REVIEWS

MEK1 and MEK2 inhibitors

and cancer therapy:
the long and winding road
Christopher J. Caunt1, Matthew J. Sale2, Paul D. Smith3 and Simon J. Cook2
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 ERK1/2 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.
e-mails: paul.d.smith@
astrazeneca.com;
simon.cook@babraham.ac.uk
doi:10.1038/nrc4000
The authors dedicate this article to Prof. Chris Marshall,
FRS (1949–2015), 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 recep-
tors, and the core components of this pathway are now
well known1,2. 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 for-
mation 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 partners1,3; these include other pro-
tein 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 cycle4. ERK1 and ERK2 can also regulate cell
survival by phosphorylating members of the apoptosis
regulating BCL‑2 protein family at the mitochondria5.
ERK1/2 signalling regulates processes that are crucial for
normal development, including cell proliferation, dif-
ferentiation, survival and cell motility; indeed, germline
deletion of some components of the ERK pathway causes
embryonic lethality 6, and a MEK1/2 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 hall-
marks and driving characteristics of the cancer cell8,9.
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, ERK1/2 signalling.
The discovery of the core components of the RAS–
ERK pathway 2 kick-started a protein kinase drug dis-
covery effort that continues today 10–12. The first ERK
pathway inhibitor to be discovered, PD98059, was
reported 20 years ago13 and was shown to act inde-
pendently of ATP as an apparent allosteric inhibitor of
MEK1 and MEK2. Since then, MEKis have proved to be

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REVIEWS

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 targets14,15. As they are not ATP-
competitive, it was correctly anticipated that MEKis
would be more selective than conventional kinase inhibi-
tors and widely hoped that this would translate into rapid
clinical success. In fact, it took a further 18 years until
trametinib16 became the first MEKi to receive regulatory
approval. In the interim, the identification of activating
BRAF mutations (most notably BRAFT1799A 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
vemurafenib18,19 and dabrafenib20,21.
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 SOS RAS GTP SOS RAS GTP –

P P P – SPRY

RAF RAF RAF RAF P RAF RAF

P

P MEK1/2

KSR1

KSR1
MEK1/2 MEK1/2 – –
P

ERK1/2 ERK1/2 ERK1/2 – DUSP6

Cytoplasm

Nucleus
ERK1/2 ERK1/2 ERK1/2 – DUSP5

P P P P P P
ETS ETS ETS

Figure 1 | Scaffolds and feedback controls underpin the normal functioning of the ERK1/2 pathway. a | A simplified
linear representation of the RAS-regulated RAF–MEK–ERK signalling cascade. Activated growth factor Naturereceptors
Reviews (for
| Cancer
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 (MEK1/2). MEK1/2 are dual-specificity protein kinases that
phosphorylate ERK1 and ERK2 (ERK1/2) on a conserved TEY motif to activate them. ERK1/2 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 cell166–168. 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 fidelity22,169,170. For example, the kinase suppressor
of RAS 1 (KSR1) scaffold assembles RAF, MEK1/2 and ERK1/2 to increase signalling efficiency; acts allosterically to activate
the RAF kinase domain171; 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 output166. 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 propagation23,101. Notably, ERK can phosphorylate CRAF and BRAF to inhibit MEK
phosphorylation172–174, and MEK1 to inhibit ERK phosphorylation41,42. 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 perturbations166,175 and explains the ERK reactivation that is observed in tumour cells with wild-type
BRAF75. 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 domain176. 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 locations24.

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 REVIEWS

The role of MEK in the ERK pathway
The ERK pathway is frequently represented as a lin-
ear 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 feed-
back regulation that are vital for the role of the ERK
pathway in information processing. Understanding
how the pathway is activated by different input stim-
uli 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 systems-
level features. First, activation of the ERK pathway
depends on scaffold proteins such as kinase suppres-
sor of RAS 1 (KSR1), which increase the efficiency of
the inter­actions between the enzyme and substrate at
each step22 (FIG. 1b). Second, ERK1/2 signalling is criti-
cally regulated by homeostatic feedback controls23 that
include the direct phosphorylation of upstream com-
ponents and increased expression of Sprouty (SPRY)
proteins and dual-specificity phosphatases (DUSPs);
the DUSPs inactivate ERK1 and ERK2 by dephosphory­
lating 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 fre-
quent loss of the ERK-specific cytoplasmic phos-
phatase 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 deter-
minants of rapid pathway adaptation and long-term
acquired resistance to MEKis27,28. 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 net-
works (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 RAS-
or 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

MEKK1 MEKK3 MAP3K8 MLK2 MLK4

Integrins MEKK2
ARAF BRAF CRAF MLK1 MLK3
RAC

P P
PAK P MEK1/2

MKK3 or MKK4 or
MEK5 IKK
MKK6 MKK7
ERK1/2

P P P P P P P P ERK5 p38 JNK1/2 IκB

BIM MCL1 RSK MNK Cytoplasm

P P P P P P P P P P P P P P Nucleus
FOS ETS MYC MSK MEF2 MEF2 JUN NF-κB

P P
CCND1 CHOP

Figure 2 | MEK1 and MEK2 are the key ‘gatekeepers’ for ERK1 and ERK2 in a wider signalling Nature Reviews |The
network. Cancer
canonical pathway for ERK activation (RAS–RAF–MEK–ERK) is shown on the left. ERK1 and ERK2 (ERK1/2) 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 (MEK1/2). 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)31–35. These ‘alternative’ MEK1/2 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-κB31–35. This reflects a key feature of ERK pathway
architecture: receptor tyrosine kinases (RTKs)177, RAS GTPases2 and MAP3Ks31–35 are relatively promiscuous, activating two
or more parallel signalling cascades; even RAF proteins may regulate non-MEK targets through scaffold functions178,179.
By contrast, MEK1/2 are exquisitely specific activators of ERK1/2. Because ERK1/2 have hundreds of binding partners and
substrates throughout the cell1,3, this represents an astonishing achievement in signal processing through MEK1/2 and
underscores their role as key ‘gatekeepers’ of ERK signalling.

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REVIEWS
Receptors Integrins
and RAS
CFC and cancer
CFC and cancer
mutations
mutations
RAF and ERK PAK
MAP3Ks
Ser218 Ser222 Thr292 Ser298
Lys97 P P P P
MEK1 1 DD NES NRR Kinase catalytic domain AL PRD
Ser222 Ser226
Lys101 P P
MEK2 1 DD NES NRR Kinase catalytic domain AL PRD
Figure 3 | Linear representation of the key functional domains of the human MEK1
NatureofReviews | Cancer
and MEK2 proteins. Human MEK1 and MEK2 encode protein kinases 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, 300–310, by permission of Oxford University Press.
these MAP3Ks are mutated in cancer 29 and can confer
resistance to BRAFis30,31. Some of these MAP3Ks also
activate the JUN N-terminal kinase (JNK), p38, ERK5
and nuclear factor-κB (NF‑κB) signalling pathways31–35,
so that activation of the ERK pathway proceeds in con-
cert 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 second-
ary structure of MEK1 and MEK2 (FIG. 3) comprises
an amino‑terminal sequence, a central protein kinase
domain (residues 68–361 in MEK1 and 72–367 in
MEK addiction MEK2) that contains the kinase activation loop and a
How dependent on MEK1/2 proline-rich segment, as well as a short carboxy‑terminal
activity a tumour cell is for sequence6,36,37. The N‑terminal region contains an ERK1
survival and proliferation; it can
broadly be measured by how
and ERK2 docking site and a strong nuclear export
sensitive a tumour cell is to a sequence that controls cytoplasmic MEK1 and MEK2
MEK inhibitor. localization and overlaps with a negative regulatory
580 | O CTOBER 2015 | VOLUME 15
© 2015 Macmillan Publishers Limited. All rights reserved
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 inter-
face between these lobes. Activation of MEK1 and MEK2
DVD 393
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
DVD 400
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 phos-
phorylation 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
loop41,42. Notably, Thr292 is absent from MEK2, adding
to a body of evidence that MEK1 and MEK2 have non-
redundant 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 defects44.
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 demon-
stration that activating mutations in the cDNAs encoding
MEK1 and MEK2 that mimicked activation loop phos-
phorylation could transform cells45,46. Subsequently, one
of the earliest allosteric MEKis, PD184352 (also known
as CI‑1040), was shown to inhibit tumour cell prolif-
eration in vitro and to inhibit tumour growth in vivo14.
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 depend-
ency of cancer cells on MEK1 and MEK2 in preclinical
models10–12,15. Such ‘MEK addiction’ seems to be strong-
est in tumours that harbour BRAFV600E (REF. 47), which
is consistent with the transforming effects of this onco-
gene 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 vivo47,48. This high-
lights a key difference between MEKis and BRAFis. The
anti-proliferative effects of BRAFis are confined to cells
that express BRAF‑V600E or similar activating muta-
tions in BRAF49. However, in the presence of RAS–GTP,
BRAFis, such as vemurafenib, promote paradoxical acti-
vation 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
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 REVIEWS

prevention of RAF-inhibitory autophosphorylation50–53. smoking-associated lung adenocarcinoma, which may

In the clinic, this paradoxical ERK activation results in
a range of secondary cutaneous lesions, including pap-
illomas, squamous cell carcinomas, keratoacanthomas
and basal cell carcinomas that profoundly limits the use
of vemurafenib in the treatment of tumours that express
BRAF‑V600E21.
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 develop-
ment 54. The first report of an amino acid-altering MEK
mutation (encoding MEK2‑P298L) was in a lung can-
cer cell line in 1997 but the functional consequences
were not defined55. 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 57–60. 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
account for up to 600 patients with lung cancer per
year in the United States60. 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 can-
cer defines a specific patient population that may ben-
efit 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 KrasG12V (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 KrasG12V-driven lung cancers in
mice61,62. 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 MEK1/2 inhibitors
MEK1/2 inhibitor Year Developer or In vitro IC50 for Ability to disrupt Clinical T0.5 (hours Refs
reported owner MEK1 (nM)* MEK phosphorylation progression or days)
PD098059 1995 Pfizer 2000 ‡ Weak Pre-clinical Not relevant 13,63,73
U0126 1998 DuPont 72‡ Weak Pre-clinical Not relevant 64,73
PD184352 (CI‑1040) 1999 Pfizer 17 ‡
Weak Phase II 20.9+/−4.8 h 14,72,180||
PD0325901 2004 Pfizer 1‡
Weak Phase II 7.8 h 10,181||
Binimetinib (MEK162, 2006 Novartis/Array 12 Weak Phase III 3.63–7.4 h 182–185
ARRY‑438162) Biopharma
Selumetinib 2007 AstraZeneca/ 14‡ Weak Phase III 5.33 h 68,73,
(AZD6244, Array Biopharma 186
ARRY‑142886)
Refametinib 2009 Bayer AG 19§ Weak Phase II 12 h 114,187,
(RDEA119, BAY 188
869766)
CH4987655 2009 Chugai 5.2 Moderate Phase I 4h 75,79,
(RO4987655) Pharmaceutical 189,190
Co
Pimasertib 2010 Merck KGaA 52‡ Not available Phase II 5h 191
(AS703026,
MSC1936369)
TAK‑733 2011 Takeda 3.2‡ Weak Phase I 48–56 h 192–194
Trametinib 2011 GlaxoSmithKline 0.7 Moderate Approved for ~4 days 16,75,195
(GSK1120212) BRAFV600E/K‑mutant
melanoma
CH5126766 2012 Chugai 160‡ Strong Phase I 60 h 75,78,196
(RO5126766) Pharmaceutical
Co
Cobimetinib 2012 Genentech 4.2 Weak Phase III 40 h 74,110,
(GDC‑0973, XL518) (Roche) 197
GDC‑0623 2013 Genentech 5 Strong Phase I 4–10 h 74,198
(Roche)
*No unified protocol was used to generate the in vitro MEK1 IC50 values stated and so these should be treated as a rough guide to potency only. IC50 values shown
for CH4987655, 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).

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Feedback relief
ERK1/2‑catalysed feedback
phosphorylation and inhibition
of RAF normally operates in
the pathway but is relieved
when MEK1/2 are inhibited
and ERK1/2 activity declines,
resulting in the activation of
RAF and further MEK1/2
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, dem-
onstrated 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 cer-
tain off-target effects65,66 stimulated the development
of further generations of MEKi (TABLE 1).
PD184352 was the first MEKi to be evaluated in vivo
and was shown to inhibit the growth of CRC tumour
xenografts14. Crystal structures of MEK1 bound to ana-
logues of PD184352 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
specific68. 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 pos-
sible partial occlusion of the ERK1 and ERK2 activation
loop binding site67–69. Although the clinical progression
of PD184352 was curtailed by poor biological availability
and potency 70, the past 10 years have seen the discov-
ery 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 mechanisms71; 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 dephosphory­
lation 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,
PD0325901, selumetinib (also known as AZD6244
and ARRY-142886) and cobimetinib 72–75. 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
RAF RAF RAF RAF
• CH5126766
• GDC-0623
P P P P P P • Trametinib
MEK1/2 MEK1/2 MEK1/2 MEK1/2

P P • PD0325901 P P
ERK1/2 • Selumetinib ERK1/2 ERK1/2
• Cobimetinib

DUSP
• Proliferation Adaptive
• Survival resistance

Figure 4 | MEKis that inhibit MEK1/2 phosphorylation suppress the rebound in ERK1/2 activation that results
Nature Reviews | Cancer
from relief of negative feedback. a | With the exception of tumour cells that harbour activating BRAF mutations, ERK1
and ERK2 (ERK1/2) signalling is subject to extensive ERK1/2-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 (MEK1/2) inhibition with a MEK inhibitor (MEKi) blocks ERK1/2 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 PD0325901,
selumetinib and cobimetinib, do not disrupt the phosphorylation of the MEK1/2 activation loop sites and so treatment
with these MEKis and concomitant relief of negative feedback typically results in the accumulation of phosphorylated
MEK1/2. This is thought to explain the rebound in phosphorylated ERK1/2 and pathway output that is observed with
these MEKis in various contexts, notably in tumour cells with mutant RAS74,75. c | A subset of newer MEKis, so-called
‘feedback busters’, including trametinib, CH5126766 and GDC‑0623, disrupt the conformation of the MEK1/2 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 MEK1/2
phosphorylation or rebound in ERK1/2 activity is observed (right), translating into more durable pathway inhibition and
superior efficacy in preclinical models75.

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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.
NATURE REVIEWS | CANCER
especially BRAF‑V600E melanoma, principally because
BRAF‑V600E is fully active as a monomer and insensi-
tive to ERK1- and ERK2‑dependent inhibitory phospho-
rylation and the inhibitory action of SPRY proteins, but
also because the activity of RAS and CRAF is typically
low in BRAF‑V600E melanoma72,76,77. Intriguingly, some
newer MEKis — including trametinib, CH5126766 and
GDC‑0623 — while inhibiting negative feedback mecha-
nisms, mitigate the consequences of this by reducing the
phosphorylationn of MEK by RAF74,78 (FIG. 4). This may
explain why these inhibitors cause more durable sup-
pression of ERK1 and ERK2 phosphorylation, cell pro-
liferation and xenograft growth in the context of RAS
mutations74,75. It is important to understand the under­
lying 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 inter­actions
between the MEKi and the MEK1 and MEK2 activation
loop residues. For example, GDC‑0623 is proposed to form
a stronger hydrogen bond inter­action with Ser212 than
other inhibitors, thereby constraining the activation loop
and disrupting phosphorylation by RAF74. However, crys-
tal structures of MEK1 bound to various allosteric MEKis
— such as CH4987655, CH5126766, and PD184352- and
PD0325901‑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 phosphory­lation
correlates with the extent to which a MEKi coordinates
with Asn221 and Ser222 to displace the activation loop75,79.
Whereas CH5126766 binds both Asn221 and Ser222 and
causes activation loop displacement, a near-identical
enantiomer of PD0325901 does not coordinate Asn221
and Ser222 or displace the activation loop, which is con-
sistent with the differing abilities of these inhibitors to
disrupt MEK1 and MEK2 phosphorylation69,75. 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 dual-
phosphorylated 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–MEK1/2 complexes. MEKis also
modulate the interaction between MEK1 or MEK2 and
RAF, and although this may influence their ability to sup-
press MEK1 and MEK2 activity, no simple correlation
is apparent. For example, selumetinib and PD0325901
induce the binding of all three RAF kinases to MEK1 and
MEK2, and this seems to attenuate the activity of these
MEKis75. By contrast, GDC‑0623 and CH5126766 also
promote the association of RAF with MEK1 and MEK2,
© 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
but inhibit their phosphorylation74,75,78. Both trametinib
and cobimetinib promote the dissociation of RAF–
MEK1/2 complexes, but whereas trametinib antago-
nizes MEK1 and MEK2 phosphorylation, cobimetinib
does not 74,75. Thus, although a firm causal relationship
between MEKi activity and the association or disso-
ciation of the RAF–MEK1/2 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 BRAFV600E
mutations. Intriguingly, MEKis that disrupt MEK1 and
MEK2 phosphorylation seem to have a relatively weaker
affinity for dual-phosphorylated MEK1/2 than do the
MEKis that permit the accumulation of phosphorylated
MEK1/2. The binding of GDC‑0623 to a constitutively
active phosphomimetic mutant of MEK1 and the binding
of trametinib to MEK1 that had been pre-phosphory­
lated in vitro were both markedly weaker than binding to
wild-type MEK1 and unphosphorylated MEK1, respec-
tively 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‑MEK1/2 levels. These observa-
tions 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 BRAFV600E-mutant
xenograft models72. 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 identifica-
tion 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 path-
ways. Primary sensitivity to MEKi correlates with the
decreased expression of cyclin D1 (CCND1), expres-
sion 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 cancers80
and can confer resistance to ERK pathway inhibitors81.
Indeed, activating mutations in RAS, BRAF or MEK1 and
MEK2 can co­operate with CDKN2A loss in a variety of
tumours82–84. Such results have led to the effective combi-
nation of BRAFis or MEKis with inhibitors of CDK4/6 in
preclinical models85,86. Other signalling pathways — such
as PI3K, adenomatous polyposis coli (APC)–β‑catenin,
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REVIEWS

PDGFRβ,
EGFR or HER2 HER3 VEGFR or AXL FGFR2 or FGFR3

RAS P PI3K

RAF RAF

STAT3
MEKi MEK1/2 ERK1/2

Cytoplasm • Proliferation
• Survival
Nucleus
ERK1/2

CtBP MYC ?
Inhibit gene
BETi BETi BETi
expression
ERBB3 PDGFRB FGFR2
NRG1 VEGFR2 FGFR3
AXL

Figure 5 | Adaptive kinome reprograming arising from MEK1/2 inhibition. Active ERK1 and ERK2 (ERK1/2) chronically
Nature Reviews
inhibit signalling from an array of receptor tyrosine kinases (RTKs) by direct phosphorylation of receptors | Cancer
at inhibitory 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 ERK1/2‑dependent phosphorylation
of transcriptional regulators such as CtBP and MYC (solid arrows). Inhibition of MEK1 and MEK2 (MEK1/2) 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 signalling28,96–99 (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 transcription100. 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 BRAFV600E,
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 resist-
ance to MEKis87,88. 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 combinations89,90 and
ongoing clinical trials91. STAT3 activation has been shown
to promote MEKi resistance; combining a STAT3 inhibi-
tor with selumetinib overcame resistance and promoted
tumour cell death92,93. 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 cells94.
Loss of feedback inhibition and MEK–ERK reactiva-
tion. Although MEKis have shown great promise in
BRAF‑V600E preclinical models16,47, as well as some
clinical activity in BRAF‑V600E melanoma95, their
more limited success in RAS-mutant tumours may well
be due in large part to the loss of ERK1/2‑dependent
feedback and reactivation of the MEK–ERK1 pathway.
For example, tumour cell lines with BRAFV600E exhibited
durable inhibition of ERK1 and ERK2 and were very
sensitive to PD0325901, whereas tumour cell lines with
mutations in KRAS were notably resistant to PD0325901
and exhibited strong ERK1 and ERK2 reactivation within
24–48 hours of drug administration75. 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 mecha-
nism — such as trametinib, CH5126766, GDC‑0623 and
G-573 — that inhibit MEK1 and MEK2 kinase activity
and prevent MEK1/2 phosphorylation by RAF74,75 (FIG. 4).
Interestingly, despite the existence of multiple non-RAF
MAP3Ks that can activate MEK1 and MEK2 (includ-
ing MAP3K8 (also known as COT), some MEKKs and
MLKs31–35) few studies have assessed whether these alter-
native MEK activators are inhibited by ERK-dependent
feedback phosphorylation and thus might contribute to
MEK reactivation following MEK inhibition.
Adaptive kinome reprogramming. ERK1/2 signal-
ling inhibits an array of RTKs such that MEK1 and
MEK2 inhibition elicits rapid RTK activation (FIG. 5).

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For example, ERK1- and ERK2‑mediated phosphory­
lation of EGFR and HER2 (also known as ERBB2) sup-
presses signalling to HER3 (also known as ERBB3) so that
MEKis promote the rapid reactivation of EGFR–HER3
and PI3K‑dependent signalling 96,97. Unbiased, non-
targeted chemical proteomic analysis demonstrated the
activation of multiple RTKs following MEK1 and MEK2
inhibition, which were otherwise repressed by the ERK1
and ERK2 target MYC28. In BRAF-mutant thyroid cancer
cells, BRAFis or MEKis promoted ERK1 and ERK2 reac-
tivation 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 resist-
ance via STAT3 activation99. 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 transcrip-
tional programmes underpin kinome reprogramming, an
alternative strategy is to combine MEKis with inhibitors of
key chromatin reader proteins such as the bromodomain-
containing protein 4 (BRD4). Certainly, JQ1, a BET
domain inhibitor, synergizes well with tyrosine kinase
inhibitors in acute myelogenous leukaemia100 (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 sam-
ples101. The first report of acquired resistance to MEKis
was the identification of a gain‑of‑function mutation
(MEK1P124L) in a metastatic focus of a patient with
melanoma with BRAFV600E, who exhibited prolonged
stable disease under treatment with selumetinib102.
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 RO4927350), as well as in samples from
patients undergoing MEKi therapy 103–105. These ‘on
target’ mutations (MEK1F129L and MEK2Q60P) activate
MEK1/2 or cluster at the allosteric inhibitor-binding
pocket and abrogate MEKi binding (for example,
MEK1L115P, MEK1G128D/L215P, MEK1F129L, MEK1V211D 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 BRAFV600E. Two stud-
ies in four CRC cell lines harbouring BRAFV600E dem-
onstrated the amplification of the mutant BRAF allele;
MEKi resistance was overcome by BRAF RNA interfer-
ence (RNAi) or co‑treatment with a RAF inhibitor 106,107.
BRAFV600E amplification has also been observed in mela-
noma cell lines selected in trametinib105, indicating that
BRAFV600E amplification is a common mecha­nism of
resistance irrespective of the MEKi used. Amplification
of KRASG13D has also been described as a mechanism of
MEKi resistance in CRC cells107, either in isolation or
concurrent with an activating MEK1F129L mutation103,
suggesting that these oncogenes may cooperate to confer
NATURE REVIEWS | CANCER
© 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
MEKi resistance. Thus, amplification of the upstream
driving oncogene (BRAFV600E or KRASG13D) drives resist-
ance 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 pres-
ence of MEKis (FIG. 6), thus highlighting a strong hard-
wired 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 mutations103
and BRAFi plus MEKi in the case of BRAFV600E ampli-
fication106,107. Indeed, the combination of dabrafenib
(a BRAFi) and trametinib (a MEKi) has now received reg-
ulatory approval in advanced-stage melanoma, in which
it increased progression-free survival (compared with
either agent alone) and reduced the incidence of cutane-
ous lesions arising from paradoxical RAF activation in
tissues with wild-type BRAF21,108. However, recent studies
have revealed that acquired resistance may still arise in
patients with BRAFV600E melanoma who are treated with
dual BRAFi plus MEKi blockade through the acquisition
of BRAFV600E ‘ultra’ amplification, MEK1 mutations and/or
loss of the CDKN2A locus, as well as loss of the nuclear
ERK-regulatory phosphatase DUSP4 (REF. 27), echo-
ing the recurring theme of ERK pathway re‑activation.
In the case of KRASG13D 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 trials89–91.
More recently, the first potent, selective ERKis have
been described, including VTX11e, an ATP-competitive
inhibitor of ERK1 and ERK2, and SCH772984, an ATP-
competitive inhibitor of ERK1 and ERK2 that also pre-
vents the activating phosphorylation of ERK1 and ERK2
by MEK1 and MEK2 (REFS 103,104). Of these selective
ERKis, SCH772984 can resensitize tumour cells with
acquired resistance to either BRAFis or MEKis, provid-
ing proof‑of‑principle for combining ERK inhibitors
with BRAFis or MEKis. SCH900353 (MK08353), an
analogue of SCH772984, has now entered clinical trials,
as have BVD‑523, RG7842 and CC‑90003. These new
potent and selective ERK inhibitors add new weapons to
the arsenal of ERK1/2 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 dif-
ferences in the mechanism of inhibition, the pharmaco­
kinetic properties of the MEKis that are currently in
clinical development vary considerably with half-lives
(T0.5) 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 bio-
logical rationale rather than pragmatic choices based
upon pharmacokinetics and tolerance109, and most clini-
cal trials are predicated upon continuous daily dosing.
The exception is cobimetinib, which is administered on
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REVIEWS

a MEK1 or MEK2 mutation prevents MEKi b BRAF amplification maintains or c KRAS amplification increases pool of active
binding or activates MEK1 or MEK2 increases pool of active MEK1 or MEK2 MEK1 or MEK2 but also activates other RAS effectors

KRAS KRAS KRAS KRAS KRAS PLCε

Ca2+

PKC
BRAF BRAF
BRAF BRAF ARAF BRAF CRAF PI3K RAL-GEF

MEK1/2 MEK1/2 MEK1/2 MEK1/2 MEK1/2 MEK1/2 MEK1/2 MEK1/2 PDK1 RAL

P P P P P P
ERK1/2 ERK1/2 ERK1/2 PKB mTOR

Mitigation Mitigation Mitigation

Combine MEKi Combine MEKi Combine MEKi with RAFi
with ERKi with RAFi and PI3Ki, PKBi or mTORi

Figure 6 | Mechanisms of acquired resistance to MEK1/2 inhibitors
(MEKis). Studies in preclinical models and analysis of clinical samples
have revealed two basic mechanisms of acquired resistance to MEK1 and
MEK2 (MEK1/2) inhibitors (MEKis; represented by a red X), and, in both
cases, tumour cells adapt to maintain or re‑activate ERK1 and ERK2
(ERK1/2) in the presence of the drug101, 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 MEK1/2 mutations confer resistance by
reducing MEKi binding or enhancing intrinsic MEK1/2 activity102–105 and
resistance can be overcome by combining MEKi with ERK1/2 inhibitors.
b | Amplification of the upstream driving oncogene, BRAFV600E (REFS 106,107)
or KRASG13D (REF. 107), has also emerged as a mechanism of acquired MEKi
resistance. Selective amplification of the mutant BRAF V600E
allele greatly
Nature Reviews | Cancer
increases the proportion of active MEK1/2, exceeding the drug-inhibited
pool, to reactivate ERK1/2; in this case, resistance can be overcome by
combining a MEKi with a RAF inhibitor (RAFi)106,107. c | Amplification of
KRASG13D can also drive MEKi resistance by the same basic mechanism107
but provides a greater therapeutic challenge. KRASG13D can activate
multiple signalling pathways (such as PI3K, RAL, phospholipase Cε (PLCε),
and so on) and even the combined inhibition of MEK1/2 and PI3K signalling
fails to reverse the acquired resistance to selumetinib that is driven by
amplification of KRASG13D (REF. 107). Hexagons indicate oncogenic
mutations, either those that are present as the primary driving oncogene
and are amplified by MEKi selection (BRAFV600E, KRASG13D) or those that
emerge upon selumetinib selection (for example, MEK1P124L and MEK1F129L).

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 T0.5 of ~40 hours110, its adverse
event profile and evidence of melanoma re‑growth using
schedules with longer drug holidays111.
Pharmacodynamics. The main biomarker for monitor-
ing 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 86–9766 and pimasertib all
reduced levels of p-ERK1 and p-ERK2 in tumour tissue
using immunohistochemistry assays, and in some cases
have shown complete inhibition112–114. On this basis and
on the basis of data from BRAF inhibitors in BRAFV600E
melanoma18 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
mono­therapy. Trametinib has gained regulatory approval
for this indication following rapid clinical develop-
ment 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 dacarbazine95. 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 first-
generation BRAFi115 — is being superseded by BRAFi
plus MEKi combinations, of which three have completed
or are in clinical trials111,116,117. 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 CRC118,119.
However, others have shown sufficient activity to prompt
Phase III trials; for example, MEK162 in NRAS-mutant
advanced-stage melanoma120 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, NSCLC124, uveal melanoma125 and acute
myeloid leukaemia (AML)126,127, and were generally not

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Plexiform neurofibromatosis
A non-cancerous
hyperproliferative disease of
the nerve sheath that is driven
by the loss of the NF1 tumour
suppressor.
Neoadjuvant
A neoadjuvant therapy is
treatment given before primary
therapy; in the context of this
Review, selumetinib is
administered first as adjuvant
therapy and continued until
the radioactive iodine therapy
has been administered.
NATURE REVIEWS | CANCER
considered to warrant further monotherapy trials. For
example, trametinib failed to show superiority over
docetaxel in a trial in KRAS-mutant-positive NSCLC128.
One conclusion from monotherapy trials is that most
KRAS-mutant tumours are not highly addicted to ERK
signalling, at least to the degree that it can be inhibited
by MEKi monotherapy at tolerated doses using chronic
dosing schedules. Possible solutions include better patient
selection, optimizing dosing schedules and combination
therapy. For example, there is a growing appreciation that
relief of feedback RAF phosphorylation and reactivation
of MEK and ERK may limit the monotherapy activity of
the MEKis that do not prevent the phosphorylation
of MEK by RAF (discussed above). Indeed, it is entirely
possible that such feedback relief and reactivation of MEK
is masking the true extent of ‘MEK addiction’ in KRAS-
mutant tumours. Thus, one obvious rational combination
therapy that should be tested for KRAS-mutant tumours is
intrapathway dual inhibition (MEKi plus RAFi or MEKi
plus ERKi).
Although responses to MEKi monotherapy have been
modest in adult solid tumours, emerging data from the
use of selumetinib in paediatric diseases — such as low-
grade paediatric glioma and plexiform neurofibromatosis
(neurofibromatosis type 1 (NF1) with inoperable plexi-
form neurofibromas) — show an encouraging and dura-
ble response rate129,130. Both diseases are characterized by
ubiquitous mutational activation of RAS–ERK signalling
and it is tempting to speculate that as low-grade diseases
these tumours are less able to adapt to MEKis either
through feedback ‘relief ’ or compensatory pathway acti-
vation, perhaps owing to low somatic mutation rates, as
reported recently for pilocytic astrocytoma131.
Clinical activity in drug combinations. Randomized
Phase III trials to assess MEKi in combination with first-
generation BRAFis in BRAF‑V600E‑mutant melanoma
have recently reported a clear benefit of the combination
not only in efficacy but also in tolerability compared with
a BRAFi or MEKi alone111,116,117. This successful combi-
nation paradigm is, however, an outlier due to the two
drugs synergizing in the tumour while antagonizing each
other in normal tissue. Although it is highly unlikely that
this type of combination effect can be reproduced with
other classes of drugs the result highlights the impor-
tant characteristics of a successful drug combination:
mechanistic synergy, appropriate patient population
selection and good tolerance. It is worth noting that the
combination of a MEKi and a first-generation BRAFi is
markedly less active in BRAF-mutant CRC132 in which
relief of feedback and consequent activation of EGFR
signalling is thought to limit response, prompting triple
combination trials including anti-EGFR mono­clonal
antibodies (mAbs) with early, encouraging signs of
activity 133. Beyond dual pathway inhibition in BRAF-
mutant melanoma the furthest advanced MEKi com-
bination is selumetinib and docetaxel, which showed
strong preclinical activity 134,135 and is now in Phase III
trials in KRAS-mutant NSCLC following a small ran-
domized Phase II study in which the combination
showed a benefit over docetaxel alone in terms of overall
© 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
response rate and progression-free survival136. A simi-
larly encouraging response rate for trametinib in com-
bination with docetaxel in NSCLC137 indicates that this
combination is robustly active in this disease; it remains
to be seen to what extent the antitumour efficacy is
compromised by the tolerability burden. Other combi-
nations of MEKi and standard-of-care cytotoxic drugs
have fared less well: trametinib and pimasertib failed
to progress beyond Phase II trials in combination with
gemcitabine in pancreatic cancer 138,139 and selumetinib
in combination with irinotecan in KRAS-mutant CRC
showed only modest activity 140. Combinations with
novel targeted agents, principally targeting PI3K, AKT
or mTORC1, have required dose modifications, and
although responses have been reported (for example,
in NSCLC, pancreatic cancer and low-grade ovarian
cancer) they have not yet shown definitive, confirmed
combination activity in the clinic91,141,142.
Finally, one MEKi combination concept with intrigu-
ing activity that may come close to fulfilling the criteria
of synergy, selected patient population and tolerance
is the use of a MEKi to re‑sensitize iodine-refractory
differentiated thyroid cancer (DTC) to radioactive
iodine (RAI) therapy. The predominant oncogenic
pathway in DTC, RAS–ERK, drives the loss of thyroid
differentiation-specific gene expression, including the
sodium–iodine transporter that takes up RAI. The result
— inadequate uptake of and response to RAI — can be
reversed by MEK inhibition, and this has prompted a
Phase III neoadjuvant trial in patients with DTC who are
at high risk of early relapse143,144.
Adverse clinical events associated with MEKis. Because
MEK1 and MEK2 are rarely mutated in human cancer
there is little prospect of the target-related, genetic thera-
peutic index for a MEKi that is evident with the first gen-
eration of BRAFis in BRAF‑V600E/K melanoma; thus,
normal tissue toxicity will constrain clinical activity, as
evidenced by the difference in response rate between
MEKis and BRAFis in BRAF-mutant melanoma. For
example, trametinib (a MEKi) and dabrafenib (a BRAFi)
achieved overall response rates of 22% and 53%, respec-
tively, as single agents145–147. With at least eight MEKis in
clinical development, the adverse event profile is becom-
ing clear and characteristic: acneform rash, diarrhoea
and fatigue are prevalent and generally well managed,
whereas a range of ocular toxic effects, oedema, creatine
phosphokinase elevations and cardiac complications are
rarer but more troublesome148. The range of ocular toxic
effects and their management have been reviewed else-
where in detail149, and although many of these may be
self-limiting, some effects require dose discontinuation
or dose modification. Therefore, in conclusion, the dose
and target engagement for all MEKis is currently limited
by normal tissue toxic effects.
Combinations of MEKis and first-generation BRAFis
are better tolerated than the respective monotherapies
because the paradoxical activation of ERK signalling
by the BRAFi in BRAF wild-type tissue antagonizes
the pathway inhibition by the MEKi, and the MEKi
limits the paradoxical activation of ERK signalling.
VOLUME 15 | O CTOBER 2015 | 587
REVIEWS
RASness
Refers to a transcriptomic
signature that reports
activation of the RAS pathway
and may predict responses to
RAS pathway drugs.
588 | O CTOBER 2015 | VOLUME 15
In this way, MEKi-induced skin rash and BRAFi-
induced squamous lesions are diminished in combi-
nation 21. Combining MEKis with standard-of-care
cytotoxic chemotherapy or various targeted agents has
invariably increased the toxicity burden for patients.
In the case of selumetinib in combination with cyto-
toxic chemotherapy it has been possible to maintain
the recommended monotherapy dose, albeit with
enhanced toxicity 136,140,150. By contrast, combinations
with targeted therapies have generally required dose
reduction or dosing schedule alteration of one or both
agents141,151; for example, selumetinib in combination
with MK‑2206, an allosteric AKT inhibitor, or the com-
bination of trametinib and pan-Class I PI3K inhibitor
buparlisib (BKM120) showed some signs of clinical
activity but was compromised by dose and schedule
modifications to accommodate the enhanced toxic
effects that are associated with dual-pathway inhibi-
tion91,152. In the absence of a strong and specific clini-
cal efficacy signal it is possible that combination toxic
effects will further limit the clinical development of
MEK inhibitors and agents that target PI3K–AKT
signalling. Tolerance to trametinib in combination
with cytotoxic chemotherapy is more variable: it can
result in non-tolerance when trametinib is combined
with platinum doublet chemotherapy, and it requires
growth factor support when combined with docetaxel,
and dose reduction when combined with peme-
trexed153, although it does not require dose reduction in
combination with gemcitabine138.
Patient populations. Although preclinical studies indi-
cate a preferential activity for MEKis in tumours carry-
ing BRAF and RAS mutations47,48,154,155, clinical validation
of this concept is lacking despite the demonstration of
clinical activity in selected patient populations. Even
the approval of trametinib in BRAFV600E/K-mutant mela-
noma did not demonstrate that trametinib was pref-
erentially active in BRAF-mutant disease, as patients
who did not have mutations in BRAF were not included
in randomized trials95,145. Across many disease types
MEKis have induced clinical responses in tumours
with activating mutations in KRAS, NRAS, MEK1,
GNAQ and GNA11 (guanine nucleotide-binding
protein sub­unit αq and subunit α11) and inactivation
of the RAS GTPase-activating protein NF1 as well as
in tumours in which no pathway-related mutation has
been detected109,118,120–122,125,127,144,145. Progress towards the
validation of patient selection biomarkers will require
more rigorous trials. Although small non­randomized
trials of trametinib in combination with either docet­
axel or pemetrexed in advanced NSCLC127,128,156 indi-
cate that KRAS mutation status does not condition
the response rate or progression-free survival, the
outcome of ongoing randomized studies in NSCLC
with prospective KRAS mutation testing should help
to demonstrate with greater certainty the degree to
which overall clinical benefit, compared with stand-
ard of care, is dependent upon KRAS mutational sta-
tus (ClinicalTrials.gov identifiers NCT01750281 and
NCT01933932).
© 2015 Macmillan Publishers Limited. All rights reserved
Future perspectives
The past decade of MEKis clinical development has thrown
into sharp focus the issues that will need to be addressed
if their potential is to be realized. The use of MEKis with
a shorter T0.5 at higher intermittent doses should test the
hypothesis that a higher degree of pathway inhibition (with
an inhibitor alone or in combination) can improve anti­
tumour efficacy, delay time to resistance and also main-
tain a therapeutic margin by sparing normal tissue157. The
advent of next-generation sequencing platforms and more
sensitive and quantifiable means for testing transcriptome
signatures (such as those that measure ERK pathway out-
put and RASness)48,158 will provide a step change in the
breadth and depth of molecular information around which
to develop patient selection biomarkers. Alternative com-
bination partners are currently progressing rapidly in clini-
cal trials. The success of intrapathway combinations to treat
BRAF-V600E/K melanoma in the clinic is mirrored by pre-
clinical data on KRAS-mutant tumours combining MEKis
with RAF dimer inhibiting BRAFis159 (WO2008120004)
or with ERKis. Indeed, the entry of ERK1 and ERK2
inhibitors into clinical trials means that ‘on-target’ muta-
tions in ERK1 and ERK2 should be anti­cipated as possible
mechanisms of acquired resistance160. Intrapathway com-
binations will be more challenging to develop clinically
owing to the probable added toxicity burden, although
intermittent dosing may mitigate this added burden.
MEK1 and MEK2 inhibition ultimately regulates the G1/S
transition, and therefore efficacy may be compromised
in tumours with lesions on the cyclin D–CDK4/6–RB
pathway (for example, loss of CDKN2A)86,88,161. Indeed,
early clinical data indicate that the combination of MEKis
and CDK4/6is holds some promise162. As MEKis predomi-
nantly exert cytostatic effects, identifying drug combina-
tions that harness ‘MEK addiction’ to promote tumour cell
synthetic lethality will prove increasingly important and
hopefully fruitful. Indeed, this has already been achieved
as a preclinical proof‑of‑principle with BH3 mimetics.
MEKis modulate the apoptotic threshold by stabiliz-
ing the pro-apoptotic protein BIM; in combination with
BH3 mimetics that antagonize either BCL-2 or BCL-XL,
BIM is liberated to neutralize anti-apoptotic BCL‑2 fam-
ily members such as MCL1, or to directly activate BAX,
thus driving tumour cell death163,164 and thereby delaying
the onset of MEKi resistance163. Most importantly, given the
rapid recent advancement and success of tumour immu-
notherapies, it is vital that the combination of MEKis with
immune checkpoint inhibitory antibodies (for example,
programmed cell death protein 1 ligand 1 (PDL1)-specific
antibodies that antagonize the immune-suppressive effects
of PDL1, thereby boosting tumour immunity) is evaluated.
The complex effects of MEKis on the relationship between
the tumour and the immune environment are formida-
ble, comprising both positive (for example, tumour cell
death, release of antigens, increased MHC expression and
increased immune system priming) and negative effects
(for example, inhibitory effects on T cell activation and pro-
liferation)165. Optimization of dose and schedule and the
identification of patient populations in which RAS–ERK
signalling may exert an immune-suppressive effect will be
the key to success.
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© 2015 Macmillan Publishers Limited. All rights reserved
Acknowledgements
The authors thank J. Sebolt-Leopold who provided informa-
tion as a personal communication and apologize to those
whose work was not included owing to space limitations.
Competing interests statement
The authors declare competing interests: see Web version for
details.
DATABASES
NCT01750281:
https://clinicaltrials.gov/ct2/show/NCT01750281
NCT01933932:
https://clinicaltrials.gov/ct2/show/NCT01933932
SUPPLEMENTARY INFORMATION
See online article: S1 (figure)
ALL LINKS ARE ACTIVE IN THE ONLINE PDF
www.nature.com/reviews/cancer