Rationale for dual inhibition of the MAPK pathway

BRAF targets in melanoma. Biological mechanisms, resistance, and drug discovery. Cancer drug discovery and development. Volume 82. Ed. Ryan J. Sullivan. Springer (2015)

The strong correlation between MAPK pathway inhibition and clinical benefit observed in the clinical development of the selective BRAF inhibitors led to the hypothesis that resistance could be due to reactivation of signaling by the pathway. Due to the highly selective effects of the BRAF inhibitors in melanoma cells with V600 BRAF mutations, and the paradoxical pathway activation and growth observed in cells without these mutations, one explanation for the emergence of resistance could be the selective depletion of BRAF-mutant cells from molecularly heterogeneous tumors. Indeed, some studies have suggested that different regions of individual tumors vary in the relative frequency of cells with and without BRAF mutations [68]. However, sequencing analyses of melanoma samples collected at the time of resistance in multiple studies have demonstrated in all cases the continued presence of the same activating BRAF mutation that was present before the start of therapy [69, 70]. Similar results were also observed in cell lines that were selected in vitro for secondary resistance through chronic exposure to increasing doses of the BRAF inhibitors.

A second potential mechanism that could potentially cause resistance to the BRAF inhibitors would be the acquisition of secondary mutations in the BRAF gene. Secondary mutations in drug targets are a common finding in CML and gastrointestinal stromal tumors (GISTs) that have developed resistance to imatinib. Preclinical studies demonstrated that artificially introducing mutations at the Thr529 gatekeeper residue of BRAF could negate the inhibitory effects of vemurafenib and other selective BRAF inhibitors in melanoma cell lines [71]. However, despite this demonstration, and the experience with other targeted therapies, to date no secondary mutations in the BRAF gene have been identified in resistant melanoma tumors or cell lines [69].

While new mutations in BRAF have not been identified as a mechanism of resistance, two other alterations have: copy number gain and alternative splicing. Copy number gain of the mutant BRAF allele was identified in 4 of 20 (20%) progression samples by whole exome sequencing, with corresponding increased BRAF protein expression [72]. Resistance in cell lines with BRAF copy number gain could be overcome by treating the cells with increased doses of the selective BRAF inhibitors, suggesting a therapeutic strategy for patients with resistance due to this mechanism. However, this strategy will likely not be effective in patients with resistance due to aberrant splicing of BRAF. This phenomenon was identified in 6 of 19 (32%) progression samples from patients, as well as in several cell lines selected for resistance, which demonstrated expression of a smaller (61 kDa) form of the BRAF protein [73]. This truncated form of the protein efficiently forms heterodimers with CRAF, which subsequently activates MEK and ERK. This interaction between CRAF and the truncated BRAF was not prevented by treatment with increased doses of the selective inhibitors of BRAF. However, the continued dependence on MAPK pathway signaling was demonstrated by the fact that the cells remained sensitive to MEK inhibitors. The utilization of heterodimers by BRAF with other RAF isoforms at the time of resistance was also identified by another group of investigators, although the mechanism underlying the switch to this capability was not identified [74]. Those studies demonstrated that treatment of the studied resistant cell lines with MEK inhibitors was able to block activation of the pathway and induce growth inhibition. However, in contrast to the parental (sensitive) cells from which the resistant clones were selected, MAPK pathway inhibition alone was not sufficient to induce apoptosis, suggesting the potential for additional aberrations to be driving resistance concurrently.

In addition to alterations in BRAF, alterations in other members of the MAPK pathway produce reactivation of MEK and ERK signaling in spite of continued exposure to BRAF inhibitors. As mentioned previously, co-occurrence of BRAF V600E and activating NRAS mutations is detected in less than 1% of newly diagnosed melanomas. However, this overlap is more common after exposure to selective BRAF inhibitors. The presence of activating NRAS mutations was initially described in 2 progressing tumors derived from the same patient; interestingly, the tumors actually had different NRAS mutations (Q61K and Q61R), implying that they had arisen from independent clones [69]. NRAS mutations were also identified in 4 of 19 (21%) progressing lesions in another study, and were mutually exclusive with aberrant splicing of BRAF [73]. In vitro studies demonstrated that the presence of a concurrent NRAS mutation results in re-activation of ERK via CRAF and remains sensitive to MEK inhibitors. Whole exome sequencing of a single patient with acquired resistance to a BRAF inhibitor identified acquisition of a somatic mutation that resulted in a C121S substitution in MEK1 as a cause of resistance [75]. A subsequent sequencing analysis of MEK in clinical samples obtained before the start of treatment with vemurafenib and at the time of progression identified several mutations in the gene. Interestingly, some of the mutations (i.e. P124L substitution) were identified in the pre-treatment samples in patients who achieved clinical responses, suggesting that they were not sufficient to cause resistance. However, other mutations (i.e. Q56P) were identified only in progressing lesions, and thus likely causative of disease progression [70]. This heterogeneity implies that additional studies will be needed over time to classify the functionality and clinical significance of various MEK mutations [76]. Finally, overexpression of COT, a serine-threonine kinase that is capable of activating downstream components of the MAPK pathway, was observed following BRAF inhibition in 2 of 3 patients samples obtained early in their treatment with vemurafenib, and in 1 patient was highest at the time of disease progression [77]. While in vitro studies suggested that COT may be able to phosphorylate ERK directly, BRAF inhibitor-resistant cells with enforced COT expression remained sensitive to MEK inhibitors.

The identification of multiple molecular aberrations that cause reactivation of MAPK pathway signaling supports the rationale to target this pathway at multiple levels (Fig. 6.3) [78]. Analysis of tumor biopsies obtained after 2 weeks of treatment in the phase II clinical trial of vemurafenib demonstrated that patients who did not achieve clinical responses had significantly less inhibition of ERK activation than patients who responded [70]. This demonstration of early, incomplete inhibition of the pathway in some patients suggested that combined inhibition may not only be an effective strategy to use after acquired resistance develops, but also potentially as a way to improve the magnitude, and hopefully duration, of the initial responses to therapy. These hypotheses are now supported by the clinical experience with combinatorial therapy with BRAF and MEK inhibitors.

Trametinib is an orally available potent inhibitor of MEK1/2 [79]. Clinical testing has demonstrated that trametinib has activity as a single agent in metastatic melanoma patients with BRAF V600 mutations who have not previously been treated with BRAF inhibitors. In a randomized phase III trial of trametinib versus chemotherapy that allowed cross-over at the time of progression, trametinib treatment produced significant improvements in response rate (22 versus 8%, p = 0.01), PFS (4.8 versus 1.5 months, HR 0.45, p < 0.0001), and OS (6 month OS 81 versus 67%, HR 0.54, p = 0.01) [80]. While the enthusiasm about these results were dampened specifically in the melanoma field in light of the parallel development and results of selective BRAF inhibitors, this trial represents the first positive phase III trial for a MEK inhibitor in any cancer type, thus confirming the potential for clinical utility for these agents. However, even more impressive results were observed when trametinib was combined with the selective BRAF inhibitor dabrafenib. A randomized phase II study was conducted in BRAF inhibitor-naпve metastatic melanoma patients with BRAF V600 mutations (V600E or V600K) [81]. All patients received the standard dose of vemurafenib (150 mg twice daily), and then were randomized to receive placebo, half-dose (1 mg per day; referred to as “150/1” treatment) or full-dose (2 mg per day; “150/2”) trametinib after these combinations were demonstrated to be safe and well-tolerated. Consistent with preclinical studies implicating paradoxical activation of the MAPK pathway as the mechanism of cutaneous SCCs and KAs from BRAF inhibitor therapy, the incidence of these lesions was markedly reduced in patients who received MEK combination therapy (2% with 150/1, 7% with 150/2) compared to those who received dabrafenib alone (19%). Patients who received the combination were also less likely to develop rashes, although other toxicities (i.e. acneiform dermatitis, fevers/chills, nausea/vomiting, diarrhea, neutropenia) were more frequent. However, these toxicities were generally manageable with supportive care or interruption of treatment. More importantly, the combination demonstrated significant improvements in multiple clinical outcomes. The clinical response rates were 54% for dabrafenib monotherapy, 50% for the 150/1 combination, and 76% (including 9% complete responses) for the 150/2 combination. The median PFS was 5.8 months for dabrafenib monotherapy, 9.2 months for 150/1 (HR 0.56, p = 0.006), and 9.4 months for 150/2 (HR 0.39, p < 0.001). At 12 months only 9% of patients treated with dabrafenib alone remained progressionfree, compared to 26% with 150/1 and 41% with 150/2.

 BRAF Targets in Melanoma_ Biological Mechanisms, Resistance, and Drug Discovery-Springer-Verlag New York (2015) 6.3

Fig 6.3. Resistance mechanisms and combinatorial strategies for BRAF-mutant melanomas. Schema of described mechanisms of resistance to selective inhibitors of mutant BRAF in melanoma. a MAPK-pathway dependent mechanisms. b MAPK-pathway independent mechanisms. Classes of agents that may be used to target components of the pathways are indicated to the side in each panel.

Trametinib has also undergone early evaluation in patients who have progressed on BRAF inhibitors. Despite preclinical evidence that cells with acquired resistance to BRAF inhibitors often remain sensitive to MEK inhibition, to date these results have been relatively disappointing. Treatment with single agent trametinib in failed to result in a clinical response in 37 patients who had developed resistance to a selective BRAF inhibitor, although 2 of 3 patients who had stopped BRAF inhibitor therapy due to toxicities did respond [82]. The median PFS of the patients overall was only 1.8 months. Combined treatment (150/2) with dabrafenib and trametinib achieved clinical responses in 4 out of 21 (20%) patients who had previously progressed on a BRAF inhibitor, and in 1 of 5 (20%) patients who had progressed on a MEK inhibitor [83]. Although full interpretation of the results will require additional follow-up to allow for meaningful assessment of time-dependent outcomes, one implication of the results is that it may be more effective to continue BRAF inhibitors and add other agents to this therapy in patients who progress on BRAF inhibitors than to simply change them to different targeted agents. This finding, if confirmed, would be similar the clinical experience in HER2/neu-positive breast cancer patients following progression on trastuzumab.

While the slight improvement observed with the combination of dabrafenib and trametinib treatment compared to trametinib alone in the progressing patients is interesting, overall the relatively low activity has been disappointing in the face of the results observed in BRAF inhibitor naпve patients, and the multiple studies supporting reactivation of MEK at the time of resistance. The evidence of significant benefit in some patients, however, does suggest that it may be possible to predict which patients this regimen is effective in by comparison of the clinical outcomes to the underlying resistance mechanisms. Alternatively, early assessment of the regimen’s ability to inhibit ERK activation may predict benefit. However, no data has been presented to date testing either possibility. In turn, while combined inhibition of BRAF and MEK as front-line therapy has been very impressive, it is clear that many patients are still developing resistance in a relatively short period of time, and it is unclear how many, if any, of the patients treated with the combination are achieving the durable disease control that is seen in some patients treated with immunotherapies.

Despite these limitations, the rapid advances in outcomes that have been achieved again demonstrate the dramatic potential clinical benefit for rational combinatorial treatment approaches. Additional testing is currently ongoing with other BRAF and MEK inhibitors to determine whether differences in pharmacological properties may result in greater efficacy. Alternative dosing regimens have also been proposed as another strategy to prevent or delay resistance in preclinical models, but there is no clinical data yet addressing this hypothesis [84]. Evaluation of inhibitors of other targets in the MAPK pathway, including ERK, is also ongoing [85]. However, multiple lines of evidence also suggest that strategies that combine MAPK pathway inhibition with targeting of other pathways may be an effective clinical strategy for some patients.

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