Oncogenic RAF: a brief history of time
Article first published online: 13 OCT 2010
© 2010 John Wiley & Sons A/S
Pigment Cell & Melanoma Research
Volume 23, Issue 6, pages 760–762, December 2010
How to Cite
Solit, D. and Rosen, N. (2010), Oncogenic RAF: a brief history of time. Pigment Cell & Melanoma Research, 23: 760–762. doi: 10.1111/j.1755-148X.2010.00779.x
- Issue published online: 13 OCT 2010
- Article first published online: 13 OCT 2010
- Accepted manuscript online: 27 SEP 2010 09:52AM EST
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 was observed among the 48 BRAF mutant patients who received 240 mg twice daily or more of the drug. This level of activity far exceeds that observed with any prior agent in melanoma and has prompted the initiation of a Phase 3 trial comparing PLX4032 to chemotherapy in patients with melanoma whose tumors express the V600E BRAF mutation. Overall, the drug was well tolerated; however, upwards of a third of patients develop keratoacanthomas or squamous cell carcinomas on treatment.
What accounts for the greater clinical activity of PLX4032 compared to that of MEK inhibitors such as AZD6244? The PLX4032 study represents the first trial of a RAF or MEK inhibitor in which the patient cohort was enriched for those whose tumors harbored V600E BRAF mutations. The importance of this enrichment is highlighted by recent Phase 1 data with the MEK inhibitor GSK1120212 (Infante et al., 2010). In this trial, patients were genotyped for BRAF mutations upon study entry and, in a cohort of 20 patients with V600E BRAF mutant melanoma, a total RECIST response rate of 40% was observed. In contrast, in a cohort comprising patients with melanoma and pancreatic cancer with wild-type BRAF, only three of 37 patients had a partial response. These preliminary results are consistent with the preclinical data that almost all tumors with mutant BRAF are sensitive to MEK inhibition, whereas only a minority of those with wild-type BRAF, including those with mutant RAS, exhibit a similar degree of drug sensitivity (Joseph et al., 2010; Solit et al., 2006).
While patient enrichment probably accounts for some of the incremental increase in activity observed with PLX4032 over inhibitors of MEK, this is likely to be only part of the story. As expected, PLX4032 treatment of cells with V600E BRAF mutations results in potent inhibition of BRAF signaling and ERK phosphorylation, resulting in induction of cell cycle arrest and in some models cell death (Joseph et al., 2010). In contrast, in cells with wild-type BRAF, PLX4032 fails to inhibit MAPK pathway activity and instead causes a paradoxical induction of ERK phosphorylation (Hatzivassiliou et al., 2010; Heidorn et al., 2010; Poulikakos et al., 2010). This preclinical observation that PLX4032 inhibits MAP kinase pathway activation only in mutant BRAF tumor cells and not in normal cells suggests that high doses of this agent will not cause toxicity because of ERK inhibition. It therefore may exhibit a wider therapeutic index than inhibitors of MEK, which inhibit ERK in normal tumor cells as well. The observation does suggest that PLX4032 could cause toxicity by activating ERK in normal tissues.
As shown in these reports, these predictions have been born out. High serum levels of PLX4032 (>40 μM) were achieved with limited toxicity. The adverse effects observed were dissimilar to those of MEK inhibitors, suggesting that whereas those associated with the latter were likely because of ERK inhibition, toxicities associated with PLX4032 may be attributable to ERK activation. It is tempting to speculate that the appearance of keratoacanthomas and squamous cell carcinomas in these patients is because of induction of ERK signaling, but this is unproven. One can infer that the more potent antitumor activity of PLX4032 compared to MEK inhibitors is because of the ability of the drug to inhibit ERK signaling in the tumor more profoundly as it does not inhibit the pathway in normal cells. This is supported by the data of Bollag et al. (2010) in which phosphorylation of cytoplasmic ERK was typically inhibited more than 80% in tumors that underwent regression in response to the drug.
In our view, then, the clinical effects of PLX4032 can be attributed to its selective inhibition of ERK signaling in tumor cells and paradoxical activation in normal cells. The mechanism underlying this phenomenon is controversial. Heidorn et al. (2010) have observed that selective BRAF inhibition activates CRAF activity and signaling. Our data (Poulikakos et al., 2010) show that paradoxical activation of signaling occurs with all RAF inhibitors, including those that inhibit all the RAF family members. RAS isoforms dimerize in cells in a manner dependent on RAS activation. Drugs that compete with ATP for binding to RAF, including PLX4032, transactivate these dimers. Thus, binding of the drug to one member of the dimer activates the other (Poulikakos et al., 2010). This paradoxical activation of ERK by PLX4032 is not observed in cells expressing V600E BRAF, because, in these cells, RAS-GTP levels are too low to support dimerization and transactivation.
These models have several implications. They suggest that the clinical activity of PLX4032 will be restricted to tumors harboring activating mutations of BRAF and that PLX4032 may in fact enhance the growth of tumors in which the ERK pathway is activated by other mechanisms. Furthermore, molecular lesions that activate RAS or enhance the dimerization of wild-type RAF in any way might attenuate or prevent the effects of this drug. These might include RAS mutation (Poulikakos et al., 2010), activation of receptor tyrosine kinases, or elevation of the expression of wild-type RAF (Montagut et al., 2008).
The development of effective RAF inhibitors is a revolutionary event for the treatment of metastatic melanoma. The clinical response rates achieved with this drug and a similar compound from GlaxoSmithKline currently in early clinical trial are unprecedented in this disease. However, this is only a first step. The responses are often very impressive, but they are rarely complete. Furthermore, they are temporary with a median time to progression of only approximately 9 months. Work now must concentrate on increasing both the magnitude and duration of the responses and on understanding the mechanisms underlying acquired resistance with a focus on the development of novel strategies effective after tumor progression. These may include combination therapies that inhibit the function of other dysregulated signaling pathways in melanoma, such as the PI3K pathway.
It also may be possible to develop improved, later generation RAF inhibitors. Some have suggested that pan-RAF inhibitors that effectively inhibit wild-type RAF dimers would inhibit the pathway more effectively, would not induce keratoacanthomas, and would be active in some RAS tumors. In our view, these inhibitors would likely be similar to MEK inhibitors and would have a narrower therapeutic index than PLX-4032. It may be possible to develop an inhibitor that selectively binds to V600E BRAF. Such an inhibitor would not affect ERK at all in normal cells and would likely not be associated with keratoacanthomas. In our view, in the short term, the greatest benefit would be the development of a drug or strategy for drug administration that effectively inhibits ERK signaling in melanoma brain metastases as a significant number of patients with melanoma develop CNS disease. These patients were not studied in the PLX4032 clinical trial and at this time have yet to benefit from this therapeutic advance.
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