METHODS AND PHARMACEUTICAL COMPOSITIONS FOR ENHANCING CD8+ T CELL-DEPENDENT IMMUNE RESPONSES IN SUBJECTS SUFFERING FROM CANCER

Abstract

The present invention relates to methods and pharmaceutical compositions for enhancing CD8+ T cell-dependent immune responses in subjects suffering from cancer. In particular, the present invention relates to a method of enhancing the CD8+ T cell-dependent immune response in a subject suffering from cancer comprising administering to the subject a therapeutically effective amount of an agent capable of increasing intra-tumoral ceramide content.

Claims

1. A method of enhancing the CD8+ T cell-dependent immune response in a subject suffering from cancer comprising administering to the subject a therapeutically effective amount of an agent capable of increasing intra-tumoral ceramide content.

2. A method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective combination of an immune checkpoint inhibitor with an agent capable of increasing intra-tumoral ceramide content, wherein administration of the combination results in enhanced therapeutic efficacy relative to the administration of the immune checkpoint inhibitor alone.

3. The method of claim 1 wherein the subject suffers from a cancer selected from the group consisting of neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coil; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemanigipendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

4. The method of claim 1 wherein the subject suffers from a melanoma, a melanoma resistant to BRAF inhibitors, or a melanoma with elevated plasma lactate dehydrogenase (LDH).

5. The method of claim 1 wherein the cancer is characterized by a low tumor infiltration of CD8+ T cells.

6. The method of claim 2 wherein the immune checkpoint inhibitor is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.

7. The method of claim 1 wherein the agent capable of increasing intra-tumoral ceramide content is a neutral sphingomyelinase 2 (nSMase 2) polypeptide or a polynucleotide encoding for a neutral sphingomyelinase 2 (nSMase 2) polypeptide.

8. The method of claim 7 wherein the nSMase 2 polypeptide comprises an amino acid sequence of a nSMase2 variant having at least 90% of identity with SEQ ID NO:1.

9. The method of claim 7 wherein the polynucleotide encoding for nSMase2 is delivered with a vector.

10. The method of claim 1 wherein the agent capable of increasing intra-tumoral ceramide content is selected from the group consisting of DNA methyltransferase inhibitors and histone deacetylase inhibitors.

11. A method of treating cancer in a subject in need thereof comprising i) quantifying the density of CD8+ T cells in a tumor tissue sample obtained from the subject ii) comparing the density quantified at step i) with a predetermined reference value and iii) administering to the subject a therapeutically effective amount of an agent capable of increasing intra-tumoral ceramide content when the density quantified at step i) is lower than the predetermined reference value.

12. The method of claim 1 wherein the agent capable of increasing intra-tumoral ceramide content is a carrier that is suitable to deliver an amount of ceramide intra-tumorally.

13. The method of claim 12 wherein the carrier is a liposome.

14. A method of treating cancer in a subject in need thereof comprising i) quantifying the density of CD8+ T cells in a tumor tissue sample obtained from the subject ii) comparing the density quantified at step i) with a predetermined reference value and iii) administering to the subject a therapeutically effective combination of an immune checkpoint inhibitor with an agent capable of increasing intra-tumoral ceramide content when the density quantified at step i) is lower than the predetermined reference value.

15. A method of enhancing the potency of an immune checkpoint inhibitor administered to a subject as part of a treatment regimen, the method comprising administering a pharmaceutically effective amount of an agent capable of increasing intra-tumoral ceramide content to a subject in combination with the immune checkpoint inhibitor.

Description

FIGURES

[0056] FIG. 1: nSMase 2 over-expression in B16K1 cells does not alter cell growth in vitro.

[0057] A, B16K1 cells expressing (2+) or not (2?) V5-tagged nSMase 2 were analyzed by Western blot using anti-V5 and anti-I3-actin antibodies. B, Neutral SMase activity in B16K1 nSMase 2+ (2+) and nSMase 2? (2?) as well as in mock-transfected B16K1 cells. C, Intracellular ceramide levels in B16K1 nSMase 2+(2+) and nSMase 2? (2?) as well as in mock-tranfected B16K1 cells. Data are expressed as the % of values obtained with mock-transfected B16K1 cells. Values are shown as means?sem of 3 independent experiments (B and C). D, In vitro B16K1 cell growth was evaluated in 10% FCS medium (left panel) or 0% FCS medium (left panel). Data are displayed as means?sem of triplicates from one representative experiment out of three. E, B16K1 cells were stably transduced with a control retroviral vector (B16K1 mock), a retroviral vector encoding WT (nSMase2 WT) or a catalytically inactive (nSMase2 C.I.) VS-tagged nSMase 2. Cells were analysed by Western blot using anti-V5 and anti-?-actin antibodies. F, Neutral SMase activity in B16K1 transduced with a control retroviral vector (B16K1 mock), a retroviral vector encoding WT (nSMase2 WT) or a catalytically inactive nSMase 2 (nSMase2 C.I.). Data are shown as means?sem of 3 independent experiments. (*: p<0.05; **: p<0.01). G, B16K1 melanoma cells were incubated or not with 1 ?M of 5 aza-2 deoxycitidine (5-aza) during 72 hours or with 0.1 ?M Trichostatine A (TSA) during the last 16 hours. Smpd3 expression was analysed by RT-qPCR. Data are shown as means?sem of 3 independent experiments.

[0058] FIG. 2: Impact of nSMase 2 over-expression on B16K1 tumor growth in mice.

[0059] A, WT (left panel) or CD8 KO (right panel) mice were intradermally and bilaterally injected with 3?10.sup.5 B16K1 nSMase 2+ (2+) or nSMase 2? (2?) cells. Tumor volume was determined at the indicated days with a calliper. Data are shown as means?sem of a minimum of 6 tumors per group (*: p<0.05; ***: p<0.001). B, B16K1 cells expressing or not (mock) a WT or catalytically inactive (C.I.) nSMase2 were intradermally and bilaterally injected in WT (left panel) or CD8 KO (right panel) mice and tumor volumes were determined at the indicated days. Data are displayed as means?sem of 8 tumors in CD8 KO and WT mice per group (*: p<0.05; **: p<0.01).

[0060] FIG. 3: Analysis of TILs and cytokines mRNA in B16K1 tumors over-expressing nSMase 2.

[0061] One million B16K1 cells transduced with a retroviral vector encoding a wild-type (WT) or catalytically inactive (C.I.) nSMase2 were intra-dermally injected in wild-type mice and 12 days later, tumors were collected. A, Tumors were weighed. B, In some experiments, tumors were dissociated and the tumor TIL content was analysed by flow cytometry. The proportion of total CD4+ (left panel) and CD8+ (right panel) TIL was determined. Data are shown as means?sem of 18 tumors per group. C, CD8+T cells specific for Trp2 peptides were quantified using the dextramer technology. Representative staining (left panel) and proportion of total Trp2-specific CD8+T cells are depicted. Data are shown as means?sem of 6 tumors per group. D, In additional experiments, RNA from tumors were purified and CXCL9 and IFNy transcripts were analysed by RT-qPCR. Data are shown as means?sem of 8 determinations per group. (*: p<0.05; **: p<0.01).

[0062] FIG. 4: nSMase 2 enzyme activity enhances exosome immunogenicity.

[0063] Exosomes from B16K1 cells transduced with a retroviral vector encoding a wild-type (WT) or catalytically inactive (C.I.) nSMase2 were purified by ultracentifugation. A, Exosome preparation was observed by electronic microscopy. B, RNA was purified from exosomes produced by B16K1 cells expressing WT or C.I nSMase2 and the presence of miR-155 and miR-21a was analysed by RT-qPCR. Data are shown as means?sem of 4 independent experiments carried out with 4 independent exosome preparations. C, Bone marrow-derived dendritic cells were incubated with 10 ?g/mL exosomes from B16K1 expressing WT or C.I nSMase2. After 24h of co-culture, CXCL9, IL-12 and SOCS1 transcripts were analyzed by RT-qPCR. Data are displayed as means?sem of 3 independent experiments carried out with 2-3 independent exosome preparations. D, WT mice were intradermally co-injected with B16K1 cells and exosomes purified from B16K1 expressing WT or C.I nSMase2. 12 days after B16K1 inoculation, tumor volumes were measured at the indicated days with a calliper (left panel). The proportion of total CD8+ TIL was determined by flow cytometry (right panel). Data are shown as means?sem of 9 to 15 mice per group. (*: p<0.05; **: p<0.01).

[0064] FIG. 5: nSMase2 enhances the response to immunotherapies.

[0065] WT mice were intradermally and bilaterally injected with 3?10.sup.5 B16K1 melanoma cells expressing or not the wild type (WT) or catalytically inactive (C.I) nSMase2. Mice received intraperitoneal injection of anti-PD-1 antibodies (?PD-1, 200 ?g) or anti-CTLA-4 antibodies (?CTLA-4, 200 ?g for the first injection and then 100 ?g) or vehicle (PBS) at days 6, 10 and 13 (n=10 tumors per group). A, Diagram representing the experimental protocol. B, Tumor volumes were measured using a calliper at day 17. (*: p<0.05; **: p<0.01; ***: p<0.001).

[0066] FIG. 6: Melanoma nSMase2 enhances CD8+ T cell-dependent immune responses. A, Analysis of overall survival in metastatic melanoma patients from the TCGA melanoma cohort, exhibiting high (80th percentile) and low (20th percentile) SMPD3 expression in melanoma samples. B, Heatmap for a selected list of genes in samples with highest (SMPD3.sup.high) and lowest (SMPD3.sup.low) SMPD3 expression. Genes were clustered using a Euclidean distant matrix and average linkage clustering. C, Correlation analyses of SMPD3 expression with the indicated genes. D-E, WT mice injected with B16K1 nSMase2.sup.low (black bars) or nSMase2.sup.high (white bars) were sacrificed and tumor-infiltrating leukocytes were analysed by flow cytometry (D). Alternatively, the levels of total (left panel) and specific subtypes (right panel) of ceramide were determined in tumors (E). (*: p<0.05; ***: p<0.001).

[0067] FIG. 7: Melanoma nSMase2 enhances the response to immunotherapies. A, Upper panel, heatmap for a selected list of genes in human metastatic melanoma samples exhibiting the highest (SMPD3.sup.high) and lowest (SMPD3.sup.low) SMPD3 expression. Genes were clustered using a Euclidean distant matrix and average linkage clustering. Lower panel, correlation analysis of SMPD3 and PDCD1 expression. B-D, WT mice were intradermally injected with B16K1 cells expressing high (nSMase2.sup.high) or low (nSMase2.sup.low) levels of nSMase2. At Day 12, tumors were collected, dissociated and the content of PD-1+CD8+ TILs was analysed by flow cytometry (B). Alternatively, mice received intraperitoneal injection of anti-PD-1 (?PD-1, 200 ?g) or vehicle (PBS) at days 6, 10 and 13 (n=10 tumors per group). Individual tumor curves are depicted. Inserts, numbers indicate the number of total regression(s) out of total number of tumors (C). Overall survival was determined for each group (D) (*: p<0.05; **: p<0.01; ***: p<0.001).

EXAMPLES

Example 1

[0068] Material & Methods

[0069] Cells: B16K1 is a genetically modified cell line obtained from B16F10 cells, which stably express the MHC-I molecule H-2Kb (41-43). Cells were cultured in DMEM medium containing 10% heat-inactivated fetal calf serum (FCS). To study cell proliferation, B16K1 cells overexpressing or not WT VS-tagged nSMase 2 were cultured in DMEM medium containing 0 or 10% FCS. Cells were counted at the indicated times by using a cell counter (Beckman coulter). For dendritic cell (DC) preparation, bone morrow derived cells were cultured in complete RPMI medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, 50 ?M ?-mercaptoethanol and 20 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) at 37? C. with 5% CO2. Medium was changed every 2-3 days. After at least 7 days of culture, DC differentiation was analyzed by FACS. In some experiments, DCs were cultured during 24 h with 10 ?g/mL of exosomes.

[0070] B16K1 cell transfection: B16K1 cells were transfected (Superfect reagent, Qiagen) with a plasmid (pEF6-V5-TOPO) containing the cDNA encoding the mouse nSMase 2. Transfected cells were selected for their resistance to blasticidin (7 ?g/mL) and subjected to limit dilution. Resistant cells were cultured in DMEM containing 7 ?g/mL blasticidin and analysed by Western blot. Two cell populations were selected for the present study: B16K1 nSMase2+ and B16K1 nSMase2?, which overexpressed or not the VS-tagged nSMase 2, respectively. Mock-transfected B16K1 cells have been obtained by transfecting a plasmid conferring resistance to blasticidin.

[0071] Cloning of the His-nSMase2 WT and catalytically-inactive (D428A) in pMSCV-Puro: Retroviral expression vectors encoding wild-type (WT) or the mutant mouse nSMase-2 were obtained by cloning the product of the partial BamHI and Pmel digestion of pEF6-V5-His donor expression vectors encoding a WT or a catalytically inactive (D428A) mouse nSMase-2 into pMSCV-Puro (44) linearized with BglII and HpaI.

[0072] Retrovirus production and cell transduction: The generation of viruses has been described previously (45). Viral particles of WT and catalytically inactive (D428A) nSMase-2 derived from pMSCV-Puro vectors were produced to transduce 1 to 3?10.sup.6 mouse B16K1 cells for 16 h in 6-well plates in the presence of Polybrene (8 ?g/ml). Cells were then washed in phosphate-buffered saline (PBS), harvested, plated in complete medium containing puromycin (2.5 ?g/ml) and incubated for 3 days before amplification and subsequent analysis of the polyclonal populations.

[0073] Mice: WT C57BL/6 mice were from Janvier laboratories. CD8-deficient C57BL/6 mice were a gift from Prof. J. van Meerwijk (INSERM U1043, Toulouse, France). Mice were housed in temperature-controlled rooms in the specific pathogen-free animal facility (Anexplo platform, Toulouse, France), kept on a 12-h light/dark cycle, and had unrestricted access to food and water. All animal studies were conducted according to national and international policies and were approved by the local committee for animal experimentation.

[0074] In vivo tumorigenesis: 3?10.sup.5 B16K1 cells overexpressing or not the WT or catalytically-inactive V5-tagged nSMase 2 were intra-dermally injected in WT and CD8.sup.?/? mice. In some experiments, 1 ?g of exosomes purified from B16K1 cells overexpressing the WT or catalytically-inactive nSMase 2 were co-injected with 3?10.sup.5 parental B16K1 cells. Tumor volumes were measured using a caliper at the indicated days.

[0075] Immunotherapy protocol: 3?10.sup.5 B16K1 cells were intra-dermally and bilaterally injected in wild-type mice (n=5 mice per condition). Mice received intraperitoneal injections of anti-PD-1 antibodies (?aPD-1, 200 ?g) or anti-CTLA-4 antibodies (?CTLA-4, 200 ?g for the first injection and then 100 ?g) or vehicle (PBS) at days 6, 10 and 13. Tumor volumes were measured using a caliper at the indicated days.

[0076] Analysis of lymphocyte content in tumors: One million B16K1 cells overexpressing or not the WT or catalytically-inactive VS-tagged nSMase 2 were intra-dermally injected in WT mice. In some experiments, 3.3 ?g exosomes purified from B16K1 cells overexpressing the WT or catalytically-inactive nSMase 2 were co-injected with 1?10.sup.6 parental B16K1 cells. At day 12, mice were sacrificed and tumors were collected and digested with the Tumor Dissociation Kit, mouse (miltenyi). Cells were stained with the antibodies or MHC-I dextramers and live-dead reagent (Invitrogen) before flow cytometry analysis. Antibodies used in this study were anti-mouse CD45 (BD Biosciences, BUV395), anti-mouse Thy1 (Biolegend, APC-Cy7), anti-mouse CD8 (BD Biosciences, BV605) and anti-mouse CD4 (eBioscience, FITC).

[0077] Sphingomyelin analysis from B16K1 cell lines: 3?10.sup.6 B16K1 cells were incubated in the presence of 1 ?Ci/mL [.sup.3H]choline for 48 h. Cells were collected and sedimented at 4? C. by low-speed centrifugation, and cell pellets were immediately frozen at ?20? C. Cell pellets were suspended in 0.6 mL of distilled water, and disrupted at 4? C. by brief sonication. Lipids were extracted, and [.sup.3H]choline-labeled SM was quantified as previously reported (46).

[0078] Sphingolipid analysis from tumors: Tumors were collected and disrupted using the FastPREP technology (MP Biomedicals). Lipids were extracted from 5 mg of tumor samples. SM levels were quantified by measuring the lipid phosphorus content (47). Ceramide mass was measured essentially as described (48), using recombinant Escherichia coli diacylglycerol kinase (Calbiochem, Meudon, France) and [?-32P]ATP. Radioactive ceramide-1-phosphate was isolated by TLC using chloroform/acetone/methanol/acetic acid/water (50:20:15:10:5, by volume) as developing solvent. Alternatively, SLs were measured by mass spectrometry on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring positive ionization mode as described previously (49). Results from mass spectrometry analysis were normalized to total protein concentration as determined using a Bradford assay.

[0079] Neutral sphingomyelinase activity measurement: Cellular and tumor nSMase activities were assayed as described previously (50) using [choline-methyl-.sup.14C]SM (100,000 dpm/assay) as substrate.

[0080] Western blot analysis: Cells were washed and harvested in PBS containing 20 mM NaF, 20 mM sodium pyrophosphate, 1 mM NaVO.sub.4, and 5 mM EDTA. Cells were lysed in a buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 0.5% deoxycholate, 1 mM NaVO.sub.4, 10 mM ?-glycerophosphate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 ?g/ml leupeptin, 2 ?g/ml pepstatin A, and 10 ?g/ml aprotinin, for 30 min on ice. Equal amounts of proteins were separated in a 7.5% SDS-polyacrylamide gel and blotted onto nitrocellulose membranes (Hybond-C, Amersham Pharmacia Biotech). Proteins were detected using anti-V5 and anti-actin antibody and an ECL detection system (Amersham Pharmacia Biotech).

[0081] Confocal microscopy analysis: B16K1 cells were cultured on glass coverslips for 24 h and fixed in PBS-paraformaldehyde. After permeabilization with saposin, cells were stained with anti-giantin and anti-V5 (Invitrogen) antibodies and dye-coupled secondary antibodies and analysed by confocal microscopy (Zeiss, LSM510).

[0082] Exosome purification. Cells were cultured in medium with exosome-free FCS, which was prepared by centrifugations to remove existing exosomes. Cell culture medium was collected after 5 days of culture and exosomes were isolated by differential centrifugations. Briefly, the culture medium was centrifuged at 10,000?g for 45 min. The supernatant was then centrifugated at 110,000?g for 70 min at 4? C. to pellet exosomes. Exosome pellet was then washed with PBS, and further centrifuged at 110,000?g for 70 min at 4? C. The resulting pellet was resuspended in PBS.

[0083] RNA isolation and qRT-PCR. RNA isolation from cells and exosomes was performed by using the Qiagen RNeasy mini kit and Qiagen miRNeasy kit respectively, according to manufacturer's instructions. Mature miRNA cDNA was made with a miRCURY LNA universal RT miRNA PCR kit using 20 ng of RNA from each sample (Exiqon). qPCR of mature miRNA was performed with the miRCURY LNA universal RT miRNA PCR kit SYBR green master mix with LNA primers for mmu-miR155-5p, mmumiR-21a-5p, miR-146-5p, RNU1A1 and 5S RNA (Exiqon). RNU1A1 and 5S RNA were used to normalize expression. cDNA from total RNA was made with SuperScript II Reverse Transcriptase by using 1 ?g of RNA from each sample (Thermofischer). qPCR was performed with SYBR Green Master Mix (Takara) and primers for transcript encoding murine ?-actin, HPRT, CXCL9, IFN?, IL-12 and SOCS1 (Qiagen). For RNA isolation from tumors, one million B16K1 cells overexpressing WT or catalytically-inactive V5-tagged nSMase 2 were intra-dermally injected in WT mice. At day 12, mice were sacrificed, tumors were collected and dissociated using the homogenizer Precellys evolution of bertin technologies at 6,500 rpm during 2 cycles of 30 s in vials containing ceramic beads. RNA purification was performed using the RNeasy Midi Kit (Qiagen).

[0084] SMPD3 expression and mutations in human melanoma: SMPD3 expression was analysed using the TCGA melanoma cohort.sup.32. TCGA genomic and clinical data were downloaded from the UCSC cancer genome browser project website (https://genomecancer.ucsc.edu). The analysed population consisted in 342 patients with distant metastasis for whom RNAseq and clinical data overlap. Gene expression was measured experimentally using the Illumina HiSeq 2000 RNA Sequencing platform and log2(x+1) transformed. The strength of relationship between genes was assessed using the

[0085] Spearman rank correlation coefficient. Date of origin for computation of overall survival was the date to specimen procurement. Survival rates were estimated using Kaplan-Meier method and comparison between groups (low expression vs high expression) was performed using log-rank test. SMPD3 mutation analysis in human melanoma was assessed on cBioportal (http://www.cbioportal.org/).sup.33,34 and polyphen2 (http://genetics.bwh.harvard.edu/pph2/).

[0086] Statistical analyses: Results are expressed as means of at least three independent determinations per experiment. Mean values were compared using Student's t-test with Prism software (Graph-Pad). Differences were considered to be statistically significant when P<0.05 (*p<0.05; **p<0.01; ***p<0.001; n.s.: not significant).

Example 2

[0087] Results

[0088] nSMase 2 is Expressed at Low Levels in Melanoma

[0089] We initially performed a meta-analysis on the Oncomine database to evaluate the levels of nSMase 2 transcripts. Data from two independent studies (51, 52) indicated that mRNA encoding nSMase 2 is expressed at low levels in melanoma. Indeed, from the Riker and coworkers' study (51), nSMase 2 was less expressed in cutaneous melanoma than in normal skin or skin carcinomas (data not shown). Interestingly, nSMase 2 mRNA levels were even lower in metastatic melanoma when compared with in situ or cutaneous melanoma (data not shown), indicating that nSMase 2 downregulation is likely associated with melanoma progression. According to the Wagner et al. study (52), nSMase 2 expression is lower in melanoma cell lines as compared to lung, colorectal or pancreatic cancer cell lines (data not shown). In murine (B16F10 and B16K1) melanoma cell lines, mRNA encoding nSMase 2 was expressed at low levels as evaluated by RT-qPCR and Smpd3 expression was greatly enhanced upon trichostatin A but not 5-Azacytidine treatment (FIG. 1G), indicating that Smpd3 is likely downregulated in B16 melanoma cell lines in an HDAC-dependent manner.

[0090] nSMase 2 Over-Expression Triggers Ceramide Accumulation in B16K1 Melanoma Cell Line.

[0091] To evaluate the role of nSMase 2 in B16 melanoma growth, a plasmid encoding a V5-tagged nSMase 2 was transfected into B16K1 melanoma cells, which stably overexpress MHC I molecules. Transfected cells were selected for their resistance towards blasticidin. Clones were isolated by limiting dilution and further characterized for VS-tagged nSMase 2 expression and nSMase activity. One clone, called 2?, did not express VS-tagged nSMase 2, as evaluated by Western blot (FIG. 1A), despite its resistance to the antibiotic. In sharp contrast, another clone, called 2+, robustly expressed VS-tagged nSMase 2 (FIG. 1A). V5-tagged nSMase 2 expression did not alter MHC I expression at the cell surface of B16K1 cells (data not shown). In accordance with Western blot experiments, 2+ cells exhibited a strongly increased nSMase specific enzyme activity as compared to mock-transfected cells and 2? cells (FIG. 1B). Confocal microscopy experiments indicated that VS-tagged nSMase 2 proteins were localized at the plasma membrane in 2+ cells (data not shown). Moreover, 2+ cells displayed a 3-fold increase in intracellular ceramide level as compared to mock-transfected cells or 2? cells (FIG. 1C). Thus, expression of stable VS-tagged nSMase 2 in B16K1 cells is accompanied by a significant accumulation of ceramide.

[0092] nSMase 2 Over-Expression Does Not Sffect B16K1 Melanoma Tumorigenic Properties In Vitro.

[0093] The production of ceramide, derived from SM breakdown as a consequence of increased nSMase 2 expression, has been reported to inhibit cell growth (26, 27). To evaluate the consequence of nSMase 2 over-expression on B16K1 cell proliferation, we evaluated the cell growth of 2+ and 2? cells under 10% FCS or serum starvation conditions. Cell growth of both clones was similar under both conditions (FIG. 1D). Moreover, their ability to form colonies on soft agar and grow into spheroids was not impaired by nSMase 2 over-expression (data not shown).

[0094] To further evaluate the effect of nSMase 2 overexpression in mouse melanoma, B16K1 cells were transduced with a retroviral vector encoding either a WT or catalytically inactive nSMase 2. This approach allowed the generation of cell lines expressing either WT or catalytically inactive nSMase 2, which did not derive from single clones. The transduced cell lines potently expressed WT and catalytically inactive nSMase2 as evaluated by Western blot (FIG. 1E). Immunofluorescence analysis indicated that transduced B16K1 expressed different levels of either WT or catalytically inactive nSMase 2 (data not shown), consistent with the fact that transduced cells are heterogeneous cell populations. Both the WT nSMase 2 and its mutant form were located at the plasma membrane but not at the Golgi apparatus (data not shown). As compared to mock-transduced cells, neutral SMase activity was increased in cells expressing the WT, but not the catalytically inactive nSMase 2 (FIG. 1F). Sphingolipidomic analysis disclosed ceramide accumulation in WT nSMase 2 overexpressing cells (data not shown). However, none of the other SL species, including SM, displayed significant changes in B16K1 nSMase 2 WT cells as compared to their mock counterparts. In vitro growth was comparable for the different cell lines (data not shown), further indicating that nSMase 2 is not a major modulator of B16K1 cell proliferation.

[0095] Collectively, our data indicate that nSMase 2 over-expression in B16K1 cells had no consequence on cell growth and tumorigenic properties in vitro despite the intracellular ceramide increase.

[0096] nSMase 2 Over-Expression Impairs B16K1 Melanoma Cell Growth In Vivo.

[0097] We next evaluated the impact of nSMase 2 over-expression on B16K1 tumor growth in vivo. Wild-type C57B.sup.1/ 6 mice were challenged by intradermal injection of either 2? or 2+ cells. Interestingly, 2+ cell growth was reduced by 70% as compared to 2? cells, indicating that nSMase 2 over-expression greatly impaired B16K1 growth in WT mice (FIG. 2A, left panel). Three weeks after inoculation, tumors were collected and proteins were extracted to evaluate VS-tagged nSMase 2 expression and nSMase specific enzyme activity. As expected, tumors derived from 2+ cells, but not 2? cells, expressed the VS-tagged nSMase 2 and nSMase activity was consequently increased (data not shown).

[0098] To further evaluate the consequence of VS-tagged nSMase 2 expression on SL metabolism, intra-tumor SL levels were determined by mass spectrometry (data not shown). Importantly, VS-tagged nSMase 2 expression was associated with a significant increase of intra-tumor ceramide and sphingosine levels; however, this effect did not extend to the levels of sphingosine-1-phosphate, which remained unaltered (data not shown). Among the different ceramide species, both C16:0 and C24:1 ceramides were significantly increased in tumors expressing VS-tagged nSMase 2 (data not shown).

[0099] Since B16K1 cells do express high levels of MHC-I, which restricts antigen recognition by CD8+ T cell, we sought to evaluate whether CD8+ T cells are responsible for the alteration of B16K1 tumor growth upon V5-tagged nSMase 2 expression. Thus, we grafted 2? and 2+ cells in nude and CD8-deficient mice. In sharp contrast to the above observations in WT mice (i.e., immuno-competent mice), nSMase 2 over-expression did not impair the B16K1 tumor growth in nude mice (data not shown) and CD8-deficient mice (FIG. 2A, right panel).

[0100] To evaluate whether the nSMase 2-dependent alteration of SL composition is involved in the inhibition of B16K1 melanoma growth, we grafted WT mice with B16K1 cells, which have been transduced with a control retroviral vector (mock) or with retroviral vectors encoding either WT (nSMase 2 WT) or catalytically-inactive nSMase 2 (nSMase 2 C.I.). Whereas the in vivo tumor growth of WT nSMase 2 over-expressing cells was reduced (by more than 50%) as compared to mock transduced B16K1 cells or catalytically-inactive nSMase 2 expressing cells in immunocompetent mice, WT nSMase 2 overexpression did not compromise B16K1 tumor growth in CD8-deficient mice (FIG. 2B).

[0101] Collectively, our data indicate that nSMase 2 expression and enzyme activity impair B16K1 melanoma growth in immunocompetent but not immunodeficient mice.

[0102] nSMase 2 Over-Expression Enhances T Cell-Dependent Immune Response Towards B16K1 Cells.

[0103] We thus hypothesized that nSMase2 over-expression in B16K1 cells enhances the CD8 T cell-dependent immune response towards melanoma. To evaluate this tenet, we initially analyzed the immune response by monitoring the tumor-infiltrating leukocytes (TILs) by flow cytometry. The tumor content of leukocytes (CD45+), T lymphocytes (Thy1+) and, albeit to a lesser extent, Natural Killers (NK1.1+) was significantly enhanced in tumors that over-expressed nSMase 2 (data not shown). In contrast, B lymphocytes (CD19+) were poorly infiltrated into the B16K1 tumors and nSMase 2 over-expression did not modify CD19+ TIL content (data not shown). Moreover, analysis of myeloid cells indicated that tumor-infiltrating macrophages (CD11b+Gr1-F480+) and myeloid-derived suppressor cells (MDSC, Gr1+CD11b+) remained unchanged following nSMase 2 over-expression in melanoma cells (data not shown). Interestingly, among the T cells, whereas the tumor infiltration of CD4+ T cells was slightly increased, the proportion of CD8+ TILs was 3-fold higher in tumors over-expressing nSMase 2 as was the ratio of CD8+ to CD4+ TILs (data not shown).

[0104] As ceramide is a putative bioactive molecule in cell death signaling, we evaluated whether nSMase 2 over-expression sensitized B16K1 cells to cell-mediated cytotoxicity. As a matter of fact, 2+ and 2? cells were equally sensitive to cell-mediated cytotoxicity, indicating that nSMase 2 over-expression did not enhance the B16K1 cell death under our experimental conditions (data not shown). Moreover, nSMase 2 overexpression did not sensitize B16K1 cells to some effector molecules of cell-mediated cytotoxicity (i.e., the death receptor ligands CD95L, TRAIL and TNF) (data not shown).

[0105] Collectively, our data indicate that nSMase 2 over-expression in B16K1 cells (i) alters tumor SL composition, (ii) facilitates the CD8+ T cell tumor infiltration and, consequently, (iii) inhibits B16K1 tumor growth.

[0106] The nSMase 2 Enzyme Activity is Required for Enhancing T Cell-Dependent Anti-Melanoma Response.

[0107] The immune response was next analyzed by evaluating TIL content in B16K1 tumors overexpressing either WT or catalytically inactive nSMase 2 (FIG. 3). At day 12 post-injection, the tumor weight was significantly reduced by WT nSMase 2 overexpression (FIG. 3A) and this was associated with an increased CD45+ TIL content (data not shown). Both CD8+ and CD4+ TILs were increased in tumors overexpressing wild-type nSMase 2 (FIG. 3B). We next evaluated the tumor content in CD8+ T cells specific for tyrosinase-related protein 2 (TRP2), a differentiation antigen of melanocytic cells. Using the MHC-I dextramer technology, we showed that the TRP2-specific CD8+ T cell content was higher in tumors overexpressing WT nSMase 2 (FIG. 3C). Analysis of mRNA expression evaluated by RT-qPCR in B16K1 tumors overexpressing WT or CI nSMase 2 showed that mRNA encoding CXCL9 and IFN?, two major Th1-related cytokines, were significantly increased upon WT nSMase 2 overexpression (FIG. 3D).

[0108] Altogether, our data indicate that nSMase 2 catalytic activity is required for enhancing T cell-dependent immune responses towards B16K1 melanoma cells.

[0109] The nSMase 2 Enzymatic Activity Enhances the Immunogenicity of Exosomes Produced by Melanoma Cells.

[0110] nSMase 2 has recently been shown to facilitate the budding of exosomes, which likely contribute to the modulation of the anti-melanoma immune response. Thus, we have evaluated the consequences of WT or CI nSMase 2 overexpression in B16K1 melanoma cells on exosome secretion and molecular composition. Exosomes were purified from the culture medium of B16K1 cells overexpressing either WT or CI nSMase 2. The quantity of secreted exosomes, as evaluated by total protein determination, the ultra-structural morphology analysed using electron microscopy, as well as the protein composition (tetraspanins, melanoma antigens as evaluated by using western blot and FACS analysis) were similar for both exosome types (FIG. 4A and data not shown). Since nSMase 2 is involved in the exosomal secretion of some miRNA, we next evaluated the exosomal miRNA content, and found that miR-155 was greatly enriched in exosomes secreted by B16K1 cells overexpressing WT nSMase 2 (FIG. 4B). In contrast, the exosomal content of miR-21a and miR-146a was similar in both exosome types (FIG. 4B and data not shown). Considering that exosomes are efficiently uptaken by dendritic cells and miR-155 is a major pro-inflammatory miRNA modulating dendritic cell differentiation, we next analysed the capacity of the exosomes to facilitate dendritic cell maturation. We initially analysed, by flow cytometry, the expression level of dendritic cell surface maturation markers such as CD80, CD86, MHC-I and MHC-II. All those markers were up-regulated to the same extent at the dendritic cell surface following incubation with exosomes from B16K1 overexpressing either WT or C.I nSMase 2 (data not shown). In sharp contrast, the exosomes from B16K1 overexpressing WT nSMase 2 greatly enhanced the intracellular levels of mRNA encoding IL-12, a major pro-Th1 cytokine, and CXCL9, a chemokine facilitating T cell tumor infiltration (FIG. 4C), both of them being induced by IFN?. This phenomenon was associated with a decrease in cellular amounts of mRNA encoding SOCS1, a major IFNy signaling repressor, which is a well-known miR-155 target (53) (FIG. 4C). This data indicates that the exosomal miR-155, which is enriched upon WT nSMase 2 overexpression, is biologically active and facilitates the expression of Th1 cytokines. Thus, we hypothesize that nSMase 2 enhances the immunogenicity of exosomes secreted by melanoma cells. To evaluate this tenet, exosomes from B16K1 overexpressing WT or C.I. nSMase 2 were co-injected with parental B16K1 cells in immunocompetent mice. Exosomes from B16K1 overexpressing WT nSMase 2 significantly reduced tumor growth and enhanced CD8+ TIL content as compared to the exosomes from B16K1 overexpressing CI nSMase 2 (FIG. 4D).

[0111] Collectively, our data indicate that the nSMase 2 enzymatic activity enhances the immunogenicity of exosomes produced by B16K1 melanoma cells.

[0112] nSMase 2 Enzymatic Activity Enhances the Response to Immune Checkpoint Inhibitors.

[0113] To further evaluate the role of nSMase 2 in the anti-melanoma immune response, we analysed the consequences of nSMase 2 over-expression on the response to emerging immune therapies (i.e., anti-CTLA-4, anti-PD-1) (FIG. 5). Under our experimental conditions (FIG. 5A), whereas anti-PD-1 and anti-CTLA-4 have limited anti-tumor effects when mice were injected with B16K1 overexpressing the CI nSMase 2, WT nSMase 2 overexpression in B16K1 cells significantly enhanced the response to both antibody treatments (FIG. 5B). The effect was greater towards anti-CTLA-4 antibody and 4 out of 5 mice, which have been grafted with B16K1 overexpressing WT nSMase 2, displayed total tumor regression. Interestingly, those mice did not develop melanoma tumors upon a novel B16K1 injection two months after the first B16K1 graft, indicating that they were fully vaccinated (data not shown).

[0114] Altogether, our data indicate that the enzymatic activity of nSMase 2 in B16K1 melanoma enhances the therapeutic response to immune checkpoint inhibitors.

[0115] Discussion:

[0116] The present study provides evidence for the first time that expressing nSMase 2 in B16K1 mouse melanoma cells facilitates the CD8+ T cell tumor infiltration, thereby slowing down melanoma growth. NSMase 2 overexpression in B16K1 cell lines enhanced CD8+ TIL content and impaired B16K1 tumor growth in wild-type mice (i.e., immuno-competent) but not in mice lacking CD8+ T cells (i.e., nude and CD8-deficient mice).

[0117] The mechanisms by which nSMase 2 facilitates the CD8+ T cell-dependent immune response most likely depends on the alteration of intratumor SL content since overexpression of a catalytically inactive nSMase 2 mutant had no effect on B16K1 tumor growth and CD8+ T cell-dependent immune response. Analysis of tumor SL content indicated a significant increase in ceramide levels (from 1 to 1.5 nmol/mg) in tumors overexpressing nSMase 2. Moreover, intra-tumor sphingosine levels also increased upon nSMase 2 overexpression, albeit to a lesser extent (from 20 to 30 pmol/mg). Taking into account that sphingosine facilitates the secretion of RANTES/CCLS (54, 55), which is a potent chemoattractant towards CD8+ T cells, the possibility that the nSMase 2-induced sphingosine increase is involved in CD8+ T cell infiltration cannot be ruled out. In addition, sphingosine is the substrate of sphingosine kinases, which produce S1P, a critical mediator of lymphocyte traffic (33). One should note however, that the levels of intratumor S1P remained unchanged upon nSMase 2 overexpression. Hence, it is unlikely that S1P directly mediates the nSMase 2-triggered increase of CD8+ TIL content.

[0118] Detailed analyses of the intracellular SL content in transduced B16K1 cells over-expressing or not wild-type nSMase 2 demonstrated that only the intracellular levels of ceramide increased upon nSMase 2 over-expression. The intracellular concentration of all other SL metabolites, including SM, glycosphingolipids as well as sphingosine and sphingosine-1-phosphate, did not change upon nSMase 2 over-expression. As a matter of fact, SM reduction was observed in B16K1 nSMase 2+ clone, which exhibits a strong neutral SMase activity (180 nmol/h/mg), but not in B16K1 nSMase 2 WT cell lines, which display a much lower neutral SMase activity (20 nmol/h/mg). The increase in CD8+ T cell tumor infiltration and the subsequent tumor growth reduction were found not only for B16K1 nSMase 2+ clone but also for B16K1 nSMase 2 WT cell lines, indicating that the SM reduction, which is only observed in B16K1 exhibiting the highest neutral SMase activity, is unlikely responsible for both phenomena.

[0119] The mechanisms by which nSMase 2 facilitates CD8+ TIL content have been investigated. We provide evidence for the first time that nSMase 2 expression enhances the immunogenicity of melanoma cell-derived exosomes by increasing their content in miR-155, a major pro-inflammatory miRNA, which silences SOCS1 mRNA (53), thereby facilitating the increase in IL-12 and CXCL9 mRNA content in dendritic cells. Consequently, we observed an increased level in mRNA encoding IFN? and CXCL9 in B16K1 tumors overexpressing WT nSMase 2. The increased immunogenicity of melanoma cell-derived exosomes is further documented by the increased CD8+ TIL content and the decreased B16K1 tumor weight upon injection of exosomes derived from B16K1 overexpressing WT nSMase 2. As a matter of fact, nSMase 2 overexpression did not facilitate the exosomal secretion of miR-21a and miR-146, indicating that nSMase 2, and putatively ceramide, may enhance the budding of exosomes, which are enriched in selective miRNA, including miR-155 as documented here as well as miR-210 and miR-10b as reported by others. The mechanisms by which nSMase 2 facilitates the selectivity of the miRNA association to exosomes, remain to be determined.

[0120] We provide evidence for the first time that nSMase 2 enzyme activity in melanoma enhances the therapeutic response to emerging immunotherapies (i.e., anti-PD-1, anti-CTLA-4). Monoclonal antibodies inhibiting CTLA-4 (ipilimumab) or PD1 (nivolumab, pembrolizumab) have demonstrated significant efficacy in the treatment of metastatic melanoma, promoting high response rate and long-lasting tumor control. Despite promising results, about 40% of patients do not display therapeutic response and a significant proportion of responders experience tumor relapse within 2 years following treatment induction. It is tempting to speculate that increasing SMPD3 expression and/or the intratumor ceramide level in melanoma tumors may constitute an original therapeutic strategy to improve the efficacy of emerging immunotherapies.

Example 3

[0121] Results:

[0122] NSMase2 Expression Enhances CD8+ Tumor-Infiltrating Lymphocytes in Melanoma.

[0123] Analysis from the Oncomine and TCGA databases indicated that mRNA encoding nSMase2 is expressed at low levels in human metastatic melanoma as compared to primary tumors, suggesting that nSMase2 downregulation is likely associated with melanoma progression. The clinical outcome in metastatic melanoma patients exhibiting high (80th percentile) and low (20th percentile) SMPD3 expression was next analysed. Low SMPD3 expression was statistically associated with shortened overall survival (FIG. 6A), further arguing that SMPD3 downregulation is associated with a bad prognosis in melanoma.

[0124] We next analysed the gene signatures in patients from the TCGA melanoma cohort exhibiting high and low SMPD3 expression in melanoma samples. Of great interest was the finding that high SMPD3 expression was mostly associated with the Immune system process and Lymphocyte activation according to Gene ontology classification. We next identified the genes that were differentially expressed in human melanoma exhibiting either high or low SMPD3 expression in melanoma samples from patients affected with metastatic melanoma (FIG. 6B). High SMPD3 expression was associated with high expression of CD3G, CD3D and CD3E, which reflect tumor-infiltrating T lymphocytes (TIL). Among T cell genes, we found that CD8A, CD8B and CD4 were enriched in melanoma samples expressing SMPD3 at high levels. Moreover, various Thl-related genes such as IFNG, TNF, CXCL9, CXCL10 and CCL5 as well as cell-mediated cytotoxicity genes were highly expressed in melanoma samples exhibiting high SMPD3 expression (FIG. 6B). Accordingly, SMPD3 expression was significantly correlated with the expression of diverse genes, which likely reflects CD8+ T cell infiltration (FIG. 6C). This observation was not restricted to metastatic melanoma since similar correlations were found in triple negative breast cancers (Table 1). Of note, the expression levels of genes encoding the other known sphingomyelinase isoforms were not associated with a gene signature of CD8+ TIL in metastatic melanoma patients, except SMPD2 the expression of which poorly, yet significantly, correlated with that of CD8B. As a matter of fact, SMPD4 was anti-correlated with T cell-related genes. Thus, SMPD3 expression is associated with a signature of CD8+ T cell tumor infiltration in human melanoma samples, and this cannot be extended to the other sphingomyelinase isoforms.

[0125] We hypothesized that SMPD3 downregulation contributes to melanoma immune escape with the expression level of nSMase2 being critical for the CD8+ T cell-dependent immune response towards melanoma. To evaluate this tenet, we selected B16K1 (MHC-I.sup.high) mouse melanoma cell line, which express endogenous nSMase2 at low levels, due to HDAC-dependent epigenetic mechanism. We first generated B16K1 melanoma cell lines overexpressing or not nSMase2. Overexpressed enzyme was mainly located at the plasma membrane and led to robust increase in intracellular neutral sphingomyelinase activity and ceramide level without affecting two- and three-dimensional cell growth in vitro (FIG. 1 and data not shown). We next analysed the immune response in mice grafted with B16K1 melanoma cells expressing nSMase2 at low or high levels. Twelve days after B16K1 cell injection, the tumor content of leukocytes (CD45+) and T lymphocytes (Thy1+) was significantly enhanced in tumors that expressed nSMase2 at high levels (FIG. 6D). Among the T cells, the proportion of CD8+ TILs was 3-fold higher in tumors expressing nSMase2 at high levels (FIG. 6D).

[0126] Interestingly, nSMase2 overexpression as evaluated by western blot triggered an intra-tumor increase (i) in the nSMase activity, C16 and C24-ceramides (FIG. 6E) and sphingosine (data not shown) and (ii) a reduction of B16K1 tumor growth in WT mice (FIG. 2A). Of note, no significant changes were noticed for tumor sphingomyelin and S1P content in nSMase2 overexpressing tumors (data not shown). A similar trend was observed in B16F10 melanoma cells in which nSMase2 overexpression significantly reduced tumorigenesis in WT mice without affecting their proliferation rate in vitro (data not shown). Importantly, nSMase2 overexpression failed to impair B16K1 melanoma growth in CD8-deficient mice (FIG. 2A).

[0127] Collectively, our data indicate that (i) SMPD3 expression is associated with a CD8+ T cell gene signature in human metastatic melanoma samples, which may translate into improved overall survival and (ii) nSMase2 overexpression in mouse melanoma enhances CD8+ T cell-dependent immunity, which impairs tumor growth.

[0128] The nSMase2 Enzyme Activity is Required for Enhancing T Cell-Dependent Anti-Melanoma Immune Response

[0129] Analysis of the SMPD3 nucleotide sequence from 5 independent studies indicated mutations in the coding sequence, ranging from 2.5% to 20% mutation frequency depending on the study. The highest mutation frequency was observed in desmoplastic melanoma, whereas the lowest being in uveal melanoma. Most of the mutations were missense mutations and half of them affected residues in the catalytic domain. Moreover, twelve mutations are predicted to be probably damaging (HumDiv score>0.85) according to PolyPhen-2 analysis. We next evaluated whether a single missense mutation (D428A) into the catalytic domain, which abolished enzyme activity.sup.39, had a putative impact on nSMase2 biological activity in CD8+ T cell-dependent immune response and melanoma growth in mice. B16K1 cells were transduced with a retroviral vector encoding either WT or catalytically-inactive nSMase2. This approach induced a mild expression of both WT and catalytically-inactive nSMase2, leading to significant increase of nSMase activity in WT nSMase2 expressing cells without affecting cell proliferation capacity in vitro nor subcellular localisation. The in vivo tumor growth of WT nSMase2 expressing cells was reduced (by more than 50%) as compared to mock-transduced B16K1 cells or catalytically-inactive nSMase2 expressing cells in syngeneic mice.

[0130] The immune response was next analyzed at day 12 post-melanoma B16K1 cell injection. T cells (Thy1+) as well as dendritic cells (DC) (CD11c+) were increased in draining lymph nodes and tumors upon WT nSMase2 expression. Of note, whereas the content of Tregs was increased in lymph nodes, the Treg tumor infiltration was slightly, yet not significantly, enhanced by WT nSMase2 expression. WT nSMase2 overexpression significantly increased CD45+ leukocytes, CD4+ and CD8+ T cells, and DC content in both lymph nodes and tumors and reduced the tumor weight. We next evaluated the tumor content of CD8+ T cells specific for tyrosinase-related protein 2 (TRP2), a differentiation antigen of melanocytic cells. Using the MHC-I dextramer technology, we show that TRP2-specific CD8+ T cell content was higher in tumors expressing WT nSMase2. Of note, the levels of mRNA encoding CXCL9 and IFN?, two major Th1-related cytokines, were significantly increased upon WT nSMase2 expression in melanoma tumors but not in B16K1 cell culture.

[0131] Altogether, our data indicate that nSMase2 catalytic activity is required for enhancing T cell-dependent immune response towards melanoma cells.

[0132] nSMase2 Expression in Melanoma Synergises with Immune Checkpoint Inhibitors.

[0133] In human melanoma samples, high SMPD3 levels were associated with an increased expression of immunosuppressive genes, such as PDCD1, which encodes the immune checkpoint PD-1 (FIG. 7A). Similar findings were observed in triple negative breast cancers (Table 2). In good agreement with this finding, we observed in mouse melanoma an increased proportion of CD8+ TILs and, albeit to a lesser extent, CD4+ TILs expressing PD-1 in B16K1 tumors expressing WT nSMase2 (FIG. 7B). We next evaluated the therapeutic activity of anti-PD-1 towards melanoma cells expressing nSMase2 at low and high levels. Whereas anti-PD-1 significantly delayed nSMase2.sup.low melanoma growth (FIG. 7C), all tumors relapsed presumably due to immune escape mechanisms and, consequently, all mice died within 40 days post-B16K1 injection (FIG. 7D). Moreover, nSMase2 overexpression delayed melanoma growth and slightly, yet significantly, increased the overall survival (FIGS. 7C and 7D). Of major interest, the therapeutic efficacy of anti-PD-1 was dramatically enhanced by nSMase2 overexpression (FIG. 7C). In the group of mice injected with B16K1 nSMase2.sup.high and anti-PD-1, all mice survived (FIG. 7D) and none of them developed melanoma upon a second B16K1 cell injection, indicating that they were fully vaccinated against melanoma cells. Our observation was unlikely restricted to anti-PD-1 since WT nSMase2 expression greatly enhanced the therapeutic effect of anti-CTLA-4 blocking antibodies against melanoma.

[0134] Altogether, our data indicate that expression of WT nSMase2 in B16K1 melanoma synergizes with immune checkpoint blockade therapies in mice.

[0135] Discussion:

[0136] The present study provides the first evidence that (i) SMPD3 is expressed at low levels in most human metastatic melanoma samples and (ii) low SMPD3 expression is associated with shortened overall survival in patients. Noteworthy, high SMPD3 expression was associated with Immune system process and Lymphocyte activation. Accordingly, melanoma samples expressing SMPD3 at high levels exhibited gene signature of TILs, including genes encoding cytotoxic CD8+ T cell markers such as CD8A/B, GZMA/B and GNLY. As a matter of fact, the expression of genes (SMPD1, SMPD2, SMPD4) encoding the other sphingomyelinase isoforms did not correlate with TCR signaling pathway. Thus, the distinctive biological properties of nSMase2 in melanoma do not extend to the other sphingomyelinases, presumably due to different subcellular localisation and/or biochemical properties as well as different role in cell signaling.sup.2. One should note, however, that enforced expression of acid SMase, encoded by Smpd1 in B16F1 melanoma, is associated with an augmentation of CD8+ TIL.sup.15. The lack of correlation between SMPD1 expression and immune-related gene signature in human metastatic melanoma samples indicates that acid SMase is unlikely a critical modulator of CD8+ T cell-dependent immune response in melanoma patients.

[0137] In good agreement with data from human melanoma, nSMase2 heightens the CD8+ T cell dependent immune response, thereby slowing down melanoma growth in mice. Strikingly, nSMase2 overexpression in mouse melanoma cell lines enhanced CD8+ TIL content and impaired melanoma growth in WT animals (i.e., immuno-competent) but not in mice lacking CD8+ T cells (i.e., CD8-deficient mice), demonstrating that nSMase2 anti-tumorigenic properties are fully dependent on its ability to stimulate adaptive immunity. Collectively, our data reveal that SMPD3 downregulation or mutation likely contributes to melanoma immune escape, facilitating melanoma progression.

[0138] The mechanisms by which nSMase2 facilitates the CD8+ T cell-dependent immune response most likely rely on the alteration of intratumor SL content since expression of a catalytically inactive nSMase2 mutant had no effect on B16K1 tumor growth. Accordingly, intra-tumor ceramide and sphingosine content was significantly increased in nSMase2-overexpressing melanoma tumors. Taking into account that sphingosine facilitates the secretion of RANTES/CCL5.sup.44,45, which is a potent chemoattractant towards CD8+ T cells, the possibility that the nSMase 2-induced sphingosine increase is involved in CD8+ T cell infiltration cannot be ruled out. In addition, sphingosine is the substrate of sphingosine kinases, which produce S1P, a critical mediator of lymphocyte traffic.sup.46. One should note however that the levels of intratumor S1P remained unchanged upon nSMase2 overexpression. Hence, it is unlikely that S1P directly mediates the nSMase2-triggered increase of CD8+ TIL content. Another interesting hypothesis is that ceramide, which exhibits some analogy with Lipid A, the biologically active core of lipopolysaccharide .sup.47, may mimic pathogen-associated molecular patterns, facilitating DC maturation and ultimately priming the adaptive immune response.

[0139] SMPD3 expression in patients was also associated with the expression of genes encoding immune checkpoints such as PD-1, presumably leading to melanoma immune escape. Accordingly, we observed an increased proportion of CD4+PD-1+ and CD8+PD-1+ TILs in mouse melanoma tumors, which overexpressed nSMase2. Consequently, whereas nSMase2 overexpression in mouse melanoma significantly delayed melanoma growth, all mice died within 40 days post-melanoma cell injection, which strongly suggests melanoma immune escape. In addition, whereas immune checkpoint inhibitors had limited therapeutic effects towards B16K1 melanoma, both anti-PD-1 and anti-CTLA4 greatly suppressed tumor growth of WT nSMase2 expressing melanoma. These observations demonstrate that melanoma nSMase2 enhances the therapeutic response to emerging immunotherapies.

[0140] It is tempting to speculate that targeting SL metabolism in melanoma tumors may constitute an original therapeutic strategy to overcome resistance of melanoma, and possibly other cancer types, to emerging immunotherapies. In addition, SMPD3 expression in melanoma samples may serve as a novel biomarker to predict survival and response to immunotherapy.

[0141] Tables:

TABLE-US-00002 TABLE 1 correlation between SMPD3 and various genes of immunoactivation in human TNBC (* p < 0.05; ** p < 0.01; *** p < 0.001): Spearman's correlation coefficient in human TNBC between SMPD3 and: IFNG IRF1 TBX21 CD8A CD8B CXCL9 CXCL10 0.296 *** 0.445 *** 0.454 *** 0.472 *** 0.409 *** 0.412 *** 0.217 * CCL5 CXCL13 PRF1 GNLY GZMA GZMB 0.356 *** 0.388 *** 0.379 *** 0.336 *** 0.458 *** 0.312 ***

TABLE-US-00003 TABLE 2 correlation between SMPD3 and various genes of immune escape in human TNBC (* p < 0.05; ** p < 0.01; *** p < 0.001): Spearman's correlation coefficient in human TNBC between SMPD3 and: FOXP3 IDO1 IDO2 CTLA4 PDCD1LG1 PDCD1LG2 PDCD1 IL10 LAG3 TIGIT 0.365 *** 0.317 *** 0.399 *** 0.355 *** 0.352 *** 0.222 * 0.415 *** 0.289 ** 0.263 ** 0.418 ***

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