Inhibition of UNG / UDG in BRCA-associated cancers

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of resistant HRD cancer, particularly resistant BRCA-associated cancer, chemotherapy-resistant HRD cancer, and chemotherapy-resistant BRCA-associated cancer.

Description

[Technical Field]

[0001] Field of the present invention The present invention relates to methods and pharmaceutical compositions for the treatment of resistant HRD cancer, particularly resistant BRCA-associated cancer, chemotherapy-resistant HRD cancer, and chemotherapy-resistant BRCA-associated cancer. [Background technology]

[0002] Background of the present invention Over the past several decades, numerous efforts have been made to gain mechanistic insights into the synthetic lethality between PARP1 and the BRCA1 / 2 genes (Maya-Mendoza et al., Nature 559, pages 279-284, 2018; Hanzlikova et al., Nature 559, pages 279-284, 2018). Defects in homologous recombination (HR)-mediated DNA repair are primarily caused by gene inactivation of the BRCA1 and BRCA2 (BRCA1 / 2) genes and play a role in the onset and progression of many tumor types. Defective HR (HRD) leads to genetic instability and over-reliance on alternative DNA repair mechanisms for survival, and provides a starting point for synthetic lethal-based targeted therapies, as exemplified by the extreme sensitivity of HRD tumors to poly(ADP-ribose) polymerase inhibitors (PARPi) (Bryant et al., Nature 434, pages 913-917, 2005; Farmer et al., Nature 434, pages 917-921, 2005). This lethal interaction has been successfully utilized in clinical practice since the approval of PARP inhibitors (PARPi) for the treatment of BRCA1 / 2 mutant tumors, also known as homologous recombination (HR)-deficient tumors (HRD) (Patel et al., Oncogene 40, pages 3001-3014, 2021). Various trials have been designed to evaluate the efficacy of PARPi in patients with breast, ovarian, and prostate tumors carrying BRCA1 / 2 mutations (Pujade-Lauraine et al., The Lancet. Oncology 18, Issue 9, pages 1274-1284, 2017). However, PARPi and other chemotherapy agents have shown limited efficacy in achieving remission in HRD cancer, particularly due to the development of drug resistance and, as a significant impairment of clinical efficacy, resistance to chemotherapy (Gogola et al., Annual Review of Cancer Biology 3, pages 235-254, 2019).Therefore, for these patients, there are no treatment options left, and the need for alternative treatment options is urgently needed (Konstantinopoulos et al., Cancer Discov 5, pages 1137-1154, 2015).

[0003] The inventors of this invention have identified NMNAT1 (nicotinamide mononucleotide adenylyltransferase 1) and NAD + We previously identified that both SIRT6-dependent deacetylases are synthetically lethal to BRCA1 / 2 through their function in base excision repair. The enzyme NMNAT1, key to nuclear NAD biogenesis, maintains genomic stability in BRCA1 / 2 mutant cells. By targeting the SIRT6 axis, we demonstrated that the cells are sensitive to PARPi and that this pathway is not superior to PARP1.

[0004] As a result, inhibition of NMNAT1 or SIRT6 not only killed HRD cells, but also PARPi-resistant and platinum-resistant cells, regardless of resistance mechanisms, and demonstrated a remarkable effect in killing resistant HRD cells, including those with acquired drug resistance. To find alternative therapeutic options for HRD cancers, including BRCA1 / 2 mutant tumors, and cancers that have acquired PARPi resistance, the inventors further investigated the action of the NMNAT1 / SIRT6 pathway.

[0005] Uracil DNA glycosylase (UNG) is a protein member of the uracil DNA glycosylase (UDG) family. Four UDGs (i.e., UNG, SMUG1, TDG, and MBD4) are characterized in human cells by their different cellular localization and catalytic efficiency to ensure effective uracil recognition and removal. One of the important functions of uracil DNA glycosylase is to prevent mutagenesis by removing uracil from DNA molecules by cleaving N-glycosidic bonds and initiating the base excision repair (BER) pathway. UNG is a key player in locating DNA damage and repairing uracil accumulation in telomeres (Baquero et al, Mol Oncol 13, Issue 5, pages 1110-1120, 2019). UNG initiates BER activity, and its loss has been reported to restore sensitivity to several DNA damaging agents, such as fork-inducing agents, which stalled in BRCA2-deficient cells (Pathania, Grantome NIH, project 1R15CA235436-01A1). In addition, UNG knockdown can induce apoptosis, reduced cell proliferation, and increased cellular sensitivity to genotoxic stress in prostate cancer cell lines. Furthermore, it has been observed that UNG-deficient colon cancer cells become highly sensitive to pemetrexed-induced uracil accumulation, which can lead to cell cycle arrest, DNA double-strand break formation, and apoptosis (Pulukuri et al., Mol Cancer Res 7, pages 1285-1293, 2009; Weeks et al., Mol Cancer Ther 12, pages 2248-2260, 2013).

[0006] The inventors demonstrated that UNG plays a crucial role in preserving genomic stability for the survival of BRCA1 / 2-deficient cells, and that UNG is also synthetically lethal with BRCA1 / 2 through its function in base excision repair. Loss of UNG reduces the survival of HRD cells and PARPi-resistant cells and confers sensitivity to cells with high HR activity against PARPi. UNG is a target of the NMNAT1 / SIRT6 pathway and functions downstream of SIRT6 in the survival of BRCA1 / 2-mutated tumors. Since inhibition of NMNAT1 or SIRT6 kills HRD cells, as well as PARPi and platinum-resistant cells, regardless of the resistance mechanism, inhibition of UNG should also target PARPi and platinum-resistant HRD cells. [Overview of the project]

[0007] The present invention relates to methods and pharmaceutical compositions for the treatment of resistant HRD cancer, particularly resistant BRCA-associated cancer, chemotherapy-resistant HRD cancer, and chemotherapy-resistant BRCA-associated cancer. In particular, the present invention is defined by the claims. [Modes for carrying out the invention]

[0008] Detailed description of the invention The inventors investigated the role of UNG in HRD cancer, particularly chemotherapy-resistant BRCA-associated cancer, and in the NMNAT1 / SIRT6 pathway.

[0009] Previously, the inventors demonstrated that SIRT6 inhibition kills BRCA1 and BRCA2 mutant tumor cells but does not affect the survival of non-BRCA mutant cells. The inventors also demonstrated SIRT6 deletion in PARPi-sensitive cells. In particular, SIRT6 inhibition kills PARP inhibitor and cisplatin-resistant BRCA1 and BRCA2 mutant tumors, including those with somatic reversion of BRCA1 / 2 mutations. Thus, targeting SIRT6 kills chemotherapy-resistant HRD cells, especially PARPi-resistant HRD cells.

[0010] The inventors hereby demonstrate that uracil DNA glycosylase (UNG) is also synthetically lethal to BRCA1 / 2 through its function in base excision repair, and that UNG is a target of the NMNAT1 / SIRT6 pathway. UNG functions downstream of SIRT6 in the survival of BRCA1 / 2 mutant tumors.

[0011] The inventors further demonstrated that targeting UNG-mediated uracil removal leads to chromatin capture of SMUG1 and HMCES (5-hydroxymethylcytosine linkage, specific to ES cells) crosslinking to SMUG1-generating debase (AP) sites (which prevent their processing by APE1). This hinders the progression of the replication fork (RF) and creates DNA damage that triggers HR-mediated repair. Blocking or overcoming UNG removal capacity leads to a dramatic causal relationship in HRD cells, where SMUG1 capture—when propagated by mitosis—results in under-replicated DNA, leading to chromosomal fragmentation, rearrangement, and ultimately cell death. These results highlight UNG-mediated uracil removal as a novel vulnerability in HRD carcinomas with some potential to address PARPi resistance.

[0012] In short, the present invention focuses on the role of UNG inhibitors in HRD cancer, and the use of UNG inhibitors in the treatment of resistant HRD cancer, particularly resistant BRCA-associated cancer, chemotherapy-resistant HRD cancer, and chemotherapy-resistant BRCA-associated cancer, including BRCA-associated cancer with acquired drug resistance to monotherapy or combination therapy using PARPi.

[0013] Accordingly, the present invention relates to the targeting of UNG in the treatment of drug-resistant HRD cancer, particularly drug-resistant BRCA-associated cancer, chemotherapy-resistant HRD cancer, and chemotherapy-resistant BRCA-associated cancer.

[0014] UNG inhibitor Accordingly, in a first embodiment, the present invention relates to a uracil DNA glycosylase (UNG) inhibitor for use in the treatment of drug-resistant homologous recombination repair deficiency (HRD) cancer, wherein the UNG inhibitor is a molecule capable of silencing a gene expressing the UNG enzyme, or a molecule that inhibits the properties of the UNG enzyme.

[0015] In some embodiments, the present invention relates to a UNG inhibitor in which the resistant HRD cancer is a resistant BRCA-associated cancer, a chemotherapy-resistant HRD cancer, a chemotherapy-resistant BRCA-associated cancer, or a metastatic resistant HRD cancer.

[0016] In some embodiments, the present invention relates to a UNG inhibitor in which the resistant HRD cancer is a PARPi-resistant BRCA-associated cancer or a cisplatin-resistant BRCA-associated cancer, including those with somatic reversion of BRCA mutations and HR repair.

[0017] As used herein, the terms “subject,” “individual,” and “patient” are interchangeable and refer to mammals. Typically, the subject according to the present invention refers to any subject, preferably a human. In certain embodiments, the term “subject” refers to a subject who has or is at high risk of developing cancer. In certain embodiments, the term “subject” refers to a subject who has or is at high risk of developing HRD cancer, particularly BRCA-associated cancer. In certain embodiments, the term “subject” refers to a subject who has or is at high risk of developing resistant HRD cancer. In some embodiments, the term “subject” refers to a subject who has or is at high risk of developing resistant BRCA-associated cancer. In some embodiments, the term “subject” refers to a subject who has or is at high risk of developing chemotherapy-resistant HRD cancer. In some embodiments, the term “subject” refers to a subject who has or is at high risk of developing chemotherapy-resistant BRCA-associated cancer. In some embodiments, the term “subject” refers to a subject who has or is at high risk of developing metastatic resistant HRD cancer.

[0018] In some embodiments, the term “subject” refers to subjects who have or are at high risk of developing chemotherapy-resistant HRD cancer and / or BRCA-deficient cancer (basal cell-like, luminal, and HER2-overexpressing breast cancer, and other cancers) and chemotherapy-resistant BRCA-related cancers such as breast, ovarian, prostate, pancreatic, or other types of tumors carrying BRCA1 / 2 mutations or BRCA expression defects. In certain embodiments, the term “subject” refers to subjects who have or are at high risk of developing PARPi-resistant BRCA-related cancer or cisplatin-resistant BRCA-related cancer.

[0019] As used herein, the terms “treatment” or “to treat” include both prophylactic or preventive treatments, as well as curative or disease-modifying treatments, which encompass treatments for subjects at risk of developing a disease, suspected of having a disease, or diagnosed with a disease or medical disorder, and include treatments for subjects with a medical disorder or who are likely to develop a medical disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or improve one or more symptoms of a disorder or recurrent disorder, or to extend the subject’s survival time beyond what would be expected without such treatment. “Treatment regimen” means a pattern of treatment for a disease, for example, a pattern of medication used during treatment. A treatment regimen may include an induction regimen and a maintenance regimen. The phrase “induction regimen” or “induction period” means a treatment regimen (or part of a treatment regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide the subject with a high level of medication at the beginning of a treatment regimen. An introductory regimen may employ a “loading regimen” (in part or in whole), which may include administering a larger dose of the drug than the physician would use during the maintenance regimen, administering the drug more frequently than the physician would use during the maintenance regimen, or both. The phrase “maintenance regimen” or “maintenance period” refers to a treatment regimen (or part of a treatment regimen) used to maintain a subject during treatment for a disease, for example, to keep the subject in remission for an extended period (several months or several years). A maintenance regimen may employ continuous treatment (e.g., administering the drug regularly, e.g., weekly, monthly, yearly, etc.) or intermittent treatment (e.g., interrupted treatment, intermittent treatment, treatment on relapse, or treatment upon achievement of specific predetermined criteria [e.g., signs of the disease]).

[0020] As used herein, the term "cancer" refers to the following tissues or organs: breast; liver; kidney; heart; mediastinum, pleura; floor of mouth; lips; salivary glands; tongue; gums; oral cavity; palate; tonsils; larynx; trachea; bronchi, lungs; pharynx, hypopharynx, oropharynx, nasopharynx; esophagus; digestive organs such as stomach, intrahepatic bile ducts, biliary tract, pancreas, small intestine, colon, rectum; urinary organs such as bladder, gallbladder, ureters; rectosigmoid junction; anus, anal canal; skin; bone; joints; limbs. This refers to any cancer that may develop in any one of the following: articular cartilage; eyes and adnexa; brain; peripheral nerves, autonomic nervous system; spinal cord, cranial nerves, meninges; and various parts of the central nervous system; connective, subcutaneous and other soft tissues; retroperitoneum, peritoneum; adrenal glands; thyroid gland; endocrine glands and related structures; female reproductive organs such as ovaries, uterus, cervix, uterine body, vagina, and vulva; male reproductive organs such as penis, testes, and prostate; hematopoietic and reticuloendothelial systems; blood; lymph nodes; or thymus.

[0021] The term "cancer" according to the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, non-Hodgkin lymphoma, neuroblastoma, glioma, adenocarcinoma, mesothelioma (including pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, and end-stage mesothelioma), rectal cancer, endometrial cancer, thyroid cancer (including papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, undifferentiated thyroid carcinoma, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid carcinoma, pheochromocytoma, and paraganglioma), skin cancer (malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, keratosporinus, lentigo, dysplastic nevus, lipoma, hemangioma, and skin cancer). (including dermatofibroma), nervous system cancer, brain cancer (including astrocytoma, medulloblastoma, glioma, low-grade glioma, ependymoma, germ cell tumor (pineal glandoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumor, spinal nerve fibroma, glioma or sarcoma), skull cancer (including osteoma, hemangioma, granuloma, xanthomas or degenerative osteitis), meningeal cancer (including meningioma, meningiosarcoma or gliomatosis), head and neck cancer (including squamous cell carcinoma of the head and neck and oral cancer (e.g., buccal oral cancer, lip cancer, tongue cancer, oral cancer or pharyngeal cancer, etc.)), lymph node cancer, gastrointestinal cancer, liver cancer (hepatocellular carcinoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, (including angiosarcoma, hepatocellular adenoma and hemangioma), colon cancer, gastric cancer, esophageal cancer (including squamous cell carcinoma, laryngeal, adenocarcinoma, leiomyosarcoma or lymphoma), colorectal cancer, intestinal cancer, small intestine cancer (e.g., adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma or fibroma), large intestine cancer (e.g., adenocarcinoma, tubular adenoma, chorioadenoma, hamartoma or leiomyoma), pancreatic cancer (including pancreatic ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor or bipoma), ENT cancer, breast cancer (HER2-enriched breast cancer, luminal A breast cancer, luminal Breast cancer (including B breast cancer and triple-negative breast cancer), uterine cancer (including endometrial cancer such as endometrial cancer, endometrial stromal sarcoma and malignant mixed Müllerian duct tumor, uterine sarcoma, leiomyosarcoma and gestational trophoblastic disease), ovarian cancer (including undifferentiated germ cell tumor, granulosa-follicular cell tumor and Sertoli-Leydig cell tumor), cervical cancer, vaginal cancer (including vaginal squamous cell carcinoma, vaginal adenocarcinoma, clear cell vaginal adenocarcinoma, vaginal germ cell tumor, vaginal staphyloid sarcoma and vaginal melanoma), vulvar cancer (including vulvar squamous cell carcinoma, vulvar verrucosa, vulvar melanoma, basal cell vulvar carcinoma, Bartholin's gland carcinoma, vulvar adenocarcinoma and Keiler's erythroplasia), urogenital cancer,Kidney cancer (including clear cell carcinoma, chromophobic renal cell carcinoma, papillary renal cell carcinoma, adenocarcinoma, Wilms' tumor, nephroblastoma, lymphoma, or leukemia), adrenal cancer, bladder cancer, urethral cancer (e.g., squamous cell carcinoma, transitional cell carcinoma, or adenocarcinoma), prostate cancer (e.g., adenocarcinoma or sarcoma), and testicular cancer (e.g., seminoma, teratoma, embryonic carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenoid tumor, or lipoma), lung cancer (including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCL), including squamous cell carcinoma of the lung). C) Lung adenocarcinoma (LUAD), and including large cell lung cancer, bronchogenic carcinoma, alveolar carcinoma, bronchiolar carcinoma, bronchial adenoma, pulmonary sarcoma, chondrotoxic hamartoma and pleural mesothelioma), sarcomas (including Askin tumor, staphyloid sarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant Schwannoma, osteosarcoma and soft tissue sarcoma), soft tissue sarcomas (alveolar soft tissue sarcoma, angiosarcoma, phyllodes cystic sarcoma, dermatofibrosarcoma protuberans, tendonoid, fibroplastic round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal Stromal tumors (GIST), hemangioemyloidoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, prickly fibrous histiocytic tumor, rhabdomyosarcoma, synovial sarcoma and undifferentiated pleomorphic sarcoma, cardia cancer (including sarcomas such as angiosarcoma, fibrosarcoma, rhabdomyosarcoma or liposarcoma, myxoma, rhabdomyomas, fibromas, lipomas and teratomas), bone cancer (osteogenic sarcoma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma) This includes sarcomas, malignant lymphomas and reticulum sarcomas, multiple myeloma, malignant giant cell tumors of bone, chordomas, osteochondromas, osteochondral exostosis, benign chondrommas, chondroblastomas, chondromyxofibromas, osteoid osteomas and giant cell tumors), hematological and lymphatic cancers, hematological malignancies (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma and myelodysplastic syndromes), Hodgkin's disease, non-Hodgkin's lymphoma, and hair cell and lymphatic disorders, as well as their metastases.

[0022] The terms “homologous recombination repair deficiency cancer” or “HRD cancer” have their general meaning in the art and refer to cancers exhibiting defective homologous recombination (HRD)-mediated DNA repair, which leads to genetic instability and over-reliance on alternative DNA repair mechanisms for survival. Thus, the term “HRD cancer” refers to cancers exhibiting defective homologous recombination resulting from genetic modifications, including PALB2, CHEK2, ATM, BARD1, RAD51C, RAD51D, FANCC, BRIP1, FANCM, XRCC2, or other genes involved in the HR DNA repair pathway. The HR status of cancer can be measured by any well-known method in the art, e.g., genomic probing for HRD-related genomic identification characteristics, or by using commercially available HRD diagnostic tests such as Myriad myChoice®. The term “HRD cancer” includes, but is not limited to, BRCA-related cancers. Conversely, BRCA-related cancers are not necessarily HRD cancers.

[0023] The term “BRCA-associated cancer” has its general meaning in the art and refers to cancer associated with BRCA mutations or BRCA expression defects. The term “BRCA-associated cancer” refers to cancers selected from cancers associated with BRCA1 and / or BRCA2 mutations, cancers associated with BRCA1 and / or BRCA2 expression defects, and homologous recombination repair deficiency (HRD) cancers with BRCA deficiency (e.g., basal cell-like, luminal, HER2-overexpressing carcinomas, breast, ovarian, and prostate tumors carrying BRCA1 / 2 mutations, and other cancers). As used herein, “BRCA1 / 2” means BRCA1 and / or BRCA2, more specifically BRCA1 and BRCA2. In some embodiments, the term “BRCA-associated cancer” refers to breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, head and neck cancer, and melanoma associated with BRCA1 and / or BRCA2 mutations or BRCA1 and / or BRCA2 expression defects. In some embodiments, the term “BRCA-associated cancer” refers to metastatic BRCA-associated cancer.

[0024] The term “resistant HRD cancer” has its general meaning in the art and refers to HRD cancer that is resistant to treatment, such as HRD cancer resistant to chemotherapy, radiotherapy, and other cancer therapies. The term “resistant HRD cancer” also refers to resistant BRCA-associated cancers, chemotherapy-resistant HRD cancers, chemotherapy-resistant BRCA-associated cancers, such as PARP inhibitor (PARPi)-resistant BRCA-associated cancers, PARPi-resistant HRD tumors including tumors with somatic reversion of BRCA1 / 2 mutations and subsequent HR repair, cisplatin-resistant BRCA-associated cancers, and cisplatin-resistant BRCA1 and BRCA2 mutant tumors including tumors with somatic reversion of BRCA1 / 2 mutations and subsequent HR repair. In some embodiments, the term “resistant HRD cancer” refers to metastatic resistant HRD cancer.

[0025] The term "PARP inhibitor" or "PARPi" has its general meaning in the art and refers to PARP inhibitors such as olaparib, lucaparib, niraparib, and talazoparib. The term "PARP inhibitor" also refers to PARP inhibitors such as iniparib, veliparib, pamiparib (BGB-290), CEP9722, E7016, and 3-aminobenzamide.

[0026] The term "SIRT6" refers to the NAD-dependent protein deacetylase Sirtuin-6. Previously, the inventors demonstrated that SIRT6 inhibition kills BRCA1 and BRCA2 mutant tumor cells but does not affect the survival of non-BRCA mutant cells. The inventors also demonstrated SIRT6 deletion in PARPi-sensitive cells. The inventors also demonstrated that SIRT6 inhibition kills PARP inhibitor and cisplatin-resistant BRCA1 and BRCA2 mutant tumors, including those with somatic reversion of BRCA1 / 2 mutations, and showed that targeting SIRT6 kills chemotherapy-resistant HRD cells, particularly PARPi-resistant HRD cells.

[0027] The term "NMNAT1" has its general meaning in the relevant technical field and refers to nicotinamide nucleotide adenylyltransferase 1 (protein registry number Q9HAN9), also known as nicotinamide / nicotinic acid mononucleotide adenylyltransferase 1.

[0028] The terms "UNG" or "UDG" refer to the uracil DNA glycosylase enzyme and its coding gene. This gene codes for one of several uracil DNA glycosylases. One of the key functions of uracil DNA glycosylase is to remove uracil from DNA molecules by cleaving N-glycosidic bonds and preventing mutagenesis by initiating the base excision repair (BER) pathway. The use of alternative promoters and splicing of this gene leads to two distinct isoforms: mitochondrial UNG1 and nuclear UNG2. As used herein, "UNG" means either one or both of the two UNG types (i.e., UNG1 and / or UNG2).

[0029] In some embodiments, the UNG inhibitor is an organic small molecule, polypeptide, aptamer, oligonucleotide, or antibody.

[0030] As used herein, the term “UNG inhibitor” refers to any compound selected from the group consisting of compounds that target uracil DNA glycosylase, but is not limited thereto. The term “UNG inhibitor” refers to a compound that binds to UNG and functions as a potent antagonist of UNG. The term “UNG inhibitor” has its general meaning in the art and refers to a compound that selectively inactivates UNG. Typically, UNG inhibitors are small organic molecules, polypeptides, aptamers, oligonucleotides (antisense oligonucleotides, siRNA, shRNA, DNA and RNA aptamers), or antibodies. Several UNG inhibitors are known in the art, such as those described in WO2021 / 087246A1, WO2006 / 135763, WO2006 / 135763, US6,177,437, and WO1998 / 039334.

[0031] The term "UNG inhibitor" refers to any compound selected from the following non-proteinogenic uracil DNA glycosylase inhibitors (npUGIs), although this is not the only one that fits the description:

[0032] In some embodiments, a small molecule inhibitor of UDG is expressed by the following formula (I): [ka] {In the formula, R1 is H, furanose carbohydrate, pyranose carbohydrate, carbohydrate mimetic, C1-16 alkyl, C1-16 alkenyl, C1-16 alkynyl, C1-16 alkoxy, or C6-20 aryl. Here, the furanose carbohydrate or its derivative, pyranose carbohydrate or its derivative, carbohydrate mimetic, C1-16 alkyl, C1-16 alkenyl, C1-16 alkynyl, C1-16 alkoxy, or C6-20 aryl is independently and optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, cyano, NO2, N(R4)(R5), C1-16 alkoxy, or C6-20 aryl. Here, the C6-20 aryl is further optionally substituted with one or more substituents selected from the group consisting of C1-16 alkyl, C1-16 alkenyl, C1-16 alkynyl, C1-16 alkoxy, hydroxyl, halo, cyano, NO2, or N(R4)(R5); ·L is O, S, or *-N(R3)** (wherein R3 is H or C1-16 alkyl, ** indicates an attachment point to the R2 portion, and * indicates an attachment point to the rest of the molecule); R2 is H, N(R4)(R5), or C6-20 aryl, where the C6-20 aryl is independently substituted with one or more substituents selected from the group consisting of C1-16 alkyl, C1-16 alkenyl, C1-16 alkynyl, or C1-16 alkoxy; R4 and R5 are each independently H or C6-20 aryl; and R6 is either H or a halo. It is a compound of or a pharmaceutically acceptable salt thereof.

[0033] In some embodiments, the small molecule inhibitor of UDG is expressed by the following formula (II): [ka] {In the formula, L is a linker, for example, a linker containing an imine or oxime portion; • SB is a binding element, for example, a binding element containing a phenyl group. It is a compound of [the compound].

[0034] In some embodiments, the small molecule inhibitor of UDG is expressed by the following formula (IIa): [ka] {In the formula, A is independently -O-, -CH2-, or a single bond; Ar is an aromatic or heteroaromatic group. It is a compound of [the compound].

[0035] In some embodiments, the small molecule inhibitors of UDG are the following compounds: [ka] It is a compound derived from [source].

[0036] In some embodiments, the small molecule inhibitor of UDG is given by the following formula (III): [ka] {In the formula, X is O, NR1, S, or CH2; R1 is hydrogen or (C1-C2) alkyl; R2 is a hydrogen; a (C2-C6) alkyl optionally substituted with one or more hydroxyl, amino, or carboxyl groups; or a phenyl optionally substituted with a (C1-C4) alkyl, carboxyl, or amino group; a (C1-C4) alkoxy(C2-C6) alkyl; an N-(C1-C4) alkyl-carboxyamide(C1-C2) alkyl; a benzyl whose phenyl portion is optionally substituted with a (C1-C4) alkyl, carboxyl, or amino group; a pyrrolyl(C1-C2) alkyl whose pyrrole is optionally substituted with a (C1-C4) alkyl; or an imidazolyl(C1-C2) alkyl whose imidazole is optionally substituted with a (C1-C4) alkyl group; R3 and R5 are either the same or different, and are hydrogen; carboxyamide; N-(C1-C2) alkylcarboxamide; carboxamidino; or N-(C1-C2) alkylcarboxamidino, respectively; and R4 is a (C6-C10) alkyl group optionally substituted with one or more hydroxyl, amino, or carboxyl groups; the alkyl portion is a (C6-C10) alkyl group forming part of a (C4-C8) cycloalkyl ring; a (C6-C10) alkenyl group; or a (C1-C14) alkoxy group. A compound of; or a pharmaceutically acceptable salt thereof, provided that R2 is not hydrogen when X is NH, R3 and R5 are hydrogen, and R4 is n-propyl, n-butyl, n-pentyl, i-pentyl, n-hexyl, or n-octyl.

[0037] In some embodiments, the small molecule inhibitor of UDG is expressed by the following formula (IV): [ka] {In the formula, B is a nucleoside purine or pyrimidine base, or a heterocyclic analog thereof; X is independently O, N, S, or CH2; Y represents N(R1)2, C(R)2, O, S, P, Se, B, Al, or As; R is independently hydrogen or a lower alkyl group; R1 is either independently absent, or is a hydrogen atom or an amino protecting group; R2 may be hydrogen, nucleotides or oligonucleotides (e.g., 3' linked), phosphoryls (e.g., phosphates, e.g., mono, di, or triesters), phosphonates, phosphoramidates, carbamates, phosphorothioates, phosphorodithioates, hydroxyl blocking groups, or, if valence and stability are acceptable, halogens, lower alkyls, lower alkenyls, lower alkynyls, carbonyls (e.g., esters, carboxylates, or formates (formatc)), thiocarbonyls (e.g., thiol esters, thiol carboxylates, or thiol formates), ketyls, aldehydes, etc. Mino, acylarnino, amide, amidino, cyano, nitro, azide, sulfonyl, sulfoxide, sulfate, sulfonate, sulfamoyl, sulfonamide, phosphoryl, phosphonate, phosphinate, -(CH2)m-R8, -(CH2)m-OH, -(CH2)mO-lower alkyl, -(CH2)mO-lower alkenyl, -(CH2)mO-(CH2)n-R8, -(CH2)m-SH, -(CH2)mS-lower alkyl, -(CH2)mS-lower alkenyl, -(CH2)mS-(CH2)n-R8, or solid or polymeric support; R3 may be hydrogen, nucleotide or oligonucleotide (e.g., 5' linked), phosphoryl, phosphonate, phosphoramidate, carbamate, phosphorothioate, phosphorodithioate, hydroxyl blocking group, or, if valence and stability are acceptable, halogen, lower alkyl, lower alkenyl, lower alkynyl, carbonyl fzc / gq an ester (carbonyl fzc / gq an ester), carboxylate, or formate), thiocarbonyl (e.g., thiol ester, thiol carboxylate, or thiol formate), ketyl, aldehyde, amino, acylamino, amide, amidino, cyano, nitro, azide, sulfonyl, sulfoxide, sulfate, sulfonate, sulfamoyl, sulfonamide, phosphoryl, phosphonate, phosphinate, -(CH2)m-R8, -(CH2)m-OH, -(CH2)mO-lower alkyl, -(CH2)mO-lower alkenyl, -(CH2)mO-(CH2)n-R8, -(CH2)m-SH, -(CH2)mS-lower alkyl, -(CH2)mS-lower alkenyl, -(CH2)mS-(CH2)n-R8, or solid or polymeric support; and R4, R5, R6, and R7 are each generated independently and, if valence and stability are acceptable, hydrogen, halogens, lower alkyls, lower alkenyls, lower alkynyls, carbonyls (e.g., esters, carboxylates, or formates), thiocarbonyls (e.g., thiol esters, thiol carboxylates, or thiol formates), ketyls, aldehydes, aminos, acylaminos, amides, amidinos, cyanos, nitros, azides, sulfonyls, and sulfonyls. These are sulfates: sulfate, sulfate, sulfonate, sulfamoyl, sulfonamide, phosphoryl, phosphonate, phosphinate, -(CH2)m-R8, -(CH2)m-OH, -(CH2)mO-lower alkyl, -(CH2)mO-lower alkenyl, -(CH2)mO-(CH2)n-R8, -(CH2)m-SH, -(CH2)mS-lower alkyl, -(CH2)mS-lower alkenyl, and -(CH2)mS-(CH2)n-R8; R8 is a compound where each generation is independent and consists of a substituted or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl or heterocyclic compound, carbonyl, sulfonyl or phosphoryl; n and rn are independent occurrences and are either zero or integers between 1 and 6; p is zero, 1 or 2; and • q and s(5) are independently zero or integers between 1 and 4, provided that the sum of q and s is zero or an integer between 1 and 4, and Y is 0, then p is 1 or 2. It is a compound of [the compound].

[0038] An "aptamer" refers to a class of molecules that represent an antibody substitute in terms of molecular recognition. Aptamers are oligonucleotide sequences that have the ability to recognize substantially any class of target molecules with high affinity and specificity. Such ligands can be isolated through in vitro evolution of a random sequence library (SELEX) as described in Turek C. and Gold L., 1990. A random sequence library is obtained by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer that has been ultimately chemically modified from a unique sequence. The possible modifications, uses, and advantages of this class of molecules are outlined in Jayasena SD, 1999. Peptide aptamers consist of a conformationally restricted antibody-variable region displayed by a platform protein (e.g., E. coli thioredoxin A) selected from the combinatorial library by a two-hybrid method (Colas et al., 1996). Once an aptamer directed to the target of the present invention as described above has been constructed, those skilled in the art can easily select or inactivate the target.

[0039] "Antibody" means an antibody directed against a target (the term also includes "antibody portion"). The aforementioned antibody is a monoclonal antibody. In one embodiment of the antibody or portion thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibody or portion thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibody or portion thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibody or portion thereof described herein, the antibody portion includes the light chain of the antibody. In one embodiment of the antibody or portion thereof described herein, the antibody portion includes the heavy chain of the antibody. In one embodiment of the antibody or portion thereof described herein, the antibody portion includes the Fab portion of the antibody. In one embodiment of the antibody or portion thereof described herein, the antibody portion includes the F(ab')2 portion of the antibody. In one embodiment of the antibody or portion thereof described herein, the antibody portion includes the Fc portion of the antibody. In one embodiment of the antibody or portion thereof described herein, the antibody portion includes the Fv portion of the antibody. In one embodiment of the antibody or portion thereof described herein, the antibody portion includes the variable domain of the antibody. In one embodiment of an antibody or portion thereof described herein, the antibody portion comprises one or more CDR domains of the antibody.

[0040] As used herein, "antibody" includes both natural and non-natural antibodies. Specifically, "antibody" includes polyclonal antibodies and monoclonal antibodies, as well as their monovalent and bivalent fragments. Furthermore, "antibody" includes chimeric antibodies, fully synthetic antibodies, single-chain antibodies, and their fragments. Antibodies may be human antibodies or non-human antibodies. Non-human antibodies may be humanized by recombinant methods to reduce their immunogenicity in humans.

[0041] Antibodies are prepared according to conventional methodologies. Monoclonal antibodies may be produced using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the present invention, mice or other suitable host animals are immunized with antigenic targets at appropriate intervals (e.g., twice weekly, once weekly, twice monthly, or once monthly). The animals may be given a final "boost" of the antigen within one week of sacrifice. It is often desirable to use immunological adjuvants during immunization. Suitable immunological adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter Titermax, saponin adjuvants (e.g., QS21 or Quil A), or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well known in this art. Animals may be immunized subcutaneously, intraperitoneally, intramuscularly, intravenously, intranasally, or via other routes. A given animal may be immunized using multiple forms of antigens via multiple routes.

[0042] In short, the antigen may be provided as a synthetic peptide corresponding to the antigenic region of interest within the target. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph nodes, or other organs of an animal and fused with a suitable myeloma cell line using a drug (e.g., polyethylene glycol) to form a hydridoma. Following the fusion, the cells are placed in a medium permissible for the growth of the hybridoma (but not the fusion partner) using standard methods as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following the culture of the hybridoma, the cell supernatant is analyzed for the presence of an antibody of desired specificity (i.e., selectively binding to the antigen). Appropriate analytical techniques include ELISA, flow cytometry, immunoprecipitation, and Western blotting. Other screening techniques are well known in this field. Preferred techniques are those that confirm the binding of antibodies to structurally intact, naturally folded antigens (e.g., non-denaturing ELISA, flow cytometry, and immunoprecipitation).

[0043] Importantly, as is well known in this art, only the paratope, a small part of the antibody molecule, is involved in the binding of the antibody to its epitope (see, in general, Clark, WR (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions are, for example, effectors of the complement cascade, but are not involved in antigen binding. Antibodies produced with the pFc' region enzymatically cleaved or without the pFc' region are named F(ab')2 fragments and retain both antigen-binding sites of the intact antibody. Similarly, antibodies produced with the Fc region enzymatically cleaved or without the Fc region are named Fab fragments and retain one of the antigen-binding sites of the intact antibody molecule. Further analysis reveals that a Fab fragment consists of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted as Fd. The Fd fragment is a major determinant of antibody specificity (a single Fd fragment can be associated with up to 10 different light chains without altering antibody specificity), and the Fd fragment retains its epitope-binding ability in isolation.

[0044] Within the antigen-binding region of an antibody, as is well known in the art, there is a complementation-determining region (CDR) (which directly interacts with the antigen's epitope) and a framework region (FR) (which maintains the paratope's tertiary structure) (see Clark, 1986; Roitt, 1991 in general). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulin, there are four framework regions (FR1 to FR4) separated by three complementation-determining regions (CDR1 to CDRS). The CDR, and especially the CDRS region, and especially the heavy chain CDRS, are significantly involved in antibody specificity.

[0045] It is now well established in the art that the non-CDR region of a mammalian antibody may be replaced with a similar region of an allospecific or heterospecific antibody while maintaining the epitope specificity of the original antibody. This is most clearly demonstrated in the development and use of "humanized" antibodies, in which the non-human CDR is covalently linked to the human FR region and / or Fc / pFc' region to produce a functional antibody.

[0046] The present invention provides compositions and methods comprising humanized antibodies in certain embodiments. As used herein, “humanized” describes an antibody in which some, most, or all of the amino acids outside the CDR region are substituted with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Patents No. 4,816,567, No. 5,225,539, No. 5,585,089, No. 5,693,761, No. 5,693,762, and No. 5,859,205 (which are incorporated herein by reference). U.S. Patents No. 5,585,089 and No. 5,693,761, as well as International Publication No. 90 / 07861, also propose four possible criteria that may be used in the design of humanized antibodies. The first proposal was to use, for the acceptor, a framework from a specific human immunoglobulin that is abnormally homologous to the donor immunoglobulin being humanized, or a consensus framework from many human antibodies. The second proposal was that if the amino acids in the human immunoglobulin framework are abnormal and the donor amino acid at that position is typical for the human sequence, then the donor amino acid may be selected rather than the acceptor. The third proposal was that the donor amino acid may be selected rather than the acceptor amino acid at a position directly adjacent to three CDRs in the humanized immunoglobulin chain. The fourth proposal was to use the donor amino acid at a framework position where the amino acid is predicted to have a side chain atom within 3A of the CDR in the three-dimensional model of the antibody and is predicted to be able to interact with the CDR. The above methods are merely examples of some of the methods that persons skilled in the art can use to produce humanized antibodies. Persons skilled in the art will be familiar with other methods for humanizing antibodies.

[0047] In one embodiment of a humanized antibody, some, most, or all of the amino acids outside the CDR region are substituted with amino acids from a human immunoglobulin molecule, however, some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not impede the antibody's ability to bind to a given antigen. Suitable human immunoglobulin molecules may include IgG1, IgG2, IgG3, IgG4, IgA, and IgM molecules. "Humanized" antibodies retain similar antigen specificity to the original antibody. However, using specific methods of humanization, the affinity and / or specificity of antibody binding may be increased using a method of "directional evolution," as described by Wu et al., / . Mol. Biol. 294:151, 1999 (the contents of which are incorporated herein by reference).

[0048] Fully human monoclonal antibodies can also be prepared by immunizing mice that are transgenic for most of the heavy and light chain loci of human immunoglobulins. See, for example, U.S. Patents Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, and 6,150,584, and the references cited herein (these are incorporated herein by reference). These animals are genetically modified to have a functional deletion in the production of endogenous (e.g., mouse) antibodies. The animals are further modified to include all or part of the human germline immunoglobulin loci, so that immunization of these animals results in the production of fully human antibodies against the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex / GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma techniques. These monoclonal antibodies possess human immunoglobulin amino acid sequences and, therefore, do not induce a human anti-mouse antibody (KAMA) response when administered to humans.

[0049] In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Patent Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Patent Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

[0050] Thus, as will be apparent to those skilled in the art, the present invention also provides F(ab')2 Fab fragments, Fv fragments, and Fd fragments; chimeric antibodies in which the Fc region and / or FR region and / or CDR1 region and / or CDR2 region and / or light chain CDR3 region are substituted with homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR region and / or CDR1 region and / or CDR2 region and / or light chain CDR3 region are substituted with homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR region and / or CDR1 region and / or CDR2 region and / or light chain CDR3 region are substituted with homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR region and / or CDR1 region and / or CDR2 region are substituted with homologous human or non-human sequences. The present invention also includes so-called single-chain antibodies.

[0051] Various antibody molecules and fragments may be derived from any of the generally known immunoglobulin classes (including, but not limited to, IgA, secretory IgA, IgE, IgG, and IgM). The IgG subclass is also well known to those skilled in the art and includes, but is not limited to, human IgG1, IgG2, IgG3, and IgG4. In a preferred embodiment, the compound of the present invention is human IgG4.

[0052] In another embodiment, the antibody according to the present invention is a single-domain antibody. The terms “single-domain antibody” (sdAb) or “VHH” refer to a single heavy-chain variable domain of an antibody of a type that can be found in camelid mammals that naturally lack a light chain. Such a VHH is also called “nanobody®”. According to the present invention, the sdAb may be a llama sdAb in particular. The term “VHH” refers to a single heavy chain having three complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3. The terms “complementarity-determining region” or “CDR” refer to a hypervariable amino acid sequence that defines the binding affinity and specificity of the VHH.

[0053] The VHH according to the present invention can be readily prepared by those skilled in the art using certain experiments. The VHH variants and modified forms may be produced using any of the techniques known in the art, such as in vitro maturation.

[0054] VHHs or sdAbs are typically produced by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA derived from immunized animals into phage display vectors such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries against immobilized antigens, e.g., antigens coated on the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often exhibit lower affinity for their antigens compared to VHHs derived from animals that have undergone multiple immunizations. The high affinity of VHHs from immune libraries is attributed to natural selection of mutant VHHs during the clonal development of B cells in the lymphoid organs of immunized animals. The affinity of VHH from non-immune libraries is often improved by mimicking this strategy in vitro, i.e., by site-directed mutagenesis of the CDR region and further rounds of panning against immobilized antigens under enhanced stringency conditions (higher temperature, high or low salt concentration, high or low pH, and low antigen concentration). Camelid-derived VHH are readily expressed at very high levels in E. coliperiplasm compared to the corresponding domain of conventional antibodies and can be purified from there. VHH generally exhibit high solubility and stability and can be readily produced in yeast, plant, and mammalian cells. For example, the "Hamers Patents" describe methods and techniques for producing VHH against any desired target (see, e.g., US5,800,988, US5,874,541, and US6,015,695).The "Hamers Patent" describes, in more detail, the production of VHH in bacterial hosts such as E. coli (see, e.g., US6,765,087) and lower eukaryotic hosts such as fungi (e.g., Aspergillus or Trichoderma), or yeasts (e.g., Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see, e.g., US6,838,254).

[0055] In another embodiment, the present invention provides an antibody that competes with the antibody of the present invention in terms of binding to a target.

[0056] As used herein, the term “binding” in the context of antibody binding to a given antigen or epitope typically refers to binding with affinity corresponding to a KD of approximately 10⁻⁷ M or less, e.g., approximately 10⁻⁸ M or less, e.g., approximately 10⁻⁹ M or less, approximately 10⁻¹⁰ M or less, or approximately 10⁻¹¹ M or less, as determined by surface plasmon resonance (SPR) technology in the BIAcore 3000 instrument, using, for example, the antigen in a soluble form as ligand and the antibody as analyte. BIACORE® (GE Healthcare, Piscaataway, New Jersey) is one of a variety of surface plasmon resonance assay formats routinely used for epitope bin panels of monoclonal antibodies. Typically, an antibody binds to a given antigen with an affinity corresponding to a KD at least 10 times lower than its KD for binding to a nonspecific antigen (e.g., BSA, casein) (which is not identical to or closely related to the given antigen), for example, at least 100 times lower, for example, at least 1,000 times lower, for example, at least 10,000 times lower, for example, at least 100,000 times lower. If the antibody's KD is very low (i.e., the antibody has high affinity), then its KD for binding to the antigen is typically at least 10,000 times lower than its KD for the nonspecific antigen. An antibody is said to not essentially bind to an antigen or epitope if such binding is undetectable (for example, using plasmon resonance (SPR) techniques in a BIAcore 3000 instrument with the antigen in a soluble form as the ligand and the antibody as the analyte), or if the binding is 100, 500, 1000, or more than 1000 times less than the binding detected by the antibody and the antigen or epitope having a different chemical structure or amino acid sequence.

[0057] Additional antibodies can be identified based on their ability to cross-compete with other antibodies of the Invention in a standard target binding assay (e.g., competitively inhibit binding in a statistically significant manner). The ability of a test antibody to inhibit the binding of the antibodies of the Invention to a target demonstrates that the test antibody can compete with that antibody for binding to the target; such an antibody may, according to a non-limiting theory, bind to the same or related (e.g., structurally similar or spatially proximal) epitopes on the target as the antibody it competes with. Thus, another aspect of the Invention provides an antibody that binds to the same antigen as the antibody disclosed herein and competes with it. As used herein, an antibody is "competing" for binding if the competing antibody inhibits the target binding of the antibody or antigen-binding fragment of the present invention by more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% in the presence of an equimolar concentration of the competing antibody.

[0058] In other embodiments, the antibody or antigen-binding fragment of the present invention binds to one or more target epitopes. In some embodiments, the epitope to which the antibody or antigen-binding fragment of the present invention binds is a linear epitope. In other embodiments, the epitope to which the antibody or antigen-binding fragment of the present invention binds is a nonlinear stereochemical epitope.

[0059] In some embodiments, the present invention relates to UNG inhibitors in which the oligonucleotide is an antisense oligonucleotide, siRNA, shRNA, DNA aptamer, or RNA aptamer. The UNG inhibitor is a UNG expression inhibitor.

[0060] The term "expression," when used in relation to the expression of a gene or nucleic acid, refers to the conversion of information contained within a gene into a gene product. A gene product may be the direct transcript of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or other types of RNA) or a protein produced by the translation of mRNA. Gene products also include messenger RNA modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by processes such as methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristylation, and glycosylation.

[0061] "Expression inhibitor" refers to a natural or synthetic compound that has a biological effect of inhibiting gene expression. "Expression inhibitor" refers to any compound that has a biological effect of inhibiting the expression of a target gene and / or a target protein. In one embodiment of the present invention, the expression inhibitor is short hairpin RNA (shRNA), small inhibitory RNA (siRNA), or antisense oligonucleotide. Preferably, the expression inhibitor is siRNA or shRNA.

[0062] The target expression inhibitors used in the present invention may be based on antisense oligonucleotide constructs. Antisense oligonucleotides, which include antisense RNA molecules and antisense DNA molecules, act to directly inhibit the translation of target mRNA by binding to the target mRNA, thereby preventing protein translation or increasing mRNA degradation, and thereby reducing the level of the target protein in the cell and decreasing its activity. For example, an antisense oligonucleotide of at least about 15 nucleotides complementary to a specific region of the mRNA transcription sequence encoding the target can be synthesized, for example, by conventional phosphodiester technology and administered, for example, by intravenous injection or intravenous infusion. Methods using antisense techniques to specifically reduce the gene expression of genes with known sequences are well known in the art (see, for example, U.S. Patents 6,566,135, 6,566,131, 6,365,354, 6,410,323, 6,107,091, 6,046,321 and 5,981,732).

[0063] Small inhibitory RNAs (siRNAs) can also function as targeted gene expression inhibitors for use in the present invention. Target gene expression is reduced by contacting the target or cell with small double-stranded RNA (dsRNA), or a vector or construct that induces the production of small double-stranded RNA, resulting in specific inhibition of gene expression (i.e., RNA interference or RNAi). Methods for selecting a suitable dsRNA or dsRNA-coding vector are well known in the art for genes with known sequences (see, for example, Tuschi, T. et al. (1999); Elbashir, SM et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, ​​TR. et al. (2002); U.S. Patents 6,573,099 and 6,506,559; and International Publications 01 / 36646, 99 / 32619 and 01 / 68836).

[0064] Short hairpin RNA (shRNA) or small inhibitory RNA (siRNA) can function as gene expression inhibitors for use in the present invention. Gene expression can be reduced using short double-stranded RNA (dsRNA) that specifically inhibits gene expression, or using vectors or constructs that induce the production of short double-stranded RNA (i.e., RNA interference or RNAi). Methods for selecting appropriate dsRNA or dsRNA-encoding vectors for genes with known sequences are well known in the art.

[0065] Ribozymes can also function as target expression inhibitors for use in the present invention. Ribozymes are enzymatic RNA molecules that have the ability to catalyze the specific cleavage of RNA. The mechanism of action of ribozymes involves sequence-specific hybridization of the ribozyme molecule with complementary target RNA, followed by endonuclease cleavage.

[0066] Therefore, genetically engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze the endonuclease-like cleavage of target mRNA sequences are useful within the scope of this invention. Specific ribozyme cleavage sites within any potential RNA target are first identified by scanning the target molecule for ribozyme cleavage sites, typically encompassing sequences GUA, GUU, and GUC. After identification, short RNA sequences of approximately 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structures that could render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated, for example, by testing their reach to hybridization with complementary oligonucleotides using ribonuclease protection assays.

[0067] Both antisense oligonucleotides (ODNs) and ribozymes useful as target inhibitors can be prepared by known methods. These include, for example, chemical synthesis techniques such as solid-phase phosphoramidite chemosynthesis. Alternatively, antisense RNA molecules can be generated by in vitro or in vivo transcription of the DNA sequence encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors incorporating suitable RNA polymerase promoters, such as the T7 or SP6 polymerase promoter. Various modifications to the oligonucleotides of the present invention can be introduced as means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of ribonucleotide or deoxyribonucleotide flanking sequences to the 5' and / or 3' ends of the molecule, or the use of phosphorothioates or 2'-O-methyl molecules rather than phosphodiesterase binding within the oligonucleotide backbone.

[0068] The antisense oligonucleotides, siRNAs, and ribozymes of the present invention can be delivered in vivo, either alone or in association with a vector. In its broadest sense, “vector” is a vehicle capable of facilitating the transport of antisense oligonucleotides, siRNAs, or ribozyme nucleic acids to cells, and preferably cells expressing a target. Preferably, the vector transports the nucleic acid to the cell with less degradation than would occur in the absence of the vector. Generally, useful vectors in the present invention include, but are not limited to, plasmids, phagemids, viruses, and other vehicles derived from viral or bacterial sources manipulated by insertion or incorporation of antisense oligonucleotides, siRNAs, or ribozyme nucleic acid sequences. Viral vectors are preferred types of vectors and include, but are not limited to, the following viral nucleic acid sequences: retroviruses such as Moloney's mouse leukemia virus, Harvey's mouse sarcoma virus, mouse mammary cancer virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; SV40 virus; polyomavirus; Epstein-Barr virus; papillomavirus; herpesvirus; vaccinia virus; poliovirus; and RNA viruses such as retroviruses. Other vectors known in the art but not named can readily be used.

[0069] Preferred viral vectors are based on non-cellular eukaryotic viruses in which non-essential genes are replaced with genes of interest. Examples of non-cellular viruses include retroviruses (e.g., lentiviruses) whose life cycle involves reverse transcription of genomic viral RNA into DNA and subsequent proviral integration into host cell DNA. Retroviruses are approved for human gene therapy trials. Retroviruses with replication defects (i.e., those capable of directing the synthesis of the desired protein but lacking the ability to produce infectious particles) are most useful. Such genetically modified retroviral expression vectors are generally useful for highly efficient gene transduction in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfection of a packaging cell line with the plasmid, production of recombinant retroviruses by the packaging cell line, recovery of viral particles from tissue culture medium, and infection of target cells with the viral particles) are provided by KRIEGLER ("A Laboratory Manual," WH Freeman CO, New York, 1990) and MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, NJ, 1991).

[0070] Preferred viruses for specific applications are adenoviruses and adeno-associated viruses, which are double-stranded DNA viruses already approved for human use in gene therapy. Adeno-associated viruses can be modified to have replication defects and have infectivity to a wide range of cell types and species. Furthermore, they have advantages such as thermal and lipid solvent stability; high transduction frequency to diverse cell lineages, including hematopoietic cells; and lack of re-infection inhibition, which consequently allows for transduction of multiple series. Reports indicate that adeno-associated viruses can be site-specifically integrated into human cell DNA, thereby minimizing the possibility of insertional mutations and variability in the expression of inserted genes characteristic of retroviral infections. In addition, wild-type adeno-associated virus infection has been tracked for more than 100 passages in tissue culture in the absence of selective pressure, which means that adeno-associated virus genome integration is a relatively stable event. Adeno-associated viruses can also function outside of chromosomes.

[0071] Other vectors include plasmid vectors. Plasmid vectors are well described in the art and are well known to those skilled in the art. See, for example, SANBROOK et al., "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In recent years, plasmid vectors have been used as DNA vaccines for delivering antigen-coding genes to cells in vivo. They are particularly advantageous in this regard because, like many viral vectors, they do not pose safety problems. However, these plasmids, which have promoters that are compatible with host cells, can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUCl9, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those skilled in the art. In addition, plasmids can be custom-designed to use restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids can be delivered by a variety of parenteral, mucosal, and local routes. For example, a DNA plasmid may be injected via an intramuscular, intradermal, subcutaneous, or other route. The plasmid may also be administered by an intranasal spray or drops, a rectal suppository, or orally. The plasmid may also be administered to epithelial or mucosal surfaces using a gene gun. The plasmid may be provided in aqueous solution, dried on gold particles, or in association with other DNA delivery systems, including but not limited to liposomes, dendrimers, cocreates, and microencapsulation.

[0072] In some embodiments, UNG inhibitors are used in combination with PARP inhibitors. In some embodiments, the PARP inhibitor is selected from the group consisting of olaparib, lucaparib, niraparib, talazoparib, iniparib, veliparib, pamiparib (BGB-290), CEP9722, E7016, E7449, and 3-aminobenzamide. In some embodiments, the UNG inhibitor is used in combination with cisplatin or a Polθ inhibitor such as novobiocin. In some embodiments, UNG inhibitors are used in combination with NMNAT1 inhibitors.

[0073] As used herein, the term “NMNAT1 inhibitor” refers to any compound selected from the group consisting of compounds that target nicotinamide nucleotide adenylyltransferase 1, but is not limited to these. The term “NMNAT1 inhibitor” refers to a compound that binds to NMNAT1 and functions as a potent antagonist of NMNAT1. The term “NMNAT1 inhibitor” has its general meaning in the art and refers to a compound that selectively inactivates NMNAT1. Typically, NMNAT1 inhibitors are small organic molecules, polypeptides, aptamers, oligonucleotides (antisense oligonucleotides, siRNA, shRNA, DNA, and RNA aptamers), or antibodies. NMNAT1 inhibitors are well known in the art, for example, as described in WO2013 / 130672; Kusumanchi et al., 2013.

[0074] The term "NMNAT1 inhibitor" is not limited to this, but refers to any compound selected from siRNAs such as s34981 (hNMNAT1 siRNA) (Kusumanchi et al., 2013; WO2013 / 130672), siRNA ID130437, siRNA ID130438, siRNA ID130439, siRNA ID212976, and siRNA ID212977 (Thermo Fisher Scientific).

[0075] In some embodiments, the NMNAT1 inhibitor is an antisense oligonucleotide, siRNA, shRNA, DNA aptamer, or RNA aptamer. In some embodiments, UNG inhibitors are used in combination with SIRT6 inhibitors. In some embodiments, the SIRT6 inhibitor is selected from the group consisting of OSS128167, Trichostatin A, and SIRT6 inhibitory quercetin derivatives luteolin, catechin gallate, and gallocatechin gallate.

[0076] Pharmaceutical composition In a further embodiment, the present invention relates to a pharmaceutical composition comprising a uracil DNA glycosylase (UNG) inhibitor for use in the treatment of resistant homologous recombination repair deficiency (HRD) cancer in a subject requiring such treatment, and a pharmaceutically acceptable carrier, wherein the UNG inhibitor is a molecule that enables silencing of a gene expressing the UNG enzyme, or a molecule that inhibits the properties of the UNG enzyme.

[0077] Part of the present invention also comprises a pharmaceutical composition comprising (i) a uracil DNA glycosylase (UNG) inhibitor, which is a molecule that enables silencing of a gene expressing the UNG enzyme, or a molecule that inhibits the properties of the UNG enzyme; (ii) at least one of the PARP inhibitors, cisplatin, Polθ inhibitors, NMNAT1 inhibitors, and SIRT6 inhibitors as described above, and (iii) a pharmaceutically acceptable carrier.

[0078] In some embodiments, resistant HRD cancer is resistant BRCA-associated cancer, chemotherapy-resistant HRD cancer, or chemotherapy-resistant BRCA-associated cancer. Typically, the compounds of the present invention may be combined with pharmaceutically acceptable excipients and, optionally, with a sustained-release matrix such as a biodegradable polymer to form a therapeutic composition.

[0079] Typically, the compounds according to the present invention described above are administered to the subject in a therapeutically effective dose. The “therapeutic effective dose” of the compounds of the present invention as described above means a sufficient amount of the compound that can be applied to any medical treatment with a reasonable benefit / risk ratio. However, naturally, the total daily dose of the compounds and compositions of the present invention is determined by the attending physician within the scope of appropriate medical judgment. A specific therapeutically effective dose level for a particular subject is determined by a variety of factors, including the disorder being treated and its severity; the activity of the specific compound used; the specific composition used, the subject’s age, weight, overall health, sex, and diet; the time of administration, the route of administration, and the rate of excretion of the specific compound used; the duration of treatment; drugs used in combination or concurrently with the specific compound used; and similar factors well known in the medical field. For example, it is well within the scope of the art to start administration of the compound at a level lower than the dose required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dose of the product can vary widely from 0.01 to 1,000 mg / day per adult. Typically, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the compound of the present invention for symptomatic dose adjustment to the target of treatment. The pharmacopoeia typically contains about 0.01 mg to about 500 mg of the compound of the present invention, preferably 1 mg to about 100 mg of the compound of the present invention. The effective dose of the drug is usually supplied at a dose level of 0.0002 mg to about 20 mg per kg of body weight per day, and in particular, about 0.001 mg to 7 mg per kg of body weight per day.

[0080] In certain embodiments, the compounds according to the present invention may be used at concentrations of 0.01 μM to 20 μM, and more specifically, the compounds of the present invention may be used at concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, and 20.0 μM.

[0081] In accordance with the present invention, the compounds of the present invention are administered to a subject in the form of a pharmaceutical composition. Typically, the compounds of the present invention can be combined with pharmaceutically acceptable excipients and, optionally, with a sustained-release matrix such as a biodegradable polymer to form a therapeutic composition. "pharmaceutically acceptable" or "pharmaceutically acceptable" means, as necessary, molecular entities and compositions that do not cause adverse reactions, allergic reactions or other adverse reactions when administered to mammals, in particular humans. A pharmaceutically acceptable carrier or excipient means a non-toxic solid, semi-solid or liquid extender, diluent, encapsulating material or any type of formulation aid.

[0082] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, or rectal administration, the active ingredient may be administered to animals and humans alone or in combination with another active ingredient in unit dosage forms or as mixtures with conventional pharmaceutically supporting agents. Preferred unit dosage forms include oral route dosage forms such as tablets, gel capsules, powders, granules and oral suspensions or liquids, sublingual and buccal dosage forms, aerosols, indwellings, subcutaneous dosage forms, transdermal dosage forms, topical dosage forms, intraperitoneal dosage forms, intramuscular dosage forms, intravenous dosage forms, subcutaneous dosage forms, transdermal dosage forms, subarachnoid and intranasal dosage forms, and rectal dosage forms.

[0083] Typically, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These may be, in particular, isotonic sterile saline (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride and the like, or mixtures thereof), or a dry composition, especially a lyophilized composition, that allows for the composition of an injectable solution after the addition of sterile water or saline, as is optional. Suitable pharmaceutical dosage forms for injection include sterile aqueous solutions or suspensions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or suspensions. In all cases, the dosage form must be sterile and fluid enough to pass through an injection needle easily. The dosage form must be stable under manufacturing and storage conditions and protected from microbial contamination such as bacteria and fungi. Solutions containing the compounds of the present invention as free bases or pharmaceutically acceptable salts can preferably be prepared with water mixed with a surfactant such as hydroxypropylcellulose. Dispersion formulations can also be prepared with glycerol, liquid polyethylene glycol, and mixtures thereof, and with oils. Under normal storage and use conditions, these formulations contain preservatives that prevent microbial growth. The compounds of the present invention can be formulated into compositions in neutral or salt form. Medicinally acceptable salts include acid addition salts (formed with free amino groups of proteins) and acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is desirable to include isotonic agents, such as sugars or sodium chloride. Extension of absorption of the injectable composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin in the composition. Sterile injectable formulations are prepared by compounding the required amount of the active compound in a suitable solvent with some of the other components listed above as needed, and then performing sterile filtration. Generally, dispersion formulations are prepared by incorporating various sterilized agents of the present invention into a sterile vehicle containing a dispersion base and other necessary components selected from those listed above. For sterile powders for the preparation of sterile injectable solutions, a typical preparation method is vacuum drying and freeze-drying techniques, where powders of the compound of the present invention and further desired components are obtained from pre-prepared sterile filtered solutions thereof. Preparation of more concentrated or very concentrated solutions for direct injection is also conceivable to deliver high concentrations of the activator to small tumor areas, where the use of DMSO as a solvent is assumed to result in extremely rapid penetration. After formulation, the solution is administered in a manner suitable for the administration formulation and in an amount that is therapeutically effective. The formulation is readily administered in various dosage forms, such as the types of injectable solutions described above, but drug-release capsules and similar forms can also be used. For parenteral administration in aqueous solutions, for example, the solution should be appropriately buffered as needed, and the liquid diluent is first isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be apparent to those skilled in the art in light of this specification. Some variation in dosage is inevitable depending on the condition of the patient being treated. In any case, the person administering the medication will determine the appropriate dose for each individual patient.

[0084] The pharmaceutical composition of the present invention may include any further compounds used for cancer treatment as described above. These compounds include, as described above, PARP inhibitors, cisplatin, Polθ inhibitors, NMNAT1 inhibitors, or SIRT6 inhibitors in particular. Preferably, the pharmaceutical composition comprises a UNG inhibitor and a PARP inhibitor or Polθ inhibitor as described above.

[0085] In some embodiments, the pharmaceutical compositions of the present invention may include any further compounds used for the treatment of HRD cancer, BRCA-associated cancer, resistant HRD cancer, or resistant BRCA-associated cancer. In one embodiment, the aforementioned additional active compounds may be included in the same composition or administered separately.

[0086] In another embodiment, the pharmaceutical composition of the present invention relates to combination preparations for simultaneous, separate, or sequential use in the treatment of resistant HRD cancer in a subject requiring it. In some embodiments, the pharmaceutical compositions of the present invention relate to combination preparations for simultaneous, separate, or sequential use in the treatment of resistant BRCA-associated cancers in subjects requiring such treatment. In some embodiments, the pharmaceutical compositions of the present invention relate to combination preparations for simultaneous, separate, or sequential use in the treatment of chemotherapy-resistant HRD cancer in a target that requires them. In some embodiments, the pharmaceutical compositions of the present invention relate to combination preparations for simultaneous, separate, or sequential use in the treatment of chemotherapy-resistant BRCA-associated cancers in subjects requiring such treatment.

[0087] treatment In a further embodiment, the present invention relates to a method for treating a patient in need of such treatment for a resistant homologous recombination repair deficiency (HRD) cancer, such as a resistant BRCA-associated cancer, a chemotherapy-resistant HRD cancer, or a chemotherapy-resistant BRCA-associated cancer, comprising administering a therapeutically effective amount of a uracil DNA glycosylase (UNG) inhibitor to the patient.

[0088] The treatment method of the present invention may include further administration of a PARP inhibitor, cisplatin, Polθ inhibitor, NMNAT1 inhibitor, or SIRT6 inhibitor, as described above. Typically, the administration of the compounds of the present invention may be combined with the administration of other active compounds, pharmaceutically acceptable excipients, and optionally a sustained-release matrix, such as a biodegradable polymer. Typically, the additional active compounds described above are administered to the subject in therapeutically effective doses.

[0089] In one embodiment, the aforementioned additional compounds may be included in the same composition or administered separately.

[0090] The present invention is further illustrated by the following drawings and embodiments. [Brief explanation of the drawing]

[0091] [Figure 1a] a. Quantification and representative images of clonal formation in wild-type (WT) and BRCA2- / -(clones C1 and C2)RPE-1 cells with and without BRCA2 cDNA complementation after transfection with the indicated NMNAT1 siRNA. [Figure 1b-c] b. Clonal genicity and WB analysis of BRCA2 mAID HeLa cells in the presence or absence of auxin (IAA) after transduction using NMNAT1 shRNA or control-carrying viral particles. c. Clonal genicity of WT and BRCA1- / - RPE-1 cells after transfection with siNMNAT1 #2. [Figure 1d] d. Clonal formation of WT and NMNAT1- / - RPE-1 cells after transfection with the indicated BRCA1 / 2 siRNA. [Figure 1e] e. Quantification of clonal formation in HR-non-deficient and HR-deficient cells displayed using siNMNAT1, and representative images. [Figure 1f-g]f. Quantification of DNA damage in P53- / - RPE-1 cells by alkaline COMET assay in the presence or absence of NMNAT1. g. Survival assay of WT and NMNAT1- / - RPE-1 cells exposed to the indicated dose of the PARP inhibitor rucaparib (PARPi). [Figure 2] a, b. Proliferation curves of WT and NMNAT1- / - cells in P53- / -(a) and P53+ / +(b)RPE-1. c. Quantification of DNA damage by alkaline COMET assay of the same cells as a and b. [Figure 3a] a. WB analysis showing PARP1 activation in wild-type (WT) cells, NMNAT1- / - RPE-1 cells (G9) supplemented with catalytically inactive (MUT, W169A) NMNAT1 cDNA or empty vector (EV) after exposure to PARG inhibitors and MMS. [Figure 3b-c] b. Quantification of DNA damage by alkaline COMET assay in wild-type (WT) or NMNAT1- / - (clone G9) RPE-1 cells with or without supplementation of catalytically inactive (MUT, W169A) NMNAT1 cDNA (EV). c, d. Clonal formation of the same cells as in b, corresponding to transfection with PARPi(c) or subsequently with BRCA2 or control (siCTRL) siRNA (left panel of d). Representative images are shown in the right panel of d. [Figure 3d] d. Clonal formation of the same cells as in b, corresponding to transfection with PARPi(c) or subsequent BRCA2 or control (siCTRL) siRNA (left panel of d). Representative images are shown in the right panel of d. [Figure 4a-b] a. Illustration of the drug-resistant cell lines used in this procedure. b. Clonal formation of wild-type (WT), BRCA2- / - (clones C1 and C2), and BRCA2- / - induced PARPi-resistant RPE-1 cells after control or NMNAT1 (siNMNAT1 #2) siRNA transfection. [Figure 4c-d]c. Clonal formation of parental and induced PARPi-resistant CAPAN-1 cells after transduction using shNMNAT1 lentiviral particles. Representative images are shown in the right panel. d. Clonal formation of PEO-1 and PEO-4 after transduction using shNMNAT1 lentiviral particles. Lentiviral particles carrying scrambled shRNA (shSCR) were used as controls in all experiments shown in c and d. [Figure 5a-b] a. Viability assay of RPE-1 cells corresponding to PARPi after transfection with siRNA targeting the indicated sirtuin. b, c. Quantification of clonal genicity assays of WT and SIRT6- / - RPE-1 cells corresponding to transfection with PARPi(b), siBRCA2, or control (siCTRL)(c). Representative images are shown in the panel below. [Figure 5c] c. Quantification of clonal genicity assays of WT and SIRT6- / - RPE-1 cells after transfection with PARPi(b) or siBRCA2 or control (siCTRL)(c). Representative images are shown in the panel below. [Figure 6a-b] a. Illustration of the drug-resistant cell lines used in this procedure. b. Clonogenicity formation of parental and induced PARPi-resistant CAPAN-1 cells after transduction using shSIRT6 lentiviral particles. [Figure 6c-d] c. Clonal formation of PEO-1 and PEO-4 cells after transduction using shSIRT6 lentiviral particles. d. Clonal formation of parental and induced cisplatin-resistant CAPAN-1 cells after transduction using shSIRT6 virus particles. Lentiviral particles carrying scrambled shRNA (shSCR) were used as controls in all experiments shown in b, c, and d. [Figure 7a-b]a. Clonal formation of wild-type (WT) and BRCA2- / - (clones C1 and C2) cells after exposure to the indicated dose of the SIRT6 inhibitor OSS_128167. b. Clonal formation of wild-type (WT), BRCA2- / - (clones C1 and C2), and BRCA2- / induced PARPi-resistant RPE-1 cells after exposure to 0.5 mM of the SIRT6 inhibitor OSS_128167. [Figure 7c] c. Clonal formation of parental and induced PARPi-resistant CAPAN-1 clones after exposure to the indicated dosage of the SIRT6 inhibitor OSS_128167. Normal mammary epithelial cells MCF10A were included as an HR-non-deficient cell line. [Figure 8] A plot of PARPi-resistant BRCA1 mutant PDX HBCx-11 tumors at day 32 after treatment with either the PARP inhibitor olaparib (PARPi) or olaparib in combination with the SIRT6 inhibitor OSS_128167 (SIRT6i) (a). The number of tumors classified as stable or progressive disease is plotted in the lower panel (b). [Figure 9]UNG functions downstream of SIRT6 in the survival of BRCA1 / 2 mutant tumors. a. Scatter plots of SILAC forward (FWD) and reverse (REV) proteomics experiments showing that UNG is significantly downregulated in the nucleus of SIRT6 knockout cells (SIRT6- / -). b. Immunoblot validation of MS-proteomics data showing decreased UNG protein levels in SIRT6- / - cells. c. Survival clonal genicity assay in wild-type (WT) and BRCA2 knockout RPE_1 clones (cl.1 and cl.2) due to loss of UNG protein. UNG protein was downregulated by two independent siRNA sequences (si#6 and si#7). d. Survival clonal genicity assay of parental (PARPi untreated) and PARPi-resistant OVCAR8 cells. UNG protein was downregulated by two independent siRNA sequences (si#6 and si#7). OVCAR8 is a BRCA1-hypermethylated ovarian tumor cell line. e, f. Quantification of ssDNA gaps by DNA fiber assay in cells lacking either SIRT6(e) or UNG(f). [Figure 10] UDG deficiency kills PARPi-untreated and PARPi-resistant HRD cells. a. Survival assay of WT and UNG-deficient cells after exposure to the indicated dose of the PARP inhibitor (PARPi) rucaparib. b. Quantification of clonal positivity of WT cells and two BRCA2- / - clones (C1 and C2) in UNG deficiency using two separate siRNAs (#6 and #7). c. Quantification of clonal positivity of WT and UNG- / - clones after BRCA2 deficiency using siRNA. Western blotting shows UNG knockout efficiency. d. Quantification of clonal positivity of PARPi-untreated and PARPi-resistant BRCA1 and BRCA2 mutant cancer cell lines (OVCAR8 and CAPAN-1) in UNG deficiency using two separate siRNAs (#6 and #7). [Figure 11-1]Uracil removal is regulated by nuclear NAD+. a. Quantification of post-replication ssDNA gaps using DNA fiber assay in P53- / - RPE-1 cells 48 hours after transfection with the indicated siRNA. b. Illustration of the 2'-deoxyuridine (dUTP) / thymidine (dTTP) biosynthesis pathway. The biological processes involved are shown in italics. c. Nuclear fractionation and immunoblot profiling of the indicated protein after 24 hours of drug exposure (0.1 μM FdUrd + 5 μM TAS-114) in WT and UNG- / - cells. For washout samples (wo), cells were collected 24 hours after drug removal. Western blot images are representative of the three experiments. [Figure 11-2] d. Left side, quantification of DNA damage (left side, -rUNG) and gDNA-uracil (right side, +rUNG) in wild-type (WT) and NMNAT1- / - cells after 24 hours of FdUrd treatment (20 μM) and indicated time of drug washout (wo). UNG- / - cells before and after treatment with rUNG are also shown. Right side, representative image of cells treated in the same way as (left side). e. Top, diagram of the iPOND workflow. Left side, immunoblot analysis of EdU-labeled DNA before (introduction) and after capture in WT and NMNAT1- / - cells. EdU pulse-free (EdU-) and thymidine chase (Thym+) were used as controls for specificity and RF proximity, respectively. Right side, quantification of captured UNG protein levels as (left side), normalized for captured PCNA and H3. [Figure 11-3]f. Quantification of γH2AX focus formation before and after 24-hour treatment of WT (gray) and NMNAT1- / - (orange) cells using the indicated dosage of γTAS-114 (μM). g. Quantification of clonal viability assays for 12 days after siRNA transfection and drug treatment of WT and NMNAT1- / - cells as shown (left) and representative images (right). 48 hours after siRNA transfection, 5 μM TAS-114 was used or not used in combination with the indicated dosage of FdUrd over 24 hours, followed by drug washout. [Figure 12-1] UNG inhibition leads to SMUG1-mediated DNA damage and HR activation. a. Quantification (left) and representative images (right) of cell cycle profiles of treated and untreated WT and UNG- / - cells as shown in (Figure 1c). Quantification (left) is based on gates highlighted in red (right). b. Nuclear fractionation and immunoblot profiling of shown proteins following drug exposure of WT and UNG- / - cells as shown in (Figure 1c). Western blot images are representative of three experiments, and the protein lysates are the same as those shown in (Figure 1c). [Figure 12-2] c. Immunoblotting profiling of HMCES DNA adducts following RADAR in WT and UNG- / - cells treated as shown in (Figure 2b). MG132 10 μM was added 3 hours prior to cell collection. [Figure 12-3] d. Immunofluorescence-based quantification (top) and representative images (bottom left) of SMUG1 (left), HMCES (center), and γH2AX (right) foci in WT and UNG- / - cells processed as shown in (Figure 2b). Bottom right: Quantification of HMCES / γH2AX colocalized foci. [Figure 12-4]e. Immunoblotting profiling of the indicated protein in the chromatin fraction of UNG- / - cells following siRNA transfection and drug treatment as shown. 48 hours after siRNA transfection, cells were treated with the indicated drug (0.1 μM FdUrd + 5 μM TAS-114 over 24 hours). f. Quantification of 12-day clonal viability assay in P53- / - RPE-1 cells transfected with the indicated siRNA and followed by continuous exposure to the indicated dose of rucaparib (PARPi) or without it after 48 hours. g. Immunofluorescence-based quantification (left) and representative image (right) of the RAD51 focus in WT (left) and UNG- / - (right) cells treated as shown in (Figure 2b). Representative images of the γH2AX (top) and SMUG1 (bottom) focuses are also shown. [Figure 13-1] dU misuptake results in low-replicating DNA and chromosomal fragmentation in HR-deficient cells. a. Representative images (n=3) of pulverized / fragmented chromosomes and chromosomal translocations in BRCA2- / - cells treated with 0.1 μM FdUrd + 5 μM TAS-114 for 24 hours and analyzed 48 hours after drug washout. b. Quantification (left) and representative image (right) of late ultrafine crosslinks (UFB) in BRCA2- / - cells treated as shown in (Figure 3a) and analyzed 24 hours after drug washout (n=3). c. Quantification (left) and representative image (right) of 53BP1 NB (top) and micronuclei (bottom) in BRCA2- / - cells treated as shown in (Figure 3a) and analyzed 48 hours after drug washout (n=4). [Figure 13-2] d. Quantification of clonal survival assay over 12 days in BRCA2- / - cells transfected with the indicated siRNA and treated with 1 μM 1TAS-114 for the duration of the experiment, or untreated cells, 48 ​​hours later. [Figure 14-1]Targeted uracil removal by UNG kills HR-deficient tumors regardless of their resistance to PARPi. a. Bottom, quantification of 12-day clonal survival assay in WT and two BRCA2- / - clones (C1 and C2) following transfection with the shown siRNA. Top, immunoblot showing UNG deficiency. Arrows indicate UNG1 (bottom) and UNG2 (top) (n=3). b. Quantification of 12-day clonal survival assay in BRCA2- / - cells following transfection with the shown siRNA (bottom) and representative image (top). [Figure 14-2] c. Quantification of clonal survival assay for 12 days in the indicated PARPi-resistant HR-deficient cell line after transfection with the indicated siRNA. [Figure 15-1] The interaction between UNG and SMUG1 in uracil removal is regulated by nuclear NAD+. a. Top: Immunoblot profiling of WT and NMNAT1- / - RPE-1 cells for the indicated proteins. Bands corresponding to UNG1 (lower MW) and UNG2 (upper MW) are shown. Asterisks (*) indicate nonspecific bands. Cells were treated with the indicated doses of FdUrd for 24 hours with or without 5 μM TAS-114. Bottom: Cell cycle distribution (n=2) of WT (+ / +) and NMNAT1- / - (- / -) RPE-1 cells treated as (top). b. Quantification of gDNA-uracil and DNA damage in WT RPE-1 cells treated with or without 5 μM TAS-114 for 24 hours using modified comet assays with or without recombinant UNG (rUNG) (right) or (left), respectively. The results are presented as tail moments. At least 80 comets were measured using CometScore for each condition. The results shown are mean ± standard error of the mean (SEM) (n=3). [Figure 15-2]c. Top: The role of NMNAT1 (red) in the regulation of the NAD+ salvage pathway and nuclear NAD+ homeostasis. Involved enzymes are shown in bold and italics. Bottom: Immunoblot profiling of NMNAT1, ADP-ribosylation (PAR), and PARP1 (short-term and long-term exposure) in WT and NMNAT1- / - cells supplemented with or without catalytically inactive mutant (MUT) NMNAT1 (W169A). PARP1 was detected after membrane stripping with anti-ADP-ribosylation antibody. Cells were treated with PDD 00017273 (PARGi, 10 μM) for 2 hours, or untreated, and 10 mM MMS was added 10 minutes prior to cell lysis. Vinculin was used as a loading control. [Figure 15-3] d. Quantification of 12-day clonal survival assays (n=3) in the same cells as (c, bottom) treated with or without the indicated dose of rucaparib (PARPi) over the duration of the experiment. e. Quantification of 12-day clonal survival assays (n=3) in WT (black) and NMNAT1- / - (red) cells following exposure to the indicated dose of the indicated DNA-damaging drug. [Figure 15-4]f. Quantification of clonal survival assays for 12 days in WT, NMNAT1- / , and UNG- / - cells following 24-hour exposure to the indicated dose of FdUrd with or without 5 μM TAS-114 (n=3). g. Quantification of clonal survival assays for 12 days in WT (black), NMNAT1- / -, and UNG- / - cells following 24-hour exposure to the indicated dose of TAS-114 (n=3). For WT cells, the data are the same as in (Figure 14c). h. Quantification of DUT mRNA levels by RT-qPCR following 72-hour transfection of WTRPE-1 cells with upper, DUT siRNA (siDUT) or associated control (siCTRL). Results are expressed as a fold change relative to siCTRL. (Bottom), quantification of clonal viability assays over 12 days in WT (left), NMNAT1- / - (center), and UNG- / - (right) cells following transfection with DUT siRNA and associated control (siCTRL). Data are expressed as the percentage of colonies formed in each cell line relative to appropriate siCTRL (mean ± sem) (n=3). i. Quantification of gDNA-uracil and DNA damage, as shown in (b), in WT and NMNAT1- / - cells by treatment with or without 5 μM TAS-114 and 0.08 μM FdUrd over 24 hours. [Figure 16]SMUG1 produces double-segment breaks (DSBs) during replication in the absence of NMNAT1 / UNG-mediated uracil removal. a. Cell cycle distribution of WT (+ / +) and NMNAT1- / - (- / -) RPE-1 cells by 10 μM FdrU over 24 hours and 24 and 48 hours of drug washout (wo). Data are expressed as the percentage of cells in different cell cycle phases (n=2). b. Mass spectrometry-based quantification of indicated metabolites (n=3). c. Quantification of γH2AX foci per nucleus in WT and UNG- / - RPE-1 cells, after or without exposure to TAS-114, quantified by immunofluorescence. Foci were scored separately in cells in S phase (EdU-positive, right side) or other cell cycle phases (EdU-negative, left side). At least 50 EdU-positive cells and 50 EdU-negative cells were scored (n=3). d. Quantification of γH2AX foci per nucleus in WT, NMNAT1- / -, and UNG- / - RPE-1 cells with or without 24-hour exposure to 5 μM FdUrd, followed by drug washout (wo) or no drug washout (wo) over the indicated duration (n=3). e. Immunoblotting of indicated proteins after nuclear separation of WT and NMNAT1- / - cells into soluble (nucleosol) or insoluble (chromatin) fractions, following a washout (wo) of several hours after 24-hour drug exposure. f. Immunoblotting of SMUG1, TDG, and MBD4 in WT RPE-1 cells 72 hours after transfection with the indicated siRNA pool. Vinculin was used as a loading control. [Figure 17]Overcoming UNG removal ability leads to capture of SMUG1-HMCES into chromatin, persistent PARP1 overactivation, and HR activation. a. Immunoblotting of HMCES and histone H3 before (introduction) capture using iPOND technology. All samples (WT and UNG- / - cells) were treated with 0.001 μM FdrU and 5 μM TAS-114 for 24 hours, followed by a 24-hour drug washout (wo) or no treatment. After treatment, cells were pulsed with EdU for 10 minutes, and then, if indicated, followed by a thymidine (Thym) chase for an additional 30 minutes. EdU was removed from negative controls. Protein samples before capture (introduction) represent 0.13% of the material used for EdU-labeled DNA pulldown (capture) (n=2). b. Immunofluorescence-based quantification of co-localization between indicated protein foci after 24-hour combined treatment with or without a 24-hour drug washout (wo) of 5 μM TAS-114 and 0.1 μM FdrU in WT and UNG- / - cells. Percentages of co-localized foci between SMUG1 and HMCES (left), SMUG1 and γH2AX (center), and HMCES and SMUG1 (right) are shown, respectively. [Figure 18] dU misuptake results in low-replicating DNA and chromosomal fragmentation in HR-deficient cells. a. Immunoblot profiling of BRCA2 protein in WT and BRCA2- / - RPE-1 cells (clones C1 and C2). Clone C2 was used to perform BRCA2 complementation with wild-type BRCA2 cDNA. Vinculin was used as a loading control. b. Immunofluorescence-based quantification of ionizing radiation (IR)-induced RAD51 focus in WT and BRCA2- / - clones. Cells were irradiated with 8 Gy, fixed, and stained 6 hours after irradiation (n=3). [Figure 19-1]Synthetic lethality between NMNAT1 / UNG inhibition and BRCA1 / 2 mutations. a. Quantification of 12-day clonal survival assay in WT and BRCA2- / - cells following transduction with viral particles carrying either pLentiCRISPRv2 Neo_UNG gRNA (UNG gRNA) or related control (CTRL gRNA) (top) and representative image (bottom right) (n=3). Cells were seeded for clonal assay after G418 sorting. Bottom left, immunoblot analysis of UNG after G418 sorting. b. Left side, Quantification of 12-day clonal survival assay in WT and BRCA2- / - RPE-1 cells following transduction with retroviral particles carrying either UGI or related control (EV) (n=3). Right side, qPCR-based quantification of UNG activity in the same cell line as (left side). c. Left: Quantification of 12-day clonal survival assay in WT and UNG- / - cells following transfection with siBRCA2 (n=3). Right: Immunoblot analysis of UNG cells in the same samples as (left). Arrows indicate UNG1 (bottom) and UNG2 (top). Vinculin was used as a loading control. [Figure 19-2] d. Quantification of 12-day clonal survival assays in WT and NMNAT1- / - clones (G9 and D7) following transfection with the indicated siRNA (n=3). e. Left side, quantification of 12-day clonal survival assays in wild-type (WT) or catalytically inactive mutant (MUT:W169A) NMNAT1 cDNA-supplemented, or unsupplemented (EV, empty vector) WT and NMNAT1- / - cells following transfection with siBRCA2 (left side) and representative images (right side) (n=3). [Figure 19-3]f. Quantification of 12-day clonal survival assays in HR-deficient cancer cell lines CAPAN1 (BRCA2 mutation) and OVCAR8 (highly methylated BRCA1 promoter) following transfection with siUNG#7 (n=3). g. Quantification of 12-day clonal survival assays in a panel of high-HR-potency, HR-deficient, and normal (MCF10A) cell lines following transfection with siNMNAT1 (left) and representative images (right) (n=3). [Figure 20-1] Targeted UNG-mediated uracil removal kills a subset of PARPi-resistant HR-deficient tumors. a. Quantification of clonal viability assays over 12 days (n=3) of WT, PARPi-untreated (C1 and C2), and resistant (C1-3, C1-5, C2-1, and C2-2) BRCA2- / - RPE-1 cells continuously exposed to indicated doses of rucaparib (PARPi). b. Replication fork degradation assay by DNA combing. WT, PARPi-untreated (C2), and resistant BRCA2- / - RPE-1 clones (C2-1 and C2-3) were sequentially labeled with thymidine analogs IdU and CldU over 30 minutes, followed by treatment with 4 mM hydroxyurea over 3 hours to stall the replication fork. The CldU / IdU ratio was used as a measure of replication fork degradation (n=3). [Figure 20-2] c. Quantification of clonal viability assays for PARPi-untreated parental CAPAN-1 cells and induced PARPi-resistant clones over 12 days. All cell lines were continuously exposed to the indicated dose of rucaparib during the assay (n=3). [Figure 20-3] d. Quantification of per-nucleus RAD51 foci in cells (c) 6 hours after or without exposure to 8 Gy of ionizing radiation (IR) (n=2). [Figure 20-4]e. 12-day clonal survival assay of WT cells, quantification of PARPi-untreated (C1 and C2) and resistant (C1-3, C1-5, C2-1, and C2-2) BRCA2- / - RPE-1 clones following NMNAT1 (siNMNAT1) knockdown. Survival rates of each cell line were normalized against the associated control (siCTRL) (n=3). [Figure 20-5] f. Quantification (left) and representative images (right) of a 12-day clonal viability assay of normal mammary MCF10A (HR non-deficient) and indicated CAPAN1 cells, including parent (PARPi untreated) and PARPi-resistant induced clones (C1-C17), following transduction using either shRNA-targeted NMNAT1 (shNMNAT1) or untargeted shRNA (shSCR)-supported particles. The viability of each cell line is normalized against the associated control (shSCR) (n=3). Representative images (PARPi) of the PARPi response and associated control (DMSO) for each cell line are also shown. [Figure 20-6] g. Survival curves for relative tumor volume (RTV) of subcutaneous OVCAR8 xenografts in nude mice. Mice carrying parental (black) or PARPi-resistant (red) OVCAR8 xenografts were treated twice a week with rucaparib (PARPi, dashed line) or vehicle (solid line). h. Left side, growth of PARPi-resistant OVCAR8 (BRCA1-deficient) xenografts in vivo. Top, immunoblot showing the efficacy of NMNAT1 silencing. Right side, relative tumor volume (RTV) for individual mice treated as shown on the left side after 5 weeks of growth in or without doxycycline (doxy). [Figure 20-7] i. Overall survival rate for mice treated with vehicle or doxycycline as shown in (h). [Examples]

[0092] Example 1: NMNAT1 inhibition kills BRCA1 and BRCA2 mutant tumor cells. NMNAT1-producing nuclear NAD in homologous recombination repair deficiency (HRD) cells +To study the role of, the inventors generated several BRCA1 / 2 isogenic cell lines, namely, P53 - / - BRCA1 in RPE-1 cells - / - and BRCA2 - / - knockout clones, as well as BRCA2 mini auxin-inducible degron (mAID) in HeLa cells. In P53 - / - RPE-1 cells, two separate small interfering RNA sequences (siRNAs) that impaired the colony-forming activity of BRCA2 - / - clones but had no effect on the survival of parental HRP cells were used to knockdown NMNAT1 (Figure 1a). Complementation of BRCA2 - / - cells (C2+BRCA2) with full-length BRCA2 cDNA rescued the survival of BRCA2 - / - cells deficient in NMNAT1 (Figure 1a). Similarly, in the mAID BRCA2 HeLa isogenic model, knockdown of NMNAT1 by short hairpin RNA (shRNA) reduced cell proliferation only in the presence of auxin, i.e., by BRCA2 proteolysis (Figure 1b). Furthermore, NMNAT1 silencing also impaired the colony-forming activity of BRCA1 - / - P53 - / - RPE-1 cells but was harmless to parental HRP cells (Figure 1c). These data suggest that NMNAT1 is synthetically lethal with BRCA1 / 2.

[0093] To confirm these findings, the inventors used a complementation approach by silencing BRCA1 / 2 in NMNAT1 - / - RPE-1 cells generated by CRISP-Cas9 genome editing. While having only a minor effect on P53 - / - RPE-1 cells, knockdown of BRCA1 / 2 by siRNA completely impaired the colony-forming activity of NMNAT1 - / - P53 - / - RPE-1 cells (Figure 1d).

[0094] Next, the inventors compared the cytotoxicity of siNMNAT1 between HRP and HR-deficient (HRD) cancer cells. siNMNAT1 significantly affected the colony-forming activity of HRD cells, while it had only a minor effect on HRP cells, including immortalized normal mammary epithelial MCF-10A cells (Figure 1e). In short, the data indicate that NMNAT1, along with BRCA1 / 2, is synthetically lethal.

[0095] The inventors quantified DNA damage in RPE-1 cells with and without NMNAT1 using the alkaline COMET assay. P53 - / - NMNAT1 - / - The clones showed more DNA damage compared to the parent cells, indicating that NMNAT1 contributes to maintaining genomic stability (Figure 1f).

[0096] The accumulation of DNA damage can lead to enhanced susceptibility to genotoxic drugs. + Assuming that NMNAT1 functions upstream of PARP1 by providing a mechanism, the reduced PARP1 activity due to NMNAT1 loss would be the primary cause of the observed synthetic lethality between NMNAT1 and BRCA. Interestingly, NMNAT1 - / - Cells are sensitized to the PARP inhibitor rucaparib (PARPi) (Figure 1g), and the role of NMNAT1 is not entirely superior to that of PARP1, but rather, nuclear NAD + However, it was suggested that it may also be used by other downstream enzymes to support HRD cell survival.

[0097] NMNAT1 loss is normal immortalized P53 + / + It is not harmful to cells. To rule out all possible adverse effects of NMNAT1 loss in HRP cells, the inventors administered P53 over a one-week period. - / - NMNAT1 - / - The proliferation rates of RPE-1 and parental cells were observed. Three NMNAT1 - / - All clones are from parent P53. - / -Compared to RPE-1 cells, a gradual decrease in proliferation was observed (Figure 2a). To better evaluate the effect of NMNAT1 loss in normal tissue, the inventors considered that normal cells have high P53 activity, and therefore P53 + / + We created an NMNAT1 knockout clone in RPE-1. Conversely, P53 + / + NMNAT1 - / - When comparing the RPE-1 clone to the parent cell, the inventors found that P53 + / + NMNAT1 - / - Clones (C12, C14, C17, C19, and C22) and parent P53 + / + As demonstrated by the similar proliferation of cells, we observed that NMNAT1 loss had no effect whatsoever on cell proliferation (Figure 2b). These data suggest that NMNAT1 loss is associated with P53 - / - While it slightly reduces cell proliferation, it is P53 + / + Without any adverse effects on cells, this suggests that targeted NMNAT1 does not have toxic effects in normal tissues.

[0098] NMNAT1 - / - P53 - / - Observed accumulation of DNA damage in cells (Figure 1g) is P53 - / - NMNAT1 loss is likely the main cause of reduced proliferation in RPE-1 cells (Figure 2a). However, the inventors found that NMNAT1 - / - P53 + / + When DNA damage was measured in cells using the alkaline COMET assay, the increase in DNA damage was compared to the parent cells of NMNAT1. - / - P53 - / - The loss was considerably less than that of RPE-1 (Figure 2c). These results indicate that the NMNAT1 loss was less than that of NMNAT1 - / - P53 + / + This is consistent with the observation that it did not affect cell proliferation. The catalytic activity of NMNAT1, i.e., nuclear NAD +To evaluate whether NMNAT1 is essential for the survival of HRD cells, the inventors used either the wild-type (WT) or catalytically inactive version of the enzyme (W169A, MUT) to test NMNAT1 - / - We supplemented RPE-1 cells and tested cell survival with BRCA2 knockdown. Supplementation with WT NMNAT1 enhanced PARP1 activation, thereby increasing NMNAT1 activity to the same level as in parental cells. - / - The amount of DNA damage in cells could be rescued, but not in the W169A mutant (Figure 3a, b). These results indicate that both PARPi sensitivity and synthetic lethality with BRCA2 were rescued by WT NMNAT1 supplementation, but not by W169A NMNAT1 (Figure 3c, d). In short, these data demonstrate that nuclear enzymes other than NMNAT1 and PARP1 are important for HRD cell survival. These results indicate that NMNAT1 is a key factor whose activity is required for HRD cell survival.

[0099] Inhibition of NMNAT1 kills PARP inhibitor and cisplatin-resistant BRCA1 / 2 mutant tumors, including those with somatic reversion of BRCA1 / 2 mutations. The inventors developed BRCA2 - / - Sequential exposure of RPE-1 to rucaparib induced resistance cells. The inventors did not observe HR repair in all induced clones, but resistance occurred due to fork stabilization. In those clones, NMNAT1 knockdown with siRNA was followed by drug-untreated BRCA2 - / - Colony formation activity was impaired to a level comparable to that of cells, but HRP RPE-1 was unharmed (Figure 4b).

[0100] Next, using a similar approach, we obtained several clones derived from the BRCA2-mutated pancreatic cancer cell line CAPAN-1. After becoming resistant to rucaparib, these clones did not restore HR, but rather developed resistance through other mechanisms that need further investigation. Knockdown of NMNAT1 impaired colonization activity in all CAPAN-1-induced resistant clones (Figure 4c). In summary, these data indicate that targeted NMNAT1 also kills PARPi-resistant HRD cells.

[0101] Nevertheless, HR repair via secondary mutations in the BRCA gene is, to date, the only mechanism of resistance to PARPi demonstrated in clinical practice. For this reason, the inventors evaluated the effect of NMNAT1 inhibition in HRD cells that have developed chemotherapy resistance due to secondary mutations in BRCA2, which repairs the open reading frame of the gene and thereby repairs HR. In particular, the inventors tested the chemotherapy-resistant HR-restored ovarian cancer cell line PEO4 together with BRCA2 mutant-versus-parent PEO1 cells, as well as five clones derived from long-term in vitro cisplatin exposure of CAPAN-1 cells, each carrying a different secondary mutation in the BRCA2 gene. Surprisingly, shRNA-mediated knockdown of NMNAT1 impaired the colony-forming ability of both PEO4 cells (Figure 4d) and resistant CAPAN-1 clones, suggesting that targeted NMNAT1 kills chemotherapy-resistant cells regardless of the mechanism of drug resistance.

[0102] Example 2: SIRT6 inhibition kills BRCA1 and BRCA2 mutant tumor cells. In addition to PARP1, nuclear NAD +Furthermore, sirtuins, a class of enzymes involved in many processes including DNA repair, are used to deacetylate and mono-ADP-ribosylate these substrates. To evaluate the roles of nuclear sirtuins (SIRT1, SIRT2, SIRT3, SIRT6, and SIRT7), nuclear sirtuins were silenced with specific siRNAs, and the PARPi response was tested. Deletion of SIRT6, rather than SIRT1, SIRT2, SIRT3, or SIRT7, sensitized cells to PARPi (Figure 5a).

[0103] To demonstrate these findings, the inventors used SIRT6 - / - RPE-1 cells were generated, and their survival in response to PARPi or BRCA2 knockdown was evaluated. While there was only a slight effect on RPE-1 cells, both PARPi and BRCA2 knockdown affected SIRT6 - / - The colony-forming activity of cells was impaired (Figure 5b, c). To determine that the catalytic activity of SIRT6 is important for the survival of HRD cells, the inventors used either wild-type or the previously described (data not shown) functionally dissociated SIRT6 mutant to stimulate SIRT6 - / - Cells were complemented. While S56Y SIRT6 lacking both activities did not rescue either PARPi-sensitive or BRCA2 synthetic lethal cells, it partially rescued the MAR-inactive mutant G60A and the deacetylase-inactive mutant R65A, but only to a slightly smaller extent than wild-type SIRT6 (data not shown). In summary, these data indicate that both catalytic activities of SIRT6 are essential for HRD cell survival via PARP-independent mechanisms.

[0104] Inhibition of SIRT6 kills PARP inhibitor and cisplatin-resistant BRCA1 / 2 mutant tumors, including those with somatic reversion of BRCA1 / 2 mutations. Despite the shocking cytotoxic effects of PARPi in BRCA mutant cells, resistance disturbances are ubiquitous in clinical settings, necessitating the development of alternative therapies for treating progressive disease. Our finding that SIRT6 inhibition kills HRD cells in a PARP1-independent manner suggests that targeting this axis would also address BRCA mutant cells that develop resistance to PARPi. To test this, the inventors used several cell models (repeatedly generating major known resistance mechanisms, including fork stabilization and HR repair) (Figure 6a).

[0105] Several clones were obtained from the BRCA2 mutant pancreatic cancer cell line CAPAN-1. After becoming resistant to rucaparib, these clones did not restore HR, but rather developed resistance through other mechanisms. Knockdown of SIRT6 impaired colonization activity in all CAPAN-1 induced resistant clones (Figure 6b). In summary, these data indicate that targeted SIRT6 also kills PARPi-resistant HRD cells.

[0106] Nevertheless, HR restoration via secondary mutations in the BRCA gene is, to date, the only mechanism of resistance to PARPi demonstrated in clinical practice. For this reason, the inventors evaluated the effect of SIRT6 inhibition in HRD cells that have developed chemotherapy resistance due to secondary mutations in BRCA2, which repairs the open reading frame of the gene and thereby repairs HR. In particular, the inventors tested the chemotherapy-resistant HR-restored ovarian cancer cell line PEO4 together with BRCA2 mutant-versus-parental PEO1 cells, as well as five clones derived from long-term in vitro cisplatin exposure of CAPAN-1 cells, each carrying a different secondary mutation in the BRCA2 gene. Surprisingly, shRNA-mediated knockdown of SIRT6 impaired the colony-forming ability of both PEO4 cells (Figure 6c) and resistant CAPAN-1 clones (Figure 6d), suggesting that targeted SIRT6 kills chemotherapy-resistant cells regardless of the mechanism of drug resistance.

[0107] The SIRT6 inhibitor OSS_128167 kills PARPi-untreated and resistant tumor cells. Inhibition of SIRT6 by OSS_128167 is BRCA2 - / - The colony-forming activity of RPE-1 cells was impaired, but the survival of parental HRP cells was not affected (Figure 7a), thus confirming the synthetic lethality between SIRT6 and BRCA. Furthermore, SIRT6 inhibitors also reduced PARPi-resistant BRCA2 to the same extent as in parental drug-untreated cells. - / - RPE-1 cells were also killed (Figure 7b). Similarly, SIRT6 inhibition also resulted in cell death of PARPi-resistant clones created by successive drug exposure of the BRCA2 mutant pancreatic cancer cell line CAPAN-1 (Figure 7c). Importantly, at the same dosage, OSS_128167 did not affect the colonization activity of normal mammary epithelial cells MCF10A, demonstrating that SIRT6 inhibition is not harmful to normal cells. In short, these data suggest that targeting SIRT6 catalytic activity with OSS_128167 may be a useful strategy for addressing PARP inhibitor-untreated and resistant HRD tumor cells.

[0108] SIRT6 inhibitors kill chemotherapy-resistant BRCA-associated cancer cells in an in vivo mouse model of patient-derived xenograft (PDX) BRCA1-mutated triple-negative breast cancer (TNBC) that is resistant to PARPi. The inventors used a patient-derived xenograft (PDX) HBCx-11 model established from PARPi-resistant BRCA1-mutated triple-negative breast cancer (TNBC). HBCx-11 tumors were transplanted into nude mice, which were then further classified into groups receiving either vehicle, PARPi, or a combination of PARPi and SIRT6i. PARPi alone had little effect on tumor regression (1 in 10 tumors responded), but the inventors found that adding SIRT6i to chemotherapy dramatically increased the tumor response (8 in 10 tumors) (Figure 8).

[0109] Example 3: UNG functions downstream of SIRT6 in the survival of BRCA1 / 2 mutations in parental (PARPi untreated) and PARPi-resistant tumors. To discover the mechanism by which SIRT6 supports HRD cell survival, the inventors attempted a comprehensive SILAC (stable isotope labeling with amino acids in cell culture)-based proteomics approach to identify novel SIRT6 targets. WT and SIRT6 - / - The comparison of nuclear proteomes is shown in SIRT6. - / - This leads to the identification of proteins whose expression is either downregulated or upregulated in cells (Figure 9a, b). SIRT6 may support genomic stability through the regulation of key DNA repair / replication proteins whose function is likely essential for HRD cell survival. To test this hypothesis, the inventors performed Gene Ontology (GO) analysis of our proteomics dataset and found that DNA glycosylase UNG is associated with SIRT6 - / - We observed that it was downregulated in the cells.

[0110] The inventors developed SIRT6 - / - They found that the cells exhibited more anucleotide-free regions within their genomes, which could be adapted to the reduced UNG activity in these cells (Figure 9c). Surprisingly, they found that deletion of either NMNAT1 or SIRT6 strongly sensitized the cells to FdUrd, a thymidylate synthase inhibitor, which led to uracil misuptake in the genome (Figure 9d). These data suggest defects in uracil removal (i.e., UNG activity) in the absence of NMNAT1 and / or SIRT6. Finally, they found that UNG deletion by siRNA affected the stability of the replication fork, as indicated by an increase in the number of ssDNA gaps (Figure 9f). It should be noted that ssDNA gaps also affect SIRT6. - / - Enhancement in cells further suggested that UNG is a target of SIRT6 (Figure 9e).

[0111] Having found that UNG activity is deficient in the absence of SIRT6, the inventors then wondered whether UNG inhibition phenotypicly mimics NMNAT1 / SIRT6 inhibition in responses to PARPi and HRD cell survival. They investigated RPE-1 P53 cells with UNG knockout or deficiency. - / - They found that the cells were sensitized to rucaparib compared to parent cells (Figure 10a). Next, they examined the role of UNG in the fitness of HRD cells, and they compared WT and BRCA2 - / - UNG was knocked down in both RPE-1 clones using two separate siRNAs, and cell viability was tested in a clonogenicity assay. The results showed that UNG silencing was effective against BRCA2 - / - This demonstrates that it kills clones but does not harm HR-non-deficient (HRP) cells (Figure 10b). Similarly, UNG - / - The cells were sensitive to BRCA2 deficiency (Figure 10c). Based on the data, they tested whether targeted UNG also kills HRD cancer cells that have acquired resistance to PARPi. For this purpose, they generated rucaparib-resistant ovarian cancer cells (OVCAR8: clone RR14) and evaluated their survival after UNG knockdown. They found that UNG siRNA kills both parental OVCAR8 and clone RR14 with acquired PARPi resistance. In addition, they found that UNG inhibition kills BRCA2 - / - We found that it kills PARPi-resistant clones derived from cells, as well as those derived from BRCA2 mutant CAPAN1 tumor cell lines (Figure 10d).

[0112] Overall, the data reveal a crucial role of UNG in HRD cell survival and cellular response to PARPi, which phenotypicly mimics SIRT6 / NMNAT1 function. Functional results suggest that targeted UNG may represent a potentially beneficial therapeutic approach for HRD tumors, including those with acquired PARPi resistance.

[0113] Example 4: UNG-mediated uracil removal is performed using nuclear NAD + It is adjusted by [the specified method]. The uracil DNA glycosylase SMUG1 has recently been shown to create single-stranded DNA (ssDNA) gaps following inhibition of translation synthesis. Therefore, the inventors investigated whether basal gDNA-uracil processing by either UNG or SMUG1 also results in ssDNA gap formation. They quantified ssDNA gaps using a modified DNA fiber assay based on the use of ssDNA-specific endonuclease S1. The inventors found that siRNA-mediated deficiency of UNG slowed the replication fork (RF) speed and resulted in post-replication ssDNA gaps, while SMUG1 deficiency did not. Nevertheless, SMUG1 knockdown rescued the ssDNA gaps observed with UNG loss, demonstrating that SMUG1 creates gaps in the absence of UNG (Figure 11a). Typically, ssDNA gaps arise from replication repriming following impairment that hinders RF progression. The inventors hypothesized that the observed SMUG1-mediated gap formation was due to SMUG1 binding to gDNA-uracil accumulation in the absence of UNG, which could lead to a cycle of RF stall and repriming.

[0114] To investigate this hypothesis, the inventors developed the RPE-1TP53 - / - UNG knockout in cells (UNG - / -They created a 2D molecule and examined the distribution of the two UDGs in the soluble (nucleosol) and insoluble (chromatin) fractions. To force dU misincorporation in gDNA without inducing thymidine starvation, they combined a low dose of the thymidylate synthase (TS) inhibitor 5-fluorodeoxyuridine (FdUrd) with the dUTPase inhibitor TAS-114 (Figure 11b). The low dose of FdUrd (0.1 μM) that we used in part inhibited TS, thereby moderately reducing the dTTP pool, but—combined with TAS-114 which simultaneously increased dUTP levels—it resulted in gDNA-uracil accumulation without cell cycle arrest (Figure 15a left panel and 15b). Due to the increase in gDNA-uracil, the inventors were able to induce UNG - / - The absence of UNG recruitment to chromatin in cells was accompanied by the continued retention of SMUG1 in chromatin, whereas in WT cells, we observed accumulation of SMUG1 in the soluble nuclear fraction (Figure 11c).

[0115] UNG and SMUG1 are nuclear NADs. + They interact during controlled uracil removal. To investigate the underlying mechanism of interaction between UNG and SMUG1, the inventors investigated nuclear NAD, a molecule with an essential—yet not yet fully characterized—function in DNA repair. + The role of (nicotinamide adenine dinucleotide) was investigated. In the nucleus of mammalian cells, NAD + It is synthesized by NMNAT1 (nicotinamide mononucleotide adenylyltransferase 1) and stimulates the activity of PARP1 and sirtuins (Figure 15c). The inventors have developed an NMNAT1 knockout (NMNAT1 - / - RPE-1 cells were generated, and their sensitivity to a panel of DNA damage reagents was evaluated (Table 1). NMNAT1 - / -The cells were more sensitive to drugs targeting the TS pathway (i.e., FdUrd and MTX) compared to WT cells, and were further sensitized to TS inhibitors when combined with TAS-114 (Figures 15d-f). NMNAT1 - / - The observation that cells were more than 100 times more sensitive to simultaneous treatment with FdUrd+TAS-114 compared to WT cells suggests that nuclear NAD plays a role in regulating gDNA-uracil homeostasis, rather than being part of a general response to replication stress. + This more strongly suggests a specific role of (Figure 15f). Further strengthening this, the inventors proposed -UNG - / - Cells, as well as -NMNAT1 - / - We found that cells exhibit enhanced gDNA-uracil and reduced viability due to DUT targeting, either through siRNA-mediated knockdown (siDUT) or pharmacological inhibition (TAS-114) (Figure 15g-i).

[0116] [Table 1]

[0117] Nuclear NADs in the control of uracil removal + To further investigate its role, the inventors used a modified alkaline COMET assay in which permeabilized nuclei were incubated with recombinant UNG (rUNG), and the resulting DNA damage was used as a measure of gDNA-uracil, thereby comparing WT and NMNAT1 - / - The gDNA-uracil in cells was quantified. The inventors observed an increase in uracil content after treatment with a high dose of FdUrd (20 μM), which led to cell cycle arrest in both cell lines due to thymidine starvation (Figure 16a). This increase was attributed to NMNAT1 - / - This is more pronounced in WT than in cells, and NMNAT1 - / -This tended to be due to reduced cell proliferation (and consequently a lower rate of dU misuptake). Nevertheless, after drug washout, uracil content returned to the baseline level of wild-type (WT) cells, but on the other hand, it was NMNAT1 - / - Further increases were observed in cells (Figure 11d).

[0118] NMNAT1 - / - Uracil accumulation in cells is due to a causal relationship between persistent dU misuptake and defective uracil removal. Mass spectrometry-based metabolic analysis compares NMNAT1 levels with those of WT cells. - / - We revealed a slight increase in free dTTP and a decrease in dUMP in this region, eliminating the higher probability of dU misincorporation as the cause of the increase in gDNA-uracil (Figure 16b).

[0119] The nuclear isoform of UNG2 migrates along the active replication fork (RF) along with PCNA and RPA, and then cleaves newly incorporated uracil during DNA replication. Therefore, nuclear NAD + To test the possibility that RF specifically modulates the presence of UNG2, the inventors performed immunoblotting analysis of UNG after protein isolation using nascent DNA (iPOND). The inventors compared NMNAT1 with WT cells. - / - We observed a significant decrease in UNG2 levels in cells with active RF, while PCNA levels remained unchanged (Figure 11e). Furthermore, FdUrd treatment induced the recruitment of the nuclear isoform UNG2 into chromatin, which in turn triggered NMNAT1 - / - Loss occurred in the cells (Figure 15j, upper panel). This was not due to differences in either TS inhibition or cell cycle distribution (Figure 15j).

[0120] In summary, these data reveal important interactions between two major UDGs required for maintaining genomic stability. UNG is important for efficient DNA replication, and in its absence, the resulting accumulation of gDNA-uracil is linked to SMUG1 on chromatin. Nevertheless, attempts at neutralization engineered by SMUG1 lead to stalled forks, accumulation of ssDNA post-replicative gaps, and genetic instability. Finally, the inventors found that this interaction is regulated by nuclear NAD + and that it maintains the presence of UNG in RF and the suppression of SMUG1 in non-replicating chromatin to ensure effective uracil removal.

[0121] SMUG1 creates DSBs during replication in the absence of UNG-mediated uracil removal. The inventors next investigated the causal relationship of gDNA-uracil accumulation to genomic stability. In cells lacking either NMNAT1 or UNG, increased gDNA-uracil by TAS-114 induced exclusive DNA double-strand break (DSB) formation during DNA replication (as shown by the increased number of γH2AX foci in EdU-positive cells) (Figs. 11f and 16c, d). These data are in stark contrast to previous investigations showing that the mere presence of uracil in DNA does not have detrimental consequences for RF progression and genomic stability. Thus, the inventors hypothesized that rather than the presence of uracil, recognition and / or removal of uracil by other UDGs leads to toxic DNA intermediates that impair RF progression in cells with reduced UNG.

[0122] UNG - / - Similar to what was observed in cells, the inventors found that impairment of UNG mobilization to chromatin in NMNAT1 - / - cells was accompanied by persistent retention of SMUG1 on chromatin (Fig. 16e). Notably, the inventors found that knockdown of SMUG1 restored NMNAT1 to gDNA-uracil enhancing drugs - / -We found that it completely rescued the cellular sensitivity, whereas knockdown of other human UDGs did not (Figure 11g).

[0123] In short, these data indicate that when UNG-mediated removal of uracil is lost or reduced, chromatin capture by SMUG1 interferes with RF progression, leading to ssDNA gap and DSB formation, which in turn results in increased sensitivity to gDNA-uracil-induced drugs.

[0124] Example 5: UNG inhibition leads to SMUG1-mediated DNA damage and HR activation. Overcoming UNG removal ability leads to the capture of SMUG1-HMCES on chromatin, continuous PARP1 overactivation, and HR activation. To investigate the mechanism by which SMUG1 exerts its toxicity in the absence of UNG, the inventors investigated UNG - / - Cells were inoculated with the gDNA-uracil-enhancing drug (FdUrd+TAS-114) as an antigen. Cell cycle analysis showed that gDNA-uracil accumulation was observed in WT and UNG cells. - / - We revealed that it slows down DNA replication in both RPE-1 cell lines. However, UNG - / - Unlike WT cells, the cells were unable to resume DNA replication after drug washout, as quantified by the percentage of BrdU-negative cells (Figure 12a).

[0125] SMUG1 has a stronger affinity than UNG for the aprine / apyrimidine (AP) site created by uracil excision. Therefore, persistent binding of SMUG1 to the AP site would attenuate their processing through base excision repair (BER) and impede RF progression. Notably, the inventors found that the capture of SMUG1 on chromatin is related to its 5-hydroxymethylcytosine (5hmC) binding, ES cell-specificity (HMCES), and the recently described AP site sensor (Figure 12b).

[0126] HMCES covalently binds to AP sites during replication at ssDNA-dsDNA junctions (i.e., RFs) to form stable DNA-protein crosslinks (DPCs), and it protects AP sites from aberrant processing to maintain the integrity of the genome. However, HMCES levels at RFs only slightly increase in UNG - / - cells, suggesting that the observed HMCES retention mainly occurs in non-replicating chromatin (Figure 17a). Whether HMCES forms DPCs in UNG - / - cells was evaluated by the inventors by using a rapid approach to the DNA adduct recovery (RADAR) assay to quantify DNA-protein adducts. Notably, UNG - / - cells showed higher levels of HMCES-DNA adducts both at basal levels and after drug exposure (Figure 12c).

[0127] Next, the inventors investigated whether the increases in SMUG1 and HMCES occur in the same genomic regions (i.e., AP sites). To date, it has not been reported that HMCES and SMUG1 form foci. Surprisingly, the inventors detected large-scale formation of co-localization foci of SMUG1 and HMCES only after dU misincorporation in UNG - / - cells, and it was suggested that HMCES crosslinks to AP sites created by SMUG1 (Figures 12d and 17b). To confirm this, the inventors knocked down SMUG1 in UNG - / - cells and evaluated chromatin-bound HMCES levels. SMUG1 deficiency completely abolished HMCES recruitment to chromatin, confirming that HMCES is associated with SMUG1-generated AP sites (Figure 12e).

[0128] For proper uracil processing by BER, the AP site must be cleaved by APE1, an AP site-selective endonuclease. The inventors found that retention of SMUG1 and HMCES on chromatin is associated, on the one hand, with defective recruitment of APE1, and on the other hand, with prolonged PARP1 activation (identified by ADP-ribosylation) and enhanced DSB formation (identified by γH2AX) (Figure 12b). The inventors then reasoned that the prolonged activation of PARP1 and chromatin retention resulting from SMUG1 capture would sensitize cells to PARPi. Based on this observation, the inventors found that UNG deficiency sensitizes HR-non-deficient (HRP) cells to the PARPi rucaparib, and that concomitant SMUG1 deficiency rescues drug sensitization (Figure 12f).

[0129] Defective APE1 recruitment suggests that BER cannot contact and process SMUG1 / HMCES-binding DNA damage. Notably, we found that UNG cells, compared to WT cells, - / - We found the basis for RAD51 focus (measurement of HR activity) and increased gDNA-uracil-mediated activity in (Figure 12g left). Importantly, most SMUG1 focus coexists with RAD51 and γH2AX (Figure 12g right), and SMUG1 deficiency is associated with UNG - / - ADP-ribosylation and γH2AX induction were eliminated in cells (Figure 12e). This suggests that the HR pathway is activated to rescue DNA damage caused by SMUG1 capture. These results indicate that in the absence of effective uracil removal by UNG during RF, SMUG1-producing AP sites are protected by HMCES, and their processing by APE1 via BER is impaired. Therefore, persistent SMUG1 / HMCES-DNA adducts create impairments in RF progression and lead to DNA damage that triggers HR-mediated repair. From this perspective, SMUG1 capture does not offset UNG loss, but rather leads to genomic instability and death in cells with high gDNA-uracil levels.

[0130] dU misuptake leads to low replication of DNA and chromosomal fragmentation in HR-deficient cells. Next, the inventors investigated the causal relationship between SMUG1-HMCES capture in misuptake dU and the fitness of BRCA1 / 2-deficient (i.e., HRD) cells. The inventors used RPE-1TP53 - / - BRCA2 previously produced in cells - / - Clones (C1 and C2) were used (Figure 18a, b).

[0131] To enhance the replication stress induced by dU, the inventors combined TAS-114 with a low dose of FdUrd. First, they found that BRCA2 was affected by the induction of immature chromatin aggregation by calcium phosphate A upon dU misuptake. - / - Chromosome breaks in cells were measured. Multicolor FISH was used to analyze BRCA2 - / - We demonstrated that FdUrd+TAS-114 treatment of cells induces a strong increase in chromosomal rearrangement, including rupture and fragmentation (Figure 13a). Chromosomal fragmentation due to immature DNA condensation tends to arise from low-replicating DNA (UR-DNA) regions. We showed that cell division initiation in the presence of UR-DNA initiates mitotic DNA synthesis (MiDAS). UR-DNA that escapes MiDAS resolution forms late DNA crosslinks known as ultrafine (UFB) crosslinks, which can then be inherited by daughter G1 cells through inclusion in the 53BP1 nucleolus (53BP1 NB) and micronucleus. The inventors also found BRCA2 - / - We observed that simultaneous treatment of cells with FdUrd + TAS-114 resulted in a 3- to 4-fold increase in UFB, 53BP1 NB, and the number of micronuclei, as quantified in the late and cyclin A-negative G1 phases, respectively (Figure 13b, c). In addition, UNG deficiency was associated with BRCA2 deficiency in response to low doses of TAS-114. - / - We sensitized the cells, and it was also rescued by SMUG1 knockdown, as well as by none of the other UDGs, further reinforcing our data that SMUG1 capture creates DNA damage that activates HR-mediated repair (Figure 13d).

[0132] In short, these data suggest that fine-tuning the UNG / SMUG1 interaction during uracil removal is crucial for maintaining genomic stability in BRCA2-deficient cells.

[0133] Example 6: Targeting uracil removal with UNG kills HR-deficient tumors regardless of their resistance to PARPi. Synthetic lethality between UNG inhibition and BRCA1 / 2 mutations. Next, the inventors evaluated the potential synthetic lethality between UNG deficiency and BRCA1 / 2 mutations. The inventors found that inhibition of UNG (or NMNAT1) by siRNA, CRISPR sgRNA, or the peptide inhibitor UGI was associated with BRCA2 - / - We found that it selectively kills cells (clones C1 and C2) (Figures 14a and 19a, b). Similarly, siRNA-mediated BRCA1 / 2 deficiency leads to NMNAT1 - / - and UNG - / - The clonal survival of cells was selectively impaired (Figure 19c, e). In particular, the inventors observed that loss of UNG (or NMNAT1) killed HR-deficient cancer cell lines but was unharmed in HRP cancer cells, and further confirmed the synthetic lethal interactions between UNG and BRCA1 / 2, and between NMNAT1 and BRCA1 / 2 (Figure 19f, g). Finally, SMUG1 deficiency rescued the synthetic lethality between BRCA2 and NMNAT1 / UNG (Figure 14B).

[0134] In short, these data indicate that the accumulation of gDNA-uracil, either due to increased dU misuptake or inhibition of UNG-mediated removal, leads to SMUG1-dependent UR-DNA, chromosomal fragmentation, and ultimately HRD cancer cell death.

[0135] Targeted UNG-mediated uracil removal kills a subset of PARPi-resistant HR-deficient tumors. Despite the initial success of PARPis in treating BRCA-deficient tumors, the development of resistance has become a clinical challenge. The inventors investigated the potential of targeted uracil removal as a novel strategy to overcome PARPi resistance. The inventors targeted two different BRCA2 deficiencies against rucaparib. - / - Continuous exposure to RPE-1 clones induced PARPi-resistant clones. First, they confirmed that these generated clones possessed drug resistance, and we found that this was related to enhanced RF stability (Figure 20a, b).

[0136] To evaluate whether disruption of UNG-mediated uracil removal is a beneficial strategy for killing PARPi-resistant HRD cells, the inventors generated several PARPi-resistant clones from the BRCA1-deficient (promoter-highly methylated) ovarian cell line OVCAR8 and the pancreatic BRCA2 mutant cell line CAPAN1. None of the CAPAN1 clones acquired PARPi resistance via HR repair, as quantified by RAD51 focus formation (Figure 20c, d). Clonal survival assays showed that knockdown of either UNG or NMNAT1 clearly reduced the survival of all PARPi-resistant HRD cancer cells tested (Figures 14c and 20e, f). Next, to apply these findings to the translational environment, the inventors utilized organoids obtained from patient-derived xenografts (PDX) established from triple-negative breast cancer (TNBC). Finally, the inventors evaluated the effect of -NMNAT1 knockdown on -UNG inhibition on PARPi-resistant HRD tumor growth in vivo. PARPi-resistant BRCA1-deficient tumor cells (OVCAR8, clone 14) expressing either doxycycline-inducible NMNAT1 or scrambled (Scr)shRNA were xenotransplanted into athymic nude mice. Tumor growth was observed under PARPi treatment and / or NMNAT1 deficiency. PARPi slowed the growth of parental OVCAR8 tumors but did not affect the growth of PARPi-resistant clones (Figure 20f). Nevertheless, NMNAT1 deficiency significantly impaired the growth of PARPi-resistant OVCAR8 tumors and increased the survival rate of tumor-bearing mice (Figure 20h, i).

[0137] In summary, these data indicate that a subset of PARPi-resistant HRD tumors, including some with acquired RF stability or HR repair, retain susceptibility to gDNA-uracil accumulation. Therefore, the inventors demonstrate that UNG-mediated uracil removal represents a novel vulnerability in HRD tumors that may overcome PARPi resistance.

Claims

1. A uracil DNA glycosylase (UNG) inhibitor for use in the treatment of resistance homologous recombination repair deficiency (HRD) cancer, wherein the UNG inhibitor is a molecule capable of silencing a gene expressing the UNG enzyme or a molecule that inhibits the properties of the UNG enzyme.

2. The UNG inhibitor for use according to claim 1, wherein the resistant HRD cancer is a resistant BRCA-associated cancer, a chemotherapy-resistant HRD cancer, a chemotherapy-resistant BRCA-associated cancer, or a metastatic resistant HRD cancer.

3. The UNG inhibitor for use according to claim 1, wherein the resistant HRD cancer is a PARPi-resistant BRCA-associated cancer or a cisplatin-resistant BRCA-associated cancer, including those with somatic reversion of BRCA mutations and HR repair.

4. A UNG inhibitor for use according to any one of claims 1 to 3, wherein the UNG inhibitor is an organic small molecule, polypeptide, aptamer, oligonucleotide, or antibody.

5. The UNG inhibitor for use according to claim 4, wherein the oligonucleotide is an antisense oligonucleotide, siRNA, shRNA, DNA aptamer, or RNA aptamer.

6. A UNG inhibitor for use according to any one of claims 1 to 5, in combination with a PARP inhibitor.

7. The UNG inhibitor for use according to claim 6, wherein the PARP inhibitor is selected from the group consisting of olaparib, lucaparib, niraparib, talazoparib, iniparib, veliparib, pamiparib (BGB-290), CEP9722, E7016, E7449, and 3-aminobenzamide.

8. A UNG inhibitor for use according to any one of claims 1 to 5, in combination with cisplatin.

9. A pharmaceutical composition comprising a uracil DNA glycosylase (UNG) inhibitor for use in the treatment of resistant homologous recombination repair deficiency (HRD) cancer in a subject requiring such treatment, and a pharmaceutically acceptable carrier, wherein the UNG inhibitor is a molecule that enables silencing of a gene expressing the UNG enzyme or a molecule that inhibits the properties of the UNG enzyme.

10. The pharmaceutical composition for use according to claim 9, wherein the resistant HRD cancer is a resistant BRCA-associated cancer, a chemotherapy-resistant HRD cancer, or a chemotherapy-resistant BRCA-associated cancer.

11. A pharmaceutical composition comprising a uracil DNA glycosylase (UNG) inhibitor, comprising a PARP inhibitor and a pharmaceutically acceptable carrier.

12. A method for treating a patient with a resistant homologous recombination repair deficiency (HRD) cancer, such as a resistant BRCA-associated cancer, a chemotherapy-resistant HRD cancer, or a chemotherapy-resistant BRCA-associated cancer, the method comprising administering a therapeutically effective amount of a uracil DNA glycosylase (UNG) inhibitor to the patient.

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