RAF conformational autoinhibition and 14-3-3 proteins promote paradoxical activation

RAF kinase inhibitors can, in some conditions, increase RAF kinase signaling. This process, which is commonly referred to as “paradoxical activation” (PA), is incompletely understood. RAF kinases are regulated by autoinhibitory conformational changes, and the role of these conformational changes in PA is unclear. Our mathematical investigations reveal that a dynamic equilibrium between autoinhibited and non-autoinhibited forms of RAF, along with the RAF inhibitor stabilization of the non-autoinhibited form, can be sufficient to create PA. Using both computational and experimental methods we demonstrate that 14-3-3 proteins, which stabilize both RAF autoinhibition and RAF dimerization, potentiate PA. Our model led us to hypothesize that increased 14-3-3 expression would amplify PA for the third generation RAF inhibitors that normally display minimal to no PA. Our subsequent experiments find that 14-3-3 overexpression increases PA, increases RAF dimerization, and promotes resistance to these inhibitors, effectively “breaking” these “paradox breaker” and pan-RAF inhibitors. Overall, this work reveals a robust mechanism for PA based solely on equilibrium dynamics of canonical interactions in RAF signaling and identifies conditions which allow PA to occur.