Structural and Mechanistic Analysis of the Regulation and Pharmacology of BRAF and MEK1

Abstract

The Ras/RAF/MEK/ERK pathway is a nexus in growth factor signal transduction, integrating and relaying signals from membrane-bound receptors to effect growth and proliferation programs in the cell. Given this central role, it is commonly a source of oncogenic potential, as activating mutations anywhere along this axis drive different malignancies, including melanoma, pediatric low-grade gliomas, and colorectal cancers. Since specific inhibition of mutants of the small GTPase Ras eluded us until last year, the field went in search of anti-cancer drugs one step down the line, to RAF. The three isoforms of RAF (A, B, and CRAF) share high sequence similarity, but have at least some non-overlapping functions. Since the vast majority of cancer-causing BRAF mutations occur at position V600 (particularly V600E), a battery of BRAFV600E inhibitors made their way to the clinic. Of these, Dabrafenib, Vemurafenib, and Encorafenib were the most successful. Unfortunately, when melanoma patients were treated with these drugs, they developed secondary skin malignancies, such as squamous cell carcinomas and keratoacanthomas. In 2010, it was discovered that inhibition of BRAFV600E activates wildtype B and CRAF kinases by inducing membrane-localized dimerization. This phenomenon has subsequently been termed paradoxical activation, and it leads to phosphorylation of ERK and a burst of aberrant growth and proliferation. Third- and fourth-generation RAF inhibitors, which have dual specificity for B and CRAF, and also inhibit monomeric or dimeric species of RAF kinases with roughly equal potency, show promise in the clinic. Another approach actively being pursued is inhibition of the next step down the axis: MEK. Virtually all current MEK inhibitors (MEKis) are allosteric, and their binding sites have been determined by X-ray crystallography. Intriguingly, a number of MEKis exhibit differential potency depending on the mutational background of the upstream pathway (KRASmut, BRAFV600E, KIAA:BRAF truncation/fusion). More recently, the structure of a BRAF:MEK complex with MEKi bound, as well as a new structure of full-length BRAF and MEK in complex with 14-3-3 proteins, have begun to offer clues to the mechanisms underlying the differential effect of MEKis. In the work described here, I show how we have expanded our understanding of the basis of MEK inhibitor efficacy for shutting down MAPK signaling. Structural studies of a panel of eight clinically-relevant MEKis, coupled with corresponding inhibition and binding data, show that they work through at least one of four different mechanisms: 1) preventing MEK1 recruitment to BRAF, 2) preventing phosphorylation of MEK1, 3) preventing release of phosphorylated MEK1 from BRAF, and 4) direct inhibition of phosphorylated MEK1 activity (Graphical abstract). I also present a structure of the BRAF kinase in complex with TAK580, a new Type II inhibitor. These insights can help us develop new treatments of MAPK-driven malignancies.