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

Abstract

RAF kinase inhibitors can actually 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 find that PA can result from a dynamical equilibrium between autoinhibited and non-autoinhibited forms of RAF, along with the RAF inhibitor stabilizing the non-autoinhibited form. We also investigate whether PA is influenced by 14-3-3 proteins, which can both stabilize RAF autoinhibition and RAF dimerization. Using both computational and experimental methods we demonstrate that 14-3-3 proteins potentiate PA. Third generation RAF inhibitors normally display minimal to no PA. Our mathematical modeling led us to hypothesize that increased 14-3-3 expression should also amplify PA for these agents. Subsequent experiments support our hypothesis and show that 14-3-3 overexpression increases PA in these third generation RAF inhibitors, effectively "breaking" these "paradox breakers" and pan-RAF inhibitors. We have therefore created and experimentally validated a robust mechanism for PA based solely on equilibrium dynamics of canonical interactions in RAF signaling.

A Systematic Analysis of Signaling Reactivation and Drug Resistance

“…Paradox-breaking RAF inhibitors are thought to avoid paradoxical pathway activation by not inducing RAF dimerization (Karoulia et al, 2016). However, recent experiments showed that the paradox-breaking RAF inhibitor PLX8394 still induces paradoxical activation of ERK when cells overexpress 14-3-3 proteins, which promote RAF dimerization (Mendiratta et al, 2019). Our model recapitulates these data.…”

A systematic analysis of signaling reactivation and drug resistance

“…Paradox-breaking RAF inhibitors are thought to avoid paradoxical pathway activation by not inducing RAF dimerization ( Karoulia et al, 2016 ). However, recent experiments showed that the paradox-breaking RAF inhibitor PLX8394 still induces paradoxical activation of ERK when cells overexpress 14-3-3 proteins, which promote RAF dimerization ( Mendiratta et al, 2019 ). Our model recapitulates these data.…”