Oncogenic RAF: a brief history of time.

Remarkable clinical activity with the RAF inhibitor PLX-4032 in melanomas containing mutant BRAF has recently been described in the New England Journal of Medicine by Flaherty et al. (2010). In a second manuscript published in Nature, the discovery and preclinical development of the drug is chronicled by the scientist at Plexxicon who led this effort, Gideon Bollag (Bollag et al., 2010). The initial preliminary report of the Phase 1 data revealing the activity of this drug was first presented at the Annual Meeting of the American Society of Clinical Oncology Annual Meeting in 2009, 7 years after the discovery of BRAF mutations in human cancer (Davies et al., 2002). In a disease in which the development of therapies against novel targets is often frustrating and slow, this is an amazing achievement, the origins of which can be traced back over 25 years. In 1983, Rapp et al. cloned v-raf as the transformative factor in a replication-defective type C murine virus (3611-MSV) but no mutations were detected in the homologous human RAF genes (ARAF, BRAF and CRAF) until 2002 (Rapp et al. 1983). At that time, Davies et al. (2002) at the Sanger identified BRAF as commonly mutated in human tumors. These mutations were particularly common in melanoma where they are found in upwards of 50% of patients but were also identified in approximately 10% of colorectal tumors and a third of papillary thyroid cancers. BRAF mutations are typically missense substitutions found clustered in two regions – the glycine rich loop in exon 11 and the activation segment within exon 15. The most common mutations disrupt an inactive conformation of the protein and lead to constitutive hyperactivation of the kinase (Wan et al., 2004). Notably, a single mutation within codon 600 (V600E) is found in over 90% of cases. RAF activation occurs in response to activation of RAS proteins by upstream growth factor-induced signals. Activated RAF in turn phosphorylates and activates MEK1 and 2 [mitogen-activated protein ⁄ extracellular signal-regulated kinase kinase (MAPKK)] which in turn phosphorylate and activate the extracellular signal-related kinases 1 and 2 (ERK1 ⁄2). The activation of this signaling cascade (the ‘classical’ MAP kinase pathway) regulates a number of effector kinases and transcription factors that drive key cellular processes including cell proliferation and survival. Phosphorylation of other RAF substrates may also be important in normal physiology and cellular transformation; however, this remains to be firmly established. Tumor cells with BRAF mutations depend on expression of BRAF for proliferation (Wellbrock et al. 2004). Furthermore, melanoma cell lines with the mutation are almost universally sensitive to inhibition of ERK signaling with MEK inhibitors (Solit et al., 2006). These findings suggested that inhibitors of RAF, MEK or ERK could have major clinical activity in patients whose tumors harbored activating BRAF mutations. Unfortunately, sorafenib (Nexavar, Bayer), the first RAF inhibitor to be tested clinically, lacked meaningful clinical activity in melanoma. This result suggested to some that mutant RAF did not play an important role in these tumors. In retrospect, this conclusion was unwarranted. Sorafenib is an unselective inhibitor of many kinases, and it inhibits tumors with mutant and wild-type RAF with equal potency. It is likely that the toxicity of sorafenib precluded administration of a high enough dose to inhibit ERK signaling adequately. In contrast to sorafenib, allosteric MEK inhibitors are exceptionally selective. These drugs potently inhibit ERK signaling in tumors. In patients, the major dose-limiting toxicity of these agents is skin rash. In clinical trials, the MEK inhibitor AZD6244 exhibited activity in melanoma, with a 13% partial response rate in tumors harboring a BRAF mutation. However, given the expectations from trials of oncoprotein-targeted therapy in other diseases, these results were disappointing. They suggested to many that BRAF ⁄ERK signaling was dispensible for maintenance of most melanomas, possibly because of the cellular heterogeneity and genetic complexity of the disease, as well as to postulated inherent resistance of melanoma stem cells. The two reports by Flaherty et al. (2010) and Bollag et al. (2010) dispel these concerns and validate the dependence of these tumors on BRAF. In the study by Flaherty et al., the authors report the Phase 1 results of the RAF inhibitor PLX4032 (also known as RG7204). PLX4032 is a potent nanomolar, ATP-competitive inhibitor of all three RAF isoforms as well as the V600E mutant form of BRAF. In this trial, a 77% response rate