BRAF AND MAPK PATHWAY MOLECULES FOR TARGETED THERAPY OF MALIGNANT MELANOMA

The clinical activity of the BRAF inhibitor PLX4032 (vemurafenib) in patients with BRAFV600E mutant melanoma is limited primarily by the development of resistance leading to tumor progression. Strategies to overcome primary and acquired resistance are required. In a panel of 27 genetically characterized patient-derived melanoma cell lines the sensitivity to PLX4032 was dependent on BRAFV600E and independent from other gene alterations that commonly occur in melanoma, such as CDKN2A, and mutations of TP53, PTEN loss, and BRAF and MITF gene amplification. To investigate the molecular basis underlying acquired resistance to BRAF inhibitor, PLX4032-resistant cells were derived from a high sensitive BRAFV600E melanoma cell line, and used as a model. The resistant variant line showed increased AKT and ERK phosphorylation and enhanced IGF-1R/PI3K signaling. Combined treatment with PLX4032 plus PI3K inhibitors resulted in significant cell growth inhibition by decreasing pAKT and pERK signaling. To explore molecular mechanisms underlying primary resistance two melanoma cell lines lacking sensitivity to PLX4032 were used as models. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA, indicating that CRAF is not involved in the activation of ERK in the resistant cell lines. Treatment with the MEK inhibitor UO126 inhibited cell growth and decreased ERK phosphorylation indicating alternative activation of MEK-ERK signaling. Genetic characterization by MLPA and analysis of pTyr signaling by MALDI-TOF mass spectrometry revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. Testing of co-inhibition of the MET, SRC and MAPK signaling pathways by the combined treatment with the MET inhibitor, SU11274 or the SRC inhibitor, BMS-354825 plus PLX4032 resulted in a significant inhibitory effect on melanoma cell proliferation, survival, migration and invasive capacity. These results support combinatorial approaches targeting MAPK pathway at different nodes and intercepting parallel signal transduction pathways as a strategy to override resistance to BRAF inhibitors.