Identification of the molecular mechanisms by which pigmentation limits Vemurafenib activity in melanoma cells

Melanoma is the most aggressive type of skin cancer and remains a therapeutic challenge. BRAFV600E is the most common mutation in melanoma and causes the hyperactivation of the MAPK pathway. BRAF and MEK inhibitors have substantial therapeutic efficacy in patients with BRAF mutant melanoma. However, they still have significant limitations, including short-lasting efficacy, hence short-lived responses, due to early onset of acquired resistance. An early phase exists, in which cells are in a drug tolerant state that can be used in our favor before genetic resistance occurs. We showed that one of the mechanisms that cells implement to find a strategy of survival from targeted therapy is an increase in pigmentation upon vemurafenib treatment, which confers to pigmentable melanoma cells higher resistance to BRAF inhibitors compared to non-pigmentable cells. Here we build on our previous data to confirm that melanin induction negatively affects therapy outcome in vitro, in vivo and in patients. We also demonstrate that intracellular melanosomes are the source of resistance to MAPKi in pigmentable cells. Crucially, we show that melanin acts as scavenger of ROS induced by MAPK inhibitors (MAPKi). Pigmentable cells present lower levels of ROS, as well as DNA damage, compared to non-pigmentable cells and by chemically or genetically removing melanin, we obtain an increase in ROS levels and a rescue in the efficacy of MAPKi treatment. Conversely, when we turn non-pigmentable into pigmentable cells, by adding melanin nanoparticles, we obtain opposite results. In addition, we demonstrate that melanosomes-mitochondria physical contacts favor the melanin-mediated scavenging of vem-induced ROS and we propose RAB27A as a possible candidate that can mediate the connections among the two organelles. Finally, we expand on our previous findings that the efficacy of vem plus pigmentation inhibitors on pigmentable cells is further increased by blocking a second adaptive response, i.e. vem-induced shift toward oxidative phosphorylation. In conclusion, we affirm that pigmentation is an adaptive cellular response that limits the efficacy of MAPKi. We also propose the combination of MAPKi with pigmentation inhibitors as a way to tame the resistance of pigmentable melanoma cells. Finally, we emphasize the necessity to stratify metastatic melanoma tumors according to their pigmentation status.