Abstract 78: A strategy for effective targeting of oncogenic BRAF signaling with increased therapeutic index using conformation-selective RAF and MEK inhibitors
Authors: Adamopoulos Christos , Ahmed Tamer A. , Tucker Maxwell R. , Ung Peter M. , Xiao Min , Karoulia Zoi , Wu Xuewei , Aaronson Stuart A. , Ang Celina , Rebecca Vito , Schlessinger Avner , Herlyn Meenhard , Wang Qi , Shaw David E. , Poulikakos Poulikos I. 
Published: July 2021

Abstract
BRAF kinase, a critical component of the RAS/MAPK signaling pathway, requires dimerization for its catalytic activity, whereas the oncoprotein BRAF(V600E) can be catalytically active as a monomer. Current clinical RAF inhibitors selectively bind and inhibit monomeric over dimeric BRAF providing a high therapeutic index and are now standard practice in combination with MEK inhibitors in the treatment of patients harboring BRAF(V600E) tumors. However adaptive resistance due to RAF dimerization limits their effectiveness. Recently, RAF inhibitors that equipotently inhibit both monomeric and dimeric RAF have been developed, but they are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues. Here we identify and characterize a third class of RAF inhibitors that preferentially binds and inhibits dimeric over monomeric BRAF(V600E). Using long-timescale molecular dynamics (MD) simulations, we observed distinct dynamic features of RAF inhibitors bound to monomeric and dimeric BRAF, thus providing a structural explanation for the selectivity of these drugs. Biochemical analysis using RAF dimer selective inhibitors revealed that the two forms of dimeric and monomeric BRAF(V600E) differ in their conformation in their active site and their strength of interaction with MEK. Further, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index, we assessed the triple combination of RAF monomer- plus RAF dimer selective inhibitor plus a MEK inhibitor that potently disrupts the BRAF-MEK complex. The triple combination potently suppressed tumor growth in multiple colorectal and melanoma BRAF(V600E) cell line- and in vivo models that were resistant to the current clinical RAF and MEK inhibitor combination. Strikingly, while the double combination treatment with the RAF dimer-selective inhibitor and MEK inhibitor was associated with a significant gradual weight loss in vivo, the triple combination showed no weight loss or other apparent toxicities, indicating a higher therapeutic index. Finally, off-label use of the triple combination in a patient with a stage IV BRAF(V600E) colorectal cancer that progressed on standard therapies achieved durable tumor control with minimal toxicities. Thus, a rationally designed combinatorial approach of conformation-selective RAF and MEK inhibitors may be a highly effective and well tolerated therapeutic strategy for patients with BRAF(V600E) tumors.
Citation Format: Christos Adamopoulos, Tamer A. Ahmed, Maxwell R. Tucker, Peter M. Ung, Min Xiao, Zoi Karoulia, Xuewei Wu, Stuart A. Aaronson, Celina Ang, Vito Rebecca, Avner Schlessinger, Meenhard Herlyn, Qi Wang, David E. Shaw, Poulikos I. Poulikakos. A strategy for effective targeting of oncogenic BRAF signaling with increased therapeutic index using conformation-selective RAF and MEK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 78.