This indicated that ERK re\activation occurs upstream of MEK, and the most prominent candidate for this activation is CRAF. MITF\high populations through the endothelin receptor B (EDNRB), but also AXL\high populations through EDNRA, making it a master regulator of phenotype heterogeneity. Endothelin receptor antagonists suppress AXL\high\expressing cells and AOH1160 sensitize to BRAF inhibition, suggesting that targeting EDN1 signalling could improve BRAF inhibitor responses without selecting for AXL\high cells. gene, express higher levels of additional oncogenic drivers that confer intrinsic MAPK inhibitor resistance. These melanomas are characterized by gene signatures, which correlate with enhanced expression of the receptor tyrosine kinase AXL (Sensi cultures were analysed for MITF expression by Western blot and immunofluorescence (magenta). Nuclei were stained with DAPI. Scale bar: 20?m (white arrows, high MITF; black arrows, low MITF). Relative AXL and MITF expression in a panel of melanoma cell lines that have been characterized for their response to BRAF inhibition (Barretina situation stroma\derived signals from the local tumour microenvironment could have differing effects on MITF expression (Smith cultures in the absence of a microenvironment, but intriguingly MITF heterogeneity prevailed, and stronger and weaker MITF\expressing cells were detected (Fig?1B). Importantly, the presence of weaker MITF\expressing cells was not due to enrichment for a AXL\high/MITF\low populationconsidered the most resistant phenotypeas this fraction was rather reduced in cultures responding to BRAF inhibitor (Fig?EV1C and D). We therefore attempted to monitor the dynamics of individual cells within one MITF\high cell line in the response to MAPK inhibition in more detail. To identify a representative cell line, we assessed the AXL and MITF expression status in a panel of melanoma cell lines and their link to response to BRAF inhibition. In agreement with previous reports, we found a correlation with high AXL expression and low MITF expression and resistance to BRAF inhibition (Fig?1C). The group of MITF\expressing cell lines displayed a considerable distribution of MITF expression levels, and whereas weaker expression correlated with BRAF inhibitor sensitivity, increased MITF expression protected from BRAF inhibition (Fig?1C). We chose WM164 cells as they express intermediate MITF and AXL levels and respond to BRAF inhibition (Fig?1C). In untreated WM164 cells, MITF expression is heterogeneous (Fig?1D), which allowed us to assess whether high MITF expression will be selected for over the time of treatment. Using the FUCCI system, which can report on the individual phases of the cell cycle, we followed single FUCCI\WM164 cells (Haass test); ***test); ***test); ** 0.05, ***cultures. DMSO\treated A375 cells were set at 100%. A Western blot for pERK and ERK under the respective conditions is shown. test); **test); ns 0.05, **test); ***cultures isolated from tumours that had regressed on BRAF inhibitor (Fig?EV3D), as well as with generated A375\T cells (Fig?EV3E). MEK inhibition could overcome the paracrine protection and ERK re\activation mediated by soluble factors (Fig?EV3E). This indicated that ERK re\activation occurs upstream of MEK, and the most prominent candidate for this activation is CRAF. We thus used the pan\RAF inhibitor RAF265, which abolished the re\activation of ERK phosphorylation (Fig?3E) and completely overcame the protective effect produced by A375\T cells (Fig?3F). A similar effect was observed in other melanoma cell lines when they were treated with conditioned medium (Fig?EV3F). Using specific inhibitors to identify the upstream activator of CRAF revealed that the pan\PKC inhibitor GO\6983 (PKCi) was able to overcome ERK re\activation and the protective effect AOH1160 produced by co\culturing A375 cells with A375\T cells (Fig?3E and F). These data strongly suggest that prolonged BRAF inhibition AOH1160 triggers the production of secreted factors capable of re\activating the pathway via PKC and CRAF. Indeed, treatment of melanoma cell lines with conditioned medium derived from corresponding cell lines treated for 14?days with BRAF inhibitor, resulted in an increase in the phosphorylation of proteins recognized as PKC substrates (Fig?3G). The secretome of BRAF inhibitor pre\treated cells is enriched in?Endothelin\1 A quantitative proteomics analysis of conditioned medium AOH1160 from untreated A375 and A375\T cells (applying a cut\off of twofold change) identified 387 proteins that were enriched in the conditioned medium of pre\treated A375\T cells; amongst these were 77 secreted/extracellular space\signalling proteins (Fig?4A). Ingenuity Pathway Analysis identified 27 of these 77 proteins as activators of ERK (Fig?4B). Amongst these was EGF, which together with its receptor Pdgfra has been previously implicated in BRAF inhibitor resistance through reactivation of ERK (Girotti test); ns 0.05, **test); ***test);.