ARTICLE Received 1 Oct 2013 | Accepted 16 Apr 2014 | Published 22 May 2014 DOI: 10.1038/ncomms4901 OPEN Mixed lineage kinases activate MEK independently of RAF to mediate resistance to RAF inhibitors Anna A. Marusiak1, Zoe C. Edwards1, Willy Hugo2,*, Eleanor W. Trotter1,*, Maria R. Girotti3, Natalie L. Stephenson1, Xiangju Kong2, Michael G. Gartside4, Shameem Fawdar1, Andrew Hudson1, Wolfgang Breitwieser5, Nicholas K. Hayward4, Richard Marais3, Roger S. Lo2 & John Brognard1 RAF inhibitor therapy yields significant reductions in tumour burden in the majority of V600E-positive melanoma patients; however, resistance occurs within 2–18 months. Here we demonstrate that the mixed lineage kinases (MLK1–4) are MEK kinases that reactivate the MEK/ERK pathway in the presence of RAF inhibitors. Expression of MLK1–4 mediates resistance to RAF inhibitors and promotes survival in V600E-positive melanoma cell lines. Furthermore, we observe upregulation of the MLKs in 9 of 21 melanoma patients with acquired drug resistance. Consistent with this observation, MLKs promote resistance to RAF inhibitors in mouse models and contribute to acquired resistance in a cell line model. Lastly, we observe that a majority of MLK1 mutations identified in patients are gain-of-function mutations. In summary, our data demonstrate a role for MLKs as direct activators of the MEK/ERK pathway with implications for melanomagenesis and resistance to RAF inhibitors. 1 Signalling Networks in Cancer Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester M20 4BX, UK. 2 Division of Dermatology, Department of Medicine, Jonsson Comprehensive Cancer Center and the University of California, Los Angeles, California 90095-1750, USA. 3 Molecular Oncology Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester M20 4BX, UK. 4 Oncogenomics Research Group, QIMR Berghofer Medical Research Institute, Herston, Brisbane QLD 4006, Australia. 5 Cell Regulation Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester M20 4BX, UK. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.B. (email: [email protected]). NATURE COMMUNICATIONS | 5:3901 | DOI: 10.1038/ncomms4901 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4901 he MLKs are MAP3Ks that regulate both the JNK and p38 (Fig. 1b). Activation of the MEK/ERK pathway was enhanced by MAPK pathways1. They directly phosphorylate MKK4/7 to expression of a constitutively active MLK3 and MLK4b (Fig. 1b), Tactivate the JNK pathway and MKK3/6 to activate the p38 where a proline residue is mutated to an alanine (referred to as pathway in response to extracellular stimuli, leading to regulation P469A for MLK3 and P491A for MLK4b hereafter). This muta- of a diverse array of cellular fates1. The MLK family contains tion results in a constitutively active MLK mutant, by relieving primary family members (MLK1–4, also known as MAP3K9, autoinhibitory binding of this conserved proline to the SH3 MAP3K10, MAP3K11 and KIAA1804, respectively) that have Src domain1. Homologous mutations in MLK1 and MLK2 did not homology-3 (SH3) and CRIB (Cdc42/Rac-interactive binding increase pathway activation, indicating that MLK1/2 are regulated region) domains, and subfamily members (DLK, LZK and ZAK) in a unique manner and autoinhibition may occur by binding to that lack these regulatory domains1. Previous studies have other carboxy-terminal regions of the kinases (Fig. 1b). To indicated that one family member (MLK3) can activate the confirm that activation of the MEK/ERK pathway was dependent RAF/MEK/ERK pathway; however, this activation appeared on MLK kinase activity, rather than scaffolding effects, we independent of kinase activity and the authors suggested MLK3 generated constitutively active KD mutants for MLK3 and MLK4 acted as a scaffold to promote BRAF activation2,3. Consistent that would serve as an open scaffold, but lack kinase activity (KD/ with MLK3 regulating the RAF/MEK/ERK pathway, depletion of P469A or KD/P491A, respectively). Both mutants were unable to endogenous MLK3 suppressed activation of the pathway, and activate the MEK/ERK pathway (Fig. 1c), indicating a functional mice deficient in both MLK2 and MLK3 have reduced activation kinase domain is required for activation of the ERK pathway. of ERK in response to tumour necrosis factor stimulation2,4.In Evaluation of other distant MLK family members and another addition, various studies suggest that MLKs are oncogenic and MAP3K indicated that this function is specific to MLK1–4, as mutations in this family of kinases have been observed in several exogenous expression of LZK, ZAKb or ASK1 did not alter different cancers5–7. MAP3K9 (MLK1) has been identified as a signalling through the MEK/ERK pathway (Fig. 1d). This implies gene that is frequently mutated in melanoma (12 of 85, or 14%, of that regulatory domains present on MLK1–4, such as the SH3 melanoma patients evaluated had MLK1 mutations)8. Recently, domain or the CRIB domain, are required for regulation of the genetic alterations in MLKs have been reported by cancer MEK/ERK pathway. genomics data sets at a frequency of 15, 18 and 25% in cutaneous To determine the mechanism used by MLK1–4 to activate the skin melanomas9–12. However, the role of the MLKs in MEK/ERK pathway, we overexpressed the MLKs in HEK293T melanomagenesis or resistance to RAF inhibitors has not been cells and treated the cells with a pan-RAF inhibitor (L779450) or investigated to date. a MEK inhibitor (U0126). MLK1–4 activated the MEK/ERK Aberrant activation of the MEK/ERK pathway leads to pathway in the presence of the RAF inhibitor but not the MEK tumorigenesis and the role of mutationally activated BRAF as a inhibitor, indicating that MLKs directly phosphorylate MEK driver of metastatic melanoma has been well established13–15. (Fig. 2a and Supplementary Fig. 1a in H157 cells; note both Inhibition of mutationally activated BRAFV600E by vemurafenib L779450 and U0126 blocked EGF-stimulated ERK activation in or dabrafenib results in significant clinical response rates in cells, Supplementary Fig. 1b). These results were verified with V600E-positive metastatic melanoma patients. However, most additional RAF and MEK inhibitors, sorafenib and AZD6244, responses are incomplete (due to innate and adaptive drug respectively (Supplementary Fig. 1c,d). To assess whether the resistance) and, among those patients with objective tumour MLKs directly phosphorylate MEK, we performed in vitro kinase responses, the median duration of response is B6 months due to assays using purified inactive MEK1. Immunoprecipitated full- acquired drug resistance16,17. RAF inhibitor resistance can be length MLK1–4 directly phosphorylated KD MEK1 and the achieved through several mechanisms, including amplification or activity of the kinases was not altered by the presence of RAF or mutations in upstream kinases (RAFs, MEK1 or COT kinases MEK inhibitors (Fig. 2b and Supplementary Fig. 1e). To rule out or genetic alteration in upstream activators such as NRAS, KRAS the possibility that other kinases could co-precipitate with MLKs or epidermal growth factor receptor), ultimately leading to and phosphorylate MEK1, we used purified GST-MLK4 kinase reactivation of the MEK/ERK pathway in a majority of cases18–25. domain in an in vitro kinase assay and observed that the MLK4 Other mechanisms of resistance have also been identified, kinase domain directly phosphorylated MEK1 and was not including activation of the PI3K (phosphoinositide 3-kinase)/ inhibited by RAF or MEK inhibitors (Fig. 2c). This is consistent AKT pathway23,26,27. Thus, there is an intense effort to further with our previous report that purified GST-MLK1 kinase domain understand mechanisms of innate, adaptive and acquired can directly phosphorylate KD MEK1 in vitro28. resistance. Here we describe that MLK1–4 directly phospho- rylate MEK and activate the MEK/ERK pathway independently of RAF kinases. Moreover, we find that increased expression of MLKs reactivate the ERK pathway in melanoma cells. Based on MLKs correlates with drug resistance in patients, implicating our proposed mechanism whereby MLKs can activate the MEK/ their potential role as mediators of resistance to RAF inhibitors in ERK pathway in a manner independent of the RAF kinases, we melanoma. sought to determine whether MLKs may mediate reactivation of this pathway in the presence of RAF inhibitors in V600E-positive melanoma cells. We transiently expressed MLK1–4 and their Results respective KD mutants in A375 cells and treated the cells with MLKs are direct MEK kinases that activate the ERK pathway. vemurafenib (PLX4032). We observed that expression of MLKs In an effort to evaluate the role of the mixed lineage family of reactivated the MEK/ERK pathway in the presence of vemur- kinases (Fig. 1a) in regulating downstream signalling pathways, afenib in a kinase-dependent manner (Supplementary Fig. 2a). we overexpressed WT (wild type), KD (kinase dead) and con- Next, we generated melanoma cell lines (both with V600E stitutively active MLK1–4 in HEK293T cells and assessed mito- mutations: A375 and A2058) where MLK expression could be gen-activated protein kinase (MAPK) pathway activation. induced in response to tetracycline. Vemurafenib effectively Exogenous expression of MLK1–4 resulted in activation of inhibited phosphorylation