DUT inhibition in homologous recombination deficiency cancer

Abstract

The invention relates to methods and pharmaceutical compositions for use of a deoxyuridine 5'-triphosphate nucleotidohydrolase (DUT) inhibitor for the treatment of resistant HRD cancer, particularly resistant BRCA-associated cancer.

Description

[0001] DUT INHIBITION IN HOMOLOGOUS RECOMBINATION DEFICIENCY CANCER

[0002] FIELD OF THE INVENTION

[0003] The invention relates to methods and pharmaceutical compositions for use of a deoxyuridine 5'-triphosphate nucleotidohydrolase (DUT) inhibitor for the treatment of resistant HRD cancer, particularly resistant BRCA-associated cancer.

[0004] BACKGROUND OF THE INVENTION

[0005] In the last decades, many efforts have been undertaken to gain mechanistic insights into the synthetic lethality between PARP1 and BRCA1 / 2 genes (Maya-Mendoza et al., Nature 559, pages 279-284, 2018; Hanzlikova et al., Nature 559, pages 279-284, 2018). Deficiencies in homologous recombination (HR)-mediated DNA repair occur mainly through genetic inactivation of BRCA1 and BRCA2 (BRCA1 / 2) genes and play a role in the initiation and progression of many tumor types. Defective HR (HRD) causes genomic instability and hyper-dependence on alternative DNA repair mechanisms for survival, setting the stage for synthetic lethality-based targeted therapy, 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 is successfully exploited in the clinic since the approval of PARP inhibitors (PARPi) for the treatment of BRCA1 / 2-mutated tumors (Patel et al., Oncogene 40, pages 3001-3014, 2021), also referred as homologous recombination (HR)-deficient tumors (HRD). Various trials have been devised to evaluate PARPi effectiveness for patients with breast, ovarian, and prostate tumors harboring BRCA1 / 2 mutations (Pujade-Lauraine et al., The Lancet. Oncology 18, Issue 9, pages 1274-1284, 2017). However, PARPi and other chemotherapeutics have shown limited effectiveness in achieving HRD cancer remission, notably because drug resistance emerges and resistance to chemotherapy is emerging as the major obstacle to clinic effectiveness (Gogola et al., Annual Review of Cancer Biology 3, pages 235-254, 2019). Hence, no therapeutic options are left for these patients, stressing the need for alternative therapeutic options (Konstantinopoulos et al., Cancer Discov 5, pages 1137-1154, 2015).

[0006] Enzymes preventing DNA damage also contribute to chemotherapy resistance. Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is a catabolic enzyme in the pyrimidine metabolic pathway that degrades deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dllMP) and prevents the incorporation of aberrant nucleosides such as deoxyuridine (dll) into DNA. Therefore, dUTPase maintains the intracellular dUTP pool at an extremely low level to minimize deoxyuridine misincorporation into DNA. Once deoxyuridine is misincorporated into DNA, the respective base uracil gets efficiently excised from DNA by uracil-DNA glycosylases (UDGs) such as UNG or SMUG1 as a backup mechanism.

[0007] Uracil-DNA glycosylase (UNG) is a protein member of the uracil-DNA glycosylases family. One important function of uracil-DNA glycosylases is to prevent mutagenesis by eliminating uracil from DNA molecules by cleaving the N-glycosylic bond and initiating the base-excision repair (BER) pathway. UNG is an important player in DNA damage recognition and repair of uracil accumulation in telomeres (Baquero et al, Mol Oncol 13, Issue 5, pages 1110-1120, 2019). UNG initiates BER activity, and its depletion has been reported to restore sensitivity to some DNA damaging agents such as stalled fork inducing agents in BRCA2-deficient cells (Pathania, Grantome NIH, project 1 R15CA235436-01A1). In addition, UNG knockdown can induce apoptosis in prostate cancer cell lines, reduce cell proliferation and increase cellular sensitivity to genotoxic stress. Others have observed that colon cancer cells lacking UNG can be hypersensitive to pemetrexed-induced uracil accumulation, resulting in 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).

[0008] The inventors have demonstrated that DUT, the only human dUTPase, is also synthetically lethal with BRCA1 / 2 deficiency. Inhibition of DUT reduces survival of HRD cells including those showing resistance to PARPi. The inventors have demonstrated that DUT inhibition leads to SMUG1-dependant damage, which synergizes with PARP inhibition to kill cancer cells. DUT inhibition thus sensitizes cancer cells to PARPi, in particular PARPi resistant HRD tumors.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention relates to a DUT inhibitor for use in the treatment of resistant Homologous Recombination Deficiency (HRD) cancer.

[0011] The present invention also relates to a pharmaceutical composition comprising a DUT inhibitor and a pharmaceutical acceptable carrier for use in the treatment of resistant HRD cancer in a subject in need thereof. The present invention also relates to a pharmaceutical composition comprising a DUT inhibitor in combination with a PARPi or UNG inhibitor (UNGi) and a pharmaceutical acceptable carrier.

[0012] The present invention further relates to a method of treating resistant HRD cancer such as resistant BRCA-associated cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a DUT inhibitor.

[0013] DETAILED DESCRIPTION OF THE INVENTION

[0014] The inventors investigated the role of DUT in HRD cancer, particularly in PARPi- resistant BRCA-associated cancer.

[0015] The inventors have demonstrated that DUT is synthetically lethal with BRCA1 / 2 deficiency through its function in genomic stability and that DUT inhibition leads to SMUG1- dependant damage such as accumulation of double-stand breaks (DSBs) in replication forks (RF). PARPi-resistant cells have been associated with increased RF stability. The inventors have demonstrated that DUT inhibition sensitizes PARPi-resistant cells by causing DSB at RF similarly to what is observed in PARPi-sensitive cells.

[0016] The present invention highlights the potential of targeting uracil misincorporation as a novel strategy to overcome PARPi resistance and the use of DUT inhibitors in the treatment of resistant HRD cancer, particularly resistant BRCA-associated cancer and BRCA-associated cancer with acquired drug resistance to mono- or combination therapy with PARPi.

[0017] Accordingly, the invention relates to the targeting of DUT in the treatment of resistant HRD cancer, particularly resistant BRCA-associated cancer and PARPi-resistant BRCA- associated cancer.

[0018] DUT inhibitors

[0019] The present invention thus relates to a DUT inhibitor for use in the treatment of resistant Homologous Recombination Deficiency (HRD) cancer. The DUT inhibitor is a molecule capable of silencing the gene expressing the DUT enzyme, or a molecule inhibiting DUT enzymatic properties.

[0020] In some embodiments, the invention relates to a DUT inhibitor for use in the treatment of HRD cancer, wherein the resistant HRD cancer is resistant BRCA-associated cancer.

[0021] In some embodiments, the invention relates to a DUT inhibitor for use in the treatment of HRD cancer, wherein the resistant HRD cancer is PARPi resistant BRCA-associated cancer. Said PARPi resistant BRCA-associated cancer may include those with somatic reversion of the BRCA mutation and HR restoration.

[0022] As used herein, the terms “subject”, “individual” or “patient” are interchangeable and refer to a mammal. Typically, a subject according to the invention refers to any subject, preferably human. In a particular embodiment, the term “subject” refers to a subject afflicted or at risk to be afflicted with cancer. In a particular embodiment, the term “subject” refers to a subject afflicted or at risk to be afflicted with HRD cancer, particularly BRCA-associated cancer. In a particular embodiment, the term “subject” refers to a subject afflicted or at risk to be afflicted with resistant HRD cancer. In some embodiments, the term “subject” refers to a subject afflicted or at risk to be afflicted with resistant BRCA-associated cancer. In some embodiments, the term “subject” refers to a subject afflicted or at risk to be afflicted with chemo-resistant HRD cancer. In some embodiments, the term “subject” refers to a subject afflicted or at risk to be afflicted with chemo-resistant BRCA-associated cancer. In some embodiments, the term “subject” refers to a subject afflicted or at risk to be afflicted with metastatic resistant HRD cancer.

[0023] In some embodiments, the term “subject” refers to a subject afflicted or at risk to be afflicted with chemo-resistant BRCA-associated cancer such as chemo-resistant HRD cancer and / or BRCA-deficiency cancer (basal-like, luminal, and HER2-overexpressing breast carcinomas and other cancers) and breast, ovarian, prostate, pancreatic or any other type of tumors harboring BRCA1 / 2 mutations or BRCA expression deficiency. In a particular embodiment, the term “subject” refers to a subject afflicted or at risk to be afflicted with PARPi resistant BRCA-associated cancer or cisplatin resistant BRCA-associated cancer.

[0024] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0025] As used herein, the term “cancer” refers to any cancer that may affect any one of the following tissues or organs: breast; liver; kidney; heart, mediastinum, pleura; floor of mouth; lip; salivary glands; tongue; gums; oral cavity; palate; tonsil; larynx; trachea; bronchus, lung; 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, ureter; rectosigmoid junction; anus, anal canal; skin; bone; joints, articular cartilage of limbs; eye 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 gland; thyroid gland; endocrine glands and related structures; female genital organs such as ovary, uterus, cervix uteri; corpus uteri, vagina, vulva; male genital organs such as penis, testis and prostate gland; hematopoietic and reticuloendothelial systems; blood; lymph nodes; thymus.

[0026] The term “cancer” according to the invention comprises leukemias, seminomas, melanomas, teratomas, lymphomas, non-Hodgkin lymphoma, neuroblastomas, gliomas, adenocarninoma, 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 cancer, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma and paraganglioma), skin cancer (including malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi’s sarcoma, keratoacanthoma, moles, dysplastic nevi, lipoma, angioma and dermatofibroma), nervous system cancer, brain cancer (including astrocytoma, medulloblastoma, glioma, lower grade glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors, spinal cord neurofibroma, glioma or sarcoma), skull cancer (including osteoma, hemangioma, granuloma, xanthoma or osteitis deformans), meninges cancer (including meningioma, meningiosarcoma or gliomatosis), head and neck cancer (including head and neck squamous cell carcinoma and oral cancer (such as, e.g., buccal cavity cancer, lip cancer, tongue cancer, mouth cancer or pharynx cancer)), lymph node cancer, gastrointestinal cancer, liver cancer (including hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma and hemangioma), colon cancer, stomach or gastric cancer, esophageal cancer (including squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma or lymphoma), colorectal cancer, intestinal cancer, small bowel or small intestines cancer (such as, e.g., adenocarcinoma lymphoma, carcinoid tumors, Karposi’s sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma or fibroma), large bowel or large intestines cancer (such as, e.g., adenocarcinoma, tubular adenoma, villous adenoma, hamartoma or leiomyoma), pancreatic cancer (including ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors or vipoma), ear, nose and throat (ENT) cancer, breast cancer (including HER2-enriched breast cancer, luminal A breast cancer, luminal B breast cancer and triple negative breast cancer), cancer of the uterus (including endometrial cancer such as endometrial carcinomas, endometrial stromal sarcomas and malignant mixed Mullerian tumors, uterine sarcomas, leiomyosarcomas and gestational trophoblastic disease), ovarian cancer (including dysgerminoma, granulosa-theca cell tumors and Sertoli-Leydig cell tumors), cervical cancer, vaginal cancer (including squamous-cell vaginal carcinoma, vaginal adenocarcinoma, clear cell vaginal adenocarcinoma, vaginal germ cell tumors, vaginal sarcoma botryoides and vaginal melanoma), vulvar cancer (including squamous cell vulvar carcinoma, verrucous vulvar carcinoma, vulvar melanoma, basal cell vulvar carcinoma, Bartholin gland carcinoma, vulvar adenocarcinoma and erythroplasia of Queyrat), genitourinary tract cancer, kidney cancer (including clear renal cell carcinoma, chromophobe renal cell carcinoma, papillary renal cell carcinoma, adenocarcinoma, Wilm’s tumor, nephroblastoma, lymphoma or leukemia), adrenal cancer, bladder cancer, urethra cancer (such as, e.g., squamous cell carcinoma, transitional cell carcinoma or adenocarcinoma), prostate cancer (such as, e.g., adenocarcinoma or sarcoma) and testis cancer (such as, e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors or lipoma), lung cancer (including small cell lung carcinoma (SCLC), non-small cell lung carcinoma (NSCLC) including squamous cell lung carcinoma, lung adenocarcinoma (LLIAD), and large cell lung carcinoma, bronchogenic carcinoma, alveolar carcinoma, bronchiolar carcinoma, bronchial adenoma, lung sarcoma, chondromatous hamartoma and pleural mesothelioma), sarcomas (including Askin's tumor, sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and soft tissue sarcomas), soft tissue sarcomas (including alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, plexiform fibrohistiocytic tumor, rhabdomyosarcoma, synovial sarcoma and undifferentiated pleomorphic sarcoma, cardiac cancer (including sarcoma such as, e.g., angiosarcoma, fibrosarcoma, rhabdomyosarcoma or liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma), bone cancer (including osteogenic sarcoma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing’s sarcoma, malignant lymphoma and reticulum cell sarcoma, multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma, osteocartilaginous exostoses, benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumors), hematologic and lymphoid cancer, blood cancer (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma and myelodysplasia syndrome), Hodgkin’s disease, non-Hodgkin’s lymphoma and hairy cell and lymphoid disorders, and the metastases thereof.

[0027] The term “homologous recombination deficiency cancer” or “HRD cancer” has its general meaning in the art and refers to cancer displaying defective homologous recombination (HRD)-mediated DNA repair which causes genomic instability and hyperdependence on alternative DNA repair mechanisms for survival. Thus, the term “HRD cancer” refers to a cancer displaying alteration of genes including BRCA1 , BRCA2, PALB2, CHEK2, ATM, BARD1 , RAD51C, RAD51 D, FANCC, BRIP1 , FANCM, XRCC2 or any other genes involved in HR DNA repair pathway. The HR status of a cancer can be determined by any well-known methods in the art, for example by probing the genome for HRD-related genomic signatures or by using a commercial HRD diagnostic test such as Myriad myChoice®. The term “HRD cancer” include but is not limited to BRCA-associated cancer. Conversely, a BRCA-associated cancer is not necessarily a HRD cancer.

[0028] The term “resistant HRD cancer” has its general meaning in the art and refers to HRD cancer resistant to treatment such as HRD cancer resistant to chemotherapy, radiotherapy and other cancer therapy. The term “resistant HRD cancer” also refers to resistant BRCA-associated cancer, chemo-resistant HRD cancer, chemo-resistant BRCA- associated cancer such as PARP inhibitor (PARPi) resistant BRCA-associated cancer, PARPi-resistant HRD tumors including tumors with somatic reversion of BRCA1 / 2 mutation and subsequent HR restoration. In some embodiments, the term “resistant HRD cancer” refers to metastatic resistant HRD cancer.

[0029] The term “BRCA-associated cancer” has its general meaning in the art and refers to cancer associated with BRCA mutation, or BRCA expression deficiency. The term “BRCA- associated cancer” refers to cancer selected from cancer associated with BRCA1 and / or BRCA2 mutation, cancer associated with BRCA1 and / or BRCA2 expression deficiency, homologous recombination deficiency (HRD) cancer and / or BRCA-deficiency cancer (such as basal-like, luminal, and HER2-overexpressing carcinomas, breast, ovarian, and prostate tumors harboring BRCA1 / 2 mutations and other cancers). The term “BRCA-associated cancer” does not refer to a cancer with a functional BRCA1 / 2. In some embodiments, the term “BRCA-associated cancer” refers to breast cancer, ovary cancer, cervix cancer, pancreas cancer, IDIIT cancer, head and neck cancer and melanoma with BRCA1 and / or BRCA2 mutation, or BRCA1 and / or BRCA2 expression deficiency. In some embodiments, the term “BRCA-associated cancer” refers to metastatic BRCA-associated cancer. As used herein “BRCA1 / 2” denotes BRCA1 and / or BRCA2, more particularly BRCA1 and BRCA2. In some embodiments, the BRCA-associated cancer is a cancer with a BRCA1 expression deficiency. In some embodiments, the BRCA-associated cancer is a cancer with a BRCA2 expression deficiency. In some embodiments, the BRCA-associated cancer is a cancer with BRCA1 and BRCA2 expression deficiencies. Said BRCA1 or BRCA2 expression deficiency may be caused, respectively, by a BRCA1 or BRCA2 mutation.

[0030] The term “PARP inhibitor” or “PARPi” has its general meaning in the art and refers to PARP inhibitor such as olaparib, rucaparib, niraparib and talazoparib. The term “PARP inhibitor” also refers to PARP inhibitor such iniparib, veliparib, Pamiparib (BGB-290), CEP 9722, E7016 and 3-Aminobenzamide.

[0031] The term “DUT” refers to the human deoxyuridine 5'-triphosphate nucleotidohydrolase (dllTPase), also known as dllTP pyrophosphatase or deoxyuridine phosphatase. DUT is a catabolic enzymes that degrades dUTP to dUMP and prevents uracil misincorporation into DNA. Uracil misincorporation is a mechanism of cytotoxicity induced by chemotherapeutic agents in the treatment of different tumors. Thus, DUT is essential for DNA integrity but also acts as a survival factor for tumor cells. In some embodiments, DUT presents synthetic lethality with BRCA1 mutations. In some embodiments, DUT presents synthetic lethality with BRCA2 mutations. By “synthetic lethality”, it is meant the cellular lethality caused by the combined alteration of DUT and BRCA1 / 2, which alterations are otherwise individually tolerable for cell viability.

[0032] As used herein, the term “DUT inhibitor” refers to any compound selected from the group consisting of but not limited to compounds targeting human dUTPase. The term “DUT inhibitor” refers to compounds that bind to DUT and function as potent antagonists of DUT. The term “DUT inhibitor” has its general meaning in the art and refers to a compound that selectively inactivates DUT. In some embodiments, the DUT inhibitor is a small organic molecule, a polypeptide, an aptamer, an oligonucleotide or an antibody. Said oligonucleotide may be an antisense oligonucleotide, a siRNA, a shRNA, a DNA aptamer or a RNA aptamer. In some embodiments, the small organic inhibitor of DUT is TAS-114 (N-[(1 R)-1-[3-(cyclopentyloxy)phenyl]-ethyl]-3-[(3,4-dihydro-2,4-dioxo-1 (2H)- pyrimidinyl)methoxy]-1-propanesulfonamide).

[0033] By “aptamers” is meant class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Then after raising aptamers directed against the target of the invention as above described, the skilled man in the art can easily select those blocking or inactivating the target.

[0034] By “antibody” is meant antibody (the term including “antibody portion”) directed against the target. Said antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.

[0035] As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

[0036] Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice- monthly or monthly) with antigenic forms of the target. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.

[0037] Briefly, the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in the target. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners 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 culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e. , that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.

[0038] Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (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, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

[0039] Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

[0040] It is now well-established in the art that the non CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and / or Fc / pFc' regions to produce a functional antibody.

[0041] This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761 , 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761 , and WO 90 / 07861 also propose four possible criteria, which may be used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.

[0042] In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGI, lgG2, lgG3, lgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and / or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al. , / . Mol. Biol. 294:151 , 1999, the contents of which are incorporated herein by reference. Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591 ,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to 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 technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.

[0043] In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

[0044] Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and / or FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and / or CDR1 and / or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so- called single chain antibodies.

[0045] The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGI, lgG2, lgG3 and lgG4. In a preferred embodiment, the compound of the invention is a Human lgG4.

[0046] In another embodiment, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals, which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1 , CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.

[0047] The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.

[0048] VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto 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 show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

[0049] In another aspect, the invention provides an antibody that competes for binding to the target with the antibody of the invention.

[0050] As used herein, the term "binding" in the context of the binding of an antibody to a predetermined antigen or epitope typically is a binding with an affinity corresponding to a KD of about 10-7 M or less, such as about 10-8 M or less, such as about 10-9 M or less, about 10-10 M or less, or about 10-11 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin panels of monoclonal antibodies. Typically, an antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1 ,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD for binding to a non-specific antigen (e.g., BSA, casein), which is not identical or closely related to the predetermined antigen. When the KD of the antibody is very low (that is, the antibody has a high affinity), then the KD with which it binds the antigen is typically at least 10,000-fold lower than its KD for a non-specific antigen. An antibody is said to essentially not bind an antigen or epitope if such binding is either not detectable (using, for example, plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte), or is 100 fold, 500 fold, 1000 fold or more than 1000 fold less than the binding detected by that antibody and an antigen or epitope having a different chemical structure or amino acid sequence.

[0051] Additional antibodies can be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with other antibodies of the invention in standard antigen binding assays. The ability of a test antibody to inhibit the binding of antibodies of the present invention to the target demonstrates that the test antibody can compete with that antibody for binding to the target; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on the target as the antibody with which it competes. Thus, another aspect of the invention provides antibodies that bind to the same antigen as, and compete with, the antibodies disclosed herein. As used herein, an antibody “competes” for binding when the competing antibody inhibits the target binding of an antibody or antigen binding fragment of the 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 competing antibody.

[0052] In other embodiments the antibodies or antigen binding fragments of the invention bind to one or more epitopes of the target. In some embodiments, the epitopes to which the present antibodies or antigen binding fragments bind are linear epitopes. In other embodiments, the epitopes to which the present antibodies or antigen binding fragments bind are non-linear, conformational epitopes.

[0053] In some embodiments, the invention relates to DUT inhibitor wherein said oligonucleotide is an antisense oligonucleotide, a siRNA, a shRNA, a DNA aptamer or a RNA aptamer. Said DUT inhibitor is an DUT expression inhibitor.

[0054] The term “expression” when used in the context of expression of a gene or nucleic acid refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include messenger RNAs, which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.

[0055] An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. An "inhibitor of expression" refers to any compound that has a biological effect to inhibit the expression of a target gene and / or the expression of target protein. In one embodiment of the invention, said inhibitor of expression is a short hairpin RNA (shRNA), a small inhibitory RNA (siRNA), or an antisense oligonucleotide. Preferably, the inhibitor of expression is a siRNA or a shRNA.

[0056] The target expression inhibitors for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the target mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the target proteins, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding the target can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131 ; 6,365,354; 6,410,323; 6,107,091 ; 6,046,321 ; and 5,981 ,732).

[0057] Small inhibitory RNAs (siRNAs) can also function as a target expression inhibitors for use in the present invention. The target gene expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that the target expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01 / 36646, WO 99 / 32619, and WO 01 / 68836). Examples of siRNAs targeting DUT include a siRNA comprising or consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a combination thereof.

[0058] Short hairpin RNA (shRNA) or Small inhibitory RNAs (siRNAs) can function as inhibitors of gene expression for use in the invention. Gene expression can be reduced with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known.

[0059] Ribozymes can also function as target expression inhibitors for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of the target mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUO. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

[0060] Both antisense oligonucleotides (ODNs) and ribozymes useful as target inhibitors can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and / or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

[0061] Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing the target. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0062] Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KRIEGLER (A Laboratory Manual," W.H. Freeman C.O., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991).

[0063] Preferred viruses for certain applications are the adeno-viruses and adeno- associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

[0064] Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al., "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUCI9, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

[0065] In some embodiments, the DUT inhibitor causes SMUG1 -dependant damage at replication forks (RF), such as RF blockade and / or accumulation of DSBs, and subsequently leads to HRD cancer cell death. In some embodiments, the DUT inhibitor is used in combination with a PARP inhibitor or UNGi. In some embodiments, the DUT inhibitor is used in combination with a PARPi. In some embodiments, the DUT inhibitor is used in combination with an UNGi.

[0066] In some embodiments, the PARP inhibitor is selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, iniparib, veliparib, Pamiparib (BGB-290), CEP 9722, E7016, E7449 and 3-Aminobenzamide.

[0067] The term “UNG” refers to Uracil DNA glycosylase enzymes and coding gene. This gene encodes one of several uracil-DNA glycosylases. One important function of uracil- DNA glycosylases is to prevent mutagenesis by eliminating uracil from DNA molecules by cleaving the N-glycosylic bond and initiating the base-excision repair (BER) pathway. Alternative promoter usage and splicing of this gene leads to two different isoforms: the mitochondrial UNG1 and the nuclear UNG2. As used herein, by “UNG”, it is meant any one of the two UNGs, or both (i.e. UNG1 and / or UNG2).

[0068] As used herein, the term “UNG inhibitor” refers to any compound selected from the group consisting of but not limited to compounds targeting Uracil DNA glycosylase. The term “UNG inhibitor” refers to compounds that bind to UNG and function as potent antagonists of UNG. The term “UNG inhibitor” has its general meaning in the art and refers to a compound that selectively inactivates UNG. Typically, a UNG inhibitor is a small organic molecule, a polypeptide, an aptamer, an oligonucleotide (antisense oligonucleotides, siRNA, shRNA, DNA and RNA aptamers), or an antibody. Some UNG inhibitors are known in the art as such as described in WO 2021 / 087246 A1 , WO 2006 / 135763, WO 2006 / 135763, US 6,177,437 and WO 1998 / 039334.

[0069] In some embodiments, the DUT inhibitor sensitizes PARPi-resistant cells. By “sensitize”, it is meant that the treatment with a DUT inhibitor and a PARPi induces cell death and / or reduces cell viability in cells previously resistant to a treatment with a PARPi alone. In particular, said PARPi resistant cells may be associated with increased RF stability and be sensitive to the accumulation of gDNA-uracil. DUT inhibition may thus sensitize cells to PARPi by increasing DSB at RF.

[0070] Pharmaceutical composition

[0071] In a further aspect, the invention relates to a pharmaceutical composition comprising a DUT inhibitor and a pharmaceutical acceptable carrier for use in the treatment of resistant Homologous Recombination Deficiency (HRD) cancer in a subject in need thereof. In some embodiments, the DUT inhibitor is a molecule capable of silencing the gene expressing the

[0072] DUT enzyme or a molecule inhibiting DUT enzymatic properties.

[0073] Also part of the invention is a pharmaceutical composition comprising (i) a DUT inhibitor, which is a molecule capable of silencing the gene expressing the DUT enzyme or a molecule inhibiting DUT enzymatic properties, (ii) a PARP inhibitor, and (iii) and a pharmaceutical acceptable carrier.

[0074] In some embodiments, the resistant HRD cancer is resistant BRCA-associated cancer. In a preferred embodiment, the resistant HRD cancer is PARPi resistant BRCA- associated cancer.

[0075] Typically, the compound of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

[0076] Typically, the compounds according to the invention as described above are administered to the subject in a therapeutically effective amount.

[0077] By a "therapeutically effective amount" of the compound of the present invention as above described is meant a sufficient amount of the compound at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1 ,000 mg per adult per day. Typically, the compositions contain 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 the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the compound of the present invention, preferably from 1 mg to about 100 mg of the compound of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0078] In a particular embodiment, the compound according to the invention may be used in a concentration between 0.01 pM and 20 pM, particularly, the compound of the invention may be used in a concentration of 0.01 , 0.05, 0.1 , 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 pM.

[0079] According to the invention, the compound of the present invention is administered to the subject in the form of a pharmaceutical composition. Typically, the compound of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0080] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0081] Typically, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The compound of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized agents of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the compound of the present invention plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0082] Pharmaceutical compositions of the invention may include any further compound which is used in the treatment of cancer such as described above. These compounds include in particular a PARP inhibitor, 5-fluoro-2'-deoxyuridine (Fdllrd) or 5-Fluorouracil (5- Fll). Preferably, the pharmaceutical composition comprises a DUT inhibitor and a PARP inhibitor, as described above.

[0083] In some embodiments, the pharmaceutical compositions of the invention may include any additional active compound which is used in the treatment of HRD cancer, BRCA-associated cancer, resistant HRD cancer or resistant BRCA-associated cancer.

[0084] In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.

[0085] In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of resistant HRD cancer in a subject in need thereof.

[0086] In some embodiments, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of resistant BRCA-associated cancer in a subject in need thereof.

[0087] In some embodiments, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of chemo-resistant HRD cancer in a subject in need thereof.

[0088] In some embodiments, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of PARPi-resistant BRCA-associated cancer in a subject in need thereof.

[0089] In some embodiments, the pharmaceutical composition according to the invention comprises a DUT inhibitor in combination with a PARPi or UNGi and a pharmaceutical acceptable carrier. In some embodiments, the pharmaceutical composition according to the invention comprises a DUT inhibitor in combination with a PARPi and a pharmaceutical acceptable carrier. In some embodiments, the pharmaceutical composition according to the invention comprises a DUT inhibitor in combination with an UNGi and a pharmaceutical acceptable carrier.

[0090] Treatment

[0091] In a further aspect, the invention relates to a method of treating resistant Homologous Recombination Deficiency (HRD) cancer such as resistant BRCA-associated cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a DUT inhibitor.

[0092] In some embodiments, the resistant BRCA-associated cancer is a PARPi-resistant BRCA-associated cancer.

[0093] The method of treating of the invention may include further administration of a PARP inhibitor or an UNG inhibitor, as described above. In some embodiments, the DUT inhibitor is administered in combination with a PARPi. In some embodiments, the DUT inhibitor is administered in combination with an UNGi. Typically, the administration of the compound of the invention may be combined with administration of other active compound, pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers. Typically, the additional active compounds as described above are administered to the subject in a therapeutically effective amount.

[0094] In one embodiment, said additional compounds may be contained in the same composition or administrated separately.

[0095] The invention is further illustrated by the following figures and examples.

[0096] FIGURES

[0097] Figure 1. Quantification of gH2AX foci formation in WT and BRCA2'~ cells treated with 5 mM TAS-114 as indicated.

[0098] Figure 2. Immunofluorescence-based quantification (top) and representative images (bottom) of gH2AX / pRPAS33 colocalizing foci in WT and BRCA21' cells treated with 5 mM TAS-114 as indicated.

[0099] Figure 3. Quantification of the 12-day clonogenic survival assay in the indicated cell lines in response to continuous TAS-114 exposure.

[0100] Figure 4. Quantification of the 12-day clonogenic survival assay in WT cells upon transfection with siCTRL or siBRCA2. Twenty-four hours after transfection, cells were exposed for 24 hours to the indicated doses of Fdllrd and then the drug was washed out and cells grown in drug-free medium for the rest of the experiment.

[0101] Figure 5. Quantification of 12-day clonogenic survival assay following drug treatment of WT and BRCA2- / - cells as indicated.

[0102] Figure 6. Quantification (left) and representative images (right) of the 12-day clonogenic survival assay in WT and BRCA2- / - cells following siRNA-mediated knockdown of DUT. Cells were seeded 24h after transfection.

[0103] Figure 7. Immunofluorescence-based quantification of gH2AX / pRPAS33 colocalizing foci in BRCA2~'~ cells following siRNA transfection and TAS-114 treatment (5 mM, 24 hours) as indicated.

[0104] Figure 8. Quantification of the 12-day clonogenic survival assays in BRCA2'~ cells transfected with indicated siRNAs and treated or not with 1 mM TAS-114.

[0105] Figure 9. Quantification (top) and representative images (bottom) of chromosomal breaks (in grey) and pulverized chromosomes (in white) in BRCA21' cells upon 24- and 48- hours washout of TAS-114 + Fdllrd. UT means untreated.

[0106] Figure 10. Quantification of the 12-day clonogenic survival assay in RPE-1 cells transfected with the indicated siRNAs and continuously exposed to rucaparib (PARPi).

[0107] Figure 11. Top, quantification of gH2AX foci in the indicated RPE-1 cell lines treated or not with TAS-114 (C2: BRCA2~'~ clone; C2-1 , C2-3: PARPi-resistant BRCA2'~ clones). Bottom, quantification and representative images of gH2AX / pRPAS33 colocalizing foci in the PARPi-resistant BRCA2- / - clone C2-1 treated as in top.

[0108] Figure 12. Quantification of the 12-day clonogenic survival assay in cells as in Figure 11 continuously exposed or not to the indicated doses of TAS-114.

[0109] Figure 13. Quantification (left) and representative images (right) of the survival assay in the organoids derived from the indicated breast tumors following TAS-114 + FdUrd combined treatment. UT means untreated.

[0110] Figure 14. Quantification of the 12-day clonogenic survival assay of WT, PARPi- naive (C1 and C2) and -resistant (C1-3, C1-5, C2-1 , and C2-2) BRCA2- / - RPE-1 cells continuously exposed to the indicated doses of rucaparib (PARPi).

[0111] Figure 15. Replication fork degradation assay by DNA combing. WT, PARP-naive (C2) and -resistant BRCA2- / - RPE-1 clones (C2-1 and C2-3) were labelled consecutively with the thymidine analogs IdU and CldU for 30 minutes followed by 3 hours of 4 mM hydroxyurea treatment to stall the replication forks. The CldU / IdU ratio was used as a measure of replication fork degradation.

[0112] Figure 16. Top, Representative images of the 12-day clonogenic survival assay in PARP-naive and -resistant OVCAR-8 cells (clone 14) continuously exposed or not to the indicated doses of rucaparib (PARPi). Bottom, quantification of the 12-day clonogenic survival assay in the same cells as in (top) continuously exposed or not to the indicated doses of TAS- 114.

[0113] EXAMPLES

[0114] Example 1 : Methods

[0115] Cell culture and treatments hTERT-RPE-1 (human retinal pigment epithelium, female) and derivatives were cultured in Dulbecco’s Modified Eagle’s Medium / Nutrient Mixture F-12 Ham (Sigma) supplemented with 10% fetal bovine serum, 100 U / rnL penicillin and 100 U / rnL streptomycin. The ovarian cancer cell line OVCAR8 and derivatives were cultured in RPMI 1640 (ThermoFisher) supplemented with 10% fetal bovine serum, 100 U / rnL penicillin and 100 U / rnL streptomycin. Cells were tested every month by qPCR for mycoplasma. 5-Fluoro- 2'-deoxyuridine (FdUrd, Sigma, F0503), TAS-114 (MedChemExpress, HY-124062), rucaparib (Selleckchem, S1098), olaparib (Selleckchem, S1060) were used at the indicated concentrations. siRNA sequences and transfection

[0116] All siRNA-mediated knockdown experiments were performed using RNAiMax (Thermo Fisher Scientific) the cells following manufacturer’s instructions. All the following siRNAs were purchased from Dharmacon (Horizon Discovery): DUT (LQ-003540-0002 (ON-Target plus, set of 4 siRNA of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 as detailed in Table 1)), UNG (UNG #6 LQ-011795-06-0005, and UNG #7 LQ- 011795-07-0005), SMUG1 (LQ-012838-01 -0002, (ON-Target plus, set of 4)), TDG (LQ- 003780-01-0002 (ON-Target plus, set of 4)) , MBD4 (LQ-011554-0002 (ON-Target plus, set of 4)). For all the experiments, AllStars negative control siRNA (Cat. #1027280, Qiagen) served as the negative control.

[0117] Table 1. Sequence listing Generation of drug-resistant cells hTERT-RPE- TPSS-7BRCA2~!~ clones were made resistant to the PARPi rucaparib by two different strategies as follows: clone C1 was grown for 3 months in 0.5 pM rucaparib containing medium; clone C2 was subjected to increasing concentration of rucaparib from 0.01 pM to 0.1 pM by progressively increasing the dose of 0.1 pM each week for 10 weeks. After long-term drug exposure, C1 and C2-derived resistant cells were diluted for single clone selection and assessed for their PARPi sensitivity and RAD51 foci formation upon ionizing radiation (IR). C1-3 and C1-5 PARPi resistant clones were derived from BRCA2~!~ C1 clone, whereas C2-1 and C2-3 were generated from the C2 clone. Rucaparib-resistant CAPAN-1 cells were generated by growing cells in 1 pM PARPi, whereas OVCAR8- resistant clones in 2 pM PARPi-containing medium for 3 months prior to single clone selection. The PARPi-resistant cells were always kept in culture in the presence of the drug, whereas for all the experiments cells were seeded in the absence of the drug.

[0118] Clonogenic assays

[0119] For clonogenic survival, cells were seeded into 6-well plates and treated with cytotoxic drugs the next day. For PARPi and TAS-114, cells were grown continuously in the presence of the indicated drug concentrations. For MTX, hydroxyurea, Fdllrd and Fdllrd+TAS-114 combined treatment, cells were treated for 24 hours at the indicated doses and then cultured for 12 days in drug-free medium. For MMS and H2O2, cells were treated for 90 min and then cultured for 12 days in drug-free medium. For IR, cells were seeded into 6-well plates and irradiated at the indicated doses the day after. For siRNA and gRNA, cells were seeded for clonogenic assay into 6-well plates 24 hours after transfection and - where indicated- treated with drugs the following day.

[0120] Colony formation was scored 12 days after treatment by fixing and staining with 0.5% (w / v) crystal violet in methanol. Survival curves were expressed as a percentage (mean ± s.e.m.) of colonies formed relative to the appropriate control in at least three independent experiments. Statistical significance was calculated using a two-tailed paired t-test.

[0121] Establishment of patient-derived xenograft organoids (PDxO)

[0122] PDX cells were embedded in 100-pl Matrigel domes (Corning, #354230), which were plated in six-well tissue culture plates onto a 70-pl Matrigel base layer. After a 30min incubation period, Matrigel domes were solidified, and a subtype-specific culture medium was added. The PDxO medium was as follows: Advanced DMEM / F12 with 5% FBS, 10 mM HEPES, 1 * Glutamax, 1 pg ml-1 hydrocortisone, 50 pg ml-1 gentamicin, 10 ng ml-1 hEGF, 100 ng ml-1 FGF2 and 1 mM NAC. Medium was exchanged every 3 to 4 days, and, once mature, cultures were passaged by a dissociation step in TrypLE Express. Single cells were seeded at 200,000-400,000 cells per dome.

[0123] Drug screening of PDxOs

[0124] Mature organoids were collected from culture using TrypleExpress (Life technologies, #12605010) for 20 min. Twenty thousand to fifty thousand cells (-200 organoids) were seeded per well in 96-well tissue culture plates in 10% matrigel. Fdllrd and TAS+114 were added to the wells after 48h. at the indicated concentrations. To quantify drug responses, cell viability data were be obtained 10 days post-treatment using CellTiter- Glo 3D (Promega, #G9682) (v / v). The plates were mixed and incubated 20 min at room temperature. Then luminescence was monitored using Tecan Infinite 200. The drug response was represented by plotting the dose-response curve and the IC50 values are also indicated.

[0125] Statistics

[0126] All data are represented as mean ± s.e.m. over at least three independent experiments, unless otherwise stated. For statistical analysis of survival data, unpaired two- tailed t-test and Mann-Whitney test were used as indicated. Statistical significance is represented on figures by asterisks, (* = p < 0.05, ** = p < 0.01 , *** = p < 0.001 , **** = p < 0.0001).

[0127] Example 2: Uracil excision by UNG is essential for the survival of HR-deficient cells

[0128] Recently, ssDNA gaps have emerged as a major contributor to genomic instability and chemosensitivity in BRCA 7 / 2-deficient cells. To study the consequences of 2’- deoxyuridine (dU) misincorporation and impaired uracil excision in the survival of BRCA1 / 2- deficient cells, the inventors generated BRCA2~!~ clones (C1 and C2) in RPE-1 TP53~'~ cells. The inventors found that exposure to TAS-114 (a selective inhibitor of DUT, the only human dUTPase) increases the number of gH2AX foci prominently in the S phase ( / .e., EdU- positive fraction) of BRCA2~!' cells (clone C2) (Fig. 1). Additionally, they observed a robust colocalization of gH2AX foci with phosphorylated RPA2 on serine 33 (pRPA), indicative of massive resection events at DNA breaks ( / .e., damaged RFs) (Fig. 2). Finally, TAS-114 and / or FdUrd, as well as depletion of DUT, selectively killed BRCA2'1' cells (Fig. 3, 4, 5 and 6). Knockdown of SMUG1 -and none of the other UDGs- rescued RF stability and BRCA2 cell survival upon TAS-114 treatment (Fig. 7). In addition, UNG depletion sensitized BRCA2~I~ cells to low dose of TAS-114, which was also rescued by SMUG1 knockdown (Fig. 8).

[0129] Next, the inventors assessed the impact of SMLIG1 -mediated damage at replication forks (RFs) on the genomic stability of BRCA2'1' cells. Chromosomal breaks in BRCA21' cells were measured by inducing premature chromatin condensation by calyculin A upon dll misincorporation. FdUrd+TAS-114 treatment induced chromosomal breaks and pulverized chromosomes, likely arising from toxic DNA repair intermediates and underreplicated DNA stretches, respectively (Fig. 9).

[0130] Altogether, the inventors show that accumulation of gDNA-uracil -by dU (2’- deoxyuridine) misincorporation - leads to SMUG1-dependent damage at RFs, accumulation of DSBs, and ultimately HRD cancer cell death.

[0131] Example 3: Targeting uracil excision kills PARPi-resistant HR-deficient tumors

[0132] Despite the initial success of PARPis in the treatment of BRCA-deficient tumors, the development of resistance has become a clinical challenge. The cytotoxicity of PARPis in HRD cells has been attributed to impaired HR-mediated repair of stalled RFs resulting from PARP1 -trapping lesions. The inventors hypothesized that the observed SMUG1 -mediated RF blockade might synergize with the trapping effect of PARPi. Surprisingly, the inventors found that depletion of UNG sensitizes HR-proficient (HRP) cells to the PARPi rucaparib and that concomitant depletion of SMUG1 rescues drug sensitization (Fig. 10). These data suggest that SMUG1 activity unleashed by UNG depletion synergizes with PARPi.

[0133] To corroborate these findings, the inventors assessed the potential of targeting uracil excision as a novel strategy to overcome PARPi resistance by generating PARPi-resistant clones by continuous exposure of two different BRCA2'~ RPE-1 clones to rucaparib. First, the inventors confirmed that these generated clones developed drug resistance, which we found to be associated with increased RF stability (Fig. 14 and 15) or drug efflux. Nonetheless, they found that TAS-114 treatment causes DSB increase at RFs in PARPi- resistant cells similarly to what is observed in PARPi-naive BRCA2'1' cells (Fig. 11). This was associated with an increased number of gH2AX / pRPA colocalizing foci, suggesting the formation of damaged forks downstream of the uracil machinery in PARPi-resistant cells (Fig. 11). Notably, TAS-114 treatment killed PARPi-resistant cells (Fig. 12 and 16), indicating that there is no cross-resistance between PARPis and uracil-increasing drugs.

[0134] Next, the inventors assessed the therapeutic potential of disrupting UNG-mediated uracil excision to kill PARPi-resistant HRD cells. To this end, several PARPi-resistant clones from either the BRCA1 -deficient (promoter hypermethylation) ovarian cancer cell line OVCAR8 and the BRCA2-mutated pancreatic cancer cell line CAPAN-1 were generated. None of the CAPAN-1 clones acquired PARPi resistance through HR restoration, as demonstrated by the inability of RAD51 foci formation upon IR. Clonogenic survival assays showed that knockdown of either UNG or NMNAT1 specifically decreased the survival of all tested PARPi-resistant HRD cancer cells. The PARPi-resistant 0VCAR8 clone 14 (C14) generated showed higher level of BRCA1 mRNA in comparison to the parental cell line while there was no increase in drug efflux pumps, suggesting BRCA1 overexpression as the mechanism of PARPi resistance. This was associated with RAD51 foci formation and acquired resistance to PARPi. Survival assays showed that clone C14 remains sensitive to FdUrd + TAS-114 similar to the parental cell line, further supporting the concept that there is no cross-resistance between PARPis and gDNA-uracil increasing drugs.

[0135] Then, to examine these results in a translational setting, the inventors assessed the therapeutic potential of targeting uracil excision using organoids derived from patient- derived xenografts (PDXs) established from triple negative breast cancers (TNBCs). The FdUrd + TAS-114 response was compared among organoids derived from HR-proficient (HBCx-232-Org), PARPi-naive BRCA1-mutated (HBCx-153-Org) and PARPi-resistant BRCA1-mutated (HBCx-8-Org and HBCx-246-Org) tumors. In HBCx-8-Org, PARPi resistance is driven by the restoration of RAD51 foci formation without a secondary mutation in BRCA151. The HBCx-246 tumor showed cross-resistance to talazoparib, carboplatin and gemcitabine, and the resistance was associated with a BRCA1 secondary mutation, a frequent chemoresistance mechanism observed in patients. Strikingly, all PARPi-resistant organoids showed sensitivity to the combination treatment, as compared to the HR- proficient organoid (Fig. 13), Next, the inventors evaluated the effect of dU misincorporation by FdUrd + TAS-114 treatment in five CAPAN-1 clones derived from prolonged in vitro cisplatin exposure, with each clone bearing a different secondary mutation in BRCA252. They confirmed that the clones both restored HR (i.e. RAD51 foci formation) and developed PARPi cross-resistance. Nonetheless, all the resistant CAPAN-1 clones remained sensitive to gDNA-uracil (by FdUrd + TAS-114 combination or NMNAT1 depletion) like the parental CAPAN-1 cells.

[0136] Finally, the inventors transplanted the HBCx-246 tumor in nude mice and tested the tumor response to dU misincorporation. Notably, FdUrd is unstable and has poor pharmacokinetic (PK) properties, making it unsuitable for in vivo use. As a substitute, they used capecitabine, a prodrug that similarly to FdUrd inhibits thymidylate synthase. Mice were separated in groups, receiving either a vehicle or capecitabine + TAS-114, or the two drugs separately. While either TAS-114 or capecitabine alone had no effect on tumor growth, the combination of the two drugs significantly reduced tumor growth. Collectively, these data show that PARPi-resistant HRD tumors are sensitive to the accumulation of gDNA-uracil.

[0137] Conclusion

[0138] In conclusion, DUT inhibition is synthetic lethal with BRCA mutations in cancer cells. DUT inhibition also kills PARPi-resistant cancer cells. The inventors have demonstrated that DUT inhibition leads to SMUG1 -dependant damage, which synergizes with PARP inhibition to kill cancer cells. DUT inhibition thus sensitizes cancer cells to PARPi, in particular PARPi resistant HRD tumors.

Claims

REVENDICATIONS1. A deoxyuridine 5'-triphosphate nucleotidohydrolase (DUT) inhibitor for use in the treatment of PARP inhibitor (PARPi) resistant Homologous Recombination Deficiency (HRD) cancer.

2. The DUT inhibitor for use according to claim 1 , wherein the DUT inhibitor is a molecule capable of silencing the gene expressing the DUT enzyme or a molecule inhibiting DUT enzymatic properties.

3. The DUT inhibitor for use according to claim 1 or 2, wherein the PARPi resistant HRD cancer is a PARPi resistant BRCA-associated cancer.

4. The DUT inhibitor for use according to any one of claims 1 to 3, wherein said DUT inhibitor is a small organic molecule, a polypeptide, an aptamer, an oligonucleotide or an antibody.

5. The DUT inhibitor for use according to claim 4, wherein said small organic molecule is TAS- 114.

6. The DUT inhibitor for use according to any one of claims 1 to 5, in combination with a PARP inhibitor or UNG inhibitor (UNGi).

7. The DUT inhibitor for use according to claim 6, wherein the PARP inhibitor is selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, iniparib, veliparib, Pamiparib (BGB-290), CEP 9722, E7016, E7449 and 3-Aminobenzamide.

8. A pharmaceutical composition comprising a DUT inhibitor and a pharmaceutical acceptable carrier for use in the treatment of PARPi resistant HRD cancer in a subject in need thereof.

9. The pharmaceutical composition for use according to claim 8, wherein the PARPi resistant HRD cancer is a PARPi resistant BRCA-associated cancer.

10. A pharmaceutical composition comprising a DUT inhibitor in combination with a PARPi or UNGi and a pharmaceutical acceptable carrier.

11. A method of treating PARPi resistant HRD cancer such as PARPi resistant BRCA- associated cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a DUT inhibitor.

12. The method of claim 11 , wherein the DUT inhibitor is administered in combination with a PARPi or UNGi.

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