Inflammation promotes phenotypic plasticity in melanoma, a source of non-genetic heterogeneity, but the molecular framework is poorly understood. Here we use functional genomic approaches and identify a reciprocal antagonism between the melanocyte lineage transcription factor MITF and c-Jun, which interconnects inflammation-induced dedifferentiation with pro-inflammatory cytokine responsiveness of melanoma cells favouring myeloid cell recruitment. We show that pro-inflammatory cytokines such as TNF-α instigate gradual suppression of MITF expression through c-Jun. MITF itself binds to the c-Jun regulatory genomic region and its reduction increases c-Jun expression that in turn amplifies TNF-stimulated cytokine expression with further MITF suppression. This feed-forward mechanism turns poor peak-like transcriptional responses to TNF-α into progressive and persistent cytokine and chemokine induction. Consistently, inflammatory MITFlow/c-Junhigh syngeneic mouse melanomas recruit myeloid immune cells into the tumour microenvironment as recapitulated by their human counterparts. Our study suggests myeloid cell-directed therapies may be useful for MITFlow/c-Junhigh melanomas to counteract their growth-promoting and immunosuppressive functions.

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Background: Upregulation of the histone methyltransferase enzyme EZH2 and its histone modification H3K27me3 has been linked to melanoma progression, metastasis, and resistance to immune checkpoint blockade (ICB). In clinical trials, EZH2 inhibitors are currently tested to overcome resistance to ICB. The aim of this study is to evaluate expression patterns and the predictive value of H3K27me3 and EZH2 in metastatic melanoma samples prior to ICB. As H3K27me3 expression has been associated with a dedifferentiated, invasive melanoma phenotype, we also investigated the prognostic value of H3K27me3 expression in primary melanomas. Results: H3K27me3 and EZH2 expression were evaluated in a cohort of 44 metastatic melanoma samples before ICB using immunohistochemistry (IHC). 29/44 (66%) of melanomas showed H3K27me3 expression, and 6/44 (14%) showed EZH2 expression. No predictive value for therapeutic response to anti-PD-1 therapy could be found for H3K27me3 or EZH2 expression on melanoma cells. To investigate the prognostic significance of H3K27me3, we analyzed H3K27me3 expression in a representative cohort of 136 primary melanomas with known sentinel lymph node status. H3K27me3 expression is associated with increased tumor thickness and nodal involvement. Melanoma metastases showed a higher expression of H3K27me3 in comparison to primary melanomas. In human melanoma cell lines, TNFα and INFγ could not induce H3K27me3 expression. Conclusion: Our study shows that H3K27me3 expression is more frequent than EZH2 and is associated with a more invasive and metastatic melanoma cell phenotype. We suggest that H3K27me3 expression by IHC might be a suitable method to evaluate the activity of EZH2 inhibitors in clinical trials.

Antigen loss is a key mechanism how tumor cells escape from T-cell immunotherapy. Using a mouse model of melanoma we directly compared antigen downregulation by phenotypic adaptation with genetically hardwired antigen loss. Unexpectedly, genetic ablation of Pmel, the melanocyte differentiation antigen targeted by adoptively transferred CD8 + T-cells, impaired melanoma cell growth in untreated tumors due to competitive pressure exerted by the bulk wild-type population.This established an evolutionary scenario, where T-cell immunotherapy imposed a dynamic fitness switch on wild-type melanoma cells and antigen loss variants, which resulted in highly variable enrichment of the latter in recurrent tumors. Stochastic simulations by an individual-based continuous-time Markov process suggested variable fitness of subclones within the antigen loss variant population as the most likely cause, which was validated experimentally. In summary, we provide a framework to better understand how subclone heterogeneity in tumors influences immune selection of genetic antigen loss variants through stochastic events.