Critical Review Targeting RAS/RAF/MEK/ERK Signaling in Metastatic Melanoma

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1 Critical Review Targeting RAS/RAF/MEK/ERK Signaling in Metastatic Melanoma Ao-Xue Wang* Xiao-Yi Qi Department of Dermatology, The Second Affiliated Hospital of Dalian Medical University, Dalian, People s Republic of China Abstract The RAS/RAF/MEK/ERK pathway has been reported to be activated in over 80% of all cutaneous melanomas, making it the focus of many scientific studies in the melanoma field. Discoveries of mutations and aberrant expression of components in this cascade, in particular, BRAF and NRAS render a deeper understanding of the mechanisms responsible for oncogenesis and provide new therapeutic strategies for this deadly disease. This review starts with a comprehensive discussion on the role of this pathway in initiation and progress of melanoma. Mechanistically, mutated BRAF and NRAS exert most of the oncogenic effects through the activation of the MAPK pathway, which both drive the uncontrolled growth of melanoma cells and regulate the cell survival. In a subsequent section, clinical efficacy of targeted small-molecule inhibitors is highlighted. BRAF-targeted therapies (e.g., vemurafenib, dabrafenib) have showed impressive results in systemic therapy for melanoma harboring activating BRAF V600E mutations. MEK inhibitors show limited activity in phase I trials, and inhibitors directly targeting mutated NRAS, to date, have not been realized. Furthermore, the emerging mechanisms underlying both intrinsic and acquired drug resistance as well as approaches to prevent or abrogate the onset of therapeutic escape are addressed. Finally, the promising vistas and major challenges involving small-molecule inhibitors targeting this MAPK pathway in melanoma therapy are briefly discussed. It can be envisaged that disseminated melanoma is no longer such a bleak prognosis in future given the research and development of new signal transduction inhibitors based on our evolving understanding of melanoma genetics and intracellular signaling. VC 2013 IUBMB Life, 65(9): , 2013 Keywords: melanoma; BRAF inhibitor; resistance; MEK; signaling Introduction VC 2013 International Union of Biochemistry and Molecular Biology Volume 65, Number 9, September 2013, Pages *Address correspondence to: Ao-Xue Wang, Department of Dermatology, The Second Affiliated Hospital of Dalian Medical University, 467 Zhongshan Road, Dalian , Liaoning Province, People s Republic of China. Tel.: (Mobile). Fax: wangaxdl@hotmail.com Received 28 March 2013; Accepted 3 June 2013 DOI /iub.1193 Published online 29 July 2013 in Wiley Online Library (wileyonlinelibrary.com) Although much progress has been made in the management of advanced tumors in recent years, cutaneous melanoma remains a challenging cancer (1). As the most devastating form of skin cancers, cutaneous lesions detected as little as 1 mm in Breslow thickness could have microscopic evidence of lymph node metastasis which, when present, confers a significantly increased risk for metastasis and results in a precipitous drop in survival rate (2). Advanced melanoma with dissemination to distant sites and visceral organs leads to a median survival time of only 6 9 months and a 3-year survival rate of only 10 15% (3). For many years, metastatic melanoma is assumed to be refractory to all tested systemic therapeutic interventions and little to no improvement on survival rate has been made to date in spite of increased response rates achieved with combinations of chemotherapeutics or with the combination of chemotherapy and cytokines (1,4). For instance, dacarbazine-or temozolomide-based chemotherapies, the standard treatments in the setting of metastatic melanoma, are ineffective in most patients with melanoma despite 5 10% response rates achieved. In addition, the cytotoxic chemotherapies are amenable to the development of drug resistance and disease relapse (1). Interferon (IFN)-a2b and interleukin-2 (IL-2) are two FDA-approved biological response modifiers for metastatic melanoma. High doses of IFN-a2b used in the adjuvant treatment of high risk melanoma has presented a better progression-free survival (PFS) than untreated patients. IL-2, a conventional cytokine for metastatic melanoma, has been considered preferred initial treatment although it induces only 748 IUBMB Life

2 limited responses. However, combination chemotherapy regimens with or without IFN-a2b and/or IL-2 have demonstrated durable responses in only a small percentage of patients (10 20%) and none is thought to improve survival outcomes for the overall population of patients (5). Recent advances in molecular profiling and genome sequencing have shown melanoma to be a heterogeneous group of malignancies whose initiation depends on distinct patterns of oncogenic mutation (6,7). Importantly, the persistent activation of oncogene-driven signaling cascades is reported to involve in the sustained proliferation, survival, invasion, and metastases of melanoma cells. The discoveries may thus lead to the idea that targeted inhibition of the mutant oncogenes and downstream signaling pathways would be of utmost significance for melanoma therapy. On the basis of these principles, a variety of pharmacological inhibitors targeting the recently identified mutated signal transduction molecules are currently being developed and exhibit unprecedented clinical activities (8). In this commentary, we will review the role of RAS/RAF/ MEK/ERK signaling in malignant melanoma and discuss the latest progress toward targeting the deregulated oncogene products involving the signaling pathway. RAS/RAF/MEK/ERK Signaling Pathway in Melanoma The RAS/RAF/MEK/ERK signaling pathway is central to the pathogenesis of cutaneous melanoma (9).It is typically activated by oncogenic mutations, such as NRAS and BRAF, as well as mutations at upstream membrane receptors (e.g., KIT). Following activation of the pathway, signals are relayed from cell surface receptors to transcription factors via a series of protein phosphorylation, which result in profound effects on the regulation of cellular proliferation, cell survival, and apoptosis. Mutations in BRAF (v-raf murine sarcoma viral oncogene homolog B1) appear to be the most common genetic alteration and major driver of this pathway, which occur in 50 70% of cutaneous melanomas (9). There are three main isoforms of the RAF protein: ARAF, BRAF, and CRAF. The reasons for mutations at BRAF and not ARAF or CRAF in melanoma patients are not entirely clear. Activating mutations of BRAF require only one genetic mutation compared to two genetic events in either ARAF or CRAF, which may explain why BRAF is mutated at a higher frequency (9). Moreover, 89% of these mutations occur within the kinase activation loop, of those 80% are a valine to glutamic acid mutation at codon 600 (GTG to GAG, known as V600E) in exon 15 (10). Substitution in the amino acid sequence at this position creates a constitutively active BRAF molecule with elevated kinase activity, leading to the cascade activation of MEK (mitogen-activated protein/erk kinase) and ERK (extracellular signal-related kinase) independent of upstream RAS activation. A number of other clinically relevant, but less common mutations identified from melanoma specimens are V600K and V600G/R, which has been reported in 16% and 3% of patients with BRAF-mutant metastatic melanoma, respectively (11). The frequency of non- V600E mutations is particularly important when interpreting the BRAF inhibitors with a variety of mutation subtypes in clinical trials. A minor subgroup of melanomas has also been identified with BRAF mutations of low activity in positions other than 600, that is, D594G, G469E, and G469A mutation (12). Melanoma cells expressing a BRAF mutant of G469E/ D594G are shown to signal through CRAF rather than BRAF to activate the mitogen-activated protein kinases (MAPK) pathway (12,13). Targeting CRAF with its inhibitor sorafenib in low-activity mutants of BRAF induces melanoma cell death by apoptosis. Interestingly, BRAF activating mutations are also present in up to 80% of benign nevi. In spite of the high frequency in nevi, the role of BRAF mutations in oncogenesis has been well demonstrated in preclinical and clinical trials. The gain-offunction mutations in BRAF play a causal role in reproducing the cancer phenotype in both cell-based studies and in vivo mouse models (14,15). Conversely, small-molecule and RNA interference-based therapeutics targeting oncogenic BRAF induce melanoma cell death and tumor regression in some settings (16,17). The dependence of melanomas on BRAF kinase activity is further supported by the antitumor activity of BRAF inhibitors that have recently been tested in genetically defined subgroups of melanoma patients (8,18). However, studies investigating the specific role of BRAF mutations in disease initiation and progress have also shown conflicting results. The introduction of V600E mutated BRAF into primary human melanocytes does not induce oncogenic transformation (19). Likewise, mice with conditional melanocyte-specific expression of BRAFV600E fail to progress to melanoma over months (20). Indeed, as a genetically complex disease, melanoma development has been increasingly shown to be associated with both BRAF/MAPK and PI3K (phospho-inositide 3- kinase)/akt pathway activation. This is supported by current evidence indicating that cotargeting of the PI3K pathway and the MAPK pathway results in full inhibition the proliferation of many melanomas (21,22). Mutations of NRAS are the other major oncogenic event in melanoma development and occur in approximately 20 25% of all cases. The most common site of mutation is codon 61 in exon 2, with C181A (Q61K) and A182G (Q61R) being found most frequently (23). These mutations result in the constitutive activation of the protein which no longer requires ligand for activation. NRAS activating mutations result in prolongation of the GTP-bound state of NRAS. GTP binding triggers a permanent activation of RAF proteins, leading to enhanced signalings through the MAPK pathway, followed by promotion of proliferation, survival, invasion, and angiogenesis of melanoma. Intriguingly, activation of ERK induces persistent phosphorylation of BRAF which prevents BRAF binding to NRAS and, in turn, inhibits BRAF kinase activity. To overcome this negative feedback inhibition of BRAF, melanoma cells containing an oncogenic NRAS use CRAF to activate the MAPK pathway via a Wang and Qi 749

3 IUBMB LIFE mutant BRAF and CRAF complex (13,24). Alternatively, about 50% of such cells have acquired an activating mutation in the BRAF gene (usually V600E), which makes the BRAF kinase activity independent of RAS (13). A study involving microinjection of the RAS-neutralizing antibody into melanoma cell lines with the V600D/E mutation found that the antibody failed to block proliferation of melanoma cells in culture, inferring that BRAFV600 mutant melanomas uncoupled the requirement for BRAF signaling on NRAS (10). Of note, constitutive expression of Q61K NRAS at levels that are optimal for oncogenic transformation in melanocytes has been demonstrated to induce tumors efficiently only when Cdkn2a (p16 INK4a ) is deleted, implicating additional oncogenic events in the induction of melanoma (25). Although studies on the role of the MAPK pathway in melanoma place emphasis on the more upstream components such as RAS and RAF, the importance of MEK or ERK in MAPK pathway activation in melanoma has also been documented (26,27). As a major mediator of oncogene-induced melanoma formation, MEK plays a role in promoting cellular transformation and inhibiting apoptosis through transcriptional/post-translational mechanisms. This is supported by the evidence that inhibition of MEK reduces proliferation and invasion of BRAFV600E mutant human melanoma cells in vitro along with growth of tumors in vivo (26). While MEK mutations were previously not proved in melanoma, it is of note that a recent sequencing of melanoma exomes has identified activating mutations in MAP2K1 or MAP2K2 (MEK1 and MEK2, respectively), which results in constitutive ERK1/2 phosphorylation and higher resistance to MEK inhibitors (6). Activated ERK, the only known substrate for MEK phosphorylation, is found in 90% of human melanomas (27). Activated ERK phosphorylates a variety of nuclear and cytoplasmic substrates that mediate the pleiotropic effects of the pathway, including activation of nuclear transcription factors, regulation of the expression and function of proapoptotic and antiapoptotic proteins (such as BIM, BMF, BAD, and Mcl-1), as well as expression of many proteins involved in cell cycle regulation (e.g., upregulation of cyclin D1 and suppression of the cell cycle inhibitor p27 KIP1 ) (28,29). This, in turn, leads to cellcycle progression and regulation of cellular apoptosis/survival. Interestingly, despite the similar functions, ERK1 and ERK2 control different signaling events in melanoma cells. For example, reducing ERK2, but not ERK1 increased levels of specific proapoptotic proteins (i.e., Noxa), whereas knockdown of ERK1 plus ERK2 reduced Noxa below constitutive levels (27). Similarly, reducing ERK1 but not ERK2 increased XIAP, which is a member of the inhibitor of apoptosis (IAP) family and blocks both the endogenous (mitochondrial) and exogenous (death receptor-related) apoptosis pathways. Moreover, ERK regulates melanoma cell invasion through the RND3- mediated Rho/ROCK/LIM pathway and through a novel phosphodiesterase PDE5A pathway (30). In addition to the direct effects upon melanoma cell behavior, ERK can phosphorylate and activate inhibitor jb kinase through an indirect mechanism and lead to activation of the nuclear factor j-b (NF-jB) transcription factor, another pathway that melanoma tumors use to achieve survival, proliferation, resistance to apoptosis and metastasis (29). Targeting the RAS/RAF/MEK/ERK Signalings The importance of aberrant activation of the RAS/RAF/MEK/ ERK pathway in tumorigenesis has been demonstrated in over 80% of cutaneous melanomas. Among the abnormalities at various levels along the MAPK pathway, BRAF and NRAS activation comprise two major oncogenic events for driving melanoma development. Thus, for many years, there has been great enthusiasm to pursue drugs that selectively target this pathway. Development of RAF Inhibitors BRAF-targeted therapies represent the first major breakthrough in systemic therapy for metastatic melanoma. Following the discovery of BRAF mutations in cancer, several smallmolecule inhibitors of BRAF have been prospectively developed over the last decade, including nonselective ones and more recently highly selective ones. Selective BRAF inhibitors are gaining exclusively interest as being significantly more effective clinically and therefore are the focus of further discussion. Besides BRAF, CRAF may be another target for therapy development for melanoma, and CRAF inhibitors may be more relevant for NRAS mutant melanoma and low activity BRAF mutants. Nonselective RAF Inhibitors One of the earlier BRAF inhibitors is the multikinase inhibitor sorafenib (BAY ), which targets not simply BRAF but also CRAF as well as the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinases (RTKs) (31). However, preclinical investigations have showed sorafenib to be a relatively weak inhibitor of BRAF and that sorafenib as monotherapy only led to minor levels of regression in BRAFV600E mutated melanoma (31,32). Although subsequent clinical phase-i or -II studies found sorafenib in combination with carboplatin and paclitaxel (CP), or dacarbazine stabilized disease and suggested an improvement in median PFS (33,34), a randomized phase-iii trial showed the median PFS of 17.4 weeks for sorafenib plus CP arm compared to 17.9 weeks for placebo plus CP (hazard ratio [HR], 0.91; 99% CI, ; two-sided log-rank test P ) (35). Response rate was 12% with sorafenib versus 11% with placebo. Toward this end, sorafenib as a combination agent eventually failed to demonstrate a sufficient survival benefit in patients with advanced melanoma. RAF265 is a novel nonselective small-molecular inhibitor of mutant BRAFV600E, which inhibits both BRAF and VEGFR2 with dose-dependent module (36). A recent report showed that the introduction of RAF265 in mutant BRAFV600E tumor xenografts resulted in inhibition 750 RAS/RAF/MEK/ERK Signaling in Metastatic Melanoma

4 of tumor growth and regression (37). Similarly, one study using orthotopic implants of patient tumors in mice revealed that RAF265 treatment led to reduced proliferation and induction of apoptosis (38). Selective BRAF Inhibitors The latest generation of highly specific and potent BRAF inhibitors offers a significant improvement over sorafenib against mutant BRAF. These drugs show a greater selectivity for mutant BRAF and have fewer off-target effects. Compounds that have been evaluated currently under preclinical or clinical investigation include AZ628, XL281, GDC-0879, SB590885, LGX818, dabrafenib (GSK ), vemurafenib (formerly known as PLX4032 or RG704), and its analog PLX4720 (39 41). Of these, vemurafenib and dabrafenib are the most comprehensively investigated. Vemurafenib is a potent ATP-competitive RAF inhibitor (40). Both in vitro and in vivo preclinical studies using BRAF mutant melanoma cell lines and xenograft models have demonstrated very impressive single-agent activity in melanoma management (15,42). The underlying mechanisms include inhibiting phospho-erk expression, inducing G1-phase cell cycle arrest and increasing apoptosis-related BIM expression. Of note, the effects of vemurafenib have been realized only in melanoma cells with activating BRAF mutations, and equivalent responses are seen in melanoma models with both heterozygous and homozygous BRAF mutations (40). In fact, vemurafenib and other small-molecule BRAF inhibitors have been shown to activate MAPK signaling pathway in such cell types that have wild-type BRAF but have other oncogenic events, such as a RAS mutation or upstream constitutive RTK activations (43,44). The mechanistic explanation for the unexpected finding has recently been provided. In cells with wild-type BRAF, BRAF and the closely related RAF kinase CRAF both signal as dimers. While an ATP-competitive RAF inhibitor binds to one RAF molecule in the nonmutated RAF dimer, it induces transition of the other inhibitor-free RAF molecule to the active state via direct interaction, causing a marked increase in catalytic activity (43). In this process, activating RAS is crucial for RAF dimerization and transactivation induced by inhibitors. Phase I, II, and III clinical trials with vemurafenib have shown unprecedented response rates of tumor regression and remarkably improved PFS compared to previously available chemotherapy for melanoma. In a phase I clinical trial, approximately 81% (26 of 32 patients) of patients with BRAF V600E-mutated melanoma responded to therapy when treated at the recommended phase II dose of 960 mg twice daily. The estimated median PFS was more than 7 months with duration of response ranging from 2 months to over 18 months. In all cases, responses were associated with marked reduction of intratumoral phosphorylated ERK, cyclin D1, and Ki-67, demonstrating a very impressive single-agent clinical activity on inhibition of the MAPK pathway (18). The most common adverse events of vemurafenib were rash, arthralgia, photosensitivity, and fatigue. Importantly, more than 23% of patients rapidly (mostly <12 weeks of treatment) developed cutaneous squamous cell carcinomas (SCC) of the keratoacanthoma type on areas of sun exposed skin. After the phase-i trial with an expansion cohort in melanoma patients (BRIM-1), a large phase-ii trial of vemurafenib enrolled 132 patients (122 with the V600E mutation and 10 with the V600K mutation) who had a median follow-up of 12.9 months (45). A high response rate of 53% was achieved (6% with a complete response and 47% with a partial response). Of note, among the 10 patients with BRAF V600K mutations, 4 (40%) had a partial response, 3 (30%) had stable disease, 2 had primary progressive disease (20%). The median PFS rate was 6.8 months and the median overall survival (OS) was 15.9 months. A pivotal phase-iii trial assessed 675 patients with untreated metastatic melanoma (BRIM-3) who were randomly assigned to receive either vemurafenib (960 mg orally twice daily) or dacarbazine (1,000 mg per square meter of bodysurface area intravenously every 3 weeks) (46). This study included patients with unresectable tumors, aged 18 years or older, with a life expectancy of 3 months or longer, and an Eastern Cooperative Oncology Group Performance Status Scale (ECOG PS) score of 0 or 1. Patients with noncontrolled brain metastases were excluded. The study was terminated at the time of the first interim analysis when a significant OS benefit was seen in vemurafenib, with a HR of 0.44 favoring vemurafenib over dacarbazine. A survival benefit occurred in each prespecific subgroup of the vemurafenib arm, according to age, gender, ECOG PS, tumor stage, and geographic region. Survival data were impressive: at 6 months, the estimated OS was 84% for vemurafenib as compared to 64% for dacarbazine. The median PFS assessed in 549 patients was 5.3 months in the vemurafenib arm and 1.6 months in the dacarbazine arm, respectively. Median survival seemed to be significantly improved by about 2.5 months, but it is too early for definitive assessment (5). Also, among 439 patients who were evaluated for tumor response, the overall response rate was 48% for vemurafenib compared with the 5% response rate for dacarbazine. Furthermore, common side effects associated with vemurafenib were evaluated in the BMIR3 study, including arthralgia, rash, fatigue, alopecia, keratoacanthoma or SCC, photosensitivity, nausea, and diarrhea. However, only 38% of patients required dose modification because of the toxic events in the vemurafenib group. An updated survival data were further presented recently (47). Compared with the initially published results in 2011 (48%), the overall response rate with vemurafenib was higher (57%), with higher complete response rate (5.6%) and partial response rate (51%). Another selective BRAF inhibitor dabrafenib has also been reported recently showing similarly excellent responses to what have seen in vemurafenib trials. Dabrafenib is a reversible, highly potent RAF inhibitor that selectively inhibits BRAF V600E kinase with IC50 much lower than that for wild-type BRAF or CRAF (48). Preclinical data showed that dabrafenib inhibited the MAPK pathway in BRAF V600E mutated Wang and Qi 751

5 IUBMB LIFE melanoma cells leading to tumor regression in xenograft mouse models (48). In a phase I/II trial of dabrafenib, objective responses were seen in 25 of 36 patients with Val600 BRAFmutant melanoma (69%, 95% CI ) and confirmed responses in 18 (50%, ) when given the recommended phase 2 dose (49). The responses were improved, with 17 patients (47%) on treatment for more than 6 months. In 27 patients with Val600Glu BRAF-mutant melanoma, responses were reported in 21 (78%, ), and 15 (56%, ) had a confirmed response. Impressive activities were also observed in patients with previously untreated brain metastases in the same study, proving reductions in size of brain lesions in 9 of 10 patients. This preliminary result is of great significance considering the present dilemma in treating melanomas with brain metastases. Furthermore, activity of dabrafenib was assessed in a multicenter, open-label phase II trials in patients with BRAF mutant melanoma metastatic to the brain (50). In 74 patients with Val600Glu BRAF-mutant melanoma who had not received previous local treatment for brain metastases (cohort A), 29 (39.2%, 95% CI ) achieved an overall intracranial response, as did 20 (30.8%, ) of 65 who had not received previous local treatment (cohort B). One (6.7%, ) of 15 patients with Val600Lys BRAF-mutation in cohort A achieved an overall intracranial response, as did four (22.2%, ) of 18 in cohort B. Median PFS for patients with Val600Glu BRAFmutation was longer than 16 weeks in both cohorts, and 8.1 and 15.9 weeks in cohort A and cohort B, respectively. The result suggested dabrafenib was active in patients irrespective of whether or not the brain metastases have been previously treated. More recently, a randomized phase-iii trial of dabrafenib was undertaken comparing its activity with that of standard dacarbazine chemotherapy (51). PFS was the primary endpoint, which allowed dacarbazine patients to crossover at the time of progression. The trial showed significantly improved PFS for dabrafenib as compared to dacarbazine (5.1 months versus 2.7 months), with a HR of Dabrafenib was generally well-tolerated and side effects were mild, including skin-related toxic effects, fever, fatigue, arthralgia, and headache. Development of MEK Inhibitors MEK is a major downstream mediator of oncogenic BRAF, and several MEK inhibitors are currently under active development for treatment of patients with advanced melanoma, such as PD , selumetinib (AZD6244, ARRY ), and trametinib (GSK ). A phase I trial of PD showed limited evidence of activity, with 3 of 48 patients having partial responses and 10 patients having stable disease for more than 4 months (52). The common side effects of rash, diarrhea, and in particular, visual disturbances including retinal vein occlusion limited the delivery of sufficient drug amounts required to adequately suppress the MAPK pathway in tumor cells. Selumetinib is a selective and non-atp-competitive inhibitor of MEK. Preclinical studies have shown that this drug can inhibit tumor cell growth by suppression of phosphorylated ERK1/2, especially in melanoma cell lines harboring BRAF mutations (53). In a phase I study, selumetinib showed moderate effect among patients with metastatic melanoma harboring BRAFV600E mutations (54). At the recommended phase II dose selumetinib was well tolerated with target inhibition. Rash was the most frequent and dose-limiting toxicity. More recently, an open-label, multicenter, randomized phase II trial comparing selumetinib monotherapy with temozolomide, however, did not show a benefit in terms of response rates or impact on PFS (55). Of note, five of the six objective responses to selumetinib were observed in BRAF-mutated melanoma. A randomized phase II study of first-line treatment in BRAFmutated melanomas comparing selumetinib plus dacarbazine with dacarbazine alone is in progress (clinicaltrials.gov registry number NCT ). The development of trametinib, a novel allosteric MEK inhibitor, has changed the interest of MEK inhibitor identification in BRAF mutant melanoma. Trametinib represents the best activity in any MEK inhibitor evaluated thus far, allowing more potent inhibition of MAPK signaling (56,57). A multicentre, phase I study was undertaken in 97 patients with melanomas (57). The study design included cohort expansion, pharmacodynamic assessment and identification of drug activity in BRAF-mutant tumors. Of 36 patients with BRAF V600E or V600K mutations, 2 had complete responses and 10 partial responses (confirmed response rate, 33%). Median PFS in this subgroup was 5.7 months. Of 39 patients with BRAF wild-type melanoma, 4 partial responses were confirmed (confirmed response rate, 10%). The data showed substantial clinical activity of trametinib in melanoma and suggested MEK was a potential therapeutic target. More recently, a phase III openlabel trial of 322 patients with V600E or V600K BRAF mutation melanoma who received either trametinib or chemotherapy was randomly assigned (58). The median PFS assessed in the trametinib group was 4.8 months and 1.5 months in the chemotherapy group, respectively. At 6 months, the rate of OS was 81% in the trametinib group and 67% in the chemotherapy group despite crossover (HR 0.54, P ). The research results demonstrated improved rates of PFR and OS in patients who received trametinib treatment, as compared with chemotherapy. Notably, the combination with trametinib and BRAF inhibitor dabrafenib, has shown additive or even synergistic effects on PFS and may avoid the CRAS stimulation of the MEK pathway in a preclinical research (56). More recently, the clinical activity of combination therapy with trametinib and dabrafenib was tested in a randomized, openlabel phase II study (59). Patients receiving therapy with 150 mg of dabrafenib and 2 mg of trametinib (combination 150/2) showed significantly improved median PFS of 9.4 months, as compared with 5.8 months in dabrafenib monotherapy group (HR for progression or death, 0.39; 95% CI, ; P < 0.001). The combination therapy (150/2) also induced greater extent of tumor regression, with an objective response rate of 76%, as compared with 54% in monotherapy 752 RAS/RAF/MEK/ERK Signaling in Metastatic Melanoma

6 (P ). The result also showed an acceptable safety profile with combination treatment when each agent was administered at its full single-agent dose. Targeting NRAS The prominent role of NRAS as an oncogene in melanoma necessitates evaluation of therapeutic targeting of NRAS for the treatment of advanced and high-risk melanoma. However, to date, RAS remains an elusive target in cancer, and no therapeutic strategy is available that can directly inhibit its signaling activity (60,61). A nonrandomized phase II study of MEK162, a small-molecule MEK 1/2 inhibitor, presented the efficacy and safety data in NRAS-mutated advanced melanomas (60). After 3.3 months of median follow-up period, 6 (20%) of 30 patients with NRAS-mutant melanoma had a partial response including three confirmed. Median time and median duration to response was 7.9 weeks and 7.6 weeks, respectively. Median PFS was 3.7 months (95% CI ). This study suggested a promising targeted agent with clinical activity in patients with NRAS-mutant melanoma and provided a justification of a phase 3 randomized trial for MEK162 as first-line therapy in such patients. Dual inhibition of target downstream signaling through the MAPK and PI3K/AKT pathways might antagonize the effect of RAS mutation if these drug combinations can be safely delivered at clinically achievable doses (61). Mechanisms of Resistance to BRAF and MEK Inhibitors and Strategies to Manage Resistance Mechanisms Underlying Resistance to BRAF Inhibitors Various BRAF inhibitors almost always cause tumor regression in patients who harbor the activating BRAF mutations and other genetic events. Although very encouraging, the clinical responses seen so far to these drugs are typically short-lived. Tumors eventually progress or grow again in nearly every case, with a median time to progression of approximately 7 months (18). A major problem with the use of these inhibitors and the further advancement of melanoma therapies is de novo or acquired drug resistance, which has been ascribed to complex mutational profiles and concurrent gene alterations (43,62 64) (Fig. 1). These clinical observations make it imperative to understand the nature of this resistance and to identify potential therapies that will overcome this resistance. De novo resistance denotes immediate resistance to initial treatment through pre-existing mutations (intrinsic) or interactions between tumor cells and microenvironments (extrinsic) (63,65). Intrinsic resistance to BRAF inhibitors has been well documented in both preclinical and clinical studies (18,66). Emerging evidence suggest that tumor heterogeneity, genomic mutation, or epigenetic alterations involved in intrinsic bypass survival pathways, in particular, PI3K/AKT pathway, may lead to the causal contribution (67). Impaired PTEN expression/function, observed in more than 10% of melanoma specimens, confers in part intrinsic resistance to BRAF inhibitors in melanoma through genetic activation of the PI3K/AKT pathway (21). In the context of PTEN loss, increased AKT signaling in association with inhibition of BRAF limits the nuclear accumulation of the transcription factor FOXO3a, leading to a decrease in BIM-mediated apoptosis. It was shown that the combination of a BRAF inhibitor and a PI3K inhibitor could overcome intrinsic BRAF inhibitor resistance through a mechanism involving the activation of FOXO3a and increase in BIM expression (67). Moreover, upregulation of PTEN protein and mrna expression restored the sensitivity of a limited melanoma cell line groups to a BRAF inhibitor. This provides a rationale for considering combination regimens of BRAF inhibitors with PI3K pathway inhibitors for this subset of BRAFmutated melanomas. A role for AKT activation in intrinsic BRAF inhibitor resistance was further demonstrated by the research showing that overexpression of AKT3 prevented PLX4720- induced apoptosis through the downregulation of both BIM and BMF (68). Genetic inactivation of p16 INK4A is also detected in association with activating mutations of BRAF in some melanomas (25,69). Typically, p16 status is significantly inversely associated with cyclin D1 levels, showing that p161 tumors generally are cyclin D1- and vice versa. CyclinD1amplificationinBRAF mutated cells has been demonstrated to confer intrinsic resistance to BRAF inhibition and to drive cell cycle entry with BRAF being inhibited. In addition, loss of p16 leads to unopposed activation of cyclin-d-dependent kinases CDK4/6 which enhances cyclin D1-induced resistance to BRAF inhibitors (65). Although activation of these bypass signaling pathways represents putative mechanisms of primary resistance to the BRAF and MEK inhibitors, there is some suggestion that exogenous growth-factor support from other cell types in the tumor microenvironment may be involved (62,63). Insulin like growth factor (IGF)-I signaling is highly active in metastatic melanoma cells (62). Somewhat surprisingly, IGF-1 is not expressed in melanoma cells although IGFR is closely associated with melanoma progression, suggesting that paracrine-derived signals may be responsible for activation of IGFR during advancement of melanocytic disease. The IGF-1 signaling axis originates from the IGF-1 receptor. After ligand binding, IGF-1R initiates a series of signaling events that positively affect downstream PI3K-AKT and MAPK pathways involving innate drug resistance to BRAF inhibitor. In this context, intrinsic resistance could be overcome by treating the melanoma cells with IGFR inhibitors plus AKT or MEK inhibitor. The latter view is evidenced by mechanistic studies that the combination of a PI3K and a MEK inhibitor or an IGFR and a MEK inhibitor reverse the cell resistance to vemurafenib therapy (70). More recently, hepatocyte growth factor (HGF) secreted by tumor stromal cells has been uncovered to activate MAPK and PI3K-AKT signaling pathways via binding the HGF receptor MET, which resulted in immediate resistance to RAF inhibitor treatment (63). Dual inhibition of RAF and either HGF or MET restore drug sensitivity, suggesting RAF in combination with HGF or Wang and Qi 753

7 IUBMB LIFE FIG 1 Mechanisms underlying drug resistance to BRAF inhibitors. Multiple resistance mechanisms of BRAFV600E melanomas to BRAF inhibitors have been identified: 1) RAS activation via RAS mutations or RTK/RTK ligand activation leads to binding between BRAF/CRAF and increased stimulation of CRAF, thereby promoting downstream MEK and ERK signaling; 2) BRAF, MEK, or ERK gene amplification/mutation, a BRAFV600E splice variant, and activation of another MEK kinase, COT, have all been shown to confer resistance; 3) RTK activation also allows for resistance by activating PI3K/AKT signaling as well as other pathways; 4) inactivation of PTEN and p16 render ERK signaling refractory to RAF inhibition via inhibiting Foxo3a-BIM and CDK 4/6 in the nucleus. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. Acquired resistance to BRAF inhibitors, when melanomas begin to grow again following an initial response to therapy, occurs inevitably in most of the patients treated. Unlike the resistance to ABL, EGFR, and KIT inhibitors reported in oncogene-directed therapies for other cancers, new gatekeeper mutations as a mechanism of acquired resistance to BRAF inhibitors have not yet been identified in melanoma patients failing this therapy (71). Instead, it has already been shown that the acquisition of BRAF inhibitor resistance was mainly associated with mutations in NRAS or the downstream kinase MEK1, BRAF amplification or splicing, overexpression of cancer osaka thyroid (COT), and activation of RTKs (64,72,73). BRAF V600E melanomas can flexibly switch among the three different RAF isoforms by a yet unidentified mechanism. Inhibition of BRAF kinase activity promotes altered scaffolding and activation of CRAF, which causes hyposensitivity of melanoma cells to BRAF inhibitor by phosphorylating MEK/ ERK signaling. This contention is supported by a genetic analysis of biopsies from a patient failing vemurafenib therapy, upon which the melanoma cells switched RAF isoform usage, using CRAF to activate the MAPK pathway (72). Intriguingly, knockdown of CRAF alone could not restore drug sensitivity. Therefore, targeting CRAF together with PI3K or MAPK which reveals a link to a common set of signaling pathways involving resistance mechanisms could delay the emergence of resistance to BRAF inhibitors (73,74). Likewise, a new activating mutation at codon 121 in MEK1 that was absent in the corresponding pretreatment tumor was identified in a melanoma 754 RAS/RAF/MEK/ERK Signaling in Metastatic Melanoma

8 patient who developed resistance to vemurafenib after an initial response (7). The MEK1 mutation was shown to increase kinase activity and mediate cross-resistance to both BRAF and MEK inhibitor in vitro. Thus, MEK1 or functionally similar mutations are predicted to confer resistance to combined MEK/RAF inhibition. Recently, a 61-kDa variant form of BRAF V600E (p61braf V600E) was identified in both cells resistant to vemurafenib and tumors from patients with acquired vemurafenib resistance (75). This splicing variant of BRAF V600E lacks the RASbinding domain and is expressed endogenously or ectopically, which creates constitutive ERK signaling through RAF dimerization, as compared to full length BRAF V600E. Knockdown of p61braf V600E (but not of full-length BRAF or CRAF) and a mutation to abolish the dimerization of p61braf V600E both restore the sensitivity to vemurafenib. BRAF amplification identified by whole exome sequencing has also been reported to be associated with vemurafenib resistance (73). This genomic copy number gain results in BRAF V600E overexpression and ERK reactivation, which is necessary and sufficient for acquired resistance to BRAF inhibitor. Intriguingly, the amplification-driven BRAF inhibitor resistance, in contrast to mutant NRAS-mediated resistance where BRAF V600E may be bypassed by the alternative use of CRAF, functions largely independently of CRAF. Most importantly, along with dosing escalation of vemurafenib, cells with amplified BRAF V600E revealed concentration-dependent sensitivity to this BRAF inhibitor, suggesting a potential therapeutic rationale for dose escalation studies in the selected melanoma patients. The upregulated expression of multiple RTKs is proved to be associated with acquired vemurafenib resistance. Of the RTKs investigated, constitutive activation of IGFR-1 and increased level of phosphorylated PDGF receptor (PDGFR)-b are most extensively identified that both activate PI3K and other compensatory pathways and attenuate the effects of the RAF inhibitor on ERK signaling by elevating RAS-GTP (62,76). The mechanism underlying the overexpression of PDGFRb was not clear but was not noted to be the result of an activating mutation or genomic amplification. Somewhat surprisingly, although knockdown of the receptor inhibited proliferation of resistant cells with elevated PDGFRb phosphorylation, these resistant cell lines were not sensitive to the PDGFR inhibitor imatinib (72). The identification of COT/MAP3K8 as a novel candidate for MAPK pathway activation in a RAF-independent manner promises current treatment of acquired resistance to BRAF and MEK inhibitor (77). Indeed, COT activated ERK through MEK-dependent and MEK-independent mechanisms. This is especially important because for the first time, it suggests the possibility that activation of alternative kinases other than RAF can phosphorylate MEK and maintain ERK signaling. It was also shown that genomic amplification of COT was associated with acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibition. Further studies demonstrated that both COT short hairpin RNA (shrna) knockdown and small molecule COT inhibitors could reduce the MAPK signaling and survival of COT amplified cell lines (77). Most importantly, this COT-driven drug resistance was overcome by treating the cells with a combination of a BRAF and MEK inhibitor or COT inhibitor. Mechanisms Underlying Resistance to MEK Inhibitors MEK inhibitors are still in early stages of clinical investigation and the limited clinical samples available from patients exposed to these drugs make it difficult to establish the mechanisms of acquired drug resistance. Nevertheless, several potential mechanisms of resistance to MEK inhibitors have been identified in the case of BRAF-mutant tumors. Similar to the resistance mechanisms to BRAF inhibitors, increased CRAF activity, activating NRAS mutations or point mutations in MEK1 which uniformly promotes MAPK reactivation is unraveled to confer resistance to the MEK inhibitor (56). MEK inhibitor resistance causes the impaired activation of FOXO3a and a subsequent reduction in BIM promoter activity in a few melanoma cell lines (78). In this instance, the dual inhibition with the combination of MEK and AKT inhibitor restore the nuclear localization of FOXO3a, upregulate BIM expression and enhance the levels of apoptosis. These findings have led to the development of strategies being used in the clinic to overcome the resistance. Intriguingly, it was shown that the MEKresistant cell lines retained their addiction to the MAPK pathway, as evidenced by their sensitivity to inhibitors that act downstream of the mutated MEK target, ERK1/2 kinase (79). Thus, targeting RAF and ERK by small-molecule inhibitors may act to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Future Perspective The recent years have seen incredible progress in the treatment of advanced melanoma with the success to identify distinct pathways and targets. The RAS/RAF/MEK/ERK signaling pathway, one of the most well-known MAPK pathways, is known to be vitally important for melanoma tumor growth and maintenance. Targeting the members of the pathway with small-molecule inhibitors, and BRAF inhibitors in particular, offers clinically relevant promise that durable responses could be achieved. However, only 50% of metastatic melanoma patients who have the V600 BRAF mutation demonstrate a satisfactory benefit and most responses are transient. Considering the context that two other mutually exclusive genetic subsets may occur in cutaneous melanomas, including tumors with mutated NRAS and wild type at both loci (wt/wt), new approaches to treatment of metastatic melanomas are still needed. One of the major challenges facing the melanoma field is how to develop strategies to overcome de novo and acquired drug resistance to small-molecule inhibitors. Tremendous strides in the mechanisms of resistance and comprehensive Wang and Qi 755

9 IUBMB LIFE understanding of the biology of MAPK signaling provide insight into rational combination regimens and sequences of molecularly targeted therapies. Although the resistance mechanisms identified so far are diverse, most appear to rely directly upon reactivation of MEK/ERK signaling and upon increased signaling output through the PI3K/AKT/mTOR pathway (8). Correspondingly, rationally designed drug combinations may be able to significantly extend the life span of patients with BRAF mutant melanoma. The most promising combination strategy under evaluation is the simultaneous inhibition of BRAF and MEK. Several groups have already shown preclinically that dual BRAF and MEK inhibition may prevent or delay the onset of resistance. Clinical trials are also currently underway to combine the BRAF inhibitors or MEK inhibitors with PI3K pathway inhibitors in melanomas. A phase II clinical trial is opening soon to assess the clinical efficacy of BRAF inhibitor LGX818 single agent and to further evaluate the efficacy and safety of the drug combinations of LGX818 with the MEK inhibitors MEK162, or PI3-kinase inhibitor BKM120 as well as other target drugs in patients with BRAF mutant melanoma (NCT ). Current standard evaluation and management arena highlight the importance of understanding the molecular underpinnings and cell signaling diversity of melanoma to assess the best strategy for the patients treatment. To achieve this will require continued efforts of basic scientists and clinicians and the ongoing support of the pharmaceutical companies. If progress continues as we expect, a future can be envisaged in which disseminated melanoma is no longer such a bleak prognosis and can instead be reduced to the level of a manageable, chronic disease. References [1] Mimeault, M. and Batra, S. K. (2012) Novel biomarkers and therapeutic targets for optimizing the therapeutic management of melanomas. World J. Clin. Oncol. 3, [2] Yee, V. S., Thompson, J. F., McKinnon, J. G., Scolyer, R. A., Li, L. X., et al. (2005) Outcome in 846 cutaneous melanoma patients from a single center after a negative sentinel node biopsy. Ann. Surg. Oncol. 12, [3] Berwick, M., Erdei, E., and Hay, J. (2009) Melanoma epidemiology and public health. Dermatol. Clin. 27, , viii. [4] Patel, P. M., Suciu, S., Mortier, L., Kruit, W. H., Robert, C., et al. (2011) Extended schedule, escalated dose temozolomide versus dacarbazine in stage IV melanoma: final results of a randomised phase III study (EORTC 18032). Eur. J. Cancer 47, [5] Eggermont, A. M. and Robert, C. (2011) New drugs in melanoma: it s a whole new world. Eur. J. Cancer 47, [6] Nikolaev, S. I., Rimoldi, D., Iseli, C., Valsesia, A., Robyr, D., et al. (2012) Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma. Nat. Genet. 44, [7] Wagle, N., Emery, C., Berger, M. F., Davis, M. J., Sawyer, A., et al. (2011) Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J. Clin. Oncol. 29, [8] Flaherty, K. T. (2012) Targeting metastatic melanoma. Annu. Rev. Med. 63, [9] McCubrey, J. A., Steelman, L. S., Chappell, W. H., Abrams, S. L., Wong, E. W., et al. (2007) Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim. Biophys. Acta 1773, [10] Davies, H., Bignell, G. R., Cox, C., Stephens, P., Edkins, S., et al. (2002) Mutations of the BRAF gene in human cancer. Nature 417, [11] Long, G. V., Menzies, A. M., Nagrial, A. M., Haydu, L. E., Hamilton, A. L., et al. (2011) Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J. Clin. Oncol. 29, [12] Smalley, K. S., Xiao, M., Villanueva, J., Nguyen, T. K., Flaherty, K. T., et al. (2009) CRAF inhibition induces apoptosis in melanoma cells with non- V600E BRAF mutations. Oncogene 28, [13] Dumaz, N. (2011) Mechanism of RAF isoform switching induced by oncogenic RAS in melanoma. Small GTPases 2, [14] Wellbrock, C., Ogilvie, L., Hedley, D., Karasarides, M., Martin, J., et al. (2004) V599EB-RAF is an oncogene in melanocytes. Cancer Res. 64, [15] Yang, H., Higgins, B., Kolinsky, K., Packman, K., Go, Z., et al. (2010) RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models. Cancer Res. 70, [16] Pritchard, C. A., Hayes, L., Wojnowski, L., Zimmer, A., Marais, R. M., et al. (2004) B-Raf acts via the ROCKII/LIMK/cofilin pathway to maintain actin stress fibers in fibroblasts. Mol. Cell Biol. 24, [17] Hoeflich, K. P., Gray, D. C., Eby, M. T., Tien, J. Y., Wong, L., et al. (2006) Oncogenic BRAF is required for tumor growth and maintenance in melanoma models. Cancer Res. 66, [18] Flaherty, K. T., Puzanov, I., Kim, K. B., Ribas, A., McArthur, G. A., et al. (2010) Inhibition of mutated, activated BRAF in metastatic melanoma. N. Engl. J. Med. 363, [19] Michaloglou, C., Vredeveld, L. C., Soengas, M. S., Denoyelle, C., Kuilman, T., et al. (2005) BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436, [20] Dankort, D., Curley, D. P., Cartlidge, R. A., Nelson, B., Karnezis, A. N., et al. (2009) Braf(V600E) cooperates with Pten loss to induce metastatic melanoma. Nat. Genet. 41, [21] Gopal, Y. N., Deng, W., Woodman, S. E., Komurov, K., Ram, P., et al. (2010) Basal and treatment-induced activation of AKT mediates resistance to cell death by AZD6244 (ARRY ) in Braf-mutant human cutaneous melanoma cells. Cancer Res. 70, [22] Jiang, C. C., Lai, F., Thorne, R. F., Yang, F., Liu, H., et al. (2011) MEK-independent survival of B-RAFV600E melanoma cells selected for resistance to apoptosis induced by the RAF inhibitor PLX4720. Clin. Cancer Res. 17, [23] Ellerhorst, J. A., Greene, V. R., Ekmekcioglu, S., Warneke, C. L., Johnson, M. M., et al. (2011) Clinical correlates of NRAS and BRAF mutations in primary human melanoma. Clin. Cancer Res. 17, [24] Ji, Z., Flaherty, K. T., and Tsao, H. (2010) Molecular therapeutic approaches to melanoma. Mol. Aspects Med. 31, [25] VanBrocklin, M. W., Robinson, J. P., Lastwika, K. J., Khoury, J. D., and Holmen, S. L. (2010) Targeted delivery of NRASQ61R and Cre-recombinase to post-natal melanocytes induces melanoma in Ink4a/Arflox/lox mice. Pigment Cell Melanoma Res. 23, [26] Byron, S. A., Loch, D. C., Wellens, C. L., Wortmann, A., Wu, J., et al. (2012) Sensitivity to the MEK inhibitor E6201 in melanoma cells is associated with mutant BRAF and wildtype PTEN status. Mol. Cancer 11, 75. [27] Qin, J., Xin, H., and Nickoloff, B. J. (2012) Specifically targeting ERK1 or ERK2 kills melanoma cells. J. Transl. Med. 10, 15. [28] Young, A., Lyons, J., Miller, A. L., Phan, V. T., Alarcon, I. R., et al. (2009) Ras signaling and therapies. Adv. Cancer Res. 102, [29] Balmanno, K. and Cook, S. J. (2009) Tumour cell survival signalling by the ERK1/2 pathway. Cell Death Differ. 16, [30] Arozarena, I., Sanchez-Laorden, B., Packer, L., Hidalgo-Carcedo, C., Hayward, R., et al. (2011) Oncogenic BRAF induces melanoma cell invasion by downregulating the cgmp-specific phosphodiesterase PDE5A. Cancer Cell 19, [31] Wilhelm, S. M., Carter, C., Tang, L., Wilkie, D., McNabola, A., et al. (2004) BAY exhibits broad spectrum oral antitumor activity and targets 756 RAS/RAF/MEK/ERK Signaling in Metastatic Melanoma