EP4658269A1

EP4658269A1 - Compound for use in the prevention and/or treatment of cancer

Info

Publication number: EP4658269A1
Application number: EP24702536.4A
Authority: EP
Inventors: Agnès COSTE; Godefroy JACQUEMIN; Clément BLOT; Laurent ALRIC; Kimberley COULSON
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Etablissement Francais du Sang; Centre Hospitalier Universitaire de Toulouse; Universite Toulouse III Paul Sabatier
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Etablissement Francais du Sang; Centre Hospitalier Universitaire de Toulouse; Universite de Toulouse
Priority date: 2023-02-02
Filing date: 2024-01-29
Publication date: 2025-12-10
Also published as: JP2026505695A; WO2024160746A1

Abstract

The present invention belongs to the field of disease therapy, and more specifically to the field of cancer therapy. It relates more specifically to compound inhibiting the liver receptor homolog-1 (LRH-1) for use in the prevention and/or treatment of cancer involving myeloid-derived suppressor cells (MDSC) The present invention also relates to an in-vitro method for detecting a cancer involving myeloid-derived suppressor cells (MDSC) from a biological sample, to an in-vitro method for determining the susceptibility of a cancer from a biological sample to a compound.

Description

Compound for use in the prevention and/or treatment of cancer

Technical field

The present invention belongs to the field of disease therapy, and more specifically to the field of cancer therapy. It relates more specifically to compound inhibiting the liver receptor homolog-1 (LRH-1 ) for use in the prevention and/or treatment of cancer involving myeloid-derived suppressor cells (MDSC)

The present invention also relates to an in-vitro method for detecting a cancer involving myeloid-derived suppressor cells (MDSC) from a biological sample, to an in-vitro method for determining the susceptibility of a cancer from a biological sample to a compound.

In the description below, references between [ ] refer to the list of references at the end of the examples.

Technical background

Colorectal cancer (CRC) is the third most common cancer and, as for most cancers, tumor metastasis represent the major cause of death (Bray et al., 2018 ). Twenty five percent of patients who are diagnosed with CRC develop metastases with tumor spread in the peritoneum and abdominal organs defined as peritoneal carcinomatosis of colorectal cancer (PCCR). Despite an increasing number of treatment options, the average survival is still less than three years (Gasser et al., 2020).

There is therefore a real need to find a method and/or a compound which allows more efficient treatment and/or effective treatment of cancer, in particular colorectal cancer. In particular there is a real need to find new strategies, i.e. new targets/pathways, in the treatment of cancer.

The development of new treatments for PCCR appears to be a relevant approach in the management of CRC considering the significative mortality related to peritoneal carcinomatosis. However, there is not specific PCCR therapies available and the therapies for CRC have to be improved since they do not allow to effectively increase the average survival. In addition, the therapies do not allow to reduce or limit the development of metastases.

Such as most solid cancers, PCCR is associated with immune infiltrates exhibiting strong immunosuppressive properties (Meirow et al., 2015). In recent years, increasing evidence highlighted myeloid-derived suppressor cells (MDSC) as one of the players mediating immunosuppression in the tumor microenvironment (Umansky and Sevko, 2013). MDSC frequency in the blood and in the tumor of CRC patients is negatively correlated with overall survival (Wang et al., 2018).

There is therefore a real need to find a method and/or a compound which allows to the treatment of cancer, for example by inhibiting the immunosuppression in the tumor microenvironment.

MDSC are a heterogeneous population of immature myeloid cells (IMC) that fail to terminally differentiate and present a remarkable ability to suppress CD8 and CD4 T cell response (Gabrilovich, 2017; Gabrilovich and Nagaraj, 2009a; Gabrilovich et al., 2001 ; Veglia et al., 2018). Under inflammatory conditions, IMC expand and differentiate mainly into monocytes and activated neutrophils (Gabrilovich, 2017). This process, known as myelopoiesis, is a fundamental mechanism to protect the host from pathological events. However, in most cancers and other chronic conditions, the stimuli inducing the immune response fail to be cleared resulting in a persistent release of low stimulatory intensity signals, perpetuating the inflammatory response (Colotta et al., 2009). These stimuli induce abnormal myelopoiesis known as “emegency” myelopoiesis during which the accumulating IMC fail to completely differentiate into activated neutrophils and monocytes. Instead, the long-duration inflammatory signals lead to the expansion and activation of MDSC (Fleming et al., 2018; Veglia et al., 2018). Based on their phenotype and morphology, MDSC can be divided into two subtypes: monocytic-MDSC (M-MDSC) and granulocytic- or polymorphonuclear-MDSC (G-MDSC or PMN-MDSC). In mice, M-MDSC are defined as CD11 b⁺Ly6G’Ly6C⁺ and share phenotypical and morphological characteristics with monocytes. G-MDSC are described as CD1 1 b⁺Ly6G⁺Ly6C’ cells and are closely related to neutrophils (Bronte et al. , 2016; Veglia et al. , 2018). Moreover, MDSC usually lack surface markers of monocytes like CD11 c and major histocompatibility complex class II (MHCII) (Dolcetti et al., 2010; Movahedi et al., 2008) and can be readily separated from macrophages, since macrophages have high expression of F4/80 and low or undetectable expression of Ly6C (Gabrilovich and Nagaraj, 2009a; Gabrilovich et al., 2012; Veglia et al., 2018).

Functionally, MDSC use a wide range of mechanisms to prevent tumor-infiltrating lymphocyte activation and function, thereby supporting tumor progression and metastasis dissemination (Gabrilovich and Nagaraj, 2009a; Kumar et al., 2016a; Noman et al., 2014; Ostrand-Rosenberg and Fenselau, 2018). One of the main immunosuppressive mediators produced by MDSC is the arginase-1 (ARG1 ), which is an essential enzyme leading to the depletion of L-arginine (Bronte and Zanovello, 2005a; Bronte et al., 2003). The resulting lack of L-arginine causes a translational blockade in infiltrating T cells leading to cell cycle arrest in G0-G1 (Rodriguez et al., 2007). Moreover, ARG1 induces T cell anergy by downregulating the T cell receptor (TCR) ^-chain (Baniyash, 2004). Besides ARG1 , MDSC also express the inducible nitric oxide synthase (iNOS), which is involved in L- arginine catabolism and nitric oxide (NO) production. NO induces T cell anergy by nitrating mediators of the interleukin (IL)-2 signaling pathway (Gabrilovich et al., 2012; Mazzoni et al., 2002). Furthermore, MDSC secrete immunosuppressive cytokines and growth factors such as Transforming Growth Factor-[3 (TGF-P), IL-10, C-C Motif Chemokine Ligand-17 (CCL17) and CCL22 that reduce antitumor activity of effector T cells and recruit regulatory T cells (Treg) (Veglia et al., 2018). MDSC also express high levels of programmed death-ligand 1 (PD-L1 ) which, upon the binding to its receptor, induces T cell anergy resulting in the loss of interferon (IFN)-y and IL-2 production (Berger and Pu, 2018; Noman et al., 2014). Factors that induce MDSC expansion through the induction of “emergency” myelopoiesis include the cyclooxygenase-2 (COX2)/prostaglandin E2 (PGE2) axis (Castellone et al., 2005; Obermajer et al., 2011 a; Rodriguez et al., 2005; Veltman et al., 2010), Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF) (Serafini et al., 2004), Vascular Endothelial Growth Factor (VEGF) (Gabrilovich and Nagaraj, 2009a) and activation of the CCAAT/enhancer binding proteins [3 (C/EBP[3) transcription factor (Ostrand- Rosenberg and Fenselau, 2018). Moreover, it is now clear that the mechanisms by which MDSC acquire immunosuppressive properties require not only factors that promote their expansion but also those that induce their activation. These factors activate several different signaling pathways in MDSC which involve nuclear factor-KB (NF-KB) and Signal transducer and activator of transcription 1 (STAT1 ) and STAT6. Among them, STAT6, which is activated by IL-4 or IL-13, appears to be a pivotal factor in MDSC activation. Indeed, Bronte et al demonstrated that IL-4 upregulates ARG1 expression, thus promoting MDSC suppressive functions (Bronte et al., 2003). Consistent with these observations, STAT6 deficiency prevents IL-4Ra downstream signaling and thus blocks ARG1 production by MDSC (Sinha et al., 2005). In addition, the IL-4Ra-STAT6 pathway was also found to be involved in IL-13-induced MDSC differentiation further contributing to block immune surveillance against metastasis (Sinha et al., 2005).

Similarly, in macrophages, the activation of STAT6 by IL-13 or IL-4 promotes their alternative polarization and ARG1 upregulation (Heydeck et al., 1998; Huang et al., 1999; Odegaard et al., 2007; Takeda et al., 1996).

There is therefore a real need to find a method and/or a compound which allows to the treatment of cancer, for example by inhibiting the immunosuppression in the tumor microenvironment. There is therefore a real need to find a method and/or a compound which allows to the treatment of cancer involving MDSC, for example by inhibiting the recruitment and the immunosuppressive properties of MDSC.

Detailed description of the invention

The present invention allows to overcome the drawback and inconvenient of the prior art by providing a compound inhibiting the liver receptor homolog-1 (LRH-1 ) for use in the prevention and/or treatment of cancer involving myeloid-derived suppressor cells (MDSC).

An object of the invention is a compound inhibiting the liver receptor homolog-1 (LRH-1 ) for use in the prevention and/or treatment of cancer involving myeloid-derived suppressor cells (MDSC).

Another object of the invention is a compound inhibiting the peroxisome proliferator-activated receptors gamma (PPARy) for use in the prevention and/or treatment of cancer involving myeloid-derived suppressor cells (MDSC).

The inventors have surprisingly and unexpectedly demonstrated, in colorectal carcinoma bearing mice specifically deleted for the liver receptor homolog-1 (Lrh-1 ) in the myeloid lineage, a strong decrease in tumor burden associated with a decrease in the accumulation and activation of myeloid- derived suppressor cells (MDSC) in the tumor microenvironment. Subsequently to the MDSC decline and inactivation, the inventors have demonstrated a reactivation of the adaptive anti-tumor immune response. In particular, the inventors have demonstrated an increase of Th1 CD4 T cell and cytotoxic CD8 T cell proportions, as well as a decrease in regulatory T cells.

The inventors have also unexpectedly demonstrated that pharmacological treatment of cancer, for example colorectal carcinoma bearing mice with a compound inhibiting LRH-1 , for example an inverse agonist of LRH-1 , for example ML-180, support the protumor role of LRH-1 through its immunosuppressive activity and identify this nuclear receptor as innovative therapeutics targets in peritoneal carcinomatosis of colorectal origin. Altogether these results highlight PPARy and LRH-1 as critical actors allowing to counteract immunosuppression by preventing MDSC differentiation and demonstrate that compound inhibiting LRH-1 allow to prevent the development of the peritoneal carcinomatosis of colorectal origin and also allow to the treatment of cancer, for example colorectal cancer.

Using two mouse models specifically deleted for Lrh-1 in the myeloid lineage, the inventors have surprisingly identified this nuclear receptor as major players of PCCR progression through their role in the control of MDSC expansion and activation. The inventors have surprisingly and unexpectedly demonstrated the in vivo efficiency of compound inhibiting of LRH-1 , for example a reverse agonist, on tumor growth inhibition. In others words, the inventors have surprisingly and unexpectedly demonstrate that inhibitors of LRH-1 allow to treat cancer by inhibiting the growth of tumor. In addition the inventors have surprisingly demonstrate that the present invention, in particular compounds inhibiting LRH-1 allow to inhibit the tumor growth by preventing MDSC accumulation.

The inventors have also surprisingly and unexpectedly demonstrated that compound inhibiting LRH-1 allows to inhibit the tumor- induced immunosuppression and also allows to inhibit the tumor growth and to treat cancer.

The inventors have also surprisingly demonstrated that the crucial role of LRH-1 and PPARy in MDSC differentiation in humans.

Definitions

To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

As used herein other than the claims, the terms "a," "an," "the," and/or "said" means one or more. As used herein in the claim(s), when used in conjunction with the words "comprise," "comprises" and/or "comprising," the words "a," "an," "the," and/or "said" may mean one or more than one. As used herein and in the claims, the terms "having," "has," "is," "have," "including," "includes," and/or "include" has the same meaning as "comprising," "comprises," and "comprise." As used herein and in the claims "another" may mean at least a second or more. As used herein and in the claims, "about" refers to any inherent measurement error or a rounding of digits for a value (e.g., a measured value, calculated value such as a ratio), and thus the term "about" may be used with any value and/or range.

The phrase "a combination thereof" "a mixture thereof" and such like following a listing, the use of "and/or" as part of a listing, a listing in a table, the use of "etc." as part of a listing, the phrase "such as," and/or a listing within brackets with "e.g.," or i.e. , refers to any combination (e.g., any subset) of a set of listed components, and combinations and/or mixtures of related species and/or embodiments described herein though not directly placed in such a listing are also contemplated. Such related and/or like genera(s), sub-genera(s), specie(s), and/or embodiment(s) described herein are contemplated both in the form of an individual component that may be claimed, as well as a mixture and/or a combination that may be described in the claims as "at least one selected from," "a mixture thereof" and/or "a combination thereof."

As used herein, the term “and/or” means any one of the items, any combination of the items, or all of the items with which this term is associated.

As used herein, the term “about” refers to a variation of ±5-10% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and/or less than a recited integer. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment. As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible subranges and combinations of subranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percents) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.

As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than," "or more," and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into subranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio

An "effective amount" refers to an amount effective to treat a disease, disorder, and/or condition, or to bring about a recited effect. For example, an amount effective can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect.

The terms "treating", "treat" and "treatment" include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and/or (iv) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms "treat", "treatment", and "treating" extend to prophylaxis and include prevent, prevention, preventing, lowering, stopping or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" includes medical, therapeutic, and/or prophylactic administration, as appropriate.

Liver receptor homolog 1 (LRH-1 , LRH1 ; also known as NR5A2) refers to an orphan nuclear receptor that belongs to the NR5A or the Ftz-F1 subfamily of nuclear receptors. Members of this subfamily all bind DNA as monomers, and the specificity of DNA recognition is to a large extent dictated by the Ftz-F1 box, a unique domain at the C terminus of the DNA-binding domain of NR5A members. LRH-1 display constitutive transcriptional activity. This nuclear receptor is highly expressed in the intestine, liver, pancreas and ovary.

Peroxisome proliferator activated receptor y (PPARy) refers to a member of a family of ligand-activated nuclear transcription factors which, after ligand binding, form a heterodimer with the retinoic X receptor. This heterodimer settles then to PPAR-responsive elements (PPREs) in the promoter regions of target genes. This nuclear receptor has been linked to the regulation of adipogenesis and to the maintenance of glucose and lipid metabolisms. It also controls the inflammation response through the regulation of myeloid cells activation/differentiation. (Heikkinen S, Auwerx J, Argmann CA. PPARgamma in human and mouse physiology. Biochim Biophys Acta. 2007 Aug;1771 (8):999-1013. doi:

10.1016/j.bbalip.2007.03.006. Epub 2007 Mar 27. PMID: 17475546; PMCID: PMC2020525).

MDSC refers to a heterogeneous population of immature myeloid cells that fail to terminally differentiate and present a remarkable ability to suppress CD8 and CD4 T cell response (Gabrilovich DI. Myeloid-Derived Suppressor Cells. Cancer Immunol Res. 2017 Jan;5(1 ):3-8. doi: 10.1158/2326-6066. C I R-16-0297. PMID: 28052991 ; Veglia F, Perego M, Gabrilovich D. Myeloid-derived suppressor cells coming of age. Nat Immunol. 2018 Feb; 19(2): 108-119. doi: 10.1038/s41590-017-0022-x. Epub 2018 Jan 18. PMID: 29348500; PMCID: PMC5854158). Based on their phenotype and morphology, MDSC can be divided into two subtypes: monocytic-MDSC (M-MDSC) and granulocytic- or polymorphonuclear- MDSC (G-MDSC or PMN-MDSC). M-MDSC are myeloid cells expressing for example CD11 b⁺Ly6G’Ly6C⁺ in mice or CD11 b⁺CD15’ CD14⁺CD33⁺HLA-DR’ in human and share phenotypical and morphological characteristics with monocytes. G-MDSC are myeloid cells expressing CD11 b⁺Ly6G⁺Ly6C- in mice CD11 b⁺CD15⁺CD14-CD33⁺HLA-DR- in human and are closely related to neutrophils as described in Bronte V, Brandau S, Chen SH, Colombo MP, Frey AB, Greten TF, Mandruzzato S, Murray PJ, Ochoa A, Ostrand-Rosenberg S, Rodriguez PC, Sica A, Umansky V, Vonderheide RH, Gabrilovich DI. Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards. Nat Commun. 2016 Jul 6;7:12150. doi: 10.1038/ncomms12150. PMID: 27381735; PMCID: PMC4935811. Functionally, MDSC use a wide range of mechanisms to prevent tumor-infiltrating lymphocyte activation and function, thereby supporting tumor progression and metastasis dissemination as disclosed in Ostrand-Rosenberg S, Fenselau C. Myeloid-Derived Suppressor Cells: Immune-Suppressive Cells That Impair Antitumor Immunity and Are Sculpted by Their Environment. J Immunol. 2018 Jan 15;200(2):422-431 . doi: 10.4049/jimmunol.1701019. PMID: 29311384; PMCID: PMC5765878. MDSC frequency in the blood and in the tumor of patients with colon cancer is negatively correlated with overall survival (Wang PF, Song SY, Wang TJ, Ji WJ, Li SW, Liu N, Yan CX. Prognostic role of pretreatment circulating MDSCs in patients with solid malignancies: A meta-analysis of 40 studies. Oncoimmunology. 2018 Jul 30;7(10):e1494113. doi: 10.1080/2162402X.2018.1494113. PMID: 30288362; PMCID:

PMC6169582.).

In the present, “inhibiting the liver receptor homolog-1” means inhibiting liver receptor homolog-1 formation and/or function. The terms "inhibition of", "to inhibit", "inhibiting" or "inhibited" liver receptor homolog-1 formation mean that the formation of liver receptor homolog-1 is delayed, reduced or, preferably, entirely prevented in the presence of the inhibiting compound. “Inhibiting the liver receptor homolog-1” also means completely or partially inhibiting the biological activity of liver receptor homolog-1 by any mode of action, including but not limited to preventing the expression product of the liver receptor homolog-1 gene from being produced (interrupting the liver receptor homolog-1 gene transcription and/or blocking the translation of the mRNA coming from the liver receptor homolog-1 gene expression) and directly inhibiting the liver receptor homolog-1 biological activity, for example, and among others, by binding to the receptor.

Methods for decreasing/abrogating the expression of the gene encoding the liver receptor homolog-1 protein include, without being limited to, editing technologies such as CRISPR/cas9 or Cas9 nickase technology. Preferably inhibition of liver receptor homolog-1 , as defined herein, corresponds with inhibition of its biological function. Inhibition of the biological function of liver receptor homolog-1 may be for example reducing its biological function by altering the conformational dynamics of LRH-1 .

For instance, a compound inhibiting the liver receptor homolog-1 according to the invention may bind to the liver receptor homolog-1 in a fashion that interferes or effectively abrogates the function, e.g., by altering the conformational dynamics of the receptor. The compound inhibiting the liver receptor homolog-1 may also interact with another factor capable of binding to the liver receptor homolog-1 to indirectly inhibit liver receptor homolog-1 function. For example, the compound may cause dissociation of co-activator of LRH-1 . It may for example enhance LRH-1 interaction with NR co-repressors such as SHP as disclosed in Busby S, Nuhant P, Cameron M, Mercer BA, Hodder P, Roush WR, Griffin PR. Discovery of Inverse Agonists for the Liver Receptor Homologue-1 (LRH1 ; NR5A2). 2010 Oct 12 [updated 2011 Dec 12], In: Probe Reports from the NIH Molecular Libraries Program [Internet], Bethesda (MD): National Center for Biotechnology Information (US); 2010-. PMID: 23166964.

Methods for determining the inhibition of the liver receptor homolog- 1 may be any method from one skilled in the art. It may be for example the method disclosed in Busby S, Nuhant P, Cameron M, Mercer BA, Hodder P, Roush WR, Griffin PR. Discovery of Inverse Agonists for the Liver Receptor Homologue-1 (LRH1 ; NR5A2). 2010 Oct 12 [updated 2011 Dec 12], In: Probe Reports from the NIH Molecular Libraries Program [Internet], Bethesda (MD): National Center for Biotechnology Information (US); 2010- . PMID: 23166964. For example, the inhibition of LRH-1 can be determined for example by studying the expression of its target genes by RT-qPCR and by a dose response curve of aromatase activity as disclosed in Busby S, Nuhant P, Cameron M, Mercer BA, Hodder P, Roush WR, Griffin PR. Discovery of Inverse Agonists for the Liver Receptor Homologue-1 (LRH1 ; NR5A2). 2010 Oct 12 [updated 2011 Dec 12], In: Probe Reports from the NIH Molecular Libraries Program [Internet], Bethesda (MD): National Center for Biotechnology Information (US); 2010- PMID: 23166964.

In the present, a compound inhibiting the liver receptor homolog-1 (LRH-1 ) may be any compound known from one skilled in the art and/or commercially available adapted to inhibit the liver receptor homolog-1. For example, compound inhibiting the liver receptor homolog-1 inhibits its biological function. “Compound inhibiting the liver receptor homolog-1 (LRH- 1 )” refers herein to any inhibitor, inverse agonist, antagonist of LRH-1 that is likely to limit or to avoid, reversibly or irreversibly, selectively or not selectively, the biological normal effect of LRH-1 . More particularly, it may refer to any antagonist of LRH-1 , that is likely to bind, reversibly or irreversibly, LRH-1 , thereby limiting or avoiding, reversibly or irreversibly, the biological normal effect of LRH-1 . By “limit” or “limiting” by an antagonist it is understood a decrease of at least 50%, for example 60%, 70%, 80%, 90% or 99%, of the biological effect of LRH-1 compared to the normal biological effect of LRH-1 receptor, i.e. the biological effect of LRH-1 receptor that is not inhibited by the inhibiting compound. More particularly, it may refer to any inverse agonist of LRH-1 , that is likely to bind, reversibly or irreversibly, LRH-1 , thereby limiting or avoiding, reversibly or irreversibly, the biological normal effect of LRH-1. By “limit” or “limiting” by an inverse agonist it is understood a decrease of at least 50%, for example 60%, 70%, 80%, 90% or 99%, of the biological effect of LRH-1 compared to the normal biological effect of LRH-1 receptor, i.e. the biological effect of LRH-1 receptor that is not inhibited by the inhibiting compound.

Compound inhibiting the liver receptor homolog-1 may be any compound whatever its form. It may be for example a chemical compound, a peptide, a polynucleic acid, an antibody. It may be for example an inhibitor of liver receptor homolog-1 (LRH-1 ), an inverse agonist of liver receptor homolog-1 (LRH-1 ), an antagonist of liver receptor homolog-1 (LRH-1 ). It may be for example an inverse agonist selected from the group comprising ML-180, ML179. It may be for example an antagonist selected from the group comprising 7-[4-(2-Piperidinyl)ethoxy]benzoyl Raloxifene, 1 -(3'-(1 -(2- Morpholinoethyl)-1 H-pyrazol-3-yl)biphenyl-3-yl)ethanone, 1 -(3'-( 1 -(2-(4- Morpholinyl)ethyl)-1 H-pyrazol-3-yl)-3-biphenylyl)ethenone. It may be for example an antagonist for example 3D2 as disclosed in Benod C, Carlsson J, Uthayaruban R, Hwang P, Irwin J J, Doak AK, Shoichet BK, Sablin EP, Fletterick RJ. Structure-based discovery of antagonists of nuclear receptor LRH-1. J Biol Chem. 2013 Jul 5;288(27): 19830-44. doi: 10.1074/jbc.M112.411686. Epub 2013 May 10. PMID: 23667258; PMCID: PMC3707686.

Compound inhibiting the liver receptor homolog-1 may be a compound as mentioned in table 1 below.

Table 1 : Compounds inhibiting the liver receptor homolog-1

In the present, “inhibiting the peroxisome prol iterator-activated receptors gamma (PPARy)” means inhibiting the peroxisome proliferator- activated receptors gamma (PPARy) formation and/or function. The terms "inhibition of", "to inhibit", "inhibiting" or "inhibited" the peroxisome prol iterator-activated receptors gamma (PPARy) formation mean that the formation of the peroxisome proliferator-activated receptors gamma (PPARy) is delayed, reduced or, preferably, entirely prevented in the presence of the inhibiting compound. “Inhibiting the peroxisome proliferator- activated receptors gamma (PPARy)” also means completely or partially inhibiting the biological activity of the peroxisome proliferator-activated receptors gamma (PPARy) by any mode of action, including but not limited to preventing the expression product of the peroxisome proliferator-activated receptors gamma (PPARy) gene from being produced (interrupting the peroxisome proliferator-activated receptors gamma (PPARy) gene transcription and/or blocking the translation of the mRNA coming from the peroxisome proliferator-activated receptors gamma (PPARy) gene expression) and directly inhibiting the peroxisome proliferator-activated receptors gamma (PPARy) biological activity, for example, and among others, by binding to the receptor. Methods for decreasing/abrogating the expression of the gene encoding the peroxisome proliferator-activated receptors gamma (PPARy) protein include, without being limited to, editing technologies such as CRISPR/cas9 or Cas9 nickase technology. Preferably inhibition of the peroxisome proliferator-activated receptors gamma (PPARy), as defined herein, corresponds with inhibition of its biological function. Inhibition of the biological function of the peroxisome proliferator- activated receptors gamma (PPARy) may be for example reducing its biological function for example by blocking interaction with natural binding partner, for example by inhibiting the formation of heterodimer between PPARy and retinoid X receptor or inhibiting coactivator binding to PPARy for example by affecting the conformation of PPARy, for example of its ligandbinding domain with retinoid X receptor (: Biochemistry 2002, 41 , 21 , 6640- 6650 ; Journal of Biological Chemistry 2002, 277 (22), 19649-19657). For instance, a compound inhibiting the peroxisome proliferator-activated receptors gamma (PPARy) according to the invention may bind to the peroxisome proliferator-activated receptors gamma (PPARy) in a fashion that interferes or effectively abrogates the function. The compound inhibiting the peroxisome proliferator-activated receptors gamma (PPARy) may also interact with another factor capable of binding to the peroxisome proliferator- activated receptors gamma (PPARy) to indirectly inhibit the peroxisome proliferator-activated receptors gamma (PPARy) function.

Methods for determining the inhibition of the peroxisome proliferator- activated receptors gamma (PPARy) may be any method from one skilled in the art, for example a competition binding assay against the human ligand binding domain. It may be for example the method as disclosed in Biochemistry 2002, 41 , 21 , 6640-6650.

In the present, a compound inhibiting the peroxisome proliferator- activated receptors gamma (PPARy) may be any compound known from one skilled in the art and/or commercially available adapted to inhibit the peroxisome proliferator-activated receptors gamma (PPARy). For example, compound inhibiting the peroxisome proliferator-activated receptors gamma (PPARy) inhibits its biological function. “Compound inhibiting the peroxisome proliferator-activated receptors gamma (PPARy)” refers herein to any inhibitor, inverse agonist, antagonist of PPARy that is likely to limit or to avoid, reversibly or irreversibly, selectively or not selectively, the biological normal effect of PPARy. More particularly, it may refer to any antagonist of PPARy, that is likely to bind, reversibly or irreversibly, PPARy, thereby limiting or avoiding, reversibly or irreversibly, the biological normal effect of PPARy. By “limit” or “limiting” by an antagonist it is understood a decrease of at least 50%, for example 60%, 70%, 80%, 90% or 99%, of the biological effect of PPARy compared to the normal biological effect of PPARy, i.e. the biological effect of PPARy that is not inhibited by the inhibiting compound. More particularly, it may refer to any inverse agonist of LRH-1 , that is likely to bind, reversibly or irreversibly, PPARy, thereby limiting or avoiding, reversibly or irreversibly, the biological normal effect of PPARy. By “limit” or “limiting” by an inverse agonist it is understood a decrease of at least 50%, for example 60%, 70%, 80%, 90% or 99%, of the biological effect of PPARy compared to the normal biological effect of PPARy, i.e. the biological effect of PPARy that is not inhibited by the inhibiting compound.

Compound inhibiting the peroxisome proliferator-activated receptors gamma may be any compound whatever its form. It may be for example a chemical compound, a peptide, a polynucleic acid, an antibody. It may be for example an inhibitor of peroxisome proliferator-activated receptors gamma (PPARy), an inverse agonist of peroxisome proliferator-activated receptors gamma (PPARy), an antagonist of peroxisome proliferator- activated receptors gamma (PPARy). It may be for example a compound selected from the group comprising GW 9662, T-0070907, FH-535, SR-16832, SR-202. It may be for example a compound of following formula:

Compound inhibiting peroxisome proliferator-activated receptors gamma (PPARy) may be a compound as mentioned in table 2 below.

Table 2: Compounds inhibiting the peroxisome proliferator-activated receptors gamma (PPARy)

In the present, the term “peptide” refers to a linear molecule which is formed by the manner in which the amino acid residues bind together by a peptide bond . In the present, the term “polynucleic acid” refers to polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetic thereof including those composed of naturally-occurring nucleobases, sugars and covalent inter-nucleoside (backbone) linkages including linked poly- heterocyclic bases having non-naturally-occurring portions that function similarly.

The term "antibody" is used herein in the broadest sense. According to the present invention, "antibody" refers to any polypeptide which at least comprises (i) a Fc region and (ii) a binding polypeptide domain derived from a variable domain of an immunoglobulin. The said binding polypeptide domain is able to bind specifically one given target antigen or a group of target antigens. In the present, the antibody according to the invention has a binding affinity to liver receptor homolog-1 (LRH-1 ) or to peroxisome prol iterator-activated receptors gamma (PPARy). A binding polypeptide domain which derives from a variable region of an immunoglobulin comprises at least one or more CDRs. Herein, antibodies include, but are not limited to, full-length immunoglobulins, monoclonal antibodies, a VHH domain (also named single heavy chain domain or Nanobody®), multispecific antibodies, Fc-fusion protein comprising at least one variable region, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies. Antibodies also encompass antibody-fusion proteins, antibody conjugates and fragments of each respectively. Accordingly, a variant antibody of the invention comprises, in its Fc region, at least one amino acid modification that increase its binding affinity for Matrix Binding Motif (M-motif) on Tenascin-C (TNC) as compared to its parent antibody. Of particular interest are antibody variants which have increased in vivo halflives as compared to parent polypeptides.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught.

The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology, and refers to an antibody that may be derived from a single B cell, a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with an antigen of interest or a cell expressing such an antigen.

Covalent modifications of antibodies are also included within the scope of this invention, and are generally, but not always, done post-translationally. Such modifications include, but are not limited to, glycosylates, labelling and conjugation. The term "labeling group" means any detectable label, which is a compound and/or element that can be detected due to its specific functional properties, and/or chemical characteristics, the use of which allows the antibody to which it is attached to be detected, and/or further quantified if desired. In some embodiments, the labeling group is coupled to the antibody via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and may be used in performing the present invention.

In the present “cancer involving myeloid-derived suppressor cells (MDSC)” means cancer wherein myeloid-derived suppressor cells are present for example in the microenvironment of a cancer (e.g., tumor) or for example, are found in cancerous tissue (e.g., tumor tissue). It may be any cancer of any organ known from one skilled in the art. It may be for example solid cancer. It may be for example any disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. It may be for example any cancer of any organ known from one skilled in the art involving myeloid-derived suppressor cells (MDSC). It may be for example cancer of any organ or tissue of a human or of an animal. It may be for example a cancer of any organ of the digestive system. It may be for example a cancer of any organ selected from the group comprising mouth, liver, esophagus, trachea, stomach, colon, pancreas, cervical, uterine, gall bladder, bladder, prostate, rectum, and lymphomas. It may be for example a cancer of any organ selected from the group comprising mouth, esophagus, trachea, pancreas, stomach, gall bladder, small intestine, colon, rectum, liver. It may be for example cancer associated with MDSC immune infiltrates mediating immunosuppression in the tumor microenvironment as disclosed in Meirow Y, Kanterman J, Baniyash M. Paving the Road to Tumor Development and Spreading: Myeloid-Derived Suppressor Cells are Ruling the Fate. Front Immunol. 2015 Oct 12;6:523. doi: 10.3389/fimmu.2015.00523. PMID: 26528286; PMCID: PMC4601280., Umansky V, Sevko A. Tumor microenvironment and myeloid-derived suppressor cells. Cancer Microenviron. 2013 Aug;6(2): 169-77. doi: 10.1007/sl 2307-012-0126-7. Epub 2012 Dec 16. PMID: 23242672; PMCID: PMC3717060. It may be for example colorectal cancer, for example associated with MDSC immune infiltrates mediating immunosuppression in the tumor microenvironment as disclosed in Meirow Y, Kanterman J, Baniyash M. Paving the Road to Tumor Development and Spreading: Myeloid-Derived Suppressor Cells are Ruling the Fate. Front Immunol. 2015 Oct 12;6:523. doi: 10.3389/fimmu.2015.00523. PMID: 26528286; PMCID: PMC4601280., Umansky V, Sevko A. Tumor microenvironment and myeloid-derived suppressor cells. Cancer Microenviron. 2013 Aug;6(2): 169- 77. doi: 10.1007/s12307-012-0126-7. Epub 2012 Dec 16. PMID: 23242672; PMCID: PMC3717060. MDSC frequency in the blood and in the tumor of patients with colon cancer is negatively correlated with overall survival (PMID : 30288362). It may be for example a peritoneal carcinomatosis, for example a peritoneal carcinomatosis of colorectal origin.

In the present, the compound of the invention may be used in a combination therapy, for example with a therapeutic agent.

In the present, the compound of the invention may be used in a combination, for example with a therapeutic agent.

In the present, the compound of the invention may be used in a combination, for example with anti-cancer drug, anti-angiogenic drug, antiinflammatory drug, anti-oxidative drug, immunotherapy.

“Combination therapy" (or "co-therapy") includes the administration of a compound inhibiting the liver receptor homolog-1 (LHR-1 ) and/or pharmaceutical composition of the invention, and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents.

“Combination therapy" (or "co-therapy") also includes the administration of a compound inhibiting peroxisome proliferator-activated receptors gamma (PPARy) and/or pharmaceutical composition of the invention, and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).

"Combination therapy" may, but generally is not, intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention. "Combination therapy" is intended to embrace administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents.

Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, topical routes, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination selected may be administered by injection while the other therapeutic agents of the combination may be administered topically.

A “therapeutic agent” is any compound known in the art that is used in the detection, diagnosis, or treatment of a condition or disease. Such compounds may be naturally-occurring, modified, or synthetic. Non-limiting examples of therapeutic agents may include drugs, therapeutic compounds, genetic materials, metals (such as radioactive isotopes), proteins, peptides, carbohydrates, lipids, steroids, nucleic acid-based materials, or derivatives, analogues, or combinations thereof in their native form or derivatized with hydrophobic or charged moieties to enhance incorporation or adsorption into a cell. Non-limiting examples of therapeutic agents may include immune- related agents, thyroid agents, respiratory products, antineoplastic agents, anti-helmintics, anti-malarials, mitotic inhibitors, hormones, anti-protozoans, anti-tuberculars, cardiovascular products, blood products, biological response modifiers, anti-fungal agents, vitamins, peptides, anti-allergic agents, anti-coagulation agents, circulatory drugs, metabolic potentiators, anti-virals, anti-anginals, antibiotics, anti-inflammatories, anti-rheumatics, narcotics, cardiac glycosides, neuromuscular blockers, sedatives, local anesthetics, general anesthetics, or radioactive atoms or ions. A therapeutic agent may be a toxin, a small therapeutic molecule, a therapeutic nucleic acid, or a chemotherapeutic agent. A chemotherapeutic agent refers to a chemical compound that is useful in the treatment of cancer. The compound may be a cytotoxic agent that affects rapidly dividing cells in general, or it may be a targeted therapeutic agent that affects the deregulated proteins of cancer cells.

In the present, the therapeutic agent may be preferably selected from group comprising anticancer drug, anti-inflammatory drug, anti-angiogenic drug, anti-oxidative drug, immunotherapy.

In the present, the therapeutic agent may be anticancer drug. It may be for example 5 fluoro-uracile (5FU), oxaliplatine. It may be a cytotoxic drug. As used herein, the term “cytotoxic drug” refers to a molecule that when entering in contact with a cell, optionally upon internalization into the cell, alters a cell function (for example cell growth and/or proliferation and/or differentiation and/or metabolism such as protein and/or DNA synthesis) in a detrimental way or leads to cell death. As used herein, the term “cytotoxic drug” encompasses toxins, in particular cytotoxins. It may be, for example, a compound selected from the group comprising calicheamycin, dolastin 10, dolastin 15, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin F (MMAF), monomethylauristatin-D (MMAD), monomethyl auristatin E (MMAE), and 5-benzoylvaleric acid-AE ester (AEVB) and duocarmycin; nitrogen mustard analogues for example cyclophosphamide, melphalan, ifosfamide or trofosfamide; ethylenimines such as thiotepa; nitrosoureas for example carmustine; alkylating agents for example temozolomide or dacarbazine; folate-like metabolic antagonists such as methotrexate or raltitrexed; purine analogues for example thioguanine, cladribine or fludarabine; pyrimidine analogues for example fluorouracil, tegafur or gemcitabine; vinca alkaloids for example vinblastine, vincristine or vinorelbine and analogues thereof; podophyllotoxin derivatives for example etoposide, taxans, docetaxel or paclitaxel; anthracyclines for example doxorubicin, epirubicin, idarubicin and mitoxantrone, and analogues thereof; other cytotoxic antibiotics for example bleomycin and mitomycin; platinum compounds for example cisplatin, carboplatin and oxaliplatin; pentostatin, miltefosine, estramustine, topotecan, irinotecan and bicalutamide, and toxins for example ricin toxin, liatoxin and Vero toxin

In the present, the therapeutic agent may be an anti-angiogenic drug. It may be for example any anti-angiogenic drug known from one skilled in the art. It may be for example an anti-angiogenic drug selected from the group comprising bevacizumab, itraconazole, carboxyamidotriazole, TNP- 470, IFN-a, IL-12, suramin, SU5416, thrombospondin, angiostatin, endostatin, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, prolactin, linomide, ramucirumab, tasquinimod, ranibizumab, sorafenib, sunitinib, pazopanib and everolimus.

In the present, the therapeutic agent may be an anti-inflammatory drug. It may be for example any an anti-inflammatory drug known from one skilled in the art. It may be for example Nonsteroidal anti-inflammatory drugs, It may be for example anti-inflammatory drug selected from the group comprising aspirin, ibuprofen, naproxen ibuprofen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin.

In the present, the therapeutic agent may be an anti-oxydative drug. It may be for example any anti-oxydative drug known from one skilled in the art. It may be for example an anti-oxydative drug selected from the group comprising ascorbic acid, vitamin a, vitamin e, lipoic acid, masoprocol, pramipexole, nitric oxide, allopurinol, pentoxifylline, melatonin, dimethyl sulfoxide, probucol, 3,4-dihydroxycinnamic acid, resveratrol, 3- hydroxyanthranilic acid, dihydrolipoic acid, p-coumaric acid, quercetin, aeol 10150, transcrocetinate, acetylcysteine, nicaraven, lodoxamide, ferulic acid, uric acid, idebenone, chromic chloride, thiosulfuric acid.

In the present, the therapeutic agent may be an immunotherapy, It may be for example any immunotherapy known from one skilled in the art. It may be for example an immunotherapy selected from the group comprising pembrolizumab, nivolumab, pidilizumab, avelumab

In the present, the administration of the medicament may be carried out by any way known to one skilled in the art. It may, for example, be carried out directly, i.e. pure or substantially pure, or after mixing of a compound inhibiting the liver receptor homolog-1 (LHR-1 ) or a compound inhibiting the peroxisome prol iterator-activated receptors gamma (PPARy) with a pharmaceutically acceptable carrier and/or medium. According to the present invention, the medicament may be an injectable solution, a medicament for oral administration, for example selected from the group comprising a liquid formulation, a multiparticle system, an orodispersible dosage form. According to the present invention, the medicament may be a medicament for oral administration selected from the group comprising a liquid formulation, an oral effervescent dosage form, an oral powder, a multiparticle system, an orodispersible dosage form. The medicament may be in any form that can be administered to a human or an animal. It may for example be a pharmaceutical composition as defined below.

Another object of the present invention is a pharmaceutical composition comprising a compound inhibiting the liver receptor homolog-1 (LRH-1 ) for use in the prevention and/or treatment of cancer involving myeloid-derived suppressor cells (MDSC).

Another object of the present invention is a pharmaceutical composition comprising a compound inhibiting the peroxisome proliferator- activated receptors gamma (PPARy) for use in the prevention and/or treatment of cancer involving myeloid-derived suppressor cells (MDSC).

The pharmaceutical composition may be in any form that can be administered to a human or an animal. The person skilled in the art clearly understands that the term “form” as used herein refers to the pharmaceutical formulation of the medicament for its practical use. For example, the medicament may be in a form selected from the group comprising an injectable form, an oral suspension, a pellet, a powder, granules or topical form (e.g. cream, lotion, collyrium).

The pharmaceutical composition may comprise a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be any known pharmaceutical support used for the administration of a drug to a human or animal, depending on the subject to be treated. It may be for example a pharmaceutically acceptable carrier selected from the group comprising, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxylpropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxylpropylstarch, sodium- glycol-starch, sodium hydrogen carbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, Aerosil, talc, and sodium lauryl sulfate; flavoring agents such as citric acid, menthol, glycyrrhizin- ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as surfactants; diluents such as water, physiological saline, and orange juice; base waxes such as cacao butter, polyethylene glycol, and kerosene; and the like.

According to the invention, the pharmaceutical composition may be administrated by any adapted route of administration known to one skilled in the art. For example, the pharmaceutical composition may be administrated by oral administration and/or parenteral administration.

According to the present invention, the pharmaceutical composition may comprise any pharmaceutically acceptable and/or therapeutically effective amount of a compound inhibiting the liver receptor homolog-1 (LRH-1 ).

The compound inhibiting the liver receptor homolog-1 (LRH-1 ) is as defined above.

For example, in the case of cancer, the therapeutically effective amount of compound inhibiting the liver receptor homolog-1 (LRH-1 ) may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e. slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e. slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.

According to the present invention, the pharmaceutical composition may comprise any pharmaceutically acceptable and/or therapeutically effective amount of a compound inhibiting the peroxisome proliferator- activated receptors gamma (PPARy). The compound inhibiting the peroxisome prol iterator-activated receptors gamma (PPARy) is as defined above.

For example, in the case of cancer, the therapeutically effective amount of the peroxisome proliferator-activated receptors gamma (PPARy) may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e. slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e. slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.

The inventors have surprisingly demonstrated that it is possible to determine from a biological sample whether a cancer involve or not myeloid- derived suppressor cells (MDSC).

Accordingly, another object of the invention is a method for detecting a cancer involving myeloid-derived suppressor cells (MDSC) from a biological sample comprising: searching in the biological sample of cells expressing CD45, CD11 b, CD14, CD15, CD33, HLADR when the biological sample comprises cells expressing CD45, CD11 b, CD14, CD15, CD33, HLADR respectively CD45^high, CD11 b^high, CD14^high, CD15^high, CD33^high, HLADR^|OW, the cancer involved myeloid-derived suppressor cells (MDSC).

The presence of CD45^high, CD11 b^high, CD14^high, CD15^high, CD33^high, HLADR^|OW MDSC in the biological sample may be determined using detecting agents which can be for example antibodies, specifically, anti- CD45 antibodies, anti-CD11 b antibodies, anti-CD14 antibodies, anti-CD33 antibodies and anti-HLADR antibodies. It may be any anti-CD45 antibodies known from one skilled in the art and or commercially available. It may be any anti-CD11 b antibodies known from one skilled in the art and or commercially available. It may be any anti-CD14 antibodies known from one skilled in the art and or commercially available. It may be any anti-CD33 antibodies known from one skilled in the art and or commercially available. It may be any anti-HLADR antibodies known from one skilled in the art and or commercially available. The presence or absence of each of the cell markers may be denoted by a + or a - sign, respectively. Thereby HLADR’ or CD33+ MDSC are MDSC which lack HLADR and express CD33. The designation HLADR^|OW or HLADR^high relates to cells which show a relatively low or high expression level of HLADR respectively. Myeloid-derived suppressor cells (MDSC) are as defined above.

Cancer involving myeloid-derived suppressor cells (MDSC) is as mentioned above.

"Biological sample" means any biological liquid, for example, it may be a sample of blood, for example peripheral blood, including whole blood or fractioned blood, plasma, serum, ascites, a spleen biopsy, tissue biopsy, tissue section, for example colon tissue section or a tumor sample. Preferably, it can be a serum or synovial fluid sample, advantageously a synovial fluid sample. The biological sample may be a biological sample previously taken from a patient or subject. According to the invention, when the biological sample is a blood sample, it may be a biological sample previously taken from a vein on a patient or subject. It may be, for example, ascites previously collected at abdominal cavity or abdomen on a patient or subject.

CD45 means Cluster differentiation 45 is a protein tyrosine phosphatase, receptor type, C.

CD11 b means cluster differentiation 11 b is a type I transmembrane glycoprotein of 170 kDa.

CD14 means cluster of differentiation 14, is a lipopolysaccharide- binding protein, which functions as an endotoxin receptor.

CD15 means cluster of differentiation 15, is a carbohydrate adhesion molecule. CD33 means cluster of differentiation 33, is also designated siglec-3 (sialic acid binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67) is a transmembrane receptor.

HLADR or HLA-DR means Human Leukocyte Antigen - DR isotype, is a MHC class II cell surface receptor encoded by the complex on chromosome 6 region 6p21 .31 .

According to the invention, the biological sample may be a sample taken from 5 minutes to 48 hours, for example from 1 hour to 24 hours, for example from 1 hour to 6 hours prior to its use in the method according to the invention. According to the invention the volume of the biological sample can be from 10pl to 10 ml, for example from 12pl to 5 ml.

Searching in a biological sample cells expressing CD45, CD11 b CD14, CD15, CD33, HLADR may be carried out by any method adapted known from one skilled in the art. It may be for example a method disclosed in Bronte et al, Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards. Nat Commun 7, 12150.

In the present, the measure of expression may be carried out by any adapted method known from one skilled in the art. It may be for example a method including or be based on amplification, for example Whole Transcriptome Assay (WTA), followed by high-throughput sequencing (NGS), Polymerase Chain Reaction (PCR), real-time polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), realtime quantitative polymerase chain reaction (RT-qPCR), hybridization methods, e.g. hybridization arrays, and quantitative polymerase chain reaction (qPCR), real-time quantitative polymerase chain reaction (RT- qPCR), hybridization methods, e.g. hybridization arrays (chips), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc. ), and processes including sequencing steps, for example, using Illumina or lonTorrent platforms, fluorescence activated cell sorter (FACS), immunohistology, ELISA, RIA or Western blotting of the suitable MDSC cell markers. For example, it may be a commercially available method, for example, commercialized by Myltenyi Biotec.

CD45^high means that the cells show a relatively high expression level of CD45.

CD1 1 b^high means that the cells show a relatively high expression level of CD11 b.

CD14^high means that the cells show a relatively high expression level of CD14.

CD15^high means that the cells show a relatively high expression level of CD15.

CD33^high means that the cells show a relatively high expression level of CD33.

HLADR^|OW means in a cell population that comprises MDSC with anti- HLADR antibodies, a cell population can be identified according to the intensity of staining HLADR^high.

The definitions of "high" or "low" staining levels are relative in each tested sample and are well within the knowledge of the skilled person in the art of the invention. The level of expression high or low may be as disclosed in Bronte et al, Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards. Nat Commun 7, 12150.

The process may further comprise the following steps:

- measuring the expression level of gene selected from the group comprising Arginase-1 , Inos, IL-10 and Pdl-1 from cells expressing cluster of differentiation (CD) CD45 and CD11 b,

- comparison respectively of the measured expression level with a referenced expression level of Arginase-1 , Inos, IL-10 and Pdl-1 , when the measured expression level of Arginase-1 , Inos, IL-10 and /or Pdl-1 is superior to the referenced level of expression, the cells expressing cluster of differentiation (CD) CD45 and CD11 b are myeloid-derived suppressor cells (MDSC). The measure of expression may be carried out by any process known from one skilled in the art. For example, the measure of level of expression of Arginase-1 may be carried out with the process as disclosed in Ochoa JB, Bernard AC, O'Brien WE, Griffen MM, Maley ME, Rockich AK, Tsuei BJ, Boulanger BR, Kearney PA, Morris SM Jr. Arginase I expression and activity in human mononuclear cells after injury. Ann Surg. 2001 Mar;233(3):393-9. doi: 10.1097/00000658-200103000-00014. PMID: 11224628; PMCID: PMC1421256. For example, the measure of level expression of Inos may be carried out with the process as disclosed in Dabbeche-Bouricha E, Hadiji- Abbes N, Abdelmaksoud-Damak R, Alaya N, Ayadi W, Charfi S, Khabir A, Sellami-Boudawara T, Mokdad-Gargouri R. Quantitative measurement of iNOS expression in melanoma, nasopharyngeal, colorectal, and breast tumors of Tunisian patients: comparative study and clinical significance. Tumour Biol. 2016 Apr; 37(4):5153-64. doi: 10.1007/s13277-015-4303-4. Epub 2015 Nov. For example, the measure of IL-10 may be carried out with the process disclosed in Itakura, E., Huang, RR., Wen, DR. et al. IL-10 expression by primary tumor cells correlates with melanoma progression from radial to vertical growth phase and development of metastatic competence. Mod Pathol 24, 801-809 (2011 ). https://doi.Org/10.1038/modpathol.2011.5. In the present the comparison of expression level may be carried out with any adapted method known from one skilled in the art.

According to the invention, the referenced expression level of Arginase-1 , Inos, IL-10, TGF[3, CCL4, CCL22 and Pdl-1 may be the expression level of Arginase-1 , Inos, IL-10, TGF[3, CCL4, CCL22 or Pdl-1 measured in a biological sample of healthy subjects, or the mean expression level of Arginase-1 , Inos, IL-10, TGF[3, CCL4, CCL22 or Pdl-1 measured in a group of healthy subjects. Preferably, the referenced expression level of Arginase-1 , Inos, IL-10, TGF[3, CCL4, CCL22 or Pdl-1 value may be the expression level of Arginase-1 , Inos, IL-10, TGF[3, CCL4, CCL22 or Pdl-1 measured in a pool of biological samples from healthy subjects. Another object of the invention is a method for detecting a cancer involving myeloid-derived suppressor cells (MDSC) from a biological sample comprising:

Determining the expression level of LRH1 , comparing the expression level measured with a referenced expression level of LRH1 , when the expression level of LRH-1 , is superior than the referenced expression level of LRH-1 the cancer involved myeloid-derived suppressor cells (MDSC).

Cancer involving myeloid-derived suppressor cells (MDSC) is as mentioned above.

"Biological sample" is as defined above.

LRH1 is as defined above.

According to the invention, the referenced expression level of LRH-1 may be the expression level LRH-1 measured in a biological sample of healthy subjects, or the mean expression level LRH-1 measured in a group of healthy subjects. Preferably, the referenced expression level of LRH-1 value may be the expression level of LRH-1 measured in a pool of biological samples from healthy subjects.

In the present, “healthy subject” is intended to mean a mammal, for example a human being, that has not been subject to a disease and/or a cancer as mentioned above. It may be for example a human being, which has not been subject to a cancer, as defined above.

“Group of healthy subjects” or “group of reference healthy subjects” is intended to mean a group making it possible to define a reliable reference value or reliable reference intervals. It may for example be a group comprising at least 2 reference subjects as defined above, for example at least 10, at least 40, at least 60, at least 100 reference subjects or healthy subjects. It may for example be a group comprising from 30 to 500 subjects, from 40 to 200, from 45 to 110 reference subjects or healthy subjects. The inventors have also surprisingly demonstrated that it is possible to determine from a biological sample the susceptibility of a cancer to a compound inhibiting the liver receptor homolog-1 (LRH-1 ).

Accordingly, another object of the invention is a method for determining the susceptibility of a cancer from a biological sample to a compound inhibiting the liver receptor homolog-1 (LRH-1 ) comprising: a. isolating monocytes from the biological sample, b. culturing the isolated monocytes with a compound inhibiting the liver receptor homolog-1 (LRH-1 ), c. observation of the obtained culture of b) where when the obtained culture of b) does not comprise MDSC, the cancer is susceptible to a compound inhibiting the liver receptor homolog-1 (LRH-1 ).

Compound inhibiting the liver receptor homolog-1 (LRH-1 ) is as defined above.

Myeloid-derived suppressor cells (MDSC) are as defined above.

The biological sample is as defined above.

Isolated Monocyte means isolated cells expressing CD14⁺ and CD16⁺

In the present, isolating monocyte from a biological sample may be carried out by any method adapted know from one skilled in the art. It may be for example a method involving a centrifugation step.

In the present the monocyte may be cultured in any adapted cultured medium known from one skilled in the art. It can be for example a commercially available culture medium, for example PRMI medium.

According to the invention, monocyte culture step b. can be carried out in a culture medium comprising at least one differentiation factor. It may be for example a culture medium comprising at least one differentiation factor selected from the group comprising IL-4, GM-CSF and PGE-2. It may be for example a culture medium comprising at least two differentiation factors selected from the group comprising IL-4, GM-CSF and PGE-2. It may be for example a culture medium comprising IL-4, GM-CSF and PGE-2.

According to the invention, monocyte culture step b. can be carried out in PRMI culture medium.

According to the invention, monocyte culture step b. can be carried out in PRMI culture medium comprising at least one differentiation factor selected from the group comprising Granulocyte Macrophage Colony- Stimulating Factor (GM-CSF), Interleukin 4 (IL4) and/or Prostaglandin E2 (PGE2), for example monocyte culture step b. can be carried out in PRMI culture medium comprising Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin 4 (IL4) and/or Prostaglandin E2 (PGE2).

According to the invention the concentration of differentiation factor in the monocyte culture medium may be from 1 ng/ml to 2000ng/ml.

According to the invention the monocyte culture medium may comprises a Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) concentration from 1 ng/mL to 20ng/mL, for example of 20ng/mL.

According to the invention the monocyte culture medium may comprises an Interleukin 4 (IL4) concentration from 1 ng/mL to 10ng/mL, for example 10 ng/ml.

According to the invention the monocyte culture medium may comprises a Prostaglandin E2 (PGE2). concentration from 0.2 to 5 pmol/L, for example 2.6 pmol/L.

In the present, the monocyte culture step b. can be carried out at a temperature comprised from 36 to 38°C, or equal to 37°C.

In the present, monocyte culture of step b can be comprised from 2 to 7 days, from 3 to 6 days, of 5 days.

In the present, the observation of culture of isolated monocyte may be carried out by any adapted method known from one skilled in the art. It may be for example flow cytometry or RT-qPCR.

The process may further comprise the following steps: d. measuring the expression level of at least one among CCAAT/enhancer binding protein beta (C/EBP[3), LRH-1 , PPARy and Interferon regulatory factor 8 (IRF8) of cultured cell of step b, e. comparing respectively of the expression level measured at step d with a referenced expression level of C/EBP[3, LRH-1 , PPARy and IRF8, when the expression level of C/EBP[3, LRH-1 , PPARy measured at step d is superior than the referenced expression level of C/EBP[3 LRH-1 , PPARy and/or when the expressions level of IRF8 measured at step d is less to the referenced expression level of IRF8, the cancer is susceptible to a compound inhibiting the liver receptor homolog-1 (LRH-1 ).

In the present, the measure of expression level may be carried out by any adapted method known from one skilled in the art. It may be for example a method including or be based on amplification, for example Whole Transcriptome Assay (WTA), followed by high-throughput sequencing (NGS), Polymerase Chain Reaction (PCR), real-time polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), realtime quantitative polymerase chain reaction (RT-qPCR), hybridization methods, e.g. hybridization arrays, and quantitative polymerase chain reaction (qPCR), real-time quantitative polymerase chain reaction (RT- qPCR), hybridization methods, e.g. hybridization arrays (chips), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc. ), and processes including sequencing steps, for example, using Illumina or lonTorrent platforms.

In the present, the referenced expression level of C/EBP[3 or IRF8 may be the expression level of C/EBP[3 or IRF8 measured in a biological sample of healthy subjects, or the mean expression level of C/EBP[3 and IRF8 measured in a group of healthy subjects. Preferably, the reference expression level of C/EBP[3 or IRF8 may be the level expression of C/EBP[3 or IRF8 measured in a pool of biological samples from healthy subjects.

In the present the comparison of expression level may be carried out with any adapted method known from one skilled in the art.

The inventors have also surprisingly demonstrated that it is possible to determine from a biological sample the susceptibility of a cancer to a compound inhibiting the peroxisome prol iterator-activated receptors gamma (PPARy).

Accordingly, another object of the invention is a method for determining the susceptibility of a cancer from a biological sample to a compound inhibiting the peroxisome proliferator-activated receptors gamma (PPARy) comprising: i. isolating monocytes from the biological sample, ii. culturing the isolated monocyte with a compound inhibiting the peroxisome proliferator-activated receptors gamma (PPARy), iii. observation of the obtained culture of b) where when the obtained culture of ii) does not comprise MDSC, the cancer is susceptible to a compound inhibiting the peroxisome proliferator-activated receptors gamma (PPARy)

Compound inhibiting the peroxisome proliferator-activated receptors gamma (PPARy) is as defined above.

Myeloid-derived suppressor cells (MDSC) are as defined above.

The biological sample is as defined above.

Isolated Monocyte means isolated cells expressing CD14⁺ and CD16⁺ In the present, isolating monocyte, for example from a biological sample, may be carried out by any method adapted know from one skilled in the art. It may be for example a method involving a centrifugation step. In the present the monocyte may be cultured in any adapted cultured medium known from one skilled in the art. It can be for example a commercially available culture medium, for example PRMI medium.

According to the invention, monocyte culture step ii. can be carried out in PRMI culture medium. According to the invention, monocyte culture step ii. can be carried out in PRMI culture medium comprising Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin 4 (IL4) and/or Prostaglandin E2 (PGE2).

According to the invention, monocyte culture step ii. can be carried out in a culture medium comprising at least one differentiation factor. It may be for example a culture medium comprising at least one differentiation factor selected from the group comprising IL-4, GM-CSF and PGE-2. It may be for example a culture medium comprising at least two differentiation factors selected from the group comprising IL-4, GM-CSF and PGE-2. It may be for example a culture medium comprising IL-4, GM-CSF and PGE-2.

According to the invention, monocyte culture step ii. can be carried out in PRMI culture medium comprising at least one differentiation factor selected from the group comprising Granulocyte Macrophage Colony- Stimulating Factor (GM-CSF), Interleukin 4 (IL4) and/or Prostaglandin E2 (PGE2), for example monocyte culture step b. can be carried out in PRMI culture medium comprising Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin 4 (IL4) and/or Prostaglandin E2 (PGE2).

According to the invention the concentration of differentiation factor in the monocyte culture medium may be from 1 to 2000 ng/ml.

According to the invention the monocyte culture medium may comprises a Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) concentration from 1 to 20ng/mL, for example of 20ng/mL.

According to the invention the monocyte culture medium may comprises an Interleukin 4 (IL4) concentration from 1 to 10 ng/ml, for example 10 ng/ml. According to the invention the monocyte culture medium may comprises a Prostaglandin E2 (PGE2). concentration from 0.2 to 5pmol/L, for example 2.6 pmol/L.

In the present, the monocyte culture step ii. can be carried out at a temperature comprised from 36 to 38°C, or equal to 37°C.

In the present, monocyte culture of step ii can be comprised from 2 to 7 days, from 3 to 6 days, of 5 days.

In the present, the observation of culture of isolated monocyte may be carried out by any adapted method known from one skilled in the art. It may be for example flow cytometry.

The process may further comprise the following steps: f. measuring the expression level of at least one among CCAAT/enhancer binding protein beta (C/EBP[3), LRH-1 , PPARy and Interferon regulatory factor 8 (IRF8) of cultured cell of step b, g. comparing respectively of the expression level measured at step f with a referenced expression level of C/EBP[3, LRH-1 , PPARy and IRF8 when the expression level of C/EBP[3, LRH-1 , PPARy measured at step f is superior than the referenced expression level of C/EBP[3 LRH-1 , PPARy and/or when the expressions level of IRF8 measured at step d is less to the referenced expression level of IRF8, the cancer is susceptible to a compound inhibiting the peroxisome proliferator-activated receptors gamma (PPARy).

The inventors have also surprisingly demonstrated that it is possible to determine from a biological sample the susceptibility of a cancer to a test compound.

Accordingly, another object of the invention is a method for determining the susceptibility of a cancer involving involved myeloid-derived suppressor cells (MDSC) from a biological sample to a test compound: i. isolating from the biological sample a monocyte, j. culturing the isolated monocyte with a test compound, k. observation of the obtained culture of step j where when the obtained culture of j does not comprise MDSC, the cancer is susceptible to said test compound.

Myeloid-derived suppressor cells (MDSC) are as defined above.

The biological sample is as defined above.

According to the invention, monocyte culture step j. can be carried out in PRMI culture medium. According to the invention, monocyte culture step j. can be carried out in PRMI culture medium comprising Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin 4 (IL4) and/or Prostaglandin E2 (PGE2).

In the present, the monocyte culture step j. can be carried out at a temperature comprised from 36 to 38°C, or equal to 37°C.

In the present, monocyte culture of step j can be comprised from 2 to 7 days, from 3 to 6 days, of 5 days.

The process may further comprise the following steps: h. the expression level of at least one among CCAAT/enhancer binding protein beta (C/EBP[3), LRH-1 , PPARy and Interferon regulatory factor 8 (IRF8) of cultured cell of step j, i. comparing respectively of the expression level measured at step h with a referenced expression level of C/EBP[3, LRH-1 , PPARy and IRF8 when the expression level of C/EBP[3 measured at step h is less than the referenced expression level of C/EBP[3 and/or when the expressions level of IRF8 measured at step h is superior to the referenced expression level of IRF8, the cancer is susceptible to a test compound.

In the present, the measure of expression level may be carried out by any adapted method known from one skilled in the art. It may be for example a method including or be based on amplification, for example Whole Transcriptome Assay (WTA), followed by high-throughput sequencing (NGS), Polymerase Chain Reaction (PCR), real-time polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), realtime quantitative polymerase chain reaction (RT-qPCR), hybridization methods, e.g. hybridization arrays, and quantitative polymerase chain reaction (qPCR), real-time quantitative polymerase chain reaction (RT- qPCR), hybridization methods, e.g. hybridization arrays (chips), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc. ), and processes including sequencing steps, for example, using Illumina or lonTorrent platforms. For example, it may be a commercially available method.

In the present a test compound may be any compound known from one skilled in the art and/or commercially available. It may be any compound whatever its form. It may be for example a chemical compound, a peptide, a polynucleic acid, an antibody, an aptamer.

Other advantages may still be apparent to those skilled in the art by reading the examples below, illustrated by the accompanying figures, given by way of illustration.

Brief description of the figures:

Figure 1: Figure 1 A represents a scheme illustrating the time in days between the intra peritoneal (i.p.) injection of MC38 cells (day 0) in 9-12 week-old C57BL/6 mice in which the Lrh-1 or Ppary gene were selectively disrupted in myeloid derived cells (noted Lrh-1 ^M’^/_ and Pparg^M’^/_ respectively) and their wild type littermates (noted Lrh-1 ^M+/+ and Pparg^M+/+ respectively) and their sacrifice (day 15 post tumor cell injection) to collect Tumors and peritoneal liquids. Figure 1 B are pictures of peritoneal tumors 15 days post tumor cell injection of Lrh-1 ^M-/’’ Pparg^M’^/_, Lrh-1 ^M+/+ and Pparg^M+/+ mice, the arrows indicate tumors. Figure 1 C represents diagram of the peritoneal tumor weights in mg (ordinate) 15 days post tumor cell injection from Lrh-1 ^M’^/_’ Pparg^M’^/_, Lrh-1 ^M+/+ and Pparg^M+/+ mice (abscissa) Data are represented as mean +/- s.e.m. ****P<0.0001 compared to the corresponding wild-type littermates. Data pool represent three independent experiments (n=15 mice per group).

Figure 2. Figure 2 A represents pictures of Dot-Plot of myeloid cell populations in the tumor microenvironment obtained from 9-12 week-old C57BL/6 mice in which 1.10⁷ MC-38 cells were intraperitoneally injected and in which the Lrh-1 or Pparg gene were selectively disrupted in myeloid derived cells (Lrh-1^M'^!' and Pparg^M'^!' respectively) and their wild type littermates (Lrh-1^M+I+ and Pparg^M+l+ respectively). The myeloid cell populations in the tumor microenvironment were harvested and evaluated 15 days post tumor cell injection by flow cytometry after staining with appropriated markers i.e. CD45-PEVio770, CD11 b-FITC, F4/80- PerCPVio700, Ly6C-Vioblue, Ly6G-APC and Siglec-F-PE (Myltenyi Biotec). On this figure SSC means side scatter (granularity), CD11 b means antibodies against CD11 b. Myeloid cells were gated as CD45+ CD11 b+ (gate A) . Histogram represents the percentage of CD45+ myeloid cells (ordinate) obtained from Lrh-1^M'^!' Pparg^M'^!', Lrh-1^M+I+ and Pparg^M+l+ mice. Figure 2 B represents pictures of Dot-Plot of myeloid cell populations in the tumor microenvironment obtained from 9-12 week-old C57BL/6 mice in which 1 .10⁷ MC-38 cells were intraperitoneally injected and in which the Lrh- 1 or Pparg gene were selectively disrupted in myeloid derived cells (Lrh-1^M~ ^/_ and Pparg^M'^!' respectively) and their wild type littermates (Lrh-1^M+I+ and Pparg^M+l+ respectively). The myeloid cell populations in the tumor microenvironment were harvested and evaluated 15 days post tumor cell injection by flow cytometry after staining with appropriated markers i.e. CD45-PEVio770, CD11 b-FITC, F4/80-PerCPVio700, Ly6C-Vioblue, Ly6G- APC and Siglec-F-PE (Myltenyi Biotec). M-MDSC/monocytes were gated as CD1 1 b⁺ Ly6G’ Ly6C^hi (gate B), G-MDSC/neutrophils were gated as CD11 b⁺ Ly6G⁺ Ly6C^l0 (gate C) and macrophage/DC were gated as CD11 b⁺ Ly6C’ Ly6G’ (gate D). Figure 2 C represents histograms of the proliferation index (ordinate) of CD4 or CD8 T cells after 3 days of co-culture from the three myeloid subpopulations sorted and co-cultured with splenocytes from wild type mice labeled with CFSE and activated by CD3/CD 28 antibodies (T cell/sorted cell ratio of 2:1 ). The cells are CD4⁺alone (control), CD4⁺ Ly6G⁺ Ly6C^l0, CD4⁺ Ly6G’ Ly6C^hi , CD4⁺ Ly6C’ Ly6G; CD8⁺ alone (control), CD8⁺ Ly6G’ Ly6C^hi , CD8⁺ Ly6G⁺ Ly6C^l0, CD8⁺ Ly6C’ Ly6G’.Data are represented as mean +/- s.e.m *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 , ## P<0.01 , ### P<0.001 compared to the corresponding control.

Figure 3 : figure 3A is a schematic representation of the in vitro MDSC differentiation assay. Figure 3 B represents histograms showing the T cell proliferation index (ordinate) after 2 days of co-culture of MDSC were cocultured with CD3/CD28 activated splenocytes from wild-type mice (Lrh-1 M^+/+) or Lrh-1 M ^/_ mice).

Figure 4: Figure 4 A represents a schematic representation of the injection kinetics: 1.10⁷ MC-38 cells were intraperitoneally injected in 9-12 week-old C57BL/6 Lrh-1^M+I+ and Pparg^M+l+ mice in which the Lrh-1 or Pparg gene were selectively disrupted in myeloid derived cells (Lrh-1^M'^!' and Pparg^M'^!' respectively) and their wild type littermates (Lrh-1^M+I+ and Pparg^M+l+ respectively). Lrh-1^M+I+ and Pparg^M+l+ were i.p injected with ML-180 (30 mg/kg) or GW-9662 (5 mg/Kg). Tumors and peritoneal liquids were collected 15 days post-tumor cell injection. Figure 4 B represents the Peritoneal tumor weights in mg (ordinate) of Lrh-1^M'^!', Pparg^M'^!' ,Lrh-1^M+l+ and Pparg^M+l+ mice at day 15 injected (+) or not (-) with ML-180, GW-9662 or a vehicle (control). Figure 5 C represents histograms showing myeloid cell subpopulations (CD11 b⁺ Ly6G⁺ Ly6C^Lo, CD11 b⁺ Ly6G’ Ly6C^hi, CD11 b⁺ Ly6G’ Ly6C^Lo (percentage of CD11 b+ cells (ordinate)) in the tumor microenvironment of Lrh-1^M'^!', Pparg^M'^!' ,Lrh-1^M+l+ and Pparg^M+l+ mice at day 15 injected (+) or not (-) with ML-180, GW-9662 or a vehicle (control) (abscissa) evaluated by flow cytometry after staining with appropriated markers. Figure 4 D represents histograms showing the percentage of CD4 cells (ordinate), in particular the percentage of Th 1 -related T cells identified as CD4⁺ CD183⁺ CCR6’ and the percentage of cytotoxic related T cells identified as CD8⁺ CD183⁺ in the tumor microenvironment of Lrh-1^M'^!', Pparg^M'^!' ,Lrh-1^M+l+ and Pparg^M+l+ mice at day 15 injected (+) or not (-) with ML-180, GW-9662 or a vehicle (control) evaluated by flow cytometry after staining with appropriated markers. Figure 4 E represents histograms showing the percentage of cell viability (ordinate, in particular MC-38 viability at 24 h and 48 h after injection of 3,7pM, 10pM or 100pM of ML-180 or of 5 pM, 10pM or 100pM of GW-9662 or without injection of ML-180 or GW-9662 (Ctr). Data are represented as mean +/- s.e.m *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 compared to the corresponding control.

Figure 5 :Histograms (Figure 6 A-C) of mRNA relative expression (ordinate) from 9-12 week-old C57BL/6 mice in which the Lrh-1 or Pparg gene were selectively disrupted in myeloid derived cells (Lrh-1^M'^!' and Pparg^M'^!' respectively) and their wild type littermates (Lrh-1^M+I+ and Pparg^M+l+ respectively) in which 1.10⁷ MC-38 cells were intraperitoneally injected. 15 days post tumor cell injection, peritoneal cells were harvested. The three myeloid subpopulations were sorted for phenotypic studies using fluorescent activated cell sorting (FACS). Gene expression of immunosuppressive and differentiation markers: Arginase-1 , Inos, IL-10, Pdl-1 , F4/80, MHCII, C/EBP[3, IRF8, Ccl22 and Ccl4 was evaluated by RT qPCR Data are represented as mean +/- s.e.m. ns: not significant, *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 compared to the corresponding control.

Figure 6 : LRH-1 in myeloid cells promote the accumulation of immunosuppressive G-MDSC subpopulation in the tumor microenvironment and peritoneal carcinomatosis from pancreatic origin development. Figure 6A represents diagram of the tumor weights in mg (ordinate) from Lrh-1 ^+/+_’ Lrh-T^/_ and Pparg^M+/+ mice (abscissa). (A-B) 15.104 R211 -luc cells were intraperitoneally injected in 9-12-week-old C57BL/6 mice in which the Lrh-1 gene were selectively disrupted in myeloid-derived cells (Lrh-1 M^_/_) and their wild-type littermates (Lrh-1 M^+/+). Tumors and peritoneal liquids were collected 15 days post-tumor cell injection. (A) Peritoneal tumor weights 15 days post-tumor cell injection (n=4 for each group). Figure 6B representspictures of Dot-Plots and histogram quantifications showing myeloid cell populations in the tumor microenvironment, evaluated by flow cytometry after staining with appropriated markers. Doublets were exclued and CD45+ viable cells were gated (not shown). M-MDSC/monocytes were gated as CD11 b+ Ly6G- Ly6Chi (gate B), G-MDSC/neutrophils were gated as CD11 b+ Ly6G+ Ly6Clo (gate C) and macrophage/DC were gated as CD1 1 b+ Ly6C- Ly6G- (gate D). Figure 6 B represents pictures of Dot-Plot of myeloid cell populations in the tumor microenvironment in which the Lrh-1 was selectively disrupted and their wild type littermates (Lrh-1^M+I+).

Histogram represents the percentage of CD11 b+ cells (ordinate) of M- MDSC/monocytes Ly6G- Ly6Chi, G-MDSC/neutrophils Ly6G+ Ly6Clo and macrophage/DC Ly6C- Ly6G-. Data are represented as mean +/- s.e.m. *P<0.05, **P<0.01 , ***P<0.001 compared to the corresponding control.

Figure 7 : LRH-1 and PPARy promotes the differentiation of MDSC from human monocytes (A) Human MDSC from healthy or pathology-proven peritoneal carcinomatosis from colorectal origin blood samples were characterized through flow cytometry. Figure 7A represents Dot-Plot showing MDSC (CD45⁺ CD11 b⁺ CD33⁺ HLA-DR-) and histogram quantification of MDSC (ordinate represents the percentage of CD45+CD1 1 b+CD33+HLA-DR-) (n=6 healthy donors (gray) and 6 patients (“cancer patients” (black)). (B-C) Human MDSC from healthy or pathology- proven peritoneal carcinomatosis from colorectal origin biopsies were isolated. Figure 7B represents histograms of gene expression of (B) MDSC immunosuppressive markers i.e. ARG1 , NOS2, HLA-DR; IL-10, TGF-beta, PD-L1 , CCL4 and CCL22 (ordinate : Relative mRNA level) and Figure 7C represents histograms of transcription factors (i.e. CERPbeta, IRF8, PPARy and NR5A2 regulating MDSC differentiation was analyzed through Real- Time qPCR of healthy biopsies (gray) and tumor biopsies (black) (n=10 for each group). Figures 7 D-E Bone marrow cells from healthy donors were isolated. These cells were lineage depleted to isolate monocytes which were then cultured with MDSC differentiation factors (IL-4, GM-CSF and PGE2). After 5 days of culture, cells were phenotyped by flow cytometry. Figure 7 D is a) Schematic representation of the in vitro human MDSC differentiation assay. Figure 7 E is an histogram showing the percentage of in vitro differentiated human MDSC (CD45⁺ CD11 b⁺ CD33’ HLA-DR’) (ordinate) regarding the culture conditions : without differentiation factors (untreated) with IL-4, GM-CSF and PGE2, with IL-4, GM-CSF and PGE2 and ML180 (2,5pM) or with IL-4, GM-CSF and PGE2 and GW9662 (10pM): . Data are represented as mean +/- s.e.m. *P<0.05, **P<0.01 , ***P<0.001 ,

****p<0.0001 compared to the corresponding control.

EXAMPLES

Example 1 :

EXPERIMENTAL PROCEDURES

MC-38 cell culture

The MC-38 murine colon carcinoma cell line (Kerafast) was cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen), supplemented with L-glutamine (Invitrogen), penicillin, streptomycin (Invitrogen), and 10% heat-inactivated fetal calf serum.

Mice

All mouse experiments were performed according to protocols approved by the institutional ethics committee (CEEA122) with permit number 6555- 2016082912056664 v3 in accordance with European legal and institutional guidelines (2010/63/UE) for the care and use of laboratory animals. Lrh-1 ^M- ^/_ and Ppary^M’^/_ mice have been described earlier (Lefevre et al., 2013) and the corresponding floxed littermates were used as controls throughout all the experiments (Gales et al., 2010; Lefevre et al., 2013).

In vivo MC-38 tumors, CD4 and CD8 T cell depletion, ML-180 and GW- 9662 treatments.

Syngeneic MC-38 cells were administered intra-peritoneally (i.p) (1.10⁷ cells/mouse in 500 pL phosphate-buffered saline (PBS)) in 9-12-week-old C57BL/6 mice. 12- or 15-days post-tumor cell injection, mice were euthanized using CO2 asphyxia. Tumors and peritoneal liquids were collected for further analysis. For CD4 and CD8 T cells depletion, mice were i.p injected with Mab CD8a clone YTS 169.4 and Mab CD4 clone GK1.5 (BioXcell). (200pg/mouse, 2 days before MC-38 injection and then every 3 days) or with a vehicle solution.

ML-180 was purchase to Cayman chemical. ML-180 is also designated SR1848.

GW-9662 was purchase to Cayman chemical.

For ML-180 treatment, mice were i.p injected with SR1848 (SIGMA) formulated in 10% DMSO and 10% Tween 80 in Phosphate Buffer Saline (PBS) (30mg/kg, 3 days after MC-38 injection and then every 3 days) or with a vehicle solution.

For GW-9662 treatment, mice were i.p injected daily with GW-9662 (SIGMA) formulated in 5% Tween 80 in PBS (5mg/kg, 1 day after MC-38 injection) or with a vehicle solution.

Assessment of tumor burden.

Mice were monitored daily for signs of tumor progression and evaluation of body weight. After mice euthanasia, peritoneal tumors were removed and weighed.

Flow cytometry

After mice euthanasia, peritoneal cells were harvested, centrifuged and red blood cells were lysed with ACK lysing buffer. All analyses were gated on viable cells after a Live/Dead Staining (Molecular Probes® LIVE/DEAD Fixable Violet, Aqua or Yellow Dead Cell Stain Kit, Life technologies™).

For myeloid cells analysis, cells were labeled with the following antibodies: CD45-PEVio770, CD11 b-FITC, F4/80-PerCPVio700, Ly6C-Vioblue, Ly6G- APC and Siglec-F-PE (Myltenyi Biotec)

For lymphocyte infiltration/activation and Th1 profile studies, peritoneal cells were labeled with the following antibodies: CD45-PEVio770, CD3-PE, CD3- Percp, CD4-APCVio700, CD8-VioGreen, CD25-VioBrightFITC, NK1.1- PerCPVio700, Foxp3-APC, CD183-APC, IFN-y-FITC, IL-2-PE, TNF-a-APC. (Myltenyi Biotec). For cytokine intracellular stainings, cells were stimulated for 1 hour with tetradecanoyl phorbol acetate (TPA) (50ng/ml) and ionomycin (5pg/ml) followed by 4 hours with TPA (50ng/ml), ionomycin (5pg/ml) and monensin (1/1000). The intracellular stainings were performed with the inside stain kit (Miltenyi Biotec) following manufacturer’s recommendations. Appropriate fluorochrome-matched isotype antibodies (Miltenyi Biotec) were used to determine nonspecific background stainings. All stainings were performed on 100 pL of PBS ^/_ 1 % heat-inactivated fetal calf serum. All analyses were performed using a BD Fortessa flow cytometer with the Diva software.

Myeloid cell sorting

Peritoneal cells were stained with the following antibodies: CD45- PEVio770, CD11 b-FITC, Ly6C-Vioblue, Ly6G-APC (Miltenyi Biotec). Ly6C means Lymphocyte antigen 6 complex; Ly6G means Lymphocyte antigen 6 Complex locus G6D. The three myeloid subpopulations (Ly6G⁺ Ly6C^l0, Ly6G’ Ly6C^hi and Ly6G’ Ly6C’) were sorted with a BD InfluxTM cell sorter. The presence or absence of each of the cell markers may be denoted by a + or a - sign, respectively. The designation low or high relates to cells which show a relatively low or high expression level respectively. in vitro MDSC differentiation assay

Bone marrow cells were harvested from the femur of Lrh-1 ^M_/_ and Pparg^M’^/_ mice and their wild-type littermates (Lrh-1 ^M+/+and Pparg^M+/+ respectively). B cells, T cells, dendritic cells, macrophages, granulocytes, and red blood cells were depleted using the mouse lineage cell depletion kit and LS columns from Milteniy Biotec according to the manufacturer’s directions. Lineage- depleted bone marrow cells were >50% ckit+ (CD117) as assayed by flow cytometry. After depletion, cells were cultured between 1 ,10⁵ cells/mL and 5.10⁵ cells/mL in 24-well plates at 37°C in 5% CO2 for 5 days in RPMI medium (supplemented with 3% FCS, 1 % penicillin, streptomycin (Invitrogen)) containing GM-CSF (Invitrogen, 20ng/mL), IL-4 (Clinisciences, 10 ng/ml) and PGE2 (Cayman, 2.6 pmol/L) (Sinha et al., 2007). At the end of the assay, myeloid cell subpopulations were analyzed by flow cytometry.

T cell inhibition assay

Sorted myeloid subpopulations were cocultured in RPMI medium (Invitrogen) at a 1 :2 ratio with splenocytes extracted from wild-type mice, previously labeled with Carboxyfluorescein succinimidyl ester (CFSE) (BD Biosciences, 5pmol/L) and activated by CD3e monoclonal antibody (eBioscience 10pg/ml) and CD28 monoclonal antibody (eBioscience 2pg/ml). T cell proliferation was analyzed by flow cytometry by CFSE dilution measurement after 2 or 3 days of co-culture.

Tumor cell viability assay

MC-38 cells were seeded in 96-well plates (5.10³ cells/well) and incubated at 37 °C, 5% CO2. After 24h, tumor cells were treated with ML-180 and GW- 9662 (from 0 to 100 pM) and incubated for additional 24h or 48h. After drug exposure, culture medium was removed and adherent cells were incubated for 2h with 50 pl of medium containing for example PBS and a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 12mM) (Sigma-Aldrich). The reduced formazan crystals were then dissolved in 50 pl DMSO by incubating at 37°C for 30 min in the dark. Cell viability was measured by reading the absorbance at 540 nm using a microplate reader (Envision, PerkinElmer).

Reverse Transcription and Real-Time PCR

Sorted cell mRNA were isolated using the RNAqueous®-Micro Total RNA Isolation Kit (Thermofisher) using the manufacturer’s protocol. Synthesis of cDNA was performed according to the manufacturer’s recommendations (Verso Kit, Thermo electron). RT-qPCR was performed on LightCycler 480 system using LightCycler SYBR Green I Master (Roche Diagnostics). Amplification reactions were performed in a total volume of ten microliters using the following cycling conditions: 60 cycles (10 s at 95°C, 10 s at 60°C and 10 s at 72°C). The primers (at a final concentration of 5 pM) were designed with the software Primer 3. Glyceraldehyde-3-phosphate deshydrogenase (GAPDH) mRNA was used as the invariant control. Serially diluted samples of pooled cDNA were used as external standards in each run for the quantification. Primer sequences are listed in table 1 below. Table 1 : primer sequences

Statistical analysis

For each experiment, data were subjected to one-way analysis of variance followed by the means multiple comparison method of Bonferroni-Dunnett. P < 0.05 was considered as the level of statistical significance.

LRH-1 and PPARy in myeloid cells contribute to the progression of the peritoneal carcinomatosis. To determine the impact of LRH-1 and PPARy in myeloid cells on the development of the peritoneal carcinomatosis, the syngeneic MC-38 murine adenocarcinoma cells were intraperitoneally (i.p) injected in 9-12-week-old mice in which the Lrh-1 or Pparg gene were selectively disrupted in myeloid- derived cells (Lrh-1 ^M_/_ and Pparg^M’^/_ respectively) (n=15 mice per group). In particular, 1.10⁷ MC 38 cells were intraperitoneally injected in 9-12 week-old C57BL/6 mice in which the Lrh-1 or Pparg gene were selectively disrupted in myeloid derived cells (Lrh-1 ^M_/_ and PpargM-/- respectively) and their wild type littermates (Lrh-1 M+/+ and PpargM+/+ respectively). Tumors and peritoneal liquids were collected 15 days post tumor cell injection. (Figure 1A). MC-38 cell intraperitoneal injection (Figure 1A) resulted in the development of solid tumors into the peritoneal cavity (Figure 1 B). Figure 1 B represents pictures of peritoneal tumors 15 days post tumor cell injection, the arrows indicate tumors. Figure 1 C represents the peritoneal tumor weights 15 days post tumor cell injection Data are represented as mean +/- s.e.m. ****P<0.0001 compared to the corresponding wild-type littermates. Data pool represent three independent experiments. Interestingly, the strongly significant lower tumor weight observed in Lrh-1^M'^!' and Pparg^M'^!' at day 15 post tumor cell injection compared to their respective wild-type littermates (Lrh-1^M+I+ and Pparg^M+/+) demonstrated that Lrh-1 and Pparg in myeloid cells promote tumor development (Figure 1 B-C). Thus, these first results clearly establish that LRH-1 and PPARy in myeloid cells are involved in the progression of the peritoneal carcinomatosis of colorectal origin.

LRH-1 and PPARy in myeloid cells promote the accumulation of immunosuppressive G-MDSC and M-MDSC subpopulations in the tumor microenvironment.

To determine how LRH-1 and PPARy deficiency in myeloid cells reduces tumor development in mice, an evaluation of the myeloid cell infiltration into the tumor microenvironment from Lrh-1^M'^!' and Pparg^M'^!' mice and their wildtype littermates was carried out (Figure 2). Although LRH-1 and PPARy did not impact the proportion of total CD11 b⁺ myeloid cells (Figure 2A), myeloid subpopulation proportions were drastically altered (Figure 2B). Indeed, LRH- 1 and PPARy deficiency in myeloid cells induced a decrease in the proportion of CD11 b⁺ Ly6G⁺ Ly6C^Lo subpopulation (Figure 2B). This was mirrored by an increase of both CD11 b⁺ Ly6G’ Ly6C^hi and macrophages/DC (CD11 b⁺ Ly6G’ Ly6C’) populations (Figure 2B).

Given that phenotypic and morphological criteria are not sufficient to clearly distinguish G-MDSC from neutrophils, as both exhibit a CD11 b⁺ Ly6G⁺ Ly6C^Lo phenotype, and M-MDSC from monocytes, as both display a CD11 b⁺ Ly6G’ Ly6C^hi profile, a co-culture of these sorted subpopulations with T cells was carried out and an evaluation of their ability to specifically suppress CD4 and CD8 T cell proliferation was done (Figure 2C). In Lrh-1^M+I+ and Pparg^M+l+ tumor-bearing mice, the strong immunosuppressive activity of sorted CD11 b⁺ Ly6G⁺ Ly6C^Lo and CD11 b⁺ Ly6G’ Ly6C^hi subpopulations allowed the identification of these cell subsets as respectively G-MDSC and M- MDSC (Figure 2C). Consistent with the strong immunosuppressive activity of these two subpopulations in Lrh-1^M+I+ and Pparg^M+l+ tumor bearing mice, immunosuppressive genes such as Arginase-1, Inos, 11-10 and Pdl-1 were highly expressed (Figure 6A).

Interestingly, CD11 b⁺ Ly6G⁺ Ly6C^Lo and CD11 b⁺ Ly6G’ Ly6C^hi subpopulations completely lost their immunosuppressive capacity in Lrh-1^M~ ^/_ and Pparg^M'^!' mice when co-cultured with T cells (Figure 2C) and displayed a weak expression of Arginase-1, Inos, 11-10 and Pdl-1 compared to their wild-type littermates (Figure 5A). These findings demonstrate that the lack of LRH-1 and PPARy in myeloid cells rather promotes their differentiation towards monocytes and Macrophages/DC. Supporting these data, the expression of characteristic markers of macrophage/DC differentiation such as F4/80 and MHCII in CD11 b⁺ Ly6G⁺ Ly6C^Lo and CD11 b⁺ Ly6G’ Ly6C^hi subpopulations was significantly increased in Lrh-1^M'⁷' Pparg^M ~ tumorbearing mice (Figure 5B). Moreover, macrophages/DC populations did not show any immunosuppressive activity but rather promoted CD4 T cell proliferation (Figure 2C). Their immunosuppressive ability was not modified in Lrh-1^M' and Pparg^M'' mice (Figure 2C).

These results highlight LRH-1 and PPARy in myeloid cells as critical contributors in the accumulation of immunosuppressive G-MDSC and M- MDSC subpopulations instead of non-immunosuppressive monocytes and macrophages/DC during the development of peritoneal carcinomatosis of colorectal origin.

LRH-1 induces the differentiation of MDSC through PPARy ligand synthesis.

To further investigate the mechanisms by which LRH-1 and PPARy promotes MDSC accumulation into the tumor microenvironment, an evaluation of their impact on an in vitro MDSC differentiation assay was carried out. Bone marrow cells were isolated from the femur of Lrh-1^M'^!' and Pparg^M-^!- _{m jce anc}| their _W||_C| _typ_e ijttermates. These cells were lineage depleted and the remaining progenitor cells were cultured with MDSC differentiation factors (IL-4, GM-CSF and PGE-2). After 5 days of culture, cells were phenotyped by flow cytometry and their immunosuppressive activities were evaluated (Figure 3A). Surprisingly, in absence of LRH-1 or PPARy, the ability of bone marrow progenitor cells to differentiate into immunosuppressive MDSC (CD11 b⁺ Ly6G’ Ly6C^hi) was significantly reduced (Figure 3B), supporting the central role of these two nuclear receptors in the differentiation of bone marrow progenitor cells into MDSC. This was reinforced by the decreased expression of C/EBP[3, a transcriptional regulator of MDSC differentiation (Marigo et al., 2010), and by the upregulation of IRF8, a well-described transcriptional regulator of macrophage/DC differentiation (Gabrilovich and Nagaraj, 2009b), in CD11 b⁺ Ly6G⁺ Ly6C^Lo and CD11 b⁺ Ly6G’ Ly6C^hi cells from Lrh-1 ^M-/_ and Pparg^M’^/_ tumor-bearing mice (Figure 5). Pharmacological inhibition of LRH-1 reduces tumor growth in vivo by promoting myeloid cell differentiation toward non immunosuppressive monocytes and macrophages/ DCs.

LRH-1 and PPARy were investigated as relevant therapeutic targets for the treatment of cancer, in particular the peritoneal carcinomatosis of colorectal origin. The efficiency of both a reverse LRH-1 agonist (ML-180) and a specific PPARy antagonist (GW-9662) were evaluated in vivo on the development of peritoneal carcinomatosis of colorectal origin (Figure 4A). ML-180 and GW-9662 treatments in Lrh-1^M+I+ or Pparg^M+l+ tumor-bearing mice significantly reduced the tumor load similarly to what was observed in Lrh-1^M'^!' or Pparg^M'^!' mice (Figure 4B). In line with the tumor decrease, the pharmacological treatments induced in Lrh-1^M+I+ and Pparg^M+l+ tumorbearing mice a strong reduction of the G-MDSC (CD11 b⁺ Ly6G⁺ Ly6C^Lo) which was mirrored by which was mirrored by an increase of the monocytelike (CD11 b⁺ Ly6G’ Ly6C^hi) and macrophage/DC (CD1 1 b⁺ Ly6G’ Ly6C’) as observed in Pparg^M'^!' and Lrh-1^M'^!' (Figure 4C). Interestingly, the decrease of G-MDSC subpopulation correlates with a drastic increase of the percentage of Th1 -related T cells (CD4⁺ CD183⁺ CCR6’) and cytotoxic- related T cells (CD8⁺ CD183⁺) in Lrh-1^M+I+ and Pparg^M+l+ tumor-bearing mice treated with GW-9662 and ML-180 compared to Lrh-1^M+I+ and Pparg^M+l+ untreated mice (Figure 4C-D). These results demonstrate that the inhibition of both LRH-1 and PPARy, by ML-180 or GW-9662 respectively, promotes the differentiation of myeloid cells toward non-immunosuppressive monocytes/macrophages/DC, resulting in the activation of CD4 and CD8 T cells and hence in the inhibition of the peritoneal carcinomatosis of colorectal origin development. To determine the direct impact of ML-180 and GW-9662 on colon tumor cells, an evaluation of their ability to modify the viability of colon adenocarcinoma cells (MC-38) was carried out. In accordance with the well-established role of LRH-1 in the induction of intestinal cell proliferation through the [3-Catenin dependent pathway (Botrugno et al., 2004), ML-180 significantly decreased the viability of MC- 38 at 24h and 48h at concentrations ranging from 10 pM to 100pM (Figure 4E). Moreover, the GW-9662 treatment had no impact on MC-38 viability at 5 and 10pM, while it increased the tumor cell viability at 100pM (Figure 4E). This improved viability was in line with several studies identifying PPARy as a tumor suppressor gene (Park and Kwak, 2012). Altogether these results demonstrate that both ML-180 and GW-9662 treatments decrease the peritoneal carcinomatosis development by preventing MDSC accumulation in the tumor microenvironment. Thus, these data highlight for the first time the two nuclear receptors LRH-1 and PPARy as critical targets in the progression of peritoneal carcinomatosis of colorectal origin. Furthermore, the direct antiproliferative effect of ML-180 on colon tumor cells strengthens the use of LRH-1 inhibitors in anti-cancer therapy.

To investigate whether LRH-1 -induced immunosuppressive tumor microenvironment can be extended to another type of tumor, the progression of peritoneal carcinomatosis of pancreatic origin in Lrh-1^M+I+ and Lrh-1^M'^!' mice was evaluated (Figure 6). Interestingly, the tumor burden in peritoneal cavity, as reflected by the tumor weight, was significantly decreased in Lrh- at day 15 post pancreatic tumor cells (R211 ) injection compared to their respective wild-type littermates (Lrh-1^M+I+ mice) (Figure 6A). In agreement with the data observed in colorectal peritoneal carcinomatosis, LRH-1 deficiency in myeloid cells induced a decrease in the proportion of both G-MDSC (gate C) and M-MDSC (gate B) accompanied by an increase of macrophages/DC populations (gate D) (Figure 6B). Overall, these data support the extension of LRH-1 targeting as a therapeutic strategy in various MDSC infiltrating peritoneal carcinomatosis.

LRH-1 and PPARy promotes the differentiation of MDSC from human monocytes

The level of circulating MDSC (CD45⁺, CD11 b⁺, CD33⁺, HLA-dr) was investigated in peripheral blood of patients with colonic peritoneal carcinomatosis (Figure 7A). Although healthy subjects presented an insignificant proportion of circulating MDSCs, cancer patients showed a high frequency of MDSCs in the peripheral blood (30% of the CD45+ population). In order to confirm the accumulation of MDSCs in the tumor microenvironment of patients with colonic carcinomatosis, the immunosuppressive status of infiltrating CD11 b cells from tumor biopsies or biopsies of healthy subjects were analysed. The co-expression of Arginase- 1 and NOS2, characteristic of MDSCs, was found only in CD11 b cells from biopsies of cancer patients (Figure 7B).

In addition, the loss of HLA-DR expression, also characteristic of MDSCs, was also observed in CD11 b+ cells from biopsies of patients with colonic carcinomatosis. The level of ARNm encoding anti-inflammatory and immunosuppressive genes (IL-10, Tgf|3, Pd-11 , Ccl22, Ccl4) increased significantly in CD11 b cells from tumor biopsies compared to CD11 b cells from biopsies of healthy subjects (Figure 7B). Altogether, these data demonstrated an elevated frequency of MDSCs in the blood and in the tumor microenvironment of patients with colonic carcinomatosis. This accumulation of MDSCs in tumor biopsies was also supported by the increase of CEBP[3 mRNA expression, a key factor in MDSC differentiation and the decrease of IRF8, a regulator of macrophages/DC differentiation (Figure 7C).

To confirm the involvement of LRH-1 and PPARy in the control of myeloid cell differentiation into MDSCs in humans, the in vitro differentiation of MDSCs from human bone marrow monocytes was evaluated. The use of a reverse LRH-1 agonist (ML180) and a specific PPARy antagonist (GW- 9662) significantly reduced the ability of bone marrow-derived monocytes to differentiate into GM-CSF, IL-4 and PGE2-induced MDSCs (Figure 7D), confirming the crucial role of these two nuclear receptors in MDSC differentiation in humans. Increased gene expression of PPARy and LRH-1 in CD11 b cells from tumor biopsies (Figure 7C), further support PPARy and LRH-1 as major contributors to MDSC differentiation and activation and hence their accumulation during the development of peritoneal colonic carcinomatosis.

Discussion

Nuclear receptors are critical for a variety of processes including cell proliferation, differentiation and immunity (Dhiman et al., 2018; Yang et al., 2021 ; Zhao et al., 2019). Notably, some of them are involved in the regulation of tumor immunology and even serve as promising immune checkpoint (Hermann-Kleiter et al., 2015; Klepsch et al., 2018). Moreover, their activation is regulated by small lipophilic molecules that can easily be substituted by synthetic ligands as illustrated by the number of nuclear receptor-based drugs that have been used in clinical trials (Yang et al., 2021 ).

In the present example conducted in two mouse models specifically deleted for Lrh-1 or Ppary in the myeloid lineage, it is demonstrated for the first time that these two nuclear receptors play a crucial role in the promotion of peritoneal carcinomatosis of colorectal origin, as reflected by the strong tumor load decrease in Lrh-1^M'^!' and Pparg^M'^!' tumor-bearing mice compared to their wild-type littermates. No study reports any effect of LRH-1 specific deletion in the myeloid lineage on tumor development. Several studies have highlighted the controversial role of PPARy in myeloid lineage, which appears to be dependent on tumor stage. Indeed, Raquel et al. reported that PPARy specific deletion in myeloid cells exacerbates intestinal inflammation by increasing CD8 and Th1 CD4 activation and recruitment, suggesting a protective role of PPARy in myeloid cells in inflammation-induced colorectal cancer initiation (Hontecillas et al., 2011 ).

The results demonstrate and reveal that LRH-1 and PPARy nuclear receptors as major players in advanced colorectal cancer murine model through, for example their ability to promote both MDSC differentiation and activation. Indeed, in absence of LRH-1 or PPARy, ex vivo a complete loss of immunosuppressive properties of both CD11 b⁺Ly6G^high Ly6C^l0W G-MDSC and CD11 b⁺Ly6G’Ly6C^high M-MDSC was observed. These two populations displayed a strong decrease in the expression of ARG1 and iNOS, key effectors of their immunosuppressive activities (Bronte and Zanovello, 2005b; Mazzoni et al., 2002).

In addition to the loss of immunosuppressive properties in Lrh-1^M'^!' and Pparg^M'^!' mice, a drastic change in the accumulation of myeloid subpopulations illustrated by a shift from immature G-MDSC and M-MDSC towards differentiated monocyte and macrophage/DC populations was demonstrated. Moreover, in vitro a lower ability of bone marrow progenitor cells from Pparg^M'^!' and Lrh-1^M'^!' mice to generate MDSC compared to wildtype m ice-derived bone marrow progenitor cells was shown. Altogether, these results demonstrate that PPARy and LRH-1 are involved in MDSC generation through the control of “emergency” myelopoiesis.

This example also shed light on the central role of myeloid PPARy and LRH- 1 -mediated T cell suppression in the control of tumor progression. Indeed, on the one hand, the decreased tumor burden and MDSC accumulation observed in Pparg^M'^!' and Lrh-1^M'^!' mice correlates with a reactivation of the antitumor adaptive immune system. This was illustrated by an increase of Th1 -related CD4 T cells and cytotoxic-related CD8 T cells which was mirrored by a Treg decrease in Pparg^M'^!' and Lrh-1^M'^!' mice. On the other hand, T cell depletion induced by CD4 and CD8 depleting antibodies, completely abolished the decreased tumor burden observed in Pparg^M'^!' and Lrh-1^M'^!' mice. These results are further corroborated by the strong reduction of ex vivo immunosuppressive activity of MDSC deleted for PPARy or LRH- 1 and the loss of their immunosuppressive transcriptional profile. Supporting our data, the role of PPARy and LRH-1 in the control of adaptive immunity has already been demonstrated in different pathophysiological contexts. Although several studies report that PPARy and LRH-1 induce the expansion of Th2 CD4 and Tregs as well as a decrease in CD4 Th1 and Th17 T cells (Cobo-Vuilleumier et al., 2018; Hontecillas et al., 2011 ; Li et al., 2011 ), none of them reported the role of PPARy and LRH-1 in the control of adaptive immunity through MDSC biology.

Overall, in accordance with the close link between the profile of tumor infiltrating immune cells and the prognostic of colorectal cancer (Galon et al., 2014; Mlecnik et al., 2011 ; Wang et al., 2018), the obtained results clearly demonstrate that PPARy and LRH-1 are therapeutic targets to reactivate an effective anti-tumor immune system.

Tumor-bearing mice were treated with selective pharmacological inhibitors of both LRH-1 and PPARy. Interestingly, the inhibition of LRH-1 and PPARy, by respectively ML-180 and GW-9662, reduced tumor growth, prevented MDSC accumulation and promoted CD8 and CD4 T cell activation. Besides the protumor role of LRH-1 in myeloid cells, the results clearly demonstrate that LRH-1 inhibition by ML-180 reduced MC-38 viability. Consistently with the anti-proliferative role of ML-180 on MC-38, this nuclear receptor was identified in colonic epithelial cells to induce their proliferation through a [3- catenin-dependent mechanism (Botrugno et al., 2004; Schoonjans et al., 2005). Thus, the in vivo antitumor effect of ML-180 treatment associated with its direct anti proliferative activity on MC-38 demonstrate a dual benefit to use LRH-1 inhibitors, by targeting both myeloid cells and colon tumor cells. The obtained results clearly demonstrate that LRH-1 and PPARy nuclear receptors are targets in colorectal cancer and demonstrate that their inhibition allow to counteract immunosuppression by preventing MDSC generation. The results also demonstrate that LRH-1 and PPARy are two immune-checkpoints and pave the way for the development of new nuclear receptors-targeting immunotherapies.

This example clearly demonstrates that compound inhibiting LRH-1 allows the treatment of cancer, in particular colorectal and pancreatic cancers.

This example also clearly demonstrates that compound inhibiting LRH-1 allow to reactivate an effective anti-tumor immune system and also allow to reduce the immuno-suppressive properties of disease, for example of cancer, for example of the tumor microenvironment (TME).

This example also clearly demonstrate that compound inhibiting LRH- 1 allow to treat cancer, for example cancer involving MDSC, for example by preventing MDSC differentiation and inhibit the growth of tumor.

This example also clearly demonstrated that compound inhibiting LRH-1 allows to inhibit the tumor-induced immunosuppression and also allows to inhibit the tumor growth and to treat cancer.

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Claims

1 . Compound inhibiting the liver receptor homolog-1 (LRH-1 ) for use in the prevention and/or treatment of cancer involving myeloid-derived suppressor cells (MDSC).

2. Compound for use according to claim 1 , wherein the compound is selected from the group comprising inhibitor, antagonist and inverse agonist of the liver receptor homolog-1 (LRH-1 ).

3. Compound for use according to claim 1 , wherein the compound is selected from the group comprising ML-180, ML179, 7-[4-(2- Piperidinyl)ethoxy]benzoyl Raloxifene, 1 -(3'-(1 -(2-Morpholinoethyl)-1 H- pyrazol-3-yl)biphenyl-3-yl)ethanone, and 1 -(3'-(1-(2-(4- Morpholinyl)ethyl)-1 H-pyrazol-3-yl)-3-biphenylyl)ethenone.

4. Compound for use according to any one of the preceding claims wherein the compound is in combination with anti-cancer, anti-angiogenic drug, anti-inflammatory drug, anti-oxidative drug, immunotherapy.

5. Compound for use according to any one of the preceding claims wherein the cancer is selected from the group comprising colorectal cancer, pancreas cancer, digestive system cancer, stomach cancer.

6. Compound for use according to any one of the preceding claims wherein the cancer is a peritoneal carcinomatosis of colorectal origin.

7. A pharmaceutical composition comprising a compound as defined according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier, for use in the prevention and/or treatment of cancer involving myeloid-derived suppressor cells (MDSC).

8. In-vitro method for detecting a cancer involving myeloid-derived suppressor cells (MDSC) from a biological sample comprising: searching in the biological sample of cells expressing CD45, CD11 b CD14, CD15, CD33, HLADR, when the biological sample comprises cells expressing CD45, CD11 b, CD14, CD15, CD33, HLADR respectively CD45^high, CD11 b^high, CD14^high, CD15^high, CD33^high, HLADR^|OW, the cancer involved myeloid-derived suppressor cells (MDSC).

9. In-vitro method for determining the susceptibility of a cancer from a biological sample to a compound as defined in claims 1 to 5 comprising: a. isolating monocytes from the biological sample, b. culturing the isolated monocyte with a compound as defined in anyone of claims 1 to 5, c. observation of the obtained culture of b) where when the obtained culture of b) does not comprise MDSC, the cancer is susceptible to said compound as defined in claims 1 to 6.

10. Method for determining the susceptibility of a cancer involving involved myeloid-derived suppressor cells (MDSC) from a biological sample to a test compound: i. isolating from the biological sample a monocyte, j. culturing the isolated monocyte with a test compound, k. observation of the obtained culture of step j) where when the obtained culture of j) does not comprise MDSC, the cancer is susceptible to said test compound.