Targeting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond

Abstract

The identification of mutationally activated BRAF in many cancers altered our conception of the part played by the RAF family of protein kinases in oncogenesis. In this Review, we describe the development of BRAF inhibitors and the results that have emerged from their analysis in both the laboratory and the clinic. We discuss the spectrum of RAF mutations in human cancer and the complex interplay between the tissue of origin and the response to RAF inhibition. Finally, we enumerate mechanisms of resistance to BRAF inhibition that have been characterized and postulate how strategies of RAF pathway inhibition may be extended in scope to benefit not only the thousands of patients who are diagnosed annually with BRAF-mutated metastatic melanoma but also the larger patient population with malignancies harbouring mutationally activated RAF genes that are ineffectively treated with the current generation of BRAF kinase inhibitors.

Figure 1. BRAF mutations in cancer

BRAF codon positions (1 through 766) are depicted on the × axis. Graphs from top to bottom show the number of mutations reported for each codon (top panel), the spectrum of BRAF mutations compiled from multiple studies in thyroid, skin^,, colon cancers^, and lung^,, (second panel), the position of putative phosphorylation sites that are reported to have a functional consequence on kinase activity, stability or localization (third panel), and BRAF functional domains: RAS binding domain (RBD) and kinase domain are highlighted in blue, phosphate binding loop (P-loop) highlighted in orange, activation loop (A-loop) highlighted in yellow, fusion points highlighted in magenta (lower graph).

Figure 2. Biochemistry of RAF inhibitors in BRAF-V600E and BRAF wild-type cells

BRAF oncoproteins promote proliferation by constitutively activating the MAP kinase pathway. (a) ATP competitive RAF inhibitors (I) disrupt MEK activation and prevent proliferation of BRAF-V600E mutated melanomas in a dose dependent manner. (b) Wild-type RAF proteins exist largely in an auto-inhibited state. In the context of activated RAS, RAF inhibitors stimulate MEK activation at sub-saturating inhibitor concentrations by relieving auto-phosphorylation (P) of wild-type RAF, promoting RAF dimerization and association with RAS. Saturating inhibitor concentrations prevent MEK phosphorylation by blocking all RAF catalysis.

Figure 3. Loss of feedback inhibition and activation of the PI’3 kinase pathway mediates primary resistance to BRAF-V600E inhibition

Resistance to BRAF inhibitors in metastatic colorectal and thyroid cancers is mediated by loss of a feedback loop (EGF→EGFR signaling in colorectal cancers or NRG1→HER2 and HER3 signaling in papillary thyroid cancers) whereby the MAP kinase pathway activity inhibits mitogen dependent signaling (highlighted in blue). The negative feedback loop is disrupted when treated with a RAF inhibitor and proliferation is restored through RTK, RAS and PI3K signaling (highlighted in red).

Figure 4. Mechanisms of acquired resistance to BRAF-V600E inhibition leading to reactivation of the MAP kinase pathway

Multiple mechanisms of acquired resistance (highlighted in blue) to BRAF-V600E inhibitors lead to reactivation of the MEK-ERK pathway (highlighted in red) in the presence of a RAF inhibitor. The mechanisms of resistance include: activating mutations (*) of NRAS, overexpression (↑) of CRAF, overexpression of BRAF-V600E itself, alternative splicing of BRAF that renders the protein immune to inhibitors (p61), overexpression of COT, mutational activation of MEK1 and MEK2, ligand dependent RAS signaling through PDGFRβ or IGF-1R, or stromal cell secretion of HGF to activate through c-MET signaling.

Figure 5. Intermittent BRAF inhibitor therapy to forestall the onset of drug resistant melanoma

Prior to the initiation of BRAF inhibitor therapy to BRAF mutated melanomas, the bulk of the cells have optimal BRAF-V600E→MEK→ERK activity (A) and are drug sensitive (blue) with only rare variant cells with intrinsic drug resistance (black). The addition of BRAF inhibitor leads to insufficient BRAF-V600E→MEK→ERK activity in the bulk of the tumor (A) leading to tumor regression (B, response 1). However, BRAF inhibitor therapy immediately starts to select for expansion of melanoma cells with elevated BRAF-V600E expression and therefore intrinsic drug resistance, because they have an optimal level of BRAF-V600E→MEK→ERK activity—therefore a fitness benefit—in the presence of drug. Upon cessation of drug administration, drug resistant melanoma cells with elevated BRAF-V600E expression experience an excess of BRAF-V600E→MEK→ERK activity (A) that confers a fitness deficit on the cells (B, response 2). However, cessation of drug administration also allows residual drug sensitive cells to restore their optimal level of BRAF-V600E→MEK→ERK activity (A) and therefore re-initiate their proliferation (B, response 2).