Methods and pharmaceutical composition for treating prostate cancer

Abstract

Inventors have firstly demonstrated that the transcriptomic signature of PTENpc- / - LSCmed cells is enriched in human MSPC signature, associated to metastases and ADT resistance. Using scRNAseq specifically with sorted PTENpc- / - LSCmed cells, they identified three distinct LSCmed clusters and showed that castration favors the emergence of the most stem-like LSCmed subpopulation by cell plasticity. In the latter, they identified the transcription factor FOSL1 / AP-1 and the kinase PIM1 as relevant therapeutic targets, and they showed that combined inhibition using JQ1, a BET / AP-1 inhibitor, and CX-6258, a pan-PIM kinase inhibitor, efficiently prevents PTENpc / - LSCmed cell growth in organoids. These findings were confirmed in the human prostate HPV10 cell line here identified as a robust model of Club / Hillock cells. Combined drug delivery to castrated PTEN-null mice induced a significant prostate weight decrease associated with the reduction of histopathological phenotypes and dramatically altered organoid-formation capacity of LSCmed cells sorted from these tumors. Accordingly, the drug combination significantly delayed tumor growth of MSPC-like human PC-3 cells subcutaneously injected into castrated immunodeficient mice. Altogether, this work shows a new therapeutic potential of combined FOSL1 and PIM1 targeting to prevent, or at least delay, the growth of MSPC-like cells. Accordingly, the invention relates to i) an inhibitor of FOSL1 and ii) an inhibitor of PIM1, as a combined preparation for use in the treatment of prostate cancer in a subject in need thereof.

Description

[0001] METHODS AND PHARMACEUTICAL COMPOSITION FOR TREATING

[0002] PROSTATE CANCER

[0003] FIELD OF THE INVENTION:

[0004] The present invention is in the field of medicine, in particular oncology.

[0005] BACKGROUND OF THE INVENTION:

[0006] Prostate Cancer (PCa) is the second most frequent male cancer worldwide and the fifth in terms of death (Bray et al., 2018). At the aggressive stage, PCa invades adjacent tissues and metastasizes to distant sites such as bone, liver or lung (Bubendorf et al., 2000). At this stage, gold-standard treatment is androgen deprivation therapy (ADT, i.e. chemical castration). A clinical response is initially observed in virtually all patients, reflecting the death of androgendependent tumoral cells. Within 1 to 3 years, however, castration-resistant prostate cancer (CRPC) will develop in most patients, leading to metastatic dissemination and, ultimately, patient death. Second generation anti-androgen therapies targeting extra-gonadic testosterone production (Abiraterone) or Androgen Receptor (AR) activation (Enzalutamide, Apalutamide) improve overall survival of CRPC patients by a few months (Wang et al., 2021). However, -30% of the latter show initial therapeutic resistance and the others develop resistance after a few months of treatment which has been linked to emergence of aggressive variants of prostate cancer such as Neuroendocrine Prostate Cancer (NEPC) (Han et al., 2022) or Double-Negative Prostate Cancer (DNPC) (Bluemn et al., 2017). The development of efficient therapies able to prevent the progression of prostate cancer towards CRPC is hampered by our limited understanding of the cellular and molecular mechanisms driving this critical shift in the course of the disease.

[0007] The inventors earlier identified in the mouse prostate a population of castration-tolerant luminal cells that they named LSCmed according to their cell sorting profile (Lin-, Scal+ et CD49fined) (Sackmann-Sala et al., 2014, Sackmann-Sala et al., 2017). LSCmed cells correspond to the luminal progenitor cluster subsequently identified in WT mice by single cell RNA sequencing (scRNAseq) (Crowley et al., 2020, Guo et al., 2020, Mevel et al., 2020, Joseph et al., 2020, Graham et al., 2022), and are the mouse equivalent of Club and Hillock epithelial cells in the human prostate (Henry et al., 2018, Baures et al., 2022A and B). Low androgen pathway activation and enrichment in stem-related transcriptional programs characterize all these cells (Sackmann-Sala et al., 2017, Baures et al., 2022A, Yan et al., 2022). Accordingly, sorted LSCmed cells generate prostaspheres, organoids and prostatic structures after heterotopic engraftment (Sackmann-Sala et al., 2014, Kwon et al., 2016, Barros Silva et al., 2018, Crowell et al., 2019, Crowley et al., 2020, Karthaus et al., 2020). Furthermore, their prevalence increases after castration at the expense of androgen-dependent cells, which experimentally assessed their castration-tolerance predicted in silico (Sackmann-Sala et al., 2014, Kwon et al., 2016, Barros Silva et al., 2018). Deletion of the tumor suppressor PTEN gene in prostatic luminal cells (PTEN110'7' mice) generates metastatic castration-resistant prostate tumors (Wang et al., 2003, Mulholland et al., 2011). In these tumors, LSCmed cells represent up to 80% of the epithelium and this compartment is quantitively unaffected by the castration (Sackmann-Sala et al., 2017). Indeed, many LSCmed cells survive castration and even continue to proliferate (Sackmann-Sala et al., 2017). Sorted PEEN1’1-" LSCmed cells generate tumors in heterotopic engraftment (Sackmann-Sala et al., 2017) and organoids irrespective of the presence of androgens (Baures et al., 2022B). In this model, LSCmed cells are assumed to drive CRPC.

[0008] Human CRPC has heterogeneous phenotypes (Robinson et al., 2015). Mesenchymal stem-like prostate cancer (MSPC) has been recently identified as a novel intrinsic transcriptional subtype of metastatic CPRC distinct from ARPC (AR positive prostate cancer) and NEPC subtypes (Han et al., 2022). MSPC is associated with high metastatic potential and poor prognosis. The defining features of MSPC are enrichment in Epithelial-Mesenchymal Transition (EMT) and luminal progenitor (LSCmed / Hillock / Club) signatures, including low AR signaling. Club and Hillock cells have also been identified in treatment-naive primary tumors (Chen et al., 2021, Song et al., 2022), where the MSPC subtype identifies a small subset of tumors enriched with TP53 and PTEN deletion and associated with accelerated progression and patient death (Han et al., 2022). These data suggest that the PTENpc" mouse is a good model for studying MSPC pathobiology, in particular to delineate the pivotal role of LSCmed-like cells in generating this particular tumor subtype.

[0009] Epithelial remodeling is emerging as a major mechanism driving castration-resistance. In mouse models, castration was shown to trigger luminal cell reprogramming into luminal progenitor LSCmed-like cells in both healthy and cancer contexts (Karthaus et al., 2020, Chan et al., 2022). In patients, the MSPC subtype has been proposed to result from Enzalutamide / abiraterone-induced lineage plasticity of ARPC (Bluemn et al., 2017, Han et al., 2022, Tang et al., 2022). These data indicate that LSCmed / Club / Hillock cells are not only intrinsic components of the prostatic epithelium, but also represent a specific cell state achieved by cell reprogramming in response to castration. As -30% naive tumors highlighted a mixed ARPC / MSPC phenotype (Han et al., 2022), it is therefore important to determine the role of this particular cell type during the early steps of castration-resistance in order to identify therapeutic strategies able to interfere with tumor progression towards CRPC.

[0010] SUMMARY OF THE INVENTION:

[0011] The invention relates to i) an inhibitor of FOSL1 and ii) an inhibitor of PIM1, as a combined preparation for use in the treatment of prostate cancer in a subject in need thereof.

[0012] The invention is defined by the claims.

[0013] DETAILED DESCRIPTION OF THE INVENTION:

[0014] In this study, inventors have firstly demonstrated that the transcriptomic signature of PTENP' ' LSCmedcells is enriched in human MSPC signature, associated to metastases and enzalutamide / abiraterone-resistance. Using scRNAseq specifically with sorted PTENpc" LSCmedcells, they identified three distinct LSCmedclusters and showed that castration favors the emergence of the most stem-like LSCmedsubpopulation by cell plasticity. In the latter, they identified the transcription factor FOSL1 / AP-1 and the kinase PIM1 as relevant therapeutic targets, and they showed that combined inhibition using JQ1, a BET / AP-1 inhibitor, and CX- 6258, a pan-PIM kinase inhibitor, efficiently prevents PTENpc -LSCmedcell growth in organoids. These findings were confirmed in the human prostate HPV 10 cell line here identified as a robust model of Club / Hillock cells.

[0015] Altogether, this work shows a new therapeutic potential of combined FOSL1 and PIM1 targeting to prevent, or at least delay, the growth of MSPC-like cells.

[0016] In a first aspect, the present invention relates to i) an inhibitor of FOSL1 and ii) an inhibitor of PIM1, as a combined preparation for use in the treatment of prostate cancer in a subject in need thereof.

[0017] More particularly, the present invention relates to a method for treating prostate cancer in a subject in need thereof comprising a step of administrating the subject with a therapeutically effective amount of an inhibitor of FOSL1 and an inhibitor of PIM1.

[0018] As used herein, the term “subject” denotes a vertebrate such as mammal, bird, fish, amphibian or reptile. In some embodiments, the vertebrate is a warm-blooded vertebrate (i.e. mammal, bird, fish). In some embodiments, the subject is a mammal, such as a rodent, a feline, a canine (e.g. a dog), an equine (e.g. horse), a bovine (e.g. a beef), a sheep or a primate.

[0019] More particularly, the subject according to the invention is a human.

[0020] In some embodiments, the subject suffers from prostate cancer.

[0021] In some embodiments, the subject suffers from prostate cancer in which the FOSL1 and PIM1 expression and / or activity are overexpressed.

[0022] In some embodiments, the subject suffers from castration-resistant prostate cancer (CRPC).

[0023] In some embodiments, the subject suffers from Neuroendocrine Prostate Cancer (NEPC).

[0024] In some embodiments, the subject suffers from Double-Negative Prostate Cancer (DNPC).

[0025] As used herein, the term “prostate cancer” refers to uncontrolled growth of cells in the prostate, a gland in the male reproductive system below the bladder. Early prostate cancer causes no symptoms. Abnormal growth of prostate tissue is usually detected through screening tests, typically blood tests that check for prostate-specific antigen (PSA) levels. Those with high levels of PSA in their blood are at increased risk for developing prostate cancer.

[0026] In another embodiment, the prostate cancer is resistant cancer, metastatic cancer, Castration-Resistant Prostate Cancer (CRPC), Neuroendocrine Prostate Cancer (NEPC) or Double-Negative Prostate Cancer (DNPC).

[0027] In another embodiment, the prostate cancer is resistant cancer.

[0028] In another embodiment, the prostate cancer is metastatic cancer.

[0029] In a particular embodiment, the prostate cancer is castration-resistant prostate cancer (CRPC). CRPC refers to disease progression despite androgen depletion therapy (ADT) and may present as either a continuous rise in serum prostate-specific antigen (PSA) levels, the progression of pre-existing disease, and / or the appearance of new metastases.

[0030] In a particular embodiment, the prostate cancer is Neuroendocrine Prostate Cancer (NEPC). NEPC refers to an aggressive histologic subtype of prostate cancer that most commonly arises in later stages of prostate cancer as a mechanism of treatment resistance.

[0031] In a particular embodiment, the prostate cancer is Double-Negative Prostate Cancer (DNPC). DNPC is defined by prostate cancer exhibiting low / no detection of Androgen Receptor (AR) and neuroendocrine markers (e.g. synaptophysin) by immunohistochemistry. As used herein, the term “metastatic cancer” refers to the spread of cancer from one organ or tissue to another location. The term also refers to tumor tissue that forms in a new location as a result of metastasis. A "metastatic cancer" is a cancerthat spreads from its original, or primary, location, and may also be referred to as a "secondary cancer" or "secondary tumor". Generally, metastatic tumors are named for the tissue of the primary tumor from which they originate.

[0032] As used herein, the term “resistant cancer” refers to a cancer which does not respond to a treatment. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. The resistance to drug leads to rapid progression of metastatic cancer. The resistance of cancer for the medication is caused by mutations in the gene which are involved in the proliferation, divisions or differentiation of cells.

[0033] In a particular embodiment, the resistant cancer is resistant to androgen depletion therapy (ADT).

[0034] In a particular embodiment, the resistant cancer is resistant to anti-androgen therapies targeting extra-gonadic testosterone production (Abiraterone) or Androgen Receptor (AR) activation (Enzalutamide, Apalutamide).

[0035] In a further embodiment, the resistant cancer is resistant to a combined preparation comprising androgen depletion therapy (ADT) and anti-androgen therapies targeting extra- gonadic testosterone production.

[0036] The resistant and metastatic prostate cancer is associated with mesenchymal stem-like prostate cancer (MSPC) and is considered as a novel intrinsic transcriptional subtype of metastatic CPRC. In the context of the invention, the transcriptomic signature of PTEN^" LSCmed cells is enriched in human MSPC signature, associated to metastases and ADT resistance.

[0037] More particularly, a decrease of Androgen Receptor (AR) signaling was observed in LSCmedcells after castration, indicating they are sensitive, but resistant, to castration. Differential gene expression (DEG) analysis identified 91 genes that exhibited >1.5-fold (adj. pval<0.05) difference after castration and the 20 most discriminative ones were reported here.

[0038] In a further embodiment, the resistant and metastatic prostate cancer is characterized by PTEN^" ' LSCmed cells which are markedly enriched in Mesenchymal Stem-like Prostate Cancer (MSPC).

[0039] In a particular embodiment, the resistant cancer is resistant to androgen deprivation therapy. In a further embodiment, the resistant cancer is resistant to anti-androgen therapies targeting extra-gonadic testosterone production (Abiraterone) or Androgen Receptor (AR) activation (Enzalutamide, Apalutamide).

[0040] As used herein, the term "treatment" or "treat" refers to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at 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.]).

[0041] As used herein, the term “FOSL1” also called FRA1, refers to Fos-like 1, is a transcription factor. It is a subunit of the transcriptional complex API. It plays a crucial role in cell differentiation, response to environmental stresses, and tumorigenesis. The human gene FOSL1 corresponds to NCBI Entrez Gene No. 8061. Gene FOSL1 encodes the protein Fos- related antigen 1 (FRA1), which corresponds to Uniprot reference P15407. As used herein, the term "PIM1” refers to proviral integration site for moloney murine leukemia virus 1 is proto-oncogene serine / threonine-protein kinase. This enzyme in humans is encoded by the PIM1 gene. It plays a role in signal transduction in blood cells, contributing to both cell proliferation and survival, and thus provides a selective advantage in tumorigenesis. The human gene PIM1 corresponds to NCBI Entrez Gene No. 5292 and the human protein PIM1 corresponds to Uniprot reference Pl 1309.

[0042] As used herein, the term “inhibitor of FOSL1” denotes a molecule that partially or totally inhibits FOSL1 biological activity or expression. The term also encompasses a molecule able to decrease or inhibit FOSL1 gene expression.

[0043] As used herein, the term “inhibitor of PIM1” denotes a molecule that partially or totally inhibits PIM1 biological activity or expression. The term also encompasses a molecule able to decrease or inhibit PIM1 gene expression.

[0044] In the context of the present invention, “FOSL1 inhibitor” and “PIM1 inhibitor” refers to an inhibitor which neutralizes, blocks, inhibits, abrogates, reduces or interferes with the biological activity ofFOSLl and PIMl.

[0045] In some embodiments, the FOSL1 and PIM1 inhibitors are selected from the group consisting of but not limited to: small organic molecule, peptide, peptidomimetic, antibody, aptamers, siRNA or antisense oligonucleotide.

[0046] In a particular embodiment, the FOSL1 and / or PIM1 inhibitor according to the invention is a low molecular weight compound, e. g. a small organic molecule (natural or not).

[0047] The term "small organic molecule" refers to a molecule (natural or not) 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 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

[0048] In a further embodiment, the FOSL1 inhibitor is selected from the group consisting of but not limited to: JQ1, Trametinib, BIX02189, SP600125, GSK2126458, BYL719, IKK-16, IQ2, OTX015, Molibresib (I-BET762), Bosutinib, Verdinexor , LY-1816, SRI 1302, T5224, I- BET 151, CPI-0610, ABBV-075, or GS-5829.

[0049] In a further embodiment, the FOSL1 inhibitor is JQ1. In a further embodiment, the FOSL1 inhibitor is T5224. In particular the FOSL1 inhibitor is the T5224-PROTAC. The term “PROTAC” refers to bifunctional molecules that simultaneously bind a target protein and an E3 -ubiquitin ligase. This causes poly-ubiquitination of the target protein which is thus degraded into small peptides and amino acids by the proteasome complex.

[0050] In a further embodiment, the PIM1 inhibitor is selected from the group consisting of but not limited to: CX-6258, SGI-1776, SMI-4a, AZD-1208, LGH447 (doi: 10.1038 / s41388-023- 02914-0, doi: 10.1016 / j.celrep.2023.112882, doi: 10.1083 / jcb.202208136, doi: 10.1158 / 0008- 5472.), TP-3654, AZD1208, PIM447, TCS PIMl-l, Quercetagetin, LY294002, Hispiludin, SMI-16A, DHPCC-9, SEL24, AUM302, M-110, INCB053914, GNE-955, HS-56, CDK6 / PIM1-IN-1, PIM1-1 / HDAC-IN-1, SEL24-B489, MNK / PIM1-IN-1, GDC-0339, CK2 / PIM1-IN-1, Uzansertib, K00135, R8-T198wt, HTH-01-091, MCX 28, XL413, 10-DEBC, FD1024, LGB321, or Multi-Kinase-IN-6.

[0051] In a particular embodiment, the inhibitor of PIM1 is CX-6258 as described in Haddach et al 2011 (https: / / doi.org / 10.1021 / ml200259q).

[0052] In a further embodiment, the present invention relates to i) JQ1 and ii) CX-6258, as a combined preparation for use in the treatment of prostate cancer in a subject in need thereof.

[0053] In some embodiments, the inhibitor of FOSLlor PIM1 is an antibody.

[0054] As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv) , dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMTP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93 / 1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06 / 030220 and WO 06 / 003388.

[0055] In a particular embodiment, the inhibitor of FOSL1 or PIM1 is scFv fragments.

[0056] In a particular embodiment, the inhibitor is a monoclonal antibody. 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, the human B-cell hybridoma technique and the EBV-hybridoma technique.

[0057] In a particular, the inhibitor is an intrabody having specificity for FOSL1 or PIM1. As used herein, the term "intrabody" generally refers to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking / localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, 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. In some embodiments, the inhibitor of FOSL1 or PIM1 expression is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of FOSL1 or PIM1. In a particular embodiment, the inhibitor of FOSL1 or PIM1 expression is siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometiOmes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. In a particular embodiment, the inhibitor of FOSL1 or PIM1 expression is an anti-sense oligonucleotides (ASO). Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; 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.

[0058] In a particular embodiment, the inhibitor of FOSL1 or PIM1 is bispecific antisense oligonucleotides. In the context of the invention, the bispecific antisense oligonucleotides contain binding sites for both the FOSL1 and PIM1 .

[0059] In some embodiments, the inhibitor of FOSL1 or PIM1 expression is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

[0060] In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

[0061] In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR / Cas9 system has been described in US 8697359 Bl and US 2014 / 0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffmi, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038 / cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi:10.1534 / genetics. H3.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038 / cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129 ). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR / Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

[0062] In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0063] In another aspect, the invention relates to a kit of part comprising an inhibitor FOSL1 and at least one further therapeutic agent as a combined preparation for simultaneous, separate or sequential use in the treatment of prostate cancer.

[0064] In another aspect, the invention relates to a kit of part comprising an inhibitor FOSL1 and an inhibitor PIM1 as a combined preparation for simultaneous, separate or sequential use in the treatment of prostate cancer.

[0065] In a particular embodiment, the invention relates to i) an inhibitor of FOSL1, ii) an inhibitor of PIM1 and iii) a classical treatment, as a combined preparation for simultaneous, separate or sequential use in the treatment of prostate cancer in a subject in need thereof.

[0066] As used herein, the term “simultaneous use” refers to use of at least 2 or 3 active ingredients by the same route and at the same time or at substantially the same time. The term “separate use” refers to use of at least 2 or 3 active ingredients at the same time or at substantially the same time by different routes. The term “sequential use” refers to use of at least 2 or 3 active ingredients at different times, the administration route being identical or different.

[0067] As used herein, the term “classical treatment” refers to treatments well known in the art and used to treat prostate cancer. In the context of the invention, the classical treatment refers to targeted therapy, radiation therapy, immunotherapy, ferroptosis therapy, chemotherapy or conventional androgen deprivation therapy (ADT).

[0068] In a particular embodiment, the classical treatment refers to a combined treatment to treat prostate cancer.

[0069] In a particular embodiment, the invention relates to i) an inhibitor of FOSL1, ii) an inhibitor of PIM1 and iii) a targeted therapy used as a combined preparation for use in the prevention and / or treatment of prostate cancer in a subject in need thereof.

[0070] In a particular embodiment, the invention relates to to i) an inhibitor of FOSL1, ii) an inhibitor of PIM1 and iii) a radiation therapy for use by simultaneous, separate or sequential administration in the prevention and / or treatment of prostate cancer in a subject in need thereof.

[0071] As used herein, the term “radiation therapy” or “radiotherapy” have their general meaning in the art and refers the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow. One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy. The use of machines to focus radiation (such as x-rays) on a cancer site is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, the radiation therapy is external radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction is given but fewer fractions.

[0072] In a particular embodiment, the invention relates to to i) an inhibitor of FOSL1, ii) an inhibitor of PIM1 and iii) a chemotherapeutic agent for use by simultaneous, separate or sequential administration in the prevention and / or treatment of prostate cancer in a subject in need thereof.

[0073] As used herein, the term “chemotherapy” refers to use of chemotherapeutic agents to treat a subject. As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth.

[0074] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].), cabazitaxel and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0075] In a particular embodiment, the chemotherapeutic agent is paclitaxel, cabazitaxel or docetaxel. In a particular embodiment, the invention relates to i) an inhibitor of FOSL1, ii) an inhibitor of PIM1 and iii) an androgen deprivation therapy (ADT) for use by simultaneous, separate or sequential administration in the prevention and / or treatment of prostate cancer in a subject in need thereof.

[0076] As used herein, the term “androgen deprivation therapy” (ADT) also called androgen ablation therapy or androgen suppression therapy refers to an antihormone therapy. ADT refers to physical (surgery) or chemical therapy.

[0077] In a particular embodiment, the ADT includes but is not limited to: surgery (bilateral orchiectomy), LHRH agonists and antagonist such as leuprorelin (leuprolide), goserelin, triptorelin, and degarelix or relugolix; antiandrogen therapy such as cyproterone acetate, flutamide, nilutamide, bicalutamide, darolutamide, apalutamide and enzalutamide and pharmaceutically acceptable salts thereof.

[0078] In a particular embodiment, the invention relates to i) an inhibitor of FOSL1, ii) an inhibitor of PIM1 and iii) an immunotherapy used as a combined preparation for use in the prevention and / or treatment of a cancer in a subject in need thereof.

[0079] As used herein, the term “immunotherapy” refers to the treatment disease by activating or suppressing the immune system. Immunotherapies are designed to elicit or amplify an immune response.

[0080] In a particular embodiment, the immunotherapy is performed with immune checkpoint inhibitors (ICI). It has become an important armamentarium against cancers in recent years, which can specifically activate immune cells by targeting immune checkpoints. Immune checkpoints are a type of immunosuppressive molecules expressed on immune cells, which can regulate the degree of immune activation and avoid autoimmune responses. Over the past few decades, numerous immune checkpoint molecules have been identified, including but not limited to PD-1 / PD-L1, CTLA-4, lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3). Currently, FDA-approved checkpoint inhibitors block CTLA4, PD-1 and PD-L1. PD1 and its ligand PDL1 are two important targets for ICI therapy.

[0081] In a particular embodiment, the invention relates to i) an inhibitor of FOSL1, ii) an inhibitor of PIM1 and iii) an immune checkpoint inhibitor used as a combined preparation for use in the prevention and / or treatment of a cancer in a subject in need thereof.

[0082] As used herein, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins. 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).

[0083] 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 stimulatory checkpoint include CD27 CD28 CD40, CD122, CD137, 0X40, GITR, and ICOS. 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. The Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also called CD276, was originally understood to be a co-stimulatory molecule but is now regarded as co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumour escape. B and T Lymphocyte Attenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA. CTLA-4, Cytotoxic T-Lymphocyte-Associated protein 4 and also called CD152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDO, Indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis. KIR, Killercell Immunoglobulin-like Receptor, is a receptor for MHC Class I molecules on Natural Killer cells. LAG3, Lymphocyte Activation Gene-3, works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells. PD-1, Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014. An advantage of targeting PD- 1 is that it can restore immune function in the tumor microenvironment. TIM-3, short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines. TIM-3 acts as a negative regulator of Thl / Tcl function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti-tumor T-cell response.

[0084] In some embodiments, an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In some embodiments, the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands.

[0085] In a particular embodiment, the immune checkpoint inhibitor is an antibody.

[0086] Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, PD-L1, LAG-3, TIM-3 or VISTA.

[0087] In a particular embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302. Examples of anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK).

[0088] In some embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody such as described in WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897. Examples of anti-PD-Ll antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech / Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS).

[0089] In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.

[0090] In the context of the invention, the immune checkpoint inhibitor inhibits Tim-3 or its ligand.

[0091] In a particular embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, W02011155607, WO2015117002, W02010117057 and WO2013006490.

[0092] In a particular embodiment, the immunotherapy is performed with an anti-PD-Ll.

[0093] In a particular embodiment, the immunotherapy is performed with Avelumab.

[0094] In some embodiments, the immune checkpoint inhibitor is a small organic molecule. The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, 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.

[0095] Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

[0096] In a particular embodiment, small organic molecules interfere with transduction pathway of PD-1 and Tim-3. For example, they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway.

[0097] In a particular embodiment, the small organic molecules interfere with Indoleamine- pyrrole 2, 3 -di oxygenase (IDO) inhibitor. IDO is involved in the tryptophan catabolism (Liu et al 2010, Vacchelli et al 2014, Zhai et al 2015). Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), 0- (3-benzofuranyl)-alanine, 0-(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-diacetate, 9- vinylcarbazole, acemetacin, 5- bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a 0- carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1-methyl-tryptophan, 0-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and 0-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.

[0098] In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4- fluorophenyl)-N' -hydroxy -4-{[2-(sulfamoylamino)-ethyl]amino}-l, 2, 5-oxadiazole-3 carboximidamide : In a particular embodiment, the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-l,2,4-Triazole-3,5-diamine, l-(6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)- 5H-benzocyclohepten-2-yl]- and has the following formula in the art:

[0099] In a particular embodiment, the inhibitor is CA-170 (or AUPM-170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand-1 (PD-L1) and V- domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015).

[0100] In some embodiments, the immune checkpoint inhibitor is an aptamer.

[0101] Typically, the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

[0102] In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration Thus, aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti-PD-1 aptamer. Particularly, the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015.

[0103] In a particular embodiment, the invention relates to i) an inhibitor of FOSL1, ii) an inhibitor of PIMland iii) a ferroptosis inducer, used as a combined preparation for use in the prevention and / or treatment of a cancer in a subject in need thereof.

[0104] As used herein, the term “ferroptosis inducer” denotes a compound able to increase ferroptosis occurrence. Ferroptosis inducers are well-known in the art. As example, the ferroptosis inducer may be salinomycin (AM5, Mai, Hamai et al Nat Chem 2017 10.1038 / nchem.2778), APR-246, Ras Synthetic Lethal 3 (RSL3), ML162, ML210, acrolein, erastin, Imidazole Ketone Erastin (IKE), Piperazine Erastin (PE), sulfasalazine, sorafenib, Ferroptosis Inducer 56 (FIN56), Ferroptosis inducer endoperoxide (FIN02), Caspase- Independent Lethal 56 (CIL56), mevalonate-derived coenzyme Q10, buthionine sulfoximine (BSO), amentoflavone, dihydroartemisinin (DHA), typhaneoside, artesunate, Withaferin A (WA), auranofin.

[0105] In a particular embodiment, the ferroptosis inducer is erastin.

[0106] In a particular embodiment, the ferroptosis inducer is salinomycin (AM5).

[0107] In a particular embodiment, the invention relates to i) an inhibitor of FOSL1, ii) an inhibitor of PIMland iii) an inhibitor of senescence, used as a combined preparation for use in the prevention and / or treatment of a cancer in a subject in need thereof.

[0108] As used herein, the term "senescence" has its general mean in the art and refers to the permanent cessation of DNA replication and cell growth that is not reversible by growth factors. Senescence can be characterized by certain morphological features including, but not limited to, increased size, flattened morphology, increased granularity, and senescence-associated b- galactosidase activity (SA- b-gal).

[0109] As used herein, the term “inhibitor of senescence” refers to a compound that selectively (preferentially or to a greater extent) destroys, kills, removes, or promotes selective destruction of senescent cells i.e., the compound destroys senescent cells in a biologically, clinically, and / or statistically significant manner as compared to its ability to destroy or kill non-senescent cells, or Annihilate. The senescent cell-depleting compound is sufficient to selectively kill established senescent cells, but is in an insufficient amount to kill non-senescent cells in a clinically or biologically significant manner.

[0110] In a particular embodiment, the inhibitor of senescence is selected from the group consisting of but not limited to: dasatinib, quercetin, fisetin or navitoclax.

[0111] Typically the FOSL1, PIM1 inhibitors and / or in combination with a classical treatment (targeted therapy, radiation therapy, immunotherapy, chemotherapy or ferroptosis therapy) according to the invention as described above are administered to the subject in a therapeutically effective amount.

[0112] By a "therapeutically effective amount" of FOSL1, PIM1 inhibitors and / or in combination with a classical treatment (targeted therapy, radiation therapy, immunotherapy or chemotherapy) of the present invention as above described is meant a sufficient amount of the FOSL1 and PIMlinhibitor for treating prostate cancer at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the FOSL1 and PIM1 inhibitors 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 the FOSL1 and PIM1 protein or fragment thereof and / or of the agent for FOSL1 and PIM1 inhibitors 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 FOSL1 and PIM1 inhibitors employed; the duration of the treatment; drugs used in combination or coincidental with the FOSL1 and PIMlinhibitors employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the FOSL1 and PIMlinhibitors 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 FOSL1 and PIM1 inhibitors of the present invention 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 FOSL1 and PIM1 inhibitors of the present invention, preferably from 1 mg to about 100 mg of the FOSL1 and PIM1 inhibitors of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day. In a particular embodiment, the FOSL1 and PIM1 inhibitors according to the invention may be used in a concentration between 0.01 pM and 20 pM, particularly, the FOSL1 and PIM1 inhibitors of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2 5, 5.0, 10.0, 15.0, 20.0 pM. According to the invention, the FOSL1 and PIMlinhibitors of the present invention is administered to the subject in the form of a pharmaceutical composition. Thus, the invention also relates to a therapeutic composition comprising the FOSL1 and PIM1 inhibitors for use in the treatment of prostate cancer in a subject in need thereof.

[0113] Pharmaceutical composition

[0114] The inhibitors of FOSL1 and PIM1 as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

[0115] Accordingly, in a third aspect, the invention relates to a pharmaceutical composition comprising inhibitors of FOSL1 and PIM1 and pharmaceutically acceptable excipients.

[0116] In some embodiments, the invention relates to pharmaceutical composition comprising an inhibitor of FOSL1 and PIM1 and a classical treatment (targeted therapy, radiation therapy, immunotherapy or chemotherapy) and a pharmaceutically acceptable excipient. In some embodiments, the invention relates to pharmaceutical composition comprising inhibitors of FOSL1 and PIM1 and a classical treatment (targeted therapy, radiation therapy, immunotherapy or chemotherapy) and a pharmaceutically acceptable excipient.

[0117] In some embodiments, the invention relates to pharmaceutical composition comprising inhibitors of FOSL1 and PIM1 and a classical treatment and a pharmaceutically acceptable excipient.

[0118] 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 inj ectable 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 isopropyl amine, 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.

[0119] Method of screening

[0120] A further object of the present invention relates to a method of screening a drug suitable for the treatment of a cancer comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the activity and / or expression of FOSL1 and PIM1.

[0121] Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit the activity of FOSL1 and PIM1. In some embodiments, the assay first comprises determining the ability of the test compound to bind to FOSL1 and PIM1. In some embodiments, a population of cells is then contacted and activated so as to determine the ability of the test compound to inhibit the activity of FOSL1 and PIM1. In particular, the effect triggered by the test compound is determined relative to that of a population of cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. It is to be understood that test compounds capable of inhibiting the activity of FOSL1 and PIM1, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, peptidomimetics, small organic molecules, aptamers or nucleic acids. For example, the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules.

[0122] 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:

[0123] Figure 1: Identification of Fosll / AP-1 and Pim family as validated targets of the PTENpc- / - LSCmed cells. A. Organoid-forming capacity as the number of organoids (left) and size of organoids (right) generated by sorted PTENpc" LSCmed after 10 days of culture in the presence of DMSO (vehicle) or InM JQ1 / InM CX-6258 / InM JQ1 + InM CX-6258. Data are normalized to the DMSO condition (n=4). B. Schematic representation (created with BioRender.com) of in vivo treatment of PTENpc' / " mice (left). Castrated mice received vehicle (n=8) or a combination of JQ1 (daily intraperitoneal injection) and CX-6258 (bi-weekly oral administration) (n=8) during 28 days. After 28 days of treatment, prostates were microdissected and weighed (right). **: p<0.01; ***=p<0.001; ****: p<0.0001

[0124] Figure 2 : JQ1 and CX-6258 strongly affect tumoral MSPC-like cell viability. A. RT-qPCR in HPV10 cellular fraction for Fosll and Piml. Data are normalized to the LNCaP expression (dashed line). Tumorsphere-forming capacity of HPV10 cells after 10 days of culture in DMSO (control) or IpM JQ1 / IpM CX-6258 / IpM JQ1 + IpM CX-6258 conditions. Data are normalized to the DMSO condition (n=3) (nd. = not detected). Manually counted cell number (B - left) and FACS-estimated cell death (B - right) after 72h of DMSO or IpM JQ1 / IpM CX-6258 / IpM JQ1+ IpM CX-6258 treatment. For manual counting, data are normalized to the DMSO condition (n = 3).

[0125] Figure 3: In vivo therapeutic efficacy of targeting FOSL1 and PIM1. 1.106PC3 cells were injected subcutaneously in castrated immunodeficient NSG mice. Mice received vehicle (n=12) or a combination of JQ1 (daily intraperitoneal injection) and CX-6258 (biweekly oral administration) (n=7) during 28 days. After 28 days of treatment, prostates were microdissected and weighed (right). ***=p<0.001

[0126] Figure 4 : the FosLl degrader T5224-PROTAC prevents the growth of MSPC-like PC3 cells A. Manually counted cell number after 48h treatment with DMSO (control) or 5 and 15 pM T5224-PROTAC. B. Same as in A showing treatment with IpM CX-6258 / 5 pM T5224-PROTAC / IpM CX-6258 + 5 pM T5224-PROTAC. Data are normalized to the DMSO condition. **: p<0.01; ****: p<0.0001 Figure 5 : FosLl and PIM1 expression is upregulated by enzalutamide in human prostate cancer. Three-month neoadjuvant treatment of naive high-risk prostate cancer patients with enzalutamide (D ARANA study) leads to upregulation of FosLl (A), FosLl regulon (B) and PIM1 (C) expression. *p<0.05; ****: p<0.0001.

[0127] EXAMPLE:

[0128] Material & Methods :

[0129] Animals mice ('PFENlo'<1> lo':l>mice crossed with Pb-Cre4 transgenic males) were generated as described previously and maintained on a C57BL / 6 and Sv / 129 mixed genetic background. Experiments were performed using 8- to 11-month-old mice, i.e. when aggressive malignant phenotypes were well established. Colonies were housed in controlled conditions, on a 12 / 12-h light / dark cycle with normal food and water provided ad libitum. Where indicated, mice were surgically castrated and analyzed between 5 days and 2 months after castration. Prostate samples were obtained by microdissection immediately after sacrifice by cervical dislocation. Under a dissection microscope, adipose tissues were removed from the urogenital tract. The bladder, the ampullary gland and the urethra were removed in order to isolate the prostate lobes.

[0130] Animal experiments were approved by the local ethics committee for animal experimentation (authorizations n°2022100315521514 and n°2022071116158470).

[0131] For in vivo treatments, PTEN mice were aged to 7 months and and given oral doses of CX-6258 biweekly and daily intraperitoneal doses of JQ1 over 4 weeks. Concentrations were lOOmg / kg and 50mg / kg, respectively.

[0132] Prostate dissociation

[0133] Prostates were minced using razor blades and digested in a solution of Dulbecco’s modified eagle medium (DMEM) (Gibco) containing 10% FBS (Eurobio), 1% Pen / Strep (Gibco) and 1 mg / mL Collagenase Type 1 solution (Gibco) for Ih at 37°C, followed by an incubation for 5 minutes at 37°C in 0.25% Trypsin (Gibco). The digestion is stopped by a solution of DMEM containing DNase I (Roche). Cells were passed 10 times through a 20G syringue and then through a 40 pm cell strainer to generate a single-cell suspension. Cells were subjected to differential centrifugation by using Histopaque-1119 (Sigma-Aldrich) to reduce the global level of secretion in the sample. An aliquot of cells was stained with Trypan blue (Gibco) and counted using a hemocytometer in order to assess the cell viability. Prostate subpopulation sorting by FACS

[0134] The procedure for cell sorting was performed as previously described. Isolated cells were stained for FACS on ice for 30 minutes. Antibodies (eBioscience) used for FACS were fluorescein isothiocyanate-coupled lineage (Lin) antibodies (anti-CD31, CD45 and TER-119), phosphatidyl ethanolamine-Cyanine7-coupled anti-EpCAM, phosphatidylethanolamine- coupled anti-CD49f (integrin alpha-6) and allophycocyanin-coupled anti-Scal (lymphocyte antigen 6A-2 / 6E-1). Dead cells were stained using the SYTOX Blue (Life Technologies). Cell sorting was performed on a BD FACS Aria III (BD Biosciences) in DMEM containing 2% FBS and 1% Pen / Strep. Lin antibodies were used to deplete hematopoietic, endothelial and immune cells and EpCAM antibody was used to separate epithelial versus stromal cells. CD49f and SCA-1 markers were used to select LSCmed cells among the other epithelial cell types. Sorted cells were collected in DMEM medium supplemented with 50% FBS and 1% Pen / Strep.

[0135] Single-cell RNA-Sequencing and Analyses

[0136] FACS sorted LSCmed cancer cells were sorted (BD FACS Aria III) in 96 well plates (VWR, DNase, RNase free) containing 2 pL of lysis buffer (0.2% Triton X-100, 4U of RNase inhibitor, Takara) per well. Plates were properly sealed and spun down at 2000 g for 1 min before storing at -80°C. Whole transcriptome amplification was performed with a modified SMART-seq2 protocol as described previously (Picelli et al. 2014) using 23 instead of 18 cycles of cDNA amplification. PCR purification was realized with a 0.8:1 ratio (ampureXP beads:DNA). Amplified cDNA quality was monitored with a high sensitivity DNA chip (Agilent) using the Bioanalyzer (Agilent). Sequencing libraries were performed using the Nextera XT kit (Illumina) as described previously (Picelli et al., 2014) using l / 4th of the recommended reagent volumes and l / 5th of input DNA with a tagmentation time of 9 min. Library quality was monitored with a high-sensitivity DNA chip (Agilent) using the Bioanalyzer (Agilent). Indexing was performed with the Nextera XT index Kit V2 (A-D). Up to 4 x 96 single cells were pooled per sequencing lane. Samples were sequenced on the Illumina NextSeq 500 platform using 75bp single-end reads.

[0137] Single-cell transcriptome analysis

[0138] The merged gene expression matrix (raw counts) containing all samples was analyzed with the Seurat3 package in R. Cells were fdtered by nFeature_RNA (genes detected) >3000 and <7000, percent.mt (percentage of mitochondria genes) < 10. This resulted in n=306 valid cells for downstream analysis. After filtering cells, log-normalization was performed using the default NormalizeData function in Seurat. The scaled data was regressed for cell cycle phases (S. Score and G2M. Score) and number of genes per cell (nFeature_RNA) before performing UMAP based dimension reduction (dims=l :20, resolution =0.4).

[0139] StemChecker Analysis

[0140] Seurat cluster (0,1,2) characteristic murine genes (adj.pval>0.05) were analyzed for “sternness” enrichment using the stemchecker tool (http: / / stemchecker.sysbiolab.eu / ), while masking cell cycle genes.

[0141] Pseudotime-ordering analysis

[0142] The raw count matrix (n=306 cells) was used as input for Monocle analysis (2.14.0) and normalized by the M3Drop function. Ordering of the cells was performed by using highly variable genes (n=1483). After Dimension reduction (max_components = 2, method = 'DDRTree') the resulting trajectory was colored by pseudotime, cell type and Seurat cluster information.

[0143] Differentiation Trajectory Analysis Using Velocyto and ScVelo

[0144] To infer the directionality of differentiation, we employed the Velocyto pipeline (La Manno et al., 2018). Utilizing the *.bam files and cell metadata, we computed the spliced and unspliced RNA matrices. Differentiation traj ectories were visualized using UMAP embeddings, which were generated with Seurat in R. The trajectories, represented by arrows, were plotted using ScVelo (Bergen et al., 2020) in Python.

[0145] Cell lines and treatment

[0146] HPV-10 (ATCC, CRL-2220) and LNCaP cell lines were grown at 37°C and 5% CO2, in Keratinocyte Serum Free Medium (K-SFM) (Gibco) supplemented with 0.05mg / mL Bovine Pituitary Extract (BPE) (Gibco), 5ng / mL EGF (PeproTech) and 1% Pen / Strep or DMEM supplemented with 10% FBS and 1% Pen / Strep, respectively. Medium was changed every 2 days.

[0147] Cells were plated at the appropriate density according to the performed test. After 3 days, cells were treated and analyzed according to the protocols described below.

[0148] For cell death analysis, supernatant and attached cells were collected after 3 days, resuspend in HBSS and dead cells were stained with SYTOX Blue. Proportion of dead cells was measured by flow cytometry using a Fortessa cytometer (BD Biosciences). Data were then analyzed with Flowjo software.

[0149] Organoid culture and treatment

[0150] We used the reference protocol for prostate organoid culture described by Clevers’ lab (Drost et al., 2016). LSCmed cells sorted from PTENr’c" mouse and human tumoral HPV-10 cells were plated in triplicate on a Low Growth Factor-containing Matrigel (Corning) layer in a 96-well plate (Starsted) at a concentration of 3000 or 1000 cells / well respectively, and were cultured at 37°C and 5% CO2 during 10 days. After one day of incubation, the medium was removed and cells were covered by a new layer of Matrigel in order to perform 3D culture. The organoid-forming capacity did not differ from the efficacy obtained using 3D droplet culture. Drugs were added at this stage. Medium was changed every 2 days. After 10 days of culture, organoids were fixed in 4% PFA and photos were taken with a 4x objective under a M5000 EVOS inverted microscope (Invitrogen) in order to cover the entire surface of the well. Counting and surfacing were performed on Fiji software by manually surrounding the organoid surface. The number of organoid in the various experimental condition was normalized to the mean value obtained in the non-treated condition. The size of organoid was normalized to the median value obtained in the non-treated condition.

[0151] Immunohistochemistry (IHC) and Immunofluorescence (IF)

[0152] For the IHC, all murine prostate samples were fixed in 4% PFA, paraffin wax- embedded, and sections underwent heat-induced antigen retrieval in citrate buffer at pH 6 (95°C, 30 min). IHC was performed as described previously (Baures et al., 2022B) using antibodies directed against KI-67. Signal was amplified and detected thanks to the Vector Elite ABC HRP kit with DAB substrate (Vector Laboratories, Burlingame, CA, USA) and nuclei were counterstained with haematoxylin. Slides were scanned with a Nanozoomer 2.0 (Hamamatsu, Massy, France) and analyzed using NDP.view 2.5.14 software (Hamamatsu). Positive nuclei were quantified on 10 field / slide using QuPath Software.

[0153] For the IF, cells were fixed in PFA 4% for 15 minutes at room temperature and permeabilized during 15 minutes in PBS supplemented with 0.2% Tween-20. IF was performed using antibodies directed against CK4, CK13 and PTEN. Primary antibodies were incubated overnight at 4°C and secondary antibodies were incubated during Ih at room temperature. Nuclei were stained with Hoechst dye. Samples were analyzed with a 40x objective under an Apotome 2 microscope (Zeiss). Antibodies reference are summarized in the following table :

[0154] Terminal deoxynucleotidyl transferase dUTP nick end (TUNEL) labeling

[0155] TUNEL assay was performed with the In Situ Cell Death Detection Kit (Roche, Cat. No. 11 684 809 910) according to the manufacturer’s instructions. Briefly, samples underwent dewaxation, rehydratation. Slides were heated in Citrate Buffer during 5 minutes at 350W and then incubated for Ih at 37°C, with the TUNEL reaction mixture. Fluorescence was observed and pictures were taken with a 20x objective under a M5000 EVOS inverted microscope.

[0156] Reverse Transcription-quantitative PCR (RT-qPCR)

[0157] RNA extractions from PTLNpc" LSCmed cells and human tumoral HPV-10 were performed with the Nucleospin RNA XS and Nucleospin RNA (Macherey-Nagel, Duren, Germany), respectively, as described in the manufacturer’s protocols. The reverse transcription was performed using the SuperScript™ VILO™ cDNA Synthesis Kit (Invitrogen) and the GoScript™ Reverse Transcriptase (Promega), respectively, according to the manufacturer’s instructions.

[0158] For qPCR, iTaq Universal SYBR Green Supermix (Promega) was used, and reactions were run on a qTower 2.0 real-time thermal cycler (Analytik Jena).

[0159] Expression data were normalized to Cyclophilin A for murine samples and Actin B for human samples.

[0160] Results

[0161] LSCmedcells enriched from PTEN-null prostate tumors are a model of MS PC human CRPC

[0162] As previously reported (Sackmann-Sala et al., 2017), PTENPC'ALSCmedcells are castration-tolerant. To study whether castration affects LSCmed cell fate, we performed Smart- seq2 single-cell RNA-sequencing (scRNAseq) on 384 LSCmed cells sorted from intact and 2- month-castrated mice (data not shown). By using Louvain-based clustering prior to Uniform Manifold Approximation and Projection (UMAP), we first annotated the resulting single-cell space according to the corresponding castration profiles (data not shown). The identity of the cells analyzed was assessed by the expression of the LSCmed marker Krt4 (Sackmann-Sala et al., 2017) in addition to the surface markers used in cell sorting (data not shown). Despite of their intrinsically low AR signaling in intact mice (Sackmann-Sala et al., 2017), a decrease of AR signaling was observed in LSCmed cells after castration (data not shown), indicating they are sensitive to castration. Differential gene expression (DEG) analysis identified 91 genes that exhibited >1.5-fold (adj. pval<0.05) difference after castration and the 20 most discriminative ones were reported here (data not shown). These included, after castration, an overexpression of Krt5, a basal / Hillock-specific marker, and a decrease of PSCA which has already be described to be downregulated by the androgen signaling inhibition (Mevel et al., 2020).

[0163] We then compared PTENr" LSCmedcells to recently reported signatures of human prostate cancer subtypes (Han et al., 2022, Tang et al., 2022). This analysis revealed that PTEM1’"' ' LSCmed cells are markedly enriched in Mesenchymal Stem-like Prostate Cancer (MSPC) (data not shown) and Stem Cell-Like CRPC (CRPC-SCL) (data not shown) signatures, as compared to the other prostate cancer subtypes including AR-sensitive (ARPC) and neuroendocrine (NEPC) prostate cancer. MSPC (Han et al., 2022) and CRPC-SCL (Tang et al., 2022) are double-negative prostate cancer subtypes that exhibit reduced AR signature and absence of neuroendrocrine features. They correspond to ADT-induced and metastasis- related PCa subtypes and are particularly enriched in basal and sternness features.

[0164] Together, these results highlight that the PTEN^' ' LSCmedcell population exhibits high molecular similarity with human ADT-resistant prostate cancer subtype enriched in sternness properties.

[0165] PTEN-null LSCmed cells exhibit heterogeneity

[0166] To investigate the heterogeneity of the PTENpc" LSCmed cell population, we performed unsupervised Louvain-clustering of our cells and identified 3 subpopulations (data not shown). The Cluster 0, highly predominant before the castration, was drastically reduced after castration (data not shown). This cluster expresses PSCA, a well-described androgen-dependent marker of in both murine and human luminal progenitors of the prostate (Crowell et al., 2019) (data not shown), and is enriched in development and KRAS oncogenic pathways (data not shown), as previously described in the PTENpc" mouse model (Pritchard and Nelson, 2008, Steiner et al., 2023). At the opposite, Cluster 1 was massively amplified (16-fold) whereas Cluster 2 exhibited a slight increase (3-fold) but remained a minority after castration (data not shown). The most differentially expressed gene in Cluster 2 is Onecut2 (data not shown), previously shown to contribute to the induction of neuroendocrine differentiation in prostate cancer (Rotinen et al., 2018, Guo et al., 2019, Chan et al., 2022). In the LSCmed cluster 2, Onecut2 expression was not associated to ChromograninA, Synaptophysin or Neuron-Specific Enolase expression, classically described as neuroendrocrine markers (data not shown). These results suggest that cluster 2 could have the potential to differentiate into neuroendrocrine cells which could be associated to tumor aggressiveness, as confirmed by its association to various oncogenic signatures (data not shown).

[0167] Finally, the Cluster 1, the most amplified cluster after castration, expresses Ly6d (data not shown), a marker of castration-resistant luminal progenitors that is expressed in primary prostate cancer, amplified in mCRPC and associated to biochemical relapse and decrease of overall survival (Barros-Silva et al., 2018, Steiner et al., 2023). Interestingly, we also noted the overexpression of Krt5, the basal / Hillock-specific marker, concordant with the activation of a basal cancer signature (data not shown), also observed in human MSPC (Han et al., 2022). This upregulation of Krt5 is associated to the increase of the global basal signature with a slight decrease in luminal score in response to castration, suggesting the acquisition of an intermediate phenotype (data not shown). LSCmed cells were shown to largely overlap with Club (luminallike) and Hillock (basal-like) cells of the human prostate (Baures et al., 2022A, B). According to the enrichment in basal features, Cluster 1 was associated to enrichment in Hillock profile (data not shown). Cluster 1 was also associated with oncogenic pathways, and in particular, with migration and stem signatures (data not shown), two typical features of human MSPC / SCL PCa subtypes (Han et al., 2022, Tang et al., 2022). The latter was confirmed in silico by a significant enrichment of sternness features highlighted with the StemChecker software (data not shown). Of note, these enrichments are specific to predefined signature and populations are not compared to each other in this study, confirming that the Cluster 1 is the only stem-enriched cluster. We aimed to address this finding experimentally. While there is currently no sorting strategy to enrich Cluster 0 versus Cluster 1, we determined the organoidforming capacity of bulk LSCmed cells sorted from prostates of intact mice (enriched in Cluster 0) versus 2-month-castrated mice (enriched in Cluster 1). Sorted PTEN110' ' LSCmed cells exhibit a slight increase in the organoid-forming capacity, paralleling the enrichment of sternness signature in Cluster 1 (data not shown).

[0168] According to the observation reported above for the global LSCmed cell population (data not shown), AR signaling was lower in Cluster 1 and 2 than in Cluster 0 (data not shown), confirming LSCmed cell sensitivity to castration. Transcriptomic cell plasticity as an adaptive mechanism in response to castration

[0169] The transcriptomic analysis identified three clusters which evolve after castration. To adapt our therapeutic strategy, it was necessary to understand by which mechanism Clusterl, and to a lesser extent Cluster 2, are amplified post-castration. We supposed that this amplification could be due to (1) positive selection, i.e. robust proliferation of Cluster 1 cells and / or the death of Cluster 0 cells, or (2) a transcriptomic switch from Cluster 0 to Cluster 1 / 2 profiles (data not shown). Proliferative Ki-67 positive cells were quantified in various fields of PTENpc" prostates from intact mice or mice sacrificed at 5, 21 or 60 days after castration. Despite of a slight increase of the positive proportion at 5 days, there was no significant proliferation induced by castration (data not shown). Similarly, TUNEL assay showed that, although dead cell staining was more focal than homogeneous in the prostate, the global level remained particularly low (-2.5%), with no significant variation after castration (data not shown). Otherwise, we showed by RT-qPCR that various DEG associated to Cluster 0, 1 and 2 quickly evolved after castration, as illustrated by the upregulation of Cluster 0 markers associated to the downregulation of Cluster 1 / Cluster 2 markers, already detected at 5 days postcastration, suggesting a very rapid onset of transcriptomic switch (data not shown).

[0170] The rapidity of these transcriptomic changes and the absence of concomitant cell proliferation and cell death argued that the Cluster 0 to Cluster 1 / 2 population switch was due to a mechanism of cell plasticity. RNA velocity analysis (scVelo), based on the inference of directed dynamic information by leveraging splicing kinetics, identified cluster 0 as root population (data not shown). This was paralleled by pseudotime-ordering analysis (Monocle). Transcriptional heterogeneity was identified by “intermediate” cells distributed along the lower trajectory before castration (data not shown). By and large, the Cluster 2 is composed of differentiated cells.

[0171] Altogether, these results support the hypothesis of cellular plasticity favoring an adaptive resistance mechanism starting shortly after castration.

[0172] In silico / in vitro identification of Fosll / AP-1 complex and Pirn family as potential therapeutic targets

[0173] LSCmed cells are enriched in progenitor features (Sackmann-Sala et al., 2017, Crowley et al., 2020, Guo et al., 2020, Mevel et al., 2020, Joseph et al., 2020, Graham et al., 2022, Yan et al., 2022, Baures et al., 2022A). In addition, we have shown that PTEN^7’ LSCmed cells act as tumor-initiating cells in grafts experiments (Sackmann-Sala et al, 2017). In light of these tumor-associated properties and of their similarity with human MSPC / SCL cancer subtypes, we used PTENpc-' '' LSCmed cells as a model to identify potential therapeutic targets. Due to its massive amplification and sternness feature enrichment after castration, we chose to preferentially target Cluster 1. We interrogated Cluster 1 signature to identify potential targets based on the following criteria: (1) master genes / transcription factors regulating the global signature of the Cluster 1; (2) proteins described in the literature as involved in functional abilities of tumoral cells. Finally, we chose protein also expressed in other clusters with the aim to preferentially target the most aggressive cluster (Cluster 1) but also to potentially counteract alternative plasticity mechanism of the other populations.

[0174] A Scenic analysis identified Fosll as a master gene of the Cluster 1 (data not shown). It is amplified after castration, in Cluster 1 and 2, but is already highly expressed before castration in Cluster 0 (data not shown), as is also the Fosll -regulated network (data not shown). This gene has been described as involved in various processes such as sternness or EMT (Eferl and Wagner, 2003). Interestingly, we noted that several other members of the AP- 1 family of transcription factors are also highly expressed in all subpopulations (data not shown). The second target identified in this work, Piml, is drastically enriched after castration (data not shown). This kinase has been described as a functional regulator of cell cycle, apoptosis and prostate cancer cell aggressiveness (Wang et al., 2001, Santio et al., 2015, Rebello et al., 2016). Interestingly, we noted that Pim3, but not Pim2, is also highly expressed in all PTENP';" LSCmed cell subpopulations, both before and after the castration (data not shown). RT-qPCR on sorted LSCmed cells confirmed increased expression of Fosll and Piml by 4.5- and 3.5- fold, respectively, at 21 days post-castration (data not shown).

[0175] We used the organoid assay to determine the effects of pharmacological inhibition of the two selected targets in LSCmed cells. FOSL1 has been described to mainly act in the AP-1 complex as a heterodimer (Eferl and Wagner, 2003). Therefore, we chose to target it using JQ- 1, a global BET / AP-1 inhibitor. Regarding PIM1, to avoid that specific Pirn isoform inhibition be hampered by functional redundancy of other PIM members as described in other types of diseases (van der Lugt et al., 1995, Mikkers et al., 2002), we used the Pan-PIM kinase inhibitor CX-6258 (Haddach et al., 2011). We first determined the dose-response of each drug on the organoid-forming capacity of LSCmed cells sorted from intact mice. Both drugs were active in the nanomolar range, indicating that inhibition reflected high sensitivity of LSCmed cells to JQ-1 and CX-6258 rather than a non-specific cytotoxic effect (data not shown). By treating our cells with the chosen concentration (InM for each), we observed a decrease by half both the number and the size of the organoids. In addition, our results highlight a higher efficiency when drugs are combined (Fig. 1A), with approximately 70% and 60% decrease of number and size of organoids, respectively, compared to the DMSO condition.

[0176] Since all of our in vitro results suggest a high efficacy of the drug combination, we proceeded with in vivo validation of this strategy in the PTEN-null mouse model. Patients with aggressive prostate cancer are typically treated with chemical castration but often experience recurrence after a few years. We reasoned that therapy -induced reprogramming to Cluster 1 provides a window of opportunity for combination therapy involving castration alongside both Fosll / APl and Pirn inhibition. Mice were castrated and then treated for 4 weeks with daily doses of JQ-1 and bi-weekly administrations of CX-6258 (Fig. IB left). Following treatment, we observed a decrease in global prostate weight (Fig. IB right) compared to the vehicle castrated group. This decrease was associated with a reduction in the proportion of advanced histologically-classified structure and a decrease in proliferation with no observed changes in terms of cell death (data not shown). After dissection, cells were sorted from vehicle or treated mouse and cultured in organoid medium for 10 days in the absence of pharmacological inhibitor. Surprisingly, we observed a drastic inhibition of the overall progenitor ability of the PTEN-null LSCmed cells when sorted from treated mice (data not shown). Histopathological analysis of kidney, liver and lung harvested from castrated mice treated with vehicle or a combination of JQ1 and CX-6258 (Fig. IB) was performed by pathologists blind of treatments. This analysis did not reveal major difference between the treated group and the vehicle group. So, treatment of PTEN-null mice with JQ1 and CX6258 does not induce toxicity.

[0177] This result suggests that the treatment either inhibited the intrinsic progenitor capacity of LSCmed cells or specifically targeted progenitor cells.

[0178] In vitro therapeutic targeting of a Club / Hillock-like cell line.

[0179] The next objective was to target the human counterparts of LSCmed cells. In 2018, Strand and colleagues published an atlas of the healthy human prostate and described two new epithelial cell populations (Henry et al., 2018). Called Club and Hillock cells, due to their transcriptomic proximity with eponymous pulmonary cells, these prostatic cell populations are enriched in LSCmed signature (Henry et al., 2018, Baures et al., 2022). To date, there is no established procedures to sort, culture and expand these cells from fresh human tissue samples, therefore we sought for a relevant cell model matching this phenotype. By comparing cell line signatures (GSE9633) to murine LSCmed, Club and Hillock signatures, we found that HPV10 cells are enriched in these 3 signatures. This cell line is not commonly used in the field. In comparison, PC3 / DU145 exhibited an intermediate enrichment and LNCaP / 22Rvl, classically described as luminal tumor cells, were the least enriched in luminal progenitor features among the cell panel studied (data not shown). RT-qPCR experiments confirmed expression of various LSCmed markers (e.g. KRT4, KRT7) as compared to the luminal LNCaP cell line (data not shown), as observed in silico (data not shown). By immunofluorescence, the co-expression of the cytokeratin 4 and 13 (markers of Club and Hillock, respectively) suggests the identification of an intermediate cell line (data not shown), such as murine LSCmed cells enriched in both Club and Hillock signatures. Interestingly, these cells are cultured in absence of androgens, which is reflected by the absence ofPSCA (data not shown), suggesting their intrinsic resistance to castration. In terms of cancer subtype, PC3 and DU145 were previously identified as MSPC models (Han et al., 2022, Tang et al., 2022). Here, we also showed that HPV10 cell line signature largely overlaps with the CRPC-SCL signature (Data not shown). To definitely validate this cellular model, we showed that HPV10 cells are able to generate tumor spheres in Matrigel (data not shown) and that they express FOSL1 and PIM1 (Fig. 2A). Altogether, these data validate the HPV10 cell line as the best cell model to validate the drugs of interest on human Club / Hillock-like cells.

[0180] To assess the relevance of the selected therapeutic targets, we determined the doseresponse of each drug on 2D culture, defined as the decrease by half of the cell number at 72h of treatment (data not shown) and performed culture assays to measure tumorsphere ability, cell proliferation and cell death after treatment of our cells (Fig. 2A-B). We showed that drugs drastically inhibit the tumor sphere-forming capacity of HPV10 cells (Fig. 2A - right) and decrease the number of cells after 72h of treatment (Fig. 2B - left). We showed that the decrease in cell number is mainly related to an induction of cell death (Fig. 2B - right) as almost 90% of HPV10 cells are dying after 72h with the combination of treatment. As observed on murine progenitor cells, these experiments highlight highest efficacy of drug combination (Fig. 2A-B).

[0181] To assess the in vivo efficacy of this therapeutic strategy, we employed the PC3 cell line, given that HPV10 cells have been characterized as non-tumorigenic (Weijerman et al., 1994). Drug efficacy was first confirmed in vitro, by showing a strong reduction of cell viability (data not shown). Then, PC3 cells were subsequently injected subcutaneously into the flanks of castrated NSG mice. After 24 days of treatment, tumors were excised and we observed that tumoral structures were smaller in the treated group compared to the vehicle group (Fig. 3), indicating that the drug combination inhibits human MSPC-like tumor growth. Based on cell death and proliferation assays using KI67 and TUNEL staining, respectively (data not shown), it appears that JQ1 and CX-6258 combination acts through inducing cell death in this xenograft model. We also showed that the FosLl degrader T5224-PROTAC prevents the growth of MSPC-like PC3 cells (Fig. 4A-B). Finally, we demonstrated that FosLl and PIM1 expression is upregulated by enzalutamide (DARANA study: Linder S. et al. Drug-Induced Epigenomic Plasticity Reprograms Circadian Rhythm Regulation to Drive Prostate Cancer toward Androgen Independence. Cancer Discov 2022; 12:2074-2097) in human prostate cancer (Fig. 5A-C).

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Claims

CLAIMS1. i) An inhibitor of FOSL1 and ii) an inhibitor of PIM1, as a combined preparation for use in the treatment of prostate cancer in a subject in need thereof.

2. i) An inhibitor of FOSL1, ii) an inhibitor of PIM1 and iii) a classical treatment, as a combined preparation for use in the treatment of prostate cancer in a subject in need thereof.

3. The combined preparation for use according to claims 1 to 2 wherein the prostate cancer is: resistant cancer, metastatic cancer, Castration-Resistant Prostate Cancer (CRPC), Neuroendocrine Prostate Cancer (NEPC) or Double-Negative Prostate Cancer (DNPC).

4. The combined preparation for use according to claims 1 to 2, wherein the inhibitor of FOSL1 and PIM1 is: small organic molecule, peptide, peptidomimetic, antibody, aptamers, siRNA or antisense oligonucleotide.

5. The combined preparation for use according to claims 1 to 4, wherein the inhibitor of FOSL1 is JQ1.

6. The combined preparation for use according to claims 1 to 4, wherein the inhibitor of FOSL1 is T5224.

7. The combined preparation for use according to claim 6, wherein the T5224 is a T5224- PROTAC.

8. The combined preparation for use according to claims 1 to 4, wherein the inhibitor of PIM1 is CX-6258.

9. The combined preparation for use according to claims 1 to 8, wherein the classical treatment is selected from the group consisting of but not limited to: targeted therapy, radiation therapy, immunotherapy, ferroptosis therapy, chemotherapy or conventional androgen deprivation therapy (ADT).

10. A pharmaceutical composition comprising an inhibitor of FOSL1, an inhibitor of PIM1 inhibitor and pharmaceutically acceptable excipients, as a combined preparation.

11. The pharmaceutical composition according to claim 10 for use in the treatment of prostate cancer in a subject in need thereof.

12. A kit of part comprising an inhibitor FOSL1 and an inhibitor PIMlas a combined preparation for simultaneous, separate or sequential use in the treatment of prostate cancer.

13. A method for treating prostate cancer in a subject in need thereof comprising a step of administrating the subject with a therapeutically effective amount of an inhibitor of FOSL1 and an inhibitor of PIM1.

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