Base editing to elucidate RAF regulation and signaling

Project Summary Mitogen activated protein kinase (MAPK) signaling regulates cell growth in normal cells and is frequently overactivated in cancer. Within this pathway, RAF kinases are core signaling nodes and are of particular interest as cancer drug targets. Despite extensive study, RAF kinase function and regulation remain incompletely understood. Intriguingly, several studies have shown that the expression of CRAF protein but not CRAF kinase activity is essential for the growth of KRAS mutant lung cancer. To date, only a handful of function modifying mutations have been used to study CRAF function in this context, and these are often utilized in vitro or in exogenous overexpression experiments where stoichiometry is non-physiological and native scaffolding factors are absent. Interpretation of these experimental results can also be complicated by the modular nature of RAF dimers, and it is seldom clear which RAF homo-/hetero-dimers are responsible for observed cellular phenotypes. A clearer understanding of CRAF’s oncogenic role, including homo-/hetero-dimer functions, would inform future efforts to target CRAF in KRAS-driven cancers via inhibition, targeted degradation, or other mechanisms. This proposal will utilize a combination of unbiased base editor scans and “bump-hole” methodologies to elucidate the role of CRAF in KRAS-driven cancer and understand the relative activity of various RAF dimers. Specifically, I will test the hypothesis that CRAF’s role in KRAS mutant lung cancer is to form less active RAF dimers, diminish downstream signaling, and prevent otherwise toxic levels of MAPK signaling. Confirming this hypothesis has direct implications for cancer therapeutic development and would suggest that CRAF degraders but not competitive active-site inhibitors would be effective in this tumor subtype. This project will also advance fundamental knowledge of RAF kinases and expand the methods used to study them. In confirming the central hypothesis of this proposal, I will validate the goldilocks premise of RAF signaling – that too little or too much signaling impedes cancer growth. In aim one, unbiased base editor scanning paired with rigorous follow-up characterizations will expand knowledge of CRAF’s multiple functions and interaction partners. In aim two, I will validate a co-mutational approach to reveal the relative contribution of each RAF dimer to cancer growth and MAPK signaling. This aim also has broad application in cell signaling research and could constitute a generalizable approach to studying protein homo-/hetero-dimers with conserved dimerization interfaces. While advancing knowledge of RAF biology, this fellowship will also provide a rich training environment across two cutting edge research institutions: Harvard’s department of Chemistry and Chemical Biology and the Dana Farber Cancer Institute. Training will include regular meetings with my mentors, seminars with relevant scientific experts, and opportunities to present my research internally and externally. Mentorship provided by Brian Liau and Andrew Aguirre will greatly facilitate my development as an independent cancer researcher.