Targeted therapies with MAPK inhibitors (MAPKi) are faced with severe problems of resistance in BRAF‐mutant melanoma. In parallel to the acquisition of genetic mutations, melanoma cells may also adapt to the drugs through phenotype switching. The ZEB1 transcription factor, a known inducer of EMT and invasiveness, is now considered as a genuine oncogenic factor required for tumor initiation, cancer cell plasticity, and drug resistance in carcinomas. Here, we show that high levels of ZEB1 expression are associated with inherent resistance to MAPKi in BRAFV 600‐mutated cell lines and tumors. ZEB1 levels are also elevated in melanoma cells with acquired resistance and in biopsies from patients relapsing while under treatment. ZEB1 overexpression is sufficient to drive the emergence of resistance to MAPKi by promoting a reversible transition toward a MITF low/p75high stem‐like and tumorigenic phenotype. ZEB1 inhibition promotes cell differentiation, prevents tumorigenic growth in vivo, sensitizes naive melanoma cells to MAPKi, and induces cell death in resistant cells. Overall, our results demonstrate that ZEB1 is a major driver of melanoma cell plasticity, driving drug adaptation and phenotypic resistance to MAPKi.
BackgroundThe efficacy of immunotherapies in metastatic melanoma depends on a robust T cell infiltration. Oncogenic alterations of tumor cells have been associated to T cell exclusion. Identifying novel cancer cell-intrinsic non-genetic mechanisms of immune escape, the targeting of which would reinstate T cell recruitment, would allow to restore the response to anti-programmed cell death protein 1 (PD-1) antibody therapy. The epithelial-to-mesenchymal transition (EMT)-inducing transcription factor ZEB1 is a major regulator of melanoma cell plasticity, driving resistance to mitogen-activated protein kinase (MAPK) targeted therapies. We thus wondered whether ZEB1 signaling in melanoma cells may promote immune evasion and resistance to immunotherapy.MethodsWe evaluated the putative correlation between ZEB1 expression in melanoma cells and the composition of the immune infiltrate in a cohort of 60 human melanoma samples by combining transcriptomic (RNA-sequencing) and seven-color spatial multi-immunofluorescence analyses. Algorithm-based spatial reconstitution of tumors allowed the quantification of CD8+, CD4+ T cells number and their activation state (PD-1, Ki67). ZEB1 gain-of-function or loss-of-function approaches were then implemented in syngeneic melanoma mouse models, followed by monitoring of tumor growth, quantification of immune cell populations frequency and function by flow cytometry, cytokines secretion by multiplex analyses. Chromatin-immunoprecipitation was used to demonstrate the direct binding of this transcription factor on the promoters of cytokine-encoding genes. Finally, the sensitivity to anti-PD-1 antibody therapy upon ZEB1 gain-of-function or loss-of-function was evaluated.ResultsCombined spatial and transcriptomic analyses of the immune infiltrates in human melanoma samples demonstrated that ZEB1 expression in melanoma cells is associated with decreased CD8+ T cell infiltration, independently of β-catenin pathway activation. ZEB1 ectopic expression in melanoma cells impairs CD8+ T cell recruitment in syngeneic mouse models, resulting in tumor immune evasion and resistance to immune checkpoint blockade. Mechanistically, we demonstrate that ZEB1 directly represses the secretion of T cell-attracting chemokines, including CXCL10. Finally, Zeb1 knock-out, by promoting CD8+ T cell infiltration, synergizes with anti-PD-1 antibody therapy in promoting tumor regression.ConclusionsWe identify the ZEB1 transcription factor as a key determinant of melanoma immune escape, highlighting a previously unknown therapeutic target to increase efficacy of immunotherapy in melanoma.Trial registration numberNCT02828202.
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