WO2024133723A1

WO2024133723A1 - Methods for decreasing therapeutic acquired resistance to chemotherapy and/or radiotherapy

Info

Publication number: WO2024133723A1
Application number: PCT/EP2023/087320
Authority: WO
Inventors: Luc Buee; Bruno-Georges LEFEBVRE; Thomas RICO
Original assignee: Institut National de la Santé et de la Recherche Médicale; Centre Hospitalier Universitaire De Lille; Université de Lille; Centre National De La Recherche Scientifique
Priority date: 2022-12-22
Filing date: 2023-12-21
Publication date: 2024-06-27

Abstract

Therapeutic resistance is one of the major challenges in cancer treatment. These latter may be inherently resistant or this resistance may be acquired during treatment. In this context, the tau protein, encoded by the MAPT gene, could prove to be an important contributor in resistance to cancer therapy. The inventors have been thus abled to confirm in vivo that the tau protein was a factor of resistance to radiotherapy and the chemotherapy inducing DSB (doxorubicin) and that the reduction of its expression by shRNA increased the sensitivity of tumors to these treatments. The present invention relates to a method for decreasing therapeutic acquired resistance to chemotherapy agent and/or radiotherapy agent in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Tau inhibitor.

Description

METHODS FOR DECREASING THERAPEUTIC ACQUIRED RESISTANCE

TO CHEMOTHERAPY AND/OR RADIOTHERAPY

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular oncology, for decreasing therapeutic acquired resistance to chemotherapy and/or radiotherapy in a subject.

BACKGROUND OF THE INVENTION:

Although defects in DNA damage response lead to genomic instability, this can also represent the “Achille’s heel” of cancer cells. Deficiency in any specific repair pathway makes cells more dependent on the remaining DNA-repair pathways, ren-dering them more vulnerable to certain DNA-targeting therapies. Double-strand DNA breaks (DSBs) are considered as the most lethal DNA lesions and are commonly induced in cancer radio- or chemo-therapy. However, one of the main limitations of DSB-associated therapies is the tumor resistance developed before or after treatment [1,2]. Thus, it will be important to decipher the underlying molecular mechanism of this resistance to improve existing regimens.

In cells, DSBs activate DNA damage response pathways through the kinases ATM, ATR and DNA-PKcs, that in turn activate proteins involved in two main pathways, the classical nonhomologous end-joining (cNHEJ) and the homologous recombination (HR). cNHEJ mediates repair by directly rejoining DSB ends, contrary to HR which utilizes a homologous DNA sequence to guide DNA repair [3,4], Which of the two repair routes is used depends in part of the cell cycle phase. Since HR relies on the presence of a sister chromatid, it can be effective only in the late S/G2 phase while the NHEJ pathway is effective throughout the cell cycle. Recruitment of specific factors to DNA lesions is also essential to initiate DNA repair and this is regulated, in part, by histone post-translational modifications [5], In this context, 53BP1 and BRCA1 (breast cancer 1), together with auxiliary factors, play a pivotal role in the pathway choice. They trigger either cNHEJ or HR respectively. 53BP1 blocks DNA end resection necessary for HR. cNHEJ accounts for most DSB repair in mammalian cells [6-8],

DNA repair mechanisms are multifaced but recent studies demonstrated an essential role for microtubules. First, microtubule dynamics was demonstrated to be crucial in the intracellular trafficking of DNA repair proteins [9], Second, it has been demonstrated that the interaction between microtubules and linker of the nucleoskeleton, and cytoskeleton’ complexes increase the mobility of DSBs toward repair complexes [10], Tau was first described as a neuronal microtubule-associated protein (MAPT) and when it aggregates it is a major player in neurodegenerative diseases like Alzheimer’s [11], However, Tau has many other cellular functions [12], In addition to microtubule dynamics, it interferes with several biological processes such as signaling pathways, RNA metabolism and it even contributes to the inflammatory response. Furthermore, Tau is not only found in the cytoplasm but also in the cell nucleus where it participates in chromatin organisation in neuronal and cancer cells [13,14], Several data suggested a role for Tau in DNA repair [15], In particular, Tau deletion induces an increase in DSBs and a slower DNA repair in neurons after reactive oxygen species induction triggered by hyperthermia [16,17], However, the molecular mechanism remains elusive. In this regard, it has been demonstrated in vitro that Tau protects DNA from free radical damage [18], The current explanation of this protective effect is based on Tau’s ability to bind DNA in a sequence-independent manner [19], Although Tau is known as a neuronal protein, a large body of literature has shown that its expression is increased in several types of cancer and may be associated with acquired resistance to Taxanes [20,21], Tau binds in the same binding site as paclitaxel, and consequently compete with this drug [22],

The role of Tau in genome protection and/or repair suggests that Tau expression in cancer cells could be involved in resistance to conventional anti-cancer treatments, in particular those inducing DNA damage. Starting from this hypothesis, we evaluated the role of Tau in DNA repair in breast cancer cell lines.

SUMMARY OF THE INVENTION:

The present invention is in the field of medicine, in particular oncology, for decreasing therapeutic acquired resistance to chemotherapy and/or radiotherapy in a subject. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Therapeutic resistance is one of the major challenges in cancer treatment. These latter may be inherently resistant or this resistance may be acquired during treatment. In this context, the tau protein, encoded by the MAPT gene, could prove to be an important contributor in resistance to cancer therapy. Initially described at the neural level, a literature abundant has now demonstrated that the expression of his gene is increased in several types of cancers and could be associated with resistance to taxanes. This effect has been associated with its well- known role regulator of microtubule assembly and disassembly. However, recent work has demonstrated, at the neuronal level, that tau was a pleiotropic protein, involved in several mechanisms cellular. Among them, its role in genome protection/repair could be directly related to resistance to other anti-cancer treatments, especially those inducing DNA damage.

The inventors show that DSBs from DNA disappear faster after irradiation or bleomycin treatment in MCF7 and MDA-MB-231 cells expressing MAPT in a manner endogenous than in their homologs stably transfected with a tau-directed shRNA (shTau).

This observation has been linked to an increase in the repair activities of the junctiontype DSBs of the non-homologous ends (NHEJ) and homologous recombination (HR). The inventors were able to explain this new role of tau through its ability to increase microtubular trafficking of the 53BP1 protein, a factor essential in the initiation of repair, thus improving its nuclear translocation. Tau has thus a general role in regulating the nuclear trafficking of DNA repair proteins. These results have tested in vivo using a xenograft immunodeficient (SCID) mouse model.

The inventors have been thus abled to confirm in vivo that the tau protein was a factor of resistance to radiotherapy and the chemotherapy either inducing DSB (doxorubicin) or DNA adducts (cisplatin and oxaliplatin) and that the reduction of tau expression by shRNA increased the sensitivity of tumors to these treatments.

In a first embodiment, the present invention relates to a method for decreasing therapeutic acquired resistance to chemotherapy agent and/or radiotherapy agent in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Tau inhibitor.

In a second embodiment, the present invention relates to a method for preventing and/or treating cancer with acquired resistance to treatment with chemotherapy agent and/or radiotherapy agent in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Tau inhibitor

As used herein, the term “patient” or “subject” denote a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a patient according to the invention is a human. Typically, a patient according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with a cancer.

As used herein, the term "DNA repair" refers to a collection of processes by which a cell identifies and corrects damage to DNA molecules. Single-strand defects are repaired by base excision repair (BER), nucleotide excision repair (NER), or mismatch repair (MMR). Double-strand breaks are repaired by non-homologous end joining (NHEJ), microhomology- mediated end joining (MMEJ), or homologous recombination. After DNA damage, cell cycle checkpoints are activated, which pause the cell cycle to give the cell time to repair the damage before continuing to divide. Checkpoint mediator proteins include BRCA1, MDC1, 53BP1, p53, ATM, ATR, CHK1, CHK2, and p21.

As used herein, the term "impaired DNA repair" refers to a state in which a mutated cell or a cell with altered gene expression is incapable of DNA repair or has reduced activity of one or more DNA repair pathways or takes longer to repair damage to its DNA as compared to a wild type cell.

As used herein, the terms "cancer" and "tumors" refer to or describe the pathological condition in mammals that is typically characterized by unregulated cell growth. More precisely, in the methods of the invention, diseases, namely tumors that do express/secrete Tau protein are most likely to respond to the Tau inhibitor treatment. In particular, the cancer may be associated with a solid tumor or lymphoma/leukemia (from hematopoietic cell). Examples of cancers that are associated with solid tumor formation include breast cancer, uterine/cervical cancer, oesophageal cancer, pancreatic cancer, colon cancer, colorectal cancer, kidney cancer, ovarian cancer, prostate cancer, head and neck cancer, non small cell lung cancer stomach cancer, tumors of mesenchymal origin (i.e; fibrosarcoma and rhabdomyoscarcoma) thyroid cancer.

More particular examples of such cancers include chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL), squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatocarcinoma, breast cancer, colon carcinoma, and head and neck cancer, gastric cancer, germ cell tumor, pediatric sarcoma, multiple myeloma, acute myelogenous leukemia (AML), chronic lymphocytic leukemia, mastocytosis and any symptom associated with mastocytosis.

Various cancers are also encompassed by the scope of the invention, including, but not limited to, the following: carcinoma including that of the bladder (including accelerated and metastatic bladder cancer), breast, colon (including colorectal cancer), kidney, liver, lung (including small and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate, testis, genitourinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic carcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, Tcell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burketts lymphoma; hematopoietic tumors of myeloid lineage including acute and chronic myelogenous leukemias, myelodysplastic syndrome, myeloid leukemia, and promyelocytic, leukemia; tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; other tumors including melanoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, and teratocarcinoma; melanoma, unresectable stage III or IV malignant melanoma, squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatocarcinoma, breast cancer, colon carcinoma, and head and neck cancer, retinoblastoma, gastric cancer, germ cell tumor, bone cancer, bone tumors, adult malignant fibrous histiocytoma of bone; childhood malignant fibrous histiocytoma of bone, sarcoma, pediatric sarcoma; myelodysplastic syndromes; neuroblastoma; testicular germ cell tumor, intraocular melanoma, myelodysplastic syndromes; myelodysplastic/myeloproliferative diseases, synovial sarcoma.

In a preferred embodiment, the cancer is a hematopoietic cancer, especially a leukemia or lymphoma. In another preferred embodiment of the present invention, the cancer is a solid tumor. For instance, the cancer may be sarcoma and oestosarcoma such as Kaposi sarcome, AIDS-related Kaposi sarcoma, melanoma, in particular ulveal melanoma, and cancers of the head and neck, kidney, ovary, pancreas, prostate, thyroid, lung, esophagus, breast, bladder, colorectum, liver and biliary tract, uterine, appendix, and cervix, testicular cancer, gastrointestinal cancers and endometrial and peritoneal cancers. Preferably the cancer may be sarcoma, melanoma, in particular ulveal melanoma, and cancers of the head and neck, kidney, ovary, pancreas, prostate, thyroid, lung, esophagus, breast, bladder, colorectum, liver, cervix, and endometrial and peritoneal cancers. For instance, the cancer may be selected from the group consisting of breast cancer, hepatocellular carcinoma, colorectal cancer, glioblastoma, melanoma, and head and neck cancer.

In previous study, it was shown that Tau is overexpressed in different human breast, gastric, prostate cancer cell lines and tissues (Gargini et al., 2019). In particular embodiment regarding the method of the present invention, the solid tumor is selected from the group consisting of breast cancer ((Rouzier et al., 2005;Matrone et al., 2010;Spicakova et al., 2010;Li et al., 2013), gastric cancer (Wang Q et al Pathol. Oncol. Res. (2013) 19:429-435), ovarian cancer (Smoter M. et al. Journal of Experimental & Clinical Cancer Research (2013), 32:25) , prostate cancer, glioma, Neuroblastoma, Kidney clear cell carcinoma Lung cancer, Pheochromocytoma/Paraganglioma and Neuroendocrine tumors (NETs).

As used herein, the “Double-strand breaks” (DSB) has its general meaning in the art and refers

To the most deleterious types of DNA lesions. DSB processing and repair can cause sequence deletions, loss of heterozygosity, and chromosome rearrangements resulting in cell death or carcinogenesis. There are two major pathways for repairing them: homologous recombination and nonhomologous DNA end joining (NHEJ). The diverse causes of DSBs result in a diverse chemistry of DNA ends that must be repaired. Across NHEJ evolution, the enzymes of the NHEJ pathway exhibit a remarkable degree of structural tolerance in the range of DNA end substrate configurations upon which they can act. In vertebrate cells, the nuclease, polymerases and ligase of NHEJ are the most mechanistically flexible and multifunctional enzymes in each of their classes. Unlike repair pathways for more defined lesions, NHEJ repair enzymes act iteratively, act in any order, and can function independently of one another at each of the two DNA ends being joined. NHEJ is critical not only for the repair of pathologic DSBs as in chromosomal translocations, but also for the repair of physiologic DSBs created during V(D)J recombination and class switch recombination. Therefore, patients lacking normal NHEJ are not only sensitive to ionizing radiation, but also severely immunodeficient

DNA strand breakage can be achieved by ionized radiation (radiotherapy). Radiotherapy includes, but is not limited to, y-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other radiotherapies include microwaves and UV-irradiation. Other approaches to radiation therapy are also contemplated in the present invention.

The DNA-damaging antitumor agent is preferably selected from the group consisting of an inhibitor of topoisomerases I or II, a DNA crosslinker, a DNA alkylating agent, an anti- metabolic agent and inhibitors of the mitotic spindles.

Inhibitors of topoisomerases I and/or II include, but are not limited to, etoposide, topotecan, camptothecin, irinotecan, amsacrine, intoplicine, anthracyclines such as doxorubicin, epirubicine, daunorubicine, idanrubicine and mitoxantrone. Inhibitors of Topoisomerase I and II include, but are not limited to, intoplecin. In a preferred embodiment, the DNA-damaging antitumor agent is doxorubicin.

DNA crosslinkers include, but are not limited to, cisplatin, carboplatin and oxaliplatin. In a preferred embodiment, the DNA-damaging antitumor agent is selected from the group consisting of carboplatin and oxaliplatin. Anti-metabolic agents block the enzymes responsible for nucleic acid synthesis or become incorporated into DNA, which produces an incorrect genetic code and leads to apoptosis. Nonexhaustive examples thereof include, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors, and more particularly Methotrexate, Floxuridine, Cytarabine, 6-Mercaptopurine, 6- Thioguanine, Fludarabine phosphate, Pentostatine, 5 -fluorouracil (5-FU), gemcitabine and capecitabine.

The DNA-damaging anti-tumor agent can be alkylating agents including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, metal salts and triazenes. Nonexhaustive examples thereof include Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN(R)), Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Fotemustine, cisplatin, carboplatin, oxaliplatin, thiotepa, Streptozocin, Dacarbazine, and Temozolomide.

Inhibitors of the mitotic spindles include, but are not limited to, , vinorelbine, larotaxel (also called XRP9881; Sanofi-Aventis), XRP6258 (Sanofi-Aventis), BMS-184476 (Bristol- Meyer-Squibb), BMS-188797 (Bristol-Meyer-Squibb), BMS-275183 (Bristol-Meyer-Squibb), ortataxel (also called IDN 5109, BAY 59-8862 or SB-T-101131 ; Bristol-Meyer-Squibb), RPR 109881 A (Bristol-Meyer-20 Squibb), RPR 116258 (Bristol-Meyer-Squibb), NBT-287 (TAPESTRY), Tesetaxel (also called DJ-927), IDN 5390 (INDENA), and MAC-321 (WYETH). Also see the review of Hennenfent & Govindan (2006, Annals of Oncology, 17, 735- 25 749).

Preferably, the DNA-damaging antitumor agent is an inhibitor of topoisomerases I and/or II, a DNA crosslinker, an anti-metabolic agent or a combination thereof. In a preferred embodiment, the DNA damaging antitumor agent is selected from the group consisting of doxorubicin, 5-FU, carboplatin and oxaliplatin or a combination thereof. In a most preferred embodiment, the conjugated DBait is DT01 and the DNA-damaging antitumor agent is selected from the group consisting of doxorubicin, carboplatin, 5-FU and oxaliplatin.

As used herein, the term “taxane” has its general meaning in the art and relates to a class of diterpenes. They were originally identified from plants of the genus Taxus (yews), and feature a taxadiene core. Paclitaxel (Taxol) and docetaxel (Taxotere) are widely used as chemotherapy agents. Cabazitaxel was FDA approved to treat hormone-refractory prostate cancer. In some embodiment, the DNA-damaging antitumor agent is not a taxane agent. In some embodiment, the DNA-damaging antitumor agent is not the following taxane agent: oxetaxel, paclitaxel, abraxane.

In some embodiment, the taxane is not oxetaxel. In some embodiment, the taxane is not paclitaxel. In some embodiment, the taxane is not abraxane.

In some embodiment, the method of the present invention does not comprise the administration of DNA-damaging antitumor agent does not comprise a taxane agent. In some embodiment, the method of the present invention does not comprise the administration of oxetaxel, paclitaxel, abraxane.

In some embodiment, the DNA-damaging antitumor agent of the present invention does not comprise the taxane-like compounds described in Papin S, Paganetti P. Emerging Evidences for an Implication of the Neurodegeneration -Associated Protein TAU in Cancer. Brain Sci. 2020 Nov 16;10(l 1):862. doi: 10.3390/brainscilOl 10862.

In some embodiment, Tau in cancer cells could be involved in acquired resistance to conventional anti-cancer treatment, in particular those inducing DNA damage. In some embodiment, Tau in cancer cells could be involved in acquired resistance to radiotherapy or to chemotherapy.

As used herein, the term “acquired resistance” indicates that the cancer becomes resistant and/or substantially less response to the effects of the drug after being exposed to it for a certain period of time.

As used herein, the term “radiation therapy” or “radiotherapy” has its general meaning in the art and refers the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow. One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy. The use of machines to focus radiation (such as x-rays) on a cancer site is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, the radiation therapy is external radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction is given but fewer fractions.

In some embodiment, the present methods of invention invention relates to radiotherapy inducing double-strans breaks.

Indeed, ionizing radiations cause directly or indirectly double-stranded breaks (DSBs) and trigger cell/tissue death (necrosis or apoptosis). The cytotoxic effect of ionizing radiation forms the basis for radiotherapy, which is widely used in the treatment of human cancer. The efficacy of radiotherapy is currently limited by the radio-resistance of certain tumors (for example, glioblastoma, head and neck squamous cell carcinoma) and by the side effects caused by irradiation of nearby normal tissues (for example, in the treatment of breast and cervical cancer).

As used herein, the term "radiosensitivity" refers to the relative susceptibility of cells to the harmful effect of ionizing radiation. The more radiosensitive a cell is, the less radiation that is required to kill that cell. In general, it has been found that cell radiosensitivity is directly proportional to the rate of cell division and inversely proportional to the cell's capacity for DNA repair.

As used herein, the term "radioresistant" refers to a cell that does not die when exposed to clinically suitable dosages of radiation.

As used herein, the term “radiation-induced toxicity” or “radiotoxicity” designates any toxic or adverse side effect induced by radiation which may be observed in a subject treated by radiation therapy or in an organotypic slice which was irradiated. In particular, the term radiotoxicity includes any toxic or adverse side effect of radiation, which may be observed in the irradiated organotypic slice comprising healthy tissue. The radiotoxicity also includes any toxic or adverse side effect of radiation, which may be observed in the irradiated organotypic slice comprising healthy tissue and cancer tissue.

As used herein, the term "chemotherapy" has its general meaning in the art and refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include multikinase inhibitors such as sorafenib and sunitinib, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, tri ethyl enethiophosphaorarni de and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In some embodiment, the present invention relates to chemotherapy inducing doublestranded breaks (DSBs).

The use of chemotherapeutic agents can cause DNA damages, including direct or indirect double-stranded breaks. Examples of mostly used families of chemotherapeutic agents (chemical cytotoxics) are: inhibitors of topoisomerases I or II (camptothecin/topotecan, epirubicin/etoposide), DNA crosslinkers (cisplatin/carboplatin/oxaliplatin), DNA alkylating agents (carmustine/dacarbazine) or anti-metabolic agents (5- fluorouracil/gemcitabine/capecitabine).

As used herein, the term "chemosensitivity" refers to the relative susceptibility of cancer cells to the effects of anticancer drugs. The more chemosensitive a cancer cell is, the less anticancer drug is required to kill that cell.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative, improving the patient’s condition or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients 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 patient 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 patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., daily, 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.]).

As used herein, the term “preventing” intends characterizing a prophylactic method or process that is aimed at delaying or preventing the onset of a disorder or condition to which such term applies.

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 (i.e. Tau) produced by translation of a mRNA.

As used herein, the term “Tau” denotes the Tau protein from mammals and especially from primates (and Tupaiidae). Human Tau is a neuronal microtubule-associated protein found predominantly in axons and functions to promote tubulin polymerization and stabilize microtubules. Six isoforms (isoform A, B, C, D, E, F, G, fetal-Tau) are found in the human brain, the longest isoform comprising 441 amino acids (isoform F, Uniprot P10636-8). Tau and its properties are also described by Reynolds, C. H. et al., J. Neurochem. 69 (1997) 191-198. Tau, in its hyperphosphorylated form, is the major component of paired helical filaments (PHF), the building block of neurofibrillary lesions in Alzheimer's disease (AD) brain. Tau can be phosphorylated at its serine or threonine residues by several different kinases including GSK3beta, cdk5, MARK and members of the MAP kinase family.

The protein sequence of human Tau protein, and its isoforms, may be found in Uniprot database with the following access numbers:

Tau isoform Fetal (352 Amino Acids) Uniprot Pl 0636-2

Tau isoform B (381 AA) Uniprot Pl 0636-4

Tau isoform D (383 AA) Uniprot P10636-6

Tau isoform C (410 AA) Uniprot P10636-5

Tau isoform E (412 AA) Uniprot Pl 0636-7

Tau isoform F (441 AA) Uniprot P10636-8

Tau isoform G (776 AA) Uniprot Pl 0636-9

In human Tau protein is encoded by the MAPT (Microtubule associated protein tau) gene located on chromosome 17 (Gene ID: 4137). This gene has 18 transcripts (splice variants), 1 gene allele, 255 orthologues, 1 paralogue and is associated with 13 phenotypes.

Example of human MAPT transcripts which encoded Tau protein may be found in Ensembl database with the following access number

Transcript MAPT-201 (833 AA) Ensembl ID ENST00000262410 (Protein coding)

Transcript MAPT-202 (352 AA) Ensembl ID ENST00000334239 (Protein coding Transcript MAPT-203(736 AA) Ensembl ID ENST00000344290 (Protein coding) Transcript MAPT-204 (441 AA) Ensembl ID ENST00000351559 (Protein coding) Transcript MAPT-205 (776 AA) Ensembl ID ENST00000415613 (Protein coding) Transcript MAPT-206 (412 AA) Ensembl ID :ENST00000420682 (Protein coding) Transcript MAPT-207 (410 AA) Ensembl ID ENST00000431008 (Protein coding) Transcript MAPT-208 (383 AA) Ensembl ID ENST00000446361 (Protein coding)

Transcipt MAPT-209 (381 AA) Ensembl ID ENST00000535772 (Protein coding) Transcript MAPT-212 (758 AA) Ensembl ID ENST00000571987 (Protein coding) Transript MAPT-214 (441 AA) Ensembl ID ENST00000574436 (Protein coding) Transcript MAPT-217 (412 AA) Ensembl ID ENST00000680542 (Protein coding) Transcript MAPT-218 (424 AA) Ensembl ID ENST00000680674 (Protein coding) Of course variant sequences of the Tau may be used in the context of the present invention, those including but not limited to functional homologues, paralogues or orthologues of such sequences.

The term “Tau” should be understood broadly, it encompasses the native Tau, variants thereof having binding activity with microtubule and fragments thereof having binding activity with microtubule. In particular the native Tau, variants and isoforms preferably contain at least three or four microtubule binding domains (named 3R and 4R respectively). All human Tau isoform and MAPT transcript above described contains at least three or four microtubule binding domains.

In a particular embodiment, the Tau protein used in the context of the present invention is transcript Variant (or Tau Isoform) selected from the list consisting of (1N4R) Transcript MAPT-206 (412 AA) (Protein coding Tau isoform E) and (2N4R) Transript MAPT-214 (441 AA) (Protein coding Tau isoform F).

• Tau Inhibitor

In some embodiment, the inhibitor of the present invention is Tau inhibitor.

As used herein, the term "inhibitor" as used herein includes not only drugs for inhibiting activity of target molecules, but also drugs for inhibiting the expression of target molecules.

As used herein, the term “Tau inhibitor” denotes a molecule or compound which can inhibit directly or indirectly the activity of the protein by limiting or impairing the interactions of the protein (ie with microtubules associated with translocation of DNA repair factors such as 53BP1 or with chromatin proteins and DNA), or a molecule or compound which destabilizes the protein structure, or a molecule or compound which inhibits the transcription or the translation of Tau, or accelerates its degradation. The term “Tau inhibitor” also denotes an inhibitor of the expression of the gene coding for the protein.

A specific embodiment, the Tau inhibitor is a Tau inhibitor which directly binds to tau (protein or nucleic sequence (DNA or mRNA)) and neutralizes, blocks, inhibits, abrogates, reduces or interferes with the biological activity of Tau protein such as 1) Tau protein binding activity with microtubules (associated with translocation of DNA repair factors such as 53BP1) and/or 2) initiation of repair activities of the junction-type DSBs of the non-homologous ends (NHEJ) and homologous recombination (HR) .

Accordingly, in the context of the present invention, the Tau inhibitor (i) directly binds to Tau (protein or nucleic sequence (DNA or mRNA)) and (ii) inhibits biological activity of Tau protein such as Tau protein binding activity with microtubules (associated with translocation in nucleus of DNA repair factors such as 53BP1) and/or initiation of repair activities of the junction-type DSBs of the non-homologous ends (NHEJ) and homologous recombination (HR). Examples of Tau inhibitors include but are not limited to any of the inhibitors described in “Jadhav et al. Acta Neuropathologica Communications (2019) 7:22 all of which are herein incorporated by reference.

By "biological activity" of Tau protein is meant in the context of the present invention, the translocation in nucleus of DNA repair factors such as 53BP1) and/or initiation of repair activities of the junction-type DSBs of the non-homologous ends (NHEJ) and homologous recombination (HR)

Tests for determining the capacity of a compound to be a Tau protein inhibitor are well known to the person skilled in the art. In a preferred embodiment, the antagonist/inhibitor specifically binds to Tau protein (protein or nucleic sequence (DNA or mRNA)) in a sufficient manner to inhibit the biological activity of Tau protein. Binding to Tau protein and inhibition of the biological activity of Tau protein may be determined by any competing assays well known in the art. For example, the assay may consist in determining the ability of the agent to be tested as a Tau protein inhibitor to bind to Tau protein. The binding ability is reflected by the Kd measurement. The term "Kd", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). Kd values for binding biomolecules can be determined using methods well established in the art. In specific embodiments, an antagonist / inhibitor that "specifically binds to Tau protein" is intended to refer to an inhibitor that binds to human Tau protein polypeptide with a Kd of IpM or less, lOOnM or less, lOnM or less, or 3nM or less. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of Tau protein : inhibition of Tau protein binding with microtubules (associated with translocation in nucleus of DNA repair factors such as 53BP1) and/or initiation of repair activities of the junction-type DSBs of the non-homologous ends (NHEJ) and homologous recombination (HR).

By “inhibitor of the Tau protein activity”, it is herein referred to a compound which is capable of reducing or suppressing 1) the translocation in nucleus of DNA repair factors such as 53BP1 and/or 2) initiation of repair activities of the junction-type DSBs of the non- homologous ends (NHEJ) and homologous recombination (HR).

In view of the teaching of the present disclosure, particularly of the examples, it falls within the ability of the skilled person to assess whether a compound is an inhibitor 1) the translocation in nucleus of DNA repair factors such as 53BP1 and/or 2) initiation of repair activities of the junction-type DSBs of the non-homologous ends (NHEJ) and homologous recombination (HR). A suitable test for detecting Tau protein binding activity with microtubules (associated with translocation in nucleus of DNA repair factors such as 53BP1) is described in examples hereinafter (see figure 6 and 7).

A suitable test for detecting repair activities of the junction-type DSBs of the non- homologous ends (NHEJ) is described in examples hereinafter (see figure 1 to 4) . DSBs is quantified using a phosphorylated form of H2AX as an indicator of DNA damagefsee 29],

Typically, a tau inhibitor according to the invention includes but is not limited to:

A) Inhibitor of Tau activity selected from the list consisting Anti-Tau antibody and anti- Tau aptamers, tau peptide (vaccines)

B) PROTAC (“Proteolysis Targeting Chimera”) which simultaneously bind a tau protein and an E3 -ubiquitin ligase.

C) Inhibitor of Tau gene expression selected from the list consisting of antisense, oligonucleotide, nuclease, siRNA, shRNA or ribozyme nucleic acid sequence.

In some embodiments, the Tau inhibitor is an antibody.

As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, VHH (antigen binding fragment of heavy chain only antibodies), minibodies, diabodies, bi specific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388. In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique to produce antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique. In a particular embodiment, the antibody is specific of the isoform B of Tau.

Antibodies directed against Tau can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against TAU can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-TAU single chain antibodies. Compounds useful in practicing the present invention also include anti-TAU antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to Tau. Humanized anti-TAU antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of nonhuman (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non- human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

Tau inhibitors such as anti Tau antibodies are well known in the art. Examples of patents disclosing anti Tau antibodies are. WO/2012/049570, WO/2014096321, WO/2015/004163; WO/2015200806, WO/2016/112078, WO/2018/152359, WO/2020/ 120644 (VHH anti Tau) WO/2020193520, WO/2021/010712,. . .

In the context of the invention, it could be advantageous to use a nanobody directed against Tau in order to enter the cell. Thus in another embodiment, the antibody according to the invention is a single domain antibody directed against Tau. 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. 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. 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).

Examples of patent disclosing VHH anti Tau antibodies is. WO/2020/120644

In a particular embodiment, the Tau inhibitor is a peptide, petptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.

In a particular embodiment, the Tau inhibitor is an aptamer.

The term “Aptamers” refer to a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide 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., Science, 1990, 249(4968): 505- 10. 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., Clin. Chem., 1999, 45(9): 1628-50. 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., Nature, 1996,380, 548-50).

In a particular embodiment, the Tau inhibitor is a small organic molecule.

The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

In some embodiment, the Tau inhibitor is a polypeptide. In a particular embodiment a Tau polypeptide may be used as vaccine composition in order to induce an anti Tau serum.

Accordingly, another example of Tau inhibitors according to the invention is a vaccine composition comprising an isolated peptide of Tau.

By “vaccine composition” it is herein intended a substance which is able to induce an immune response in an individual, and for example to induce the production of antibodies directed against the isolated tau polypeptide.

A vaccine is defined herein as a biological agent which is capable of providing a protective response in an animal to which the vaccine has been delivered and is incapable of causing severe disease. The vaccine stimulates antibody production or cellular immunity against the pathogen (or agent) causing the disease; administration of the vaccine thus results in immunity from the disease. Active immunization with vaccine composition is long lasting because it induces immunological memory. Active vaccines are easy to administer (different routes) and the production is cost-effective. Immunization generates polyclonal response; antibodies can recognize multiple epitopes on the target protein with different affinity and avidity. On the other hand, the immune response depends on the host immune system, there is a variability in the antibody response across patients. Like their passive immunotherapy counterparts, active vaccines targeting the mid-region, microtubule binding domain of Tau and C -terminus of Tau have been extensively investigated in preclinical studies (see table 3 of Jadhav et al. Acta Neuropathologica Communications (2019) 7:22). There are two tau active vaccines that have been tested in human clinical trials, AADvacl for Alzheimer’s disease and non-fluent primary progressive aphasia (Axon Neuroscience SE), and ACL35 vaccine for Alzheimer’s disease (AC Immune SA, Janssen). Active vaccine AADvacl consists of tau peptide (aa 294-305/4R) that was coupled to keyhole limpet haemocyanin (KLH) in order to stimulate production of specific antibodies. ACL35 vaccine is a liposome-based vaccine consisting of a synthetic peptide to mimic the phospho-epitope of tau at residues pS396/pS404 anchored into a lipid bilayer.

In some embodiment, the Tau inhibitor is a PROTAC against Tau.

As used herein, the term “PROTACs” (“Proteolysis Targeting Chimera”) means bifunctional molecules which simultaneously bind a target protein and an E3-ubiquitin ligase. This causes the poly-ubiquitination of the target protein which is thus degraded into small peptides and amino acids by the proteasome complex. The PROTAC approach is therefore a chemical protein knock-down strategy. It is therefore could be useful to provide bifunctional chimeric ligands capable of inducing targeted proteolysis of Tau according to the PROTAC strategy.

An example of PROTAC targeting Tau is describe Lu M. et al “Discovery of a Keapl- dependent peptide PROTAC to knockdown Tau by ubiquitination-proteasome degradation pathway European Journal of Medicinal Chemistry 146 (2018) 251e259. Briefly in this study authors identified Keapl, a substrate adaptor protein for ubiquitin E3 ligase involved in oxidative stress regulation, as a novel candidate for PROTACs that can be applied in the degradation of the nonenzymatic protein Tau. This peptide PROTAC by recruiting Keapl -Cul3 ubiquitin E3 ligase was developed and applied in the degradation of intracellular Tau. Peptide 1 showed strong in vitro binding with Keapl and Tau.

In another embodiment, the Tau inhibitor according to the invention is an inhibitor of Tau gene expression. In some embodiments, the Tau inhibitor is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of Tau.

In a particular embodiment, the inhibitor of Tau expression is siRNA

In a particular embodiment, the inhibitor of Tau expression is shRNA.

A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound.

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, 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 can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting 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).

Ribozymes can also function as an inhibitor of Tau expression 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 mineralocorticoid receptor 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 GUC. 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.

Antisense oligonucleotides, siRNAs, shRNAs 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, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, 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, shRNA 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 rous sarcoma virus; adenovirus, adeno-associated virus; S V40-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.

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, 1990 and in Murry, 1991.

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses. 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.

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. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding 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. These 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, eye, intradermal, subcutaneous, intra-articular 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.

In some embodiments, the Tau inhibitor is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error-prone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

In a particular embodiment, the endonuclease is CRISPR-cas.

As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038/cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534/genetics.113.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

In some embodiments, the Tau inhibitor is an aptamer.

In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration. Thus, aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti-PD-1 aptamer. Particularly, the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015.

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. a Tau inhibitor) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of drug to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for drug depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of drug employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of drug is about 0.1- 100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

In some embodiments, the patient is administered with a pharmaceutical composition comprising the therapeutically effective amount of a Tau inhibitor as active principle and at least one pharmaceutically acceptable excipient.

As used herein the term “active principle” or “active ingredient” are used interchangeably. As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and/or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

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. 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 syringeability 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. Sterile injectable solutions are prepared by incorporating the agent of the present invention 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 active ingredients 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 preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1 : Inhibition of Tau increases y -H2AX levels after bleomycin treatment in MCF7 and MDA-MB-231. (A) MCF7 and (B) MDA-MB-231 shCtrl or shTau stable clones were treated or not with bleomycin (BLM, 30 pg/ml) for 2h. Cells were then labeled with a y-H2AX antibody to quantify DNA double-strand breaks. Nuclear y-H2AX bulk fluorescence intensity was analyzed using Image J. All experiments were carried out in 3 independent experiments. Data are mean ± SD (n > 60 cells per conditions) **P<0.01; ***P<0.001..

Figure 2: Inhibition of Tau increases y -H2AX levels after X-ray treatment in MCF7 and MDA-MB-231. (A) MCF7 and (B) MDA-MB-231 shCtrl or shTau stable clones were X-irradiated with 2 Gy and then further incubated for 5 min, 2, 4 and 6h. Cells were then labeled with a y-H2AX antibody to quantify DNA double-strand breaks and the number of y-H2AX foci per cell was determined using Image J. All experiments were carried out in 3 independent experiments. Data are mean ± SD (n > 60 cells per conditions) * <0.05; ** <0.01; *** <0.001. #P<0.05 ### <0.001 compared to untreated shCtrl cells. Representative

Figure 3: Tau inhibition increases mutation frequency measured by the HPRT test. (A) Western-blot analysis of Tau expression in CHO-GFP and CHO-Tau-GFP stable clones using anti -Tau and anti-actin antibodies (B) Schematic representation of the HPRT test in CHO cells. (C) CHO cells were stably transfected with plasmids coding for GFP or GFP-Tau and treated with cisplatin (10 pM, 2 h) or oxaliplatin (20 pM, 2 h). Mutation frequency was measured as the number of colony/number of cells seeded X efficiency of plating. (D) CHO cells were stably transfected with plasmids coding for GFP or GFP-Tau and treated with X-rays (4 Gy). Mutation frequency was determined as described in B. Data are mean ± SD ***7’0.001..

Figure 4 : Tau increases HR and cNHEJ activities. (A) Schematic representation of DR-GFP reporter. (B) Western-blot analyses of a HeLa DR-GFP stable clone transiently transfected with plasmid encoding the endonuclease I-Scel with or without plasmid encoding Tau protein. (C) Tau increases HR activity. HeLa DR-GFP cells were transfected as described in (B). The percentage of GFP-positive cells was quantitated by flow cytometry (five independent experiments). Results are expressed as fold induction relative to the control. (D) Schematic representation of EJ5GFP reporter. (E) Western-blot analyses of HeLa EJ5-GFP stable clone transiently transfected with plasmid encoding the endonuclease I-Scel with or without plasmid encoding Tau protein. (F) Tau increased cNHEJ activity. HeLa EJ5-GFP cells were transfected as described in (E). The percentage of GFP-positive cells was quantitated by flow cytometry (Five independent experiments). Results are expressed as fold induction relative to the control. Data are mean ± SD **PO.OI; ***7’0.001.

Figure 5. Tau depletion confers sensitivity to doxorubicin and X-rays in mice xenografts. MCF7 cells expressing either scramble (shctrl) or shTau were injected subcutaneously (5 mice per group) and tumor volume was measured for 11 days starting from 100 mm³ and (A) treated or not with doxorubicin (6 mg/kg x 1) or (B) treated or not with X-rays (2 Gy x 2). Average tumor weight ± SD is shown. *7’0.05; **7’0.01; ***7’0.001 compared to untreated cells. ###P<Q.QQ1 compared to treated MCF7 shCtrl xenografts.

Figure 6. Tau increases 53BP1 nuclear translocation after X-ray treatment. (A). MCF7 shCtrl and shTau cells were irradiated with 2 Gy exposure and then further incubated for 15 min. Cells were then labeled with a 53BP1 antibody and the number of 53BP1 foci per cell (n >50) was determined using Image J. All experiments were carried out in 3 independent experiments. Data are mean ± SD 7’0.05; **7’0.01; ***7’0.001. (B) MCF7 shCtrl or shTau stable clones were irradiated with 2 Gy and then further incubated for 15 min. Cells were then labeled with a y-H2AX antibody to quantify DNA double-strand breaks and the number of y-H2AX foci per cell (n >50) was determined using Image J. All experiments were carried out 3 independent experiments. Data are mean ± SD ***7’0.001. (C) The graph shows the quantification of four independent experiments from 53BP1 (red) and y-H2AX (green) staining in MCF7 shCtrl or shTau stable clones irradiated or not with 2 Gy and further incubated for 15 min. Data are mean ± SD *7’0.05; **7’0.01; ***7’0.001. ##7’0.01; ###7’0.001 compared to the corresponding cytoplasmic fraction.

Figure 7. Tau regulates the 53BPl/microtubule interaction. (A) (B) The graph shows the quantification of three independent proximity ligation assays (53BP1-Dynein) experiments as in Proximity Ligation Assay showing 53BP1-Dynein interaction in MCF7shCtrl and shTau cells in control and 2 Gy treated conditions. All data are mean ± SD *7’0.05; **7’0.01; ***7’0.001.

EXAMPLE: Materials and Methodes

Materials and Plasmids pcDNA3-Tau4R and Tau-GFP have been described elsewhere [14,23,24], pimEJ5GFP was a gift from Jeremy Stark (Addgene plasmid # 44026) [25], pDRGFP (Addgene plasmid # 26475) and pCBAScel (Addgene plasmid # 26477) were a gift from Maria Jasin [26], Short hairpin Tau and RNA Ctrl vectors were purchased from Santacruz. Doxorubicin, cisplatin, oxaliplatin, 6-thioguanine and hypoxanthine were purchased from Sigma-Aldrich and bleomycin from Calbiochem. Cells were exposed to ionizing radiation (IR) using an X-ray machine (Clinac23X).

Cell Culture and Transfection

HeLa cells, MCF7, and MDA-MB-231 were cultured in Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum, 2 mM L-glutamine and 50 U/ml penicillin/ streptomycin (Gibco) at 37°C in 5% CO2 humidified air. CHO-K1 cells were cultured in RPMI1640 (Gibco) with 10% fetal bovine serum, 2 mM L-glutamine and 50 U/ml penicillin/streptomycin (Gibco). MCF7 shctrl and shTau cells have been described previously [14], Transient and stable transfection experiments were performed using the lipofectamine LTX reagent according to the manufacturer guideline (Invitrogen). To isolate stably transfected HeLa clones, cells were transfected with the pDRGFP or pimEJ5GFP plasmids and selected with puromycin (2 Dg/ml). Clones were isolated and tested for GFP expression after pCBAScel transfection. Of 6 clones tested for GFP, one was chosen for further studies. For CHO-K1 GFP or GFP-Tau, cells were transfected with plasmids encoding GFP or GFP-Tau and selected with G418 (200 pg/ml). Stably transfected cells were isolated using flow cytometry and cell sorting (Sony SH800).

Cellular extracts and western-blotting

For the preparation of whole cell extracts, cells were washed twice in ice-cold PBS and scraped in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, protease inhibitor mixture (Roche)]. The lysate was rotated fori h at 4°C and centrifuged for 15 min at 4°C. Nuclear and cytoplasmic extracts were prepared as described previously [24], Briefly, Cells were harvested, washed with PBS and resuspended in buffer A (lOmM Hepes pH=7.8, lOmM KC1, 0,5mM EDTA, ImM DTT, complete protease inhibitors [Roche]). Nuclei were recovered by centrifugation for 10 min at 2000g, resuspended in nuclear lysis buffer (50mM Hepes pH 7.4, 1.5mM MgC12, 420mM NaCl, ImM dithiothreitol [DTT] and complete protease inhibitors [Roche]), and then incubated for 1 h in a rotation wheel at 4°C to extract nuclear proteins. For western-blotting, proteins were solubilized in an SDS loading buffer and analyzed by SDS-PAGE. Primary antibodies used in Western blotting experiments were directed against Tau Cter [14], 53BP1 (Cell Signaling), H3 (Millipore), Hsp90 (Santa Cruz) and P-actin (Sigma). Secondary antibodies coupled to HRP were from Sigma-Aldrich. Immune complexes were detected using the ECL+ system from Amersham/GE Healthcare and observed with an Image Reader LAS4000 (Fujifilm). Quantification was performed by densitometry using Imaged software.

Cell fractionation into cytosolic and microtubule fractions

Cell fractionation was performed as described previously [27], Briefly, equal amounts of MCF7 shCtrl and shTau cells were recovered in buffer A (80 mM Pipes, pH 6.8, 1 mM MgCh, 2 mM EGTA, 30% glycerol, 0.1% Triton X-100, complete protease inhibitors [Roche]). After ultracentrifugation at 100,000 * g at 21 °C for 18 min, supernatants were collected as cytosolic fractions. The pellets (microtubule fraction) were recovered in RIPA buffer and sonicated. Samples were mixed with LDS buffer and equal volumes were loaded for SDS- PAGE and analyzed by immunoblotting.

Hprt mutant Frequency determination

Hrprt mutant frequency was determined by measuring the clonogenicity of cells, as previously described [28], Briefly, CHO-K1 cells stably transfected with GFP or GFP-Tau were grown in hypoxanthine medium for 5 days to eliminate preexisting Hprt mutants, then 1.5 x 10⁶ cells were plated in 75cm² flasks. After 24 h of culture, cells were treated or not with cisplatin (10 pM, 2h), oxaliplatin (20 pM, 2h) or X-rays (2 Gy). Cells were maintained for 8 days to allow the expression of the Hrprt mutant phenotype. After this period, 2x10⁵ cells were plated in 100mm petri dishes in a complete medium containing 6pg/mL of 6-thioguanine for the selection of Hprt mutants. In parallel, 200 cells were seeded in 35mm dishes (12 dishes/condition), with non-selective medium. Ten days later, colonies formed in both selective and non-selective media were fixed, stained with 4% Giemsa, and scored. Colony efficiency was expressed as a ratio between the number of colonies and the number of seeded cells. Mutant frequency was expressed as a ratio between the colony efficiency of Hprt mutants and those cells cultured in non-selective medium.

Immunofluorescence

Cells were fixed in 4% paraformaldehyde for 30 min at room temperature. Permeabilization was carried out in 0.2% Triton X-100 in phosphate-buffered saline for 10 min at room temperature. After 1 h saturation in 2% bovine serum albumin, immunostaining was performed using yH2AX (Millipore), 53BP1 (Cell Signaling), y- tubulin (Millipore). These antibodies were revealed via secondary antibodies coupled to Alexa 488 or 568 (Life Technologies). Nuclear staining was performed by adding 1/2000 DAPI (Img/mL, Life Technologies) in phosphate-buffered saline for 10 min. Slides were then analyzed with a Zeiss LSM710 confocal laser scanning microscope (60x magnification). Images were collected in the Z direction at 0.80 mm intervals and quantified using the ImageJ plug-in as described previously [14],

Proximity ligation assay

Proximity ligation assay was performed with a duolink proximity ligation assay kit (Sigma-Aldrich) according to the manufacturer's guidelines. 53BP1 (Millipore) and dynein (Abeam) were used to visualize 53BP1 -dynein proximity.

DNA repair reporter assays

HeLa cells with stably integrated HR (DR-GFP) or nonhomologous end joining (EJ5GFP) reporters were plated in 6-well plates and then transfected with a plasmid encoding the LScel endonuclease gene with or without Tau. The cells were left to grow for 48 h and then trypsinized and washed once with PBS. GFP fluorescence was analyzed using an LSR FORTESSA X20 cytometer (Becton Dickinson).

Xenograft Studies

The animals were maintained in compliance with European standards for the care and use of laboratory animals and experimental protocols approved by the local Animal Ethical Committee (agreement APAFIS# 27259-2020091810585608, Lille, France). 10 million MCF7, MCF7 shCtrl or MCF7 shTau cells were suspended in 100 pL of FBS/Matrigel v/v (Invitrogen), and then injected in the fat pad of 6-week-old female NOD/SCID mice (Jackson Laboratory). Tumors were measured with calipers, calculated by the formula volume= L x I² x A. When tumors reached - 100 mm³, mice were assigned randomly to either control, doxorubicin (6 mg/kg once via intravenous injection) or X-rays (N=10 tumors). Mice were then treated with PBS or doxorubicin (6 mg/kg). For X-rays, mice were anesthetized (ketamine, 100 mg/kg and xylazine, 20 mg/kg) and the tumor site was then exposed to radiation (2 Gy x 2) using an XRAD 320. Tumors are measured with calipers and volume was calculated as described previously.

Statistical Analysis

Data are mean ± SD. Statistical tests were carried out using GraphPad Prism software (GraphPad Inc.). Statistical significance between groups was analyzed with Wilcoxon- Mann- Whitney test and Mann-Whitney tests. A p-value less than 0.05 was considered significant.

Results Tau aids clearance of double-strand breaks.

To assess a potential role of Tau in protecting the DNA of cancer cells, we used MCF7 and MDA-MD-231 cells stably overexpressing either small hairpin RNA control (shCtrl) or targeting Tau (shTau) in which we observed 70% decrease in Tau expression [14], These control and Tau knockdown cells were treated for 2h with bleomycin (30 pg/ml), a drug known to induce double-strand breaks. We then quantified DSBs using a phosphorylated form of H2AX as an indicator of DNA damage[29]. Where we knocked down Tau, y-H2AX fluorescence intensity increased 4-fold compared to just 2-fold in both MCF7 and MDA-MD- 231 control cells (Figures 1A and IB).

Next we tested the effect of knocking down Tau on DSB clearance kinetics after a pulse of X irradiation. MCF7 and MDA-MD-231 clones were irradiated with 2 Gy and the number of y-H2AX foci was then quantified at 5 min, 2, 4 and 6h post-irradiation. A 2-fold increase in DSB foci in both MCF7-shCtrl and shTau cells clones was observed. However, while the level of y-H2AX foci decreased after 2h and had nearly returned to its initial level after 6h in MCF7- shCtrl cells, it remained unchanged 6h post-irradiation in MCF7-shTau cells (Figure 2A). We obtained similar results using another shRNA directed against Tau ruling out possible of target effects (data not shown). The same trends were observed in MDA-MD-231 shCtrl and shTau cells (Figure 2B).

Altogether, these observations suggest a role for Tau in DSB clearance/repair.

Tau decreases the mutation rate induced by DNA damaging agents

The above results suggested that Tau could be a factor that mediates resistance to DNA damaging agents. We therefore performed a classic Hprt mutation test to assess the rates of spontaneous or cisplatin-, oxaliplatin- and X-ray-induced mutations in the absence or presence of Tau. CHO KI cells, which possess only one functional Hprt allele, were stably transfected with plasmids encoding GFP or GFP-Tau fusion protein, then treated with cisplatin, oxaliplatin or X-rays (Figure 3 A). The addition of 6-thioguanine allows the selection of Hprt mutants, which reflect the mutation frequencies (Figure 3B). Figure 3C and 3D demonstrate that the frequency of spontaneous 6- thioguanine resistant mutants was not significantly different in CHO KI cells expressing Tau compared to GFP control cells. To study if Tau reduced the frequency of DNA damaging agents-induced mutations at the Hprt locus, GFP or GFP-Tau CHO KI cells were treated with cisplatin (10 pM, 2 h), oxaliplatin (20 pM, 2 h) or X-rays (4 Gy). In the three cases, the frequencies of 6- thioguanine induced by these compounds were significantly lower in cells expressing Tau (3.3-, 3.1- and 2.8-fold for cisplatin, oxaliplatin and X-rays respectively). Thus, these results confirmed a significant role for Tau in the repair of anticancer agent induced-DNA lesions.

Tau increases HR and cNHEJ activities.

The repair of DSB breaks in mammalian cells occurs mainly by the cNHEJ or the HR pathway. To ascertain if Tau acts on a specific DSB pathway, we employed reporter genes of either HR or cNHEJ. Firstly we employed the HR reporter gene (DR-GFP), which contains a non-functional GFP due to the replacement of 11 bp of the GFP sequence by the rare cutting I- Scel endonuclease recognition site. The I-Scel endonuclease generates a DSB. The HR repair restores the proper GFP coding sequence, resulting in GFP expression which is further quantified by flow cytometry (Figure 4A) [24], We established a HeLa DR-GFP stable clone and then transiently transfected a plasmid encoding the I-Scel endonuclease in the absence (Ctrl) or presence of plasmid encoding Tau (Tau). The endonuclease expression level was verified and shown to be similar between HeLa-DR-GFP-Ctrl and HeLa-DR-GFP-Tau cells (Figure 4B). Noticeably, Tau overexpression resulted in a 1.5-fold increase in the number of cells expressing GFP in HeLa-DR-GFP-Tau (Figure 4C). These results indicated that Tau promoted a more efficient HR repair.

Secondly, we evaluated the cNHEJ DNA repair process using the EJ5-GFP reporter [23], In this plasmid, the promoter is separated from the GFP by a puromycin gene containing two I-Scel sites at the 5’ and 3’ positions. Following I-Scel cleavage, the puromycin gene is excised, leading to the restoration of the GFP signal by NHEJ repair (Figure 4D). A selected HeLa EJ5-GFP clone was therefore transfected with a plasmid encoding the I-Scel in the absence or presence of plasmid encoding Tau. As above, the level of I-Scel expression was similar between the two conditions (Figure 4E). As for HR also, we observed a 1.6-fold increase in cNHEJ repair activity when Tau was expressed (Figure 4F).

Taken together, these data demonstrated that Tau increased both HR and cNHEJ repair efficiency. shTau tumors are more sensitive to DNA damage.

Finally, we sought to test the in vivo relevance of our cellular findings. For this, MCF7- shCtrl and shTau cells were injected subcutaneously into the mammary fat pads of immunodeficient SCID mice. The tumor volumes were measured every three days until they reached 100 mm³ then treated with X-rays or the widely used chemotherapy drug, doxorubicin. As shown in Figure 5A, there were no differences in the tumor growth between shCtrl and shTau xenografts during these 11 days in the absence of doxorubicin. When treated with doxorubicin, shCtrl xenografts remained at approximately the starting volume, neither growing nor shrinking. For shTau xenografts however, doxorubicin treatment led to decrease in tumor volume, of 12.7% and 28.1% on days 5 and 11 respectively (Figure 5 A). Similar results were observed after x ray treatment with X rays reducing tumor volume more in the shTau xenografts with a 29.2% and 30.2% decrease on days 5 and 11 respectively (Figure 5B).

Together, these data strongly support that Tau inhibition potentiates the therapeutic effect of doxorubicin and X-rays by inhibiting DNA repair.

Tau regulates 53BP1 nuclear localization.

While we observed that Tau increased both cNHEJ and HR, it is known that cNHEJ accounts formost ofDSB repair in mammalian cells [7,8], In addition, the recruitment of 53BP1 at DSBs is known to be critical for this mechanism [6],

We next examined the formation of 53BP1 foci in MCF7 shCtrl and shTau cells by immufluorescence. As shown in Figure 6A and 6C, we observed a 4.5-fold increase in the number of 53BP1 foci in shCtrl cells 15 min after 2 Gy irradiation. However, knocking-down Tau expression led to a decrease in 53BP1 foci number (2.1 -fold increase 15 min after 2 Gy irradiation). On the other hand Tau knockdown had no effect on the number of y-H2AX foci (Figure 6B). Similar results were found 30 min after 2 Gy irradiation.

Lower abundance of nuclear 53BP1 foci was not explained by an expression change. We therefore hypothesized that the observed differences in shCtrl and shTau conditions could be due to a perturbed nuclear localization. Indeed, it has been demonstrated that efficient DSB repair relies on nuclear translocation of DNA repair proteins such as 53BP1 [9], To test this, intracellular 53BP1 distribution was then tested using subcellular fractionation (Figure 6C). For MCF7 shCtrl cells, 53BP1 protein was found primarily in the nuclear fraction in control (66%) and irradiated conditions (75%). Although the distribution was similar in the MCF7 shTau cells in control conditions (60% in the nuclear fraction), we observed a strong decrease in the nuclear fraction after X-rays treatment (30%).

These data therefore suggest that Tau regulates the cyto-nuclear shuttling of 53BP1

Tau silencing alters 53BP1 trafficking on microtubules.

It has been shown that nuclear translocation of 53BP1 and other DNA repair proteins, relies in part on their interaction with the retrograde motor protein dynein [9], Since Tau is mostly known as a microtubule-binding protein that regulate dynein, we next wondered if its absence could impede the interaction of 53BP1 with microtubules. We first compared the microtubule-association of 53BP1. For this, 53BP1 distribution between the cytosolic and microtubule fraction was assessed in control or X-ray treated MCF7-shCtrl or -shTau cells. As shown in Figure 7A, 53BP1 is present in both cytoplasmic and microtubule fractions. Noticeably, we observed a 2-fold difference in 53BP1 -microtubule complex enrichment in nontreated MCF7-shTau cells when compared to non-treated MCF7-shCtrl. Moreover, we found that 53BPl/microtubule association was further increased in MCF7shCtrl cells after 2 Gy irradiation (1.9-fold increase) while decreasing in MCF7 shTau cells (2.1-fold decrease).

Since the higher association could indicated either an increase or a stronger association of 53BP1 with microtubules, we next examined whether there were also differences in 53BPl/dynein interaction. For this, a proximity ligation assay was performed to specifically detect 53BPl/dynein interaction in MCF7-shCtrl and shTau cells in control or 2 Gy treated conditions. As shown in Figures 7B, proximity was increased in shCtrl cells after irradiation (2.3-fold) while the signal was lowered in MCF7-shTau cells (1.2-fold increase).

Discussion

In this work, we investigated the role of Tau in repairing DNA double-strand breaks in cancer cells. We report a new function of Tau in DSB repair that it carries out by regulating DNA repair protein trafficking. Notably, we identified Tau as a major actor in regulating cNHEJ through regulating 53BP1 trafficking on microtubules

This finding has precedents in several studies suggesting a relationship between Tau and DNA damage [15,30], In particular, an increase in y-H2AX foci was observed under stress conditions, in primary neuronal cell culture in the absence of Tau or in the cortex and hippocampus of knock-out Tau mice [31,32], Although it has been suggested that Tau binds and protects DNA from damage, our observations do not support this hypothesis. First, the same level of H2AX phosphorylation was observed in shCtrl and shTau cells after irradiation, which seems to be incompatible with a direct DNA protective role for Tau. Second, we observed a delay in the resolution of □-H2AX foci in shTau cells that further suggested a role for Tau in DNA repair as opposed to DNA binding per se.

The observed kinetics of y-H2AX resolution suggested that Tau mostly assists DNA repair through cNHEJ pathway. To obtain more detailed insights into the Tau function in cNHEJ, we examined the formation of 53BP1 foci in shCtrl and shTau cells. A lower 53BP1 recruitment to DNA damage sites is known to be sufficient to hinder DSB repair by cNHEJ [33], However, while most previous studies report a decrease in 53BP1 expression upon degradation, we do not observe variations in protein level but rather an alteration in protein localization. Dysregulation of nucleocytoplasmic transport has previously been implicated in neuronal Tau pathology. Protein aggregates have been shown to interact with different components of the nuclear pore complex and alter nucleocytoplasmic transport [34,35], Using Frontotemporal Lobar Degeneration Tau mutations (FTDP-MAPT), others demonstrated that changes in microtubule dynamics caused nuclear envelope distortions and marked disruption of nucleocytoplasmic transport [36], These observations suggest that Tau loss-of-function impedes microtubule nuclear translocation.

Our results therefore suggest that 53BP1 nucleocytoplasmic shuttling is highly dependent on Tau. 53BP1 is present both in the cytoplasm and nucleus in shCtrl cells and the nuclear translocation of 53BP1 tends to increase after DSB induction. Furthermore we observed increased interaction of 53BP1 with both dynein and microtubules in the presence of Tau when treated with DNA damaging agents. Interestingly, it has been observed an enhanced nuclear targeting of the human adenovirus type 2 (Ad2) after overexpression of MAP4 and relying on directed transport along microtubules by dynein [37], These observations further suggest that Tau might exert a similar role on dynein-directed protein translocation. Indeed, Tau depletion led to strong alterations in dynein-directed protein translocation. In shTau control cells, we observed an increase in the 53BP1 microtubule fraction independently of nuclear translocation. Although the current explanation is not known, this might reflect a lower displacement rate of dynein complex along microtubules. Tau is known to increase the frequency of long dynein- directed runs [38], Altogether, these observations are in agreement with previous results showing that altering microtubule dynamics with vincristine and paclitaxel increased 53BP1 cytoplasmic retention and accentuated the effect of DNA damaging agents [9],

It is important to note that an alteration in 53BP1 protein localization is observed after DSB induction, suggesting that microtubule dynamics are regulated by DSBs themselves. It has been demonstrated that upon double-strand breakage, cytoplasmic microtubules invade the nucleus [39], This mechanism is thought to increase the mobility of damaged DNA inside the nucleus necessary for DNA repair [10,40,41], Interestingly, it has been shown recently in neurons that DSBs increased the interaction between Tau and tubulin around the nuclear membrane, and further suggesting the enhancement of tubulin polymerization [32], Based on our observations, it is tempting to speculate that Tau, responds to DSBs, by enhancing microtubule polymerization and/or stability necessary for DNA mobility and nuclear translocation of DNA repair proteins.

Transport of DNA repair proteins along microtubules is not restricted to 53BP1 but also to other proteins involved in different DNA repair pathways. p53, DNA-PK complex proteins (Ku70, Ku80, DNA-PKcs), BRCA1/2, MRN complex proteins (Mrel 1, Rad50, Nbsl), or RPA and Rad51 are also subjected to microtubule trafficking [9,42-44], In this regard, we found that the efficiency of the HR pathway in reporter gene assay is increased in the presence of Tau. This observation further suggested that Tau did not impact on DSB repair pathways choice. In this regard, we also show that Tau expressing cells are less sensitive to cisplatin and oxaliplatin treatments. Platinum drugs acts mainly by forming intrastrand diadducts, primarily repaired via the nucleotide excision repair system [45], Interestingly, cytosolic sequestration of DNA repair proteins has been linked to neuronal death in different Tauopathies such as Alzheimer’s disease, Pick’s disease, corticobasal neurodegeneration or progressive supranuclear palsy [46,47],

Conclusions

In this work, we provide evidence for a new role of Tau in DNA repair, promoting resistance to commonly used anti-cancer treatments. These findings further suggest that Tau expression may be of interest as a molecular marker for response to DNA damaging agents and as a beneficial therapeutic target in tumors.

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Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method for decreasing therapeutic resistance to chemotherapy agent and/or radiotherapy agent in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Tau inhibitor.

2. A method for preventing and/or treating cancer with acquired resistance to treatment with chemotherapy agent and/or radiotherapy agent in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Tau inhibitor.

3. The method according to claims 1 or 2, wherein the Tau inhibitor is

A) Inhibitor of Tau activity selected from the list consisting Anti-Tau antibody and anti-Tau aptamers, tau peptide (vaccines)

B) PROTAC (“Proteolysis Targeting Chimera”) which simultaneously bind a tau protein and an E3-ubiquitin ligase.

4. . The method according to claim 3 wherein the tau Inhibitor is an anti-Tau antibody.

5. The method according to claim 2 wherein the cancer is selected from the list consisting in breast cancer, gastric, cancer prostate cancer ovarian cancer, glioma, Neuroblastoma, Kidney clear cell carcinoma, Lung cancer, Pheochromocytoma/Paraganglioma and Neuroendocrine tumors (NETs).

6. The method according to claims 1 to 6 wherein the chemotherapeutic agent is cisplatin, doxorubicin, carboplatin or oxaliplatin.

7. The method according to claims 1 to 6 wherein the radiotherapeutic agent is X-rays.