The discovery that a subset of human tumours is dependent on mutationally deregulated BRAF kinase intensified the development of RAF inhibitors to be used as potential therapeutics. The US Food and Drug Administration (FDA)-approved second-generation RAF inhibitors vemurafenib and dabrafenib have elicited remarkable responses and improved survival of patients with BRAF-V600E/K melanoma, but their effectiveness is limited by resistance. Beyond melanoma, current clinical RAF inhibitors show modest efficacy when used for colorectal and thyroid BRAF-V600E tumours or for tumours harbouring BRAF alterations other than the V600 mutation. Accumulated experimental and clinical evidence indicates that the complex biochemical mechanisms of RAF kinase signalling account both for the effectiveness of RAF inhibitors and for the various mechanisms of tumour resistance to them. Recently, a number of next-generation RAF inhibitors, with diverse structural and biochemical properties, have entered preclinical and clinical development. In this Review, we discuss the current understanding of RAF kinase regulation, mechanisms of inhibitor action and related clinical resistance to these drugs. The recent elucidation of critical structural and biochemical aspects of RAF inhibitor action, combined with the availability of a number of structurally diverse RAF inhibitors currently in preclinical and clinical development, will enable the design of more effective RAF inhibitors and RAF-inhibitor-based therapeutic strategies, tailored to different clinical contexts.

SUMMARY The complex biochemical effects of RAF inhibitors account for both the effectiveness and mechanisms of resistance to these drugs, but a unified mechanistic model has been lacking. Here we show that RAF inhibitors exert their effects via two distinct allosteric mechanisms. Drug resistance due to dimerization is determined by the position of the αC-helix stabilized by inhibitor, whereas inhibitor-induced RAF priming and dimerization are the result of inhibitor-induced formation of the RAF/RAS-GTP complex. The biochemical effect of RAF inhibitor in cells is the combined outcome of the two mechanisms. Therapeutic strategies including αC-helix-IN inhibitors are more effective in multiple mutant BRAF-driven tumor models, including colorectal and thyroid BRAFV600E cancers, in which first generation RAF inhibitors have been ineffective.

SUMMARY Pharmacologic targeting of components of ERK signaling in ERK-dependent tumors is often limited by adaptive resistance, frequently mediated by feedback-activation of RTK signaling and rebound of ERK activity. Here, we show that combinatorial pharmacologic targeting of ERK signaling and the SHP2 phosphatase prevents adaptive resistance in defined subsets of ERK-dependent tumors. In each tumor that was sensitive to combined treatment, p(Y542) SHP2 induction was observed in response to ERK signaling inhibition. The strategy was broadly effective in TNBC models and tumors with RAS mutations at G12, whereas tumors with RAS(G13D) or RAS(Q61X) mutations were resistant. In addition, we identified a subset of BRAF(V600E) tumors that were resistant to the combined treatment, in which FGFR was found to drive feedback-induced RAS activation, independently of SHP2. Thus, we identify molecular determinants of response to combined ERK signaling and SHP2 inhibition in ERK-dependent tumors.

SUMMARYThe complex biochemical effects of RAF inhibitors account for both the effectiveness and mechanisms of resistance to these drugs, but a unified mechanistic model has been lacking. HereCorrespondence and requests for materials should be addressed to: Poulikos.poulikakos@mssm.edu or evripidis.gavathiotis@einstein.yu.edu. * These authors contributed equally to this work Accession numbers. Structural coordinates and parameters have been submitted to the Protein Database Bank under the following accession codes: 4RZV for BRAF R509H /VEM, 4RZW for BRAF R509H /AZ and 5ITA for BRAF WT /AZ-VEM. Other structural coordinates used in this study are the following: PDB ID: 4KSP for TAK bound to BRAF dimer, PDB ID: 3OG7 for VEM bound to BRAF V600E dimer, PDB ID: 4MNF for GDC bound to BRAF V600E dimer, PDB ID: 2FB8 for SB bound to BRAF dimer, PDB ID: 4XV2 for DAB bound to BRAF V600E dimer and PDB ID: 4XV1 for PB bound to BRAF V600E dimer and PDB 4MNE for the BRAF/MEK complex. AUTHOR CONTRIBUTIONSP.I.P., E.G., C.K., M.H., A.L., Z.K., Y.W. and T.A.A designed experiments. Z.K., T.A.A conducted biochemical and cellular studies. E.G., Y.W. performed structural determination and structural analysis. X.W. generated the CRAF-V5 CRISPR cell line. Q.X. synthesized the AZ-VEM compound. C.K., M.H., J.A.F., A.L., E.G., P.I.P. designed animal studies. Z.K., T.A.A and J.B. conducted animal experiments. C.Z. and G.B. provided reagents and analyzed data. P.I.P. and E.G. designed research, analyzed data and wrote the manuscript, which was edited by all authors.C.Z. and G.B. are employees of Plexxikon Inc. All other authors declare no competing financial interests.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. we show that RAF inhibitors exert their effects via two distinct allosteric mechanisms. Drug resistance due to dimerization is determined by the position of the αC-helix stabilized by inhibitor, whereas inhibitor-induced RAF priming and dimerization are the result of inhibitor-induced formation of the RAF/RAS-GTP complex. The biochemical effect of RAF inhibitor in cells is the combined outcome of the two mechanisms. Therapeutic strategies including αC-helix-IN inhibitors are more effective in multiple mutant BRAF-driven tumor models, including colorectal and thyroid BRAF V600E cancers, in which first generation RAF inhibitors have been ineffective. HHS Public Access