BRAF targets in melanoma. Biological mechanisms, resistance, and drug discovery. Cancer drug discovery and development. Volume 82. Ed. Ryan J. Sullivan. Springer (2015)
Early preclinical studies demonstrated that inhibition of BRAF in melanoma cell lines and xenografts with V600 BRAF mutations significantly slowed growth both in vitro and in vivo [49–51]. Based on this promising data, the effects of BRAF inhibition were tested in melanoma patients in clinical trials. Initial clinical trials mainly were performed with sorafenib. Sorafenib is a small molecule inhibitor of many kinases, including BRAF, although it actually binds to other targets (i.e. CRAF) with greater affinity. The first clinical trial of sorafenib in metastatic melanoma patients demonstrated that less than 5% of patients achieved a clinical response with this agent . Another trial in which patients were treated with paclitaxel and carboplatin, and then were randomized to receive sorafenib or a placebo, again demonstrated that sorafenib had minimal impact on clinical response rates or progression-free survival (PFS) .
While these results were disappointing, a second wave of testing was precipitated by the development of drugs that were designed to be highly selective inhibitors of BRAFand specifically of the BRAF V600E mutant protein encoded by the most common mutation of this gene. The first such agent to undergo testing was vemurafenib (also called PLX4032) . Preclinical studies demonstrated that vemurafenib potently inhibited the MAPK signaling pathway, growth, and survival of BRAF V600-mutant human melanoma cell lines, but almost no effect was seen in cell lines without such a mutation . Treatment of xenografts of these cell lines in mouse models demonstrated that the vemurafenib treatment caused tumor regression. This impressive activity accurately predicted the results seen in patients. In the phase I clinical trial of vemurafenib, approximately 80% of the patients with BRAF V600E-metastatic melanoma had significant tumor shrinkage; in contrast, none of the 5 patients who did not have this mutation responded . Subsequent preclinical studies in melanoma and other cancers by multiple groups found that treatment of cancer cells that did not have a BRAF V600 mutation, and particularly those with activation of RAS proteins, with vemurafenib and other compounds in this class caused increased tumor growth in vitro and in vivo [20, 57–59]. These studies showed that selective inhibitors of the BRAF V600-mutant protein actually caused increased activation of the MAPK pathway in these cell lines, as measured by increased phosphorylation of activation-specific sites on both MEK and ERK. This effect appears to be due to inhibitor-induced changes in the structure of the wildtype BRAF protein which results in a conformation that facilitates the formation of heterodimers with CRAF proteins. These BRAF-CRAF heterodimers activate MEK and ERK, and subsequently increase the growth of the tumor cells. Interestingly, this paradoxical activation of the MAPK pathway appears to be largely responsible for an interesting toxicity seen with vemurafenib: the development of cutaneous squamous cell carcinomas (SCCs) and/or keratoacanthomas (KAs). These lesions are observed in 20–25% of patients treated with vemurafenib, and are generally treated successfully with surgery . Molecular analyses demonstrated that these lesions frequently have mutations in RAS genes, and they demonstrate increased MAPK pathway activation following treatment with the mutant-selective BRAF inhibitors [60, 61]. This mechanism was recapitulated in animal models. Importantly, these studies demonstrated that adding a MEK inhibitor to the mutant-selective BRAF inhibitor blocked the formation of these hyperproliferative cutaneous lesions .
In addition to the critical importance of selecting patients with BRAF V600 mutations for treatment with vemurafenib, the phase I trial also demonstrated the specific relevance of MAPK pathway inhibition to the observed clinical benefit. A series of patients enrolled in the phase I trial underwent biopsies of their tumors before the start of treatment, and after 1 to 2 weeks of therapy. Analysis of P-ERK expression by immunohistochemistry (IHC) demonstrated that variable degrees of MAPK pathway inhibition were achieved in these patients with vemurafenib treatment. When the changes in P-ERK were compared to the maximal changes in tumor size, a nearly linear relationship between these two factors was observed . Greater inhibition of the pathway correlated with greater inhibition of tumor growth. This finding reinforced the importance of this pathway that was implied by the high prevalence of mutations observed in melanoma.
Subsequent clinical testing of vemurafenib was limited to patients with metastatic melanoma with V600E BRAF mutations. In the pivotal BRIM-3 phase 3 trial, such patients were randomized to treatment with vemurafenib or dacarbazine . This trial was halted at its first analysis, and it was the shortest phase III clinical trial ever conducted in oncology. Treatment with vemurafenib produced significant improvements in response rate (48 versus 5%, p < 0.001), PFS (median 5.3 versus 1.6 months, Hazard ratio [HR] 0.26, p < 0.001), and OS (6 month OS 84 versus 64%, HR 0.37, p < 0.001). Based on this data vemurafenib received regulatory approval for the treatment of metastatic melanoma patients with BRAF V600E mutations in 2011.
Dabrafenib is a structurally unrelated small molecule that also is a highly potent and selective inhibitor of V600-mutant BRAF proteins [64, 65]. In a randomized phase III trial comparing dabrafenib to dacarbazine in metastatic melanoma patients with BRAF V600E mutations, dabrafenib treatment resulted in significant improvements in response rate (50 versus 6%) and PFS (5.1 versus 2.7 months, HR 0.30, p < 0.0001) . The effects on OS did not reach statistical significance (HR 0.61, 95% confidence interval [CI] 0.25–1.48). However, in this trial patients who progressed on dacarbazine were allowed to cross-over to the dabrafenib treatment, which was not allowed in the BRIM-3 trial of vemurafenib. A third mutant-selective BRAF inhibitor, LGX818, is currently in early phase clinical testing (www.clinicaltrials.gov).
The relatively short time that elapsed from the discovery of activating BRAF mutations to the regulatory approval of vemurafenib and dabrafenib stands as a powerful example of the speed and potential impact of genomics and translational research. It is clear that the selective BRAF inhibitors have delivered tremendous clinical benefit to patients with this highly aggressive disease. Indeed, symptomatic improvement is often observed within days of starting treatment. Frustratingly, however, the clinical benefit of the BRAF inhibitors is variable, and often short-lived. For example, in the BRIM-3 trial, only ~ 3% of patients had disease progression as their best response, reinforcing that almost all patients experienced some degree of disease control. However, only 2 out of 219 patients achieved a complete response, and ~ 50% of patients achieved only minor clinical responses (< 30% reduction in tumor size) . This tremendous variability in the degree of tumor response likely reflects pre-existing heterogeneity among patients and/or tumor cells with activating BRAF mutations. Furthermore, the median duration of the responses with the BRAF inhibitors has generally been only 5–7 months in the various clinical trials with vemurafenib and dabrafenib [56, 63, 66, 67]. Approximately 90% of patients develop resistance within 1 year of starting treatment. This resumption of growth after initial responsiveness to the BRAF inhibitors reflects the development of acquired, also called secondary, resistance.
Research has now identified a variety of mechanisms that may mediate resistance to the selective BRAF inhibitors. In general terms, these mechanisms either (1) cause re-activation of MAPK pathway effectors, or (2) result in activation of other pro-survival pathways. Similar to the selective benefit of vemurafenib and dabrafenib in patients with V600 BRAF mutations, these findings support the rationale to develop personalized approaches that will overcome these various mechanisms.
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