Neutral Sphingomyelinase 2 Heightens Anti-Melanoma Immune Responses and Anti-PD-1 Therapy Efficacy

Abstract

Dysregulation of lipid metabolism affects the behavior of cancer cells, but how this happens is not completely understood. Neutral sphingomyelinase 2 (nSMase2), encoded by SMPD3, catalyzes the breakdown of sphingomyelin to produce the anti-oncometabolite ceramide. We found that this enzyme was often downregulated in human metastatic melanoma, likely contributing to immune escape. Overexpression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not CD8-deficient mice. In wild-type mice, nSMase2-overexpressing tumors showed accumulation of both ceramide and CD8+ tumor-infiltrating lymphocytes, and this was associated with increased level of transcripts encoding IFNγ and CXCL9. Overexpressing the catalytically inactive nSMase2 failed to alter tumor growth, indicating that the deleterious effect nSMase2 has on melanoma growth depends on its enzymatic activity. In vitro, small extracellular vesicles from melanoma cells overexpressing wild-type nSMase2 augmented the expression of IL12, CXCL9, and CCL19 by bone marrow-derived dendritic cells, suggesting that melanoma nSMase2 triggers T helper 1 (Th1) polarization in the earliest stages of the immune response. Most importantly, overexpression of wild-type nSMase2 increased anti-PD-1 efficacy in murine models of melanoma and breast cancer, and this was associated with an enhanced Th1 response. Therefore, increasing SMPD3 expression in melanoma may serve as an original therapeutic strategy to potentiate Th1 polarization and CD8+ T-cell-dependent immune responses and overcome resistance to anti-PD-1.

©2021 American Association for Cancer Research.

Figures

Figure 1.

Low expression of SMPD3 in patients with melanoma is associated with worse prognosis and low immune gene signatures. A,SMPD3 expression analysis in normal human skin (n = 4), primary (P. Mel., n = 14), and metastatic (M. Mel., n = 40) melanoma samples from the Oncomine database. *, P < 0.05; **, P < 0.01. B,SMPD3 expression analyzed by ISH on a skin sample from a patient with advanced melanoma. Pictures are representative of staining carried out on samples from five patients. C,SMPD3 expression analysis in tumor biopsies from patients with metastatic melanoma (TCGA melanoma cohort; n = 342). RSEM, RNA-Seq by Expectation Maximization. D, Overall survival in patients with metastatic melanoma from the TCGA melanoma cohort, exhibiting tumors with high (>80th percentile; n = 68) or low (<20th percentile; n = 68) SMPD3 expression in melanoma samples. E,, Heatmap depicting the differential expression of a selected set of genes related to immune responses in melanoma biopsies (TCGA) with high (SMPD3high) or low (SMPD3low) SMPD3 expression. Genes were clustered using a Euclidean distant matrix and average linkage clustering. F, Correlation analyses of SMPD3 expression with the indicated genes (TCGA).

Figure 2.

nSMase2 expression in mouse melanoma enhances CD8+ T-cell–dependent immune responses. A, B16K1 cells transfected to overexpress V5-tagged nSMase2 (nSMase2high) or not (nSMase2low) were analyzed by Western blot using anti-V5 and anti–β-actin antibodies. B, Confocal microscopy analysis showing V5-tagged nSMase2 (red staining) and giantin (green staining) localization in B16K1 cells expressing nSMase2 at low or high level. C, Intracellular ceramide levels in B16K1 nSMase2high and B16K1 nSMase2low cells. Data are expressed as the percentage of values obtained as compared with mock-transfected B16K1 cells. Values are mean ± SEM of four independent experiments. D–G, C57BL/6 WT (D–F) and CD8α-deficient (G) mice were intradermally and bilaterally (D, E, and G) or monolaterally (F) injected with B16K1 cells expressing high (nSMase2high; red bars, Tukey boxes and curves) or low (nSMase2low; black and white bars, Tukey boxes and curves) levels of nSMase2. D, At day 12, WT mice were sacrificed and the levels of total as well as of subspecies of ceramide were analyzed by mass spectrometry (n = 5 tumors/mice per group, Student t test and Kruskal–Wallis). E, Tumor volumes in WT mice at the indicated days (n = 4–5 mice per group). F, Tumor-infiltrating leukocytes were analyzed using flow cytometry at day 12 following tumor graft (n = 5–6 mice per group). G, Growth of B16K1 nSMase2high and nSMase2low tumor in CD8α-deficient mice. Data are mean ± SEM of five mice per group (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3.

Catalytic activity is required for nSMase2 to suppress melanoma growth and stimulate immune responses. A, Frequency of mutations and copy-number alterations in human melanoma samples from the indicated studies (www.cbioportal.org). B, Localization of missense (green dots) and splice (red dot) mutations on nSMase2 amino acid sequence from the studies depicted in A. The catalytic site corresponds to the green box. Mutations are predicted to be benign (green), possibly damaging (orange), or probably damaging (red; http://genetics.bwh.harvard.edu/pph2/). C, B16K1 cells expressing or not (mock) the WT or C.I. nSMase2 were intradermally injected in C57BL/6 WT mice, and tumor volumes were determined at the indicated days. Data are mean ± SEM of four mice per group (*, P < 0.05; **, P < 0.01; ***, P < 0.001). D–F, B16K1 cells expressing the WT or C.I. nSMase2 were intradermally and bilaterally injected in WT mice, and 12 days later, tumor-draining lymph nodes (TdLN) and tumors were collected. Tumors were weighed (D), and T-cell content was analyzed by flow cytometry in TdLNs (E, left plot) and tumors (E, right plot). Data are mean ± SEM of 18 mice per groups pooled from three independent experiments (D and E). F, CD8+ T cells specific for Trp2 peptides were quantified using dextramer technology. Representative staining and proportion of total Trp2-specific CD8+ T cells are depicted. Numbers are mean ± SEM of six mice per group (*, P < 0.05; **, P < 0.01).

Figure 4.

Melanoma nSMase2 enhances Th1-related gene expression in melanoma tumors and sEV-treated BMDCs. A, C57BL/6 mice were injected with B16K1 cells expressing the WT or C.I. form of nSMase2 as described in Fig. 3. At day 12, tumors were harvested, RNAs were purified, and transcripts encoding CXCL9 and IFNγ were analyzed by RT-qPCR. Data are mean ± SEM of eight tumors per group (*, P < 0.05). B, Correlation analyses of SMPD3 expression with the expression of genes coding for IFNG and CXCL9 (TCGA melanoma dataset). C–E, BMDCs were incubated with or without 5 to 10 μg/mL sEVs (pooled results) from B16K1 expressing either WT or C.I nSMase2 (C). After 24 hours, expression of the indicated transcripts by BMDCs was analyzed (D and E). Data are mean ± SEM of five to nine independent experiments carried out on BMDCs treated with two independent sEV preparations and depicted as the fold increase of expression as compared with untreated control BMDCs (*, P < 0.05; **, P < 0.01).

Figure 5.

nSMase 2 potentiates the efficacy of anti–PD-1 therapy in vivo. A–D, WT mice were intradermally and bilaterally injected with B16K1 expressing high (nSMase2high) or low (nSMase2low) levels of nSMase2 and received i.p. injection of anti–PD-1 (αPD-1; 200 μg) or vehicle (PBS) at days 6, 10, and 13 (5 mice per group, 8–10 tumors; A). Individual tumor curves are depicted. Inset, numbers indicate the number of total regressions out of the total number of tumors (B). Overall tumor growth analysis in each group. Values are mean ± SEM of 8 to 10 tumors per group (*, P < 0.05; **, P < 0.01; t test; C). Overall survival was determined for each group. At day 60, surviving mice were challenged with a second parental B16K1 injection (arrow). Mice did not develop tumors and survived (D; *, P < 0.05; **, P < 0.01; log-rank test).

Figure 6.

nSMase2 potentiates the PD-1–dependent immune response in mouse melanoma tumors. A–D, nSMase2high or nSMase2low B16K1 cells were bilaterally and intradermally grafted to C57BL/6 WT mice. Mice were then treated with 200 μg of anti–PD-1 or vehicle at day 7 prior to tumor immune infiltrate analysis by flow cytometry at day 10. The two tumors from each mouse were pooled prior to immune cell infiltration analysis. A–C, Infiltration of tumors by CD8+ T cells (A) and CD4+FoxP3+ regulatory T cells (B and C; n = 7 mice group). D and E, IFNγ and TNF production by tumor-infiltrating CD4+ (D) and CD8+ (E) T cells following 4-hour phorbol myristate acetate (PMA)/ionomycin incubation in the presence of a Golgi transport blocker. F, Alternatively, proportion of granzyme B+ cells was determined among CD8+ TILs (n = 10 mice per group). Statistical analyses: one-way ANOVA; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.