Thrombospondin-1 Silencing Improves Lymphocyte Infiltration in Tumors and Response to Anti-PD-1 in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is notoriously aggressive with a high metastatic potential, and targeted therapies are lacking. Using transcriptomic and histologic analysis of TNBC samples, we found that a high expression of thrombospondin-1 (TSP1), a potent endogenous inhibitor of angiogenesis and an activator of latent transforming growth factor beta (TGF-ß), is associated with (i) gene signatures of epithelial-mesenchymal transition and TGF-ß signaling, (ii) metastasis and (iii) a reduced survival in TNBC patients. In contrast, in tumors expressing low levels of TSP1, gene signatures of interferon gamma (IFN-γ) signaling and lymphocyte activation were enriched. In TNBC biopsies, TSP1 expression inversely correlated with the CD8+ tumor-infiltrating lymphocytes (TILs) content. In the 4T1 metastatic mouse model of TNBC, TSP1 silencing did not affect primary tumor development but, strikingly, impaired metastasis in immunocompetent but not in immunodeficient nude mice. Moreover, TSP1 knockdown increased tumor vascularization and T lymphocyte infiltration and decreased TGF-ß activation in immunocompetent mice. Noteworthy was the finding that TSP1 knockdown increased CD8+ TILs and their programmed cell death 1 (PD-1) expression and sensitized 4T1 tumors to anti-PD-1 therapy. TSP1 inhibition might thus represent an innovative targeted approach to impair TGF-ß activation and breast cancer cell metastasis and improve lymphocyte infiltration in tumors, and immunotherapy efficacy in TNBC.

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

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.

Combining Nivolumab and Ipilimumab with Infliximab or Certolizumab in Patients with Advanced Melanoma: First Results of a Phase Ib Clinical Trial.

PURPOSE: TNF blockers can be used to manage gastrointestinal inflammatory side effects following nivolumab and/or ipilimumab treatment in patients with advanced melanoma. Our preclinical data showed that anti-TNF could promote the efficacy of immune checkpoint inhibitors. PATIENTS AND METHODS: TICIMEL (NTC03293784) is an open-label, two-arm phase Ib clinical trial. Fourteen patients with advanced and/or metastatic melanoma (stage IIIc/IV) were enrolled. Patients were treated with nivolumab (1 mg/kg) and ipilimumab (3 mg/kg) combined to infliximab (5 mg/kg, N = 6) or certolizumab (400/200 mg, N = 8). The primary endpoint was safety and the secondary endpoint was antitumor activity. Adverse events (AEs) were graded according to the NCI Common Terminology Criteria for Adverse Events and response was assessed following RECIST 1.1. RESULTS: Only one dose-limiting toxicity was observed in the infliximab cohort. The two different combinations were found to be safe. We observed lower treatment-related AEs with infliximab as compared with certolizumab. In the certolizumab cohort, one patient was not evaluable for response. In this cohort, four of eight patients exhibited hepatobiliary disorders and seven of seven evaluable patients achieved objective response including four complete responses (CRs) and three partial responses (PRs). In the infliximab cohort, we observed one CR, two PRs, and three progressive diseases. Signs of activation and maturation of systemic T-cell responses were seen in patients from both cohorts. CONCLUSIONS: Our results show that both combinations are safe in human and provide clinical and biological activities. The high response rate in the certolizumab-treated patient cohort deserves further investigations.

Resistance of melanoma to immune checkpoint inhibitors is overcome by targeting the sphingosine kinase-1.

Immune checkpoint inhibitors (ICIs) have dramatically modified the prognosis of several advanced cancers, however many patients still do not respond to treatment. Optimal results might be obtained by targeting cancer cell metabolism to modulate the immunosuppressive tumor microenvironment. Here, we identify sphingosine kinase-1 (SK1) as a key regulator of anti-tumor immunity. Increased expression of SK1 in tumor cells is significantly associated with shorter survival in metastatic melanoma patients treated with anti-PD-1. Targeting SK1 markedly enhances the responses to ICI in murine models of melanoma, breast and colon cancer. Mechanistically, SK1 silencing decreases the expression of various immunosuppressive factors in the tumor microenvironment to limit regulatory T cell (Treg) infiltration. Accordingly, a SK1-dependent immunosuppressive signature is also observed in human melanoma biopsies. Altogether, this study identifies SK1 as a checkpoint lipid kinase that could be targeted to enhance immunotherapy.

Corrigendum: The TNF Paradox in Cancer Progression and Immunotherapy.

[This corrects the article DOI: 10.3389/fimmu.2019.01818.].

Anti-TNF, a magic bullet in cancer immunotherapy?

Immune checkpoint blockers (ICB) have revolutionized cancer therapy. However, complete response is observed in a minority of patients and most patients develop immune-related adverse events (irAEs). These include colitis, which can be treated with anti-tumor necrosis factor (TNF) antibodies such as Infliximab. In a recent issue of the Journal for ImmunoTherapy of Cancer, Badran et al. reported that co-administering Infliximab together with ICB to five cancer patients prevents colitis recurrence, with four of them exhibiting overall disease stability. The basis for this treatment strategy stemmed from our pre-clinical demonstration that TNF contributes to resistance to anti-PD-1 therapy. In agreement with this concept, we have shown that TNF blockers improve the anti-tumor therapeutic activity of ICB in mice and based on these findings we are currently evaluating the combination in melanoma patients enrolled in the TICIMEL clinical trial. Herein, (i) we discuss the scientific rationale for combining anti-TNF and ICB in cancer patients, (ii) comment on the paper published by Badran et al. and (iii) provide the TICIMEL clinical trial design.

Sphingomyelin Synthase 1 (SMS1) Downregulation Is Associated With Sphingolipid Reprogramming and a Worse Prognosis in Melanoma.

Sphingolipid (SL) metabolism alterations have been frequently reported in cancer including in melanoma, a bad-prognosis skin cancer. In normal cells, de novo synthesized ceramide is mainly converted to sphingomyelin (SM), the most abundant SL, by sphingomyelin synthase 1 (SMS1) and, albeit to a lesser extent, SMS2, encoded by the SGMS1 and SGMS2 genes, respectively. Alternatively, ceramide can be converted to glucosylceramide (GlcCer) by the GlcCer synthase (GCS), encoded by the UGCG gene. Herein, we provide evidence for the first time that SMS1 is frequently downregulated in various solid cancers, more particularly in melanoma. Accordingly, various human melanoma cells displayed a SL metabolism signature associated with (i) a robust and a low expression of UGCG and SGMS1/2, respectively, (ii) higher in situ enzyme activity of GCS than SMS, and (iii) higher intracellular levels of GlcCer than SM. SMS1 was expressed at low levels in most of the human melanoma biopsies. In addition, several mutations and increased CpG island methylation in the SGMS1 gene were identified that likely affect SMS1 expression. Finally, low SMS1 expression was associated with a worse prognosis in metastatic melanoma patients. Collectively, our study indicates that SMS1 downregulation in melanoma enhances GlcCer synthesis, triggering an imbalance in the SM/GlcCer homeostasis, which likely contributes to melanoma progression. Evaluating SMS1 expression level in tumor samples might serve as a biomarker to predict clinical outcome in advanced melanoma patients.

A Natural Variant of the Signaling Molecule Vav1 Enhances Susceptibility to Myasthenia Gravis and Influences the T Cell Receptor Repertoire.

The guanine nucleotide exchange factor Vav1 is essential for transducing T cell receptor (TCR) signals and plays an important role in T cell development and activation. Previous genetic studies identified a natural variant of Vav1 characterized by the substitution of an arginine (R) residue by a tryptophane (W) at position 63 (Vav1R63W). This variant impacts Vav1 adaptor functions and controls susceptibility to T cell-mediated neuroinflammation. To assess the implication of this Vav1 variant on the susceptibility to antibody-mediated diseases, we used the animal model of myasthenia gravis, experimental autoimmune myasthenia gravis (EAMG). To this end, we generated a knock-in (KI) mouse model bearing a R to W substitution in the Vav1 gene (Vav1R63W) and immunized it with either torpedo acetylcholine receptor (tAChR) or the α146-162 immunodominant peptide. We observed that the Vav1R63W conferred increased susceptibility to EAMG, revealed by a higher AChR loss together with an increased production of effector cytokines (IFN-γ, IL-17A, GM-CSF) by antigen-specific CD4+ T cells, as well as an increased frequency of antigen-specific CD4+ T cells. This correlated with the emergence of a dominant antigen-specific T cell clone in KI mice that was not present in wild-type mice, suggesting an impact on thymic selection and/or a different clonal selection threshold following antigen encounter. Our results highlight the key role of Vav1 in the pathophysiology of EAMG and this was associated with an impact on the TCR repertoire of AChR reactive T lymphocytes.

The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4+ T cells.

The activation of T cells requires the guanine nucleotide exchange factor VAV1. Using mice in which a tag for affinity purification was attached to endogenous VAV1 molecules, we analyzed by quantitative mass spectrometry the signaling complex that assembles around activated VAV1. Fifty VAV1-binding partners were identified, most of which had not been previously reported to participate in VAV1 signaling. Among these was CD226, a costimulatory molecule of immune cells. Engagement of CD226 induced the tyrosine phosphorylation of VAV1 and synergized with T cell receptor (TCR) signals to specifically enhance the production of interleukin-17 (IL-17) by primary human CD4+ T cells. Moreover, co-engagement of the TCR and a risk variant of CD226 that is associated with autoimmunity (rs763361) further enhanced VAV1 activation and IL-17 production. Thus, our study reveals that a VAV1-based, synergistic cross-talk exists between the TCR and CD226 during both physiological and pathological T cell responses and provides a rational basis for targeting CD226 for the management of autoimmune diseases.

TNFα blockade overcomes resistance to anti-PD-1 in experimental melanoma.

Antibodies against programmed cell death-1 (PD-1) have considerably changed the treatment for melanoma. However, many patients do not display therapeutic response or eventually relapse. Moreover, patients treated with anti-PD-1 develop immune-related adverse events that can be cured with anti-tumor necrosis factor α (TNF) antibodies. Whether anti-TNF antibodies affect the anti-cancer immune response remains unknown. Our recent work has highlighted that TNFR1-dependent TNF signalling impairs the accumulation of CD8+ tumor-infiltrating T lymphocytes (CD8+ TILs) in mouse melanoma. Herein, our results indicate that TNF or TNFR1 blockade synergizes with anti-PD-1 on anti-cancer immune responses towards solid cancers. Mechanistically, TNF blockade prevents anti-PD-1-induced TIL cell death as well as PD-L1 and TIM-3 expression. TNF expression positively correlates with expression of PD-L1 and TIM-3 in human melanoma specimens. This study provides a strong rationale to develop a combination therapy based on the use of anti-PD-1 and anti-TNF in cancer patients.

Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma.

The infiltration of melanoma tumors by macrophages is often correlated with poor prognosis. However, the molecular signals that regulate the dialogue between malignant cells and the inflammatory microenvironment remain poorly understood. We previously reported an increased expression of sphingosine kinase-1 (SK1), which produces the bioactive lipid sphingosine 1-phosphate (S1P), in melanoma. The present study aimed at defining the role of tumor SK1 in the recruitment and differentiation of macrophages in melanoma. Herein, we show that downregulation of SK1 in melanoma cells causes a reduction in the percentage of CD206highMHCIIlow M2 macrophages in favor of an increased proportion of CD206lowMHCIIhigh M1 macrophages into the tumor. This macrophage differentiation orchestrates T lymphocyte recruitment as well as tumor rejection through the expression of Th1 cytokines and chemokines. In vitro experiments indicated that macrophage migration is triggered by the binding of tumor S1P to S1PR1 receptors present on macrophages whereas macrophage differentiation is stimulated by SK1-induced secretion of TGF-ß1. Finally, RNA-seq analysis of human melanoma tumors revealed a positive correlation between SK1 and TGF-ß1 expression. Altogether, our findings demonstrate that melanoma SK1 plays a key role in the recruitment and phenotypic shift of the tumor macrophages that promote melanoma growth.

A Natural Variant of the T Cell Receptor-Signaling Molecule Vav1 Reduces Both Effector T Cell Functions and Susceptibility to Neuroinflammation.

The guanine nucleotide exchange factor Vav1 is essential for transducing T cell antigen receptor signals and therefore plays an important role in T cell development and activation. Our previous genetic studies identified a locus on rat chromosome 9 that controls the susceptibility to neuroinflammation and contains a non-synonymous polymorphism in the major candidate gene Vav1. To formally demonstrate the causal implication of this polymorphism, we generated a knock-in mouse bearing this polymorphism (Vav1R63W). Using this model, we show that Vav1R63W mice display reduced susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 peptide immunization. This is associated with a lower production of effector cytokines (IFN-γ, IL-17 and GM-CSF) by autoreactive CD4 T cells. Despite increased proportion of Foxp3+ regulatory T cells in Vav1R63W mice, we show that this lowered cytokine production is intrinsic to effector CD4 T cells and that Treg depletion has no impact on EAE development. Finally, we provide a mechanism for the above phenotype by showing that the Vav1R63W variant has normal enzymatic activity but reduced adaptor functions. Together, these data highlight the importance of Vav1 adaptor functions in the production of inflammatory cytokines by effector T cells and in the susceptibility to neuroinflammation.

Targeting TNF alpha as a novel strategy to enhance CD8+ T cell-dependent immune response in melanoma?

Tumor Necrosis Factor α (TNF) is a pleiotropic cytokine exhibiting a dual activity in oncoimmunology, either acting as a cytotoxic effector produced by leukocytes or behaving as an immunosuppressive molecule. We have just discovered that TNF signaling impairs the accumulation of tumor-infiltrating CD8+ T lymphocytes in experimental melanoma.

Glucosylceramidases and malignancies in mammals.

Sphingolipids represent a major class of lipids that are essential constituents of eukaryotic cells. They are predominantly located in plasma membrane microdomains, and play an important structural role in regulating membrane fluidity. They are also bioactive effectors involved in diverse key cellular functions such as apoptosis and proliferation. The implication of some sphingolipids in cancer is well established whereas that of some others is still a matter of intense investigation. Glucosylceramide is the backbone of more than 300 structurally different glycosphingolipids including gangliosides and sulfatides, and is essential for mammalian development. Therefore, glucosylceramidases (also named GBA1, GBA2 and GBA3 ß-glucosidases), the enzymes that hydrolyse ß-glucosylceramide, play important functions. GBA1 is a lysosomal hydrolase whose deficiency causes Gaucher disease, the most prevalent inherited lysosomal storage disorder. GBA2 is a ubiquitous non-lysosomal glucosylceramidase whose mutations have been associated with some forms of hereditary spastic paraplegia. GBA3 is a cytosolic ß-glucosidase, mostly present in the kidney, liver, spleen, intestine and lymphocytes of mammals, the function of which is still unclear. Whereas glucosylceramide synthase is implicated in multidrug resistance, the role of glucosylceramide breakdown in cancer is not yet fully appreciated. Defective GBA1 enzyme activity in humans, i.e., Gaucher disease, is associated with an increased risk of multiple myeloma and other malignancies. Putative molecular links between Gaucher disease and cancer, which might implicate the malignant cell and/or its microenvironment, are reviewed. The functions of GBA2 and GBA3 in cancer progression are also discussed.

Blocking Tumor Necrosis Factor α Enhances CD8 T-cell-Dependent Immunity in Experimental Melanoma.

TNF plays a dual, still enigmatic role in melanoma, either acting as a cytotoxic cytokine or favoring a tumorigenic inflammatory microenvironment. Herein, the tumor growth of melanoma cell lines expressing major histocompatibility complex class I molecules at high levels (MHC-I(high)) was dramatically impaired in TNF-deficient mice, and this was associated with enhanced tumor-infiltrating CD8(+) T lymphocytes. Immunodepletion of CD8 T cells fully restored melanoma growth in TNF(-/-) mice. Systemic administration of Etanercept inhibited MHC-I(high) melanoma growth in immunocompetent but not in immunodeficient (IFNγ(-/-), nude, or CD8(-/-)) mice. MHC-I(high) melanoma growth was also reduced in mice lacking TNF-R1, but not TNF-R2. TNF(-/-) and TNF-R1(-/-) mice as well as Etanercept-treated WT mice displayed enhanced intratumor content of high endothelial venules surrounded by high CD8(+) T-cell density. Adoptive transfer of activated TNF-R1-deficient or -proficient CD8(+) T cells in CD8-deficient mice bearing B16K1 tumors demonstrated that TNF-R1 deficiency facilitates the accumulation of live CD8(+) T cells into the tumors. Moreover, in vitro experiments indicated that TNF triggered activated CD8(+) T cell death in a TNF-R1-dependent manner, likely limiting the accumulation of tumor-infiltrating CD8(+) T cells in TNF/TNF-R1-proficient animals. Collectively, our observations indicate that TNF-R1-dependent TNF signaling impairs tumor-infiltrating CD8(+) T-cell accumulation and may serve as a putative target to favor CD8(+) T-cell-dependent immune response in melanoma.