Molecular Pathways: Mitogen Activated Protein Kinase (MAPK) Pathway Mutations and Drug Resistance

Home , MAP3K13, MAP3K3, MAP3K4, MAPK10, MAPK12, MAPK8

Author Manuscript Published OnlineFirst on February 13, 2013; DOI: 10.1158/1078-0432.CCR-12-0383 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Antonia L. Pritchard 1 and Nicholas K. Hayward 1*

1. Oncogenomics Research Group, CBCRC Building, Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane, QLD 4006, Australia

*Corresponding Author: Nicholas K. Hayward

Oncogenomics Research Group, Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane, QLD 4006, Australia

[email protected]

Conflict of Interest Statement: No relationship that could be construed as resulting in an actual, potential, or perceived conflict of interest are declared by either Dr. Pritchard or Prof. Hayward.

Word counts:

Abstract: 243

Background + Clinical translational advances: 3320

Number of References: 98

Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 13, 2013; DOI: 10.1158/1078-0432.CCR-12-0383 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ABSTRACT

Receptor tyrosine kinases are a diverse family of transmembrane proteins that can activate

multiple pathways upon ligation of the receptor, one of which is the series of mitogen-

activated protein kinase (MAPK) signalling cascades. The MAPK pathways play critical roles

in a wide variety of cancer types, from haematological malignancies, to solid tumours.

Aberrations include altered expression levels and activation states of pathway components,

which can sometimes be attributable to mutations in individual members. The V600E

mutation of BRAF was initially described in 2002 and has been found at particularly high

frequency in melanoma and certain subtypes of colorectal cancer. In the relatively short time

since this discovery, a family of drugs have been developed that specifically target this

mutated BRAF isoform, which, after results from phase 1 / 2 and 3 clinical trials, was granted

FDA approval in August 2011. While these drugs produce clinically meaningful increases in

progression-free and overall survival, they have not improved mortality rates, due to

acquired resistance. New drugs targeting other members of the MAPK pathways are in

clinical trials or advanced stages of development. It is hoped combination therapies of these

new drugs in conjunction with BRAF inhibitors will counteract mechanisms of resistance and

provide cures. The clinical implementation of next generation sequencing is leading to a

greater understanding of the genetic architecture of tumours, along with acquired

mechanisms of drug resistance, which will guide the development of tumour specific

inhibitors and combination therapies in the future.

BACKGROUND

Cells respond to certain growth factors, cytokines and hormones, via activation of receptor

tyrosine kinases (RTKs), with activation of subsequent signalling cascades ultimately altering

cellular processes and the expression of genes that encode proteins controlling cellular

proliferation, through regulation of the cell cycle, as well as cell death through apoptosis. The

RTKs are transmembrane glycoproteins with an intracellular tyrosine kinase domain that

activate tiers of effector proteins via an initial phosphorylation event. There are four main

signalling pathways induced by members of the RTK family, mediated by: (i) mitogen-

activated protein kinase (MAPK); (ii) the lipid kinase phosphatidylinositol 3 kinase (PI3K); (iii)

signal transducer and activator of transcription (STAT); and (iv) phospholipase Cγ (PLCγ).

The main focus of this review is the pathway mediated via MAPKs, with particular attention

to mutations that lead to dysregulation of cancer cell proliferation, and to recent advances in

pharmaceutical targeting of pathway members for cancer therapy.

The MAPK signalling cascade is highly conserved between different eukaryotic cell types

and is composed of three to five tiers of kinase families (MAP4K, MAP3K, MAP2K, MAPK

and MAPK-activated protein kinases (MAPKAPK); Figure 1), with one or more of each tier

phosphorylating and activating components of the next tier. The canonical MAPK signalling

pathway is shared by four cascades, which are classified by the MAPK family member at the

end of the phosphorylation cascade initiated by the RTK: (i) extracellular signal-related

kinases (ERK) 1 and 2 (ii) jun amino-terminal kinases (JNK) 1, 2 and 3) (iii) p38-MAPK and

(iv) ERK5 (Figure 1). While growth factors are considered to be the main regulators of the

ERK1/2 cascade, cellular stress the main inducer of the JNK and p38-MAPK cascades, and

either growth factors or stress as activators of the ERK5 cascade, considerable evidence

exists for cross-talk between various components of the pathways, meaning that ultimately

the cascades can cooperate to transmit certain signals.

The ERK1/2 signalling cascade

Signalling via small G-proteins, for example the RAS family, activates the ERK1/2 signalling

cascade. The small G-proteins are recruited by RTKs via SH2/SH3 domain containing

protein intermediates, resulting in active RAS binding to MAP3K RAF family members

(Figure 1). Upon activation, RAF subsequently phosphorylates and thus activates MAP2K1/2

(also known as MEK1/2), which thereafter act as dual specificity kinases, phosphorylating

Tyr and Thr residues of the MAPK ERK1/2 proteins (Figure 1). Importantly, thus far, the

ERK1/2 proteins are the only identified targets of MAP2K1/2, indicating there is no

redundancy in the activation of this MAPK tier, despite numerous avenues of cross-talk

higher up the cascade. Active ERK1/2 phosphorylates a number of cytoplasmic proteins,

including members of the MAPKAPK family, cytoskeletal proteins, such as vimentin (1) and

keratin-8 (2), and also translocates to the nucleus, where it activates various transcription

factors, including FOS (3), TP53 (4) and ELK1 (5).

Mutations within members of the ERK1/2 signalling cascade

Oncogenic mutations of the RAS family of genes encode amino acid alterations at three

main residues, Gly12, Gly13 and Gln61, each of which prevent the hydrolysis of bound

guanosine triphosphate to guanosine diphosphate, resulting in a constitutively active form of

the protein. Mutations in members of the RAS family are present in approximately 17% of

human cancers (COSMIC database; Table 1). Constitutive activation of RAS is likely to have

knock-on effects on other branches of the MAPK pathway due to cross-talk between the

upper tiers of the cascades. Activating mutations have also been described in BRAF in

several cancer types including colorectal cancer (6) and non-small cell lung cancer (7). The

mutation frequency is particularly high in melanoma (approximately 45%, compared to 21%

of all cancer types; Table 1), with over 90% of these being mutated at the V600 amino acid

residue (7, 8). The V600 amino acid alteration is not exclusively found in cancers, with pre-

malignant lesions, such as melanocytic nevi, also carrying this mutation (9); the implication

being that BRAF mutations alone are not sufficient to drive malignancy. Mutations in RAF1

(also known as CRAF) are less common (approximately 2%, COSMIC database; Table 1),

however, many studies have shown increased expression of RAF1 in a wide variety of

primary human cancers (10, 11). The paucity of oncogenic mutations in RAF1 has been

hypothesised to be due to 2 sites of phosphorylation being required for activation, thus the

protein would requires 2 separate mutational hits to constitutively turn on the kinase. It is

uncommon for tumours to contain both RAS and RAF mutations, suggesting that activation

of just one member of the cascade is all that is required for oncogenesis.

The JNK1/2/3 signalling cascade

The JNK branch of the MAPK pathway is primarily activated in response to stress stimuli,

such as UV-radiation, DNA damage and inflammatory cytokines; however, it can also be

weakly activated by growth factors and cytokines. These stimuli either directly activate the

G-protein RAC1, or alternatively activate the RAS family, which in turn also recruits and

phosphorylates RAC1, which then acts via the p21-protein activated kinase (PAK) family to

phosphorylate members of the MAP3K family. There are at least fourteen members of the

MAP3K family that have been linked to the activation of the JNK signalling cascade, e.g.

MAP3K4 and MAP3K12 (Figure 1). The MAP3K tier dual-phosphorylates MAP2K4 (MKK4)

at Ser257 and Thr265, or MAP2K7 (MKK7) at Ser271 and Thr275, which in turn activate

MAPK8 (JNK), MAPK9 (JNK2) and MAPK10 (JNK3). JNK and JNK2 are ubiquitously

expressed in humans, while JNK3 is primarily found in brain, heart, and testis. A wide variety

of cellular processes are activated by the JNK pathway, including apoptosis (12), signalling

molecules (13) and transcription factors, such as JUN (14), JUND (15) and TP53 (16).

Mutations in the JNK signalling pathway

Inactivating mutations in MAP2K4 have led to its classification as a tumour suppressor gene

(17); deletion and epigenetic silencing of this gene have also been reported in breast (18),

biliary (18), pancreatic (18) and prostate (19) cancers. While the main targets of MAP2K4

are the JNK family, it also has a degree of cross-talk with members of the p38 branch of

MAPK signalling (20). As JNK has been linked to apoptosis, these inactivating events have

been hypothesised to disrupt the signals intended to trigger cell death. Additionally, RAS

mutations may directly interact with JNK, by-passing the usual pathway through RAC1 (21)

(Figure 1). Recently, an activating RAC1 mutation has been found at high frequency in

melanoma (22, 23).

The p38-MAPK signalling cascade

The p38-MAPKs are strongly activated by environmental stresses and inflammatory

cytokines, however growth factors are able to weakly activate this pathway. p38-MAPKs

have been shown to play an important role in cell-cycle check-point control, at the G0, G1/S

and G2/M transitions, in a cell specific manner. The p38-MAPKs require dual

phosphorylation of the Thr-Gly-Tyr motif in the activation loop, by MAP2K3 (MEK3) or

MAP2K6 (MEK6), in order to become fully enzymatically active. These members of the

MAP2K family are highly specific for the p38-MAPK proteins, however, the MAP3K family

members that activate the MAP2K tier are tissue and stimuli specific; Figure 1. Additionally,

a role for members of the Rho family and heterotrimeric G-protein coupled receptors has

also been implicated (24, 25). There are four members of the p38-MAPK family; MAPK11

(p38-α), MAPK12 (p38-β), MAPK13 (p38-δ) and MAPK14 (p38-γ), which display tissue

specific patterns of expression (26, 27). The four subunits have additionally been shown to

differentially activate certain target substrates, for example, the microtubule-associated

protein tau (28), suggesting specialised functions for each isoform. Downstream targets of

members of the p38-MAPK pathway include the transcription factors TP53 (29), STAT1,

MEF (30) and NF-κB (31), cytoskeletal proteins and other kinases; Figure 1.

Mutations in the p38-MAPK signalling pathway

p38-MAPK cannot be classified as either a tumour suppressor or an oncogene, as it has

roles in preventing cellular proliferation in response to cellular stress causing DNA damage

(32, 33), as well as increasing angiogenesis in response to a hypoxic environment caused

by tumour mass (34). Mutations in the p38-MAPK signalling pathway are relatively rare in

comparison to the other branches of the MAPK signalling network (Table 1). Nevertheless,

studies have shown p38-MAPK dysregulation in haematological malignancies (35, 36) and

breast (37), prostate (38), gastric (39) and lung (40) cancers.

The ERK5 signalling cascade

The ERK5 signal cascade is stimulated by a variety of extracellular stimuli, such as growth

factors (41, 42), inflammatory cytokines (43) and cellular stress such as hypoxia (44) and

laminar shear-stress (45). These signals lead to a variety of cellular responses, including

angiogenesis, anti-apoptosis, cell differentiation and cell proliferation (46-48); Figure 1.

These external stimuli activate the MAP3K tier, including MAP3K2 and MAP3K3, which

specifically phosphorylate the Ser311/Thr315 residues of MAP2K5 (MEK5) (49); MAP2K5 is

the only known protein able to activate ERK5 (Figure 1). In order to fully activate ERK5,

MAP2K5 must dual-phosphorylate the Thr218 and Tyr220 residues of ERK5, which then

results in further phosphorylation of MAP2K5, as well as autophosphorylation of ERK5,

resulting in maximal activity (50, 51).

Mutations in the ERK5 signalling cascade

Oncogenically mutated RAS has been reported to activate the ERK5 pathway in certain cell

types (52), however this finding has not been confirmed by other studies (53); the wild-type

forms of RAS do not appear to stimulate this cascade (54). Mutations in the ERK5 pathway

mainly occur at the MAP4K level, with only a low proportion of mutations reported in other

tiers of the cascade (Table 1). Nonetheless, ERK5 is over-expressed in several endocrine-

related cancer types (55, 56), suggesting a role for this pathway during the malignant

process. Evidence suggests this is mediated, at least in part, via dysregulated miR-143 in

prostate cancer cells (57) and via an over-expression of STAT3 in breast cancer cells (58).

Whether there is a link between these two observations is yet to be elucidated.

CLINICAL-TRANSLATIONAL ADVANCES

Given the important roles played by the MAPK pathway in cancer development and

progression, intense focus has been directed towards therapeutically targeting these

dysregulated signals. While the RAS-protein is mutated in a high proportion of cancers and

impacts on a larger number of signalling cascades, a successful strategy to

pharmacologically target it has yet to be discovered (59). In contrast, other members of the

MAPK network downstream of RAS have proven highly tractable to pharmacologic inhibition.

Successfully developed therapies have included small molecule non‐ATP competitive

allosteric MEK inhibitors (MEKi), e.g. BAY 43‐9006 (Sorafenib); (60) and Trametinib (61),

and inhibitors of RAF family members, including Vemurafenib and Dabrafenib (62, 63). The

BRAF inhibitors (BRAFi) are a particularly novel and innovative direction for emerging

cancer therapies, as they specifically target the V600E/K mutated isoform of the protein.

These BRAFi have been subject to intense research interest and recent reviews succinctly

summarise this wealth of data (64, 65). Paradoxically, the BRAFi are capable of accelerating

growth in tumours wild-type at the V600E mutation location in BRAF (66), which emphasises

the importance of somatic mutation screening for identification of patients most suitable for

recipient of these therapies.

Clinical trials results of MAPK pathway inhibition

Phase 1 / 2 trials of the BRAFi Vemurafenib have shown good response rates in the majority

of melanoma patients (67, 68). Moreover, in a phase 3 trial, Vemurafenib increased

progression-free and overall survival, although not mortality rate, in patients with BRAF

V600E mutation positive melanomas compared to those treated with dacarbazine (62). Initial

trials excluded patients with brain metastasis, however, a recently published phase 2 trial of

Dabrafenib revealed approximately 30% of patients achieved an overall cranial response in

brain metastasis of primary melanoma (69). Stage 2 trials of Vemurafenib for patients with

brain metastases are currently recruiting (Clinical trial identifier; NCT01378975).

Further encouraging results of targeting the MAPK pathway in melanoma come from a

phase 3 trial of the MEK inhibitor Trametinib, which showed significantly improved

progression-free and overall survival compared to patients treated with either dacarbazine or

paclitaxel (70). In a subsequent phase 1 and 2 trial, combined therapy of Trametinib with a

BRAFi (Dabrafenib) showed significantly better response rates and progression-free survival

than either monotherapy (63).

Additionally, targeted agents against activated KIT, an infrequently mutated RTK in

melanoma, have also shown promise in some patients (71-73), although results from large

phase 3 randomized clinical trials of KIT inhibitors are keenly awaited (e.g. NCT01280565).

Despite the initial optimism following the introduction of molecularly targeted therapies in the

clinic, considerable reassessment has been needed, as the vast majority of patients

developed resistance to the drugs. Fortunately, in the case of BRAFi, therapeutic resistance

was identified early and the research field moved quickly to identify potential causes.

Mechanisms of resistance to BRAF inhibitors

In vivo

Investigations comparing tumours from melanoma patients pre- and post- development of

BRAFi resistance have uncovered a multitude of mechanisms leading to

regrowth/recurrence of metastatic melanoma in individuals treated with such targeted

therapies. These include increased expression and signalling through various RTKs, such as

PDGFRB (74, 75) or IGF1R (76), upregulation of the kinase MAP3K8/COT (77),

amplification of the BRAF gene resulting in higher levels of expression (78), alternative

splicing of BRAF leading to dimerization (79), NRAS mutation (74, 79, 80) and mutation of

MEK (81); although there is evidence that the latter does not always lead to development of

BRAFi resistance (75). Interestingly, Vemurafenib did not result in meaningful clinical

outcomes when treating the 8-10% of colon cancers that carry BRAF V600E mutations. The

cause of this was identified as being feedback re-activation of the MAPK pathway via the

RTK EGFR (82), implying that these tumours have innate, rather than an acquired

resistance to BRAFi. This resistance mechanism does not appear to be operative in

melanoma, which has only very low levels of EGFR expression (82). Importantly, each of the

resistance pathways in melanoma has the same endpoint – reactivation of the ERK1/2

branch of the MAPK cascade. Hence, high levels of MAPK activity appear central to, or

essential for, melanoma growth in vivo.

In vitro

A number of studies have addressed mechanisms of BRAFi resistance in vitro by generating

subclones of melanoma cell lines that develop resistance to RAF inhibitors through

prolonged culture and selection with these agents. Like the diverse mechanisms of

resistance that develop in melanoma trials patients in vivo these also have the common

theme of reactivating MAPK. They include upregulation of RAF1 (83) or upstream RTKs

such as FGFR3 (84), as well as mutation of NRAS (85) or KRAS (86).

Stromal mediated

While all of the mechanisms of BRAFi resistance mentioned above are cell autonomous,

Straussman and colleagues demonstrated an alternative paracrine resistance mechanism

(87). They showed that fibroblast secretion of hepatocyte growth factor (HGF) activates the

RTK MET on adjacent melanoma cells leading to reactivation of MAPK and resistance to the

RAF inhibitor PLX4720. Recombinant HGF was sufficient to induce BRAFi resistance in

melanoma cell lines and either HGF neutralizing antibodies or a MET inhibitor blocked

PLX4720 resistance (87). In vivo, this association was supported by a correlation with the

level of stromal cell HGF expression in tumours of BRAF mutation positive melanoma

patients who developed resistance to BRAFi (87). An important implication of this finding is

that dual inhibition of BRAF and HGF or MET could prevent the development of BRAFi

resistance in some patients. Thus clinical trials assessing the efficacy of these combined

agents seem warranted.

Future developments: peering over the horizon

The early promise but subsequent failure of single agent targeted therapies to significantly

improve mortality rates in melanoma has led to a somewhat obvious paradigm shift in

treatment approaches. Combination therapies are now becoming de rigueur. Given the

strong (and potentially necessary) involvement of MAPK signalling in melanoma growth and

the development of resistance to BRAFi, it is likely that dual use of a BRAFi with an inhibitor

of MEK or MAPK will become standard for treating patients with BRAF mutation positive

melanomas. However, this is also likely to be only the backbone of new combination

treatments, with additional agents targeting components of other pathways (e.g. PI3K, PAK,

RAC1 or CDK4), or the immune system (see below), being included. Interestingly, cell lines

with NRAS or KRAS mutations have been shown to be more sensitive to IPA3, a small

molecule inhibitor of PAK1, and sub-cytotoxic doses of IPA3 make cells more sensitive to

MEK inhibitors (88), thus offering evidence that combined therapies might have synergistic

effects and providing possible novel mechanisms by which existing drugs may still yield

clinically meaningful outcomes.

It is highly likely that other mechanisms of resistance exist than those discussed above.

Future studies will undoubtedly also investigate whether epigenetic changes, which can

control activation or silencing of gene expression, occur in cells after BRAFi exposure. Given

the recent data from Yancovitz et al showing that clonality of the BRAF V600E mutation

occurs both intra- and inter- tumours from the same patient (89), it is also highly likely that

clonal selection will play a role in resistance to BRAFi.

The advent of the “whole genome sequencing” era of genetic research has resulted in high

volume screening of melanoma cell lines and tumours for somatic mutations, which has

revealed that it is highly unlikely that driver mutations of a similar frequency to those

described in BRAF or RAS family members (Table 1) have yet to be uncovered in other

members of the MAPK pathway. It remains to be investigated whether the lower frequency

mutations could have collective effects, due to the considerable degree of cross-talk

between branches of the MAPK pathways. Further investigations are clearly required to

elucidate the effects of combinations of less-common mutations, which in turn could point to

new avenues for pharmaceutical intervention. It also remains to be determined whether

endpoints other than the dysregulation of MAPK play a vital role in melanoma tumorigenesis;

for example, GNAQ and GNA11 are frequently mutated in ocular melanoma and feed into

other pathways in addition to the MAPK cascade. If other tumorigenic mechanisms do exist,

they might also be a source of resistance to BRAFi.

An alternative to targeting components of the MAPK (and/or other) signalling pathway in

cancer treatment is the use of a new wave of immune modulators; such as antibodies to

CTLA4 (90), PD1 (91), or PD1L (91), which inhibit negative regulation of cytotoxic CD8+ T-

cells mediated by these ligands/receptors. For example, in a phase 3 trial, an anti-CTLA4

antibody (Ipilimumab) increased overall survival in patients with melanoma compared to

those treated with the glycoprotetin-100 (gp100) melanoma-antigen immunostimulatory

peptide vaccine; combined treatment with Ipilimumab and gp100 did not result in improved

survival compared to Ipilimumab alone (90). In another trial, melanoma patients treated with

a combination of Ipilimumab and dacarbazine showed significantly improved overall survival

compared to dacarbazine alone (92). Results from an ongoing combination trial of BRAFi

plus Ipilimumab (NCT01400451) are eagerly awaited.

Conclusion

In summary, the MAPK pathways activated by RTK ligation have been proven to play an

important role in tumorigenesis, as they control cell cycle and proliferation, as well as

apoptosis, which when dysregulated drives abnormal cellular responses. In 2002 the V600E

mutation in BRAF was discovered at high frequency in melanoma (7) and to a lower degree

in other cancer types (Table 1). In the intervening years, novel therapies were developed

that targeted this mutated isoform of BRAF, as rapidly as 2 years following the initial report

of V600E mutations (93). These therapies have since been refined, resulting in FDA

approval for Vemurafenib being granted in August 2011, with BRAFi being clinically available

as a therapeutic option for metastatic melanoma positive for V600E mutations. While BRAFi

do not improve mortality rates, they do result in clinically meaningful increases in

progression free and overall survival. The speed at which the laboratory findings were

translated into a meaningful clinical outcome bodes well for future driver mutations identified

in the wake of the recent technological advances allowing rapid and ever more consistent

whole genome sequencing. It is our belief that the first vital steps have been taken towards

future treatment regimens where personalised medicine based on tumour mutation status in

combination with standard chemo- and/or immune-therapies will become a regular

therapeutic course.

FIGURE LEGEND

Figure 1: The four canonical mitogen-activated protein kinase (MAPK) signalling pathways, mediating the transmission of an external signal down through the cell to effector proteins that alter cellular proliferation and survival.

Signalling pathways should not be considered as purely linear, as there is considerable cross talk, both between the various MAPK cascades and also with the other receptor tyrosine kinase (RTK) stimulated signalling pathways. This cross talk predominantly occurs between members of the upper tiers of the network, with the lower tiers providing specificity. For simplicity, this figure shows the main linear pathways only.

Additional components of the MAPK super-family exist (e.g. MAP4K2, A-RAF, MAP3K6, MAP3K9, MAP3K10, MAP3K13, MAP3K14, MAPK4, MAPK6, MAPKAPK2, and serine/threonine (Ser/Thr) kinase effector proteins, such as MNK1, MNK2, MSK1, MSK2, RPS6KB1 and RPS6KB2), but as many have not been assigned to specific pathways they have not been included in this figure, in order to aid overall interpretation of the pathways.

Similarly, since there is tissue specific expression of some members of the PAK family, not all have not been included in the Figure.

This figure was based on pathway information available from BioCarta (www.biocarta.com), in accordance with their terms and conditions and with their express permission to use the data in the creation of this diagram.

Table 1: Mutation frequencies of key molecules within the four MAPK canonical pathways reported in the Catalogue of Somatic Mutations in Cancer (COSMIC)†, or compiled from recently published exome/whole genome next generation sequencing data#.

† COSMIC: Catalogue of Somatic Mutations in Cancer (94, 95) # compiled from references (22, 23, 96-100) *Malignant Melanoma: includes all sub-tissues and all sub-histology types, including areas of low-UV exposure, mucosal tissues and those with high-UV exposure.

REFERENCES

1. Janosch P, Kieser A, Eulitz M, Lovric J, Sauer G, Reichert M, et al. The Raf‐1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments. FASEB J. 2000;14:2008‐21. 2. Ku NO, Fu H, Omary MB. Raf‐1 activation disrupts its binding to keratins during cell stress. J Cell Biol. 2004;166:479‐85. 3. Murphy LO, Smith S, Chen RH, Fingar DC, Blenis J. Molecular interpretation of ERK signal duration by immediate early gene products. Nat Cell Biol. 2002;4:556‐64. 4. Milne DM, Campbell DG, Caudwell FB, Meek DW. Phosphorylation of the tumor suppressor protein p53 by mitogen‐activated protein kinases. J Biol Chem. 1994;269:9253‐60. 5. Gille H, Sharrocks AD, Shaw PE. Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c‐fos promoter. Nature. 1992;358:414‐7. 6. Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and mismatch‐repair status. Nature. 2002;418:934. 7. Brose MS, Volpe P, Feldman M, Kumar M, Rishi I, Gerrero R, et al. BRAF and RAS mutations in human lung cancer and melanoma. Cancer Res. 2002;62:6997‐7000. 8. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949‐54. 9. Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins CM, et al. High frequency of BRAF mutations in nevi. Nat Genet. 2003;33:19‐20. 10. Schmidt CA, Oettle H, Ludwig WD, Serke S, Pawlaczyk‐Peter B, Wilborn F, et al. Overexpression of the Raf‐1 proto‐oncogene in human myeloid leukemia. Leuk Res. 1994;18:409‐13. 11. Hwang YH, Choi JY, Kim S, Chung ES, Kim T, Koh SS, et al. Over‐expression of c‐raf‐1 proto‐ oncogene in liver cirrhosis and hepatocellular carcinoma. Hepatol Res. 2004;29:113‐21. 12. Dhanasekaran DN, Reddy EP. JNK signaling in apoptosis. Oncogene. 2008;27:6245‐51. 13. Lee YH, Giraud J, Davis RJ, White MF. c‐Jun N‐terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade. J Biol Chem. 2003;278:2896‐902. 14. Kyriakis JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, et al. The stress‐activated protein kinase subfamily of c‐Jun kinases. Nature. 1994;369:156‐60. 15. Lamb JA, Ventura JJ, Hess P, Flavell RA, Davis RJ. JunD mediates survival signaling by the JNK signal transduction pathway. Mol Cell. 2003;11:1479‐89. 16. Saha MN, Jiang H, Yang Y, Zhu X, Wang X, Schimmer AD, et al. Targeting p53 via JNK pathway: a novel role of RITA for apoptotic signaling in multiple myeloma. PLoS One. 2012;7:e30215. 17. Kan Z, Jaiswal BS, Stinson J, Janakiraman V, Bhatt D, Stern HM, et al. Diverse somatic mutation patterns and pathway alterations in human cancers. Nature. 2010;466:869‐73. 18. Su GH, Hilgers W, Shekher MC, Tang DJ, Yeo CJ, Hruban RH, et al. Alterations in pancreatic, biliary, and breast carcinomas support MKK4 as a genetically targeted tumor suppressor gene. Cancer Res. 1998;58:2339‐42. 19. Royuela M, Arenas MI, Bethencourt FR, Sanchez‐Chapado M, Fraile B, Paniagua R. Regulation of proliferation/apoptosis equilibrium by mitogen‐activated protein kinases in normal, hyperplastic, and carcinomatous human prostate. Hum Pathol. 2002;33:299‐306. 20. Lin A, Minden A, Martinetto H, Claret FX, Lange‐Carter C, Mercurio F, et al. Identification of a dual specificity kinase that activates the Jun kinases and p38‐Mpk2. Science. 1995;268:286‐90. 21. Pincus MR, Brandt‐Rauf PW, Michl J, Carty RP, Friedman FK. ras‐p21‐induced cell transformation: unique signal transduction pathways and implications for the design of new chemotherapeutic agents. Cancer Invest. 2000;18:39‐50. 22. Krauthammer M, Kong Y, Ha BH, Evans P, Bacchiocchi A, McCusker JP, et al. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat Genet. 2012;44:1006‐14. 23. Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat JP, et al. A landscape of driver mutations in melanoma. Cell. 2012;150:251‐63. 24. Marinissen MJ, Chiariello M, Gutkind JS. Regulation of gene expression by the small GTPase Rho through the ERK6 (p38 gamma) MAP kinase pathway. Genes Dev. 2001;15:535‐53.

25. Zhang S, Han J, Sells MA, Chernoff J, Knaus UG, Ulevitch RJ, et al. Rho family GTPases regulate p38 mitogen‐activated protein kinase through the downstream mediator Pak1. J Biol Chem. 1995;270:23934‐6. 26. Jiang Y, Chen C, Li Z, Guo W, Gegner JA, Lin S, et al. Characterization of the structure and function of a new mitogen‐activated protein kinase (p38beta). J Biol Chem. 1996;271:17920‐6. 27. Mertens S, Craxton M, Goedert M. SAP kinase‐3, a new member of the family of mammalian stress‐activated protein kinases. FEBS Lett. 1996;383:273‐6. 28. Goedert M, Hasegawa M, Jakes R, Lawler S, Cuenda A, Cohen P. Phosphorylation of microtubule‐associated protein tau by stress‐activated protein kinases. FEBS Lett. 1997;409:57‐62. 29. Chen Y, Miao ZH, Zhao WM, Ding J. The p53 pathway is synergized by p38 MAPK signaling to mediate 11,11'‐dideoxyverticillin‐induced G2/M arrest. FEBS Lett. 2005;579:3683‐90. 30. Zhao M, New L, Kravchenko VV, Kato Y, Gram H, di Padova F, et al. Regulation of the MEF2 family of transcription factors by p38. Mol Cell Biol. 1999;19:21‐30. 31. Royuela M, Rodriguez‐Berriguete G, Fraile B, Paniagua R. TNF‐alpha/IL‐1/NF‐kappaB transduction pathway in human cancer prostate. Histol Histopathol. 2008;23:1279‐90. 32. Bulavin DV, Higashimoto Y, Popoff IJ, Gaarde WA, Basrur V, Potapova O, et al. Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase. Nature. 2001;411:102‐7. 33. Manke IA, Nguyen A, Lim D, Stewart MQ, Elia AE, Yaffe MB. MAPKAP kinase‐2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation. Mol Cell. 2005;17:37‐48. 34. Pages G, Berra E, Milanini J, Levy AP, Pouyssegur J. Stress‐activated protein kinases (JNK and p38/HOG) are essential for vascular endothelial growth factor mRNA stability. J Biol Chem. 2000;275:26484‐91. 35. Feng Y, Wen J, Chang CC. p38 Mitogen‐activated protein kinase and hematologic malignancies. Arch Pathol Lab Med. 2009;133:1850‐6. 36. Zheng B, Fiumara P, Li YV, Georgakis G, Snell V, Younes M, et al. MEK/ERK pathway is aberrantly active in Hodgkin disease: a signaling pathway shared by CD30, CD40, and RANK that regulates cell proliferation and survival. Blood. 2003;102:1019‐27. 37. Gutierrez MC, Detre S, Johnston S, Mohsin SK, Shou J, Allred DC, et al. Molecular changes in tamoxifen‐resistant breast cancer: relationship between estrogen receptor, HER‐2, and p38 mitogen‐activated protein kinase. J Clin Oncol. 2005;23:2469‐76. 38. Ricote M, Garcia‐Tunon I, Bethencourt F, Fraile B, Onsurbe P, Paniagua R, et al. The p38 transduction pathway in prostatic neoplasia. J Pathol. 2006;208:401‐7. 39. Guo X, Ma N, Wang J, Song J, Bu X, Cheng Y, et al. Increased p38‐MAPK is responsible for chemotherapy resistance in human gastric cancer cells. BMC Cancer. 2008;8:375. 40. Zhang C, Zhu H, Yang X, Lou J, Zhu D, Lu W, et al. P53 and p38 MAPK pathways are involved in MONCPT‐induced cell cycle G2/M arrest in human non‐small cell lung cancer A549. J Cancer Res Clin Oncol. 2010;136:437‐45. 41. Kato Y, Tapping RI, Huang S, Watson MH, Ulevitch RJ, Lee JD. Bmk1/Erk5 is required for cell proliferation induced by epidermal growth factor. Nature. 1998;395:713‐6. 42. Kesavan K, Lobel‐Rice K, Sun W, Lapadat R, Webb S, Johnson GL, et al. MEKK2 regulates the coordinate activation of ERK5 and JNK in response to FGF‐2 in fibroblasts. J Cell Physiol. 2004;199:140‐8. 43. Carvajal‐Vergara X, Tabera S, Montero JC, Esparis‐Ogando A, Lopez‐Perez R, Mateo G, et al. Multifunctional role of Erk5 in multiple myeloma. Blood. 2005;105:4492‐9. 44. Sohn SJ, Sarvis BK, Cado D, Winoto A. ERK5 MAPK regulates embryonic angiogenesis and acts as a hypoxia‐sensitive repressor of vascular endothelial growth factor expression. J Biol Chem. 2002;277:43344‐51. 45. Yan C, Takahashi M, Okuda M, Lee JD, Berk BC. Fluid shear stress stimulates big mitogen‐ activated protein kinase 1 (BMK1) activity in endothelial cells. Dependence on tyrosine kinases and intracellular calcium. J Biol Chem. 1999;274:143‐50.

46. Hayashi M, Fearns C, Eliceiri B, Yang Y, Lee JD. Big mitogen‐activated protein kinase 1/extracellular signal‐regulated kinase 5 signaling pathway is essential for tumor‐associated angiogenesis. Cancer Res. 2005;65:7699‐706. 47. Wang X, Tournier C. Regulation of cellular functions by the ERK5 signalling pathway. Cell Signal. 2006;18:753‐60. 48. Roberts OL, Holmes K, Muller J, Cross DA, Cross MJ. ERK5 and the regulation of endothelial cell function. Biochem Soc Trans. 2009;37:1254‐9. 49. Chao TH, Hayashi M, Tapping RI, Kato Y, Lee JD. MEKK3 directly regulates MEK5 activity as part of the big mitogen‐activated protein kinase 1 (BMK1) signaling pathway. J Biol Chem. 1999;274:36035‐8. 50. Mody N, Campbell DG, Morrice N, Peggie M, Cohen P. An analysis of the phosphorylation and activation of extracellular‐signal‐regulated protein kinase 5 (ERK5) by mitogen‐activated protein kinase kinase 5 (MKK5) in vitro. Biochem J. 2003;372:567‐75. 51. Morimoto H, Kondoh K, Nishimoto S, Terasawa K, Nishida E. Activation of a C‐terminal transcriptional activation domain of ERK5 by autophosphorylation. J Biol Chem. 2007;282:35449‐56. 52. English JM, Pearson G, Hockenberry T, Shivakumar L, White MA, Cobb MH. Contribution of the ERK5/MEK5 pathway to Ras/Raf signaling and growth control. J Biol Chem. 1999;274:31588‐92. 53. Obara Y, Nakahata N. The signaling pathway leading to extracellular signal‐regulated kinase 5 (ERK5) activation via G‐proteins and ERK5‐dependent neurotrophic effects. Mol Pharmacol. 2010;77:10‐6. 54. Fukuhara S, Marinissen MJ, Chiariello M, Gutkind JS. Signaling from G protein‐coupled receptors to ERK5/Big MAPK 1 involves Galpha q and Galpha 12/13 families of heterotrimeric G proteins. Evidence for the existence of a novel Ras AND Rho‐independent pathway. J Biol Chem. 2000;275:21730‐6. 55. Mehta PB, Jenkins BL, McCarthy L, Thilak L, Robson CN, Neal DE, et al. MEK5 overexpression is associated with metastatic prostate cancer, and stimulates proliferation, MMP‐9 expression and invasion. Oncogene. 2003;22:1381‐9. 56. Montero JC, Ocana A, Abad M, Ortiz‐Ruiz MJ, Pandiella A, Esparis‐Ogando A. Expression of Erk5 in early stage breast cancer and association with disease free survival identifies this kinase as a potential therapeutic target. PLoS One. 2009;4:e5565. 57. Clape C, Fritz V, Henriquet C, Apparailly F, Fernandez PL, Iborra F, et al. miR‐143 interferes with ERK5 signaling, and abrogates prostate cancer progression in mice. PLoS One. 2009;4:e7542. 58. Song H, Jin X, Lin J. Stat3 upregulates MEK5 expression in human breast cancer cells. Oncogene. 2004;23:8301‐9. 59. Baines AT, Xu D, Der CJ. Inhibition of Ras for cancer treatment: the search continues. Future Med Chem. 2011;3:1787‐808. 60. Wu S, Chen JJ, Kudelka A, Lu J, Zhu X. Incidence and risk of hypertension with sorafenib in patients with cancer: a systematic review and meta‐analysis. Lancet Oncol. 2008;9:117‐23. 61. Infante JR, Fecher LA, Falchook GS, Nallapareddy S, Gordon MS, Becerra C, et al. Safety, pharmacokinetic, pharmacodynamic, and efficacy data for the oral MEK inhibitor trametinib: a phase 1 dose‐escalation trial. Lancet Oncol. 2012;13:773‐81. 62. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364:2507‐16. 63. Flaherty KT, Infante JR, Daud A, Gonzalez R, Kefford RF, Sosman J, et al. Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med. 2012;367:1694‐703. 64. Jordan EJ, Kelly CM. Vemurafenib for the treatment of melanoma. Expert Opin Pharmacother. 2012;13:2533‐43. 65. Sharma A, Shah SR, Illum H, Dowell J. Vemurafenib: Targeted Inhibition of Mutated BRAF for Treatment of Advanced Melanoma and Its Potential in Other Malignancies. Drugs. 2012;72:2207‐22. 66. Hatzivassiliou G, Song K, Yen I, Brandhuber BJ, Anderson DJ, Alvarado R, et al. RAF inhibitors prime wild‐type RAF to activate the MAPK pathway and enhance growth. Nature. 2010;464:431‐5.

67. Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med. 2010;363:809‐19. 68. Sosman JA, Kim KB, Schuchter L, Gonzalez R, Pavlick AC, Weber JS, et al. Survival in BRAF V600‐mutant advanced melanoma treated with vemurafenib. N Engl J Med. 2012;366:707‐14. 69. Long GV, Trefzer U, Davies MA, Kefford RF, Ascierto PA, Chapman PB, et al. Dabrafenib in patients with Val600Glu or Val600Lys BRAF‐mutant melanoma metastatic to the brain (BREAK‐MB): a multicentre, open‐label, phase 2 trial. Lancet Oncol. 2012;13:1087‐95. 70. Flaherty KT, Robert C, Hersey P, Nathan P, Garbe C, Milhem M, et al. Improved survival with MEK inhibition in BRAF‐mutated melanoma. N Engl J Med. 2012;367:107‐14. 71. Hodi FS, Friedlander P, Corless CL, Heinrich MC, Mac Rae S, Kruse A, et al. Major response to imatinib mesylate in KIT‐mutated melanoma. J Clin Oncol. 2008;26:2046‐51. 72. Lutzky J, Bauer J, Bastian BC. Dose‐dependent, complete response to imatinib of a metastatic mucosal melanoma with a K642E KIT mutation. Pigment Cell Melanoma Res. 2008;21:492‐3. 73. Guo J, Si L, Kong Y, Flaherty KT, Xu X, Zhu Y, et al. Phase II, open‐label, single‐arm trial of imatinib mesylate in patients with metastatic melanoma harboring c‐Kit mutation or amplification. J Clin Oncol. 2011;29:2904‐9. 74. Nazarian R, Shi H, Wang Q, Kong X, Koya RC, Lee H, et al. Melanomas acquire resistance to B‐ RAF(V600E) inhibition by RTK or N‐RAS upregulation. Nature. 2010;468:973‐7. 75. Shi H, Moriceau G, Kong X, Koya RC, Nazarian R, Pupo GM, et al. Preexisting MEK1 exon 3 mutations in V600E/KBRAF melanomas do not confer resistance to BRAF inhibitors. Cancer Discov. 2012;2:414‐24. 76. Villanueva J, Vultur A, Herlyn M. Resistance to BRAF inhibitors: unraveling mechanisms and future treatment options. Cancer Res. 2011;71:7137‐40. 77. Johannessen CM, Boehm JS, Kim SY, Thomas SR, Wardwell L, Johnson LA, et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature. 2010;468:968‐72. 78. Shi H, Moriceau G, Kong X, Lee MK, Lee H, Koya RC, et al. Melanoma whole‐exome sequencing identifies (V600E)B‐RAF amplification‐mediated acquired B‐RAF inhibitor resistance. Nat Commun. 2012;3:724. 79. Poulikakos PI, Persaud Y, Janakiraman M, Kong X, Ng C, Moriceau G, et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature. 2011;480:387‐90. 80. Wilmott JS, Tembe V, Howle JR, Sharma R, Thompson JF, Rizos H, et al. Intratumoral Molecular Heterogeneity in a BRAF‐Mutant, BRAF Inhibitor‐Resistant Melanoma: A Case Illustrating the Challenges for Personalized Medicine. Mol Cancer Ther. 2012;11:2704‐8. 81. Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, Pochanard P, et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J Clin Oncol. 2011;29:3085‐96. 82. Prahallad A, Sun C, Huang S, Di Nicolantonio F, Salazar R, Zecchin D, et al. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature. 2012;483:100‐3. 83. Montagut C, Sharma SV, Shioda T, McDermott U, Ulman M, Ulkus LE, et al. Elevated CRAF as a potential mechanism of acquired resistance to BRAF inhibition in melanoma. Cancer Res. 2008;68:4853‐61. 84. Yadav V, Zhang X, Liu J, Estrem S, Li S, Gong XQ, et al. Reactivation of mitogen‐activated protein kinase (MAPK) pathway by FGF receptor 3 (FGFR3)/Ras mediates resistance to vemurafenib in human B‐RAF V600E mutant melanoma. J Biol Chem. 2012;287:28087‐98. 85. Kaplan FM, Kugel CH, 3rd, Dadpey N, Shao Y, Abel EV, Aplin AE. SHOC2 and CRAF Mediate ERK1/2 Reactivation in Mutant NRAS‐mediated Resistance to RAF Inhibitor. J Biol Chem. 2012;287:41797‐807. 86. Su F, Viros A, Milagre C, Trunzer K, Bollag G, Spleiss O, et al. RAS mutations in cutaneous squamous‐cell carcinomas in patients treated with BRAF inhibitors. N Engl J Med. 2012;366:207‐15.

87. Straussman R, Morikawa T, Shee K, Barzily‐Rokni M, Qian ZR, Du J, et al. Tumour micro‐ environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature. 2012;487:500‐4. 88. Singhal R, Kandel ES. The response to PAK1 inhibitor IPA3 distinguishes between cancer cells with mutations in BRAF and Ras oncogenes. Oncotarget. 2012;3:700‐8. 89. Yancovitz M, Litterman A, Yoon J, Ng E, Shapiro RL, Berman RS, et al. Intra‐ and inter‐tumor heterogeneity of BRAF(V600E))mutations in primary and metastatic melanoma. PLoS One. 2012;7:e29336. 90. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010;363:711‐23. 91. Chen DS, Irving BA, Hodi FS. Molecular Pathways: Next‐Generation Immunotherapy‐‐ Inhibiting Programmed Death‐Ligand 1 and Programmed Death‐1. Clin Cancer Res. 2012. 92. Robert C, Thomas L, Bondarenko I, O'Day S, M DJ, Garbe C, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med. 2011;364:2517‐26. 93. Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, et al. BAY 43‐9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res. 2004;64:7099‐109. 94. Forbes SA, Bindal N, Bamford S, Cole C, Kok CY, Beare D, et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 2011;39:D945‐ 50. 95. Forbes SA, Bhamra G, Bamford S, Dawson E, Kok C, Clements J, et al. The Catalogue of Somatic Mutations in Cancer (COSMIC). Curr Protoc Hum Genet. 2008;Chapter 10:Unit 10 1. 96. Berger MF, Hodis E, Heffernan TP, Deribe YL, Lawrence MS, Protopopov A, et al. Melanoma genome sequencing reveals frequent PREX2 mutations. Nature. 2012;485:502‐6. 97. Nikolaev SI, Rimoldi D, Iseli C, Valsesia A, Robyr D, Gehrig C, et al. Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma. Nat Genet. 2012;44:133‐ 9. 98. Pleasance ED, Cheetham RK, Stephens PJ, McBride DJ, Humphray SJ, Greenman CD, et al. A comprehensive catalogue of somatic mutations from a human cancer genome. Nature. 2010;463:191‐6. 99. Stark MS, Woods SL, Gartside MG, Bonazzi VF, Dutton‐Regester K, Aoude LG, et al. Frequent somatic mutations in MAP3K5 and MAP3K9 in metastatic melanoma identified by exome sequencing. Nat Genet. 2012;44:165‐9. 100. Wei X, Walia V, Lin JC, Teer JK, Prickett TD, Gartner J, et al. Exome sequencing identifies GRIN2A as frequently mutated in melanoma. Nat Genet. 2011;43:442‐6.

Figure 1: Extracellular

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

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

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

MAP3K4 MAP3K12 MAP3K8 MAP3K2 MAP3K3 MAP3K5 MAP3K7 MAP3K11 MAP3K

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

p38-B p38-G ERK1 ERK2 JNK JNK2 JNK3 ERK5 MAPK11 MAPK12 MAPK3 MAPK1 MAPK8 MAPK9 MAPK10 MAPK7 p38-D p38-A MAPK MAPK13 MAPK14

RSK1 RSK2 RSK3 PRAK MSK1 3PK Kinases MAPKAPK5 MAPKAPK3

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

Transcription Factors

Table 1.

Number mutations Mutations in melanoma* Gene Main MAPK (total samples analysed; (total samples analysed; (alternative name) pathway %) %) G-protein/target KRAS, NRAS and ERK-1/-2 29337 (174571; 16.8%)† 1014 (7403; 13.7%)† HRAS RAC1 JNK 8 (658; 1.2%)† 12 (247; 4.9%)# PAK1 JNK 29 (970; 2.9%)† 2 (247; 0.8%)# PAK2 JNK 12 (958; 1.3%)† 1 (247; 0.4%)# PAK4 JNK 13 (958; 1.4%)† 5 (247; 2.0%)# MAP4K tier MAP4K3 (MTK1) ERK5 44 (579; 7.6%)† 8 (247; 3.2%)# MAP4K4 (HGK) ERK5 42 (578; 7.3%)† 8 (247; 3.2%)# MAP4K1 (HPK) p38 44 (1003; 4.4%)† 6 (247; 2.4%)# MAP4K5 (KHS) p38 15 (553; 2.7%)† 2 (247; 0.8%)# MAP3K tier B-RAF ERK-1/-2 27391 (131564; 20.8%)† 5084 (11273; 45%)† RAF-1 (C-RAF) ERK-1/-2 32 (1510; 2.1%)† 3 (247; 1.2%)# MAP3K4 (MTK1) JNK 77 (600; 12.8%)† 1 (247; 0.4%)# MAP3K12 (DLK) JNK 37 (620; 5.9%)† 7 (247; 2.8%)# MAP3K2 (MEKK2) ERK5 7 (1394; 0.5%)† 1 (247; 0.4%)# MAP3K3 (MEKK3) ERK5 28 (993; 2.8%)† 2 (247; 0.8%)# MAP3K8 (MEKK1) ERK5 23 (991; 2.3%)† 4 (247; 1.6%)# MAP3K5 (ASK1) p38 58 (1030; 5.6%)† 27 (247; 10.9%)# MAP3K7 (TAK1) p38 28 (993; 2.8%)† 1 (247; 0.4%)# MAP3K11 (PTK1) p38 24 (990; 2.4%)† 9 (247; 3.6%)# MAP2K tier MAP2K1 (MEK1) ERK-1/-2 52 (1468; 3.5%)† 7 (247; 2.8%)# MAP2K2 (MEK2) ERK-1/-2 13 (516; 2.5%)† 5 (247; 2.0%)# MAP2K7 (MKK7) JNK 19 (1449; 1.3%)† 0 (247; 0%)# MAP2K4 (MEK4) JNK 100 (3658; 2.7%)† 4 (247; 1.6%)# MAP2K5 (MEK5) ERK5 16 (555; 2.9%)† 5 (247; 2.0%)# MAP2K3 (MEK3) p38 0 (781; 0%)† 11 (247; 4.5%)# MAP2K6 (MEK6) p38 20 (1420; 1.4%)† 5 (247; 2.0%)# MAPK tier MAPK3 (ERK1) ERK-1/-2 10 (1417; 0.7%)† 0 (247; 0%)# MAPK1 (ERK2) ERK-1/-2 17 (1413; 1.2%)† 4 (247; 1.6%)# MAPK8 (JNK) JNK 34 (1608; 2.1%)† 5 (247; 2.0%)# MAPK9 (JNK2) JNK 26 (1414; 1.8%)† 0 (247; 0%)# MAPK10 (JNK3) JNK 3 (1393; 0.21%)† 6 (247; 2.4%)# MAPK7 (ERK5) ERK5 30 (1414; 2.1%)† 4 (247; 1.6%)# MAPK11 (p38-β) p38 6 (1398; 0.4%)† 1 (247; 0.4%)# MAPK12 (p38-γ) p38 8 (1404; 0.6%)† 0 (247; 05)# MAPK13 (p38-δ) p38 12 (1609; 0.7%)† 2 (247; 0.8%)# MAPK14 (p38-α) p38 16 (1446; 1.1%)† 5 (247; 2.0%)# MAPKAPK tier RSK1 (RPS6KA1) ERK-1/-2 20 (978; 2%)† 4 (247; 1.6%)# RSK2 (RPS6KA3) ERK-1/-2 41 (1515; 2.7%)† 0 (247; 0%)# RSK3 (RPS6KA2) ERK-1/-2 45 (1618; 2.8%)† 8 (247; 3.2%)# MSK1 (RPS6KA5) p38 48 (1445; 3.3%)† 5 (247; 2.0%)# MAPKAPK5 (PRAK) p38 13 (1214; 1.1%)† 1 (247; 0.4%)# MAPKAPK3 (3PK) p38 2 (1601; 0.1%)† 2 (247; 0.8%)#

Molecular Pathways: MAP kinase pathway mutations and drug resistance

Antonia L Pritchard and Nicholas K Hayward

Clin Cancer Res Published OnlineFirst February 13, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-12-0383

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2013/02/13/1078-0432.CCR-12-0383. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.