Comparative Molecular Dynamics Dynamics of the BRAF Activation Loop Reveals a Biophysical Mechanism of Cancer Recurrence Under Drug Inhibition

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Oncogenic mutations in the kinase domain of the BRAF protein have long been associated with cancers involving the RAF-MEK-ERK pathway. One mutation, the V600E (valine to glutamate) replacement occurring adjacent to a site of threonine phosphorylation (T599) has been functionally well characterized and is known to occur in a large percentage of many cancers. Because both phosphorylation and the V600E mutation are both known to alter the physical behavior of the activation loop in this region. This system is especially amenable to rigorous comparative analyses of functional dynamics. Here we utilize a novel statistical method and software package; Detecting Relative Outlier Impacts in Dynamic Simulations) (DROIDS 2.0 - published last year in Biophys.J.) to compare the backbone dynamics of BRAF through an examination of Kullback-Leibler (KL) divergence of atom fluctuation distributions and subsequent multiple test corrected Kolmogorov-Smirnov (KS) tests conducted at single amino acid resolution in a large sampling set of explicitly solvated nanosecond scale molecular dynamic simulations. We compare relative BRAF dynamics in terms of phosphorylation state, RAF inhibitor drug binding and wild type vs mutant genetic states. We demonstrate that while RAF inhibitor binding in the cancer-associated mutant globally destabilizes BRAF, thereby likely disrupting growth signaling in the RAF-MEK-ERK pathway, the binding of the drug in the wildtype genetic background induces hyperactivation of the pathway by causing the dephosphorylated activation loop in the inactive BRAF to adopt a nearly identical dynamic state as the phosphorylated activation loop in the activated BRAF. We suggest that this biophysical mechanism is a likely a common cause of melanoma recurrence in the absence of secondary mutation.