Functional characterisation of dynamic BRAF signalling complexes and their modulation by tumour specific mutations and clinically relevant kinase inhibitors

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BRAF plays a central role in the activation of RAS/ERK signalling. The activation cycle of this kinase is driven by RAS induced homo- or hetero-dimerisation and tightly controlled by protein-protein interaction events and post-translational modifications (PTMs).BRAF is often dysregulated in cancer. The most common mutation, V600E, cuts the incompletely understood BRAF activation cycle short. Thereby, this mutation generates an oncoprotein in which its kinase domain maintains an active conformation that is only transiently assumed by wildtype BRAF (BRAFWT) following RAS induced activation. This allowed the development of BRAFV600E selective inhibitors that yield impressive initial response rates in various entities. Unfortunately, therapeutic responses are short-lived due to the emergence of drug resistance. BRAF inhibitor induced paradoxical ERK pathway activation represents a common resistance mechanism. This phenomenon is caused by the unforeseen property of clinically applied BRAF selective inhibitors to promote heterodimers between drug-bound BRAF and other RAF isoforms in the presence of RAS activity. Drug-bound BRAF acts as a potent allosteric activator of the drug-free RAF protomer, thereby causing ERK re-activation and tumour growth. It is likely that the paradoxical action of BRAF inhibitors exploits processes occurring during physiological RAS/ERK pathway activation. In order to develop more effective and safer inhibitors, it will be critical to understand the spatio-temporal dynamics of quaternary BRAF signalling complexes in both physiological and pharmacological settings. Using Blue Native PAGE and SEC-PCP-SILAC based mass spectrometry (MS), we demonstrated that BRAFWT and BRAFV600E organise multi-protein complexes of distinct size and composition. We also showed that RAS induces BRAFWT containing complexes of similar size as those formed by BRAFV600E. Moreover, clinically relevant drugs affect the stability of these complexes, e.g. in settings with desired or paradoxical effects of BRAF inhibitors. Based on this and other data, we posit that the activity status of the kinase domain dictates the assembly of BRAF signalling complexes. In the proposed project, we aim to confirm this hypothesis by conducting an in-depth characterisation of the composition and PTM pattern of BRAF complexes formed under physiological conditions and in the presence of kinase inhibitors of (pre)clinical relevance. We will combine our MS protocols with novel biochemical approaches to identify short-lived dynamic interactions. We will extend our studies to complexes formed by non-V600E BRAF oncoproteins, which are increasingly detected by personalised medicine programs. These mutants are hardly defined in terms of their pathomechanism and drug sensitivity, currently precluding therapeutic recommendations. Thereby, we will gain novel mechanistic insights into the BRAF signalling and can provide at the same time critical information about their druggability.