Inhibitor of ciliogenesis for use in a method of preventing therapeutic resistance in cancer

Abstract

The inventors establish a role for primary cilia in human TNBC chemotherapeutic resistance. They developed patient-derived organoids, and showed that these recapitulated the cellular heterogeneity of TNBC biopsies. They treated their TNBC organoids with chemotherapeutics and observed partial killing. The surviving cells with organoid-reconstituting capacity showed selective enrichment for the quasi-mesenchymal ciliated cell subpopulation. They developed a family of small-molecule inhibitors of ciliogenesis and show that these, or genetic ablation of primary cilia, suppress chemoresistance. In particular, the present invention relates to a method of preventing therapeutic resistance in a patient suffering from a cancer comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis. The present invention also relates to a method of treating a therapy-resistant cancer in a patient suffering from a cancer comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

Description

[0001] INHIBITOR OF CILIOGENESIS FOR USE IN A METHOD OF PREVENTING THERAPEUTIC RESISTANCE IN CANCER

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in field of medicine, in particular oncology.

[0004] BACKGROUND OF THE INVENTION:

[0005] Tumor heterogeneity and plasticity contribute to cancer therapeutic resistance which is responsible for the death of most cancer patients (Bonnefoi et al., 2014; Bray et al., 2021; Dagogo-Jack and Shaw, 2018; Marine et al., 2020). Resistance of cancer cells to treatment can result from both primary and acquired resistance to therapy (Dagogo-Jack and Shaw, 2018; Kim et al., 2018; Liau et al., 2017; Marine et al., 2020; Marsolier et al., 2022). Epithelial- Mesenchymal Transition (EMT) is a cell biological program that drives tumor heterogeneity (Bierie et al., 2017; Marine et al., 2020; Pastushenko et al., 2018), enabling epithelial cancer cells to acquire an array of mesenchymal phenotypes (Dongre and Weinberg, 2019; Pastushenko and Blanpain, 2019; Tessier et al., 2023). Tumor cells that activate this cell plasticity program can transit from early to intermediate to late hybrid epithelial-mesenchymal (E / M) states before reaching a fully elongated mesenchymal morphology, an endpoint that is rarely reached in spontaneously arising tumors (Pastushenko and Blanpain, 2019; Pastushenko et al., 2018). The distinct phenotypic states arrayed along the E-to-M axis are referred to as EMT states and cells in each of these states can display distinct functional properties (Nieto et al., 2016; Pastushenko and Blanpain, 2019; Wilson et al., 2020). Cells residing in the late hybrid E / M state, also known as the quasi-mesenchymal state, display tumor-initiating, metastatic, and therapy -resistant properties and are thought to function as cancer stem cells in many carcinomas (Bierie et al., 2017; Kroger et al., 2019; Pastushenko et al., 2018).

[0006] The degree to which EMT is activated in the neoplastic cells of a tumor is thought to rely on its cell-of-origin, which influences tumor heterogeneity and impacts response to therapy (Gupta et al., 2019; Pommier et al., 2020). Pioneering work revealed that the EMT program endows carcinoma cells with chemotherapy resistance properties, doing so by influencing the expression of ABC drug efflux pumps (Del Vecchio et al., 2014; Saxena et al., 2011), antioxidant enzymes (Del Vecchio et al., 2014), pro-apoptotic proteins (Wu et al., 2015), or by promoting DNA damage repair (Debaugnies et al., 2023). Other important studies revealed that EMT can also trigger cancer cell resistance to targeted therapies, by promoting a shift in cell signaling dependencies (Tam et al., 2013; Zhang et al., 2012), as well as to immunotherapy (Dongre et al., 2017), the latter achieved by orchestrating changes in tumor immune microenvironment (Dongre et al., 2021). Despite progress in understanding EMT -induced therapeutic resistance with the help of mouse tumor models and cancer cell lines in vitro (Shibue and Weinberg, 2017), there is still limited understanding of the degree to which EMT programs contribute to inter- and intratumor phenotypic heterogeneity during human tumor development and responses to therapy.

[0007] The research of the inventors has shown that EMT programs are activated in human triple-negative breast cancers (TNBCs) (Wilson et al., 2021). They found that EMT programs promote TNBC / claudin-low tumorigenesis by inducing primary ciliogenesis and ciliary signaling in tumor-initiating carcinoma cells (Guen et al., 2017; Wilson et al., 2021). The primary cilium is a microtubule-based structure which can be assembled as a solitary structure at the surface of cells composing both normal and neoplastic tissues where it serves as a cell signaling hub (Guen and Prigent, 2020; Hilgendorf et al., 2024). However, the role of primary cilia in cancer pathogenesis and therapeutic response remains poorly understood.

[0008] Here, the inventors used patient tumor biopsies and patient-derived cancer organoids (PDOs) to investigate the degree to which the EMT and primary cilia contribute to human TNBC heterogeneity and therapeutic response. TNBCs represent the most aggressive group of human breast tumors. Most of the highly progressed TNBCs display long-term resistance to therapy and therefore represent an unmet medical need despite recent progress in developing effective therapeutic agents (Bardia et al., 2021). Research using mouse cancer models and cell lines in vitro has suggested that TNBC heterogeneity, tumorigenesis, and therapeutic resistance is driven by EMT, notably in the claudin-low molecular subtype of this disease (Bierie et al., 2017; Del Vecchio et al., 2014; Dongre et al., 2021; Fougner et al., 2020; Grinda et al., 2021; Herschkowitz et al., 2007; Knezevic et al., 2015; Nolan et al., 2023; Pommier et al., 2020; Prat et al., 2010; Tam et al., 2013; Wilson et al., 2021; Wu et al., 2021). In the present work they show that primary cilia promote EMT-induced tumor heterogeneity and resistance to chemotherapy in TNBCs. EMT induces primary ciliogenesis in malignant cells that reside in a quasi-mesenchymal state. Moreover, primary ciliogenesis represents a vulnerability of these therapy -resistant carcinoma cells that are often the source of therapeutic failure.

[0009] SUMMARY OF THE INVENTION:

[0010] The present invention is defined by the claims. In particular, the present invention relates to a method of preventing therapeutic resistance in a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0011] DETAILED DESCRIPTION OF THE INVENTION:

[0012] Tumor heterogeneity and plasticity, driven by Epithelial-Mesenchymal Transition (EMT), enable cancer therapeutic resistance. The inventors previously showed that EMT promotes primary cilia formation, which enables sternness and tumorigenesis in triple-negative breast cancer (TNBC). Here, they establish a role for primary cilia in human TNBC chemotherapeutic resistance. They developed patient-derived organoids, and showed that these recapitulated the cellular heterogeneity of TNBC biopsies. Notably, one of the identified cell states bore a quasi-mesenchymal phenotype, primary cilia, and sternness signatures. They treated their TNBC organoids with chemotherapeutics and observed partial killing. The surviving cells with organoid-reconstituting capacity showed selective enrichment for the quasi-mesenchymal ciliated cell subpopulation. Genomic analyses argue that this enrichment reflects a combination of pre-existing cells and ones that arose through drug-induced cellular plasticity. They developed a family of small-molecule inhibitors of ciliogenesis and show that these, or genetic ablation of primary cilia, suppress chemoresistance. They conclude that primary cilia help TNBC to evade chemotherapy.

[0013] Main definitions:

[0014] As used herein the term “Epithelial-mesenchymal transition” (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties. EMT occurs in processes such as mesoderm formation, neural tube formation, wound healing, as well as the initiation of metastasis for cancer progression. EMT can be induced through several signal signaling pathways, including TGF-P, FGF, EGF, HGF, Wnt / beta-catenin, and Notch.

[0015] As used herein the term “ciliogenesis” is defined as the dynamic process of cilia (primary and motile cilia). Ciliogenesis results from a dynamic regulation of cilia assembly and disassembly. Cilia are important appendages of cells and are involved in numerous activities such as cell signaling, processing developmental signals, and directing the flow of fluids such as mucus over and around cells. Due to the importance of these cell processes, defects in ciliogenesis can lead to numerous human diseases related to non-functioning cilia known as ciliopathies. Ciliogenesis occurs through an ordered set of steps. Basal bodies migrate to the surface of the cell and attach to the cell cortex. Along the way, the basal bodies attach to membrane vesicles that fuse with the plasma membrane of the cell. The alignment of cilia is determined by the positioning and orientation of the basal bodies at this step. Once the alignment is determined, axonemal microtubules extend from the basal body and forming the cilia. Proteins must be synthesized in the cytoplasm of the cell and cannot be synthesized within cilia. For the cilium to elongate, proteins must be selectively imported from the cytoplasm into the cilium and transported to the tip of the cilium by intraflagellar transport (IFT). Once the cilium is completely formed, it continues to incorporate new tubulin at the tip of the cilia while older tubulin is simultaneously degraded. This requires an active mechanism that maintains ciliary length. Impairments in these mechanisms can affect the motility of the cell and cell signaling between cells.

[0016] There are two noted types of ciliogenesis: compartmentalized and cytosolic. Most cells undergo compartmentalized ciliogenesis in which cilia are enveloped by extensions of the plasma membrane for the entirety of development. In cytosolic ciliogenesis, the axenome must interact with proteins in the cytoplasm therefore it is directly exposed to the cytoplasm. In some cells, cytosolic ciliogenesis occurs after compartmentalized ciliogenesis.

[0017] As used herein the term “primary cilia” refers to sensory organelles. Primary cilia and flagella project from the apical side of the cells and emerge from the basal body, a modified centriole structure anchored to the plasma membrane. Their structural core is a microtubulebased cytoskeleton called the axoneme, which is surrounded by a membrane contiguous with the cell plasma membrane, but expressing specific signaling molecules. The axoneme of a primary cilium is composed of nine doublets of microtubules, whereas the axoneme of a motile cilium usually comprises two additional central microtubule doublets.

[0018] As used herein, the terms "cancer" has its general meaning in the art and refers to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non encapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malign melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brennertumor, malignant; phyllodestumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; strumaovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblasticodontosarcoma; ameloblastoma, malignant; ameloblasticfibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; ependymoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; medulloblastoma, glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocyticleukemia; mast cell leukemia; megakaryoblasticleukemia; myeloid sarcoma; and hairy cell leukemia.

[0019] In one embodiment, the subject of the present invention has a breast cancer tumor.

[0020] The term "breast cancer tumor" designates the abnormal mass of breast tissue composed of cancer, malignant cells. The American Joint Committee on Cancer (AJCC) provides two principal groups for breast cancer staging: anatomic, which is based on extent of cancer as defined by tumor size (T), lymph node status (N), and distant metastasis (M); and prognostic which includes anatomic TNM plus tumor grade and the status of the biomarkers human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR). (Kalli S., Semine A., Cohen S., Naber S. P., Makim S.S., BahlAmerican M. Joint Committee on Cancer’s Staging System for Breast Cancer, Eighth Edition: What the Radiologist Needs to Know. Radiographics. 2018 Nov-Dec;38(7): 1921-1933).

[0021] The first group called “anatomic” is divided in different stages:

[0022] Stage 0: non-invasive cancer

[0023] Stage 1, patients usually have invasive breast cancer, while 1 A refers to a tumor smaller than two centimeters that has spread to the lymph nodes but not outside the breasts, and IB can mean: no cancer is seen in the breast, but a few cancer cells are found in the lymph nodes under the arm (known as micrometastasis) or the cancer in the breast is 2cm or smaller and a few cancer cells are found in the lymph nodes under the arm (micrometastasis).

[0024] Stage 2A is when no cancer is seen in the breast but cancer is found in one to three lymph nodes under the arm or near the breastbone or the cancer in the breast is 2cm or smaller and cancer is found in one to three lymph nodes under the arm or near the breastbone or the cancer in the breast is larger than 2cm but smaller than 5cm and no cancer is found in the lymph nodes under the arm. Stage 2B can mean the cancer in the breast is larger than 2cm but smaller than 5cm. Cancer is found in one to three lymph nodes under the arm or near the breastbone or the cancer in the breast is larger than 5cm and no cancer is found in the lymph nodes under the arm.

[0025] Stage 3 describes a more aggressive form of invasive breast cancer and is divided into three sub phases (3 A, 3B and 3C). Stage 3A can mean No cancer is seen in the breast, but cancer is found in four to nine lymph nodes under the arm or near the breastbone or the cancer in the breast measures up to 5cm and cancer is found in four to nine lymph nodes under the arm or near the breastbone or the cancer in the breast is larger than 5cm, and cancer is found in up to three lymph nodes under the arm or near the breastbone. Stage 3B means the cancer in the breast can be any size and has spread to the skin of the breast or chest wall. Cancer is found in up to nine lymph nodes under the arm or near the breast bone. Stage 3C means the cancer in the breast can be any size, may have spread to the skin of the breast or chest wall and cancer is found in 10 or more lymph nodes under the arm or near the breastbone, or to nodes above or below the collarbone. Stage 4 indicates that the cancer has spread to other organs of the body (called distant metastasis) such as the lungs, the liver, distant lymph nodes, skin, or bones.

[0026] The second group “prognostic” corresponds to molecular subtypes of breast cancer that are based on the genes a cancer expresses:

[0027] - Luminal A breast cancer (luminal BC subtype) is hormone-receptor positive (estrogen- receptor and / or progesterone-receptor positive), HER2 negative, and has low levels of the protein Ki-67, which helps control how fast cancer cells grow. Luminal A cancers are low-grade, tend to grow slowly and have the best prognosis.

[0028] - Luminal B breast cancer (luminal BC subtype) is hormone-receptor positive (estrogen- receptor and / or progesterone-receptor positive), and either HER2 positive or HER2 negative with high levels of Ki-67. Luminal B cancers generally grow slightly faster than luminal A cancers and their prognosis is slightly worse.

[0029] - HER2-enriched breast cancer is hormone-receptor negative (estrogen-receptor and progesterone-receptor negative) and HER2 positive. HER2-enriched cancers tend to grow faster than luminal cancers and can have a worse prognosis, but they are often successfully treated with targeted therapies aimed at the HER2 protein, such as Herceptin (chemical name: trastuzumab), Perj eta (chemical name: pertuzumab), Tykerb (chemical name: lapatinib), Nerlynx (chemical name: neratinib), and Kadcyla (chemical name: T-DM1 or ado-trastuzumab emtansine).

[0030] - Normal-like breast cancer is similar to luminal A disease: hormone-receptor positive (estrogen-receptor and / or progesterone-receptor positive), HER2 negative, and has low levels of the protein Ki-67, which helps control how fast cancer cells grow. Still, while normal-like breast cancer has a good prognosis, its prognosis is slightly worse than luminal A cancer’s prognosis.

[0031] Triple-negative / basal-like breast cancer (TNBC) is hormone-receptor negative (estrogen-receptor and progesterone-receptor negative) and HER2 negative. This type of cancer is more common in women with BRCA1 gene mutations.

[0032] In one embodiment, the subject of the present invention has a triple-negative breast cancer (TNBC).

[0033] As used herein, the term "relapse" refers to the return of the cancer (e.g. TNBC) after a period of improvement in which no cancer cells could be detected.

[0034] As used herein, the term “therapeutic resistance” refers to a process occurring as cancers develop resistance to treatments such as chemotherapy, radiotherapy and targeted therapies, through many different mechanisms. These include specific genetic and epigenetic changes in the cancer cell and / or the microenvironment in which the cancer cell resides.

[0035] Methods of the present invention:

[0036] In a first embodiment, the present invention relates to a method of preventing therapyresistant cancer in a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0037] In one embodiment, the present invention also relates to a method of preventing resistance to chemotherapy in a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0038] In one embodiment, the present invention also relates to a method of preventing resistance to immune-checkpoint inhibitors (ICI) in a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0039] In one embodiment, the present invention also relates to a method of preventing resistance to radiotherapy in a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0040] In a second embodiment, the present invention relates to a method of treating a therapyresistant cancer in a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0041] In one embodiment, the present invention also relates to a method of treating a cancer resistant to chemotherapy in a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0042] In one embodiment, the present invention also relates to a method of treating a cancer resistant to immune-checkpoint inhibitor (ICI) in a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0043] In one embodiment, the present invention also relates to a method of treating a cancer resistant to radiotherapy in a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0044] In a third embodiment, the present invention relates to a method for enhancing the potency of chemotherapy administered to a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) as part of a treatment regimen, the method comprising administering to the patient a pharmaceutically effective amount of an inhibitor of ciliogenesis.

[0045] In one embodiment, the present invention also relates to a method for enhancing the potency of immune checkpoint inhibitor administered to a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) as part of a treatment regimen, the method comprising administering to the patient a pharmaceutically effective amount of an inhibitor of ciliogenesis.

[0046] In one embodiment, the present invention also relates to a method for enhancing the potency of radiotherapy administered to a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) as part of a treatment regimen, the method comprising administering to the patient a pharmaceutically effective amount of an inhibitor of ciliogenesis.

[0047] In a fourth embodiment, the present invention relates to a method for preventing relapse of a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) who was treated with chemotherapy comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0048] In one embodiment, the present invention also relates to a method for preventing relapse of a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) who was treated with immune checkpoint inhibitor comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis. In one embodiment, the present invention also relates to a method for preventing relapse of a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) who was treated with radiotherapy comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0049] In a fifth embodiment, the present invention relates to a method of overcoming resistance to chemotherapy in a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0050] In one embodiment, the present invention also relates to a method of overcoming resistance to immune checkpoint inhibitor in a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0051] In one embodiment, the present invention also relates to a method of overcoming resistance to radiotherapy in a patient suffering from a cancer (e.g. a triple negative breast cancer (TNBC)) comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

[0052] As used herein, the term “subject” or “patient” refer to any mammals, such as a rodent, a feline, a canine, and a primate. In some embodiments, the patient according to the invention is a human. In some embodiments, the patient according to the invention is a girl or a boy. In some embodiments, the patient according to the invention is an adult. In some embodiments, the patient according to the invention is a child (human being between the stages of birth and puberty), a teenager (human being between the stages of puberty to adulthood) or an elderly person (human being after the puberty).

[0053] In some embodiment, the subject of the present invention suffers from a resistance to chemotherapy.

[0054] As used herein, the term "chemotherapy" refers to any therapy that consists in administering a chemical agent with therapeutic usefulness in the treatment of cancer. Chemotherapeutic agents as used herein encompass both chemical and biological agents. These agents function to inhibit a cellular activity upon which the cancer cell depends for continued survival. Categories of chemotherapeutic agents include alkylating / alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most if not all of these drugs are directly toxic to cancer cells and do not require immune stimulation. Suitable chemotherapeutic agents are described, for example, in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal medicine, 14th edition; Perry et at , Chemotherapeutic, Ch 17 in Abel off, Clinical Oncology 2nd ed., 2000 ChrchillLivingstone, Inc.; Baltzer L. and Berkery R. (eds): Oncology Pocket Guide to Chemotherapeutic, 2nd ed. St. Louis, mosby-Year Book, 1995; Fischer D. S., Knobf M. F., Durivage HJ. (eds): The Cancer Chemotherapeutic Handbook, 4th ed. St. Louis, Mosby-Year Handbook.

[0055] As used herein the term "resistance to chemotherapy" is used in its broadest context to refer to the reduced effectiveness of chemotherapy to inhibit the growth of a cancer cell, kill a cancer cell or inhibit one or more cell survival functions, and to the ability of a cancer cell to survive exposure to chemotherapy designed to inhibit the growth of the cancer cell, kill the cancer cell or inhibit one or more cellular functions. The resistance displayed by a cancer cell may be complete in that chemotherapy is rendered completely ineffective against the cancer cell, or may be partial in that the effectiveness of chemotherapy is reduced. Accordingly, the term "resistant" refers to the repeated outbreak of cancer (e.g. TNCB), or a progression of cancer (e.g. TNCB) independently of whether the disease was cured before said outbreak or progression. The phrase “preventing resistance to chemotherapy” in context of the invention shall be effective if compared to a non-treated control, the cancer cells becomes more sensitive to chemotherapy. In particular, the patient will a responder. As used herein the term “responder” in the context of the present disclosure refers to a patient that will achieve a response, i.e. a patient where the cancer (e.g. TNCB) is eradicated, reduced or improved after chemotherapy. According to the invention, the responders have an objective response and therefore the term does not encompass patients having a stabilized cancer (e.g. TNCB) such that the disease is not progressing after chemotherapy. A “non-responder” or “refractory patient” includes patients for whom cancer (e.g. TNBC) does not show reduction but also patients having a stabilized cancer (e.g. TNBC) following chemotherapy. The term “non- responder” also includes patients having a stabilized cancer. Typically, the characterization of the patient as a responder or non-responder can be performed by reference to a standard or a training set. The standard may be the profile of a patient who is known to be a responder or non-responder or alternatively may be a numerical value. Such predetermined standards may be provided in any suitable form, such as a printed list or diagram, computer software program, or other media. When it is concluded that the patient is a non-responder, the physician could take the decision to administer the drug of the present invention (e.g. inhibitor of ciliogenesis).

[0056] In some embodiments the chemotherapy comprises administering to the patient at least one chemotherapeutic agent, preferably a combination of chemotherapeutic agents selected from the group consisting of alkylating agents, nitrosoureas, topoisomerase inhibitors, antimetabolites, mitotic inhibitors (plant alkaloids), and antitumor antibiotics.

[0057] Selected alkylating agents include: altretamine, bendamustine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide. mechlorethamine, melphalan, oxaliplatin, procarbazine, temozolomide, thiotepa, and trabectedin. Selected nitrosoureas include carmustine, lomustine, and streptozocin.

[0058] Selected antimetabolites include 5 -fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine, and trifluridine / tipiracil combination.

[0059] Selected topoisomerase inhibitors include etoposide, irinotecan, irinotecan liposomal, mitoxantrone, teniposide and topotecan.

[0060] Selected mitotic inhibitors include cabazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinblastine, vincristine, vincristine liposomal, and vinorelbine.

[0061] Selected anthracyclines include daunorubicin, rubicin, doxorubicin liposomal, epirubicin, idarubicin, mitoxantrone, valrubicin, bleomycin, dactinomycin, and mitomycin-c.

[0062] Other common chemotherapeutic agents include all-trans-retinoic acid, arsenic trioxide, asparaginase, eribulin, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin and vorinostat.

[0063] In some embodiments the chemotherapy comprises administering to the patient at least one chemotherapeutic agent, preferably a combination of chemotherapeutic agents selected from the group consisting of cytarabine (cytosine arabinoside, Ara-C, Cytosar-U), quizartinib (AC220), sorafenib (BAY 43-9006), lestaurtinib (CEP-701), midostaurin (PKC412), carboplatin, carmustine, chlorambucil, dacarbazine, ifosfamide, lomustine, mechlorethamine, procarbazine, pentostatin, (2'deoxycoformycin), etoposide, teniposide, topotecan, vinblastine, vincristine, paclitaxel, dexamethasone, methylprednisolone, prednisone, all- trans retinoic acid, arsenic trioxide, interferon-alpha, rituximab (Rituxan®), gemtuzumab ozogamicin, imatinib mesylate, Cytosar-U), melphalan, busulfan (Myleran®), thiotepa, bleomycin, platinum (cisplatin), cyclophosphamide, Cytoxan®)., daunorubicin, doxorubicin, idarubicin, mitoxantrone, 5-azacytidine, cladribine, fludarabine, hydroxyurea, 6-mercaptopurine, methotrexate, 6-thioguanine, or any combination thereof.

[0064] In some embodiments, the chemotherapy comprises administering a glucocorticoid. Glucocorticoids typically include cortisone, cortisol, hydrocortisone (1 ip,17-dihydroxy, 21- (phosphonooxy)-pregn-4-ene, 3,20-dione disodium), dihydroxy corti sone, dexamethasone (21- (acetyloxy)-9-fluoro-ip, 17-dihydroxy-16a-m-ethylpregna-l,4-diene-3, 20-dione), and highly derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9-chloro- 11-P, 17,21, trihydroxy-16P-methylpregna-l,4 di ene-3, 20-dione 17,21 -dipropionate). Other examples of glucocorticoids include flunisolide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort and betamethasone, glucocorticoids, for example, cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone.

[0065] In some embodiments, the patient is administered with an induction chemotherapy that typically includes i) a vinca alkaloid such as vinblastine (VBL), vinorelbine (VRL), vincristine (VCR) or vindesine (VDS), ii) a glucocorticoid such as dexamethasone or prednisone and iii) an anthracycline drug such as doxorubicin or daunorubicin. In some embodiments, the chemotherapy may include cyclophosphamide, L-asparaginase (or pegaspargase), and / or high doses of methotrexate or cytarabine (ara-C) as part of the induction phase.

[0066] In some embodiment, the subject of the present invention suffers from a resistance to immunotherapy, in particular to immune checkpoint inhibitor.

[0067] In some embodiments the immunotherapy comprises administering to the patient at least one immunotherapy agent. As used herein, the term "immunotherapy" as used herein, refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the anti-tumoral immune response of the patient. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy. Examples of common immunotherapeutic agents known in the art include, but are not limited to, cytokines, vaccines, monoclonal antibodies and non-cytokine adjuvants. Alternatively the immunotherapeutic treatment may consist of administering the patient with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells...). In some embodiments, immunotherapy regimens combine the use of non-specific and / or specific immunotherapeutic agents. Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system. Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines. Non-specific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants. In some embodiments, the immunotherapy includes CAR- T cell therapy. As used herein the term "CAR-T cell" refers to a T lymphocyte that has been genetically engineered to express a CAR. The T lymphocytes that are genetically modified may be "derived" or "obtained" from the patient who will receive the treatment using the genetically modified T cells or they may be "derived" or "obtained" from a different patient. As used herein, the term “chimeric antigen receptor” or “CAR” has its general meaning in the art and comprises one or more artificially constructed hybrid polypeptides containing an antigen binding domain linked to one or more T- cell signalling domains. Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC -restricted manner, exploiting e.g. the antigen-binding properties of monoclonal antibodies. The chimeric antigen receptor of the present invention typically comprises one more polypeptides having an extracellular domain and an intracellular domain joined by a transmembrane domain.

[0068] As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein.

[0069] As used herein the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD- 1, LAG-3, TIM-3 and VISTA. Inhibition includes reduction of function and full blockade. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. Examples of immune checkpoint inhibitor includes PD-1 antagonist, PD-L1 antagonist, PD-L2 antagonist CTLA-4 antagonist, VISTA antagonist, TIM-3 antagonist, LAG-3 antagonist, IDO antagonist, KIR2D antagonist, A2AR antagonist, B7-H3 antagonist, B7-H4 antagonist, and BTLA antagonist.

[0070] In some embodiments, PD-1 (Programmed Death-1) axis antagonists include PD-1 antagonist (for example anti-PD-1 antibody), PD-L1 (Programmed Death Ligand-1) antagonist (for example anti-PD-Ll antibody) and PD-L2 (Programmed Death Ligand-2) antagonist (for example anti-PD-L2 antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (also known as Nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and Opdivo®), Merck 3475 (also known as Pembrolizumab, MK-3475, Lambrolizumab, Keytruda®, and SCH-900475), and CT-011 (also known as Pidilizumab, hBAT, and hBAT-1). In some embodiments, the PD-1 binding antagonist is AMP -224 (also known as B7-DCIg). In some embodiments, the anti-PD-Ll antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. MDX-1105, also known as BMS-936559, is an anti-PD-Ll antibody described in W02007 / 005874. Antibody YW243.55. S70 is an anti-PD-Ll described in WO 2010 / 077634 AL MEDI4736 is an anti-PD- Ll antibody described in WO2011 / 066389 and US2013 / 034559. MDX-1106, also known as MDX-1 106-04, ONO-4538 or BMS-936558, is an anti-PD-1 antibody described in U.S. Pat. No. 8,008,449 and W02006 / 121168. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in U.S. Pat. No. 8,345,509 and W02009 / 114335. CT-011 (Pidizilumab), also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP -224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342. Atezolimumab is an anti-PD-Ll antibody described in U.S. Pat. No. 8,217,149. Avelumab is an anti-PD-Ll antibody described in US 20140341917. CA-170 is a PD-1 antagonist described in W02015033301 & WO2015033299. Other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and / or US 20120114649. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab. In some embodiments, PD-L1 antagonist is selected from the group comprising of Avelumab, BMS-936559, CA-170, Durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, Al 10, KY1003 and Atezolimumab and the preferred one is Avelumab, Durvalumab or Atezolimumab.

[0071] In some embodiments, CTLA-4 (Cytotoxic T-Lymphocyte Antigen-4) antagonists are selected from the group consisting of anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA- 4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (Ipilimumab), Tremelimumab, anti-CD28 antibodies, anti-CTLA-4 adnectins, anti-CTLA-4 domain antibodies, single chain anti-CTLA- 4 fragments, heavy chain anti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, inhibitors of CTLA-4 that agonize the co-stimulatory pathway, the antibodies disclosed in PCT Publication No. WO 2001 / 014424, the antibodies disclosed in PCT Publication No. WO 2004 / 035607, the antibodies disclosed in U.S. Publication No. 2005 / 0201994, and the antibodies disclosed in granted European Patent No. EP 1212422 B. Additional CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097; 5,855,887; 6,051,227; and 6,984,720; in PCT Publication Nos. WO 01 / 14424 and WO 00 / 37504; and in U.S. Publication Nos. 2002 / 0039581 and 2002 / 086014. Other anti-CTLA-4 antibodies that can be used in a method of the present invention include, for example, those disclosed in: WO 98 / 42752; U.S. Pat. Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al., J. Clin: Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP- 675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998), and U.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281. A preferred clinical CTLA-4 antibody is human monoclonal antibody (also referred to as MDX-010 and Ipilimumab with CAS No. 477202-00- 9 and available from Medarex, Inc., Bloomsbury, N.J.) is disclosed in WO 01 / 14424. With regard to CTLA-4 antagonist (antibodies), these are known and include Tremelimumab (CP- 675,206) and Ipilimumab.

[0072] In some embodiments, the immunotherapy consists of a combination of a CTLA-4 antagonist and a PD-1 antagonist.

[0073] Other immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211). Other immune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM-3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94). As used herein, the term “TIM-3” has its general meaning in the art and refers to T cell immunoglobulin and mucin domain-containing molecule 3. The natural ligand of TIM-3 is galectin 9 (Gal9). Accordingly, the term “TIM-3 inhibitor” as used herein refers to a compound, substance or composition that can inhibit the function of TIM-3. For example, the inhibitor can inhibit the expression or activity of TIM-3, modulate or block the TIM-3 signaling pathway and / or block the binding of TIM-3 to galectin-9. Antibodies having specificity for TIM-3 are well known in the art and typically those described in WO201 1155607, W02013006490 and WO2010117057.

[0074] In some embodiments, the immune checkpoint inhibitor is an IDO inhibitor. Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), P- (3-benzofuranyl)-alanine, P-(3-benzo(b)thienyl)- alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5 -methoxy -tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3 -di acetate, 9- vinylcarbazole, acemetacin, 5 -bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a P-carboline derivative or a brassilexin derivative. Preferably the IDO inhibitor is selected from 1-methyl-tryptophan, P-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and P-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.

[0075] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of 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 patient 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 patient 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 a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0076] As used herein, the term “inhibitor” or “antagonist” refers to a compound that decreases the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor. In some instances, an inhibitor will substantially decrease the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity or expression observed in the absence of the inhibitor. In some instances, an inhibitor will completely diminish the magnitude of at least one activity, signaling or expression of a molecule compared to the magnitude of the activity, signaling or expression observed in the absence of the inhibitor. Certain exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, aptamers, antisense oligonucleotides, interfering RNA, carbohydrates or small organic molecules.

[0077] In some present invention relates to an inhibitor of ciliogenesis for use in a method of preventing therapeutic resistance (e.g. resistance to chemotherapy or resistance to immune checkpoint inhibitor) in a patient suffering from a cancer (e.g a TNBC).

[0078] As used herein, the term "inhibitor of ciliogenesis” refers to a compound that inhibit the process of ciliogenesis.

[0079] In a particular embodiment, the inhibitors of ciliogenesis is a small organic molecule, peptide, peptidomimetic, antibody, aptamers, siRNA or antisense oligonucleotide. The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.

[0080] In a particular embodiment, the inhibitor of ciliogenesis 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.

[0081] The inventors confirmed the capability of 9 of the drugs to reduce the number and length of primary cilia without displaying major cytotoxic effect in the cells in a secondary screen Most importantly, 5 of the drugs belonged to the same chemical family that we named Naonedin (Nao-1, Nao-2, Nao-3, Nao-4, Nao-5).

[0082] In one embodiment, the inhibitor of ciliogenesis of the method of the present invention may be a compound of Formula (I):

[0083] (I), or a pharmaceutically acceptable salt thereof, wherein

[0084] X is selected from halo, Cl-C6-alkyl and halo-Cl-C6-alkyl;

[0085] R1is selected from H, -CH2-C(O)OH and -CH2-C(O)OMe;

[0086] R2and R3are independently selected from Cl-C6-alkyl and halo-Cl-C6-alkyl; and

[0087] R4, R5and R6are independently selected from -OH and -O-Cl-C6-alkyl.

[0088] Particular compounds of Formula (I), or pharmaceutically acceptable salts thereof, are those wherein one or more of X, R1, R2, R3, R4, R5and R6are defined as follows:

[0089] X is selected from halo, Cl-C6-alkyl and halo-Cl-C6-alkyl; in particular X is selected from halo, Cl-C4-alkyl and halo-Cl-C4-alkyl; more particularly X is selected from halo, Cl- C2-alkyl and halo-Cl-C2-alkyl; still more particularly X is halo; even more particularly X is Br or Cl;

[0090] R1is selected from H, -CH2-C(O)OH and -CH2-C(O)OMe; in particular R1is H or - CH2-C(O)OH;

[0091] R2and R3are independently selected from Cl-C6-alkyl and halo-Cl-C6-alkyl; in particular R2and R3are independently selected from Cl-C4-alkyl and halo-Cl-C4-alkyl; more particularly R2and R3are independently selected from Cl-C2-alkyl and halo-Cl-C2-alkyl; still more particularly R2and R3are Cl-C2-alkyl; even more particularly R2and R3are Me;

[0092] R4, R5and R6are independently selected from -OH and -O-Cl-C6-alkyl; in particular R4, R5and R6are independently selected from -OH and -O-Cl-C4-alkyl; more particularly R4, R5and R6are independently selected from -OH and-O-Cl-C2-alkyl; still more particularly R4and R5are independently selected from -OH and -O-Cl-C2-alkyl and R6is -O-C1-C2- alkyl; even more particularly R4and R5are independently selected from OH and OMe and R6is OMe.

[0093] As used herein, the term “halo” or “halogen” refers to the atoms of the group 17 of the periodic table (halogens) and includes in particular fluorine, chlorine, bromine and iodine atom. Particular halo groups in the context of the invention are chloro and bromo.

[0094] As used herein, the term “alkyl” by itself or as part of another substituent refers to a hydrocarbyl group of Formula GJBn+i wherein n is a number greater than or equal to 1. Alkyl groups may thus comprise 1 or more carbon atoms and generally, according to this invention comprise from 1 to 12, more preferably from 1 to 8 carbon atoms, and still more preferably from 1 to 6 carbon atoms. Alkyl groups within the meaning of the invention may be linear or branched. Examples of alkyl groups include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, isopentyl, sec-pentyl, tert-pentyl, n-hexyl, neohexyl, isohexyl, sec-hexyl and tert-hexyl. Particular examples of alkyl groups in the context of the invention include methyl, ethyl, n-propyl, n-butyl and tert-butyl.

[0095] As used herein, the term “haloalkyl” alone or in combination, refers to an alkyl group having the meaning as defined above wherein one or more hydrogens are replaced with a halogen as defined above. Non-limiting examples of such haloalkyl groups include chloromethyl, 1 -bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl and 1,1,1- tri fluoroethyl.

[0096] The compounds of Formula (I) containing a basic functional group may be in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts of the compounds of the invention containing one or more basic functional groups include in particular the acid addition salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulphate / sulphate, borate, camsylate, cinnamate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.

[0097] Pharmaceutically acceptable salts of compounds of Formula (I) may for example be prepared as follows:

[0098] (i) reacting the compound of Formula I with the desired acid; or

[0099] (ii) converting one salt of the compound of Formula (I) to another by reaction with an appropriate acid or by means of a suitable ion exchange column.

[0100] All these reactions are typically carried out in solution. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionization in the salt may vary from completely ionized to almost non-ionized.

[0101] In an embodiment, the inhibitor of ciliogenesis of the method of the present invention is chosen from the group consisting of: Nao-1 Nao-2 In a particular embodiment, the inhibitor of ciliogenesis of the method of the present invention is :

[0102] Nao-3

[0103] In another embodiment, the inhibitor of ciliogenesis of the present invention is chosen from but not limited to the following molecules :

[0104]

[0105] 407 H11 (chem name: 27983284) In a particular embodiment, the inhibitor of ciliogenesis is an antibody. Antibodies can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from camels, pigs, cows, horses, rabbits, goats, sheep, and mice, among others, or organotypic cultures of primary human cells from tonsils, lymph nodes or peripheral blood. 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 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 single chain antibodies. Compounds useful in practicing the present invention also include 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.

[0106] Humanized or human antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (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). Human antibodies can be generated by organotypic cultures of primary human cells from tonsils, lymph nodes or peripheral blood (see Wagar Lisa E. et al. Nature Medicine volume 27, pagesl25-135 (2021)).

[0107] In another embodiment, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.

[0108] 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.

[0109] 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).

[0110] In one embodiment, the inhibitor of ciliogenesis is an aptamer. Aptamers are 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., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

[0111] In one embodiment, the inhibitor of ciliogenesis according to the invention is a polypeptide.

[0112] In a particular embodiment the polypeptide is capable to prevent the function of ciliogenesis.

[0113] In one embodiment, the polypeptide of the invention may be linked to a “cellpenetrating peptide” to allow the penetration of the polypeptide in the cell.

[0114] The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).

[0115] The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

[0116] In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

[0117] A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

[0118] Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

[0119] Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 Daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug / linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory halflife of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomerular filtration (e.g., less than 60 kDa).

[0120] In addition, to the polymer backbone being important in maintaining circulatory half-life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

[0121] In another embodiment, the inhibitor of ciliogenesis is an inhibitor of gene expression. Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. Gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01 / 36646, WO 99 / 32619, and WO 01 / 68836).

[0122] MicroRNA (miRNA) can also function as inhibitors of expression for use in the present invention. MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRNAs base-pair to complementary sequences in mRNA molecules, then silence said mRNA molecules.

[0123] Ribozymes can also function as inhibitors of gene 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 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.

[0124] Both antisense oligonucleotides and ribozymes useful as inhibitors of gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramidite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and / or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-m ethyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

[0125] Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0126] 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).

[0127] Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wildtype 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. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on .

[0128] In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes For example, a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

[0129] 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. inhibitor of ciliogenesis) into the subject, such as by oral, 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. In a particular embodiment, a topical administration is performed to the subject. More particularly, an inhibitor of GSDMD is formulated as a cream for a topical administration. In another embodiment, an oral administration is performed to the subject. In a further embodiment, intravenous administration is performed to the subject.

[0130] By a "therapeutically effective amount" is meant a sufficient amount of an inhibitor of ciliogenesis for use in a method of preventing therapeutic resistance (e.g. resistance to chemotherapy or resistance to immune checkpoint inhibitor) in a patient suffering from a cancer (e.g a TNBC) at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic 20 adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0131] Pharmaceutical composition:

[0132] An inhibitor of ciliogenesis for use according to the invention as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

[0133] Accordingly, in a further aspect, the invention relates to a pharmaceutical composition comprising an inhibitor of ciliogenesis for use in method of preventing therapy -resistant cancer (e.g. resistance to chemotherapy or resistance to immune checkpoint inhibitor) in a patient suffering from a cancer (e.g a TNBC). Accordingly, in a further aspect, the invention relates to a pharmaceutical composition comprising an inhibitor of ciliogenesis for use in method of treating a therapy -resistant cancer (e.g. resistance to chemotherapy or resistance to immune checkpoint inhibitor) in a patient suffering from a cancer (e.g a TNBC).

[0134] In an embodiment, the inhibitor of ciliogenesis comprised in the pharmaceutical composition for use according to the invention is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein

[0135] X is selected from halo, Cl-C6-alkyl and halo-Cl-C6-alkyl;

[0136] R1is selected from H, -CH2-C(0)0H and -CH2-C(0)0Me;

[0137] R2and R3are independently selected from Cl-C6-alkyl and halo-Cl-C6-alkyl; and

[0138] R4, R5and R6are independently selected from -OH and -O-Cl-C6-alkyl.

[0139] Particular compounds of Formula (I), or pharmaceutically acceptable salts thereof, are those wherein one or more of X, R1, R2, R3, R4, R5and R6are defined as follows:

[0140] X is selected from halo, Cl-C6-alkyl and halo-Cl-C6-alkyl; in particular X is selected from halo, Cl-C4-alkyl and halo-Cl-C4-alkyl; more particularly X is selected from halo, Cl- C2-alkyl and halo-Cl-C2-alkyl; still more particularly X is halo; even more particularly X is Br or Cl;

[0141] R1is selected from H, -CH2-C(0)0H and -CH2-C(0)0Me; in particular R1is H or - CH2-C(O)OH;

[0142] R2and R3are independently selected from Cl-C6-alkyl and halo-Cl-C6-alkyl; in particular R2and R3are independently selected from Cl-C4-alkyl and halo-Cl-C4-alkyl; more particularly R2and R3are independently selected from Cl-C2-alkyl and halo-Cl-C2-alkyl; still more particularly R2and R3are Cl-C2-alkyl; even more particularly R2and R3are Me;

[0143] R4, R5and R6are independently selected from -OH and -O-Cl-C6-alkyl; in particular R4, R5and R6are independently selected from -OH and -O-Cl-C4-alkyl; more particularly R4, R5and R6are independently selected from -OH and-O-Cl-C2-alkyl; still more particularly R4and R5are independently selected from -OH and -O-Cl-C2-alkyl and R6is -O-C1-C2- alkyl; even more particularly R4and R5are independently selected from OH and OMe and R6is OMe.

[0144] In a particular embodiment, the inhibitor of ciliogenesis comprised in the pharmaceutical composition for use according the invention is chosen from the group consisting of:

[0145] Nao-1 Nao-2

[0146] In a particular embodiment, the inhibitor of ciliogenesis comprised in the pharmaceutical composition for use of the present invention is:

[0147] Nao-3 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.

[0148] As used herein, the terms "pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides 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 vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0149] 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.

[0150] 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.

[0151] FIGURES:

[0152] Figure 1. Primary cilia promote chemoresistance of hybrid E / M cancer cells. (A) The percentage of ciliated cells was quantified. ****p < 0.0001 (n=6 PDOs / patient sample / treatment condition). (B) The percentage of cleaved-caspase 3 (CC3)+ cells was measured in individual PDO for the distinct patient samples (n=6 PDOs / patient sample / treatment condition). Scale bar: 50 pm. ** p < 0.01; *** p < 0.001; ****p < 0.0001.

[0153] EXAMPLE:

[0154] Material & Methods

[0155] Methods

[0156] Human samples. Patient-derived tissues were collected from breast cancer patients that were diagnosed at the Centre Eugene Marquis and at the Institut de Cancerologie du Grand Ouest. None received therapy prior to surgery. Tissues were collected by a pathologist after resection by a surgeon. Some fragments were paraffin-embedded, other fragments were dissociated within 2 h after surgical resection (tumors). Briefly, breast tumor pieces were cut into small fragments (< 2 mm3), which were then dissociated enzymatically and mechanically as described previously (Wilson et al., 2021). Viable cells were then cryopreserved before organoid culture or directly used for organoid growth.

[0157] Patient-derived organoid culture and treatments PDO culture was performed using a protocol adapted from Sachs and colleagues (Sachs et al., 2018). Briefly, cells were seeded in advanced DMEM / F12 (Thermo-Fisher) supplemented with glutamax, HEPES, 10% R-spondin 1, 10% noggin, 2% B-27, nicotinamide (10 mM), N-acetyl-L-cysteine (500 mM), primocin (100 pg / mL), Y-27632 (5pM), heregulin Pl (5 nM), FGF-7 (5 ng / mL), FGF-10 (5 ng / mL), A83-01 (0.5 pM), EGF (5 ng / mL), SB202190 (1 pM) containing 5% matrigel. 200,000 cells were seeded per well in 24-well ultralow attachment plates (Corning). Organoids were analysed between passage 0 and passage 7.

[0158] Immunofluorescence and Image Analysis

[0159] PDOs and cells were fixed and stained according to our published protocols (Duclos et al., 2021; Dupuy et al., 2022; Wilson et al., 2021). The following primary antibodies were used: Arll3b (NeuroMab 73-287, 1 :200), acetylated tubulin (Cell Signaling Technology 5335; 1 : 1,000), E-cadherin (Cell Signaling 3195, 1 :200), a-tubulin (Sigma-Aldrich T9026; 1 :500), cleaved caspase 3 (Cell Signaling 9131; 1 :200) and vimentin (Dako M0725; 1 :500). Secondary antibodies were anti-mouse IgG2A 488 (Thermo-Fisher A21131; 1 : 1,000), anti-mouse 647 (Thermo-Fisher A21236, 1 : 1000), anti-mouse IgGl 647 (Thermo-Fisher A21240; 1 : 1,000) and anti -rabbit 546 (Thermo-Fisher Al 1035, 1 :500). Mounted coverslips with cells were examined using 60X objective and a wide-field Zeiss microscope. Organoids were embedded in low- melting point agarose and analysed using a Lightsheet Z1 Zeiss microscope. Z-stacks were deconvolved and analysed with ImageJ and Imaris.

[0160] Single-cell RNA sequencing

[0161] Single cell RNA sequencing was conducted using the Chromium Single-Cell 3’ v3.1 kit from 10X Genomics, according to the manufacturer’s protocol. Libraries were sequenced on the NovaSeq 6000 platform (Paired-end, 28 bp Readl, 90pb Read2). Raw BCL files were demultiplexing and mapped to the reference genome (refdata-cellranger-GRCh38-3.0.0) using the Cell Ranger Software Suite (v.6.1.2). The raw data were extracted from 10X format files using the ReadlOX function from the Seurat package (v4.4.0) in R v.4.1.1. We applied filtering to the feature-barcode gene expression matrix to preserve only cells of high quality based on several metrics, including the number of unique molecular identifiers (UMI), the number of genes detected and the percentage of read mapped to mitochondrial genes. Threshold were set three median absolute deviations (MAD) using the isOutlier function from the scater R package (v.1.22.0) to identify and flag cells with UMI counts and genes detected per cell bellow and above this specified threshold. Additionally, cells surpassing a threshold of three MAD for the percentage of mitochondrial reads were considered as non-viable and removed from the analysis. Doublets were identified using the scDblFinder R package (v.1.6.0), where the scDlbFinder score was calculated for each cell and the scDblFinder threshold was applied for doublet identification. Detailed quality control metrics are provided in Table S6.

[0162] Single-cell RNA sequencing data analysis

[0163] After data preprocessing, normalization was performed using the NormalizeData function, with default parameters, which implements a logarithmic normalization to the gene expression data. Data were centered and scaled using ScaleData function. During scaling step, regression of S and G2M phase scores was performed to minimized the influence of cell cycle effects in downstream analysis. For all cells from PDOs from patient sample #3 (n= 4888 cells) and cells from the three samples before (CTL n = 142 cells) and after treatment (Doxo. n= 264 cells and Taxol n= 414 cells), we used the top 2000 highly variable genes from the normalized expression matrix, to perform the principal component analysis (PCA, runPCA Seurat function), and applied Louvain graph-based clustering on the 30 first principal components. The resulting data were projected onto an Uniform Manifold Approximation and Projection (UMAP) using the runUMAP function. We applied the FindNeighbords and FindClusters functions with a resolution of 0.2 for the untreated PDOs from patient #3, and a resolution of 0.9 for the merged dataset of PDOs samples after treatment (CTL, Doxo, Taxol) to identify distinct clusters. For each defined cluster, a gene markers analysis was conducted using the Find AllMarkers function with the parameters set as follows: only.pos = TRUE, min.pct=T, logfc.threshold = 0.25. The top 5 markers genes per cluster were selected on the average log2fold change and a heatmap was generated using the DoHeatmap function. The score for the EMT, CilioUp and Lim mammary stem cell signatures was calculating using the AddModule Score function. For statistical tests comparing signature values between clusters, we utilize the stat compare means function from ggpubr R package (v.0.6.0). We employed the Wilcoxon test to compare two groups and the Kruskal-Wallis test to compare multiple groups. Statistical significance levels are indicated as follow: not significant (ns) p>0.05; * p <= 0.05; ** p<=0.01; *** p<= 0.001; **** p <= 0.0001.

[0164] Copy number variation analysis was performed using the InferCNV R package (v.1.16.0 https: / / github.com / broadinstitute / infercnv). The analysis was executed using the run function with this following parameter settings: denoise=TRUE, HMM=TRUE, leiden_resolution=l, leiden method- ’simple”, leiden function- ’modularity”, analysis mode- ’cell”. Reference cells were selected from the N-1105-epi sample, using a publicly available dataset (Pal et al., 2021) https: / / doi.org / 10.15252 / embj.2020107333

[0165] Pseudotime analysis was conducted using the R package slingshot (v.2.0.0) to perform single cell trajectory analysis, utilizing the principal curves algorithm. The analysis was conducted using the following input: cluster labels derived from the resolution of 0.9 and reduced dimensions obtained from PCA, as described previously. UMAP coordinates were used and no specific starting point was selected, default parameters were used for the analysis. ComplexHeatmap (v.2.15.4), ggplot2 (v.3.4.4), dittoSeq (v.1.4.4) R packages were used for graphical representation.

[0166] 2D Cell Culture and treatments

[0167] HMLER cells were cultured in 1 : 1 mixture ofin Dulbecco’s modified Eagle medium (DMEM / F12) supplemented with glutamax, 10% FBS, 0.01 mg / mL insulin, 0.48 pg / mL hydrocortisone, and complete mammary epithelial cell growth medium (MEGM) supplemented with bovine pituitary hormone (Lonza). For all ciliogenesis assays, cells grown until high confluence and serum starved. To repress ciliogenesis, inhibitors were added to DMEM / F12 medium without serum for 24 hours.

[0168] Drug screening

[0169] RPE1 cells were cultured in DMEM / F12 supplemented with glutamax, 10% FBS and and penicillin / streptomycin. Cells were grown until high confluence and serum starved in DMEM / F12 for 24 hours. Drugs diluted in DMEM / F12 (10 DM) were added to the cells for 24 hours. Cells were fixed and stained as discussed above. Ciliogenesis was analyzed using a custom-made Fiji plugin.

[0170] Viability analyses

[0171] Viability of cancer cells in PDOs was measured through a 3D CellTiter-Glo assay according to the manufacturer’s instructions. Viability of HMLER cells was assessed in 96- well flat-bottom plate (zell-kontakt). The cells were plated and then treated 24 h later with 0.1 pM and 1 pM Taxol or 0.1% DMSO in the presence of propidium iodide (Miltenyi Biotec). The plate was incubated in an IncuCyte Live Cell Analysis System (Essen Bioscience). Phasecontrast and fluorescence images of cells were acquired every 2 hours for 30-40 hours using the IncuCyte Zoom automated imaging system. Live cell analysis Incucyte software was used for data analysis. Western Blot Experiments

[0172] These experiments were conducted using standard procedures as described previously(Wilson et al., 2021). Western Blot were performed using primary antibodies against KIF3A (Proteintech 13930-1-AP; 1 : 1,000), IFT20 (Proteintech 13615-1-AP; 1 : 100), E- cadherin (Cell Signaling 3195; 1 : 1,000), fibronectin (BD Biosciences 610077; 1 : 11,000) and actin (Millipore MAB1501; 1 :2,000) and secondary antibodies horseradish peroxidase-coupled anti-mouse (Jackson ImmunoResearch 115-035-006; 1 :5,000) or anti-rabbit (Jackson ImmunoResearch 111-035-006; 1 :5,000).

[0173] Results

[0174] Late hybrid quasi-mesenchymal cancer cells assemble primary cilia in human TNBCs

[0175] To investigate the degree of phenotypic heterogeneity of human TNBCs associated with EMT features and primary ciliogenesis, we first used patient-derived tumor biopsies (PDBs). We collected PDBs from 13 patients who had not received therapy at the time of the biopsy. We identified cancerous lesions in PDBs through hematoxylin-eosin staining of sections of tumor biopsies (data not shown). In addition, we co-stained adjacent tumor sections for the epithelial marker E-cadherin (Ecad), the mesenchymal marker Vimentin (Vim), and the primary cilium marker ADP-ribosylation factor-like protein 13b (Ari 13b, data not shown). We found double-positive Ecad (Ecad+) / Vimentin (Vim+) cancer cells residing in hybrid epithelial- mesenchymal (E / M) states in all samples (data not shown). Importantly, the representation of Ecad+ / Vim+ cancer cells varied significantly between patient’s tumors and even between distinct areas within an individual tumor, ranging from 0 to 100 % of Ecad+ / Vim+ cancer cells (data not shown). Using the Arll3b staining, we detected primary cilia in 12 of the 13 tumors initially analyzed and at various frequencies in distinct regions of interest within individual tumors and between tumors, ranging from 0 to 17.39 % of cells that were stained (data not shown). Most importantly, we found that double-positive Ecad+ / Vim+ cancer cells are significantly more ciliated that Ecad+ / Vim- cells indicating that the propensity of cancer cells to form primary cilia reflects acquisition of a hybrid E / M state (data not shown).

[0176] We next assessed phenotypic heterogeneity linked to EMT and primary ciliogenesis in patient-derived organoids (PDOs) using 7 distinct patient samples (data not shown). To begin, we assessed inter- and intraorganoid phenotypic heterogeneity by conducting whole mount immunofluorescence co-staining and imaging of PDOs (data not shown). We detected PDOs that were comprised of double-positive Ecad+ / Vim+ cells in all breast tumor-derived samples (data not shown). The representation of Ecad+ / Vim+ double-positive cancer cells varied significantly between tumor samples and between PDOs for each tumor sample, ranging from no detectable Ecad+ / Vim+ double-positive cells in some PDOs to PDOs exclusively composed of Ecad+ / Vim+ cells (data not shown). Of note, we detected more Ecad+ / Vim+ cells in PDOs in comparison to PDBs (data not shown). We next assessed primary ciliogenesis in PDOs using Ari 13b staining (data not shown). Here, we found primary cilia in all breast tumor-derived samples but at different frequencies between PDOs (data not shown). Most importantly, we found that cancer cells that co-expressed Ecad and Vim were significantly more ciliated than cancer cells that expressed exclusively Ecad (data not shown). This revealed that the propensity of cancer cells to form primary cilia follows acquisition of a hybrid E / M phenotype in PDOs, reflecting the earlier findings in PDBs. Together, our findings in PDBs and PDOs establish that inter- and intratumor phenotypic heterogeneity in human TNBCs is linked to the epithelial- mesenchymal status of cancer cells and primary ciliogenesis. Malignant cells that reside in a hybrid E / M state have the ability to form primary cilia. However, acquisition of a hybrid E / M phenotype is not sufficient for primary ciliogenesis and only a subset of hybrid E / M cells form primary cilia.

[0177] To gain additional insight into the identity of hybrid E / M cells that form primary cilia, we conducted additional studies using PDOs. To begin, using whole-mount immunofluorescent staining and 3D-imaging studies of these structures, we found that Ecad+ cells that express high levels of Vim that reside on the outer layers of PDOs exhibited an elevated ability to form primary cilia when compared with the inner cells (data not shown). We next conducted singlecell RNA sequencing (scRNAseq) analysis of cancer cells dissociated from PDOs. Unsupervised clustering of cancer cells revealed five different cell states in the cells forming the analyzed PDOs (Cluster 0-4, data not shown). Remarkably, we found evidence for candidate genetic alterations scattered in cancer cells by inferring copy number alterations (inferCNV) from our scRNA-seq data (data not shown). Each copy-number subclone displayed distinct phenotypes corresponding to the distinct cell states previously defined, with varying proportions according to the subclone, indicating that epigenetic events contribute to transcriptional heterogeneity between cancer cells of our PDOs (data not shown). To determine whether EMT and ciliogenesis programs contribute to transcriptional heterogeneity of cancer cells, we next assessed expression of transcriptional signatures that mark EMT and ciliogenesis in the distinct cell states (data not shown). Importantly, we found differential expression of the signatures between cancer cell clusters and identified one cell cluster composed of cells that co- expressed the highest level of EMT and ciliogenesis transcriptional signatures (cluster 2, data not shown). Additional supervised and unsupervised gene set enrichment analysis revealed other significantly enriched gene sets in the list of genes that are up-regulated in cancer cells of cluster 2, including stem cell transcriptional signatures (data not shown). Altogether, our data indicate that cancer cells that reside in a late hybrid quasi-mesenchymal stem-like state represent a subpopulation of hybrid E / M cells that assemble primary cilia in human TNBCs.

[0178] Late hybrid quasi-mesenchymal ciliated cells display enhanced resistance to chemotherapy

[0179] To investigate the response of cancer cells residing in distinct states to chemotherapy, we next treated our PDOs with doxorubicin and taxol, two drugs that are in use in the oncology clinic (data not shown). We found that both chemotherapeutics induced a reduction in cancer cell viability in a dose-dependent manner (data not shown). Most importantly, a subset of cancer cells displayed enhanced chemoresistance at intermediate doses and organoid-reconstituting capacity (data not shown). Using immunostaining, we found that PDOs comprised of chemoresistant cells are enriched for hybrid E / M cells that assemble primary cilia in comparison with PDOs comprised of vehicle-treated control cancer cells (data not shown). To gain additional insights into the identity and origin of these cells, we next conducted scRNAseq analysis of cancer cells dissociated from vehicle-treated PDOs (CTL) or PDOs postchemotherapy. Unsupervised clustering of cells revealed the existence of five different cell states in these PDOs (clusters 0-4, data not shown). The percentage of cells from cluster 0 was similar in the control PDOs and PDOs post-chemotherapy (data not shown). However, the representation of cells from cluster 1 and 3 decreased and the representation of cells from cluster 2 and 4 increased in PDOs post-chemotherapy when compared with control PDOs (data not shown). Importantly, cells of cluster 2 and 4 expressed the highest levels of EMT, ciliogenesis, and stem cell transcriptional programs (data not shown). These findings revealed selective enrichment for late hybrid quasi-mesenchymal stem-like ciliated cell subpopulation after chemotherapy.

[0180] Late hybrid quasi-mesenchymal cells arise through drug-induced cellular plasticity associated with activation of EMT and ciliogenesis transcriptional programs

[0181] Using inferCNV, we found evidence that at least a subset of cells residing in late hybrid quasi-mesenchymal state after chemotherapy arose through drug-induced cellular plasticity (data not shown). Late hybrid quasi-mesenchymal cells of cluster 2 and 4 enriched post- treatment were composed of a subset of cells devoid of a candidate amplification of chrl 1 :45909669-65044828 and of another subset of cells harboring chrl 1:45909669-65044828 amplification that was only found in more early hybrid E / M cells of cluster 0-1-3 before treatment (data not shown). Using pseudotime analysis, we identified a lineage trajectory from cells residing in the early hybrid states (clusters 3-1-0) to cells residing in the late-hybrid state (4-2) associated with progressive expression of EMT and ciliogenesis transcriptional programs (data not shown). These data reveal that at least a subset of late hybrid quasi-mesenchymal ciliated cells with enhanced resistance to chemotherapy arose through drug-induced cellular plasticity associated with activation of EMT and primary ciliogenesis transcriptional programs.

[0182] Primary cilia promote chemoresistance of cancer cells that activate EMT programs

[0183] We next wished to determine whether primary cilia promote chemoresistance of cancer cells that activate EMT programs in our PDOs. Human TNBC PDOs are difficult to maintain in culture in the long-term which precluded the development of genetic strategies to genetically ablate primary cilia upon therapy in this model and incited us to use a pharmacological strategy. We could not detect any impact on ciliogenesis in PDOs of ciliobrevin A (CilA, data not shown), an inhibitor of the AAA+ ATPase motor dyneins reported to inhibit ciliogenesis among other cilium-independent processes in monolayers of cells in culture (Firestone et al., 2012; Guen et al., 2017). No other specific small molecule inhibitor of ciliogenesis has been reported.

[0184] For these various reasons, we decided to employ a microscopy -based screening assay to identify new potent small molecule inhibitors of primary ciliogenesis. The screening employed easy to grow human retinal cells (RPE1) amenable to large scale screening in monolayer culture and a library of 3271 compounds from the French National Chemical Library. We identified 12 hits in a primary screen and confirmed the capability of 9 of the drugs to reduce the number and length of primary cilia without displaying major cytotoxic effect in the cells in a secondary screen (data not shown). Most importantly, 5 of the drugs belonged to the same chemical family that we named Naonedin (Nao-1, Nao-2, Nao-3, Nao-4, Nao-5, data not shown). We next proceeded to determine whether one of the drugs represses primary ciliogenesis in our PDOs composed of cells that activate EMT to reside in hybrid E / M states. Nao-3 significantly repressed cilium assembly in all patient samples analyzed (Figure 1A). Thus, we characterized a novel family of small-molecules that represses primary ciliogenesis both in monolayer cell culture and in patient-derived samples. We next determined whether Nao-3 suppresses chemoresistance of quasi-mesenchymal cancer ciliated cells in our PDOs. We treated PDOs with a vehicle control, Nao-3 alone, taxol alone or a combination of taxol and Nao-3. Using immunostaining for cleaved-caspase 3, we found that Nao-3 induced a modest but significant increase in the number of death associated with caspase activation of cancer cells on its own, comparable to taxol (Figure IB). Most importantly, we found that Nao-3 induced a significantly more profound death of cancer cells when combined with taxol (Figure IB). Altogether, our findings reveal a novel family of small-molecule inhibitors of primary ciliogenesis that suppresses chemoresistance of malignant cells. They offer compelling evidence that primary cilia promote chemoresistance of cancer cells that activate EMT to reside in hybrid E / M states.

[0185] EMT-driven cell plasticity enables primary ciliogenesis in quasi-mesenchymal cells to mediate chemoresistance

[0186] To investigate whether EMT -induced primary ciliogenesis mediates chemoresistance in quasi-mesenchymal cells in complementary experiments, we next used experimentally transformed human mammary epithelial cells (HMLER). In general, the majority of HMLER cancer cells propagated in vitro display epithelial characteristics. In addition, we previously generated HMLER variants in which EMT can be induced experimentally by the knockdown of Ecad (Guen et al., 2017). Thus, HMLER cells acquire a quasi-mesenchymal phenotype and generate tumors that display the hallmarks of TNBC / claudin-low breast cancers when orthotopically implanted in the mouse (Guen et al., 2017; Wilson et al., 2021). Using epithelial (shCTL) and quasi-mesenchymal (shEcad) HMLER variants, we confirmed that while the majority of epithelial shCTL cells display epithelial characteristics, shEcad cells acquire a quasi-mesenchymal phenotype upon Ecad silencing, as judged by morphology and western blotting for epithelial and mesenchymal markers (data not shown). Additionally, we demonstrated that HMLER shEcad cells gain the ability to form primary cilia using immunostaining for Ari 13b (data not shown).

[0187] To determine whether EMT program that induces primary ciliogenesis also induce chemoresistance in HMLER cells, we next assessed the sensitivity of our epithelial shCTL versus quasi-mesenchymal ciliated shEcad HMLER variants to taxol by monitoring cell death through propidium iodide staining (data not shown). We found that HMLER cells that resided in a quasi-mesenchymal state and formed primary cilia are significantly more resistant to cell death in comparison to control HMLER cells in the epithelial-like state (data not shown).

[0188] To investigate whether assembly of primary cilia in response to EMT is responsible for taxol resistance, we used HMLER shEcad variants in which we inhibited ciliogenesis through the knockout of KIF3A and IFT20, two genes that are essential genes for ciliogenesis. We demonstrated that knockout of these genes resulted, as anticipated, in loss of KIF3A and IFT20 proteins and ciliogenesis inhibition in quasi-mesenchymal cancer cells (data not shown). Importantly, we found that HMLER shEcad variants in which ciliogenesis had been repressed (i.e., sgKIF3A, sgIFT20) died more of taxol relative to control ciliated HMLER shEcad cells (sgCTL, data not shown, Movie S3). Thus, primary ciliogenesis inhibition sensitize quasi- mesenchymal cancer cells to chemotherapy. Altogether, these data reveal that EMT-driven cancer cell plasticity enables primary ciliogenesis in quasi-mesenchymal cells and chemoresistance and that chemotherapy resistance is mediated by primary ciliogenesis.

[0189] Discussion

[0190] Our previously reported data established that EMT programs induce TNBC / claudin-low tumorigenesis by inducing primary ciliogenesis in tumor-initiating cells in a murine carcinoma model (Guen et al., 2017; Wilson et al., 2021). Our data now emphasize a clear connection between EMT and primary ciliogenesis in human TNBCs. We demonstrate that the propensity of cancer cells to form primary cilia follows the entrance of cancer cells into a quasi- mesenchymal state (D). The role of the EMT / primary cilium axis in tumor response to therapy had never been addressed before. We reveal that primary cilia promote EMT -induced tumor resistance to therapy in TNBCs (Data not shown). Additionally, we reveal a novel family of small molecules that repress primary ciliogenesis and suppress chemoresistance. Collectively, these findings establish novel aspects of the cell biology of tumor resistance to therapy which can be targeted pharmacologically.

[0191] Our data do not exclude the possibility that our newly identified ciliogenesis inhibitor triggers the death of malignant cells through ciliogenesis inhibition and other ciliumindependent functions. Nao-3 share similarities with microtubule targeting drugs carbazoles and could thus repress primary ciliogenesis by disrupting ciliary and non-ciliary microtubule dynamics (Peronne et al., 2020). However, our work clearly establishes that primary cilia ablation on its own induces the death of malignant chemotherapy resistant cancer cells through genetic strategies. Our work is consistent with independent recent studies that reported a role for primary cilia in resistance to therapy in lung, rhabdoid and glioblastoma cancer cell lines in vitro (Jenks et al., 2018; Kim et al., 2022; Wei et al., 2022). Jenks et al showed that in carcinoma cells of the lung, ciliogenesis is induced in response to kinase inhibitors and primary cilia mediate resistance to the drugs (Jenks et al., 2018). The underlying reason for ciliogenesis induction in this study remained unclear but EMT-driven tumor cell heterogeneity and plasticity in response to therapy could be responsible for ciliogenesis and cilium-induced therapy resistance such as in TNBCs as demonstrated in our work. The few published studies and our data on human tumor samples provide strong evidence for the notion that primary cilia promote cancer cell resistance to various cancer therapeutics in tumors. Our data reveal new insights into mechanisms of ciliogenesis induction in response to therapy in carcinomas and reveal new pharmacological tools to repress this process which drives therapy failure. It is important to consider that targeting ciliogenesis in combination to chemotherapy and not alone appears to be to a valuable strategy given heterogeneity of cancer cells.

[0192] Targeting ciliogenesis or ciliary signaling alone could also lead to therapeutic resistance. Ciliary signaling inhibition has been used in the clinic for the treatment of basal cell carcinomas and medulloblastomas (Basset-Seguin et al., 2015; Scales and de Sauvage, 2009). Some of these hedgehog-dependent tumors rely on primary cilia and smoothened ciliary localization for tumorigenesis (Conduit et al., 2017; Han et al., 2009; Wong et al., 2009; Yang et al., 2017). Inhibition of smoothened efficiently repress tumor formation but resistance can arise (Kuonen et al., 2019; Zhao et al., 2017). Interestingly, genetic screens and genomic analysis have provided evidence that loss of primary cilia due to genetic mutation in ciliary genes result in a major switch in cell signaling dependencies which can result in acquired resistance to smoothened inhibitors in these tumors (Kuonen et al., 2019; Zhao et al., 2017). Thus, combination of distinct therapies including conventional and new therapeutic strategies which target ciliogenesis / ciliary signaling and independent signaling modalities appears of great importance to successfully treat primary cilia-dependent tumors.

[0193] Pioneering work conducted in cancer cell lines in vitro and using genetically-engineered mouse cancer models suggested that EMT in cancer is a major route toward therapy resistance (Gupta et al., 2019; Marine et al., 2020; Shibue and Weinberg, 2017). Our data offer new evidence for the role of EMT in promoting inter- and intratumor phenotypic heterogeneity and therapeutic resistance in human tumors. We used PDOs to conduct our research. They recently emerged as attractive tumor models for studying tumorigenesis and therapeutic response ex vivo (Bhatia et al., 2022; Dekkers et al., 2021; Sachs et al., 2018). Our data offer new evidence which demonstrate that PDOs represent appropriate tumor avatars for studying tumor therapeutic response. They faithfully recapitulate EMT-associated inter- and intratumor phenotypic heterogeneity found in tumors which is responsible for therapy resistance. Thus, PDOs represent useful models to reinforce our understanding on the causes and consequences of cell heterogeneity and plasticity program in human tumor and for the development of new therapeutic strategies which target EMT and related processes. Our data offer new insights into the mechanisms of EMT-driven therapeutic resistance in human TNBCs. This group of breast neoplasms represent the most aggressive subtype of breast carcinomas which remain associated with an unmet medical need (Grinda et al., 2021). The recent development of new therapeutics including antibody-drug conjugates enabled important progresses for the treatment of TNBCs (Bardia et al., 2021). However, most advanced tumors remain associated with therapeutic resistance and cancer-related death (Bardia et al., 2021). Our work emphasizes the importance of considering tumor heterogeneity and plasticity of TNBCs for therapy. REFERENCES:

[0194] 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.

Claims

- 48 -CLAIMS:

1. A method of preventing therapy -resistant cancer in a patient suffering from a cancer comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

2. A method of treating a therapy -resistant cancer in a patient suffering from a cancer comprising administering to the patient a therapeutically effective amount of an inhibitor of ciliogenesis.

3. The method according to claim 1 or 2, wherein the therapy -resistant cancer is a resistance to chemotherapy.

4. The method according to claim 1 or 2, wherein the therapy -resistant cancer is a resistance to immune checkpoint inhibitor.

5. The method according to any one of claims 1 to 4, wherein the patient suffers from a breast cancer.

6. The method according to claim 5, wherein the patient suffers from a triple negative breast cancer.

7. The method according to any one of claims 1 to 6, wherein the inhibitor of ciliogenesis is a compound of Formula (I):(I), or a pharmaceutically acceptable salt thereof, whereinX is selected from halo, Cl-C6-alkyl and halo-Cl-C6-alkyl;R1is selected from H, -CH2-C(0)0H and -CH2-C(0)0Me;R2and R3are independently selected from Cl-C6-alkyl and halo-Cl-C6-alkyl; and R4, R5and R6are independently selected from -OH and -O-Cl-C6-alkyl.

8. The method according to any one of claims 1 to 7, wherein the inhibitor of ciliogenesis is chosen from the group consisting of:

9. A pharmaceutical composition comprising an inhibitor of ciliogenesis for use in a method of preventing therapy -resistant cancer in a patient suffering from a cancer.

10. A pharmaceutical composition comprising an inhibitor of ciliogenesis for use in a method of treating a therapy -resistant cancer in a patient suffering from a cancer.

11. The pharmaceutical composition for use according to claim 9 or 10, wherein the therapeutic resistance is a resistance to chemotherapy or a resistance to immune checkpoint inhibitor.

12. The pharmaceutical composition for use according to any one of claims 9 to 11, wherein the cancer is a breast cancer.

13. The pharmaceutical composition for use according to claim 12, wherein the cancer is a triple negative breast cancer.

14. The pharmaceutical composition for use according to any one of claims 9 to 13, wherein inhibitor of ciliogenesis is a compound of Formula (I):or a pharmaceutically acceptable salt thereof, whereinX is selected from halo, Cl-C6-alkyl and halo-Cl-C6-alkyl;R1is selected from H, -CH2-C(0)0H and -CH2-C(0)0Me;R2and R3are independently selected from Cl-C6-alkyl and halo-Cl-C6-alkyl; and R4, R5and R6are independently selected from -OH and -O-Cl-C6-alkyl.

15. The pharmaceutical composition for use according to any one of claims 9 to 13, wherein the inhibitor of ciliogenesis is chosen from the group consisting of: