MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road

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1 MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road Christopher J. Caunt 1, Matthew J. Sale 2, Paul D. Smith 3 and Simon J. Cook 2 Abstract The role of the ERK signalling pathway in cancer is thought to be most prominent in tumours in which mutations in the receptor tyrosine kinases RAS, BRAF, CRAF, MEK1 or MEK2 drive growth factor-independent ERK1 and ERK2 activation and thence inappropriate cell proliferation and survival. New drugs that inhibit RAF or MEK1 and MEK2 have recently been approved or are currently undergoing late-stage clinical evaluation. In this Review, we consider the ERK pathway, focusing particularly on the role of MEK1 and MEK2, the gatekeepers of activity. We discuss their validation as drug targets, the merits of targeting MEK1 and MEK2 versus BRAF and the mechanisms of action of different inhibitors of MEK1 and MEK2. We also consider how some of the systems-level properties (intrapathway regulatory loops and wider signalling network connections) of the ERK pathway present a challenge for the success of MEK1 and MEK2 inhibitors, discuss mechanisms of resistance to these inhibitors, and review their clinical progress. 2i A cocktail of two protein kinase inhibitors, one inhibiting MEK1 and MEK2, and the other inhibiting glycogen synthase kinase 3 (GSK3). 1 Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK. 2 Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK. 3 AstraZeneca, Oncology imed, Cancer Biosciences, Cancer Research UK, Li Ka Shing Centre, Cambridge Institute, Robinson Way, Cambridge CB2 0RE, UK. Correspondence to P.D.S. and S.J.C. s: paul.d.smith@ astrazeneca.com; simon.cook@babraham.ac.uk doi: /nrc4000 The authors dedicate this article to Prof. Chris Marshall, FRS ( ), an inspirational scientist whose work contributed enormously to our understanding of the RAS-regulated RAF MEK ERK pathway. The ERK signalling pathway is activated by an array of receptor types, including receptor tyrosine kinases (RTKs), G protein-coupled receptors and cytokine receptors, and the core components of this pathway are now well known 1,2. Activated RTKs recruit adaptor proteins and guanine nucleotide exchange factors (GEFs; such as SOS) to activate the HRAS, KRAS or NRAS GTPases at the inner leaflet of the plasma membrane (FIG. 1). Once activated, GTP-bound RAS (RAS GTP) drives the formation of high-activity homodimers or heterodimers of the RAF protein kinases (ARAF, BRAF or CRAF), which directly phosphorylate and activate MEK1 and MEK2 (also known as MAPKK1 and MAPKK2). MEK1 and MEK2 are dual-specificity kinases that activate ERK1 and ERK2 by phosphorylating them at conserved threonine and tyrosine residues in the T E Y motif found in their activation loop. Hundreds of proteins have been defined as ERK1 and ERK2 substrates and ERK-interacting partners 1,3 ; these include other protein kinases and transcription factors (such as ETS and the activator protein 1 complex (AP1)), which regulate the expression of immediate- and delayed-early genes such as the D type cyclins to promote G1/S progression in the cell cycle 4. ERK1 and ERK2 can also regulate cell survival by phosphorylating members of the apoptosis regulating BCL 2 protein family at the mitochondria 5. signalling regulates processes that are crucial for normal development, including cell proliferation, differentiation, survival and cell motility; indeed, germline deletion of some components of the ERK pathway causes embryonic lethality 6, and a inhibitor (MEKi) forms part of the 2i protocol that maintains embryonic stem cell pluripotency 7. The same cellular processes are deregulated in cancer and represent some of the key hallmarks and driving characteristics of the cancer cell 8,9. Many human cancers contain activating mutations in genes encoding RTKs, RAS, BRAF, CRAF, MEK1 or MEK2, which act as driving oncogenes; consequently, many cancers exhibit deregulated activation of, and an enhanced dependency on, signalling. The discovery of the core components of the RAS ERK pathway 2 kick-started a protein kinase drug discovery effort that continues today The first ERK pathway inhibitor to be discovered, PD98059, was reported 20 years ago 13 and was shown to act independently of ATP as an apparent allosteric inhibitor of MEK1 and MEK2. Since then, MEKis have proved to be NATURE REVIEWS CANCER VOLUME 15 OCTOBER

2 Allosteric inhibitor A small molecule that inhibits the activity of an enzyme by binding to a regulatory site that is distinct from the active or catalytic site. invaluable research tools, underpinning our knowledge of ERK1 and ERK2 biology and validating MEK1 and MEK2 as cancer drug targets 14,15. As they are not ATPcompetitive, it was correctly anticipated that MEKis would be more selective than conventional kinase inhibitors and widely hoped that this would translate into rapid clinical success. In fact, it took a further 18 years until trametinib 16 became the first MEKi to receive regulatory approval. In the interim, the identification of activating BRAF mutations (most notably BRAF T1799A encoding BRAF V600E) in melanoma, thyroid and colorectal cancer (CRC) in 2002 (REF. 17) galvanized the field and focused efforts on developing BRAF inhibitors (BRAFis) that proved to be BRAF V600E selective, culminating in 2008 in the description and subsequent approval of vemurafenib 18,19 and dabrafenib 20,21. In this Review, we consider the fundamental aspects of the ERK pathway from a MEK perspective, and the evidence that supports MEK1 and MEK2 as drug targets in cancer. We describe the MEKis, their unique modes of action and innate resistance mechanisms, and how tumour cells that are sensitive to MEKis can adapt and acquire resistance. We also discuss how knowledge of MEKi resistance has informed combination strategies and review the clinical experiences of MEKis, including successes and lessons learned. a b c EGFR or FGFR SOS RAS GTP RAF RAF SOS RAS GTP RAF RAF P SOS P P RAF RAS GTP RAF SPRY KSR1 P KSR1 P Cytoplasm DUSP6 Nucleus DUSP5 ETS ETS ETS Figure 1 Scaffolds and feedback controls underpin the normal functioning of the pathway. a A simplified linear representation of the RAS-regulated RAF MEK ERK signalling cascade. Activated growth factor receptors (for example, epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR)) recruit the guanine nucleotide exchange factor (GEF) SOS to promote the release of GDP from RAS, allowing GTP to bind. Active RAS (RAS GTP) drives the formation of active homodimers or heterodimers of the RAF protein kinases (ARAF, BRAF or CRAF), which directly phosphorylate and activate MEK1 and MEK2 (). are dual-specificity protein kinases that phosphorylate ERK1 and ERK2 () on a conserved TEY motif to activate them. substrates include transcription factors of the ETS family; in this way the pathway links activated receptors at the plasma membrane to changes in gene expression in the nucleus. The concentration of the core components typically increases down the pathway such that [RAF] < [MEK] < [ERK]. This signal amplification allows low-level receptor occupancy to elicit meaningful ERK signals throughout the cell b Activation of the ERK pathway is critically dependent on scaffold proteins. Scaffolds serve several roles, including increasing the efficiency of interactions between the enzyme and substrate at each step and insulating pathway components against inputs from other parallel pathways to ensure signal fidelity 22,169,170. For example, the kinase suppressor of RAS 1 (KSR1) scaffold assembles RAF, and to increase signalling efficiency; acts allosterically to activate the RAF kinase domain 171 ; controls the subcellular location of the pathway; and insulates it from other pathways. Scaffolds tend to make signal transmission more efficient but limit amplification. c The ERK pathway is extensively regulated by homeostatic negative feedback controls that fine-tune pathway output 166. Rapid and direct feedback mechanisms involve the phosphorylation of MEK1, CRAF, BRAF, KSR1, SOS and some receptor tyrosine kinases (RTKs) by ERK and downstream kinases (such as RSK) to inhibit signal propagation 23,101. Notably, ERK can phosphorylate CRAF and BRAF to inhibit MEK phosphorylation , and MEK1 to inhibit ERK phosphorylation 41,42. Loss of ERK activity (for example, by treatment with a MEK inhibitor (MEKi)) collapses these feedback loops and reactivates MEK and ERK; this confers robustness, allowing the pathway to adapt to perturbations 166,175 and explains the ERK reactivation that is observed in tumour cells with wild-type BRAF 75. The slower de novo expression of Sprouty (SPRY) proteins and the dual-specificity phosphatases (DUSPs) also regulates pathway output. SPRY proteins inhibit ERK signalling at the level of RTKs, SOS and by interfering with the RAF catalytic domain 176. The DUSPs inactivate ERK by dephosphorylating the pt E py motif. ERK-driven expression of DUSPs provides homologous intrapathway feedback to dampen pathway activation. Additionally, different DUSPs function in different locations, with DUSP5 residing in the nucleus and DUSP6 in the cytoplasm, allowing differential regulation of ERK output in these different locations OCTOBER 2015 VOLUME 15

3 The role of MEK in the ERK pathway The ERK pathway is frequently represented as a linear RAS RAF MEK ERK signalling cascade (FIG. 1a) but this ignores the non-enzymatic components of the pathway and the layers of feed-forward and feedback regulation that are vital for the role of the ERK pathway in information processing. Understanding how the pathway is activated by different input stimuli to elicit different responses (such as proliferation or differentiation) and how the pathway responds and adapts to selective inhibition (for example, with MEKis) requires some appreciation of these systemslevel features. First, activation of the ERK pathway depends on scaffold proteins such as kinase suppressor of RAS 1 (KSR1), which increase the efficiency of the inter actions between the enzyme and substrate at each step 22 (FIG. 1b). Second, signalling is critically regulated by homeostatic feedback controls 23 that include the direct phosphorylation of upstream components and increased expression of Sprouty (SPRY) proteins and dual-specificity phosphatases (DUSPs); the DUSPs inactivate ERK1 and ERK2 by dephosphorylating the pt E py motif 24 (FIG. 1c). The importance of DUSP feedback in restraining the oncogenic potential of MEK ERK signalling is exemplified by the frequent loss of the ERK-specific cytoplasmic phosphatase DUSP6 in epidermal growth factor receptor (EGFR)- and KRAS-driven non-small-cell lung cancers (NSCLCs) 25 and the demonstration that loss of DUSP5 (the nuclear counterpart of DUSP6) in mouse models accelerates HRAS-driven skin cancer 26. These negative feedback loops have additionally emerged as key determinants of rapid pathway adaptation and long-term acquired resistance to MEKis 27,28. When taking into account scaffolding proteins and feedback loops, the canonical ERK pathway diagram looks more complex (FIG. 1c) but even this complexity fails to consider the position of the ERK pathway within wider signalling networks (FIG. 2). MEK1 and MEK2 are the only activators of ERK1 and ERK2 and serve an entirely unique role as critical ERK1 and ERK2 gatekeeper kinases, processing inputs from multiple upstream kinases. Indeed, a RASor RAF-centred view ignores the fact that RAF proteins are only a subset of the MEK1 and MEK2 activators in cells (FIG. 2). Multiple MAP kinase kinase kinases (MAP3Ks) can activate MEK1 and MEK2, and some of SOS RAS GTP Integrins RAC ARAF BRAF CRAF MEKK1 MEKK3 MEKK2 MAP3K8 MLK2 MLK4 MLK1 MLK3 PAK P MEK5 MKK3 or MKK6 MKK4 or MKK7 IKK BIM MCL1 RSK MNK ERK5 p38 JNK1/2 IκB Cytoplasm FOS ETS MYC MSK MEF2 MEF2 JUN NF-κB Nucleus CCND1 CHOP Figure 2 MEK1 and MEK2 are the key gatekeepers for ERK1 and ERK2 in a wider signalling network. The canonical pathway for ERK activation (RAS RAF MEK ERK) is shown on the left. ERK1 and ERK2 () substrates include transcription factors (FOS, ETS and MYC) and other protein kinases (RSK, MNK and MSK), thereby controlling the transcription and translation of genes that promote cell cycle progression such as CCND1 (which encodes cyclin D1); other ERK substrates include regulators of apoptosis (BIM and MCL1). One key network feature is the convergence of signalling at the level of MEK1 and MEK2 (). Although ARAF, BRAF and CRAF are the best-studied MEK activators, a number of other MAP kinase kinase kinases (MAP3Ks; show on the right) can also fulfil this role, including MEKK1 (also known as MAP3K1), MEKK3 (also known as MAP3K3), MAP3K8 (also known as COT) and the mixed-lineage kinases (MLK1 4; also known as MAP3K9, MAP3K10, MAP3K11 and KIAA1804) These alternative activators can also promote the activation of ERK5, p38, JUN N-terminal kinase (JNK) and nuclear factor-κb (NF κb) (through IκB kinase or IKK) via their relevant upstream activating kinases (MEK5, MKK3 or MKK6, MKK4 or MKK7 or IκB kinase, respectively) to regulate transcription factors such as MEF2, CHOP, JUN or NF-κB This reflects a key feature of ERK pathway architecture: receptor tyrosine kinases (RTKs) 177, RAS GTPases 2 and MAP3Ks are relatively promiscuous, activating two or more parallel signalling cascades; even RAF proteins may regulate non-mek targets through scaffold functions 178,179. By contrast, are exquisitely specific activators of. Because have hundreds of binding partners and substrates throughout the cell 1,3, this represents an astonishing achievement in signal processing through and underscores their role as key gatekeepers of ERK signalling. NATURE REVIEWS CANCER VOLUME 15 OCTOBER

4 MEK1 MEK2 CFC and cancer mutations Lys97 Receptors and RAS RAF and MAP3Ks Integrins Ser218 Ser222 Thr292 Ser298 1 DD NES NRR Kinase catalytic domain AL PRD DVD 393 Lys101 CFC and cancer mutations Ser222 P Ser226 P 1 DD NES NRR Kinase catalytic domain AL PRD DVD 400 Figure 3 Linear representation of the key functional domains of the human MEK1 and MEK2 proteins. Human MEK1 and MEK2 encode protein kinases of 393 amino acids and 400 amino acids, respectively. The docking domain (DD) for ERK1 and ERK2, the nuclear export sequence (NES) and the MAP kinase kinase kinase (MAP3K) docking domain (domain of versatile docking (DVD)) are shown in red and the amino terminal negative regulatory region (NRR) domain is shown in green. The kinase catalytic domain is shown in blue and includes the highly conserved catalytic lysine residue (Lys97 or Lys101), the activation loop (AL), with sites of activating phosphorylation by RAF and other MAP3Ks, and the proline-rich domain (PRD) that in MEK1 includes Thr292, a site of negative feedback phosphorylation by ERK1 and ERK2, and Ser298, a site of phosphorylation by p21-activated kinase (PAK). Most gain-of-function mutations in MEK1 and MEK2 that are found in cancer or cardio-facio-cutaneous (CFC) syndrome cluster in the NRR or the N terminal lobe of the kinase domain (shaded in purple). Figure adapted: from REF. 36, Elsevier; from REF. 37, Springer; and from Bromberg-White, J. L., Andersen, N. J. & Duesbery, N. S. MEK genomics in development and disease. Briefings in Functional Genomics, 2012, 11, 4, , by permission of Oxford University Press. MEK addiction How dependent on activity a tumour cell is for survival and proliferation; it can broadly be measured by how sensitive a tumour cell is to a MEK inhibitor. ERK PAK these MAP3Ks are mutated in cancer 29 and can confer resistance to BRAFis 30,31. Some of these MAP3Ks also activate the JUN N-terminal kinase (JNK), p38, ERK5 and nuclear factor-κb (NF κb) signalling pathways 31 35, so that activation of the ERK pathway proceeds in concert with these other pathways (FIG. 2). Thus, specific inhibition of MEK1 and MEK2 by allosteric inhibitors may cause substantial qualitative changes to signalling networks. The extent to which these effects of MEKi are therapeutically desirable or contribute to toxicity in normal tissue is unclear. However, such qualitative changes in signalling are less likely to be observed in cells that harbour BRAF V600E and that are treated with a BRAFi, which leaves MEK ERK activation by other MAP3Ks intact (see Supplementary information S1 (figure)). Clearly, understanding the biochemistry and cell biology of MEK1 and MEK2 in the context of such wider signalling networks is essential to understand their role in oncogenic signalling and to interpret the effects of MEKis. Functional domains of MEK1 and MEK2. The secondary structure of MEK1 and MEK2 (FIG. 3) comprises an amino terminal sequence, a central protein kinase domain (residues in MEK1 and in MEK2) that contains the kinase activation loop and a proline-rich segment, as well as a short carboxy terminal sequence 6,36,37. The N terminal region contains an ERK1 and ERK2 docking site and a strong nuclear export sequence that controls cytoplasmic MEK1 and MEK2 localization and overlaps with a negative regulatory region that stabilizes an inactive kinase conformation. The extreme C terminus contains the docking site for upstream activating MAP3Ks. Like many other kinases, the MEK kinase domain consists of a small N terminal lobe and a larger C terminal lobe; conserved regions that are involved in ATP binding and hydrolysis, substrate binding, and phosphate transfer are found at the interface between these lobes. Activation of MEK1 and MEK2 requires conformational rearrangement of a C helix in the N lobe and the activation loop in the C lobe to allow the correct alignment of ATP and substrate. This rearrangement is caused by RAF- or MAP3K mediated phosphorylation of Ser218 and Ser222 within the MEK1 activation loop (Ser222 and Ser226 in MEK2) 38,39. MEK1 is also regulated by the phosphorylation of additional sites that are clustered within the kinase domain. In addition to RTKs and RAS, MEK1 integrates signals from integrins via the RAC-dependent phosphorylation of MEK1 in Ser298, which is catalysed by p21 activated kinase (PAK1) 40,41. MEK1 and MEK2 form stable heterodimers in vivo that are unable to assemble when MEK1 is phosphorylated by ERK1 and ERK2 on Thr292; this inability to form dimers decreases MEK1 and MEK2 kinase activity as part of a negative feedback loop 41,42. Notably, Thr292 is absent from MEK2, adding to a body of evidence that MEK1 and MEK2 have nonredundant roles, despite their high degree of homology and identical substrate specificity. For example, Mek2 / mice apparently develop normally 43, whereas Mek1 knockout causes embryonic death at embryonic day 10.5 (E10.5) owing to placental defects 44. MEK1 and MEK2 in cancer The importance of MEK1 and MEK2 in cancer first emerged with the recognition of their strategic position in the RAS RAF MEK ERK pathway and the demonstration that activating mutations in the cdnas encoding MEK1 and MEK2 that mimicked activation loop phosphorylation could transform cells 45,46. Subsequently, one of the earliest allosteric MEKis, PD (also known as CI 1040), was shown to inhibit tumour cell proliferation in vitro and to inhibit tumour growth in vivo 14. Since then, an array of studies have demonstrated that various MEKis block tumour cell growth both in vitro and in vivo, underscoring the broad level of dependency of cancer cells on MEK1 and MEK2 in preclinical models 10 12,15. Such MEK addiction seems to be strongest in tumours that harbour BRAF V600E (REF. 47), which is consistent with the transforming effects of this oncogene being mediated via the activation of MEK ERK. However, a considerable number of tumour cells that are driven by RAS mutations are also sensitive to MEK1 and MEK2 inhibition in vitro and in vivo 47,48. This highlights a key difference between MEKis and BRAFis. The anti-proliferative effects of BRAFis are confined to cells that express BRAF V600E or similar activating mutations in BRAF 49. However, in the presence of RAS GTP, BRAFis, such as vemurafenib, promote paradoxical activation of ERK1 and ERK2. This is a consequence of the drug-induced allosteric transactivation of one BRAF molecule within RAS-induced RAF dimers and/or the 580 OCTOBER 2015 VOLUME 15

5 prevention of RAF-inhibitory autophosphorylation In the clinic, this paradoxical ERK activation results in a range of secondary cutaneous lesions, including papillomas, squamous cell carcinomas, keratoacanthomas and basal cell carcinomas that profoundly limits the use of vemurafenib in the treatment of tumours that express BRAF V600E 21. The first demonstration of activating mutations in MEK1 or MEK2 came from cardio-facio-cutaneous (CFC) syndrome, a genetic disorder that is caused by the aberrant activation of ERK1 and ERK2 during development 54. The first report of an amino acid-altering MEK mutation (encoding MEK2 P298L) was in a lung cancer cell line in 1997 but the functional consequences were not defined 55. Activating mutations in MEK1 or MEK2 were first reported in ovarian cancer cell lines in 2007 (REF. 56); since then, gain of function mutations in MEK1 or MEK2 have been reported in melanoma, CRC and lung cancer Most of these mutations cluster together with mutations that are found in CFC syndrome in either the N terminal negative regulatory region or the ATP-binding region of the N terminal lobe (FIG. 3). Notably, activating MEK1 mutations define a subset of smoking-associated lung adenocarcinoma, which may account for up to 600 patients with lung cancer per year in the United States 60. Although MEK1 and MEK2 mutations are rare in cancer as a whole, their existence provides an important level of validation for MEK1 and MEK2 as drug targets, and their incidence in lung cancer defines a specific patient population that may benefit from MEKi therapy. In addition, MEK1 and MEK2 mutations have emerged as drivers of acquired drug resistance (see below). Finally, the deletion of both Mek1 and Mek2 in mice prevents the induction of NSCLC by endogenous Kras G12V (REF. 61). Interestingly, although CRAF is rarely mutated in human cancer its activity is strongly induced by mutant RAS proteins and Craf, but not Braf, is required for Kras G12V -driven lung cancers in mice 61,62. Therefore, both CRAF and MEK1 and MEK2 are strongly validated drug targets in mouse models of lung cancer driven by mutant Kras. Mechanism of action of MEK inhibitors The first synthetic small-molecule inhibitor of MEK1 and MEK2 kinase activity to be discovered, PD98059, was reported in 1995 (REFS 13,63) (TABLE 1). Kinetic experiments Table 1 Properties and clinical progression of some widely used allosteric inhibitors inhibitor Year reported Developer or owner In vitro IC 50 for MEK1 (nm)* Ability to disrupt MEK phosphorylation Clinical progression T 0.5 (hours or days) PD Pfizer 2000 Weak Pre-clinical Not relevant 13,63,73 U DuPont 72 Weak Pre-clinical Not relevant 64,73 PD (CI 1040) 1999 Pfizer 17 Weak Phase II 20.9+/ 4.8 h 14,72,180 PD Pfizer 1 Weak Phase II 7.8 h 10,181 Binimetinib (MEK162, ARRY ) Selumetinib (AZD6244, ARRY ) Refametinib (RDEA119, BAY ) CH (RO ) Pimasertib (AS703026, MSC ) 2006 Novartis/Array Biopharma 2007 AstraZeneca/ Array Biopharma Refs 12 Weak Phase III h Weak Phase III 5.33 h 68,73, Bayer AG 19 Weak Phase II 12 h 114,187, Chugai Pharmaceutical Co 5.2 Moderate Phase I 4 h 75,79, 189, Merck KGaA 52 Not available Phase II 5 h 191 TAK Takeda 3.2 Weak Phase I h Trametinib (GSK ) CH (RO ) Cobimetinib (GDC 0973, XL518) 2011 GlaxoSmithKline 0.7 Moderate Approved for BRAF V600E/K mutant melanoma 2012 Chugai Pharmaceutical Co 2012 Genentech (Roche) GDC Genentech (Roche) ~4 days 16,75, Strong Phase I 60 h 75,78, Weak Phase III 40 h 74,110, Strong Phase I 4 10 h 74,198 *No unified protocol was used to generate the in vitro MEK1 IC 50 values stated and so these should be treated as a rough guide to potency only. IC 50 values shown for CH , cobimetinib, GDC 0623 and trametinib were generated using methods in which MEK1 was activated after incubation with inhibitor. Apparent potency can differ greatly depending on whether constitutively active MEK1 (S218D/E S222D/E) is used ( ) or whether MEK1 is activated by RAF before inhibitor addition ( ) as well as other experimental details. J. Sebolt-Leopold, personal communication (2015). NATURE REVIEWS CANCER VOLUME 15 OCTOBER

6 Feedback relief catalysed feedback phosphorylation and inhibition of RAF normally operates in the pathway but is relieved when are inhibited and activity declines, resulting in the activation of RAF and further phosphorylation. Pathway activity also stimulates the expression of negative regulators of pathway activity such as the ERK phosphatase DUSP6; expression of these negative regulators is reduced when the pathway is inhibited. with PD98059 and U0126 (REF. 64), another MEKi, demonstrated that both molecules shared a common binding site and inhibited MEK1 and MEK2 non-competitively with respect to their substrates Mg ATP and ERK1 and ERK2. These early MEKis served as valuable research tools but their low potency, physical properties and certain off-target effects 65,66 stimulated the development of further generations of MEKi (TABLE 1). PD was the first MEKi to be evaluated in vivo and was shown to inhibit the growth of CRC tumour xenografts 14. Crystal structures of MEK1 bound to analogues of PD demonstrated the presence of a unique inhibitor-binding pocket that is separate from, but adjacent to, the Mg ATP-binding site in both MEK1 and MEK2 (REF. 67); this region of MEK1 and MEK2 exhibits little homology to other protein kinases, thus providing an explanation for why this class of drugs is so specific 68. Allosteric MEKis, such as PD184352, stabilize an inactive conformation of MEK1 and MEK2 in which helix C and the activation loop are displaced, resulting in the misalignment of catalytic residues and the possible partial occlusion of the ERK1 and ERK2 activation loop binding site Although the clinical progression of PD was curtailed by poor biological availability and potency 70, the past 10 years have seen the discovery of highly selective allosteric MEKis with far superior pharmacological and pharmaceutical properties (TABLE 1). Until recently, allosteric MEKis were thought to act through broadly equivalent mechanisms 71 ; however, recent studies have changed this view. In cells with wild-type BRAF, including those with RAS mutations, ERK-dependent feedback phosphorylation of BRAF and CRAF inhibits the binding of both BRAF and CRAF to RAS GTP and disrupts BRAF CRAF heterodimers, thus reducing RAF signalling to MEK1 and MEK2 (FIG. 1c). As a result, the loss of ERK1 and ERK2 activity following MEK1 and MEK2 inhibition results in the dephosphorylation and activation of CRAF (so called feedback relief) and a CRAF-dependent increase in phosphorylation of the MEK1 and MEK2 activation loop and reactivation of ERK1 and ERK2 (FIG. 4). This has been observed for many MEKis, including PD098059, U0126, PD184352, PD , selumetinib (also known as AZD6244 and ARRY ) and cobimetinib However, enhanced MEK1 and MEK2 phosphorylation in response to MEKis is not observed in BRAF-mutant cells, a b c EGFR or FGFR RAS GTP RAS GTP RAS GTP RAS GTP RAS GTP RAF RAF RAF RAF RAF RAF CH GDC-0623 Trametinib RAF RAF RAF RAF PD Selumetinib Cobimetinib DUSP Proliferation Survival Adaptive resistance Figure 4 MEKis that inhibit phosphorylation suppress the rebound in activation that results from relief of negative feedback. a With the exception of tumour cells that harbour activating BRAF mutations, ERK1 and ERK2 () signalling is subject to extensive -dependent negative feedback at multiple levels of the pathway, including RAF. RAS GTP drives the formation of high activity homodimers or heterodimers of the RAF protein kinases (ARAF, BRAF and CRAF), whereas ERK-dependent phosphorylation of RAF proteins inhibits the binding of BRAF and CRAF to RAS GTP and disrupts BRAF:CRAF heterodimers, thereby inhibiting phosphorylation of MEK. b MEK1 and MEK2 () inhibition with a MEK inhibitor (MEKi) blocks activation and so relieves this negative feedback and consequently allows stronger activation of upstream pathway components, including RAS and RAF (represented by increased numbers of active RAS molecules and active RAF dimers). The majority of MEKis, such as PD , selumetinib and cobimetinib, do not disrupt the phosphorylation of the activation loop sites and so treatment with these MEKis and concomitant relief of negative feedback typically results in the accumulation of phosphorylated. This is thought to explain the rebound in phosphorylated and pathway output that is observed with these MEKis in various contexts, notably in tumour cells with mutant RAS 74,75. c A subset of newer MEKis, so-called feedback busters, including trametinib, CH and GDC 0623, disrupt the conformation of the activation loop sites so that they can no longer be efficiently phosphorylated by RAF. Thus, although MEK inhibition with these MEKis is also expected to result in stronger activation of upstream pathway components, little or no increase in phosphorylation or rebound in activity is observed (right), translating into more durable pathway inhibition and superior efficacy in preclinical models OCTOBER 2015 VOLUME 15

7 Feedback buster A term adopted by the field, although something of a misnomer. All MEK inhibitors (MEKis) relieve ERK-dependent negative feedback to RAF, resulting in RAF activation. Feedback buster MEKis mitigate some, but not all, consequences of feedback relief that arise when ERK is inhibited by interfering with the phosphorylation of MEK by RAF, thereby reducing rebound activation of the pathway. By contrast, conventional MEKis do not prevent MEK phosphorylation by RAF. Phosphomimetic mutant A phosphomimetic mutant of MEK1 exhibits constitutive (MAP3K independent) activation owing to acidic substitutions at Ser218 and Ser222 in the activation loop that mimic the negative charge of phosphorylation. especially BRAF V600E melanoma, principally because BRAF V600E is fully active as a monomer and insensitive to ERK1- and ERK2 dependent inhibitory phosphorylation and the inhibitory action of SPRY proteins, but also because the activity of RAS and CRAF is typically low in BRAF V600E melanoma 72,76,77. Intriguingly, some newer MEKis including trametinib, CH and GDC 0623 while inhibiting negative feedback mechanisms, mitigate the consequences of this by reducing the phosphorylationn of MEK by RAF 74,78 (FIG. 4). This may explain why these inhibitors cause more durable suppression of ERK1 and ERK2 phosphorylation, cell proliferation and xenograft growth in the context of RAS mutations 74,75. It is important to understand the underlying mechanism of these feedback buster MEKis because KRAS-mutant tumours represent an important unmet clinical need and there is a growing interest in using MEKis as part of drug combinations in KRAS-mutant disease, including NSCLC. Inhibition of activation loop phosphorylation. Differences in the mechanism of action may reflect distinct inter actions between the MEKi and the MEK1 and MEK2 activation loop residues. For example, GDC 0623 is proposed to form a stronger hydrogen bond inter action with Ser212 than other inhibitors, thereby constraining the activation loop and disrupting phosphorylation by RAF 74. However, crystal structures of MEK1 bound to various allosteric MEKis such as CH , CH , and PD and PD like inhibitors suggest that interaction with Ser212 is invariably crucial for the binding of allosteric MEKis to MEK1 and MEK2 (REFS 67,69,75,79). Instead, the ability to disrupt MEK1 and MEK2 phosphory lation correlates with the extent to which a MEKi coordinates with Asn221 and Ser222 to displace the activation loop 75,79. Whereas CH binds both Asn221 and Ser222 and causes activation loop displacement, a near-identical enantiomer of PD does not coordinate Asn221 and Ser222 or displace the activation loop, which is consistent with the differing abilities of these inhibitors to disrupt MEK1 and MEK2 phosphorylation 69,75. Whether these MEKis antagonize phosphorylation of MEK1 at both Ser218 and Ser222 (and of MEK2 at Ser222 and Ser226) is unknown; mass spectrometry suggests that trametinib prevents the phosphorylation of MEK1 at Ser218, but not at Ser222, and this monophosphorylated form of MEK1 has severely limited kinase activity compared with dualphosphorylated MEK1 (REF. 16). Further structural analyses will determine whether, and how, distinct MEKis disrupt the phosphorylation of MEK1 and MEK2 and may guide future MEKi development. Modulation of RAF complexes. MEKis also modulate the interaction between MEK1 or MEK2 and RAF, and although this may influence their ability to suppress MEK1 and MEK2 activity, no simple correlation is apparent. For example, selumetinib and PD induce the binding of all three RAF kinases to MEK1 and MEK2, and this seems to attenuate the activity of these MEKis 75. By contrast, GDC 0623 and CH also promote the association of RAF with MEK1 and MEK2, but inhibit their phosphorylation 74,75,78. Both trametinib and cobimetinib promote the dissociation of RAF complexes, but whereas trametinib antagonizes MEK1 and MEK2 phosphorylation, cobimetinib does not 74,75. Thus, although a firm causal relationship between MEKi activity and the association or dissociation of the RAF complex has not yet been established, the modulation of these complexes may be integral to their mode of action and may influence the depth and duration of pathway inhibition. Feedback buster MEKis in the context of BRAF V600E mutations. Intriguingly, MEKis that disrupt MEK1 and MEK2 phosphorylation seem to have a relatively weaker affinity for dual-phosphorylated than do the MEKis that permit the accumulation of phosphorylated. The binding of GDC 0623 to a constitutively active phosphomimetic mutant of MEK1 and the binding of trametinib to MEK1 that had been pre-phosphorylated in vitro were both markedly weaker than binding to wild-type MEK1 and unphosphorylated MEK1, respectively 16,74. By contrast, cobimetinib bound with similar affinity to wild-type and phosphomimetic MEK1, and suppressed ERK phosphorylation more effectively than GDC 0623 in cells expressing phosphomimetic MEK1 (REF. 74). Thus, for certain feedback buster MEKis, an equilibrium may exist between the inhibition of MEK1 and MEK2 activation loop phosphorylation and the attenuation of MEKi binding by the phosphorylated activation loop, with the balance being determined by pretreatment phospho levels. These observations may be of particular relevance when constitutive MEK1 and MEK2 phosphorylation levels are high, such as in BRAF V600E positive cells; indeed, there is some support for this proposal from BRAF V600E -mutant xenograft models 72. Clearly, careful consideration of the signalling context and particular properties of a MEKi will be required to achieve optimal clinical responses, especially in RAS-mutant tumours. Resistance to MEKi and mitigation Preclinical and clinical studies have led to the identification of various modes of innate, adaptive and acquired resistance to MEKis, many of which are druggable, allowing relevant combination strategies to be tested. Intrinsic resistance through parallel oncogenic pathways. Primary sensitivity to MEKi correlates with the decreased expression of cyclin D1 (CCND1), expression of p27 (also known as KIP1) and cell cycle arrest. However, the deregulation of the cyclin-dependent kinases 4 and 6 (CDK4/6) RB axis (such as through the amplification of CCND1 or CDK4 or the loss of CDK inhibitor 2A (CDKN2A)) is common in many cancers 80 and can confer resistance to ERK pathway inhibitors 81. Indeed, activating mutations in RAS, BRAF or MEK1 and MEK2 can co operate with CDKN2A loss in a variety of tumours Such results have led to the effective combination of BRAFis or MEKis with inhibitors of CDK4/6 in preclinical models 85,86. Other signalling pathways such as PI3K, adenomatous polyposis coli (APC) β catenin, NATURE REVIEWS CANCER VOLUME 15 OCTOBER

8 EGFR or HER2 HER3 PDGFRβ, VEGFR or AXL FGFR2 or FGFR3 RAS RAF P PI3K RAF STAT3 MEKi Cytoplasm Nucleus Proliferation Survival CtBP ERBB3 BETi MYC BETi PDGFRB? FGFR2 FGFR3 BETi Inhibit gene expression NRG1 VEGFR2 AXL Figure 5 Adaptive kinome reprograming arising from inhibition. Active ERK1 and ERK2 () chronically inhibit signalling from an array of receptor tyrosine kinases (RTKs) by direct phosphorylation of receptors Nature Reviews at inhibitory Cancer sites (for example, ERK-mediated phosphorylation of epidermal growth factor receptor (EGFR) and HER2) or by repressing the transcription of the genes that encode RTKs and their cognate ligands, mediated by dependent phosphorylation of transcriptional regulators such as CtBP and MYC (solid arrows). Inhibition of MEK1 and MEK2 () collapses these feedback loops, resulting in rapid and sustained reactivation of multiple RTKs that can then sustain cell survival and proliferation by reactivation of RAF MEK ERK signalling or by activation of PI3K or signal transducer and activator of transcription 3 (STAT3)-dependent signalling 28,96 99 (dashed arrows). Such kinome reprogramming validates clinical trials in which MEK inhibitors (MEKis) are being combined with various RTK inhibitors. However, MEKis frequently cause activation of multiple RTKs so an alternative, broader approach is to combine a MEKi with BET domain inhibitors (BETis), which act on key chromatin reader proteins to inhibit transcription 100. FGFR, fibroblast growth factor receptor; NRG1, neuregulin 1; PDGFRβ, platelet-derived growth factor receptor-β; VEGFR, vascular endothelial growth factor receptor. signal transducer and activator of transcription 3 (STAT3) and NF-κB converge on the same cell cycle regulators and can drive primary MEKi resistance. For example, even in tumour cells that harbour BRAF V600E, strong PI3K dependent signalling owing to mutations in PIK3CA or the loss of PTEN can maintain CCND1 levels in the presence of MEKis and can confer resistance to MEKis 87,88. This can be overcome by combining MEKis with PI3K, mtor or AKT1 (also known as PKB) inhibitors. In addition, the frequent increase in PKB/AKT activity following treatment with MEKis has prompted numerous studies with these drug combinations 89,90 and ongoing clinical trials 91. STAT3 activation has been shown to promote MEKi resistance; combining a STAT3 inhibitor with selumetinib overcame resistance and promoted tumour cell death 92,93. More recently, the Hippo pathway effector YAP1 has been shown to promote resistance to RAFi or MEKi therapy, and combined inhibition of YAP1 and MEKi was synthetic lethal in tumour cells 94. Loss of feedback inhibition and MEK ERK reactivation. Although MEKis have shown great promise in BRAF V600E preclinical models 16,47, as well as some clinical activity in BRAF V600E melanoma 95, their more limited success in RAS-mutant tumours may well be due in large part to the loss of dependent feedback and reactivation of the MEK ERK1 pathway. For example, tumour cell lines with BRAF V600E exhibited durable inhibition of ERK1 and ERK2 and were very sensitive to PD , whereas tumour cell lines with mutations in KRAS were notably resistant to PD and exhibited strong ERK1 and ERK2 reactivation within hours of drug administration 75. Thus, hardwired homeostatic mechanisms dictate the efficacy of MEKi in tumour cells with wild-type BRAF, including those with mutations in RAS, and provide a clear rationale for dual pathway inhibition: RAFi plus MEKi to prevent MEK1 and MEK2 reactivation, or MEKi plus ERKi to prevent ERK1 and ERK2 reactivation. An alternative approach may be to use feedback buster MEKis with a dual mechanism such as trametinib, CH , GDC 0623 and G-573 that inhibit MEK1 and MEK2 kinase activity and prevent phosphorylation by RAF 74,75 (FIG. 4). Interestingly, despite the existence of multiple non-raf MAP3Ks that can activate MEK1 and MEK2 (including MAP3K8 (also known as COT), some MEKKs and MLKs ) few studies have assessed whether these alternative MEK activators are inhibited by ERK-dependent feedback phosphorylation and thus might contribute to MEK reactivation following MEK inhibition. Adaptive kinome reprogramming. signalling inhibits an array of RTKs such that MEK1 and MEK2 inhibition elicits rapid RTK activation (FIG. 5). 584 OCTOBER 2015 VOLUME 15

9 For example, ERK1- and ERK2 mediated phosphorylation of EGFR and HER2 (also known as ERBB2) suppresses signalling to HER3 (also known as ERBB3) so that MEKis promote the rapid reactivation of EGFR HER3 and PI3K dependent signalling 96,97. Unbiased, nontargeted chemical proteomic analysis demonstrated the activation of multiple RTKs following MEK1 and MEK2 inhibition, which were otherwise repressed by the ERK1 and ERK2 target MYC 28. In BRAF-mutant thyroid cancer cells, BRAFis or MEKis promoted ERK1 and ERK2 reactivation involving HER3 transcription and the autocrine secretion of neuregulin 1 (NRG1) 98, and in KRAS-mutant NSCLC cells selumetinib promoted autocrine fibroblast growth factor (FGF) production and increased expression of FGF receptors (FGFRs), which conferred MEKi resistance via STAT3 activation 99. These studies underscore the extent of such kinome re programming in response to MEKis and support ongoing trials that are combining MEKis with relevant RTK inhibitors. However, as multiple RTKs are often activated in cancer and because transcriptional programmes underpin kinome reprogramming, an alternative strategy is to combine MEKis with inhibitors of key chromatin reader proteins such as the bromodomaincontaining protein 4 (BRD4). Certainly, JQ1, a BET domain inhibitor, synergizes well with tyrosine kinase inhibitors in acute myelogenous leukaemia 100 (FIG. 5). Acquired resistance to MEKis. Various studies have now investigated acquired resistance to long-term MEKi exposure, both in tumour cell lines and in clinical samples 101. The first report of acquired resistance to MEKis was the identification of a gain of function mutation (MEK1 P124L ) in a metastatic focus of a patient with melanoma with BRAF V600E, who exhibited prolonged stable disease under treatment with selumetinib 102. Subsequently, mutations in MEK1 and MEK2 have been found in CRC, melanoma and breast cancer cell lines (with driving mutations in BRAF or KRAS) that have been treated with various MEKis (selumetinib, trametinib and RO ), as well as in samples from patients undergoing MEKi therapy These on target mutations (MEK1 F129L and MEK2 Q60P ) activate or cluster at the allosteric inhibitor-binding pocket and abrogate MEKi binding (for example, MEK1 L115P, MEK1 G128D/L215P, MEK1 F129L, MEK1 V211D and MEK2 V215E ), thereby maintaining ERK1 and ERK2 activity in the presence of MEKis (FIG. 6). Studies have also reported acquired resistance to MEKis via the amplification of BRAF V600E. Two studies in four CRC cell lines harbouring BRAF V600E demonstrated the amplification of the mutant BRAF allele; MEKi resistance was overcome by BRAF RNA interference (RNAi) or co treatment with a RAF inhibitor 106,107. BRAF V600E amplification has also been observed in melanoma cell lines selected in trametinib 105, indicating that BRAF V600E amplification is a common mecha nism of resistance irrespective of the MEKi used. Amplification of KRAS G13D has also been described as a mechanism of MEKi resistance in CRC cells 107, either in isolation or concurrent with an activating MEK1 F129L mutation 103, suggesting that these oncogenes may cooperate to confer MEKi resistance. Thus, amplification of the upstream driving oncogene (BRAF V600E or KRAS G13D ) drives resistance to MEKi by activating a greater pool of MEK1 and MEK2 to maintain ERK1 and ERK2 activity (FIG. 6). Strikingly, most, if not all, mechanisms of MEKi resistance reinstate active ERK1 and ERK2 in the presence of MEKis (FIG. 6), thus highlighting a strong hardwired addiction to ERK1 and ERK2 signalling. This provides another opportunity for rational therapeutic intervention through dual pathway inhibition; MEKi plus ERKi in the case of MEK1 and MEK2 mutations 103 and BRAFi plus MEKi in the case of BRAF V600E amplification 106,107. Indeed, the combination of dabrafenib (a BRAFi) and trametinib (a MEKi) has now received regulatory approval in advanced-stage melanoma, in which it increased progression-free survival (compared with either agent alone) and reduced the incidence of cutaneous lesions arising from paradoxical RAF activation in tissues with wild-type BRAF 21,108. However, recent studies have revealed that acquired resistance may still arise in patients with BRAF V600E melanoma who are treated with dual BRAFi plus MEKi blockade through the acquisition of BRAF V600E ultra amplification, MEK1 mutations and/or loss of the CDKN2A locus, as well as loss of the nuclear ERK-regulatory phosphatase DUSP4 (REF. 27), echoing the recurring theme of ERK pathway re activation. In the case of KRAS G13D amplification, resistance is more complex; KRAS activates multiple effector pathways, suggesting that ERK pathway inhibition may need to be combined with AKT, PI3K or mtor kinase inhibitors. Indeed, such combinations are currently being tested in preclinical disease models and clinical trials More recently, the first potent, selective ERKis have been described, including VTX11e, an ATP-competitive inhibitor of ERK1 and ERK2, and SCH772984, an ATPcompetitive inhibitor of ERK1 and ERK2 that also prevents the activating phosphorylation of ERK1 and ERK2 by MEK1 and MEK2 (REFS 103,104). Of these selective ERKis, SCH can resensitize tumour cells with acquired resistance to either BRAFis or MEKis, providing proof of principle for combining ERK inhibitors with BRAFis or MEKis. SCH (MK08353), an analogue of SCH772984, has now entered clinical trials, as have BVD 523, RG7842 and CC These new potent and selective ERK inhibitors add new weapons to the arsenal of pathway-targeted drugs for use as first-line therapies, probably in combination, and for the treatment of on pathway resistance. MEKis in the clinical setting Pharmacokinetics and dose schedule. In addition to differences in the mechanism of inhibition, the pharmacokinetic properties of the MEKis that are currently in clinical development vary considerably with half-lives (T 0.5 ) ranging from 5 hours to 4 5 days (TABLE 1). Despite this, there has been little sustained effort to optimize the dose and schedule of administration on the basis of biological rationale rather than pragmatic choices based upon pharmacokinetics and tolerance 109, and most clinical trials are predicated upon continuous daily dosing. The exception is cobimetinib, which is administered on NATURE REVIEWS CANCER VOLUME 15 OCTOBER

10 a MEK1 or MEK2 mutation prevents MEKi b BRAF amplification maintains or c binding or activates MEK1 or MEK2 increases pool of active MEK1 or MEK2 KRAS amplification increases pool of active MEK1 or MEK2 but also activates other RAS effectors KRAS KRAS KRAS KRAS KRAS PLCε Ca 2+ BRAF BRAF BRAF BRAF ARAF BRAF CRAF PI3K RAL-GEF PKC PDK1 RAL PKB mtor Mitigation Combine MEKi with ERKi Mitigation Combine MEKi with RAFi Mitigation Combine MEKi with RAFi and PI3Ki, PKBi or mtori Figure 6 Mechanisms of acquired resistance to inhibitors (MEKis). Studies in preclinical models and analysis of clinical samples have revealed two basic mechanisms of acquired resistance to MEK1 and MEK2 () inhibitors (MEKis; represented by a red X), and, in both cases, tumour cells adapt to maintain or re activate ERK1 and ERK2 () in the presence of the drug 101, providing a rational basis for treating resistance. a The emergence of mutations in MEK1 or MEK2 (indicated by a hexagon), provides an example of on target resistance to allosteric MEKis. Emergent mutations confer resistance by reducing MEKi binding or enhancing intrinsic activity and resistance can be overcome by combining MEKi with inhibitors. b Amplification of the upstream driving oncogene, BRAF V600E (REFS 106,107) or KRAS G13D (REF. 107), has also emerged as a mechanism of acquired MEKi resistance. Selective amplification of the mutant BRAF Nature V600E Reviews allele greatly Cancer increases the proportion of active, exceeding the drug-inhibited pool, to reactivate ; in this case, resistance can be overcome by combining a MEKi with a RAF inhibitor (RAFi) 106,107. c Amplification of KRAS G13D can also drive MEKi resistance by the same basic mechanism 107 but provides a greater therapeutic challenge. KRAS G13D can activate multiple signalling pathways (such as PI3K, RAL, phospholipase Cε (PLCε), and so on) and even the combined inhibition of and PI3K signalling fails to reverse the acquired resistance to selumetinib that is driven by amplification of KRAS G13D (REF. 107). Hexagons indicate oncogenic mutations, either those that are present as the primary driving oncogene and are amplified by MEKi selection (BRAF V600E, KRAS G13D ) or those that emerge upon selumetinib selection (for example, MEK1 P124L and MEK1 F129L ). Dacarbazine A DNA-alkylating agent that has commonly been used as a single agent in the treatment of metastatic melanoma. a 2 weeks on, 1 week off schedule; a pragmatic choice that is based upon its T 0.5 of ~40 hours 110, its adverse event profile and evidence of melanoma re growth using schedules with longer drug holidays 111. Pharmacodynamics. The main biomarker for monitoring pathway inhibition by MEKis, phosphorylated ERK1 and ERK2 (p ERK), has been assessed in normal tissues (such as peripheral blood mononuclear cells) and tumour biopsy samples. In multi-tumour-type Phase I studies, selumetinib, trametinib, BAY and pimasertib all reduced levels of p-erk1 and p-erk2 in tumour tissue using immunohistochemistry assays, and in some cases have shown complete inhibition On this basis and on the basis of data from BRAF inhibitors in BRAF V600E melanoma 18 the prevailing view is that 80% inhibition of ERK1 and ERK2 phosphorylation is the benchmark for clinically effective MEK1 and MEK2 inhibition. However, these measures of target inhibition are based almost entirely on single time-point paired biopsies and non-standardized assays; no study has formally evaluated a link between target inhibition and clinical response. In summary, clinical dose and schedule selection for MEKis is mostly based on establishing the maximum tolerated doses using continuous or chronic dosing regimens. Clinical activity as monotherapy. As with preclinical models, BRAF V600E mutant melanoma has proved to be the most responsive adult solid tumour to MEKi mono therapy. Trametinib has gained regulatory approval for this indication following rapid clinical development from a single-arm Phase I trial to a randomized Phase III trial in which treatment with this MEKi yielded 4.8 months progression-free survival compared with 1.5 months for dacarbazine 95. In addition, the licensing and supervisory authority of Switzerland has recently approved cobimetinib for use in combination with vemurafenib as a treatment for patients with advanced melanoma. Such is the pace of clinical development in advanced-stage melanoma that MEKi monotherapy in BRAF V600E melanoma which is relatively ineffective in patients who relapse following treatment with a firstgeneration BRAFi 115 is being superseded by BRAFi plus MEKi combinations, of which three have completed or are in clinical trials 111,116,117. MEKis have been tested in multiple tumour types with a high incidence of BRAF or RAS mutations with mixed results. Some diseases seem notably refractory; for example, KRAS-mutant CRC 118,119. However, others have shown sufficient activity to prompt Phase III trials; for example, MEK162 in NRAS-mutant advanced-stage melanoma 120 and selumetinib in serous low-grade ovarian cancer 121, a disease in which MEK162 and trametinib are currently undergoing assessment in randomized Phase III trials. Between these extremes, MEKis have shown clear but low response rates in most indications, including pancreatic cancer 109,122, biliary tract cancer 123, NSCLC 124, uveal melanoma 125 and acute myeloid leukaemia (AML) 126,127, and were generally not 586 OCTOBER 2015 VOLUME 15