Background: Current therapeutic management of advanced melanoma patients largely depends on their BRAF mutation status. However, the vast heterogeneity of the tumors hampers the success of therapies targeting the MAPK/ERK pathway alone. Dissecting this heterogeneity will contribute to identifying key players in the oncogenic progression to tailor more effective therapies. Methods: We performed a comprehensive molecular and phenotypic characterization of a panel of patient-derived BRAF^V600E-positive melanoma cell lines. Transcriptional profiling was used to identify groups of coregulated genes whose expression relates to an increased migratory potential and a higher resistance. Results: A decrease in sensitivity to MAPK/ERK pathway inhibition with vemurafenib or trametinib corresponded with an increasing quiescence and migratory properties of the cells. This was accompanied by the loss of transcriptional signatures of melanocytic differentiation, and the gain of stem cell features that conferred highly-resistant/mesenchymal-like cells with increased xenobiotic efflux capacity. Nevertheless, targeting of the implicated ABC transporters did not improve the response to vemurafenib, indicating that incomplete BRAF inhibition due to reduced drug uptake is not a main driver of resistance. Rather, indifference to MAPK/ERK pathway inhibition arose from the activation of compensatory signaling cascades. The PI3K/AKT pathway in particular showed a higher activity in mesenchymal-like cells, conferring a lower dependency on MAPK/ERK signaling and supporting stem-like properties that could be reverted by dual PI3K/mTOR inhibition with dactolisib. Conclusions: In case of MAPK/ERK independency, therapeutic focus may be shifted to the PI3K/AKT pathway to overcome late-stage resistance in melanoma tumors that have acquired a mesenchymal phenotype.

Background: Vemurafenib (PLX4032) is one of the most frequently used treatments for late-stage melanoma patients with the BRAF^V600E mutation; however, acquired resistance to the drug poses as a major challenge. It remains to be determined whether off-target effects of vemurafenib on normal stroma components could reshape the tumor microenvironment in a way that contributes to cancer progression and drug resistance. Methods: By using temporally-resolved RNA- and ATAC-seq, we studied the early molecular changes induced by vemurafenib in human dermal fibroblast (HDF), a main stromal component in melanoma and other tumors with high prevalence of BRAF^V600 mutations. Results: Transcriptomics analyses revealed a stepwise up-regulation of proliferation signatures, together with a down-regulation of autophagy and proteolytic processes. The gene expression changes in HDF strongly correlated in an inverse way with those in BRAF^V600E mutant malignant melanoma (MaMel) cell lines, consistent with the observation of a paradoxical effect of vemurafenib, leading to hyperphosphorylation of MEK1/2 and ERK1/2. The transcriptional changes in HDF were not strongly determined by alterations in chromatin accessibility; rather, an already permissive chromatin landscape seemed to facilitate the early accessibility to MAPK/ERK-regulated transcription factor binding sites. Combinatorial treatment with the MEK inhibitor trametinib did not preclude the paradoxical activation of MAPK/ERK signaling in HDF. When administered together, vemurafenib partially compensated for the reduction of cell viability and proliferation induced by trametinib. These paradoxical changes were restrained by using the third generation BRAF inhibitor PLX8394, a so-called paradox breaker compound. However, the advantageous effects on HDF during combination therapies were also lost. Conclusions: Vemurafenib induces paradoxical changes in HDF, enabled by a permissive chromatin landscape. These changes might provide an advantage during combination therapies, by compensating for the toxicity induced in stromal cells by less specific MAPK/ERK inhibitors. Our results highlight the relevance of evaluating the effects of the drugs on non-transformed stromal components, carefully considering the implications of their administration either as mono- or combination therapies.