WO2025003193A1 - Sertraline and indatraline for disrupting intracellular cholesterol trafficking and subsequently inducing lysosomal damage and anti-tumor immunity - Google Patents

Abstract

Cancer progression involves changes in the composition of the lysosomal membrane. Under those stress conditions, several adaptive responses are activated to support cell survival, such as the transcription factor EB (TFEB) to drive the expression of lysosomal and autophagy related genes. In cases where these adaptive stress responses fail to cope with lysosomal stress, the induction of lysosomal damage can result in cell death. In the present invention, the inventors used 1200 FDA approved compounds for a high-content imaged-based screen to search new TFEB inducers and autophagy activators. They identified two compounds (Sertraline and Indatraline) that, despite the activation of TFEB and the high levels of LC3 puncta, elicited a significant cytotoxic effect in cancer cell lines. The data showed that both compounds inhibited autophagy flux partially and subsequently generated a significant induction of lysosomal membrane permeabilization and cell death. Finally, the inventors demonstrated that the compounds elicited immunogenic cell death features and mice that were vaccinated with these compounds were protected against tumor growth. These results indicate that both compounds have a stimulating effect on immunity against cancer.

Description

SERTRALINE AND INDATRALINE FOR DISRUPTING INTRACELLULAR CHOLESTEROL TRAFFICKING AND SUBSEQUENTLY INDUCING LYSOSOMAL

DAMAGE AND ANTI-TUMOR IMMUNITY

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular oncology.

BACKGROUND OF THE INVENTION:

To sustain the demands of energy essential for their survival and proliferation, cancer cells heavily depend on optimal catabolic lysosomal functions. Furthermore, the progression of cancer is associated with alteration in the composition of lysosome membrane which can make them more sensitive to lysosomal damage and consequently to cell death. Under such stressful conditions, several adaptive responses are triggered to ensure cell survival. On crucial component of this response is the transcription factor EB (TFEB) along with one of its effectors known as autophagy. TFEB plays a pivotal role in driving the expression of of genes associated with lysosomal and autophagy. Together, TFEB and autophagy act as vital mechanism and the disruption and dysfunction of either can lead to cell death particularly in the context of lysosomal damage. These observations highlight that a fine-tuned understanding of lysosomal damage regulation is imperative for its use as therapeutic tool in oncology.

SUMMARY OF THE INVENTION:

The present invention is defined by the claims. In particular, the present invention relates to the use of Sertraline and Indatraline for disrupting intracellular cholesterol trafficking and subsequently inducing lysosomal damage and anti-tumor immunity.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention relates to a method for treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a selective serotonin reuptake inhibitor selected from the group consisting of sertraline and indatraline.

As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. 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 tract, 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; nonencapsulating 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; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant 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; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, 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; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; 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; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

In some embodiments, the method of the present invention is particularly suitable for the treatment of sarcoma.

As used herein, the term “sarcoma” has its general meaning in the art and refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Example of sarcomas includes chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectaltic sarcoma.

In some embodiments, the patient suffers from soft tissue sarcoma.

In some embodiments, the patient suffers from a fibrosarcoma.

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 regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). According to the present invention, the selective serotonin reuptake inhibitor of the present invention is particularly suitable for disrupting intracellular cholesterol trafficking.

More particularly, the selective serotonin reuptake inhibitor of the present invention is suitable for inducing lysosomal damage in tumor cells.

Even more particularly, the selective serotonin reuptake inhibitor is suitable for inducting antitumor immunity. As used herein, the term "anti-tumor immunity" refers to an immune response induced upon recognition of cancer antigens by immune cells.

Thus, a further object of the present invention relates to method for enhancing or inducing an anti-tumor immune response in a patient suffering from cancer, comprising administering to the patient a pharmaceutically effective amount of a selective serotonin reuptake inhibitor selected from the group consisting of sertraline and indatraline, thereby enhancing or inducing the anti-tumor immune response in the patient.

A further object of the present invention relates to a method for enhancing the potency of immunotherapy administered to a patient as part of a treatment regimen for cancer, the method comprising: administering to the patient a pharmaceutically effective amount of a pharmaceutically effective amount of a selective serotonin reuptake inhibitor selected from the group consisting of sertraline and indatraline in combination with immunotherapy.

As used herein, the expression "enhanced therapeutic efficacy" relative to cancer refers to a slowing or diminution of the growth of cancer cells or a solid tumor, or a reduction in the total number of cancer cells or total tumor burden. An "improved therapeutic outcome" or "enhanced therapeutic efficacy" therefore means there is an improvement in the condition of the patient according to any clinically acceptable criteria, including, for example, decreased tumor size, an increase in time to tumor progression, increased progression-free survival, increased overall survival time, an increase in life expectancy, a decrease of immune-adverse effects or an improvement in quality of life. In particular, "improved" or "enhanced" refers to an improvement or enhancement of 1%, 5%, 10%, 25% 50%, 75%, 100%, or greater than 100% of any clinically acceptable indicator of therapeutic outcome or efficacy. As used herein, the expression "relative to" when used in the context of comparing the activity and/or efficacy of a combination composition comprising immunotherapy with the selective serotonin reuptake inhibitor to the activity and/or efficacy of immunotherapy alone, refers to a comparison using amounts known to be comparable according to one of skill in the art.

As used herein, the term “immunotherapy” has its general meaning in the art and refers to the treatment that consists in administering an immunogenic agent i.e. an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response.

In some embodiments, the immunotherapy consists in administering the patient with at least one immune checkpoint inhibitor.

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.

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.

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.

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.

In some embodiments, the immunotherapy consists in administering to the patient a combination of a CTLA-4 antagonist and a PD-1 antagonist.

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.

A further object also relates to a method of overcoming immunotherapy resistance in patient suffering from cancer thereof comprising administering to the patent a therapeutically effective amount of a selective serotonin reuptake inhibitor selected from the group consisting of sertraline and indatraline in combination with immunotherapy.

As used herein, the term “immunotherapy resistance” refers to an acquired resistance of a cancer to the immune response induced by the immunotherapy. Therefore, a resistant tumor or tumor cell is more likely to escape and survive humoral and/or cellular immune defense mechanisms in a subject receiving the immunotherapy. The phrase “overcoming immunotherapy resistance” in context of the invention shall be effective if compared to a non-treated control, the tumor or tumor cell becomes more sensitive to an immune response induced by immunotherapy. In particular, the patient become 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 is eradicated, reduced or improved after immunotherapy. According to the invention, the responders have an objective response and therefore the term does not encompass patients having a stabilized cancer such that the disease is not progressing after immunotherapy. A “non-responder” or “refractory patient” includes patients for whom the cancer does not show reduction or improvement after immunotherapy. 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 selective serotonin reuptake inhibitor. More particularly, the method of the present invention is particularly suitable for preventing tumor escape in a patient treated with immunotherapy. As used herein, the term “tumor escape” refers to any mechanism by which tumors escape the host's immune system.

As used herein, the term “selective serotonin reuptake inhibitor” or “SSRI” has its general meaning in the art and refers to an inhibitor of the monoamine transporters which has stronger inhibitory effect at the serotonin transporter than the dopamine and the noradrenaline transporters. As used herein, the term “sertraline” has its general meaning in the art and refers to (1 S,4S)- 4-(3,4-dichlorophenyl)-N-methyl-l,2,3,4-tetrahydronaphthalen-l-amine (Cas Number: 79617- 96-2).

As used herein, the term “indatraline” has its general meaning in the art and refers to (1R,3S)- 3-(3,4-dichlorophenyl)-N-methyl-2,3-dihydro-lH-inden-l-amine (Cas Number: 86939-10-8).

As used herein, the term "therapeutically effective amount" is meant a sufficient amount of the selective serotonin reuptake inhibitor for treating or reducing the symptoms at 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 disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the 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 with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic 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.

Typically the selective serotonin reuptake inhibitor is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers 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 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. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. 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.

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

FIGURES:

Figure 1. Anti-tumor prophylactic vaccination effects of Indatraline and Sertraline.

A. Experimental schedule of prophylactic vaccination assay. MCA205 cells were treated in vitro with either indatraline (20 pM), sertraline (20 pM) or mitoxantrone (2 pM) as a positive control, to reach about 70% cell death. Dying MCA205 cells were injected by subcutaneous injection (s.c.) in the left flank of the mice. Ten days later, mice vaccinated or not with tumor cell lysates were challenged with living MCA205 cells injected s.c. into the right flank of the mice. Tumors were measured three times per week and their volume was calculated with the following formula: 4/3 * n * L/2 * 1/2 * h/2. The curves show tumor growth evolution. B-D. Individual (B) and mean (C) tumor growth, as well as tumor-free survival (D) of mice treated with indatraline. E-G. Individual (E) and mean (F) tumor growth, as well as tumor-free survival (g) of mice treated with sertraline. H. Graphical representation of tumor rechallenge in mouse survivors. Mice that did not develop tumor after the initial vaccination-tumor challenge procedure were rechallenged by s.c. injection of living and untreated MCA205 cells in the right flank and B16-F10 melanoma cells in the opposite flank. I-J. Volumes of MCA205 and B16- F10 tumors 24 days post-tumor rechallenge in mice initially vaccinated with extracts of MCA205 cells treated with indatraline (I) or sertraline (J). Data were analyzed using TumGrowth (https ://github . com/kroemerlab).

Figure 2. Immune dependent effect of Indatraline and Sertraline on MCA205 tumors growth.

A. Experimental schedule of implantation and treatment of fibrosarcoma. B-D. MCA205 cells (300,000) were injected subcutaneously in the right flank of the mice. Once the tumor was established, mice were randomized and treated intraperitoneally with indatraline (10 mg/kg) or sertraline (10 mg/kg). T cells were depleted or not by i.p. injections of anti-CD4 and anti-CD8 (100 pg per mouse of each antibody) at the indicated days. Tumors were measured three times per week and their volume was calculated with the following formula: 4/3 * 7t * L/2 * 1/2 * h/2. Data were analyzed using TumGrowth (https://github.com/kroemerlab). E. Overall survival curves from the experiment performed in B-D are reported. Statistical significance was determined by a log-rank (Mantel-Cox) test.

Figure 3. Indatraline and sertraline induce the permeabilization of the lysosomal membrane.

Top panel, a schematic representation of the galectin 3 relocalization from the cytosol to lysosomes. Bottom panel, quantification of lysosomal membrane permeabilization based on the relocalization of galectin 3 from the cytosol to lysosomes was performed. Individual data points correspond to the average value per cell from each image (n=12-16 images, corresponding to a minimum of 4,000 cells per condition). Statistical significance was determined by an unpaired Mann-Whitney U test (two-tailed).

Figure 4. Indatraline and sertraline promote the accumulation of cholesterol within lysosomes.

U2OS LAMP1-GFP cells treated with either indatraline (10 pM) or sertraline (10 pM) for 18 h. U18666A (1 pM) was used as a positive control of cholesterol accumulation in lysosomes. Cells were then stained with fllipin complex, which permits the visualization of free cholesterol.

A. The average area of fllipin positive dots indicative of the presence of cholesterol was scored.

B. The surface overlap coefficient (SOC) of fllipin positive dots and the lysosomal membrane protein LAMP1 is presented. In the box plot, mean (+), median as well as the minimum and maximum values are shown. For A and B, individual data points correspond to the average value per cell from each image (n=16-64 images, corresponding to a minimum of 3,000 cells per condition). Statistical significance was determined by an unpaired Mann-Whitney U test (two-tailed). C. Representative electron micrographs of U2OS cells after treatment with either indatraline (10 pM) or sertraline (10 pM) for 6 h. Arrows indicate the presence of multilamellar vesicles (MLVs). In the box plot, each dot represents the number of MLVs per cross-section. The mean (+), median as well as the minimum and maximum values are shown.

Figure 5. Lysosomal cholesterol accumulation is essential for the manifestation of Immunogenic Cell Death (ICD) hallmarks.

To characterize the cell death modality, murine fibrosarcoma MCA205 cells were exposed to various inhibitors of cell death pathways as described in (A-C) and then treated with either indatraline (20 pM) or sertraline (20 pM) for 24 h. Cell death was evaluated using DAPI staining followed by flow cytometry analysis as described in the methods section. A. MCA205 cells were pre-incubated with various cathepsin inhibitors during 2 h, namely odanacatib (5 pM), E64d (20 pM), and pepstatin A methyl ester (20 pM) before the addition of indatraline (20 pM) and sertraline (20 pM). B. MCA205 cells pre-treated during 2 h with specific inhibitors of cell death pathways, namely apoptosis (Q-VD-Op, 25 pM), ferroptosis (ferrostatin-1, 10 pM), or necroptosis (necrostatin-1, 10 pM) before the addition of indatraline (20 pM) and sertraline (20 pM). C. MCA205 cells were pre-treated with the cholesterol-depleting agent P- cyclodextrin (1 mM) or the medium was replaced with LDL-depleted medium supplemented with 1% BSA 2 h before the treatment with indatraline (20 pM) or sertraline (20 pM). For A, B and C, values are expressed as percentages ± SD. D. U2OS cells pre-treated with the cholesterol-depleting agent P-cyclodextrin (1 mM) or LDL-depleted medium supplemented with 1% BSA 2 h before the treatment with treated with either indatraline (10 pM) or sertraline (10 pM) for 18 h. U18666A (1 pM) was used as a positive control of cholesterol accumulation in lysosomes. Cells were then stained with fllipin complex, which permits the visualization of free cholesterol in cells. The average area of fllipin positive dots indicative of the presence of cholesterol was scored. E. Evaluation of plasma membrane exposure of calreticulin in MCA205 cells after treatment with either indatraline (20 pM) or sertraline (20 pM) for 24 h. When indicated, cells were pretreated for 2 h with 1 mM of P-cyclodextrin or the medium was replaced with LDL-depleted medium prior to the addition of indatraline or sertraline. The percentage of calreticulin-positive cells among the live population of cells was quantified. F. MCA205 cells were treated as mentioned in (E) and the concentration of ATP release in the cell supernatant was quantified. Mitoxantrone (2 pM) was used as positive controls for the induction of ATP release. G. MCA205 cells were treated as mentioned in (E), and the release of HMGB1 into the culture media was assessed. For E, F and G, statistical significance was determined by an unpaired Mann-Whitney U test (two-tailed).

EXAMPLE:

In this study, we used 1200 FDA approved compounds from the Prestwick Chemical Library for a high-content imaged-based screen to search new TFEB inducers and autophagy activators. From this screening, we identified seven compounds that simultaneously promoted TFEB nuclear translocation and LC3 puncta accumulation. Then, we particularly focused on two compounds (sertraline and indatraline) that, despite the activation of TFEB and the high levels of LC3 puncta, elicited a significant cytotoxic effect in cancer cell lines. In an attempt to understand the mechanisms involved in the cytotoxic effect of sertraline and indatraline, we explored their possible effect on autophagy and lysosomal integrity. Our data showed that both compounds partially inhibited autophagy flux and subsequently generated a significant induction of lysosomal membrane permeabilization and cell death. These processes occurred through a mechanism that required the accumulation of cholesterol at the lysosomal compartment. Finally, we demonstrated that sertraline and indatraline elicited immunogenic cell death features and their administration as a vaccine resulted in effective protection against tumor growth in mice (Figure 1). Moreover, both compounds completely lose their efficacy when T cells were depleted (via the administration of anti-CD4 and anti-CD8) in immunocompetent mice (Figure 2). These results indicate that both compounds have a stimulating effect on immunity against cancer.

Methods:

Ethics statement

All mice experiments performed in this study were carried out in agreement with the EU Directive 2010/63/EU and the specific ethical protocol APAFIS #36901-2022021116138370v4 approved by the local ethical committee no 005 registered at the French Ministry of Higher Education and Research.

Cell lines and culture conditions

U2OS (human osteosarcoma), HeLa (human cervical adenocarcinoma), HEK293 (human embryonic kidney), MCA205 (mouse fibrosarcoma), and B16-F10 (mouse melanoma) cells were obtained from the American Type Culture Collection (ATCC). CRISPR/Cas9 depleted ATG13 and ULK1 U2OS cells and stable U2OS-mCherry-LGALS3 (galectin 3) expressing cells were kindly provided by Dr. H. Wodrich (CNRS UMR 5234, Bordeaux, France). U2OS cells co-expressing GFP tagged TFEB and RFP tagged LC3, U2OS mCherry tagged LGALS3 and mGFP tagged LAMP1 cells, HeLa cells NTC (non-targeting control) and ATG5 depleted HeLa cells were generated in our laboratory. U2OS, HeLa, HEK293, and B16-F10 cells were grown in culture media composed of Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, 41966029) supplemented with 10% fetal bovine serum (Sigma- Aldrich, F7524) and 10 mM of HEPES buffer (Gibco-Invitrogen, 15630-056). For U2OS-mCherry- LGALS3 and GFP-LAMP1 cells, 200 pg/mL of hygromycin B (Invitrogen, 10687010,) and 100 pg/ml Geneticin/G418 (Gibco-Invitrogen, 10131-027) were added in cell culture media. Before each experiment, all antibiotics were removed from the media. MCA205 cells were grown in Roswell Park Memorial Institute (RPMI) (Thermo Fisher Scientific, 61870044) culture media supplemented with 10% fetal bovine serum and 10 mM HEPES buffer. All the cell lines used in this study were cultivated at 37°C in a humidified incubator with 5% CO2 and were free from mycoplasma contamination based on PCR detection.

High-throughput screening

U2OS cells co-expressing TFEB-GFP and RFP-LC3 were seeded in 384 dark-well plates at a density of 1500 cells per well 24 h prior to treatment. Compounds from the Prestwick Chemical Library were added to the cells in quadruplicate at a final concentration of 10 pM for 6 h. Torin 1 (300 nM) (positive control for TFEB translocation and LC3 puncta formation) and DMSO controls were added to each plate. At the end of the treatment, cells were fixed with 4% paraformaldehyde in PBS pH 7.4 containing 10 pg/ml of Hoechst 33342 and incubated at room temperature (RT) for 20 min. The fixed cells were washed with PBS and subjected to automated image acquisition using the ImageXpress® Micro Confocal High-Content Imaging System (Molecular Devices, San Jose, CA, USA). Four view fields were captured for each well with a Plan APO 20X objective (Nikon, Tokyo, Japan) and the appropriate filter sets. For evaluating the feasibility of the screening, the Z’ factor was calculated.

Visualization of endogenous TFEB and LC3 subcellular localization

U2OS cells were seeded in 96 dark-well plates at a density of 1 x 10⁴ cells per well in 100 pl of culture media 24 h before treatment. The next day, cells were treated with the indicated drugs for the specified time. For immunofluorescence staining, cells were washed with PBS prior to fixation with 4% PFA in PBS supplemented with 10 pg/ml of Hoechst 33342 for 20 min at RT. After fixation, cells were washed twice with PBS and permeabilized in 0.3% Triton in PBS supplemented with 5% FBS for 10 min. The cells were then incubated in blocking buffer (5% FBS in PBS) at RT for 1 h, followed by incubation with primary antibody (1 :400 TFEB antibody, in PBS 2% BSA; 1 :400 LC3) at 4°C overnight. Cells were washed twice with PBS and incubated with a secondary antibody coupled to Al exaFluor 488 or 568 (1 : 1000) diluted in 2% BSA in PBS at RT for 2 h. After three washes with PBS, automated image acquisition was performed using the ImageXpress® Micro Confocal High-Content Imaging System (Molecular Devices) with a Plan APO 20X objective (Nikon).

Autophagic flux analysis

For autophagic flux experiments, we used U2OS cells stably expressing a tandem fluorescence- tagged LC3 (GFP-RFP-LC3), which produces both GFP (“green”) and RFP (“red”) signal (“yellow” dots) within autophagosomes but emits only the RFP signal (“red only”) on autolysosomes due to the quenching of GFP-LC3 in the acidic environment of the lysosome. Quantification of double fluorescent (yellow) dots (autophagosomes) and mono fluorescent red dots (autolysosomes) per cell provided an indication of autophagic flux. For the colocalization assessment of GFP and RFP fluorescence the surface overlap coefficient (SOC) of both signals was calculated.

Intracellular cholesterol assessment

To analyze intracellular cholesterol localization, IxlO⁴ U2OS cells were seeded in 96 dark-well plates and treated with either indatraline or sertraline. At the end of the treatment, cells were washed with PBS and fixed with 4% PFA for 25 min at RT. Fixed cells were then washed with PBS prior to incubation with 2 mg glycine/ ml in PBS for 15 min to quench the rest of the paraformaldehyde. The cells were stained with filipin complex (stock concentration 5 mg/mL in DMSO, working solution 0.1 mg/ml in PBS) and incubated at RT for 1 h. Cells were washed twice with PBS and subjected to image acquisition and analysis as described below.

Galectin 3 puncta assessment

Galectin 3 puncta formation was monitored to determine lysosomal membrane permeabilization. U2OS cells that co-express mCherry-LGALS3 (galectin 3) and LAMP1-GFP were plated in 96-well plates and treated for 24 h with the indicated compounds. Cells were then washed with PBS, fixed with 4% (v/v) paraformaldehyde for 10 min at RT, and nuclei were counterstained for 20 min with Hoechst 33342. High-content image microscopy with automated image acquisition and analysis was performed as described below.

Analysis of fluorescence micrographs

Cell micrographs were segmented using either the custom module editor from MetaXpress software, or free R software with the EB Image package. First, nuclear masks were generated based on Hoechst 33342 staining using the adapted thresholding method. Then cytoplasm masks were computed using a cytosolic marker, while separating adjacent cells through a watershed with the nuclear mask being used as seed. Punctiform structures were detected by applying a top hat filter, followed by automated thresholding. All generated masks were refined using classical morphologic operations (such as opening/closing). The obtained masks were then applied to the original fluorescent images to extract relevant cell-by-cell measurements such as nuclear or cytoplasmic fluorescence intensity; TFEB subcellular localization and the area of LC3, LAMP1, LAMP2, and LGALS3 dots. Colocalization was quantified using the SOC. Following the removal of cellular debris and dead cells from the data set, the parameters of interest were subjected to normalization, statistical analysis, and graphical representation. Identical analysis pipelines have been employed for the analysis of all images. All representative images include scale bars indicating 10 pm.

Cell death analysis by flow cytometry

Cell death was evaluated by analyzing the loss of mitochondrial transmembrane potential and plasma membrane permeability using tetramethylrhodamine methyl ester (TMRM) and 4,6- diaminidino-2-phenylindole (DAPI), respectively. Briefly, cells were seeded in 24 well plates at 5 x 10⁴ cells per well. The next day, cells were treated with the indicated concentration of drugs for 24 h. Where noted, distinct inhibitors targeting various cell death pathways were added 2 h prior to drug administration. At the end of the treatment, supernatant and cells were collected and pelleted at 500 g for 5 min. Cells were then stained with 150 nM TMRM and 5 pg/ml DAPI, transferred to a u-bottom 96 well plate, and incubated at 37°C and 5% CChfor 40 min. The plate was subsequently analyzed on a MACS Quant flow cytometer using emission filters appropriate for DAPI (laser 405 nm, filter 425-475, channel VI) and for TMRM (laser 488 nm, filter 565-605, channel B2) running at least 10,000 events per condition. The percentage of dead cells (DAPI positive and TMRM negative) was calculated from the total number of cells after single-cell gating using FlowJo software (Ashland, OR, USA). RNA isolation and reverse transcription quantitative PCR

Cells were plated in 25 cm² flasks (2 x 10⁶ cells/condition). After the indicated treatment, cells were collected by centrifugation, and their pellets were subjected to RNA extraction using betamercaptoethanol and the RNeasy® Plus Mini kit (Qiagen, 74134) according to the manufacturer's instructions. Before proceeding to the reverse transcription of the samples (2 pg RNA per sample) using the Master Mix SuperScript™ IV VILO™ kit (Invitrogen, 11766050), genomic DNA was eliminated using the ezDNase™ enzyme. A 10-fold dilution of the resulting cDNA was amplified employing SsoAdvanced Univ SYBR Green Supermix in a 10 pL volume with the following program: 95°C for 30 seconds, 40 cycles of 95°C for 10 seconds, and 60°C for 20 seconds using the CFX96 Touch Real-Time PCR (Biorad, Hercules, CA, USA). Results were normalized to the expression levels of Cyclophilin A (PPIA). Quantification of mRNA levels was performed using the AACt method. Primers used for amplification are listed in Table 1 and were purchased from Eurogentec (Seraing, Belgium). All experiments were done in triplicate.

Immunoblotting

Cells were harvested, centrifuged at 500 x g, and washed twice in ice-cold PBS before lysis. Lysis buffer contained 10 mM Tris, pH 7.4, 1% SDS, 1 mM sodium vanadate, and a cocktail of protease and phosphatase inhibitors. Cell pellets were treated with benzonase endonuclease (Merck Millipore, 71205) for 5 min at RT. Protein concentration was measured by BCA protein assay kit (Thermo Fisher Scientific, 23225) using bovine serum albumin (BSA) as standard. Aliquots of cellular extracts (10 to 30 pg) were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) using a Tris/glycine buffer system based on the method of Laemmli. After SDS-PAGE, proteins were transferred to nitrocellulose membranes (Cytiva). Blots were probed with primary antibodies and then incubated with the appropriate HRP-conjugated secondary antibody. Staining for P-Actin and staining with Ponceau Red were used to validate equal protein loading. Immunostained proteins were visualized using a chemiluminescent HRP substrate on an Image Quant LAS 4000 (Cytiva, Marlborough, MA, USA). Densitometry quantifications were performed using Imaged software.

Transmission electron microscopy assay

Cells were fixed for 1 hour at 4°C in 2% glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH 7.3), post-fixed 1 h in aqueous 2% osmium tetroxide, stained en bloc in 2% uranyl acetate 30% methanol, dehydrated and finally embedded in Epon. Ultra-thin sections were stained with uranyl acetate and lead citrate. Then samples were examined with a FEI Tecnai 12 electron microscope. Digital images were taken with a SIS MegaviewIII CCD camera.

Molecular docking analysis

Structures of NPC1 and NPC2 proteins were extracted from the solved cryo-EM structure of the complete protein-protein complex (PDB ID: 6W5V). The entire structure of NPC2 has been kept (150 amino acids, residues 1 to 150), but only the cholesterol-binding domain of NPC1 has been considered for docking calculation (residues 23 to 248). Please notice that these two structures are totally equivalent to the other NCP1 and NCP2 structures in the PDB. Autodock tools software has been used to prepare the structure (hydrogens and charge addition). In addition, cholesterol and every small molecule from the PDB structure have been removed. Indatraline and sertraline ligands structures were retrieved from the ChEMBL database. As they were in SMILES format, they were converted to 3D structures by Gypsum-DL software. Docking calculations were realized with Autodock Vina software. Each protein and its ligand were put in a virtual box. It was with a grid dimension of X: 46.81 A, Y: 42.49 A and Z: 59.58 A for NPC1 and of X: 58.7 A, Y: 32.35 A and Z: 40.48 A for NPC2. Both grids were centered on the binding sites but encompassed the entire protein in order to realize blind docking simulations. Other parameters, such as exhaustiveness, were chosen by default. 10 binding modes were ranked using the Vina scoring function, and visualization of the results was made with VMD software.

NPC1 and NPC2 siRNA silencing

Human NPC1 (EHU136281) and NPC2 (EHU142601) targeting MISSION esiRNA and esiRLUC (EHURLUC) control were purchased from Sigma-Aldrich. U2OS cells were transfected with esiRNA in combination with Lipofectamine RNAiMAX according to the manufacturer’s instructions. Briefly, U2OS cells were seeded in a 12-well-plate at a density of 1.2 x 10⁵ cells per well in DMEM supplemented with 10% fetal bovine serum. The next day cells were transfected and 72 h post-transfection, the protein levels of NPC1 and NPC2 were assessed by immunoblotting.

Quantification of calreticulin cell surface exposure by flow cytometry

MCA205 cells were seeded at 1 x 10⁴ cells per well in 96-flat well plates. The next day, cells were treated for 24 h with the drugs. Indatraline and sertraline were used at 20 pM. Mitoxantrone (MTX) was used as a positive control at 2 pM. After treatment, cells were detached using trypsin for 5 min and then transferred to a 96-well plate v-bottom using a multichannel pipette. The plate was then centrifuged at 500 x g for 5 min, and the supernatant was discarded carefully to avoid losing the cell pellet. Cells were then washed with cold PBS and centrifuged (500 x g, 5 min). After, cells were stained with 50 pL per well of LIVE/DEAD staining solution (1 :1000 diluted in cold PBS) and incubated at 4°C for 30 min. After this incubation period, 150 pL of cold PBS was added, and the plate was centrifuged. The supernatant was discarded, and 40 pL of CALR antibody (ab2907) solution was added (1% BSA in PBS, CALR ab 1 :200) per well. Cells were then incubated with primary antibody for 25 min at RT. Afterwards, cells were washed with PBS and centrifuged to discard the supernatant. Cells were then fixed with 4% PFA diluted in PBS 1% BSA for 25 min at RT, washed with PBS, and centrifuged. A secondary antibody (coupled to AlexaFluor 488, antirabbit, dilution 1 :500) was added in PBS 1% BSA and incubated for 25 min at RT. Next, cells were washed with PBS, and the plate was subsequently analyzed on a MACSQuant flow cytometer using emission filters appropriate for LIVE/DEAD blue (laser 405 nm, filter 425- 475, channel VI) and for Alexa488 (laser 488 nm, filter 500-550, channel Bl). The data were analyzed utilizing FlowJo software. Data are expressed as the percentage of CALR-positive cells among the living cell population.

Quantification of extracellular ATP

The release of ATP into the extracellular medium was measured with the ENLITEN ATP bioluminescence detection kit (Promega, FF2000). MCA205 cells were seeded in 24 well plates and treated with indatraline, sertraline or MTX for 24 h. At the end of the treatment, cell supernatants were collected and centrifuged at 1000 x g for 5 min. Then, 20 pL of supernatants were transferred to a 96-well white/clear bottom plate. ATP diluted at 10'⁷ M, 10'⁸ M, 10'⁹ M, 10'¹⁰ M, and 10'¹¹ M were used as standard and water as blank. Then 100 pL of Luciferase/Luciferin (L/L) was added carefully. The plate was placed in a plate reading luminometer, and ATP concentrations were calculated using four-parameter logistics based on the standard curve and were expressed as nanomolar concentrations.

Evaluation of extracellular HMGB1

The release of HMGB1 into the extracellular media was quantified using an ELISA kit (IBL International, 30164033). After treatment with indatraline or sertraline, supernatants were collected and centrifuged at 1000 x g for 5 min. From the supernatant, 10 pL were transferred to the microtiter plate as well as HMGB1 standards. After, 100 pL per well of diluent buffer was added, and the plate was incubated for 2 h at 37°C, followed by five washes with 400 pL of washing buffer. Then, 100 pL of enzyme conjugate was added to each well and incubated for 2 h at RT. After five additional washes, 100 pL per well of color solution was added, and the plate was incubated for 30 min at RT in the dark. The development of color was stopped by adding 100 pL per well of stop solution, and the absorbance at 450 nm was measured using a spectrophotometer. HMGB1 concentrations were calculated using four-parameter logistics based on the corresponding standard curve, and concentration was represented as ng/ml.

Assessment of the prophylactic vaccination effect and antitumor efficacy of the compounds Eight-week-old wild-type C57BL/6J female mice were purchased from Envigo/Inotiv Inc (Gannat, France). All mice were housed in a specific pathogen-free environment, with controlled temperature and 12 h light/dark cycles. They had unrestricted access to food and water.

For the antitumor prophylactic vaccination experiments, MCA205 cells were exposed to the specified compounds for 24 h to achieve a cell death level of 70%. At the end of the treatment, cells were harvested and collected by 5 min of centrifugation at 400 x g. The cell pellet was washed with PBS and resuspended at 3 x 10⁶ cells/mL of PBS. Right after, 100 pL of dying cells (thus 300,000 cells per mouse) were injected subcutaneously (s.c.) in the left flank of immunocompetent C57BL/6J mice. Ten days later, mice were challenged with 300,000 living cells injected s.c. into the right flank of vaccinated mice. Tumor growth was monitored using electronic calipers 3 times/week, and volume was calculated following the formula 4/3 * n * L/2 * 1/2 * h/2 and scored as mm³. Mice were sacrificed once the tumors reached a maximum volume of 1500 mm³ or when showing any signs of discomfort. The mean tumor growth curves were generated by taking the final tumor size measurements of mice that reached the endpoint and discontinuing the analysis once over half of the group had reached the endpoint.

For studying the treatment of established fibrosarcoma tumors, MCA205 cells were injected s.c. in one flank of the mice and when tumors reached 25 mm³, mice were randomized and injected intraperitoneally (i.p.) with one dose of indatraline (10 mg/kg of body weight) or sertraline (10 mg/kg of body weight) the following day. For CD4 and CD8 depletion, 100 pL of anti-CD4, anti-CD8 and IgG were administrated intraperitoneally per mouse. One dose was injected the day of randomization and one dose the day after treatment with indatraline or sertraline. Tumor growth was monitored three times per week and tumor volume was calculated as previously described. Statistical analysis

Unless specified, results were expressed as mean ± SD. For cell death experiments, data were analyzed using R. We used the percentage of cells (DAPI positive and TMRM negative) obtained by flow cytometry from at least three independent experiments. As the data was not normally distributed, robust statistics were applied. Because the data has technical replicates (wells in plates), a mixed model was considered. To infer the significance (or p-value) upon the different conditions, robust estimation of linear mixed effects models was used within the function ‘rimer’ of R-package robustlmm. As this function provides t-values, the p-values were obtained by using the t-statistics distribution (‘pt’ function in R); for that, the effective degree of freedom was estimated by a non-robust linear model (function Tmer’ of R-package Tem4’. For fluorescence images, single-cell data were cleaned (i.e. removing outliers and aberrant events) and reduced to image units by computing population means. Normal distribution of the obtained image values was tested by graphing normal quantile-quantile plots and conducting Shapiro-Wilk tests. If data sets were normally distributed, one-way ANOVA with Tukey's multiple comparison test was applied otherwise a two-tailed unpaired Mann-Whitney test was used. For qPCR, CALR exposure, HMGB1 and ATP release experiments, a two-tailed unpaired Mann-Whitney test was applied to the values obtained from three independent experiments. In vivo data was analyzed using the freely available TumGrowth software (https://github.com/kroemerlab/TumGrowth). The statistical significance of the survival curve was determined by a log-rank (Mantel-Cox) test.

Results:

A screen of 1200 compounds identifies agents that stimulate both TFEB nuclear translocation and LC3 puncta

Our initial objective was to identify novel TFEB activators by high-content imaging of a reporter detecting the translocation of TFEB from the cytoplasm to the nucleus. The formation of LC3 puncta was implemented in the readouts of the screening process since LC3 lipidation is a standard marker of autophagy and shows an emerging role in TFEB activation following lysosomal stress. We screened the Prestwick Chemical library composed of 1200 drugs with high chemical and pharmacological diversity on human osteosarcoma U2OS cells stably coexpressing the transcription factor EB (TFEB) fused with GFP and the LC3 protein fused with RFP (U2OS GFP-TFEB RFP-LC3). The compounds from the Prestwick library have already been clinically used, hence facilitating their repurposing for cancer therapy. U2OS GFP-TFEB RFP-LC3 cells were treated with each drug at a final concentration of 10 pM for 6 h. Torin 1 (300 nM) (as a positive control of TFEB nuclear translocation and LC3 puncta formation) (data not shown) and the vehicle (DMSO, as a negative control) were included in each plate. The Z’ factor (0.7527) was calculated based on negative and positive control values to evaluate the overall quality and reliability of the screening (data not shown).

While the majority of the compounds did not have any impact on TFEB nuclear translocation, eight compounds stimulated a marked increase in TFEB nuclear translocation (data not shown). The TFEB nuclear translocation-inducing hits included four antidepressants (indatraline, maprotiline, paroxetine, and sertraline), two anti -arrhythmia agents (amlodipine and proscillaridin A), one antimalarial drug (mefloquine) and one antibiotic (monensin). Seven among these eight compounds (all with the exception of proscillaridin A) also stimulated the formation of LC3-puncta (data not shown).

Nuclear translocation of TFEB was further confirmed for the eight hits by immunofluorescence detection of endogenous TFEB in U2OS cells (data not shown) and human embryonic kidney HEK293 cells (data not shown). Moreover, these eight agents caused the nuclear translocation of the transcription factor E3 (TFE3), which, like TFEB, is a member of the MITF family of transcription factors (data not shown).

Next, we focused on the top six compounds that stimulated both TFEB translocation and LC3- puncta formation and evaluated their cytotoxic effect after 24 h of treatment. Despite the activation of TFEB and the abundance of LC3-puncta, most of the TFEB hits displayed significant cytotoxic effects on osteosarcoma cells as indicated by the loss of mitochondrial membrane potential and an increase in cell membrane permeabilization (data not shown). We noticed that the antidepressants sertraline (one of the most prescribed antidepressants in the world and indatraline exerted the most pronounced cytotoxic effects on U2OS cells (data not shown). We therefore decided to focus on these two antidepressants and to characterize their effects on lysosomes from cancer cells.

Effect of sertraline and indatraline on the autophagy/lysosomal pathway

To investigate whether TFEB nuclear translocation is associated with the transcriptional upregulation of lysosome-related genes, we evaluated mRNA expression levels of several essential autophagy and lysosomal genes, including those known to be targets of TFEB, by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Treatment of U2OS cells with either sertraline or indatraline resulted in significant upregulation of multiple genes associated with lysosomal function and integrity (data not shown). These transcripts encompassed mRNA encoding lysosomal membrane proteins (LAMP1 [lysosomal associated membrane protein 1] and LAMP2 [lysosomal associated membrane protein 2]) and hydrolases (CTSB [cathepsin B] and CTSD [cathepsin D] ) as well as for subunits of lysosomal V-ATPase (ATP6V0D1 [ATPase, H+ transporting, lysosomal VO subunit DI] and ATP6V0E1 [ATPase, H+ transporting, lysosomal VO subunit El]). Moreover, indatraline and sertraline upregulated several key autophagy genes, including genes encoding LC3, an autophagosome standard marker; SQSTMl/p62 (sequestosome 1), an autophagy cargo receptor; ATG7 (autophagy related 7), an essential protein involved in LC3 lipidation; and ATG13 (autophagy related 13), responsible for autophagy initiation. Interestingly, we noticed disparate transcriptional responses to torin 1, indatraline, and sertraline for a subset of TFEB and TFE3 target genes. These results align with previous findings suggesting that the dynamics of TFEB and TFE3 transcription response depend on the precise stimuli, as well as dosage, timing and cell type.

The upregulation of autophagy -related genes and the accumulation of LC3 puncta by sertraline and indatraline prompted us to measure autophagic flux modulated by these compounds. For this we used U2OS cells stably expressing a chimeric RFP-GFP-LC3 reporter to monitor the generation of autophagosomes (yielding yellow dots resulting from the simultaneous detection of GFP and RFP fluorescence) and autolysosomes (yielding merely red dots due to the exclusive detection of pH-resistant RFP but the quenching of pH-sensitive GFP-dependent fluorescence that vanishes in the context of the acidic pH of autolysosomes) over the course of drug treatment (data not shown). As expected, cells treated with torin 1, an inducer of autophagic flux, manifested a significant increase in autolysosomes versus autophagosomes compared to untreated cells. Conversely, when cells were exposed to bafilomycin Al (bafAl), a potent inhibitor of lysosomal and autolysosomal acidification, the opposite effect was observed, leading to a noticeable decrease in red autolysosomes associated with a significant increase in yellow autophagosomes, indicating that autophagic flux is blocked. Cells treated with either indatraline or sertraline exhibited a time-dependent accumulation of autophagosomes (data not shown). However, they did not show a significant increase in autolysosomes, suggesting that the autophagic process remains incomplete.

Autophagy is orchestrated by several autophagy-related proteins (ATG) that play an essential role in the initiation and execution of this process through the formation of various molecular complexes. These include the ULK complex (ULK1 [unc-51 like autophagy activating kinase l]-ATG13-ATG101 [autophagy related 101 B]-RB1CC1/FIP2OO [RBI inducible coiled-coil 1]) involved in the initiation step, the phosphatidylinositol 3-kinase/PtdIns3K complex (PIK3C3/Vps34 [phosphatidyl-inositol 3-kinase catalytic subunit type 3]-PIK3R4/Vpsl5 [phosphoinositide-3 -kinase regulatory subunit 4]-ATG14 [autophagy related 14]-NRBF2 [nuclear receptor binding factor 2]-BECNl [beclin 1]) required for the nucleation step and the ubiquitin-like conjugation system (ATG12 [autophagy related 12]-ATG5 [autophagy related 5]-ATG16Ll [autophagy related 16 like 1]) involved in the elongation and maturation steps (data not shown). ATG13 -depleted and ULKl-depleted cells - both failing to initiate canonical autophagy - were treated with either indatraline or sertraline. Torin 1 and starvation condition (EBSS [Earle's balanced salt solution]) were used as positive inducers of canonical autophagy. As expected, WT cells subjected to torin 1 treatment or starvation manifested endogenous LC3 puncta accumulation, while ATG13 or ULK1 depleted cells failed to manifest such an accumulation, confirming the role of ULK1 and ATG13 in canonical autophagy. Nonetheless, both WT cells and ATG13 depleted cells, as well as ULK1 depleted cells exhibited significant LC3 puncta formation in response to either sertraline or indatraline, suggesting that ULK1 and ATG13 components involved in the initiation of autophagy are not required for LC3 puncta accumulation induced by these compounds (data not shown). Moreover, LC3 co-localized with lysosomes of cells treated with sertraline or indatraline, as evidenced by the analysis of the cooccurrence of LC3 and the lysosomal marker LAMP2 (data not shown).

These findings support the idea that unconventional LC3 puncta formation, independent of autophagy, occurs in response to indatraline and sertraline. Accordingly, several recent studies highlight the implication of LC3 lipidation in processes that are not directly related to autophagy.

TFEB nuclear translocation induced by sertraline or indatraline requires an autophagyindependent function of LC3

A key step involved in the formation of LC3 puncta is orchestrated by the ubiquitin-like conjugation system (ATG12-ATG5-ATG16L1), which leads to the conjugation of LC3 to phosphatidylethanolamine (LC3-PE: the lipidated form of LC3) and its subsequent membrane localization (data not shown), correlating with an increase in electrophoretic mobility detectable by immunoblot, yielding a shift from LC3-I to LC3-II (data not shown). To address the possible role of LC3 puncta formation in TFEB nuclear translocation, we assessed TFEB nuclear translocation in U2OS cells depleted from ATG5, in which LC3 lipidation is impaired. As reported above, sertraline and indatraline both induced LC3 lipidation in WT cells. However, this effect was not observed in ATG5-depleted cells, confirming the requirement of ATG5 for LC3 lipidation (data not shown). Simultaneously, ATG5-depleted cells treated with either sertraline or indatraline manifested a significant reduction in TFEB nuclear translocation as compared to WT cells (data not shown). The dependency on ATG5 for TFEB translocation was only observed in response to sertraline and indatraline but not in response to torin 1 or starvation (data not shown). The requirement of ATG5 for TFEB nuclear translocation induced by indatraline and sertraline was also validated in HeLa cells (data not shown). To confirm if this regulation is specific for the lipidation step of LC3 but not autophagy, we analyzed TFEB nuclear localization in ULKl-depleted cells where canonical autophagy is impaired but LC3 lipidation still occurs. Indeed, ULKl-depleted cells exhibited significant TFEB nuclear translocation in response to either sertraline or indatraline as well as to torin 1 and starvation (data not shown). Altogether, these results suggest that LC3 lipidation is required for TFEB activation induced by sertraline or indatraline.

Sertraline and indatraline trigger lysosomal membrane permeabilization

Lysosomotropic agents accumulate within the lysosomal lumen and potentially impair lysosomal integrity and function. The specific requirement of LC3 lipidation for TFEB nuclear translocation by indatraline and sertraline echoes prior reports on the TFEB activation by lysosomotropic agents. Accordingly, we found that the prototypic lysosomotropic agent L- leucyl-L-leucine methyl ester (LLOMe) triggered TFEB activation through an LC3 -dependent mechanism in U2OS and HeLa cells (data not shown). This observation prompted us to test the hypothesis that sertraline and indatraline might act as lysosomotropic agents. To investigate the potential accumulation of the compounds within lysosomes, we revisited the eight TFEB hits from the screening. Among them, we selected amlodipine, which exhibits a blue fluorescence, allowing us to follow its subcellular localization. Using LAMP1-GFP cells, a lysosomal protein marker, the fluorescence confocal images revealed that the amlodipine-dependent blue fluorescence mostly overlapped with the LAMP1-GFP signal, indicating that amlodipine was capable of accumulating within lysosomes as this is typical for lysosomotropic agents (data not shown).

Lysosomotropic agents exhibit peculiar physicochemical properties, including high lipophilicity (often expressed as the partition coefficient of water and n-octanol, logP) and a high dissociation constant (pKa) value. Lipophilicity can be used to predict the extent to which a compound transfers through passive diffusion lipid bilayers, such as the lysosomal membrane. The pKa value of protonated basic sites in the molecule (basic pKa) can be used to correlate the accumulation of the compounds in an acidic environment, such as the lumen of lysosomes. Molecules with a logP > 2 and a basic pKa value between 6.5 and 11 are prone to accumulate in lysosomes. The molecular descriptors retrieved from the ChEMBL database for the eight TFEB hits allowed to computationally predict that most of the TFEB hits (6 out of 8) exhibited the molecular characteristic of lysosomotropic agents. This in silico prediction failed to identify such physicochemical properties for proscillaridin A and monensin, as well as for torin 1 (data not shown).

As lysosomotropic agents have the potential to induce lysosomal membrane permeabilization (LMP) we next investigated this possibility for the eight TFEB hits. The induction of LMP was monitored by detecting the translocation of LGALS3 (galectin 3) from the cytosol to damaged lysosomal membranes by fluorescence microscopy (data not shown). Using U2OS cells stably co-expressing LGALS3 tagged with mCherry and LAMP1 tagged with GFP, we found that the majority of TFEB hits were potent LMP inducers, as indicated by the increased abundance of LGALS3 puncta that co-localized with the lysosomal protein LAMP1. Altogether, these results revealed that six out of eight TFEB hits induced LMP. Among them are drugs that have previously been reported to induce LMP, such as amlodipine and mefloquine, as well as thus far unknown LMP inducers including indatraline, monensin, paroxetine and sertraline.

Next, we sought to determine the molecular mechanism underlying LMP induction, focusing on the two antidepressants, sertraline and indatraline. Distinct molecular pathways can initiate LMP, each resulting in excessive reactive oxygen species (ROS) production, activation of caspases or modification of the lipid composition of the lysosomal compartment. Abnormal cholesterol accumulation within lysosomes has been reported to be associated with LMP induction. To gain further insight into the mechanism involved in LMP induction, mCherry- LGALS3 U2OS cells treated with sertraline or indatraline were supplemented with the antioxidant, N-acetyl cysteine (NAC), the pan-caspase inhibitor quinolyl-valyl-O- methylaspartyl-[2,6-difluorophenoxy]-methyl ketone (Q-VD-Oph), or the cholesterol depleting agent, P-cyclodextrin. Cells were then subjected to the evaluation of galectin 3 puncta as a sign of LMP. Neither ROS scavenge by NAC nor inhibition of caspases by Q-VD-Oph did reduce LMP triggered by sertraline or indatraline (Figure 3). However, cholesterol depletion by P- cyclodextrin significantly prevented LMP induction triggered by the two compounds, suggesting a link between cholesterol and LMP (Figure 3). Sertraline and indatraline promote lysosomal cholesterol accumulation by inhibiting cholesterol binding to the lysosomal cholesterol transporters, NPC1 and NPC2

To test the hypothesis that indatraline and sertraline affect cholesterol homeostasis in cells, we next visualized free cholesterol using fllipin. Filipin, a fluorescent polyene antibiotic with a high affinity for sterol molecules with free 3P-hydroxyl groups, is commonly used to detect non-esterified (free) cholesterol. Fluorescence confocal images revealed that, as compared to untreated cells, both sertraline and indatraline caused intracellular cholesterol accumulation to a similar extent as the well-known cholesterol transport inhibitor, U18666A (Figure 4A). Moreover, we observed that cholesterol accumulated within the lysosome compartment in both sertraline and indatraline-treated cells, as suggested by the co-localization of the filipin signal with LAMP1-GFP (Figure 4B). Accordingly, transmission electron microscopy revealed the accumulation of concentric multilamellar vesicle structures (that are usually referred to as “onion-like structures” or “onionoids”) in cells treated with sertraline and indatraline but not in untreated cells (Figure 4C). Such structures are formed following the accumulation of phospholipids in multilamellar vesicles as they are detected in tissues from patients affected by the Niemann-Pick Type C lysosomal storage disorder, as well as in cells deficient for NPC intracellular cholesterol transporter 1 (NPC1) protein, arguing in favor of the impairment of NPC1 function in cells exposed to sertraline and indatraline.

Cellular cholesterol can originate from de novo biosynthesis or the uptake from the cellular microenvironment. Upon uptake, low-density lipoproteins (LDL) containing cholesteryl ester and free cholesterol are delivered to the lysosome through the endocytic pathway. Free cholesterol within the lysosome is then transferred to the endoplasmic reticulum for efflux. This process involves the coordinated action of two proteins, NPC1 and NPC intracellular cholesterol transporter 2 (NPC2). Inside the lysosome, NPC2 captures cholesterol and transports it to the membrane protein NPC1, which facilitates its transfer to the endoplasmic reticulum. The role of NPC 1 and NPC2 has been extensively studied, particularly in the context of loss-of-function mutations that cause the Niemann-Pick Type C lysosomal storage disorder. This disorder is distinguished by abnormal accumulation of cholesterol inside the lysosome, leading to disruption of intracellular trafficking and dysfunction of the lysosomal compartment. The accumulation of lysosomal cholesterol in indatraline or sertraline-treated cells is reminiscent of the abnormal lysosomal abundance of cholesterol observed in NPC disorders due to loss-of-function mutations of NPC1 or NPC2. We, therefore, hypothesized that sertraline and indatraline act as inhibitors of sterol binding sites in NPC1 and NPC2 proteins. We performed molecular docking analysis of NPC 1 and NPC2 to test this hypothesis. The results of molecular docking calculations revealed that both indatraline and sertraline preferentially bind to the same cavity to which cholesterol interacts with NPC1 and NPC2 (data not shown). Consequently, both compounds potentially inhibited the binding of cholesterol to NPC1 and NPC2. As these ligands are smaller than cholesterol, their interaction involves a more limited number of residues than the previous one. Many residues are common, notably He 205, Asn 198, Phe 193, Leu 83, and Asn 86. Most of the interactions are hydrophobic in nature, either through contact (Van der Waals' interaction) or aromatic interaction (□ -stacking). Interestingly, these residues were also found to interact with leelamine, another lysosomotropic agent, as previously shown. Moreover, the downregulation of NPC1 or NPC2 expression through siRNA (data not shown) resulted in a significant reduction of cell death induced by indatraline and sertraline. This effect was more pronounced when NPC2 expression was specifically downregulated even if 40% of NPC2 expression still remains after transfection with siRNA. This suggests that the induction of cell death by indatraline and sertraline depends, at least in part, on lysosomal cholesterol transporters, placing a specific emphasis on the role of NPC2.

In conclusion, both indatraline and sertraline can interact with the binding site of steroids in NPC1 and NPC2. It appears that sertraline has a marginally higher affinity for these proteins than indatraline (-8.83 and -8.89 kcal/mol respectively for NPC1 and NPC2 with sertraline, compared to -7.9 and -8.15 kcal/mol respectively for NPC1 and NPC2 for indatraline). More advanced calculations are required to accurately determine the binding and stability of the interaction of these compounds with NPC1 and NPC2. Nonetheless, these findings support our hypothesis that sertraline, and indatraline, may block cholesterol export from lysosomes by competitively inhibiting the binding of cholesterol to NPC1 and NPC2.

Characterization of the cell death induced by indatraline and sertraline

Next, we aimed to characterize the molecular mechanism underlying the antitumor effect of sertraline and indatraline. We employed the murine fibrosarcoma cell line MCA205, a well- established cell line that is extensively used in vitro and in vivo to assess the potential of anticancer drugs. We monitored cell death by assessing plasma membrane permeabilization using the cell-impermeable dye DAPI following flow cytometer analysis. As the induction of lysosomal membrane permeability can lead to the release of cathepsins, potentially resulting in cell death, we inhibited such catabolic enzymes to evaluate their potential contribution to cell killing. MCA205 cells were pretreated with inhibitors of various cathepsins (odanacatib, an inhibitor of cathepsin K highly expressed in fibroblasts; aloxistatin (E64d), an inhibitor of the cysteine cathepsin family; and pepstatin A, an inhibitor of the aspartic cathepsin family) before treatment with either sertraline or indatraline. None of the cathepsin inhibitors significantly impacted cell death induced by both compounds (Figure 5A).

Since cathepsin activity is dispensable for cell death induced by the two compounds, we searched for other candidates for cell death induction. To characterize the type of regulated cell death induced by indatraline and sertraline, specific cell death inhibitors that block apoptosis (Q-VD-Oph), ferroptosis (ferrostatin-1), or necroptosis (necrostatin-1) were used. While the pan-inhibitor of caspase activity (Q-VD-Oph) significantly reduced etoposide-induced apoptosis, it only partially inhibited cell death triggered by indatraline and sertraline (Figure 5B) Inhibition of ferroptosis by ferrostatin-1 markedly reduced cell death induced by erastin but failed to inhibit cell death triggered by indatraline and sertraline (Figure 5B). Similarly, inhibition of necroptosis by necrostatin-1 did not rescue cell death induced by indatraline and sertraline (Figure 5B). Collectively, these data indicate that apoptosis, but neither ferroptosis nor necroptosis, partially contributes to indatraline or sertraline-induced cell death.

Having established a role of lysosomal cholesterol accumulation for LMP, we aimed to investigate whether this response is also implicated in cell death triggered by indatraline and sertraline. The depletion of cholesterol by P-cyclodextrin or LDL-depleted medium (medium lacking serum and complemented with 1% BSA), markedly prevented cell death triggered by indatraline or sertraline (Figure 5C). As anticipated, incubating cells with either P-cyclodextrin or LDL-depleted medium suppressed the accumulation of lysosomal cholesterol induced by indatraline and sertraline, as well as for U18666A, as evidenced by fllipin staining (Figure 5D). Collectively, these findings emphasize that the accumulation of lysosomal cholesterol is a key factor responsible for the cell death induced by indatraline and sertraline.

Sertraline and indatraline induce immunogenic cell death

Antineoplastic drugs that elicit an immune response against cancer cells are particularly efficient. For example, mitoxantrone (MTX) can induce immunogenic cell death (ICD) of malignant cells, hence mediating a therapeutic effect that last beyond therapeutic discontinuation. At the molecular level, ICD is characterized by specific features including the exposure of calreticulin (CALR) on the cell surface, the secretion of adenosine triphosphate (ATP), and the release of high mobility group box 1 (HMGB1) into the extracellular space. Both indatraline and sertraline demonstrated the capability to stimulate these features of ICD on mouse fibrosarcoma MCA205 cells as effectively as the positive control MTX (Figure 5E- G). Interestingly, depletion of cholesterol by either P-cyclodextrin or LDL-depleted medium significantly inhibited CALR exposure and the release of both ATP and HMGB1 induced by the two compounds (Figure 5E-G). This observation suggests that lysosomal cholesterol accumulation is a key factor responsible for the manifestation of ICD hallmarks.

Next, we assessed the ability of indatraline and sertraline to initiate antitumor immune responses by conducting prophylactic vaccination experiments in immunocompetent C57BL/6J mice. For this, we subcutaneously (s.c.) injected C57BL/6J mice with syngeneic MCA205 cells that had previously been treated in vitro with either indatraline or sertraline. Ten days following immunization, the mice were challenged with living MCA205 cells injected into the opposite flank (Figure 1A). The inhibition of tumor growth at the challenge site was interpreted as an effective initiation of an adaptive immune response. As compared to controls, mice immunized with indatraline-treated MCA205 manifested a significant reduction in tumor growth, and this effect was as pronounced as the one observed with MTX treatment (Figure IB, C). Four out of 10 mice remained tumor-free, revealing a protective immune response (Figure ID). Similarly, mice immunized with sertraline-treated cells were significantly protected against tumor growth with 5 out of 10 mice that remained tumor-free (Figure 1E-G). Next, mice that remained tumor- free after vaccination with MCA205 cells treated either with indatraline (n=4) (Figure ID) or sertraline (n=5) (Figure 1G) were rechallenged by s.c. injections of either MCA205 fibrosarcoma or B16-F10 melanoma cells in the right and left flanks, respectively (Figure 1H). An effective protection against tumor growth was solely noticeable when mice were rechallenged with the same cancer cell type (MCA205) not with a different cell type (B16-F10) (Figure II, J). This reveals the establishment of a protective tumor-specific immune memory, confirming the immunogenicity of indatraline and sertraline-induced cancer cell death.

The anticancer efficacy of sertraline and indatraline depends on T cells

In the next series of experiments, we established subcutaneous fibrosarcomas in immunocompetent C57BL/6J mice to explore the potential anticancer effects of indatraline and sertraline in vivo (Figure 2A). Intraperitoneal (i.p.) injection of mice with a single dose of either indatraline or sertraline was sufficient to significantly delay tumor growth compared to untreated mice (Figure 2B, C). This effect was entirely lost when CD4 and CD8 T cells were depleted by means of suitable antibodies, indicating that an intact cellular immune system is required for the antitumor therapeutic efficacy of the two compounds (Figure 2D). Tumor regression triggered by indatraline or sertraline was associated with a notable extension in the survival of mice (Figure 2E). In sum, these results demonstrate that the anticancer effects of indatraline and sertraline against established sarcomas rely on a cellular adaptive immune response.

REFERENCES: 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

CLAIMS:

1. A method for treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a selective serotonin reuptake inhibitor selected from the group consisting of sertraline and indatraline.

2. The method of claim 1 wherein the cancer is sarcoma.

3. The method of claim 2 wherein the patient suffers from a soft tissue sarcoma.

4. The method of claim 3 wherein the patient suffers from a fibrosarcoma.

5. The method according to any one of claims 1 to 4 wherein the selective serotonin reuptake inhibitor induces anti-tumor immunity.

6. A method for enhancing the potency of immunotherapy administered to a patient as part of a treatment regimen for cancer, the method comprising: administering to the patient a pharmaceutically effective amount of a pharmaceutically effective amount of a selective serotonin reuptake inhibitor selected from the group consisting of sertraline and indatraline in combination with immunotherapy.

7. A method of overcoming immunotherapy resistance in patient suffering from cancer thereof comprising administering to the patent a therapeutically effective amount of a selective serotonin reuptake inhibitor selected from the group consisting of sertraline and indatraline in combination with immunotherapy.

8. The method of claim 6 or 7 wherein the immunotherapy consists in administering the patient with at least one immune checkpoint inhibitor.

9. The method of claim 8 wherein the immune checkpoint inhibitor is selected from the group consisting of PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, and BTLA antagonists.