ABSTRACT
The development of targeted inhibitors, vemurafenib and dabrafenib, has led to improved clinical outcome for melanoma patients with BRAFV600E mutations. Although the initial response to these inhibitors can be dramatic, sometimes causing complete tumor regression, the majority of melanomas eventually become resistant. Mitogen-activated protein kinase kinase (MEK) mutations are found in primary melanomas and frequently reported in BRAF melanomas that develop resistance to targeted therapy; however, melanoma is a molecularly heterogeneous cancer, and which mutations are drivers and which are passengers remains to be determined. In this study, we demonstrate that in BRAFV600E melanoma cell lines, activating MEK mutations drive resistance and contribute to suboptimal growth of melanoma cells following the withdrawal of BRAF inhibition. In this manner, the cells are drug-addicted, suggesting that melanoma cells evolve a 'just right' level of mitogen-activated protein kinase signaling and the additive effects of MEK and BRAF mutations are counterproductive. We also used a novel mouse model of melanoma to demonstrate that several of these MEK mutants promote the development, growth and maintenance of melanoma in vivo in the context of Cdkn2a and Pten loss. By utilizing a genetic approach to control mutant MEK expression in vivo, we were able to induce tumor regression and significantly increase survival; however, after a long latency, all tumors subsequently became resistant. These data suggest that resistance to BRAF or MEK inhibitors is probably inevitable, and novel therapeutic approaches are needed to target dormant tumors.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p18/metabolism , MAP Kinase Kinase 1/metabolism , Melanoma, Experimental/enzymology , PTEN Phosphohydrolase/genetics , Skin Neoplasms/enzymology , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic/metabolism , Cyclin-Dependent Kinase Inhibitor p16 , Female , Humans , Indoles/pharmacology , MAP Kinase Kinase 1/genetics , Male , Melanocytes/metabolism , Melanoma, Experimental/pathology , Mice, Inbred C57BL , Mice, Transgenic , Mutation, Missense , Neoplasm Transplantation , PTEN Phosphohydrolase/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/metabolism , Skin Neoplasms/pathology , Sulfonamides/pharmacology , VemurafenibABSTRACT
Catalytic (SH1) domains of protein tyrosine kinases (PTKs) demonstrate specificity for peptide substrates. Whether SH1 domains differentiate between tyrosines in a physiological substrate has not been confirmed. Using purified proteins, we studied the ability of Syk, Fyn, and Abl to differentiate between tyrosines in a common PTK substrate, c-Cbl. We found that each kinase produced a distinct pattern of c-Cbl phosphorylation, which altered the phosphotyrosine-dependent interactions between c-Cbl and CrkL or phosphatidylinositol 3'-kinase (PI3-K). Our data support the concept that SH1 domains determine the final sites of phosphorylation once PTKs reach their target proteins.
