Abstract A036: Oncogenic mutations in BRAF and MEK weaken the ATP-stabilized inactive conformation of RAF to promote RAF dimerization and MAPK pathway activation

Abstract

Abstract The RAS-RAF-MEK-ERK signaling axis is upregulated in many human cancers due to its central role in cell growth, differentiation and survival. Oncogenic transformation frequently arises from RAS, BRAF and MEK mutations that activate the pathway. In the clinic, similar transformations also recur as resistance mutations to KRAS.G12C inhibitor treatment. BRAF mutations at V600 (Class I) activate BRAF by releasing the kinase’s activation segment and rendering BRAF activity dimerization-independent. Yet, it remains unclear how a diverse set of dimerization-dependent BRAF mutations (Class II/III) activate the pathway. MEK mutations are assumed to bypass the RAF node entirely. We previously reported a 2.9-Å-resolution crystal structure of human BRAF kinase domain (BRAFKD) in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that stabilize an inactive, pre-signaling monomeric conformation of BRAFKD and explain how ATP breaks RAF dimers in solution. Surprisingly, the β3-αC loop of MEK provides additional stabilizing interactions to the BRAF-bound AMP-PCP molecule. Utilizing an in vitro RAF dimerization assay, we find that MEK not only modulates RAF-RAF dimerization, it also enhances both the dimer-breaking effect ATP and the dimer-forming effect of paradoxical activator molecules, such as GDC-0879. Structural analysis reveals that all common oncogenic BRAF mutations alter key interactions with ATP that stabilize the inactive monomer conformation. We find ATP is unable to break RAF dimers containing Class I, II or III mutations, confirming these mutants counteract the inhibitory effects of ATP binding by lowering the threshold for RAF dimerization and thus pathway activation. We further find that oncogenic MEK mutants, in particular β3-αC loop deletions, favor RAF-RAF dimerization, even in the presence of ATP. Our study establishes a framework for rationalizing oncogenic BRAF mutations, and suggests that oncogenic MEK mutants can act, at least in part, by promoting RAF dimerization. This model provides a better understanding of activation mechanisms in the context of constantly evolving resistance mutations where oncogenesis continues to depend on the MAPK pathway. Citation Format: Timothy J. Wendorff, Jennifer Kung, Jawahar Sudhamsu. Oncogenic mutations in BRAF and MEK weaken the ATP-stabilized inactive conformation of RAF to promote RAF dimerization and MAPK pathway activation [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A036.