An Integrated Model of RAF Inhibitor Action Predicts Inhibitor Activity against Oncogenic BRAF Signaling

Abstract

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

Mechanism of BRAF Activation through Biochemical Characterization of the Recombinant Full‐Length Protein

“…TAK-632 and SB-590885 did not exhibit paradoxical activation in vitro . However, this does not conflict with previous cell-based assays demonstrating that TAK-632 and SB-590885 display paradoxical activation in cells [22] , as they lead to the interaction of RAF with GTP-loaded RAS, followed by transactivation of RAF kinases [9, 23] . Meanwhile, TAK-632 was demonstrated to promote dimerization of purified RAF catalytic domains in vitro [24] .…”

“…Different from vemurafenib and dabrafenib that are less potent against dimeric BRAF due to negative allostery, TAK-632 and SB-590885 are representatives of RAF inhibitors that potently inhibit dimeric BRAF [22] . Both TAK-632 and SB-590885 belong to type I inhibitors that stabilizes the αC-helix IN position, thus are able to occupy both promoters within a dimer to effectively inhibit dimeric RAF [9] . The measured IC 50 values of TAK-632 (0.70 nM) and SB-590885 (0.11 nM) are ~30–200 fold lower than that of vemurafenib, consistent with our observation that purified full-length BRAF exists in the dimeric form.…”

Non‐V600E BRAF mutations and EGFR signaling pathway in colorectal cancer

“…Such mutants may be sensitive to novel RAF dimer inhibitors or MEK inhibitors (e.g., K601E, K601N, K601T, L597Q, L597V, G469A, G469V, G469R, G464V and G464E). 26,27 Class 3 BRAF mutants consist of those with low or absent kinase activity (e.g., D287H, V459L, G466V, G466E, G466A, S467L, G469E, N581S, N581I, D594N, D594G, D594A, D594H, F595L, G596D and G596R). They are RAS-dependent and sensitive to ERK-dependent feedback of RAS.…”

Establishment and application of a novel patient-derived KIAA1549:BRAF-driven pediatric pilocytic astrocytoma model for preclinical drug testing

“…We therefore conclude that the lack of MAPK inhibition upon RAFi treatment could be a class-specific aspect of our model system. One possible explanation of this finding is the described paradoxical activation of downstream ERK signaling upon RAF inhibition in conditions of elevated RAS-GTP [26], which needs to be investigated in future studies.…”