Mitogen-Activated Protein Kinase Pathway Mutations and Drug Resistance

Home , ASK1, MAP2K4, MAP2K6, MAP3K10, MAP3K12, MAP3K13

Published OnlineFirst February 13, 2013; DOI: 10.1158/1078-0432.CCR-12-0383

Clinical Cancer Molecular Pathways Research

Molecular Pathways: Mitogen-Activated Protein Kinase Pathway Mutations and Drug Resistance

Antonia L. Pritchard and Nicholas K. Hayward

Abstract Receptor tyrosine kinases are a diverse family of transmembrane proteins that can activate multiple pathways upon ligation of the receptor, one of which is the series of mitogen-activated protein kinase (MAPK) signaling cascades. The MAPK pathways play critical roles in a wide variety of cancer types, from hematologic malignancies to solid tumors. Aberrations include altered expression levels and activation states of pathway components, which can sometimes be attributable to mutations in individual members. The V600E mutation of BRAF was initially described in 2002 and has been found at particularly high frequency in melanoma and certain subtypes of colorectal cancer. In the relatively short time since this discovery, a family of drugs has been developed that speciﬁcally target this mutated BRAF isoform, which, after results from phase I/II and III clinical trials, was granted U.S. Food and Drug Administration approval in August 2011. Although these drugs produce clinically meaningful increases in progression-free and overall survival, due to acquired resistance they have not improved mortality rates. New drugs targeting other members of the MAPK pathways are in clinical trials or advanced stages of development. It is hoped that combination therapies of these new drugs in conjunction with BRAF inhibitors will counteract the mechanisms of resistance and provide cures. The clinical implementation of next-generation sequencing is leading to a greater understanding of the genetic architecture of tumors, along with acquired mechanisms of drug resistance, which will guide the development of tumor-speciﬁc inhibitors and combination therapies in the future. Clin Cancer Res; 19(9); 1–9. 2013 AACR.

Background via MAPKs, with particular attention to mutations that lead Cells respond to certain growth factors, cytokines, and to dysregulation of cancer cell proliferation and to recent hormones via activation of receptor tyrosine kinases (RTK), advances in pharmaceutical targeting of pathway members with activation of subsequent signaling cascades ultimately for cancer therapy. altering cellular processes and the expression of genes that The MAPK signaling cascade is highly conserved between encode proteins controlling cellular proliferation, through different eukaryotic cell types and is composed of 3 to 5 tiers regulation of the cell cycle, as well as cell death through of kinase families (MAP4K, MAP3K, MAP2K, MAPK, and apoptosis. The RTKs are transmembrane glycoproteins with MAPK-activated protein kinases (MAPKAPK; Fig. 1), with an intracellular tyrosine kinase domain that activate tiers of one or more of each tier phosphorylating and activating effector proteins via an initial phosphorylation event. There components of the next tier. The canonical MAPK signaling are 4 main signaling pathways induced by members of the pathway is shared by 4 cascades, which are classiﬁed by the RTK family, mediated by (i) mitogen-activated protein MAPK family member at the end of the phosphorylation kinase (MAPK), (ii) the lipid kinase phosphatidylinosi- cascade initiated by the RTK: (i) extracellular signal-related tol-3 kinase (PI3K), (iii) STAT, and (iv) phospholipase kinases (ERK) 1 and 2, (ii) c-JUN N-terminal kinase (JNK) 1, Cg. The main focus of this review is the pathway mediated 2, and 3, (iii) p38-MAPK, and (iv) ERK5 (Fig. 1). Although growth factors are considered to be the main regulators of the ERK1/2 cascade, cellular stress the main inducer of the JNK and p38–MAPK cascades, and either growth factors or Authors' Afﬁliation: Oncogenomics Research Group, CBCRC Building, Queensland Institute of Medical Research, Herston, Brisbane, Queensland, stress as activators of the ERK5 cascade, considerable evi- Australia dence exists for cross-talk between various components of Note: Supplementary data for this article are available at Clinical Cancer the pathways, meaning that ultimately the cascades can Research Online (http://clincancerres.aacrjournals.org/). cooperate to transmit certain signals. Corresponding Author: Nicholas K. Hayward, Oncogenomics Research Group, Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane, QLD 4006, Australia. Phone: 61-733-620-306; Fax: The ERK1/2 Signaling Cascade 011-61-738-453-508; E-mail: [email protected] Signaling via small G-proteins, for example the RAS doi: 10.1158/1078-0432.CCR-12-0383 family, activates the ERK1/2 signaling cascade. The small 2013 American Association for Cancer Research. G-proteins are recruited by RTKs via SH2/SH3 domain

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Pritchard and Hayward Extracellular

Growth factors, Stress, osmotic Inflammatory trophic factors shock, γ-radiation cytokines, FASL

PAK1 PAK4 RAS RAC1 PAK2 GLKHGK KHS HPK1 Intracellular MAP4K3 MAP4K4 MAP4K5 MAP4K1 MAP4K

B-RAF RAF1 MTK1 DLK MEKK1 MEKK2 MEKK3 ASK1 TAK1 PTK1

MAP3K4 MAP3K12 MAP3K8 MAP3K2 MAP3K3 MAP3K5 MAP3K7 MAP3K11 MAP3K

MEK1 MEK2 MKK7 MEK4 MEK5 MEK3 MEK6 MAP2K1 MAP2K2 MAP2K7 MAP2K4 MAP2K5 MAP2K3 MAP2K6 MAP2K

p38-β p38-γ ERK1 ERK2 JNK JNK2 JNK3 ERK5 MAPK11 MAPK12 MAPK3 MAPK1 MAPK8 MAPK9 MAPK10 MAPK7 p38-δ p38-α MAPK MAPK13 MAPK14

RSK1 RSK2 RSK3 PRAK MSK1 3PK Kinases MAPKAPK5 MAPKAPK3

c-JUN c-FOS MEF2 c-MYC SP1 STAT1 Targets of MAPK Targets

Transcription factors

Figure 1. The 4 canonical MAPK signaling pathways mediating the transmission of an external signal down through the cell to effector proteins that alter cellular proliferation and survival. Signaling pathways should not be considered as purely linear, as there is considerable cross-talk, both between the various MAPK cascades and also with the other RTK-stimulated signaling pathways. This cross-talk predominantly occurs between members of the upper tiers of the network, with the lower tiers providing specificity. For simplicity, this figure shows the main linear pathways only. Additional components of the MAPK super-family exist (e.g., MAP4K2, A-RAF, MAP3K6, MAP3K9, MAP3K10, MAP3K13, MAP3K14, MAPK4, MAPK6, MAPKAPK2, and serine/threonine (Ser/Thr) kinase effector proteins, such as MNK1, MNK2, MSK1, MSK2, RPS6KB1, and RPS6KB2), but as many have not been assigned to specific pathways they have not been included in this figure, to aid overall interpretation of the pathways. Similarly, because there is tissue-specific expression of some members of the PAK family, all of them have not been included in the figure. This figure was based on pathway information available from BioCarta (www.biocarta.com), in accordance with their terms and conditions and with their express permission to use the data in the creation of this diagram.

containing protein intermediates, resulting in active RAS thereafter act as dual speciﬁcity kinases, phosphorylating binding to MAP3K RAF family members (Fig. 1). Upon Tyr and Thr residues of the MAPK ERK1/2 proteins (Fig. 1). activation, RAF subsequently phosphorylates and thus acti- Importantly, thus far, the ERK1/2 proteins are the only vates MAP2K 1 and 2 (also known as MEK1/2), which identiﬁed targets of MAP2K1/2, indicating that there is no

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MAP Kinase Pathway: Mutations and Drug Resistance

redundancy in the activation of this MAPK tier despite Ser271 and Thr275, which in turn activate MAPK8 (JNK), numerous avenues of cross-talk higher up the cascade. MAPK9 (JNK2), and MAPK10 (JNK3). JNK and JNK2 are Active ERK1/2 phosphorylates a number of cytoplasmic ubiquitously expressed in humans, whereas JNK3 is pri- proteins, including members of the MAPKAPK family and marily found in the brain, heart, and testis. A wide variety of cytoskeletal proteins such as vimentin (1) and keratin-8 (2) cellular processes are activated by the JNK pathway, includ- and also translocates to the nucleus, where it activates ing apoptosis (12), signaling molecules (13), and transcrip- various transcription factors, including FOS (3), TP53 tion factors, such as JUN (14), JUND (15), and TP53 (16). (4), and ELK1 (5). Mutations in the JNK signaling pathway Mutations within members of the ERK1/2 signaling Inactivating mutations in MAP2K4 have led to its classi- cascade fication as a tumor suppressor gene (17); deletion and Oncogenic mutations of the RAS family of genes encode epigenetic silencing of this gene have also been reported amino acid alterations at 3 main residues—Gly12, Gly13, in breast (18), biliary (18), pancreatic (18), and prostate and Gln61—each of which prevents the hydrolysis of (19) cancers. Although the main targets of MAP2K4 are the bound guanosine triphosphate to guanosine diphosphate, JNK family, it also has a degree of cross-talk with members resulting in a constitutively active form of the protein. of the p38 branch of MAPK signaling (20). As JNK has been Mutations in members of the RAS family are present in linked to apoptosis, these inactivating events have been approximately 17% of human cancers [Catalogue of Somat- hypothesized to disrupt the signals intended to trigger cell ic Mutations in Cancer (COSMIC) database; Table 1]. death. In addition, RAS mutations may directly interact with Constitutive activation of RAS is likely to have knock-on JNK, bypassing the usual pathway through RAC1 effects on other branches of the MAPK pathway due to cross- (ref. 21; Fig. 1). Recently, an activating RAC1 mutation has talk between the upper tiers of the cascades. Activating been found at high frequency in melanoma (22, 23). mutations have also been described in BRAF in several – cancer types, including colorectal cancer (6) and non–small The p38 MAPK Signaling Cascade cell lung cancer (7). The mutation frequency is particularly The p38–MAPKs are strongly activated by environmental high in melanoma (approximately 45%, compared with stresses and inflammatory cytokines; however, growth fac- 21% of all cancer types; Table 1), with more than 90% of tors are able to weakly activate this pathway. p38–MAPKs these being mutated at the V600 amino acid residue (7, 8). have been shown to play an important role in cell-cycle The V600 amino acid alteration is not exclusively found in checkpoint control at the G0,G1–S, and G2–M transitions in cancers, with premalignant lesions, such as melanocytic a cell-specific manner. The p38–MAPKs require dual phos- nevi, also carrying this mutation (9); the implication is that phorylation of the Thr-Gly-Tyr motif in the activation loop BRAF mutations alone are not sufficient to drive malignan- by MAP2K3 (MEK3) or MAP2K6 (MEK6) to become fully cy. Mutations in RAF1 (also known as CRAF) are less enzymatically active. These members of the MAP2K family common (approximately 2%, COSMIC database; Table are highly specific for the p38–MAPK proteins; however, the 1); however, many studies have shown increased expression MAP3K family members that activate the MAP2K tier are of RAF1 in a wide variety of primary human cancers (10, tissue and stimuli specific (Fig. 1). In addition, a role for 11). The paucity of oncogenic mutations in RAF1 has been members of the Rho family and heterotrimeric G-protein– hypothesized because of 2 sites of phosphorylation being coupled receptors has also been implicated (24, 25). There required for activation; thus, the protein would require 2 are 4 members of the p38–MAPK family: MAPK11 (p38-a), separate mutational hits to constitutively turn on the kinase. MAPK12 (p38-b), MAPK13 (p38-d), and MAPK14 (p38-g), It is uncommon for tumors to contain both RAS and RAF which display tissue-specific patterns of expression (26, 27). mutations, suggesting that activation of just one member of The 4 subunits have additionally been shown to differen- the cascade is all that is required for oncogenesis. tially activate certain target substrates, for example, the microtubule-associated protein tau (28), suggesting special- The JNK1/2/3 Signaling Cascade ized functions for each isoform. Downstream targets of The JNK branch of the MAPK pathway is primarily acti- members of the p38–MAPK pathway include the transcrip- vated in response to stress stimuli, such as UV radiation, tion factors TP53 (29), STAT1, MEF (30), and NF-kB (31), DNA damage, and inflammatory cytokines; however, it can cytoskeletal proteins, and other kinases (Fig. 1). also be weakly activated by growth factors and cytokines. These stimuli either directly activate the G-protein RAC1, or Mutations in the p38–MAPK signaling pathway alternatively activate the RAS family, which in turn also p38–MAPK cannot be classified as either a tumor sup- recruits and phosphorylates RAC1, which then acts via pressor or an oncogene, as it has roles in preventing cellular the p21-protein activated kinase (PAK) family to phosphor- proliferation in response to cellular stress causing DNA ylate members of the MAP3K family. At least 14 members damage (32, 33), as well as increasing angiogenesis in of the MAP3K family have been linked to activation of the response to a hypoxic environment caused by tumor mass JNK signaling cascade, for example, MAP3K4 and MAP3K12 (34). Mutations in the p38–MAPK signaling pathway are (Fig. 1). The MAP3K tier dual-phosphorylates MAP2K4 relatively rare in comparison with the other branches of the (MKK4) at Ser257 and Thr265, or MAP2K7 (MKK7) at MAPK signaling network (Table 1). Nevertheless, studies

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Table 1. Mutation frequencies of key molecules within the 4 MAPK canonical pathways reported in the COSMICa or compiled from recently published exome/whole-genome next-generation sequencing datab

Main MAPK Number mutations Mutations in melanomac Protein (alternate name) pathway (total samples analyzed; %) (total samples analyzed; %) G-protein/target KRAS, NRAS, and HRAS ERK-1/-2 29,337 (174,571; 16.8%)a 1,014 (7,403; 13.7%)a RAC1 JNK 8 (658; 1.2%)a 12 (247; 4.9%)b PAK1 JNK 29 (970; 2.9%)a 2 (247; 0.8%)b PAK2 JNK 12 (958; 1.3%)a 1 (247; 0.4%)b PAK4 JNK 13 (958; 1.4%)a 5 (247; 2.0%)b MAP4K tier MAP4K3 (MTK1) ERK5 44 (579; 7.6%)a 8 (247; 3.2%)b MAP4K4 (HGK) ERK5 42 (578; 7.3%)a 8 (247; 3.2%)b MAP4K1 (HPK) p38 44 (1,003; 4.4%)a 6 (247; 2.4%)b MAP4K5 (KHS) p38 15 (553; 2.7%)a 2 (247; 0.8%)b MAP3K tier B-RAF ERK-1/-2 27,391 (131,564; 20.8%)a 5,084 (11,273; 45%)a RAF-1 (C-RAF) ERK-1/-2 32 (1,510; 2.1%)a 3 (247; 1.2%)b MAP3K4 (MTK1) JNK 77 (600; 12.8%)a 1 (247; 0.4%)b MAP3K12 (DLK) JNK 37 (620; 5.9%)a 7 (247; 2.8%)b MAP3K2 (MEKK2) ERK5 7 (1,394; 0.5%)a 1 (247; 0.4%)b MAP3K3 (MEKK3) ERK5 28 (993; 2.8%)a 2 (247; 0.8%)b MAP3K8 (MEKK1) ERK5 23 (991; 2.3%)a 4 (247; 1.6%)b MAP3K5 (ASK1) p38 58 (1,030; 5.6%)a 27 (247; 10.9%)b MAP3K7 (TAK1) p38 28 (993; 2.8%)a 1 (247; 0.4%)b MAP3K11 (PTK1) p38 24 (990; 2.4%)a 9 (247; 3.6%)b MAP2K tier MAP2K1 (MEK1) ERK-1/-2 52 (1,468; 3.5%)a 7 (247; 2.8%)b MAP2K2 (MEK2) ERK-1/-2 13 (516; 2.5%)a 5 (247; 2.0%)b MAP2K7 (MKK7) JNK 19 (1,449; 1.3%)a 0 (247; 0%)b MAP2K4 (MEK4) JNK 100 (3,658; 2.7%)a 4 (247; 1.6%)b MAP2K5 (MEK5) ERK5 16 (555; 2.9%)a 5 (247; 2.0%)b MAP2K3 (MEK3) p38 0 (781; 0%)a 11 (247; 4.5%)b MAP2K6 (MEK6) p38 20 (1,420; 1.4%)a 5 (247; 2.0%)b MAPK tier MAPK3 (ERK1) ERK-1/-2 10 (1,417; 0.7%)a 0 (247; 0%)b MAPK1 (ERK2) ERK-1/-2 17 (1,413; 1.2%)a 4 (247; 1.6%)b MAPK8 (JNK) JNK 34 (1,608; 2.1%)a 5 (247; 2.0%)b MAPK9 (JNK2) JNK 26 (1,414; 1.8%)a 0 (247; 0%)b MAPK10 (JNK3) JNK 3 (1,393; 0.21%)a 6 (247; 2.4%)b MAPK7 (ERK5) ERK5 30 (1,414; 2.1%)a 4 (247; 1.6%)b MAPK11 (p38-b) p38 6 (1,398; 0.4%)a 1 (247; 0.4%)b MAPK12 (p38-g) p38 8 (1,404; 0.6%)a 0 (247; 05)b MAPK13 (p38-d) p38 12 (1,609; 0.7%)a 2 (247; 0.8%)b MAPK14 (p38-a) p38 16 (1,446; 1.1%)a 5 (247; 2.0%)b MAPKAPK tier RSK1 (RPS6KA1) ERK-1/-2 20 (978; 2%)a 4 (247; 1.6%)b RSK2 (RPS6KA3) ERK-1/-2 41 (1,515; 2.7%)a 0 (247; 0%)b RSK3 (RPS6KA2) ERK-1/-2 45 (1,618; 2.8%)a 8 (247; 3.2%)b MSK1 (RPS6KA5) p38 48 (1,445; 3.3%)a 5 (247; 2.0%)b MAPKAPK5 (PRAK) p38 13 (1,214; 1.1%)a 1 (247; 0.4%)b MAPKAPK3 (3PK) p38 2 (1,601; 0.1%)a 2 (247; 0.8%)b

aCOSMIC (94, 95). bCompiled from refs. 22, 23, 96–100. cMalignant melanoma: includes all subtissues and all subhistology types, including areas of low UV exposure, mucosal tissues, and those with high UV exposure.

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have shown p38–MAPK dysregulation in hematologic wealth of data (64, 65). Paradoxically, the BRAFi are capable malignancies (35, 36) and breast (37), prostate (38), gastric of accelerating growth in tumors (wild type) at the V600E (39), and lung (40) cancers. mutation location in BRAF (66), which emphasizes the importance of somatic mutation screening for identifica- The ERK5 Signaling Cascade tion of patients most suitable for receiving these therapies. The ERK5 signal cascade is stimulated by a variety of extracellular stimuli, such as growth factors (41, 42), Results from clinical trials on MAPK pathway inflammatory cytokines (43), and types of cellular stress, inhibition such as hypoxia (44) and laminar shear stress (45). These In phase I/II trials of the BRAFi, vemurafenib has shown signals lead to a variety of cellular responses, including good response rates in the majority of patients with mel- angiogenesis, antiapoptosis, cell differentiation, and cell anoma (67, 68). Moreover, in a phase III trial, vemurafenib proliferation (refs. 46–48; Fig. 1). These external stimuli increased progression-free and overall survival, although activate the MAP3K tier, including MAP3K2 and MAP3K3, not mortality rate, in patients with BRAF V600E mutation– which specifically phosphorylate the Ser311/Thr315 resi- positive melanomas compared with those treated with dues of MAP2K5 (MEK5; ref. 49); MAP2K5 is the only dacarbazine (62). Initial trials excluded patients with brain known protein able to activate ERK5 (Fig. 1). To fully metastasis; however, a recently published phase II trial of activate ERK5, MAP2K5 must dual-phosphorylate the dabrafenib revealed that approximately 30% of patients Thr218 and Tyr220 residues of ERK5, which then results achieved an overall cranial response in brain metastasis of in further phosphorylation of MAP2K5 as well as autopho- primary melanoma (69). Stage II trials of vemurafenib for sphorylation of ERK5, resulting in maximal activity (50, patients with brain metastases are currently recruiting (Clin- 51). ical trial identifier; NCT01378975). Further encouraging results of targeting the MAPK path- Mutations in the ERK5 signaling cascade way in melanoma come from a phase III trial of the MEKi Oncogenically mutated RAS has been reported to activate trametinib, which showed significantly improved progres- the ERK5 pathway in certain cell types (52); however, this sion-free and overall survival compared with patients trea- finding has not been confirmed by other studies (53). The ted with either dacarbazine or paclitaxel (70). In subsequent wild-type forms of RAS do not seem to stimulate this phase I and II trials, combined therapy of trametinib with a cascade (54). Mutations in the ERK5 pathway mainly occur BRAFi (dabrafenib) showed significantly better response at the MAP4K level, with only a low proportion of muta- rates and progression-free survival than either monotherapy tions reported in other tiers of the cascade (Table 1). (63). Nonetheless, ERK5 is overexpressed in several endocrine- In addition, targeted agents against activated KIT, an related cancer types (55, 56), suggesting a role for this infrequently mutated RTK in melanoma, have also shown pathway during the malignant process. Evidence suggests promise in some patients (71–73), although results from this is mediated, at least in part, via dysregulated miR-143 in large phase III randomized clinical trials of KIT inhibitors prostate cancer cells (57) and via an overexpression of are keenly awaited (e.g., NCT01280565). STAT3 in breast cancer cells (58). Whether there is a link Despite the initial optimism following the introduction between these 2 observations is yet to be elucidated. of molecularly targeted therapies in the clinic, considerable reassessment has been needed, as the vast majority of – Clinical Translational Advances patients developed resistance to the drugs. Fortunately, in Given the important roles played by the MAPK pathway the case of BRAFi, therapeutic resistance was identified early in cancer development and progression, intense focus has and the research field moved quickly to identify potential been directed toward therapeutically targeting these dysre- causes. gulated signals. Although the RAS protein is mutated in a high proportion of cancers and has an impact on a larger Mechanisms of Resistance to BRAF Inhibitors number of signaling cascades, a successful strategy to phar- In vivo macologically target it is yet to be discovered (59). In Investigations comparing tumors from patients with contrast, other members of the MAPK network downstream melanoma pre- and postdevelopment of BRAFi resistance of RAS have proved highly tractable to pharmacologic have uncovered a multitude of mechanisms leading to inhibition. Successfully developed therapies have included regrowth/recurrence of metastatic melanoma in individuals small-molecule nonATP competitive allosteric MEK inhibi- treated with such targeted therapies. These include tors (MEKi), for example, BAY 43-9006 (sorafenib; ref. 60) increased expression and signaling through various RTKs, and trametinib (61) and inhibitors of RAF family members, such as platelet-derived growth factor receptor-b (74, 75) or including vemurafenib and dabrafenib (62, 63). The BRAF insulin-like growth factor I receptor (76), upregulation of inhibitors (BRAFi) represent a particularly novel and inno- the kinase MAP3K8/COT (77), amplification of the BRAF vative direction for emerging cancer therapies, as they gene resulting in higher levels of expression (78), alternative specifically target the V600E/K-mutated isoform of the splicing of BRAF leading to dimerization (79), NRAS muta- protein. These BRAFi have been subject to intense research tion (74, 79, 80), and mutation of MEK (81), although there interest, and recent reviews succinctly summarize this is evidence that the latter does not always lead to

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development of BRAFi resistance (75). Interestingly, treat- mutation–positive melanomas. However, this is also likely ment with vemurafenib did not result in meaningful clinical to be only the backbone of new combination treatments, outcomes when in the 8% to 10% of colon cancers that with additional agents targeting components of other path- carry BRAF V600E mutations. The cause of this was iden- ways (e.g., PI3K, PAK, RAC1, or CDK4), or the immune tified as being feedback reactivation of the MAPK pathway system (see below), being included. Interestingly, cell lines via the RTK EGF receptor (EGFR; ref. 82), implying that with NRAS or KRAS mutations have shown more sensitivity these tumors have innate, rather than acquired resistance to to IPA3, a small-molecule inhibitor of PAK1, and subcyto- BRAFi. This resistance mechanism does not seem to be toxic doses of IPA3 make cells more sensitive to MEKi (88), operative in melanoma, which has only very low levels of thus offering evidence that combined therapies might have EGFR expression (82). Importantly, each of the resistance synergistic effects and providing possible novel mechan- pathways in melanoma has the same endpoint—reactiva- isms by which existing drugs may still yield clinically tion of the ERK1/2 branch of the MAPK cascade. Hence, meaningful outcomes. high levels of MAPK activity seem central to, or essential for, It is highly likely that other mechanisms of resistance melanoma growth in vivo. exist than those discussed above. Future studies will undoubtedly also investigate whether epigenetic changes, In vitro which can control activation or silencing of gene expres- A number of studies have addressed mechanisms of sion, occur in cells after BRAFi exposure. Given the recent BRAFi resistance in vitro by generating subclones of mela- data from Yancovitz and colleagues showing that clon- noma cell lines that develop resistance to RAF inhibitors ality of the BRAF V600E mutation occurs both intra- and through prolonged culture and selection with these agents. intertumorally from the same patient (89), it is also Like the diverse mechanisms of resistance that develop in highly likely that clonal selection will play a role in patients with melanoma trials in vivo, these also have the resistance to BRAFi. common theme of reactivating MAPK. They include upre- The advent of the whole-genome sequencing era of gulation of RAF1 (83) or upstream RTKs such as fibroblast genetic research has resulted in high-volume screening of growth factor receptor 3 (84), as well as mutation of NRAS melanoma cell lines and tumors for somatic mutations, (85) or KRAS (86). which has revealed that it is highly unlikely that driver mutations of a similar frequency to those described in BRAF Stromal mediated or RAS family members (Table 1) have yet to be uncovered Whereas all of the mechanisms of BRAFi resistance in other members of the MAPK pathway. It remains to be mentioned above are cell autonomous, Straussman and investigated whether the lower-frequency mutations could colleagues showed an alternative paracrine resistance have collective effects, due to the considerable degree of mechanism (87). They showed that fibroblast secretion cross-talk between branches of the MAPK pathways. Further of hepatocyte growth factor (HGF) activates the RTK MET investigations are clearly required to elucidate the effects of on adjacent melanoma cells, leading to reactivation of combinations of less common mutations, which in turn MAPK and resistance to the RAF inhibitor PLX4720. could point to new avenues for pharmaceutical interven- Recombinant HGF was sufficient to induce BRAFi resis- tion. It also remains to be determined whether endpoints tance in melanoma cell lines and either HGF neutralizing other than the dysregulation of MAPK play a vital role in antibodies or a MET inhibitor blocked PLX4720 resis- melanoma tumorigenesis; for example, GNAQ and GNA11 tance (87). In vivo, this association was supported by a are frequently mutated in ocular melanoma and feed into correlation with the level of stromal cell HGF expression other pathways in addition to the MAPK cascade. If other in tumors of BRAF mutation–positive melanoma patients tumorigenic mechanisms do exist, they might also be a who developed resistance to BRAFi (87). An important source of resistance to BRAFi. implication of this finding is that dual inhibition of An alternative to targeting components of the MAPK BRAF and HGF or MET could prevent the development (and/or other) signaling pathway in cancer treatment is of BRAFi resistance in some patients. Thus, clinical trials the use of a new wave of immune modulators, such as assessing the efficacy of these combined agents seem antibodies to CTLA4 (90), PD1 (91), or PD1L (91), which þ warranted. inhibit negative regulation of cytotoxic CD8 T cells mediated by these ligands/receptors. For example, in a Future Developments: Peering Over the Horizon phase III trial, an anti-CTLA4 antibody (ipilimumab) The early promise but subsequent failure of single-agent increased overall survival in patients with melanoma targeted therapies to significantly improve mortality rates in compared with those treated with the glycoprotetin- melanoma has led to a somewhat obvious paradigm shift in 100 (gp100) melanoma antigen immunostimulatory treatment approaches. Combination therapies are now peptide vaccine; combined treatment with ipilimumab becoming de rigueur. Given the strong (and potentially and gp100 did not result in improved survival compared necessary) involvement of MAPK signaling in melanoma with ipilimumab alone (90). In another trial, patients growth and the development of resistance to BRAFi, it is with melanoma treated with a combination of ipilimu- likely that dual use of a BRAFi with an inhibitor of MEK or mab and dacarbazine showed significantly improved MAPK will become standard for treating patients with BRAF overall survival compared with dacarbazine alone (92).

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MAP Kinase Pathway: Mutations and Drug Resistance

Results from an ongoing combination trial of BRAFi plus result in clinically meaningful increases in progression- ipilimumab (NCT01400451) are eagerly awaited. free and overall survival. The speed at which the labora- tory findings were translated into a meaningful clinical Conclusions outcome bodes well for future driver mutations identified In summary, the MAPK pathways activated by RTK liga- inthewakeoftherecenttechnologic advances allowing tion have been proved to play an important role in tumor- rapid and ever more consistent whole-genome sequenc- igenesis, as they control cell cycle and proliferation as well as ing. It is our belief that the first vital steps have been taken apoptosis, which, when dysregulated, drives abnormal cel- toward future treatment regimens in which personalized lular responses. In 2002, the V600E mutation in BRAF was medicine based on tumor mutation status in combina- discovered at high frequency in melanoma (7) and to a tion with standard chemo- and/or immune therapies will lower degree in other cancer types (Table 1). In the become a regular therapeutic course. intervening years, novel therapies were developed that targeted this mutated isoform of BRAF, as rapidly as 2 Disclosure of Potential Conflicts of Interest years following the initial report of V600E mutations No potential conflicts of interest were disclosed. (93). These therapies have since been refined, resulting in U.S. Food and Drug Administration approval for Authors' Contributions vemurafenib being granted in August 2011, with BRAFi Writing, review, and/or revision of the manuscript: A.L. Pritchard, N.K. Hayward being clinically available as a therapeutic option for metastatic melanoma positive for V600E mutations. Received December 20, 2012; accepted January 11, 2013; published Although BRAFi do not improve mortality rates, they do OnlineFirst February 13, 2013.

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Molecular Pathways: Mitogen-Activated Protein Kinase Pathway Mutations and Drug Resistance

Antonia L. Pritchard and Nicholas K. Hayward

Clin Cancer Res Published OnlineFirst February 13, 2013.

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