Normal cellular function is dependent upon proper regulation of the Raf kinases, mutation of which can result in human cancer and certain developmental disorders. Inhibitors to the high activity, oncogenic V600E-B-Raf are currently in clinical use; however, caution must be taken, given that the use of these inhibitors in cells lacking V600E-B-Raf can promote heterodimerization of B-Raf and C-Raf, resulting in paradoxical pathway activation instead of inhibition. Thus, a full understanding of the Raf activation process is critical for the development of effective therapeutic strategies. Toward this end, we investigated the importance of dimerization in Raf activation and identified novel differences between B-Raf and C-Raf. In the context of normal cellular signaling, we find that growth factor stimulation induces strong B-Raf/C-Raf heterodimerization as well as some Raf homodimerization, and increases the kinase activity of both B-Raf and C-Raf. In contrast, growth factor stimulation has little effect on A-Raf. To further explore the B-Raf and C-Raf interactions, we utilized protein mutations in the dimer interface that either enhance (E586K-B-Raf and E478K-C-Raf) or prevent (R509H-B-Raf and R401H-C-Raf) Raf dimerization. Interestingly, these mutations had only a modest effect on the kinase activity of B-Raf; however, the E-K mutation greatly enhanced C-Raf activity and the R-H mutation completely abolished C-Raf kinase activity. In addition, knockdown of C-Raf was found to have little effect on growth factor-mediated B-Raf activation, whereas knockdown of B-Raf dramatically inhibited C-Raf activation following stimulation, highlighting the dependence of C-Raf activation on B-Raf. Next, we examined the effects of dimerization on mutationally-activated B-Raf and C-Raf proteins that are associated with human disease. Strikingly, we found that although alterations in the dimer interface had an impact on the ability of all the mutant proteins to heterodimerize, these alterations only affected the biological activity (as measured in focus forming assays) of B-Raf and C-Raf proteins with moderate to low kinase activity, but not B-Raf proteins possessing high kinase activity. Based on the amino acid sequence of the dimer interface region, a Raf-Dimer-Interface (RDI) peptide was designed. The RDI peptide was found to bind both B-Raf and C-Raf and could disrupt Raf heterodimerization in response to growth factor stimulation and inhibit the biological activity of mutant Raf proteins with moderate to low kinase activity. Together these data indicate that dimerization is important for Raf function under normal signaling conditions and in certain mutational settings. Moreover, targeting the Raf dimer interface represents a new inhibitor strategy with therapeutic potential.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1239. doi:1538-7445.AM2012-1239