Method for increasing PARP inhibitor sensitivity

Abstract

The present application relates to a method for increasing PARP inhibitor sensitivity, the method comprising a step for inhibiting the expression or activity of TRIM44.

Description

How to increase sensitivity to PARP inhibitors

[0001] The present invention relates to a method for increasing sensitivity to a PARP (Poly ADP-ribose polymerase) inhibitor.

[0002] Failure of DNA repair leads to genomic instability and is a major cause of cancer development. Among DNA damage, double-strand breaks (DSBs) are known to be the most lethal.

[0003] Poly(ADP-ribose) polymerase 1 (PARP1), an early sensor of DNA damage, synthesizes multibranched poly(ADP-ribose) (PAR) chains from damaged chromatin. These PAR chains then serve as recruitment platforms for downstream repair factors that initiate base excision repair (BER) at DNA single-strand breaks (SSBs). This dynamic rotation of PAR is regulated by poly(ADP-ribose) glycohydrolase (PARG).

[0004] When a DNA double-strand break occurs, the MRE11-RAD50-NBS1 (MRN) complex detects it and activates ATM kinase. Activated ATM phosphorylates MDC1 and H2AX to generate γH2AX, which induces the recruitment of various repair proteins, including BRCA1 and 53BP1. Depending on the cell cycle, either non-homologous end joining (NHEJ) or homologous recombination repair (HRR) then proceeds.

[0005] PARP1 plays a crucial role in the ATM-mediated DNA damage response and repair pathway. Specifically, it promotes the recruitment of MRE11 and NBS1 to DSB sites, and is also involved in the recruitment of BRCA1 to DSB sites. It also induces NHEJ through complex formation with DNA-PK and KU70 / 80.

[0006] Currently, PARP inhibitors (PARPi) are used clinically to treat patients with BRCA1 or BRCA2 mutations. PARPi inhibit the catalytic activity of PARP1 and induce PARP1 trapping, leading to the generation of DSBs during DNA replication. In HR-normal cells, these DSBs are repaired through HRR. However, in HR-deficient cells with BRCA1 / 2 mutations, imprecise repair leads to cell death.

[0007] However, various resistance mechanisms to PARP inhibitors are being discovered in patients with advanced cancer, including loss of PARG, acquisition of revertant mutations in BRCA1 / 2, loss of 53BP1, and loss of REV7 / MAD2L2.

[0008] Therefore, in order to enhance the therapeutic effect of PARP inhibitors, it is necessary to develop a method to overcome resistance to PARP inhibitors and increase sensitivity to PARP inhibitors.

[0009] The background technology of this application, Republic of Korea Patent Publication No. 10-2022-0125183, relates to the use of phthalazinone derivatives as anticancer agents for the treatment of cancer in a group of patients resistant to PARP inhibitors.

[0010] The present invention is intended to solve the problems of the above-mentioned prior art and provides a method for increasing sensitivity to a PARP inhibitor.

[0011] Additionally, a composition for increasing sensitivity to a PARP inhibitor is provided.

[0012] In addition, a pharmaceutical composition for preventing or treating a tumor is provided, which comprises the composition for increasing sensitivity to the PARP inhibitor as an active ingredient.

[0013] In addition, a method for screening a substance that increases sensitivity to PARP inhibitors is provided.

[0014] However, the technical tasks to be achieved by the embodiments of the present invention are not limited to the technical tasks described above, and other technical tasks may exist.

[0015] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a method for increasing sensitivity to a PARP inhibitor, comprising a step of inhibiting the expression or activity of TRIM44.

[0016] According to one embodiment of the present invention, in the presence of a PARP inhibitor, the main binding substance of TRIM44 may be converted from PARP1 to MRN (MRE11-RAD50-NBS1) complex, but is not limited thereto.

[0017] According to one embodiment of the present invention, the TRIM44 may bind to the PARP1 via a glutamate-rich domain, but is not limited thereto.

[0018] According to one embodiment of the present invention, the TRIM44 may bind to the MRN complex via a ZnF UBP (zinc finger ubiquitin-binding) domain, but is not limited thereto.

[0019] According to one embodiment of the present invention, PARP inhibitor resistance caused by 53BP1 deficiency can be overcome through inhibition of TRIM44, but is not limited thereto.

[0020] According to one embodiment of the present invention, the PARP inhibitor may include, but is not limited to, one selected from the group consisting of Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, and combinations thereof.

[0021] In addition, the second aspect of the present invention provides a composition for increasing sensitivity to a PARP inhibitor, comprising a substance that inhibits the expression or activity of TRIM44 as an active ingredient.

[0022] According to one embodiment of the present invention, TRIM44 may bind to PARP1 through the glutamate-rich domain and bind to the MRN complex through the ZnF UBP domain, but is not limited thereto.

[0023] In addition, the third aspect of the present invention provides a pharmaceutical composition for preventing or treating a tumor, comprising a composition for increasing sensitivity to a PARP inhibitor according to the second aspect of the present invention as an active ingredient.

[0024] According to one embodiment of the present invention, binding of TRIM44 may be, but is not limited to, switching from PARP1 to the MRN complex in the presence of a PARP inhibitor.

[0025] According to one embodiment of the present invention, the tumor may include, but is not limited to, a homologous recombination proficient (HR-proficient) tumor.

[0026] According to one embodiment of the present invention, the pharmaceutical composition for preventing or treating a tumor may additionally include a pharmaceutically acceptable carrier, but is not limited thereto.

[0027] According to one embodiment of the present invention, the carrier may include, but is not limited to, a group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, menthol, mineral oil, and combinations thereof.

[0028] According to one embodiment of the present invention, the pharmaceutical composition for preventing or treating a tumor may further include a component selected from the group consisting of, but not limited to, a preservative, a solubilizer, a stabilizer, a humectant, a sweetener, a colorant, a flavoring agent, a salt, a buffer, an antioxidant, a lubricant, an emulsifier, a suspending agent, a preservative, and combinations thereof.

[0029] According to one embodiment of the present invention, the pharmaceutical composition for preventing or treating a tumor may be administered by a method selected from the group consisting of intraperitoneal administration, oral administration, inhalation administration, intravenous administration, intramuscular administration, subcutaneous administration, dermal administration, intrauterine administration, tumor administration, rectal administration, and combinations thereof, but is not limited thereto.

[0030] In addition, the fourth aspect of the present invention provides a method for screening a substance for increasing sensitivity to a PARP inhibitor, comprising the steps of: treating a candidate substance to a cell; measuring the expression level or activity of TRIM44 in the cell; and selecting a candidate substance with a reduced expression level or activity of TRIM44 as a substance for increasing sensitivity to a PARP inhibitor.

[0031] The above-described problem-solving methods are merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, additional embodiments may be included in the drawings and detailed description of the invention.

[0032] The present invention provides a method for increasing PARP inhibitor sensitivity by inhibiting TRIM44 expression or activity, thereby overcoming PARP inhibitor resistance and enhancing therapeutic efficacy. Specifically, TRIM44 serves as a key link between PARP1 and the MRN complex, effectively recruiting the MRN complex to DNA double-strand breaks in the presence of PARP inhibitors. Specifically, TRIM44 binds to PARP1 via its glutamate-rich domain and to the MRN complex via its zinc finger ubiquitin-binding (ZnF UBP) domain, thereby regulating the DNA damage response process.

[0033] In addition, the present invention confirmed that PARP inhibitor resistance caused by 53BP1 deficiency can be overcome by suppressing the expression or activity of TRIM44, and in particular, by confirming that simultaneous knockdown of TRIM44 and 53BP1 can reduce the increase in cell viability caused by 53BP1 deficiency, it was demonstrated that TRIM44 can be a new therapeutic target for overcoming PARP inhibitor resistance.

[0034] Furthermore, the present invention confirmed that the therapeutic effect of PARP inhibitors can be enhanced even in HR-proficient tumors by suppressing TRIM44 expression or activity. Specifically, suppression of TRIM44 expression significantly increased sensitivity to the PARP inhibitor olaparib in HR-proficient cells, and this effect was comparable to that of suppressing BRCA1 expression. This suggests the possibility of expanding the scope of application of PARP inhibitors beyond BRCA1 / 2 mutant tumors to HR-normal tumors.

[0035] However, the effects that can be obtained from this center are not limited to the effects described above, and other effects may exist.

[0036] Figure 1a is a step-by-step schematic representation of the FokI-screening experiment process for screening 211 GFP-tagged human ubiquitin-related proteins (URPs).

[0037] Figure 1b is a representative image showing the co-localization of GFP-tagged URPs and mCherry-FokI nuclease at DSB sites, observed by fluorescence microscopy, of novel URPs (Unknown, 16 out of 211) and previously known URPs (Known, 11 out of 211) associated with DDR.

[0038] Figure 1c identifies DDR-related URPs with evolutionarily conserved motifs such as RING, HECT, and Di19 and systematically classifies them along with their domain structures.

[0039] Figure 1d systematically organizes 24 URPs by classifying them according to PARP1 dependence (11), ATM dependence (11), and PARP1 / ATM independence (2).

[0040] Figures 1e to 1g are fluorescence microscopic images showing the results of observing the accumulation of TRIMs at DNA cleavage sites over time after laser microirradiation in the control group (1e), the PARP1 inhibitor PJ34 treatment group (1f), and the ATM inhibitor KU55933 treatment group (1g), respectively.

[0041] Figure 2a shows the intracellular location and expression of TRIM44 analyzed by immunofluorescence staining after DNA damage was induced in U2OS 2-6-5 cells transfected with GFP-TRIM44 by treating them with 4-OHT and shield1.

[0042] Figure 2b shows a comparative analysis of the movement of TRIM44 to DNA damage sites after 1 hour of pretreatment of U2OS 2-6-5 cells transfected with GFP-TRIM44 with DMSO (control), ATM inhibitor, or PARP inhibitor to induce DNA damage.

[0043] Figure 2c shows the physical interaction between TRIM44 and PARP1 verified by pull-down analysis using streptavidin beads after DSBs were induced with zeocin in 293T cells expressing SFB-TRIM44.

[0044] Figure 2d schematically shows the positions and structures of the ZnF UBP, glutamate-rich (GR) domain, B-box (BB), and coiled-coil (CC) domains that constitute the structure of the human TRIM44 protein.

[0045] Figure 2e shows the binding properties of TRIM44 mutants (ΔUBP, ΔBB, ΔCC, ΔGR) with each domain deleted, analyzed by Western blot for binding properties with PARP1 and MRN complexes.

[0046] Figure 2f shows a comparative analysis of the recruitment efficiency of wild-type TRIM44 and TRIM44 with a glutamate-rich domain deletion (ΔGR) to DNA damage sites, observed and quantified by fluorescence microscopy.

[0047] Figure 3a shows the time-dependent auto-PARylation of PARP1 and histone ADP-ribosylation levels analyzed by Western blot after TRIM44 siRNA treatment in U2OS cells and DNA damage induction with H2O2.

[0048] Figure 3b shows the accumulation and disappearance of PAR chains over time after DNA damage was induced by laser microirradiation in TRIM44-deficient cells, and the results were analyzed by immunofluorescence staining.

[0049] Figure 3c shows a comparative analysis of the change in the level of PARP1 auto-PARylation according to H2O2 treatment over time by Western blot after reintroducing various TRIM44 truncation mutants into TRIM44 knockout U2OS cells.

[0050] Figure 3d shows the expression of HA-tagged ubiquitin in TRIM44-deficient cells and the ubiquitination degree of PARP1 after DNA damage was induced with H2O2, as analyzed over time by immunoprecipitation and Western blot.

[0051] Figure 3e shows the change in the ubiquitination pattern of PARP1 after DNA damage analyzed by immunoprecipitation in cells co-expressing various TRIM44 truncation mutants and HA-tagged ubiquitin.

[0052] Figure 3f shows the change in the ubiquitination level of PARP1 over time after expressing HA-tagged ubiquitin in cells treated with PARG siRNA and inducing DNA damage.

[0053] Figure 4a shows the degree of ATM activation analyzed through the level of phosphorylated ATM after reintroduction of GFP-TRIM44 into TRIM44-deficient U2OS cells and treatment with zeocin.

[0054] Figure 4b shows a comparative analysis of the degree of accumulation of MRN complex and ATM in the chromatin fraction after DNA damage was induced with zeocin in TRIM44 wild-type and knockout cells.

[0055] Figure 4c shows the binding levels of MRN complex component proteins in the chromatin fraction analyzed by Western blot after introducing various TRIM44 truncation mutants into TRIM44 knockout cells and inducing DNA damage.

[0056] Figures 4d and 4e show the recruitment of MRE11 and γH2AX to DNA damage sites over time, as observed and quantified by immunofluorescence staining, after DNA damage was induced by laser microirradiation in TRIM44-deficient cells.

[0057] Figure 5a shows the DNA repair efficiency evaluated by Comet assay at different times (0 hours, 3 hours, 6 hours, 12 hours) after inducing DNA damage with H2O2 in TRIM44-deficient cells, and verified by Western blot.

[0058] Figure 5b shows a quantitative analysis of the effect of TRIM44 deficiency on homologous recombination repair efficiency using DR-GFP-U2OS cells, as measured by the percentage of GFP-positive cells.

[0059] Figure 5c shows the cell viability evaluated by clonogenic assay according to treatment with phleomycin at various concentrations (0, 0.5, 1, 2 μg / ml) in TRIM44-deficient U2OS cells, and the results are presented in a graph and actual colony images.

[0060] Figure 6a shows the effect of TRIM44 truncation mutants on MRN complex accumulation in the chromatin fraction of zeocin-induced DNA damage sites in the presence of a PARP inhibitor (AG14361) as analyzed by Western blot.

[0061] Figures 6b and 6c show the recruitment of MRE11, NBS1, and γH2AX to DNA damage sites after laser microirradiation of TRIM44-deficient cells in the presence of a PARP inhibitor, as observed by immunofluorescence staining.

[0062] Figures 6d and 6e are graphs showing quantitative analysis of the DNA damage site accumulation of MRE11 and NBS1 observed in Figures 6b and 6c, respectively.

[0063] Figure 6f shows a comparison of changes in the interactions between TRIM44, PARP1, MRN complex, and ATM before and after PARP inhibitor treatment using streptavidin bead pull-down analysis.

[0064] Figure 6g shows the binding properties between various TRIM44 truncation mutants and MRN complex constituent proteins evaluated by streptavidin bead pull-down assay in the presence of PARP inhibitor.

[0065] Figures 7a and 7b show the cell viability evaluated by clonogenic assay according to treatment with various concentrations (0, 0.5, 1, 2 μM) of olaparib in TRIM44-deficient cells and TRIM44 / 53BP1 double-deficient cells, and the results are presented in graphs and colony images.

[0066] Figure 7c shows the analysis of TRIM44 mRNA expression levels in renal clear cell carcinoma (KIRC) using TCGA RNA-seq data.

[0067] Figure 7d shows the overall survival rate according to TRIM44 expression level in KIRC patients using Kaplan-Meier analysis.

[0068] Figure 7e shows the change in sensitivity to olaparib and phleomycin when TRIM44 is deficient in HEK293 cells, a renal cell carcinoma cell line, as assessed by clonogenic assay.

[0069] Figure 7f comprehensively schematically illustrates the functional mechanism of action of TRIM44 at DNA damage sites in the presence of PARP inhibitors.

[0070] Below, with reference to the attached drawings, embodiments of the present invention are described in detail to facilitate easy implementation by those skilled in the art. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity, and similar reference numerals have been used throughout the specification to indicate similar elements.

[0071] Throughout this specification, when a part is said to be "connected" to another part, this includes not only cases where it is "directly connected" but also cases where it is "electrically connected" with another element in between.

[0072] Throughout this specification, when it is said that a member is located “on,” “above,” “upper,” “lower,” “lower” or “lower” another member, this includes not only cases where the member is in contact with the other member, but also cases where another member exists between the two members.

[0073] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0074] The terms "about," "substantially," and the like, as used herein, are used to mean at or near the numerical value when manufacturing and material tolerances inherent to the meanings referred to are presented, and are used to prevent unscrupulous infringers from unfairly exploiting disclosures that contain precise or absolute numerical values ​​to aid understanding of the present disclosure. Furthermore, throughout the present disclosure, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0075] Throughout this specification, the term "combination thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0076] Throughout this specification, references to “A and / or B” mean “A, B, or A and B.”

[0077] Hereinafter, the method for increasing sensitivity to PARP inhibitors of the present invention will be described in detail with reference to implementation examples, examples, and drawings. However, the present invention is not limited to these implementation examples, examples, and drawings.

[0078]

[0079] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a method for increasing sensitivity to a PARP inhibitor, comprising a step of inhibiting the expression or activity of TRIM44.

[0080] The present invention provides a method for increasing PARP inhibitor sensitivity by inhibiting TRIM44 expression or activity, thereby overcoming PARP inhibitor resistance and enhancing therapeutic efficacy. Specifically, TRIM44 serves as a key link between PARP1 and the MRN complex, effectively recruiting the MRN complex to DNA double-strand breaks in the presence of PARP inhibitors. Specifically, TRIM44 binds to PARP1 via its glutamate-rich domain and to the MRN complex via its zinc finger ubiquitin-binding (ZnF UBP) domain, thereby regulating the DNA damage response process.

[0081] In addition, the present invention confirmed that PARP inhibitor resistance caused by 53BP1 deficiency can be overcome by suppressing the expression or activity of TRIM44, and in particular, by confirming that simultaneous knockdown of TRIM44 and 53BP1 can reduce the increase in cell viability caused by 53BP1 deficiency, it was demonstrated that TRIM44 can be a new therapeutic target for overcoming PARP inhibitor resistance.

[0082] Furthermore, the present invention confirmed that the therapeutic effect of PARP inhibitors can be enhanced even in HR-proficient tumors by suppressing TRIM44 expression or activity. Specifically, suppression of TRIM44 expression significantly increased sensitivity to the PARP inhibitor olaparib in HR-proficient cells, and this effect was comparable to that of suppressing BRCA1 expression. This suggests the possibility of expanding the scope of application of PARP inhibitors beyond BRCA1 / 2 mutant tumors to HR-normal tumors.

[0083] According to one embodiment of the present invention, in the presence of a PARP inhibitor, the main binding substance of TRIM44 may be converted from PARP1 to MRN (MRE11-RAD50-NBS1) complex, but is not limited thereto.

[0084] According to one embodiment of the present invention, the TRIM44 may bind to the PARP1 via a glutamate-rich domain, but is not limited thereto.

[0085] According to one embodiment of the present invention, the TRIM44 may bind to the MRN complex via a ZnF UBP (zinc finger ubiquitin-binding) domain, but is not limited thereto.

[0086] According to one embodiment of the present invention, PARP inhibitor resistance caused by 53BP1 deficiency can be overcome through inhibition of TRIM44, but is not limited thereto.

[0087] According to one embodiment of the present invention, the PARP inhibitor may include, but is not limited to, one selected from the group consisting of Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib, and combinations thereof.

[0088] In addition, the second aspect of the present invention provides a composition for increasing sensitivity to a PARP inhibitor, comprising a substance that inhibits the expression or activity of TRIM44 as an active ingredient.

[0089] Regarding the composition for increasing sensitivity to a PARP inhibitor according to the second aspect of the present application, detailed descriptions of parts overlapping with the first aspect of the present application have been omitted, but even if the descriptions have been omitted, the contents described in the first aspect of the present application may be equally applied to the second aspect of the present application.

[0090] According to one embodiment of the present invention, TRIM44 may bind to PARP1 through the glutamate-rich domain and bind to the MRN complex through the ZnF UBP domain, but is not limited thereto.

[0091] In addition, the third aspect of the present invention provides a pharmaceutical composition for preventing or treating a tumor, comprising a composition for increasing sensitivity to a PARP inhibitor according to the second aspect of the present invention as an active ingredient.

[0092] Regarding the pharmaceutical composition for preventing or treating tumors according to the third aspect of the present invention, detailed descriptions of parts overlapping with the first and second aspects of the present invention have been omitted, but even if the descriptions have been omitted, the contents described in the first and second aspects of the present invention can be equally applied to the third aspect of the present invention.

[0093] According to one embodiment of the present invention, binding of TRIM44 may be, but is not limited to, switching from PARP1 to the MRN complex in the presence of a PARP inhibitor.

[0094] According to one embodiment of the present invention, the tumor may include, but is not limited to, a homologous recombination proficient (HR-proficient) tumor.

[0095] Homologous recombination is one of the primary mechanisms for repairing DNA double-strand breaks. Tumors in which the BRCA1 / 2 proteins function normally are called HR-proficient tumors. While PARP inhibitors have typically been used primarily in HR-deficient tumors harboring mutations in the BRCA1 / 2 genes, the present invention anticipates that PARP inhibitors will also be effective in HR-proficient tumors by suppressing TRIM44 expression or activity.

[0096] HR-proficient tumors are generally less sensitive to PARP inhibitors because they can effectively repair DNA double-strand breaks through homologous recombination repair mechanisms. However, TRIM44 inhibition can enhance the therapeutic efficacy of PARP inhibitors even in these HR-proficient tumors, suggesting the potential to significantly expand the scope of application of PARP inhibitors.

[0097] In particular, inhibition of TRIM44 can promote cancer cell death by interfering with the repair of DNA damage induced by PARP inhibitors in HR-proficient tumors. This suggests that it may offer a new treatment opportunity for patients with HR-proficient tumors, for whom conventional PARP inhibitor therapy has been limited.

[0098] According to one embodiment of the present invention, the pharmaceutical composition for preventing or treating a tumor may additionally include a pharmaceutically acceptable carrier, but is not limited thereto.

[0099] According to one embodiment of the present invention, the carrier may include, but is not limited to, a group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, menthol, mineral oil, and combinations thereof.

[0100] According to one embodiment of the present invention, the pharmaceutical composition for preventing or treating a tumor may further include a component selected from the group consisting of, but not limited to, a preservative, a solubilizer, a stabilizer, a humectant, a sweetener, a colorant, a flavoring agent, a salt, a buffer, an antioxidant, a lubricant, an emulsifier, a suspending agent, a preservative, and combinations thereof.

[0101] According to one embodiment of the present invention, the pharmaceutical composition for preventing or treating a tumor may be administered by a method selected from the group consisting of intraperitoneal administration, oral administration, inhalation administration, intravenous administration, intramuscular administration, subcutaneous administration, dermal administration, intrauterine administration, tumor administration, rectal administration, and combinations thereof, but is not limited thereto.

[0102] In addition, the fourth aspect of the present invention provides a method for screening a substance for increasing sensitivity to a PARP inhibitor, comprising the steps of: treating a candidate substance to a cell; measuring the expression level or activity of TRIM44 in the cell; and selecting a candidate substance with a reduced expression level or activity of TRIM44 as a substance for increasing sensitivity to a PARP inhibitor.

[0103] Regarding the method for screening a substance for increasing PARP inhibitor sensitivity according to the fourth aspect of the present invention, detailed descriptions of parts overlapping with the first to third aspects of the present invention have been omitted. However, even if the descriptions have been omitted, the contents described in the first to third aspects of the present invention can be equally applied to the fourth aspect of the present invention.

[0104] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0105]

[0106] [Materials and Methods]

[0107] cell lines

[0108] Human U2OS, HEK293T, HEK293, and HEK293TN cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). U2OS 2-6-3 and 2-6-5 cells were provided by Dr. Roger A. Greenberg and were cultured in DMEM supplemented with 10% FBS and puromycin (1 μg / ml). U2OS-based HR reporter cells (DR-GFP U2OS) were provided by Dr. Jeremy M. Stark and were maintained in DMEM high glucose without sodium pyruvate supplemented with 10% FBS and puromycin (1 μg / ml). AsiSI-ER-U2OS (DIvA) cells were provided by Dr. Gaelle Legube and were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. All cells were cultured in a temperature-controlled chamber (37°C, 5% CO2).

[0109] Establishment of TRIM44 knockout U2OS cell lines

[0110] To generate TRIM44 knockout (KO) cell lines, single-guide RNAs (sgRNAs) targeting TRIM44 were designed using an online CRISPR design tool. The designed sgRNAs were cloned into lentiCRISPRv2 according to the protocol provided by the Zhang lab and transfected into HEK293TN cells for lentivirus production. After 48 h, the virus-containing supernatant was harvested. The virus was then infected into U2OS cells with puromycin (1 μg / ml) treatment to generate TRIM44 KO cells. Selected cells were seeded in 96-well plates and cultured for 10 days to generate single clones. At least 10 clones were tested for the presence of TRIM44 by immunoblotting with a TRIM44 antibody.

[0111] Cloning and plasmid generation of ubiquitin-related protein libraries

[0112] To generate the pENTRTM vector containing 211 human ubiquitin-related proteins (URPs), PCR amplification using individual primers was performed from a human liver cDNA library. All URPs cloned into pENTRTM were then sequenced and transferred into the pDEST53 (N-terminal GFP) vector using the Gateway LR Cloning System. Each TRIM44 truncation mutant was constructed using a classical PCR method and cloned into the pCR8 / GW / TOPO vector for entry clones, which were then subcloned into the SFB vector. All deletion regions were verified by DNA sequencing, and each primer set used in this study is described. Detailed information on other plasmids and chemicals used in this study is summarized in Supplementary Table S3.

[0113] RNA interference and plasmid transfection

[0114] siRNAs for the indicated genes purchased from Bioneer were transfected with Lipofectamine RNAiMAX for 72 h. Plasmids were transfected with polyethylenimine (PEI) for 48–72 h.

[0115] Laser microscopy and immunofluorescence

[0116] For laser microirradiation, U2OS TRIM44 WT and U2OS TRIM44 KO cells were seeded in 35-mm round glass-bottom dishes. Cells were transfected with siRNA and DNA for 48–72 h and incubated with 10 μM 5-bromo-2-deoxyuridine (BrdU) for 24 h before laser-induced DNA damage. Single- and double-strand DNA breaks were induced by laser microirradiation using a 405 nm laser on a Nikon A1R confocal microscope. Cells were subjected to laser-induced DNA damage under two different conditions: 3 s / 32 lines for live-cell imaging and 1 s / 32 lines for fixed-cell imaging using a 60x oil objective. For fixed-cell imaging, cells were damaged for the indicated times and then fixed with 2% formaldehyde in phosphate-buffered saline (PBS) at room temperature. Cells were then blocked with blocking solution (1% BSA in PBS) for 30 min at room temperature. Cells were incubated with primary antibodies overnight at 4°C. The following day, secondary antibodies were incubated for 1 hour at room temperature and mounted with VECTASHIELD containing DAPI.

[0117] Immunofluorescence for DNA damage-induced focus formation

[0118] For the DNA damage-induced RAD51 focus formation assay, U2OS cells were seeded in 35-mm glass-bottom dishes. Cells were transfected with siRNA for 48 h using RNAiMAX according to the manufacturer's instructions and then treated with 200 ng / ml neocarzinostatin (NCS) 2 h before fixation. After NCS treatment, cells were fixed with 4% PFA in PBS for 15 min at room temperature. Fixed cells were washed three times with PBS and permeabilized with 0.5% TritonX-100 in PBS for 15 min at room temperature. Nonspecific signals were blocked with blocking solution (PBS containing 1% BSA and 0.5% TritonX-100) for 1 h at room temperature. Primary antibodies diluted in blocking solution were then incubated overnight at 4°C. The following day, Alexa Fluor-conjugated secondary antibodies were incubated for 2 h at room temperature and washed three times with PBS. Cells were counterstained with 1 μg / ml DAPI diluted in PBS for 10 min at room temperature, washed with PBS, and mounted on glass using VECTASHIELD mounting solution.

[0119] To quantify focus formation, all images were acquired on a Nikon A1R confocal microscope using a 60x oil-immersion objective under identical experimental settings. Images were used to manually count foci using NIS-element AR software. Cells with ≥5 or 10 RAD51 foci per nucleus were manually counted, and the percentage of cells was calculated. Statistical analysis was performed using GraphPad Prism 8.0 software.

[0120] Homologous recombination (HR) repair analysis

[0121] The repair efficiency of homologous recombination (HR) was measured using the DR-GFP U2OS reporter cell line. To assess HR efficiency, DR-GFP U2OS cells were seeded in 12-well plates and transfected with siRNA using Lipofectamine RNAiMAX according to the manufacturer's instructions. After 24 h, I-SceI endonuclease was delivered to the cells using Lipofectamine 2000. After 72 h, the cells were trypsinized and centrifuged at 1500 × g for 5 min. The cells were then washed with PBS and resuspended in the same buffer. The cell suspension was transferred to a 5-ml round-bottom polystyrene tube for FACS analysis. GFP-positive cells were quantified using a FACSAria III.

[0122] DNA end resection analysis

[0123] To measure the efficiency of DSB end resection, a previously described method was used. AsiSI-ER-U2OS (DIvA) cells were seeded in 100-mm dishes (1.2 × 106 cells per dish) and transfected with siRNA. After 2 days, cells were treated with or without 300 nM 4-OHT for 4 h to induce DSBs.

[0124] Afterwards, cells were collected and genomic DNA was isolated using the DNeasy Blood & Tissue Kit with RNase A treatment during lysis according to the manufacturer's instructions. Then, 1000 ng of the extracted gDNA was treated with 20 units of BsrGI-HF restriction enzyme as a control or for 20 h at 37°C, and the enzyme was heat inactivated at 65°C for 20 min. Fifty ng of each treated gDNA was used as a template for quantitative PCR using SYBR Green Supermix to determine the excision site at position 335 nt. For each sample, the ΔCt was calculated by subtracting the Ct value of the treated sample from the Ct value of the control-treated sample. The percentage of single-stranded DNA (ssDNA%) in each sample was calculated using the following equation: ssDNA % 1 / (2(ΔCt-1) + 0.5) × 100.

[0125] FokI analysis

[0126] For FokI screening, the indicated plasmids were transfected with the mCherry-LacI-FokI endonuclease plasmid using Lipofectamine 2000 into U2OS-2-6-3 reporter cells integrating the Lac operator repeat (Х256). After 48 h of transfection, cells were fixed with 4% formaldehyde in PBS for 10 min at room temperature, and positive signals were analyzed and visualized using a Nikon A1R confocal microscope using a 60x oil objective. U2OS 2-6-5 cells stably expressing mCherry-LacI-FokI were seeded on glass-bottom dishes and transfected with the GFP-tagged plasmid using Lipofectamine 2000. After 48 h of incubation, DSBs were induced by treatment with 4-OHT (1 μM) and Shield1 (1 μM) for 4 h. Cells were fixed with 2% formaldehyde and mounted with VECTASHIELD containing DAPI.

[0127] Western blotting

[0128] For Western blotting analysis, samples were boiled with 1x sample buffer and separated by SDS-PAGE using gradient gels (4–20% acrylamide gels). The separated proteins were transferred to nitrocellulose membranes and blocked with 5% skim milk or 1% BSA in TBST for 1 hour at room temperature. The membranes were incubated with the indicated primary antibodies overnight at 4°C and then with secondary antibodies for 1 hour at room temperature. The immunoblotted proteins were detected with ECL reagent.

[0129] Pull-down analysis

[0130] HEK 293T cells were lysed in NETN buffer containing a cocktail of protease and phosphatase inhibitors and incubated at room temperature for 20 min. Cell lysates were centrifuged at 13,000 rpm for 15 min at 4°C.

[0131] For pull-down analysis of SFB-tagged proteins, the supernatant was incubated with streptavidin Sepharose high-performance affinity resin for 2 h at 4°C and washed four times with NETN buffer. The washed precipitate was boiled with 2X sample buffer and used for Western blotting analysis.

[0132] Whole cell extraction

[0133] U2OS cells were harvested in 1X sample buffer and sonicated using an EpiShear Probe Sonicator. Cell lysates were boiled and used for Western blotting analysis.

[0134] Immunoprecipitation

[0135] U2OS cells were lysed in NETN buffer containing a cocktail of protease and phosphatase inhibitors. Cell lysates were sonicated using an EpiShear Probe Sonicator and centrifuged at 13,000 rpm for 15 minutes at 4°C. For immunoprecipitation, the supernatant was incubated with primary antibodies overnight at 4°C. The following day, the immunoprecipitates were captured by incubation with Protein A Sepharose Fast-Flow for 1 hour, and the beads were washed four times with NETN buffer. The washed precipitates were boiled with 2X sample buffer and used for Western blotting analysis.

[0136] nuclear fractionation

[0137] U2OS cells were lysed with lysis buffer A containing a cocktail of protease and phosphatase inhibitors and incubated at 4°C for 5 minutes. The lysate was centrifuged at 1,300 rcf for 4 minutes at 4°C. For the cytosolic fraction, the soluble fraction was centrifuged again at 13,000 rpm for 15 minutes at 4°C. The nuclear fraction was isolated with lysis buffer B containing a cocktail of protease and phosphatase inhibitors and incubated at 4°C for 30 minutes. The lysate was centrifuged at 5,000 rcf for 5 minutes at 4°C.

[0138] Chromatin fraction

[0139] U2OS TRIM44 WT and TRIM44 KO cells were transfected with the indicated siRNAs and DNA. After 72 hours, cells were lysed with NETN buffer containing a cocktail of protease and phosphatase inhibitors and incubated at 4°C for 20 minutes. The lysates were centrifuged at 12,000 rpm for 10 minutes at 4°C. Chromatin fractions were separated with chromatin buffer containing a cocktail of protease and phosphatase inhibitors and incubated in a shaking incubator at 37°C for 20 minutes. Whole-cell lysates and soluble fractions isolated from the chromatin fractions were used for Western blotting analysis. Each experiment was repeated at least three times.

[0140] Alkaline comet analysis

[0141] U2OS cells were treated with 1 mM H2O2 for 30 min (release time 0 h) and then released for the indicated times (release time 3, 6, and 12 h). Alkaline Comet assays were performed using Trevigen's reagent according to the manufacturer's protocol. Imaging was performed with a fluorescence microscope, and tail moments were determined using OpenComet analysis software.

[0142] Clonogenic survival analysis

[0143] U2OS cells were transfected with the indicated siRNAs. The following day, cells were seeded in six-well plates (3,000 cells per well). The following day, cells were treated with phleomycin or olaparib at the indicated concentrations. Cells were cultured in a temperature-controlled chamber (37°C, 5% CO2) for 10–14 days. Cells were fixed with purified methanol for 10 minutes and stained with 0.01% crystal violet for 20 minutes. Colony areas were measured and analyzed using ImageJ.

[0144] Image quantification and statistical analysis

[0145] All values ​​in this study are reported as the mean ± standard error of the mean (SEM). Results represent the means of at least three independent experiments. Statistical analyses used Student's t-test or one-way ANOVA followed by Dunnett's multiple comparison test. A P value < 0.05 was considered statistically significant. Significance is indicated by an asterisk. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

[0146]

[0147] [Example 1] Identification and functional analysis of novel ubiquitin-related proteins in damaged chromatin.

[0148] Although ubiquitin-mediated signaling is essential for ATM-mediated DDR, its importance in PARP1 signaling is limited. We cloned 211 GFP-fused ubiquitin-related proteins (URPs), including ubiquitin E3 ligases, from a human liver cDNA library and examined whether these proteins translocate to DSB sites generated by an mCherry-fused FokI endonuclease reporter system in U2OS 2-6-3 cells.

[0149] Among the 211 URPs investigated (Fig. 1a), 27 (12.8%) colocalized with mCherry-FokI at DSBs (Fig. 1b). We identified 16 novel subgroups of URPs (11 of 211 URPs, 7.6%) with evolutionarily intact motifs, such as tripartite complexes, novel genes of interest (RING), homeologs to E6-AP carboxyl termini (HECT), and Di19 (Fig. 1c). We also found several URPs (11 of 211 URPs, 5.2%) with well-known functions in DDR: CHFR, PELI1, RNF2, RNF8, RNF20, RNF40, RNF138, RNF146, RNF166, RNF168, and RNF206 (Figs. 1b and 1c).

[0150] To investigate the physiological role of potential DDR-associated ubiquitin E3s, we first examined whether the ATM inhibitor KU55993 or the PARP inhibitor PJ34 altered their translocation to DNA lesions induced by laser microirradiation (mIR). Of the 24 DDR-E3s tested, 11 E3 ligases were detected at DNA lesions in the presence of KU55993 but not PJ34. Meanwhile, the other 11 E3 ligases were clearly enriched at mIR-induced DNA lesions in the presence of PJ34 but not KU55993. Two other proteins, FBXL and PELI1, translocated to DNA lesions regardless of the presence of KU55993 or PJ34 (Fig. 1d).

[0151] Consequently, we classified 24 DDR-E3s into three groups: 11 ATM-dependent, 11 PARP1-dependent, and 2 ATM- and PARP1-independent. Because the 11 E3 ligases that translocated to DNA lesions in a PARP1-dependent manner were unaffected by ATM inhibitors, and the other E3 ligases that were recruited to DNA lesions in an ATM-dependent manner were unaffected by PARP inhibitors, we concluded that these proteins are separated in their functions within the PARP1 and ATM pathways.

[0152] The present inventors were particularly interested in PARP-dependent ubiquitin signaling, focusing on the tripartite motif (TRIM) family of proteins. The TRIM family comprises approximately 90 proteins that play roles in diverse cellular processes, including signal transduction, development, protein quality control, and carcinogenesis.

[0153] We observed that TRIM31, TRIM36(v), TRIM44, and TRIM61(v) translocated to DSB sites (Figures 1b and 1e). Among these proteins, TRIM31 and TRIM36(v) translocated to DNA lesions in an ATM-dependent manner, whereas TRIM44 and TRIM61(v) translocated to DNA lesions in a PARP1-dependent manner (Figures 1f and 1g). TRIM31, TRIM36(v), and TRIM61(v) are members of the RBCC-motif subfamily, which contains a RING domain that typically functions as a ubiquitin E3 ligase. In contrast, TRIM44 belongs to the BCC-motif subfamily, which does not contain a RING domain. Although initially classified as an E3 ligase as a TRIM family member, TRIM44 has been shown to function as a ubiquitin-specific protease (USP) via its ZnF-UBP domain.

[0154]

[0155] [Example 2] Necessity of the glutamate-rich domain in TRIM44 binding to PARP1

[0156] TRIM44 translocates to DNA lesions in a PARP1-dependent manner and contains a ZnF UBP that binds to the C-terminal diglycine motif of ubiquitin. We hypothesized that PARP1-dependent TRIM44 might regulate DDR by recognizing ubiquitin chains on damaged chromatin. We observed that endogenous TRIM44 and GFP-TRIM44 translocated to single DSB sites generated by the FokI endonuclease (Fig. 2a).

[0157] Consistent with the findings in Figures 1F and 1G, TRIM44 recruitment to DSBs was not affected by ATM inhibitors, whereas treatment with AG14361, a PARP inhibitor that inhibits the catalytic activity of PARP1 but does not affect its movement to DNA lesions, was disrupted (Figure 2B). Pull-down assays using cell lysates expressing SFB-tagged TRIM44 revealed a strong interaction between TRIM44 and PARP1. This interaction was slightly enhanced by exposure to the radiomimetic agent zeocin for 2 h (Figure 2C). TRIM44 was observed to exhibit a weak interaction with ATM.

[0158] To investigate whether PARP1 loss abolishes the interaction between TRIM44 and chromatin, we performed experiments using siRNA to deplete PARP1 (siPARP1). Our findings demonstrate that TRIM44 recruitment to DNA damage sites is highly dependent on the presence and activity of PARP1. The reduced interaction between TRIM44 and H2AX in siPARP1-treated cells suggests that TRIM44 does not directly bind to H2AX but instead relies on PARP1 for its localization to damaged chromatin. Inhibition of TRIM44 translocation by PARPi further supports the idea that the catalytic activity of PARP1 is important for TRIM44's role in the DNA damage response.

[0159] We then investigated the domains of TRIM44 that are crucial for PARP1 binding. TRIM44 consists of four domains: the ZnF UBP located at the N-terminus, the glutamate-rich (GR) domain, the B-box (BB), and the coiled-coil domain (CC). It is noteworthy that the latter two domains are highly conserved among TRIM family proteins. Four TRIM44 deletion mutants (ΔUBP, ΔBB, ΔCC, and ΔGR) were generated, each of which lacked a specific domain (Fig. 2d).

[0160] These mutants were expressed in 293T cells and treated with zeocin. Pull-down assays showed that the ΔUBP, ΔBB, and ΔCC mutants bound PARP1 as efficiently as TRIM44 WT (Fig. 2e). They also maintained binding to ATM, as well as RAD50 and MRE11 of the MRN complex, similar to TRIM44 WT. In contrast, the ΔGR mutant was unable to bind to PARP1, indicating that the GR domain of TRIM44 is crucial for the interaction with PARP1.

[0161] It is noteworthy that the ΔGR mutant exhibited increased binding to ATM, RAD50, and MRE11 compared to the other three mutants. This finding suggests a potential inverse correlation between TRIM44's binding capacity to PARP1 and its association with the MRN complex. The recruitment of TRIM44 ΔGR to DSB sites in the FokI system was observed to be measurably lower than that of WT TRIM44, confirming the importance of the GR domain of TRIM44 at DSB sites (Fig. 2f).

[0162] Similarly, all deletion mutants of TRIM44, except ΔGR, migrated to damaged DNA bands generated by microirradiation. The impaired migration of TRIM44 ΔGR to DSB lesions was not due to other differences in subcellular localization, as immunocytochemistry and cell fractionation analyses revealed that WT and all deletion mutants were observed in both the nucleus and cytoplasm.

[0163] In summary, our data clearly demonstrate that TRIM44 translocates to DSBs in a PARP1-dependent manner. Furthermore, the stable localization of TRIM44 at DNA damage sites relies on physical interaction with PARP1 via its GR domain.

[0164]

[0165] [Example 3] Prevention of PARP1 hyperactivation by TRIM44

[0166] Next, we investigated the effect of TRIM44 on PARP1 activity under DNA damage. To this end, we analyzed PARP1 itself, a major target of PARP1, and the product of PARP1-mediated ADP-ribosylation of histones. Hydrogen peroxide (H2O2) induces both DNA SSBs and DSBs and activates PARP1. When U2OS cells were treated with 1 mM H2O2 for 5–30 min and subsequently probed with an anti-poly(ADP-ribose) antibody, significant auto-PARylation of PARP1 occurred within 5–10 min in control cells, along with a correlated increase in histone ADP-ribosylation (Fig. 3a).

[0167] This effect was abolished in cells co-treated with the PARP inhibitor AG14361. It is noteworthy that hyper-PARylation of PARP1 and increased histone ADP-ribosylation were observed in TRIM44-depleted cells, suggesting that TRIM44 regulates PARP1 activity. Similarly, a significant increase in PAR levels was observed in TRIM44-depleted cells treated with various doses of H2O2. When cells were exposed to methyl methanesulfonate (MMS), which induces SSB, a moderate increase in PAR levels was observed with TRIM44 siRNA.

[0168] Elevated PARylation in TRIM44 knockdown cells was confirmed by laser microirradiation experiments (Fig. 3b). Immunofluorescence staining with an anti-PAR antibody revealed rapid accumulation of PAR chains in DNA tethers within 1 minute in control cells, which almost completely disappeared within 10 minutes. However, in cells treated with TRIM44 siRNA, nascent PAR chain accumulation was more intense, and a significant amount of chains remained even after 10 minutes.

[0169] We verified that the hyper-PARylation of PARP1 in TRIM44-depleted cells was not due to impaired PARG recruitment to DNA lesions, as GFP-PARG was recruited to microirradiated DNA bands in TRIM44-depleted cells as efficiently as in control cells.

[0170] Given that TRIM44 binding to PARP1 is crucial for regulating PARP1 activity, the GR domain of TRIM44, which mediates PARP1 binding, is likely central to regulating PARP1 activity. Therefore, we reconstituted each deletion mutant in TRIM44 knockout (KO) cells and compared the levels of PARP1 auto-PARylation at 10 and 15 min after H2O2 treatment (Fig. 3c).

[0171] In TRIM44 KO cells, auto-PARylation of PARP1 persisted for 15 minutes, whereas wild-type reconstitution of TRIM44 substantially reduced PAR levels at 15 minutes. A similar decrease in PAR levels was observed in cells transfected with ΔBB or ΔCC constructs. In contrast, cells expressing ΔGR maintained PAR levels at 15 minutes, similar to TRIM44 KO cells. It is noteworthy that PAR chain accumulation was much stronger in cells transfected with the ΔUBP mutant. These data suggest that not only the GR domain but also the ZnF UBP domain of TRIM44 plays a key role in upregulating PARP1 activity.

[0172] The next question is how TRIM44's ZnF UBP regulates PARP1 activity. TRIM44's ZnF UBP is conserved among several deubiquitinating enzymes and has been shown to increase protein stability by removing K-48-linked polyubiquitin chains. Therefore, we hypothesized that TRIM44 knockdown or TRIM44 ΔUBP could lead to the accumulation of PARP1 ubiquitination, which in turn could affect the hyper-PARylation state of PARP1.

[0173] In vivo ubiquitination analysis of PARP1 immunoprecipitates using whole-cell lysates infused with the HA-ub construct demonstrated a substantial increase in PARP1 ubiquitination following H2O2 treatment. Surprisingly, however, knockdown of TRIM44 resulted in reduced PARP1 ubiquitination under DNA damage (Fig. 3d). This finding was consistently observed at different time points (1–15 min) after H2O2 treatment. PARP1 ubiquitination increased in a time-dependent manner up to 10 min, whereas TRIM44 knockdown cells consistently showed lower levels of PARP1 ubiquitination.

[0174] Consistent with Figures 3A-3C, significant accumulation of the PAR chain of PARP1 was observed in TRIM44 KD cells, which inversely correlated with ubiquitination. To determine whether these results were due to TRIM44 affecting the total level of ubiquitinated proteins, we examined the level of total ubiquitination in input lysates. Our findings demonstrate that TRIM44 deficiency does not affect the overall level of ubiquitinated proteins ( Figure 3D ).

[0175] Additionally, we assessed the basal level of PARP1 ubiquitination before H2O2 treatment. The results showed that basal PARP1 ubiquitination was comparable between siCtrl and siTRIM44 cells, indicating that TRIM44 silencing did not affect PARP1 ubiquitination under unstressed conditions. A differential effect on PARP1 ubiquitination was observed upon H2O2 treatment, highlighting the role of TRIM44 in the DNA damage response.

[0176] To clarify which domain of TRIM44 influences PARP1 ubiquitination, we performed experiments using TRIM44 WT, ΔUBP, or ΔGR mutants under overexpression conditions. As previously observed, restoration of PARP1 ubiquitination was observed in cells overexpressing TRIM44 WT, but not in cells expressing ΔUBP or ΔGR mutants ( Figure 3E ). These findings suggest that TRIM44 binds to PARP1 via the GR domain and then preserves PARP1 ubiquitination via the ZnF UBP domain, thereby limiting PARP1 hyperactivity.

[0177] Based on these results, we sought to explore the role of HPF1, known to promote serine PARylation of PARP1 after DNA damage, in PARP1 ubiquitination. To investigate this, we used three different HPF1-targeting siRNAs. We observed accumulation of PAR chains in cells treated with siRNAs targeting either TRIM44 or HPF1. Furthermore, the increase in PARP1 auto-PARylation was more pronounced in cells in which both TRIM44 and HPF1 were knocked down, indicating an additive effect. These findings suggest that TRIM44 and HPF1 target different amino acids for PARylation.

[0178] We also analyzed the PARP1 ubiquitination pattern in these cells and found that HPF1 depletion, similar to TRIM44 depletion, resulted in a decrease in PARP1 ubiquitination. Auto-PARylation of PARP1 occurs at serine (S), aspartic acid (D), glutamic acid (E), and lysine (K) residues within its BRCT and WGR domains. This suggests that there may be competition between ubiquitination and PARylation at lysine residues of PARP1 in TRIM44-depleted cells.

[0179] The relationship between PARP1 auto-PARylation and ubiquitination was further investigated by siRNA-mediated PARG knockdown (Fig. 3f) or overexpression of GFP-PARG. PARG knockdown resulted in the accumulation of PAR chains, which was accompanied by reduced PARP1 ubiquitination, whereas PARG overexpression significantly increased PARP1 ubiquitination. Taken together, these data suggest that TRIM44 maintains poly-ubiquitin chains on PARP1 to limit its hyperactivation.

[0180]

[0181] [Example 4] Hyperactivation of PARP1 inhibits the accumulation of the MRN complex at DNA lesions.

[0182] PARP1 activity has been shown to contribute to the initial recruitment of MRE11 and NBS1, components of the MRN complex, to DSB sites, and ATM activity is also influenced by PARP1 activity. We found that TRIM44 ΔGR bound more strongly to the ATM / MRN complex than the other three deletion mutants (Fig. 2e). These findings indicate that TRIM44 regulates not only PARP1 activity but also ATM-mediated DDR signaling.

[0183] Indeed, we found that p-ATM levels were reduced by TRIM44 siRNA under DNA damage, and reconstitution of GFP-TRIM44 in TRIM44 knockdown cells restored phosphorylated ATM (p-ATM) levels as well as γH2AX levels (Fig. 4a). Because ATM relies on the MRN complex to recognize and bind DNA lesions, we compared the amounts of MRN complex and ATM present in chromatin.

[0184] In TRIM44 WT cells, induction of double-strand breaks (DSBs) by zeocin resulted in increased accumulation of ATM and MRN complexes in chromatin. However, this accumulation was not observed in TRIM44 KO cells (Fig. 4b). To determine whether these results were due to TRIM44 affecting the total levels of ATM and MRN complexes, we examined the total levels of these proteins. Our findings indicate that TRIM44 deficiency does not affect the total levels of ATM and MRN complexes.

[0185] Importantly, we also found that levels of MRE11 and NBS1 were lower in TRIM44 KO cells transfected with ΔUBP than in WT-transfected cells (Fig. 4c), suggesting that PARP1 hyperactivation may affect the recruitment of the MRN complex to damaged chromatin. Interestingly, while a decrease in chromatin association of NBS1 and MRE11 was observed in PARG knockdown cells, an increase was observed in PARG overexpressing cells, supporting that PARP1 hyperactivation interferes with the recruitment of the MRN complex to damaged chromatin.

[0186] To further validate our findings, we performed a more sensitive microexamination approach. At damaged DNA fringes, MRE11 accumulation was significantly reduced in TRIM44 knockdown cells at various time points ranging from 5 to 30 minutes (Fig. 4d), and NBS1 accumulation in control cells was also higher than in TRIM44 knockdown cells (Fig. 4e).

[0187] In summary, TRIM44 limits PARP1 hyperactivation, which allows for the recruitment of the MRN complex and ATM activation at damaged chromatin. Therefore, we propose that TRIM44 is a dual regulator of PARP1 activity and ATM-mediated DDR.

[0188]

[0189] [Example 5] The Need for TRIM44 in DSB Repair

[0190] Because TRIM44 is crucial for the recruitment of the MRN complex to DNA lesions, we next determined the effect of TRIM44 on the repair efficiency of damaged chromatin using the Comet assay. In this assay, damaged tail images were automatically analyzed by the OpenComet software.

[0191] When U2OS cells were exposed to H2O2 for 30 min, the tail moment index increased, but decreased 3 h after removal (R3) and returned to control levels by 6 h, suggesting complete repair of damaged chromatin (Fig. 5a). In contrast, damaged chromatin in cells with TRIM44 siRNA was only slightly repaired 3-6 h after removal and still maintained its tail.

[0192] To further confirm the role of TRIM44 in the HR pathway, we performed homologous recombination (HR) repair assays, DNA end resection assays, and RAD51 focus formation assays (Fig. 5b). Consistent with Fig. 4, all of these processes were impaired in TRIM44 knockdown cells.

[0193] Similarly, in a clonogenic survival assay, cells harboring TRIM44 siRNA showed significantly lower survival rates upon phleomycin treatment (Fig. 5c). Approximately 30% of cells survived phleomycin treatment at 1 μg / ml, and 10% survived phleomycin treatment at 2 μg / ml. Knockdown of PARP1 by siRNA did not affect survival, suggesting that PARP1 is not actively involved in the phleomycin-responsive repair pathway.

[0194] In contrast, TRIM44 siRNA or BRCA1 siRNA resulted in nearly identical reductions in cell survival following 0.5–2 μg / ml phleomycin treatment. Approximately 10% of cells survived after 1 μg / ml phleomycin treatment in cells containing TRIM44 or BRCA1 siRNA. Therefore, these data suggest that TRIM44 is as important as BRCA1 in DSB repair.

[0195] In summary, TRIM44 is required for DSB repair. In cells with functional PARP1, TRIM44 acts to suppress PARP1 hyperactivation, leading to proper DDR and repair.

[0196]

[0197] [Example 6] Binding of TRIM44 to the MRN complex in the presence of a PARP inhibitor

[0198] PARP inhibitors represent the first approved drugs utilizing the concept of synthetic lethality for the treatment of patients with BRCA1 germline mutations. DSB induction by PARP inhibitors is resolved through ATM-dependent HR repair.

[0199] We investigated whether TRIM44 is involved in the recruitment of the MRN complex in cells treated with PARP inhibitors. U2OS cells were pretreated with AG14361 for 1 hour before zeocin addition. In chromatin fractionation analysis, we observed reduced accumulation of MRE11 and NBS1 to damaged chromatin in TRIM44 knockdown cells, regardless of the presence of PARP inhibitors (Fig. 6a), suggesting a consistent role for TRIM44 in the recruitment of the MRN complex to damaged chromatin.

[0200] This was confirmed by microirradiation experiments accompanied by immunostaining. Control and TRIM44 KD U2OS cells were treated with AG14361 for 1 hour prior to microirradiation. Indeed, the accumulation of both MRE11 and NBS1 into DNA tethers was significantly reduced in TRIM44 KD cells at 10 and 20 minutes after laser microirradiation (Figures 6b–e).

[0201] Given that the TRIM44 ΔGR mutant, which loses binding to PARP1, binds more strongly to RAD50, MRE11, and ATM than three other PARP1 interaction deletion mutants (Fig. 2e), the interaction of TRIM44 with the MRN complex may change when PARP1 activity is disrupted. In pull-down assays, a decreased interaction between TRIM44 and PARP1 was observed after AG14361 treatment. Conversely, TRIM44 showed a stronger interaction with ATM as well as with MRE11 and NBS1 (Fig. 6f).

[0202] Thus, our data demonstrate that TRIM44 switches its primary binding partner to the MRN complex in the presence of PARPi. Under these conditions, the domains of TRIM44 critical for interaction with the MRN complex were investigated. In pull-down assays, we found that TRIM44 ΔUBP almost completely lost its binding ability to ATM and the MRN complex, whereas WT and three other TRIM44 deletion mutants retained their binding capacity (Fig. 6g).

[0203] Similar to the view in Figure 2e, the ΔGR mutant completely lost binding to PARP1 but retained binding to the MRN complex, suggesting that TRIM44 can directly interact with the MRN complex in the presence of PARP inhibitors. Taken together, these data indicate that TRIM44 serves as a functional mediator of MRN recruitment to DSB sites, with the ZnF UBP domain being crucial for binding to the MRN complex in the presence of PARP inhibitors.

[0204]

[0205] [Example 7] TRIM44 is a key upstream factor determining PARP inhibitor sensitivity.

[0206] PARP inhibitors represent the first approved drugs utilizing the concept of synthetic lethality for the treatment of patients with BRCA1 germline mutations. DSB induction by PARP inhibitors is resolved through ATM-dependent HR repair. Given that TRIM44 is the molecule responsible for recruiting the MRN complex to DSBs in PARP inhibitor-treated cells, this may be a key factor in determining sensitivity to PARP inhibitors.

[0207] In a clonogenic survival assay, approximately 30% of U2OS cells survived 2 μM olaparib treatment, indicating a mild effect. PARP1 knockdown by siRNA restored survival to 70–90% (Fig. 7a). In contrast, survival in cells treated with TRIM44 or BRCA1 siRNA was significantly reduced to 5% (Fig. 7a). Thus, the efficacy of TRIM44 knockdown was comparable to that of BRCA1 knockdown.

[0208] Dual knockdown of TRIM44 and BRCA1 did not show any additive or synergistic effects on cell death, suggesting that TRIM44 and BRCA1 share a common repair pathway. Thus, our data demonstrate that suppression of TRIM44 expression increases cell sensitivity to both phleomycin (Fig. 5c) and olaparib.

[0209] To explore the potential therapeutic effects of TRIM44 in cancer, we screened for TRIM44 expression in various cancers. In the TCGA RNA-seq dataset, TRIM44 mRNA expression levels were significantly lower among patients diagnosed with renal cell carcinoma (Fig. 7c). Kaplan-Meier analysis showed that patients with low TRIM44 expression levels had worse overall survival rates compared to those with high TRIM44 expression (Fig. 7d).

[0210] As observed in U2OS cells (Fig. 7a), knockdown of TRIM44 by siRNA in HEK293 cells, a renal cell carcinoma cell line, resulted in a significant decrease in survival even at lower doses of olaparib (0.25–1 μM) (Fig. 7e). Therefore, these data suggest that KIRC patients with low TRIM44 expression may be suitable for olaparib treatment.

[0211] Previous studies have shown that resistance to PARPi therapy is due to secondary mutations that restore BRCA1 function or to BRCA2-induced HR due to dysfunction of the 53BP1-RIF1-shieldin complex. Because TRIM44 plays a role in the most upstream step of DSB repair, we determined whether TRIM44 contributes to overcoming PARPi resistance caused by 53BP1 deficiency.

[0212] We found that siRNA-mediated 53BP1 knockdown increased cell viability at olaparib concentrations of 1–2 μM, and that dual knockdown of TRIM44 and 53BP1 significantly rescued PARP inhibitor sensitivity (Fig. 7b). Specifically, as shown in Fig. 7b, cell viability treated with TRIM44 siRNA was significantly reduced by 20% after olaparib treatment at 2 μM. Conversely, siRNA-mediated 53BP1 knockdown (KD) resulted in increased cell viability at olaparib concentrations of 1–2 μM. This suggests that TRIM44 and 53BP1 do not operate within the same pathway in response to PARP inhibitors.

[0213] Notably, simultaneous knockdown of TRIM44 and 53BP1 resulted in a reduction in the enhanced cell survival observed after 53BP1 knockdown. These data indicate that TRIM44 plays a role in overcoming PARP inhibitor resistance caused by 53BP1 deficiency. Taken together, these data suggest that TRIM44 is a key upstream determinant of PARP inhibitor sensitivity. Upon PARP1 activation, TRIM44 recruits the MRN complex to damaged chromatin, initiating ATM-mediated DDR and repair.

[0214]

[0215] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0216] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. Comprising a step of inhibiting the expression or activity of TRIM44, How to increase sensitivity to PARP inhibitors.

2. In paragraph 1, In the presence of a PARP inhibitor, the main binding substance of TRIM44 is converted from PARP1 to MRN (MRE11-RAD50-NBS1) complex. How to increase sensitivity to PARP inhibitors.

3. In paragraph 2, The above TRIM44 binds to the PARP1 through the glutamate-rich domain. How to increase sensitivity to PARP inhibitors.

4. In paragraph 2, The above TRIM44 binds to the MRN complex through the ZnF UBP (zinc finger ubiquitin-binding) domain. How to increase sensitivity to PARP inhibitors.

5. In paragraph 1, Overcoming PARP inhibitor resistance caused by 53BP1 deficiency through inhibition of the above TRIM44. How to increase sensitivity to PARP inhibitors.

6. In paragraph 1, The above PARP inhibitor comprises one selected from the group consisting of Olaparib, Rucaparib, Niraparib, Talazoparib, Veliparib and combinations thereof. How to increase sensitivity to PARP inhibitors.

7. Containing a substance that inhibits the expression or activity of TRIM44 as an active ingredient, A composition for increasing sensitivity to PARP inhibitors.

8. In paragraph 7, The above TRIM44 binds to PARP1 through the glutamate-rich domain and binds to the MRN complex through the ZnF UBP domain. A composition for increasing sensitivity to PARP inhibitors.

9. A composition comprising a composition for increasing sensitivity to a PARP inhibitor according to Article 7 as an active ingredient, A pharmaceutical composition for the prevention or treatment of tumors.

10. In paragraph 9, In the presence of PARP inhibitors, binding of TRIM44 switches from PARP1 to the MRN complex. A pharmaceutical composition for the prevention or treatment of tumors.

11. In paragraph 9, The above tumor includes a homologous recombination proficient (HR-proficient) tumor, A pharmaceutical composition for the prevention or treatment of tumors.

12. In paragraph 9, A pharmaceutical composition for preventing or treating a tumor, wherein the pharmaceutical composition for preventing or treating a tumor further comprises a pharmaceutically acceptable carrier.

13. In paragraph 12, A pharmaceutical composition for preventing or treating a tumor, wherein the carrier comprises a selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, menthol, mineral oil, and combinations thereof.

14. In paragraph 12, A pharmaceutical composition for preventing or treating a tumor, wherein the pharmaceutical composition further comprises an ingredient selected from the group consisting of preservatives, solubilizers, stabilizers, wetting agents, sweeteners, coloring agents, flavoring agents, salts, buffers, antioxidants, lubricants, emulsifiers, suspending agents, preservatives, and combinations thereof.

15. In paragraph 12, A pharmaceutical composition for preventing or treating a tumor, wherein the pharmaceutical composition for preventing or treating a tumor is administered by a method selected from the group consisting of intraperitoneal administration, oral administration, inhalation administration, intravenous administration, intramuscular administration, subcutaneous administration, dermal administration, intrauterine administration, tumor administration, rectal administration, and combinations thereof.

16. Step of treating the candidate material to cells; A step of measuring the expression level or activity of TRIM44 in the above cells; and A step of selecting a candidate substance with reduced expression or activity of TRIM44 as a substance with increased sensitivity to PARP inhibitors; including, A method for screening substances that increase sensitivity to PARP inhibitors.

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