Single cell profiling of tumour biopsies and heterogeneity in response of dedifferentiated melanoma.

Abstract

64 Background: Patients with advanced BRAFV600-mutant melanoma can be treated with immune checkpoint inhibitors or combination BRAF/MEK inhibitors. The sequence of these therapies can influence response to second-line treatment. Patients who progress on first-line PD1 blockade have poorer outcomes on BRAF/MEK inhibitors compared to patients who are treatment-naïve. Thus, defining the molecular features that are modified by therapy and that influence subsequent treatment responses require thorough characterization. Methods: In this study, tumor biopsies from five BRAF-mutant melanoma patients progressing on anti-PD1 (PROG) and five BRAF-mutant treatment-naïve (NAÏVE) patients were dissociated and treated ex vivo with BRAF/MEK inhibitors (dabrafenib+trametinib (DT)) before flow cytometry profiling and single cell transcriptome sequencing. Results: DT treatment did not alter tumor or immune cell proportions but effectively inhibited MAPK signaling by decreasing phosphorylated S6 levels in tumor cells within 8/10 melanoma samples. Single cell RNA-sequencing of the tumor dissociates identified four distinct melanoma differentiation states (e.g. melanocytic, transitory, neural crest-like and undifferentiated). The proportions of these cell states differed between tumor specimens, and were not affected by DT treatment. The neural crest-like and undifferentiated melanoma cells were enriched in PROG compared to NAIVE tumor samples and all differentiation states responded, albeit variably. For instance, MAPK inhibition after DT treatment was less pronounced in undifferentiated melanoma cells. Importantly, within the undifferentiated cell state, we identified a proportion of melanoma cells capable of responding to DT, and comparison of responding vs non-responding undifferentiated cells showed enrichment of pro-inflammatory IL-6 and TNFα signaling in the non-responding population. Elevated IL-6 and TNFα pathway activity correlated with decreased potency to MAPK inhibition, indicating that increased IL-6 and TNFα signaling in undifferentiated melanoma may contribute to resistance to MAPK inhibition. Conclusions: Ex vivo treatment of tumor dissociates coupled with single cell transcriptome sequencing identified cell state-specific responses to MAPK inhibition. Resistance to MAPK inhibition may be driven, in part, by tumor-intrinsic pro-inflammatory IL-6 and TNFα signalling.