Molecular Alterations Associated with Acquired Drug Resistance during Combined Treatment with Encorafenib and Binimetinib in Melanoma Cell Lines

SIMPLE SUMMARY: Melanoma is the most aggressive, deadliest form of skin cancer. Combined BRAF-MEK inhibitor (BRAFi/MEKi) therapy was a breakthrough in the treatment of melanoma with BRAFV600-mutations. However, many patients frequently develop drug resistance to the combinatory treatment. The aim of our study was to characterize the molecular background behind acquired resistance to BRAFi/MEKi-s. After the successful development of resistant cell lines, we investigated the invasion properties, changes in gene and protein expressions, as well as the effect of the “drug holiday” of the resistant cell lines. Drug-resistant melanoma cells had a higher invasive potential and acquired a spindle-like structure, and many cancer-related proteins were overexpressed in the resistant cells. Furthermore, transcriptome analysis revealed that differentially expressed genes are functionally linked to a variety of biological functions that may lead to resistance to the inhibitors. These results may offer valuable insight into further understanding of BRAFi/MEKi resistance, as well as to the development of therapeutic tools to overcome drug resistance. ABSTRACT: Combination treatment using BRAF/MEK inhibitors is a promising therapy for patients with advanced BRAF(V600E/K) mutant melanoma. However, acquired resistance largely limits the clinical efficacy of this drug combination. Identifying resistance mechanisms is essential to reach long-term, durable responses. During this study, we developed six melanoma cell lines with acquired resistance for BRAFi/MEKi treatment and defined the molecular alterations associated with drug resistance. We observed that the invasion of three resistant cell lines increased significantly compared to the sensitive cells. RNA-sequencing analysis revealed differentially expressed genes that were functionally linked to a variety of biological functions including epithelial-mesenchymal transition, the ROS pathway, and KRAS-signalling. Using proteome profiler array, several differentially expressed proteins were detected, which clustered into a unique pattern. Galectin showed increased expression in four resistant cell lines, being the highest in the WM1617(E+BRes) cells. We also observed that the resistant cells behaved differently after the withdrawal of the inhibitors, five were not drug addicted at all and did not exhibit significantly increased lethality; however, the viability of one resistant cell line (WM1617(E+BRes)) decreased significantly. We have selected three resistant cell lines to investigate the protein expression changes after drug withdrawal. The expression patterns of CapG, Enolase 2, and osteopontin were similar in the resistant cells after ten days of “drug holiday”, but the Snail protein was only expressed in the WM1617(E+BRes) cells, which showed a drug-dependent phenotype, and this might be associated with drug addiction. Our results highlight that melanoma cells use several types of resistance mechanisms involving the altered expression of different proteins to bypass drug treatment.