilized telomeric G4s induce transcription-replication collisions, resulting in accumulation of TERRA-containing R-loops.
  • Telomeric R-loops induce liquid-liquid phase separation and BIR at telomeres. Taken together, loss of telomeric G4 dynamics induces phase separation and BIR at telomeres (Fig. 4g).
  • telomere chromatin euchromatin or heterochromatin
  • BIR a normal replication mechanism
  • MiDAS a mitotic DNA synthesis process
  • telomere ChIP was performed using antibodies to H3K9me3 and H3K27me3 in BI and TBI fibroblasts treated or not with tamoxifen. The ratio of H3K9me3 or H3K27me3 to H3 was compared to normalize for the difference 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).
  • H3K27 trimethylation is mediated by the polycomb repressive complex (PRC2), which consists of three core proteins (EZH2, SUZ12, and EED).
  • PRC2 polycomb repressive complex
  • Telomere ChIP revealed that depletion of EZH2, a methyltransferase component of PRC2 (Fig. 11a), significantly reduced H3K27me3 (Fig. 11b). Meanwhile, ⁇ 15% of H3K9me3 was reduced, while H3 was not affected (Fig. 11b).
  • telomeres in Brca2-deficient cells are in a heterochromatin state rich in H3K27me3.
  • telomeric R-loops play a direct role in H3K27me3 marking of telomeres.
  • PRC2 can bind via single-stranded non-translated RNA TERRA and influence the establishment of H3K9me3 for telomeric heterochromatin formation by catalyzing trimethylation of histone lysine 27. Since we have shown that TERRA-containing R-loops are essential for telomere LLPS formation and BIR, we additionally sought to determine whether TERRA-R-loops can bind to PRC2 and whether this interaction is essential for the maintenance of LLPS.
  • BRCA2-deficient cells are sensitive to PDS treatment, a G4 stabilizer.
  • telomere G4 is abnormally increased or stabilized by loss of dynamics when BRCA-2 is deficient.
  • the stabilization and increase of telomere G4 were further enhanced by PDS administration, and R-loop and APB were also significantly accumulated after administration.
  • telomere G4 stabilization and R-loop formation we investigated whether co-administration of PDS with EZH2 inhibitors could selectively inhibit the growth and/or viability of Brca2-deficient and telomerase-null ALT-like cells.
  • BI and TBI fibroblasts with or without Brca2 were treated with 5 ⁇ M 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 drug, while a value less than 1.0 indicates growth inhibition.
  • 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).
  • Immunofluorescence staining for rH2AX, telomeres, and telomere-FISH were performed on paraffin samples from surgical tissues of 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 to deparaffinize and rehydrate, washed three times with xylene for 15 min each/twice with 100% ethanol for 10 min each/twice with 95% ethanol for 10 min each/twice with 70% ethanol for 10 min each/twice with distilled water for 5 min each, and rehydrated with phosphate-buffered saline (PBS) for 5 min.
  • PBS phosphate-buffered saline
  • Antigen retrieval was performed by treating twice with 1x citrate buffer at 95°C for 10 min each, followed by two washes with PBS for 5 min each, and permeabilization with PBS 0.5% Triton X-100 for 1 h 30 min.
  • the sections were then blocked with 5% goat serum (PBS-T) for 1 h 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 h at room temperature.
  • the sections were washed four times for 15 min each with PBS 0.1% Triton X-100, fixed with 4% formaldehyde for 10 min, and washed four times with PBS.
  • Telomere FISH fluorescent in situ hybridization
  • Slides were dehydrated by sequential treatment with 70%, 95%, and 100% ethanol for 5 min each at room temperature, air-dried, and 200 ⁇ l of hybridization buffer (Telomere-cy3 probe 500:1) was applied onto the coverslip and denatured on a hot plate at 85°C for 20 min. Then, hybridization was performed for 2 h at room temperature or overnight at 4°C in the dark with a wet paper towel. The slides were then removed and washed twice for 15 min each in Hybridization Wash Solution #1 on a vortex.
  • telomere damage was observed in all tissues of patients with avascular necrosis of the femoral head (Fig. 13a), ovarian cancer patients (Fig. 13b), and TNBC patients (Fig. 13c), as shown in Fig. 13.
  • telomere-FISH Immunofluorescence staining and telomere-FISH for PML and telomeres were performed on ovarian cancer patient surgical tissues (paraffin samples). 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/twice with 100% ethanol for 10 min each/twice with 95% ethanol for 10 min each/twice with 70% ethanol for 10 min each/twice with distilled water for 5 min each, and rehydrating with PBS for 5 min.
  • Antigen retrieval was performed by treating with 1x citrate buffer at 95°C twice for 10 min each, 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 PBS 0.1% Triton X-100. Afterwards, they were incubated with the secondary antibody (Alexa488-goat-anti-mouse) for 2 hours at room temperature. After washing four times for 15 minutes each with PBS 0.1% Triton X-100, they were fixed with 4% formaldehyde for 10 minutes and washed four times again with PBS.
  • Telomere FISH was performed as follows: Slides were dehydrated by sequential treatment with 70%, 95%, and 100% ethanol for 5 min each at room temperature, air-dried, and 200 ⁇ l of hybridization buffer (Telomere-cy3 probe 500:1) was applied onto the coverslip and denatured on a hot plate at 85°C for 20 min. Then, hybridization was performed for 2 h at room temperature or overnight at 4°C in the dark with a wet paper towel. Slides were then removed and washed twice for 15 min each in Hybridization Wash Solution #1 on a vortex.
  • Immunofluorescence staining for TERRA according to the presence or absence of BRCA2 mutation was performed on surgical tissues (paraffin samples) of ovarian cancer patients. Paraffin tissue slides isolated from patients were baked at 55°C for 2 h to deparaffinize and rehydrate, washed three times with xylene for 15 min each/twice with 100% ethanol for 10 min each/twice with 95% ethanol for 10 min each/twice with 70% ethanol for 10 min each/twice with distilled water for 5 min each, and then rehydrated with PBS for 5 min. After antigen retrieval, washed twice with PBS for 5 min each, and permeabilized with PBS 0.5% Triton X-100 for 1 h 30 min.
  • Telomere FISH was performed as follows: Slides were dehydrated by sequential treatment with 70%, 95%, and 100% ethanol for 5 min each at room temperature, air-dried, and 200 ⁇ l of hybridization buffer (Telomere-cy3 probe 500:1) was applied onto the coverslip and denatured on a hot plate at 85°C for 20 min. Then, hybridization was performed for 2 h at room temperature or overnight at 4°C in the dark with a wet paper towel. Slides were then removed and washed twice for 15 min each in Hybridization Wash Solution #1 on a vortex.
  • telomere R-loop did not appear when the BRCA2 gene was normal, but the telomere R-loop appeared when a mutation occurred (Fig. 16, bottom right). 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.

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Abstract

La présente invention concerne une méthode de prévention ou de traitement d'un cancer positif à l'allongement alternatif des télomères par identification de facteurs impliqués dans l'allongement alternatif des télomères et des corrélations entre eux, et par régulation de l'expression des facteurs. La présente invention fournit une cible thérapeutique efficace pour un cancer positif à l'allongement alternatif des télomères, pour lesquels une méthode de traitement efficace n'a pas encore été développée en raison du mécanisme auparavant peu clair. En conséquence, la présente invention peut être efficacement utilisée pour la prévention ou le traitement efficace d'un cancer positif à l'allongement alternatif des télomères par régulation de l'expression de la cible.
PCT/KR2025/000480 2024-01-09 2025-01-09 Composition pour la prévention ou le traitement d'un cancer positif à l'allongement alternatif des télomères ou du vieillissement Pending WO2025150911A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016112374A2 (fr) * 2015-01-09 2016-07-14 The General Hospital Corporation Traitement du cancer à l'aide d'inhibiteurs de l'atr - ataxie-télangiectasie mutée and rad3 liée -
KR20160095336A (ko) * 2015-02-03 2016-08-11 서울대학교산학협력단 세포내 대안적 텔로미어 유지 활성에 관여하는 엘리먼트 및 그 용도
US20210087567A1 (en) * 2015-07-29 2021-03-25 Ifom - Fondazione Istituto Firc Di Oncologia Molecolare Therapeutic oligonucleotides
WO2022258012A1 (fr) * 2021-06-11 2022-12-15 中国科学院化学研究所 Application d'une combinaison ou d'un complexe de pyridostatine ou d'un dérivé de celle-ci et d'un composé transplatine dans la préparation d'un médicament pour le traitement du cancer
WO2023275369A2 (fr) * 2021-07-01 2023-01-05 Centre National De La Recherche Scientifique Nouvelle application de médicament

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102726869B1 (ko) 2024-04-04 2024-11-06 주식회사 덴티움 콘빔 컴퓨터 단층 촬영 영상으로부터 재구성된 가상 세팔로메트릭 영상을 이용한 치과 교정 진단에 필요한 정보를 제공하기 위한 장치 및 이를 위한 방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016112374A2 (fr) * 2015-01-09 2016-07-14 The General Hospital Corporation Traitement du cancer à l'aide d'inhibiteurs de l'atr - ataxie-télangiectasie mutée and rad3 liée -
KR20160095336A (ko) * 2015-02-03 2016-08-11 서울대학교산학협력단 세포내 대안적 텔로미어 유지 활성에 관여하는 엘리먼트 및 그 용도
US20210087567A1 (en) * 2015-07-29 2021-03-25 Ifom - Fondazione Istituto Firc Di Oncologia Molecolare Therapeutic oligonucleotides
WO2022258012A1 (fr) * 2021-06-11 2022-12-15 中国科学院化学研究所 Application d'une combinaison ou d'un complexe de pyridostatine ou d'un dérivé de celle-ci et d'un composé transplatine dans la préparation d'un médicament pour le traitement du cancer
WO2023275369A2 (fr) * 2021-07-01 2023-01-05 Centre National De La Recherche Scientifique Nouvelle application de médicament

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LEE JENNIFER J, KIM HYUNGMIN, PARK HAEMIN, LEE UKJIN, KIM CHAELIM, LEE MIN, SHIN YONGDAE, JUNG JI-JUNG, LEE HAN-BYOEL, HAN WONSHIK: "Disruption of G-quadruplex dynamicity by BRCA2 abrogation instigates phase separation and break-induced replication at telomeres", NUCLEIC ACIDS RESEARCH, vol. 52, no. 10, 10 June 2024 (2024-06-10), UK, pages 5756 - 5773, XP093335827, ISSN: 0305-1048, DOI: 10.1093/nar/gkae251 *

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