Over half of cutaneous melanoma tumors have BRAFV600E/K mutations.,Acquired resistance to BRAF inhibitors (BRAFi) remains a major hurdle in attaining durable therapeutic responses.,In this study we demonstrate that approximately 50-60% of melanoma cell lines with vemurafenib resistance acquired in vitro show activation of RhoA family GTPases.,In BRAFi-resistant melanoma cell lines and tumors, activation of RhoA is correlated with decreased expression of melanocyte lineage genes.,Using a machine learning approach, we built gene expression-based models to predict drug sensitivity for 265 common anti-cancer compounds.,We then projected these signatures onto the collection of TCGA cutaneous melanoma and found that poorly differentiated tumors were predicted to have increased sensitivity to multiple Rho kinase (ROCK) inhibitors.,Two transcriptional effectors downstream of Rho, MRTF and YAP1, are activated in the RhoHigh BRAFi-resistant cell lines, and resistant cells are more sensitive to inhibition of these transcriptional mechanisms.,Taken together, these results support the concept of targeting Rho-regulated gene transcription pathways as a promising therapeutic approach to restore sensitivity to BRAFi-resistant tumors or as a combination therapy to prevent the onset of drug resistance.
Constitutively active mutated BRAF kinase occurs in more than 40% of patients suffering from melanoma.,To block its activity, a specific inhibitor, vemurafenib, is applied as a therapy.,Unfortunately, patients develop resistance to this drug rather quickly.,Previously, we demonstrated that pairs of inhibitors directed against EGFR (epidermal growth factor receptor) and MET (hepatocyte growth factor receptor) trigger a synergistic cytotoxic effect in human melanoma cells, and decrease their invasive abilities.,In this study, we aimed to generate and characterize melanoma cells resistant to vemurafenib treatment, and then to evaluate the effectiveness of a previously developed therapy in this model.,We showed that melanoma cells resistant to the BRAF inhibitor are characterized by a lower proliferation rate and they acquire a spindle-like shape.,Using Western Blot, we also noticed increased levels of EGFR, MET, and selected markers of cancer stem cells in generated cell lines.,Resistant cells also exhibited increased invasive abilities and elevated proteolytic activity, observed using scratch wound assays and gelatin zymography.,Moreover, combination therapy reduced their viability, as measured with a colorimetric cytotoxicity test, and decreased invasiveness.,The obtained results validate the application of combination therapy directed against EGFR and MET in melanoma cells resistant to treatment with inhibitors of mutated BRAF.