MAP Kinase Signaling and Inhibition in Melanoma

REVIEW MAP kinase signaling and inhibition in melanoma

RJ Sullivan and K Flaherty

The mitogen-activated protein kinase (MAPK) pathway is critical to oncogenic signaling in the majority of patients with malignant melanoma. Driver mutations in both NRAS and BRAF have important implications for prognosis and treatment. The development of inhibitors to mediators of the MAPK pathway, including those to CRAF, BRAF, and MEK, has led to major advances in the treatment of patients with melanoma. In particular, the selective BRAF inhibitor vemurafenib has been shown to improve overall survival in patients with tumors harboring BRAF mutations. However, the duration of beneﬁt is limited in many patients and highlights the need for understanding the limitations of therapy in order to devise more effective strategies. MEK inhibitors have proven to particularly active in BRAF mutant melanomas also. Emerging knowledge about mechanisms of resistance as well as a more complete understanding of the biology of MAPK pathway signaling provides insight into rational combination regimens and sequences of molecularly targeted therapies.

Oncogene (2013) 32, 2373–2379; doi:10.1038/onc.2012.345; published online 3 September 2012 Keywords: melanoma; targeted therapy; BRAF

INTRODUCTION activation. The RAS GTPases are a critical link between activated The mitogen-activated protein kinase (MAPK) pathway is activated growth factor receptors and downstream signal transduction in the great majority of melanomas and typically occurs through pathways. In light of this central role, it is not surprising that three activating mutations of either NRAS (15–20% cutaneous melano- RAS isoforms (HRAS, KRAS and NRAS) collectively represent the mas) or BRAF (40–50%).1 These mutations can be identified in most commonly mutated oncogene in human malignancies and 13,14 benign melanocytic proliferation and all stages of invasive and distribute across a variety of cancers. In melanoma, NRAS metastatic melanoma.1 Further, the presence of a BRAF mutation in mutations have been identified in 15–30% of tumor samples and the setting of metastatic disease has been associated with a poorer are thought to be an important driver of oncogenesis in these prognosis compared with patients whose tumors lack BRAF patients, although the therapeutic value of targeting NRAS has 15–18 mutations in the era before BRAF inhibitors. In clinical trial cohorts, yet to be demonstrated. The commonly observed NRAS BRAF and MEK inhibitors appear to have neutralized the negative mutations in melanoma disable the ability of the enzyme to prognostic significance of a BRAF mutation and, in fact, their hydrolyze guanosine triphosphate to guanosine diphosphate, outcome may be superior to BRAF wild-type (BRAFWT) melanoma in leaving mutated NRAS predominantly guanosine triphosphate- the metastatic setting.2–12 The effectiveness of the BRAF inhibition in bound and capable of serial activation of the downstream 19 terms of tumor regression and improved survival has led to FDA components of the RAS effector pathways. In the absence of approval of vemurafenib (formerly PLX4032) and expected approval RAS mutation, RAS activation can be aberrantly driven in of dabrafenib (formerly GSK2118436) in 2012. As would be expected melanoma through the autocrine or paracrine hyperactivation of with single oncogene-directed therapies in genetically complex growth factor receptors, such as c-Met, epidermal growth factor 13,16,18 cancer, complete or durable responses are uncommonly seen with receptor and c-Kit. either vemurafenib or dabrafenib. Resistance to these agents In melanoma, the relative contribution of enhanced RAS typically occurs 6 to 8 months after initiation of therapy. For this effector pathway signaling in the setting of growth factor receptor reason, there is great interest in determining the mechanisms of activation or activating RAS mutation is unknown. But, there is resistance to these agents so that therapeutic strategies can be substantial evidence to support the MAPK pathway as a critical devised to prolong disease control or achieve complete responses component of oncogenic RAS signaling, although it is well more frequently. What follows is a review of MAPK pathway biology established that NRAS can signal through non-MAPK pathways, in melanoma, a brief summary of the clinical data with BRAF and such as the phospho-inositide-3-kinase (PI3K) pathway. In the MEK inhibitors in the treatment of patients with melanoma, a MAPK pathway, however, the most important downstream description of the emerging mechanisms of resistance to BRAF and mediators of activated RAS are BRAF and CRAF serine threonine 17 MEK inhibition in these patients, and a discussion about some of the kinases. For mutated NRAS, CRAF appears to be relatively more 19,20 most promising strategies to overcome this resistance. essential for downstream activation of MAPKs. Considerably less is known about the role of ARAF in cancer. Both BRAF and CRAF contain a regulatory domain, which contains a RAS-binding MELANOMA AND THE MAPK PATHWAY domain (RBD) and a cysteine-rich domain (CRD), and a kinase Under normal physiological conditions, cell growth and survival domain which interact to suppress RAF function.19,21,22 Activation is dependent on ligand-dependent growth factor receptor of RAF (either B- or C-) could occur following RAS binding, through

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA. Correspondence: Dr K Flaherty, Massachusetts General Hospital Cancer Center, Harvard Medical School, 55 Fruit Street, Boston, MA 02114, USA. E-mail: kfl[email protected] Received 20 March 2012; revised 25 June 2012; accepted 26 June 2012; published online 3 September 2012 MAP kinase signaling and inhibition in melanoma RJ Sullivan and K Flaherty 2374 a disruption in the CRD-kinase domain negative regulatory interaction, thereby allowing for kinase activity.23 However, cAMP influences which RAF isoform is predominantly activated and, in the presence of an NRAS mutation, CRAF is favored. RAF, typically in the form of either homo- or heterodimerization, interacts with MEK, initiates MEK phosphorylation, and leads to phosphorylation and activation of ERK, its only known sub- strate.17,24,25 The activation of ERK leads to a pro-growth and potentially transforming signal, through altered transcription of numerous genes. Feedback regulation of RAF kinases is mediated by Sprouty, and MEK and ERK are negatively regulated by dual- specificity phosphatases (DUSP4 and DUSP6). But in fully transformed melanoma, this feedback is dysregulated by poorly understood mechanisms and constitutive activation of ERK persists. Figure 1. Consequences of activating mutations in BRAF. Although BRAF is thought to exert its oncogenic effects almost entirely through MEK, CRAF can lead to MEK-independendent pro- particular, both a reduction of overall survival and a higher rate of survival effects, in part through its interaction with nuclear factor- brain metastases from the time of diagnosis from stage IV disease kappa B (NF-kB), as well as through inhibitory effects of critical 17,26,27 was noted. Interestingly, the prognostic significance of a BRAF regulators of apoptosis, such as ASK-1 and MST-2. mutation is confined to patients diagnosed with stage IV disease, Interestingly, unlike CRAF, activated BRAF has no known while NRAS mutation is associated with a poorer prognosis from substrates other than MEK, although it is important to note that both initial diagnosis and stage IV diagnosis. Not surprisingly, differential activation of substrates by ERK occurs depending upon given the clinical activity of BRAF-directed therapy (described the RAF isoform, and perhaps more specifically the RAF homo- or 28 below), patients with BRAF mutant, stage IV melanoma treated heterodimer, which leads to its activation. For example, BRAF with a BRAF inhibitor have a better prognosis than NRAS mutant mutation (specifically BRAFV600E), and not CRAF upregulation, is patients, BRAF mutant patients not treated with a BRAF inhibitor, associated with activating the mammalian target of rapamycin and wild-type to both NRAS and BRAF patients. (mTOR) pathway through a deactivating phosphorylation of LKB-1 by ERK and regulating beta-Trcp, which leads to activation of NF-kB.29,30 These important consequences of oncogenic BRAF Inhibitors of MAPK signaling in melanoma highlight the mechanisms by which an oncogene can influence A series of small-molecule inhibitors have been developed, which pathways outside of the canonical pathways under the control of target, with varying selectivity, BRAFV600E, other mutant BRAF the wild-type form. variants (at the 600 position), wild-type BRAF and CRAF. In In CRAF, it appears that activation requires additional activation addition, inhibitors of the downstream mediators of RAF activa- steps compared with BRAF.31,32 From an evolutionary perspective, tion, namely MEK and ERK, are also being developed. In this this may explain why an activating mutation in the BRAF kinase section, only agents for which clinical data are available are domain is the 40–60% of patients with melanoma, whereas there reviewed (Table 1). are no descriptions of activating mutations of CRAF.33 It is interesting to note that while BRAF mutations are seen in the great majority of melanocytic nevi (70–80%), vertical growth BRAF INHIBITORS phase melanomas (40–50%) and metastatic melanoma (40–50%), Sorafenib one report suggested that they are rarely detected in radial Sorafenib, a broad-spectrum tyrosine and serine-threonine kinase growth-phase melanomas (10%), although this had been thought 33–35 inhibitor has inhibitory activity in vitro against BRAF, CRAF, to be the precursor lesion before invasive tumors. This platelet-derived growth factor receptor (PDGFR), vascular suggests that BRAF mutation may actually be an acquired event, endothelial growth factor receptor (VEGFR) 2, p38 and CKIT and in some instances, in early invasive melanoma that leads to clonal was the first RAF-inhibitor actively studied in patients with expansion and tumor progression. The early occurrence of BRAF melanoma as it was available for phase II testing in the same mutations likely accounts for the lack of polyclonality at this locus 40 1,36 year in which BRAF mutations were first reported. Unfortunately, in metastatic melanoma. Somatic alterations in other pathways despite being evaluated in numerous phase I, II and III studies as a are essential for melanoma transformation. Inactivation of INK4A single agent and in combination with chemotherapy, the clinical and PTEN are the best described cooperating events in vitro and 37,38 utility of sorafenib has been disappointing, as several trials of in vivo. It is possible that heterogeneity exists within and sorafenib both alone and in combination with various cytotoxic across melanoma tumors with regarding INK4A and PTEN status, agents have revealed no substantial clinical benefit.41–47 In although this has not been thoroughly investigated. Despite the comparing the results of serial tumor biopsies in a single-agent necessity of other oncogenic events in melanoma transformation, sorafenib trial to those now available with more potent and melanomas harboring BRAF mutations remain strictly reliant on 39 selective BRAF inhibitors, it is clear that sorafenib fails to inhibit MAPK pathway activation. This cellular addiction to the pathway the MAPK pathway adequately when administered at maximum renders BRAF mutant cells susceptible to blockade of the pathway tolerated doses. The maximum tolerated dose is most likely (Figure 1). defined by toxicities arising from inhibition of kinases other than BRAF in normal tissues. This conclusion is supported by the similarity of sorafenib to other VEGF and PGDF receptor tyrosine NRAS and BRAF mutations and prognosis in melanoma kinase inhibitors with regard to common toxicities and the more Recently, it has been shown that the prognosis of patients with recent clinical experience with more selective BRAF inhibitors. melanoma is impacted depending on whether a patient0s tumor harbors either an NRAS or BRAF mutation. Specifically, both NRAS and BRAF mutational status is associated with a poorer prognosis Selective BRAF inhibitors (vemurafenib, dabrafenib) in patients with stage IV melanoma compared with patients Following the failure of sorafenib in BRAF mutant melanoma, whose tumors are wild-type for both NRAS and BRAF.6,15 In more potent and selective inhibitors of BRAF including

Oncogene (2013) 2373 – 2379 & 2013 Macmillan Publishers Limited MAP kinase signaling and inhibition in melanoma RJ Sullivan and K Flaherty 2375 Table 1. Drugs in clinical development in BRAF mutant cancers targeting components of the MAPK pathway

Agent Target Trial Findings References

Sorafenib Pan-RAF, anti- Multiple No signiﬁcant responses, comparable PFS/OS to standard 41–47 angiogenesis chemotherapy Vemurafenib BRAFV600, BRAF I, II, III High response/tumor burden reduction (B85%); improvement in PFS 3,4,8 WT and OS compared with DTIC Dabrafenib BRAFV600, BRAF I, III High response/tumor burden reduction (B85%); Improvement in PFS 2,10 WT and OS compared with DTIC Trametinib MEK 1/2 I, III Responses in BRAF mutant patients (22%), improvement in PFS and OS 5,7 compared with DTIC MEK162 MEK1/2 II 20–30% response rate in both BRAF and NRAS mutant patients 9 Selumetinib, PD- MEK 1/2 I, II Poor response rate; occasional responses in BRAF mutant patients 52–56 0325901, AS703026 described Dabrafenib plus BRAFV600, MEK I/II High disease control rate (490%) in BRAFi naive; lower RR in patients 10 trametenib 1/2 with prior BRAFi treatment (B17%) Abbreviations: MAPK, mitogen-activated protein kinase; OS, overall survival. The table summarizes clinical trials of MAPK-directed therapies that have been published or presented nationally.

vemurafenib and dabrafenib have been developed.2,4 Treatment Emerging mechanisms of resistance to BRAF-inhibition with these agents leads to a reduction in levels of pERK in tumors Virtually every patient treated with single agent BRAF or MEK V600E containing the BRAF mutation and degree of pERK inhibition inhibitors will eventually have disease progression despite initially 48,49 correlates with clinical response. And, inhibition of the MAPK profound inhibition of BRAFV600E, significant tumor regression and pathway is only observed in tumor cells harboring BRAF improved survival.3 Preliminary studies suggest that a single mutations, as other normal and cancer cells maintain MAPK resistance mechanism to vemurafenib and/or dabrafenib will not signaling through CRAF. The differential effect on signaling likely account for acquired resistance. To date, second mutations that contributes significantly to the therapeutic index of these more impair the binding of the inhibitor BRAF have not been found; a selective BRAF inhibitors. Both vemurafenib and dabrafenib are resistance mechanism in non-small cell lung cancer, chronic associated with some degree of tumor regression in 85% of myelogenous leukemia and gastrointestinal stromal tumor when patients and estimated progression-free survival rates of B5 À 6 treated with oncogene-directed therapies.57–59 The best months, which compares favorably to previously available characterized resistance mechanisms typically reactivate the 2–4,8,10,12,50 therapies for metastatic melanoma. Randomized, MAPK pathway through alternative means (Figure 2). phase III trial results are now available for both vemurafenib and The first evidence of BRAF-independent, MAPK pathway dabrafenib and each study clearly demonstrates a survival reactivating mechanisms came from BRAFV600E mutant cell lines advantage for single-agent BRAF inhibition in comparison with cultured in the presence of a selective BRAF inhibitor until 3,10 the previous cytotoxic chemotherapy standard, dacarbazine. proliferation was observed in the presence of drug. In vitro, restoration of MAPK signaling is consistently observed.60–63 This can be achieved through upregulation of receptor tyrosine kinases MEK INHIBITORS (that is, PDGFRB, ERBB2),61,62 activation of RAS,60,62 upregulation of Inhibitors of MEK have shown promise in preclinical studies in CRAF,61,63 activating mutations of MEK64,65 and activation of the melanoma and, though the MAPK pathway is activated in most Ser/Thr MAPKs (COT).61 These can be considered within-pathway melanomas, BRAFV600E mutational status correlates strongly with mechanisms that bypass BRAF as each can restore ERK activation response to MEK inhibition in murine melanoma xenograft despite ongoing BRAF inhibition. While resistance mutations in models.51 Clinically, several agents including selumetinib the kinase domain of BRAF have yet to be found either in vitro or (AZD6244), PD-0325901, trametinib (GSK1120212), AS703026 and in human tumor samples, alternative splicing of BRAFV600E has MEK162 have been evaluated in patients with melanoma with been identified in cell lines with acquired resistance and in a variable success.5,7,9,52–56 The great majority of clinical benefit subset of melanoma tumor samples from patients who responded (defined as clinical response or improved survival) has been seen in and then relapsed on BRAF inhibitor therapy.66 The kinase domain patients with BRAF mutations, though emerging clinical evidence is unaffected, but regulatory elements of BRAF, including the RAS now has been described that supports a role of MEK inhibitors in binding site, are deleted resulting in a kinase that is capable of treating patients with NRAS mutations. To date, none of the MEK dimerizing and activating MEK even in the presence of a BRAF inhibitors have resulted in clinical efficacy that is comparable to the inhibitor. Most recently, BRAF amplification in progressing tumor selective BRAF inhibitors, though trametinib and MEK162 have samples from 4 of 20 patients that was not present at baseline has recently been shown to be the most active MEK inhibitors thus far been reported. In vitro, forced overexpression of this confers in BRAF mutant melanoma.7,9 This is likely due to the fact that MEK BRAFV600E resistance to vemurafenib and other selective BRAF inhibitors block the MAPK pathway in normal tissues as well as in inhibitors, though this can be overcome with higher doses of BRAF tumor, resulting in dose limiting toxicities as a consequence of inhibitors.67 MAPK pathway. Or, oncogenic BRAF has signaling effects mediated Resistance emerges in the face of ongoing MAPK suppression by as yet undiscovered substrates other than MEK. approximately one-third of the time.48 In this setting, signaling Recently, MEK162 was shown to lead to responses in through the parallel growth and survival PI3K pathway has been approximately a quarter of patients with NRAS mutations.9 observed. Increased insulin growth factor receptor 1 (IGF-1R) Although the duration of response and progression free survival signaling or acquired loss of PTEN have been correlated were not dramatic, these findings support the continued with increased activation of AKT.68,69 These are the best- investigation of this strategy in patients with NRAS mutations. In defined examples of MAPK-independent bypass mechanisms.69 particular, MEK inhibitors will likely become the backbone of Of note, each of these mechanisms have been investigated molecularly targeted therapy combinations for these patients. and corroborated in small numbers of tumor samples from

& 2013 Macmillan Publishers Limited Oncogene (2013) 2373 – 2379 MAP kinase signaling and inhibition in melanoma RJ Sullivan and K Flaherty 2376 as persistence of the BRAFV600E mutation has been identiﬁed in all tumors analyzed and reported to date.79,80 It is possible that this mechanism favors the selection of tumor cells that harbor concomitant BRAF and NRAS mutations, a ﬁnding that has been seen in multiple series of patient samples at a rate of B15–25%.66,80 It is likely through activation of CRAF homodimers downstream of mutated NRAS that vemurafenib could contribute to growth of this subpopulation of tumor cells.81

FUTURE DIRECTIONS The documentation of single-agent efficacy of selective BRAF inhibitors and MEK inhibitors is a major advance for the treatment of patients with BRAF mutation-positive melanoma. As nearly every patient treated with these agents is predicted to progress on treatment, identifying the mechanisms of this resistance has Figure 2. Mechanisms of acquired resistance. been a major focus of melanoma researchers over the past several years. Based on the finding that MAPK pathway activity is reactivated in melanoma following selective BRAF inhibition and patients who had biopsies performed at the time of clinical that some BRAF inhibitor-associated toxicity might be abrogated progression. by MEK inhibition, the first combination trial of a selective BRAF Primary resistance to BRAF inhibition is seen in o10% of inhibitor (dabrafenib) with a MEK inhibitor (trametinib) is ongoing, patients with BRAF mutant melanoma treated with vemurafenib. appears tolerable with both agents being administered at their Emerging evidence from both preclinical studies and analysis of standard single-agent doses, and appears to be associated with a pre- and on-treatment biopsies from patients treated with BRAFi higher complete response rate, overall response rate and longer suggest that PTEN loss, elevated pretreatment levels of CRAF, progression free survival than single agent BRAF inhibitors, CCND1 amplification (with consequent cyclin D1 overexpression), though the results of a randomized phase II study comparing CDK4-activating mutations, the receptor tyrosine kinase CMET the combination to single agent dabrafenib are eagerly expression and tumor microenvironment hepatocyte growth awaited.11,48 factor expression (the ligand for CMET) are promising predictive Another approach to enhancing the effectiveness of selective biomarkers that identify relative resistance to BRAF inhibitors and BRAF and/or MEK inhibitors is through careful analysis into why are worthy of validation.63,70–73 Cyclin D and CDK4 alterations, these agents induce benefit and then augment these responses PTEN loss, and hepatocyte growth factor expression all represent with rationale combinations. For example, mutant BRAF mod- potential therapeutic opportunites given the emergence of ulates proapoptotic BCL-2 family members in melanoma, speci- selective CDK4/6 inhibitors (targeting Cyclin D and CDK4 fically triggering the inactivating phosphorylation of serine-75 of alterations), PI3K inhibitors (PTEN loss), and anti-hepatocyte BAD as well as downregulating the expression of BIM, which growth factor antibodies and/or CMET inhibitors (hepatocyte together protect the cell from undergoing apoptosis.82 Further, growth factor expression) that could be plausibly combined with inhibition of BRAF or MEK, either through siRNA or small-molecule BRAF inhibitors. inhibitors, leads to growth inhibition and apoptosis at least in part In BRAF wild-type melanoma cells, the MAPK pathway is through the upregulation of BIM and its associated suppression of activated by vemurafenib (and the analog PLX4720), leading to activity of two antiapoptotic BCL-2 family members, BCL-2 and increased proliferation.74,75 This paradoxical effect appears to be MCL-1.83–86 In particular, it appears that Bim induction and its CRAF dependent.74 Further, CRAF activation appears to be subsequent sequestration of MCL-1, thereby leading to the mediated by enhanced dimer formation: heterodimers with activation of the mitochondrial associated proteins BAK and wild-type BRAF and/or homodimers CRAF.76 This phenomenon is BAX, appears to the most critical factor for apoptosis induction most pronounced in RAS mutant cancer cell lines. Hyperactivation following inhibition of MEK (and presumably BRAF).82 of the MAPK pathway in BRAFWT non-melanoma cells is likely Interestingly, loss of expression of phophatase and tensin responsible for some, if not most, of the toxicity of BRAF homolog (PTEN), the key regulator of the PI3K pathway, is seen inhibitors.11,48 Paradoxically, it is likely this lack of MAPK in 410% of mutated BRAF melanomas and appears to abrogate pathway inhibition in BRAFWT tissues, which creates the ability the effects of BRAF inhibitors through the suppression of BIM.84 to achieve the magnitude of MAPK inhibition in BRAF mutant These findings are significant and provide the rationale for at tumors, needed to achieve clinical efficacy. Growth of cutaneous least two novel combinations. First, the addition of agents that squamous cell carcinomas (SCCs) or keratoacantomas (KAs) has might augment BRAF and MEK inhibitor associated apoptosis is been observed early in the course of therapy in B25% of important to consider. One such agent is ABT-263, and to a lesser vemurafenib-treated patients.3,10 Genetic analysis of 35 SCC/KAs extent its less bioavailable homolog ABT-737, which is a BH3- which developed in patients treated with vemurafenib showed a mimetic currently in clinical development. ABT-263 and ABT-737 mutation in either H-, K- or N-RAS in 21 (60%).77 This compares are small-molecule inhibitors of the anti-apoptotic proteins BCL-2, with a much lower rate of HRAS mutation in sporadic SCC/KAs BCL-XL and BCL-w, which have been shown to have differential arising in heavily sun-exposed individuals.78 Although not proven cytotoxicity against cell lines of several tumor types.87,88 mechanistically in these resected SCC/KAs, it is likely that the Importantly, the cell lines most resistant to these agents showed observation of MAPK stimulation in RAS mutant cells accounts for overexpression of MCL-1 and pharmacological downregulation of the appearance of these lesions in patients treated with selective MCL-1 potentiated the lethality of ABT-737 in these cell lines.89 BRAF inhibitors. Thus combination of ABT-263 or similarly targeted agents with From the perspective of acquired resistance, it is unclear what, if drugs that downmodulate or oppose MCL-1, such as BRAF or MEK any, activation of CRAF homodimers or CRAF/BRAF heterodimers inhibitors in BRAF mutant melanoma, represents an attractive might have in cells that harbor a copy of BRAFV600E and BRAFWT. therapeutic strategy. In preclinical studies, ABT-737 in It is not thought that clinically acquired resistance to vemurafenib combination with a MEK inhibitor led to enhanced lethality, occurs solely due to growth of a subset of BRAFWT melanoma cells in vitro and in vivo utilizing melanoma cell lines and xenografts,

Oncogene (2013) 2373 – 2379 & 2013 Macmillan Publishers Limited MAP kinase signaling and inhibition in melanoma RJ Sullivan and K Flaherty 2377 90 respectively, compared with either agent alone. Whether 3 Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J et al. ABT-263 in combination with MEK or selective BRAF inhibitors Improved survival with vemurafenib in melanoma with BRAF V600E mutation. will improve clinical outcomes is unknown, though appears worth N Engl J Med 2011; 364: 2507–2516. exploring in an early-phase clinical trial. 4 Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA et al. Inhibition A second compelling combination that should be evaluated in of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010; 363: patients is the combination of either a BRAF or MEK inhibitor in 809–819. combination with an inhibitor of the PI3K pathway. While this is 5 Infante JR, Gordon MS, Flaherty KT, Cox DS, DeMarini DJ, Morris SR et al. Safety logical generally, given that the MAPK and PI3K pathways are the and efficacy results from the first-in-human study of the oral MEK 1/2 inhibitor GSK1120212. J Clin Oncol 2010; 28(Suppl): abstract 8503. predominant signaling pathways utilized by human cancers, in 6 Long GV, Menzies AM, Nagrial AM, Haydu LE, Hamilton AL, Mann GJ et al. melanoma there is specific justification. First, as was discussed Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic above, PTEN loss suppresses BIM expression and appears to be an melanoma. J Clin Oncol 2011; 29: 1239–1246. important mediator of tumor cell resistance to BRAF or MEK 7 Flaherty KT, Robert C, Hersey P, Nathan P, Garbe C, Milhem M et al. inhibition.84 Further, PI3K activation via insulin growth factor Improved survival with MEK inhibition in BRAF-mutated melanoma. N Engl J Med receptor activation has been described as an acquired mechanism 2012; 367: 107–114. of resistance to BRAF inhibitors in clinical trials.57 Thus, the 8 Sosman JA, Kim KB, Schuchter L, Gonzalez R, Pavlick AC, Weber JS et al. Survival in concurrent or sequential inhibition of the PI3K pathway might be BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J Med expected to enhance the clinical effectiveness of BRAF or MEK 2012; 366: 707–714. 9 Ascierto PACB, Sanjiv SA, Dirk S, Carla Van H, Paola Q, Christian UB et al. Efficacy inhibitors. and safety of oral MEK162 in patients with locally advanced and unresectable or In addition to combination therapy with molecularly targeted metastatic cutaneous melanoma harboring BRAFV600 or NRAS mutations. J Clin agents that inhibit signaling induced by selective BRAF inhibition Oncol 2012; 30(Suppl): abstract 8511. or promote apoptosis, another promising approach to maximizing 10 Hauschild Axel JJG, VD Lev, Jouary T, Gutzmer R, Millward M, Rutkowski P et al. the benefit of BRAF or MEK inhibitors is to combine these agents Phase III, randomized, open-label, multicenter trial (BREAK-3) comparing the BRAF with immunotherapy. Recently, immune checkpoint inhibitors, kinase inhibitor dabrafenib (GSK2118436) with dacarbazine (DTIC) in patients including the anti-CTLA-4 monoclonal antibody ipilimumab and with BRAFV600E-mutated melanoma. J Clin Oncol 2012; 30(Suppl): abstract the anti-PD monoclonal antibody MDX-1106, have shown single LBA8500. agent efficacy in patients with metastatic melanoma.91–93 11 Weber JS, Flaherty KT, Jeffrey RI, Gerald SF, Richard K, Adil D et al. Updated safety and efficacy results from a phase I/II study of the oral BRAF inhibitor dabrafenib Importantly, it appears that vemurafenib does not adversely (GSK2118436) combined with the oral MEK 1/2 inhibitor trametinib (GSK1120212) affect human T-lymphocyte (T-cell) function, whereas MEK in patients with BRAFi-naive metastatic melanoma. J Clin Oncol 2012; 30(Suppl): 94 inhibitors do. Further, vemurafenib has been shown to abstract 8510. improve immune recognition by antigen-specific T-cells and to 12 DR Trefzer UM, Lebbe A, Siegfried C, Arya A, Guckert N, Schadendorf M et al. induce T-cell infiltration in melanoma tumors.95,96 These findings BREAK-2: a Phase IIA trial of the selective BRAF kinase inhibitor GSK2118436 in provide a rationale for a study evaluating the safety and efficacy of patients with BRAF mutation-positive (V600E/K) metastatic melanoma. 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