Resistance to immune checkpoint inhibitors (ICI) in cancer patients is not fully understood, and predictive biomarkers are lacking. MELANFα (NCT03348891) is an open‐label, prospective, multicenter cohort of 60 patients with advanced melanoma receiving ICI (bitherapy: ipilimumab + nivolumab; monotherapy: pembrolizumab or nivolumab). The primary objective was to evaluate whether changes in plasma TNF between baseline (W0) and week 12 (W12) identified patients with non‐progressive disease at W12. Secondary and exploratory objectives were to assess the association between plasma TNF, tumor response, and changes in circulating T cells. Plasma TNF increased along therapy, but its W12/W0 fold change was not associated with non‐progressive disease at W12. However, plasma TNF levels at W12 were significantly higher in non‐responders than in responders across therapies ( p = .0129). The remodeling of circulating T cell subpopulations was mostly triggered by bitherapy. Increased proportions of circulating central memory and effector memory CD8 T cells after bitherapy were positively and negatively associated with response to treatment, respectively. In this cohort, circulating T cells from responders and non‐responders also displayed distinct molecular characteristics. Indeed, responders showed an increased proportion of CD8 T cells with low enrichment of TNF‐related pathways and high cytotoxic potential, while non‐responders displayed increased proportions of circulating CD8 EM T cells enriched for TNF‐related pathways and directed toward cytokine expression. In conclusion, our study shows that elevated plasma TNF and enriched TNF pathways in T cells are associated with poorer clinical outcomes, reinforcing the notion that TNF may dampen ICI efficacy.

IntroductionAdvanced cutaneous melanoma is a skin cancer characterized by a poor prognosis and high metastatic potential. During metastatic spread, melanoma cells often undergo dedifferentiation toward an invasive phenotype, resulting in reduced expression of microphthalmia-associated transcription factor (MITF)-dependent melanoma antigens and facilitating immune escape. Tumor Necrosis Factor (TNF) is known to be a key factor in melanoma dedifferentiation. Interestingly, accumulating evidence suggests that TNF may play a role in melanoma progression and resistance to immunotherapies. Additionally, TNF has been identified as a potent regulator of sphingolipid metabolism, which could contribute to melanoma aggressiveness and the process of melanoma dedifferentiation.MethodsWe conducted RNA sequencing and mass spectrometry analyses to investigate TNF-induced dedifferentiation in two melanoma cell lines. In vitro experiments were performed to manipulate sphingolipid metabolism using genetic or pharmacologic alterations in combination with TNF treatment, aiming to elucidate the potential involvement of this metabolism in TNF-induced dedifferentiation. Lastly, to evaluate the clinical significance of our findings, we performed unsupervised analysis of plasma sphingolipid levels in 48 patients receiving treatment with immune checkpoint inhibitors, either alone or in combination with anti-TNF therapy.ResultsHerein, we demonstrate that TNF-induced melanoma cell dedifferentiation is associated with a global modulation of sphingolipid metabolism. Specifically, TNF decreases the expression and activity of acid ceramidase (AC), encoded by the ASAH1 gene, while increasing the expression of glucosylceramide synthase (GCS), encoded by the UGCG gene. Remarkably, knockdown of AC alone via RNA interference is enough to induce melanoma cell dedifferentiation. Furthermore, treatment with Eliglustat, a GCS inhibitor, inhibits TNF-induced melanoma cell dedifferentiation. Lastly, analysis of plasma samples from patients treated with immune checkpoint inhibitors, with or without anti-TNF therapy, revealed significant predictive sphingolipids. Notably, the top 8 predictive sphingolipids, including glycosphingolipids, were associated with a poor response to immunotherapy.DiscussionOur study highlights that ceramide metabolism alterations are causally involved in TNF-induced melanoma cell dedifferentiation and suggests that the evolution of specific ceramide metabolites in plasma may be considered as predictive biomarkers of resistance to immunotherapy.

<p>Tumor histology, age, disease stage and treatment of patients from the cohort used for tumor IgG1,2,3 and 4 dosage</p>

Supplementary Figure S1. TNF does not modulate in vitro B16 proliferation.

Supplementary Figure S3. Analysis of the immune response in spleen from WT and TNF-deficient mice.

Mean age, disease stage and treatment of the patients used to analyse the CD20 infiltrate of HGSOC metastases by IHC

Supplementary Figure S5. Analysis of Treg and MDSC content in the tumors from WT and TNF-deficient mice.

Supplementary Figure S7. Effect of Etanercept injection in mice.

<p>List of antibodies used for flow cytometry analyses of human and mouse tissues.</p>

Supplemental Figures and Supplemental Methods

<p>Age, disease stage and treatment of patients from the cohort used for plasma IgG1,2,3 and 4 dosage</p>

Sequences of primers used to perform the BCR sequencing analyses

Supplementary Tables 10 - 14

Supplementary Figure S4. Analysis of the immune response in draining lymph nodes from WT and TNF-deficient mice.

Supplementary Figure S6. Accumulation of CD8+ TIL in TNF-R1-deficient mice.

<p>Tumor histology, age, disease stage and treatment of patients from the cohort used for tumor IgG1,2,3 and 4 dosage</p>

Supplementary Tables 15 - 25

Supplementary Figure S2. Tumor growth of B16-BL6 and Yumm melanoma cell lines in TNF-deficient mice.