Mathematical modeling suggests 14-3-3 proteins modulate RAF paradoxical activation

RAF inhibitor “paradoxical activation” (PA) is a phenomenon where RAF kinase inhibitors increase RAF kinase signaling. Through mathematical modeling and experimental data analysis, we recently demonstrated that the combination of conformational autoinhibition (CA) with the disruption of CA by RAF inhibitors plays an important role in PA. 14-3-3 proteins are known to modulate RAF CA and RAF dimerization. We here extend our mathematical model to include both roles of 14-3-3 proteins, and we derive rigorous analytical expressions of RAF signal regulation as modulated by 14-3-3 proteins. We then use the model to investigate how 14-3-3 proteins may modulate PA. We mathematically show 14-3-3 protein stabilization of the autoinhibited form of RAF should potentiate PA, while 14-3-3 protein stabilization of the active RAF dimer should reduce PA. Our analysis suggests that the net-effect will often be a potentiation of PA, and that 14-3-3 proteins may be capable of inducing PA for RAF inhibitors that normally show little to no PA. We test model-based insights experimentally with two different approaches: forced increases in 14-3-3 expression (which we find amplifies PA) and evolved resistance assays (which suggest increased 14-3-3 expression may contribute to resistance to RAF inhibitors). Overall, this work supports a role for 14-3-3 in modulating RAF-inhibitor mediated paradoxical activation.Author summary: RAF kinase inhibitors demonstrate a counterintuitive behavior: these small molecules promote inhibition of isolated RAF kinase proteins, but when applied to a cellular biochemical system these small molecules cause RAF kinase activation. This phenomenon is clinically important, as it contributes to off-target toxicity and to therapeutic resistance. Mathematical models have recently helped reveal the mechanisms that underly this “paradoxical activation” phenomenon. Additionally, mathematical modeling recently demonstrated that the regulation of RAF kinase activation through conformational changes plays a major role in the paradoxical activation of RAF. In the present study, we investigate how proteins that modulate the conformational changes alter the response to RAF inhibitors. Our mathematical modeling suggests 14-3-3 proteins, which bind and stabilize an inactive form of RAF, can increase the paradoxical activation effect, and we confirm this model-based hypothesis experimentally. Consideration of the mathematical modeling results led us to hypothesize that elevated levels of 14-3-3 protein expression may contribute to RAF inhibitor resistance. Our experiments further confirm that alterations in 14-3-3 expression modulate paradoxical activation, and our experimental observations support the hypothesis that elevated 14-3-3 protein expression may contribute to RAF inhibitor resistance. Altogether, this study demonstrates how biochemically detailed mathematical models can lead to discoveries relevant to clinical pharmacology.