Abstract LB-28: RAF inhibitors activate MAPK pathway through relief of RAF auto-inhibition

Abstract

Abstract Mutations in the RAS/RAF/MEK/ERK pathway are among the most common in human cancers, and RAF small molecule kinase inhibitors are now being tested for the treatment of BRAFV600E tumors, with promising initial results. However, both clinical and pre-clinical data indicate that selective RAF inhibitors have poor efficacy in wildtype BRAF tumors. Moreover, these inhibitors actually promote phosphorylation of downstream targets, MEK and ERK when wildtype RAF is activated by upstream components of the pathway. Here, we show that biochemical activation of wildtype BRAF and CRAF by selective RAF inhibitors is exquisitely sensitive to ATP concentrations in an in vitro kinase assay. While BRAFV600E kinase activity remains high at ATP concentrations in excess of 1mM, wildtype BRAF and CRAF reach maximum kinase velocity at ∼1–10 mM ATP and are inhibited by more than 80% in the presence of 1mM ATP. Selective small molecule RAF inhibitors block the ATP concentration dependent inhibition of wildtype RAF, demonstrating that inhibition requires RAF kinase activity and suggests RAF auto-phosphorylation. Furthermore, while in its auto-inhibited state, wildtype RAF kinase activity cannot be increased by RAF inhibitors, but is rather decreased in a dose dependent manner consistent with ATP competitive inhibition kinetics, demonstrating that binding compound does not reverse RAF auto-inhibition. Together, these results suggest a novel mechanism that can account for the paradoxical activation of MAPK pathway due to wildtype RAF inhibition whereby wildtype RAF exists in an auto-inhibited state at physiological conditions through auto-phosphorylation. In the presence of a RAF inhibitor wildtype RAF auto-phosphorylation and consequential auto-inhibition of RAF is therefore relieved to activate the MAPK pathway leading to enhanced proliferation. This study offers new insight into RAF biochemistry and enzyme kinetics with significant implications for drug development strategies and therapeutic use of RAF inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-28. doi:10.1158/1538-7445.AM2011-LB-28