Supplementary Material - PDF file 1876K, Supplementary Figure S1. Characterization of mutants obtained from B-RAFV600E random mutagenesis screens Supplementary Figure S2. Biochemical characterization of C-RAF resistance alleles Supplementary Figure S3. Effects of MEK inhibition on C-RAF resistance mutants Supplementary Figure S4. 293/T cells coexpressing His/V5 tagged C-RAF resistance mutants with Flag tagged WT-C-RAF were immunoprecipitated after 48 h with His and levels of Flag tagged C-RAF interaction was assessed by immunoblotting. Input lysate represents Flag-C-RAF, V5-C-RAF, C-RAF, p-MEK, p-ERK and total ERK. Supplementary Figure S5. A model of the putative effects of pharmacological RAF inhibitors on C-RAF resistance alleles. Supplementary Figure S6. Integrated Genomics Viewer (IGV) image of a RAF1 mutation present in a primary melanoma tumor
ARTICLE ABSTRACT
Melanomas that contain B-RAFV600E mutations respond transiently to RAF and MEK inhibitors; however, resistance to these agents remains a formidable challenge. Although B- or C-RAF dysregulation represents prominent resistance mechanisms, resistance-associated point mutations in RAF oncoproteins are surprisingly rare. To gain insights herein, we conducted random mutagenesis screens to identify B- or C-RAF mutations that confer resistance to RAF inhibitors. Whereas bona fide B-RAFV600E resistance alleles were rarely observed, we identified multiple C-RAF mutations that produced biochemical and pharmacologic resistance. Potent C-RAF resistance alleles localized to a 14-3-3 consensus binding site or a separate site within the P loop. These mutations elicited paradoxical upregulation of RAF kinase activity in a dimerization-dependent manner following exposure to RAF inhibitors. Knowledge of resistance-associated C-RAF mutations may enhance biochemical understanding of RAF-dependent signaling, anticipate clinical resistance to novel RAF inhibitors, and guide the design of “next-generation” inhibitors for deployment in RAF- or RAS-driven malignancies. Cancer Res; 73(15); 4840–51. ©2013 AACR.
