Metabolic stress regulates ERK activity by controlling KSR-RAF heterodimerization

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1 Article Metabolic stress regulates ERK activity by controlling KSR-RAF heterodimerization Amandine Verlande 1,2, Michaela Krafcíková 3, David Potesil 3, Lukás Trantírek 3, Zbynek Zdráhal 3, Moustafa Elkalaf 4, Jan Trnka 4, Karel Soucek 1,5,6, Nora Rauch 7,8,9, Jens Rauch 7,8,9, Walter Kolch 7,8,9 Stjepan Uldrijan 1,2,* & Abstract Altered cell metabolism is a hallmark of cancer, and targeting specific metabolic nodes is considered an attractive strategy for cancer therapy. In this study, we evaluate the effects of metabolic stressors on the deregulated ERK pathway in melanoma cells bearing activating mutations of the NRAS or BRAF oncogenes. We report that metabolic stressors promote the dimerization of KSR proteins with in NRAS-mutant cells, and with oncogenic BRAF in BRAF V600E -mutant cells, thereby enhancing ERK pathway activation. Despite this similarity, the two genomic subtypes react differently when a higher level of metabolic stress is induced. In NRAS-mutant cells, the ERK pathway is even more stimulated, while it is strongly downregulated in BRAF V600E -mutant cells. We demonstrate that this is caused by the dissociation of mutant BRAF from KSR and is mediated by activated AMPK. Both types of ERK regulation nevertheless lead to cell cycle arrest. Besides studying the effects of the metabolic stressors on ERK pathway activity, we also present data suggesting that for efficient therapies of both genomic melanoma subtypes, specific metabolic targeting is necessary. Keywords cell cycle arrest; cell survival; melanoma; metabolic stress; RAF- ERK signaling Subject Categories Cancer; Metabolism; Signal Transduction DOI /embr Received 23 May 2017 Revised 15 November 2017 Accepted 24 November 2017 Published online 20 December 2017 EMBO Reports (2018) 19: Introduction The MAPK (mitogen-activated protein kinase)/erk (extracellular signal-regulated protein kinase) signaling pathway coordinates diverse physiological processes and plays a significant role in the development of human cancers, including melanoma [1,2]. Somatic mutations in BRAF and NRAS genes are common in melanoma. They are found in about 45 and 17% of all cases, respectively ( A glutamic acid substitution for a valine at position 600 (BRAF V600E ) accounts for more than 90% of the mutations in BRAF in cancer and results in a constitutive activation of BRAF kinase activity and downstream MEK-ERK signaling [3]. In NRAS-mutated melanomas, point mutations impair GTPase activity of RAS, locking the protein into its active conformation, or render RAS insensitive to inactivation by the RAS GTPaseactivating proteins [4,5]. Even though both BRAF V600E and mutant NRAS stimulate the ERK pathway, the routes to ERK activation differ. In contrast to the RAS-independent BRAF V600E signaling, mutant NRAS activates MEK kinases through [6]. To ensure proper signal transduction, components of the ERK module are precisely and timely assembled into multiprotein complexes through dedicated interfaces and scaffold proteins [7,8]. One of these scaffolds is the kinase suppressor of Ras ( and in mammals). KSR proteins constitutively bind MEK kinases and present them to RAF for phosphorylation and activation [9,10]. In addition to their scaffolding property, KSR proteins allosterically stimulate RAF catalytic activity independently of RAS [11 13]. Oncogenic NRAS and BRAF signaling alter metabolic pathways in cancer cells to support unrestricted neoplastic proliferation and cell survival [14]. These metabolic alterations promote the production of biosynthetic precursors and macromolecules through the glycolysis pathway in the cytosol and the tricarboxylic acid cycle (TCA) in mitochondria [15]. In proliferating cells, the TCA cycle functions predominantly as a biosynthetic pathway rather than a bioenergetic one, using glutamine as a main carbon source to produce biomass [16]. Glucose and glutamine are major contributors to energy production also in cancer cells, but unlike normal 1 International Clinical Research Center, St. Anne s University Hospital, Brno, Czech Republic 2 Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic 3 Central European Institute of Technology, Masaryk University, Brno, Czech Republic 4 Laboratory for Metabolism and Bioenergetics, Third Faculty of Medicine, Charles University, Prague, Czech Republic 5 Laboratory of Cytokinetics, Institute of Biophysics, Academy of Sciences, Brno, Czech Republic 6 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic 7 Systems Biology Ireland, University College Dublin, Dublin, Ireland 8 Conway Institute, University College Dublin, Dublin, Ireland 9 School of Medicine, University College Dublin, Dublin, Ireland *Corresponding author. Tel: ; uldrijan@med.muni.cz 320

2 Amandine Verlande et al Metabolic stress controls KSR-RAF dimers EMBO reports cells, they rely less on the oxidative phosphorylation (OXPHOS) which takes place in mitochondria [15]. The metabolic disparities between normal and cancer cells are viewed as promising targets for cancer therapy, and several drugs modifying cell metabolism were already approved as cancer treatments with many more currently undergoing clinical testing [17]. While there is a clear role for activated ERK signaling in promoting the metabolic switch supporting cancer cell proliferation [17 19], surprisingly little is known about the potential impact of metabolic perturbations on the activity of ERK signaling in cancers bearing oncogenic BRAF or NRAS mutations. In this study, we analyzed the response of the dysregulated ERK signaling to metabolic perturbations in these two most common genomic melanoma subtypes. We report that metabolic stressors promote RAF association with KSR proteins, enhancing ERK pathway activity. Nevertheless, there are significant differences in the response of the two genomic subtypes when a higher level of metabolic stress is induced, activating AMPK. The ERK pathway is even more stimulated in NRAS-mutant cells, while its activity is abrogated in BRAF V600E -mutant cells. ERK pathway downregulation correlated with BRAF V600E dissociation from KSR, an event mediated by activated AMPK. These two opposite types of ERK regulation nevertheless result in cell cycle arrest. Therefore, we evaluated the potential of metabolic targeting as a possible therapeutic strategy for melanoma. Our data indicated that NRASmutant cells could be better candidates for metabolic targeting compared to BRAF V600E -mutant cells. Results Metabolic stressors promote RAS-independent hyperactivation of the ERK pathway in NRAS-mutant melanoma cells We selected three metabolic stressors for our study that are targeting critical steps in glycolysis and OXPHOS, the two major cellular pathways of energy metabolism. To inhibit glycolysis, we used the hexokinase inhibitor 2-deoxy-D-glucose (2DG), which is currently undergoing clinical testing in phase I/II trials for the treatment of several cancer types. To inhibit OXPHOS, we chose two inhibitors of the mitochondrial complex I, rotenone (Rot) and metformin (Met). Rotenone was selected for its specificity and well-characterized mechanism of action, while metformin was selected because it is widely used in the clinic as a treatment for type 2 diabetes mellitus [17,20,21]. We tested the response of MelJuso, NRAS-mutant melanoma cells, to increasing concentrations of these drugs and observed that all three induced hyperactivation of the ERK pathway after 14 h of treatment (Fig 1A). ERK pathway activation was detectable already after 1 h of treatment (Fig EV1A), but the effect was stronger with more prolonged treatment. We also detected hyperactivation of the ERK pathway in MelJuso cells in response to other metabolic stressors such as 5-thio-D-glucose (5TG, glycolysis inhibitor), 6-aminonicotinamide (6AN, 6-phosphogluconate dehydrogenase inhibitor), oligomycin A (ATP synthase inhibitor), antimycin A (inhibitor of electron transfer at complex III), and piericidin A (NADH dehydrogenase inhibitor) (Fig 1B). Next, we evaluated ERK pathway activation after the treatment with our initial set of metabolic drugs in two other NRAS-mutant melanoma cell lines, IPC298 and SKMel30. As shown in Fig 1C, MEK1/2 and were hyperactivated in both cell lines as well. Since constitutively active RAS and are the upstream components activating MEK kinases in RAS-mutant cells, we measured kinase activity after a 4-h treatment with the metabolic stressors. Endogenous was immunoprecipitated from MelJuso cells and used in an in vitro kinase assay in the presence of recombinant kinase-dead MEK1 (K97M) as a substrate and ATP. As shown in Fig 1D, kinase activity increased upon treatment with the metabolic stressors. The phosphorylation of the N-region serine residue S338 that is required for activation [22] was also enhanced (Fig 1D). Next, we analyzed the binding of to mutant NRAS and, surprisingly, we observed that interacts less with NRAS, while MEK was more activated after 2 h of treatment with the metabolic stressors, especially with rotenone (Fig 2A). is known to be subject to negative regulation by protein kinase A (PKA), preventing its binding to RAS-GTP [23]. The adenylyl cyclase and PKA activator forskolin (Fo) was therefore used as a positive control in this experiment, efficiently disrupting the interaction between and mutant NRAS (Fig 2A). The reduced interaction of with NRAS after the treatment with the metabolic stressors was also observed 14 h post-treatment, and the effect was more pronounced compared to the 2-h time point for 2DG and metformin (Fig 2A). To confirm that the activation of the MEK- ERK pathway after the treatment with the metabolic stressors was independent of NRAS, we tested the effect of the metabolic stress in MelJuso cells depleted of NRAS by RNA interference. While the downregulation of NRAS markedly attenuated basal kinase activation, the NRAS-depleted cells treated with 2DG still exhibited increased kinase activity compared to NRAS-depleted cells (Fig 2B). Additionally, the mutant ( R89L ) that cannot bind to RAS [24] had an enhanced kinase activity in cells treated with 2DG compared to the untreated sample (Fig 2C). Lastly, the inhibitory action of forskolin on MEK activity through the disruption of binding with RAS-GTP was overcome when the metabolic stressors were co-added (Fig EV1B). Together, these data suggested that binding to NRAS is not necessary for the enhanced kinase activity by the metabolic stressors. The commonly accepted view is that the interaction with RAS- GTP serves to displace dimeric proteins from phosphorylated S259, causing conformational changes in that are necessary for its stable activation [25,26]. As we found that metabolic stressors promote activation independently of RAS, next we investigated the phosphorylation status of S259 and the binding of the proteins to the kinase. Dephosphorylation of S259 was detectable after 2 h of treatment with 2DG and rotenone and augmented over the time (Fig 2D), while an 8-h treatment with metformin was necessary to observe a decrease in phosphorylation of this site (Fig 2D). These differences between the two inhibitors might come from the fact that rotenone is an irreversible and much more potent mitochondrial complex I inhibitor than metformin and thus cellular effects are seen earlier. As 2DG and rotenone induced dephosphorylation of S259 at shorter time points than metformin, in the next step we analyzed the binding of the proteins to after a 4-h treatment with these two inhibitors. As shown in Fig 2E, 2DG and rotenone diminished the binding of proteins to. The mutant ( S259A ), which cannot bind in the CR2 domain [27], did not display any differences in binding to between control and 2DG-treated ª 2017 The Authors EMBO reports Vol 19 No

3 EMBO reports Metabolic stress controls KSR-RAF dimers Amandine Verlande et al A 2DG (mm) Rotenone (μm) Metformin (mm) B 5TG (mm) AN (μm) MelJuso : perk/erk MelJuso : perk/erk Oligomycin A (μm) Antimycin A (μm) Piericidin A (μm) MEK1/ : perk/erk C D 2DG (mm) Rotenone (μm) Metformin (mm) IPC : perk/erk MEK1/2 pmek1 SKMel 30 2DG (mm) Rotenone (μm) Metformin (mm) : perk/erk MEK1/ DG Rot Met p S338 : p/ MEK1 IP endogenous MEK1 KD ATP 2DG Rotenone Metformin Figure 1. Metabolic stressors promote kinase activity and activation of the MEK-ERK pathway in NRAS-mutant melanoma cells. A MelJuso, NRAS-mutant cells were treated with 2DG, rotenone, and metformin at the indicated concentrations for 14 h. Cell extracts were Western-blotted for phospho- (), total, phospho-mek1/2 (), and total MEK1/2. B MelJuso cells were treated with 5TG, 6AN, oligomycin A, antimycin A, and piericidin A at the indicated concentrations for 14 h. Cell extracts were Western-blotted for phospho- () and total. C IPC298 and SKMel30, NRAS-mutant cell lines were treated with 2DG, rotenone, and metformin at the indicated concentrations for 14 h. Cell extracts were Westernblotted for phospho- (), total, phospho-mek1/2 (), and total MEK1/2. D MelJuso cells were treated with 2DG (11 mm), rotenone (5 lm), and metformin (10 mm) for 4 h. Endogenous was immunoprecipitated and subjected to kinase assay in the presence of recombinant kinase-dead MEK1 (K97M) (500 ng) and ATP (20 lm). The kinase reaction was Western-blotted for endogenous, pmek1, and total MEK1. activity (%) is relative to the untreated sample. Bars show mean % SEM (n = 6). Differences between untreated and the treated samples were examined with unpaired t-test (2DG, *P = ). Below: MelJuso cells were treated with the metabolic stressors for 4 h and Western-blotted for phospho- (p) S338. levels in the lysate are shown as a loading control. Source data are available online for this figure. 322

4 Amandine Verlande et al Metabolic stress controls KSR-RAF dimers EMBO reports A 2 hrs 14 hrs B NRAS IP NRAS 2DG NRAS NRAS p α-tubulin forskolin (Fo) = Positive control C D - 2DG Rot Met Fo 2 hrs p S259 Myc- pmek1 (short exposure) pmek1 (long exposure) 4 hrs 8 hrs p S259 p S MEK1 IP myc- WT IP myc- R89L MEK1 KD ATP 2DG E F - 2DG - Rot NT FLAG- WT FLAG- S259A 2DG IP FLAG IP FLAG FLAG Figure 2. ª 2017 The Authors EMBO reports Vol 19 No

5 EMBO reports Metabolic stress controls KSR-RAF dimers Amandine Verlande et al Figure 2. Metabolic stressors promote the dissociation of the proteins from the N-terminus of and induce a RAS-independent activation of in NRAS-mutant cells. A MelJuso cells were treated with 2DG (11 mm), rotenone (Rot; 5 lm), metformin (Met; 10 mm), and forskolin (Fo; 10 lm) for 2 h or with 2DG (5.5 mm), rotenone (Rot; 5 lm), metformin (Met; 5 mm), and forskolin (Fo; 10 lm) for 14 h. Endogenous NRAS was immunoprecipitated (IP), and the immunocomplexes were Western-blotted for endogenous NRAS and. Endogenous NRAS,, phospho-mek1/2 (), and total levels in the cell lysates are also shown. B MelJuso cells were transfected with two different sirnas against NRAS (si#1, si#2) and their combination (sipool) or with a non-targeted sirna sequence (NTsiRNA). A double sirna transfection (48 and 72 h) was performed to have an optimal knockdown. Cells were treated with 2DG (11 mm) for 4 h. Cell lysates were Westernblotted for NRAS, phospho- (p), total, and a-tubulin. C Myc-epitope-tagged WT or R89L were transfected into MelJuso cells. After 24 h, cells were treated with 2DG (11 mm) for 4 h. Myc-tagged WT and myctagged R89L were immunoprecipitated and subjected to kinase assay in the presence of recombinant kinase-dead MEK1 (K97M) (500 ng) as a substrate and ATP (20 lm). R89L activity (%) is relative to the untreated R89L sample. Bars show mean % SEM (n = 3). Difference between untreated and 2DG-treated samples was examined with unpaired t-test (**P = ). D MelJuso cells were treated with 2DG (11 mm), rotenone (Rot; 5 lm), metformin (Met; 10 mm), and forskolin (Fo; 10 lm) for 2, 4, and 8 h. Cell extracts were Westernblotted for phospho- (p) S259. levels in the lysate are shown as a loading control. E MelJuso cells were treated with 2DG (11 mm) or rotenone (Rot; 5 lm) for 4 h. Endogenous proteins were immunoprecipitated (IP), and the immunocomplexes were Western-blotted for endogenous and. Endogenous and levels in the cell lysates are also shown. F FLAG-epitope-tagged WT and FLAG-epitope-tagged S259A were transfected into MelJuso cells. Cells were treated with 2DG (11 mm) for 4 h. FLAG-tagged was immunoprecipitated (IP), and the immunocomplexes were Western-blotted for endogenous and FLAG-. Endogenous and FLAG- levels in the cell lysates are also shown. Source data are available online for this figure. samples (Fig 2F). These data indicated that more molecules were in a pro-active conformation after the treatment with the metabolic stressors compared to the non-treated cells. However, it was unclear how the dissociation of the proteins from the CR2 domain of was accomplished as the depletion of NRAS did not prevent enhanced activation after 2DG (Fig 2B) and the R89L mutant was activated in response to 2DG (Fig 2C). Thus, these data suggested that further regulatory events were at play. Metabolic stressors promote interaction with KSR proteins in NRAS-mutant melanoma cells Considering the RAS-independent nature of activation under the conditions of metabolic stress, we searched the literature for RAS-independent mechanisms of RAF activation. The kinase suppressor of Ras (KSR), one of the scaffold proteins of the RAF pathway, has been described to activate RAF in a RAS-independent manner [12]. KSR proteins bind to MEK constitutively, while their binding to RAF is transient [13]. First, we tested by immunoprecipitations whether 2DG and rotenone modulate the interaction between and the KSR proteins ( and in mammals). We overexpressed -epitope-tagged WT and WT in MelJuso cells, immunoprecipitated KSRs using anti- antibody, and found that 2DG and rotenone promoted the interaction of with the KSR proteins (Fig 3A). We also tested the possibility that metabolic stressors might support dimerization of with BRAF, as -BRAF heterodimers could also contribute to enhanced MEK activation. However, we did not observe any interaction of these two kinases in response to 2DG (Fig EV2A). The multi-kinase inhibitor sorafenib was used as a positive control, as it can induce dimerization of with BRAF in NRAS-mutant cells [28]. To form dimers, KSR proteins must partially dissociate from the proteins and localize to the plasma membrane [29 31]. We immunoprecipitated endogenous and observed a significant dissociation of these proteins from upon 2DG treatment (Fig EV2B), suggesting that KSR proteins might be more capable of binding in response to metabolic perturbations. To determine whether the enhanced interaction of with KSR proteins in NRAS-mutant cells is a general mechanism that can be extended to other cell types, FLAG-epitope-tagged WT and -epitope-tagged WT and WT were overexpressed in HEK293 cells, and FLAG- WT was immunoprecipitated after treatment with 2DG or rotenone. 2DG did not promote binding to KSR proteins in HEK293 cells while rotenone did (Fig 3B). However, the binding of to the KSR proteins after 2DG in HEK293 could be induced when the MEK inhibitor PD was added (Fig 3C). PD on its own only slightly induced binding to KSR proteins, demonstrating that the enhanced heterodimer formation after 2DG and PD treatments was due to 2DG action (Fig 3C). Enhanced ERK pathway activation by 2DG probably increased negative feedback from ERK on RAF-KSR dimer formation, and the inhibition of this feedback using a MEK inhibitor stabilized 2DG-induced dimerization. These data indicated that 2DG and rotenone were able to induce the interaction between and KSR proteins in different cellular contexts. The dimerization of RAF with KSR is mediated by their kinase domains and is critical for allosteric activation of the RAF kinase activity [12]. To confirm that the dimerization of with the KSR proteins was required for the increased kinase activity in response to metabolic drugs, we engineered two KSR mutants with impaired transactivation potential: R665H and R718H. The critical arginine residue is located at the center of the dimer interface between RAF and KSR [12,32]. WT and transactivation-impaired forms of KSR were expressed in MelJuso cells and immunoprecipitated. As KSR and MEK proteins form constitutive complexes [9,33], MEK kinase phosphorylation was evaluated in the immunoprecipitates. As depicted in Fig 3D, MEK phosphorylation was enhanced in 2DG-treated WT - and WT -transfected cells, but not in 2DGtreated R665H - and R718H -transfected cells. These data suggested that KSR proteins stimulate kinase activity. Moreover, the co-expression of WT with WT or mutant KSR constructs in HEK293 treated with rotenone induced hyperactivation of the ERK pathway in WT plus WT - or WT -transfected cells, but not in WT plus R665H - or R718H -transfected cells (Fig 3E, left panel). We performed a similar experiment 324

6 Amandine Verlande et al Metabolic stress controls KSR-RAF dimers EMBO reports A B WT WT 2DG IP -KSR WT WT Rot IP -KSR FLAG- WT FLAG- WT - WT - WT FLAG IP FLAG- FLAG C FLAG- WT FLAG- WT - - 2DG PD 2DG PD - - 2DG PD 2DG PD FLAG IP FLAG- FLAG IP FLAG- FLAG FLAG D WT R665H 2DG IP -KSR E FLAG- WT FLAG- K375M Empty Empty WT R718H WT WT R718H vector vector R665H WT WT Rot FLAG- -KSR Figure 3. KSR dimerizes with and enhances its kinase activity under metabolic stress conditions. A -epitope-tagged WT and -epitope-tagged WT were transfected into MelJuso cells. After 24 h, cells were treated with 2DG (11 mm) or rotenone (Rot; 5 lm) for 4 h. -tagged WT and WT were immunoprecipitated (IP), and the immunocomplexes were Western-blotted for and endogenous. - tagged WT, WT, endogenous, phospho- (), and total levels in the cell lysates are also shown. B FLAG-epitope-tagged WT and -epitope-tagged WT or WT were transfected into HEK293 cells. After 24 h, cells were treated with 2DG (11 mm) or rotenone (Rot; 5 lm) for 4 h. FLAG-tagged was immunoprecipitated (IP) and the immunocomplexes were Western-blotted for and FLAG. -tagged WT, WT, FLAG-tagged, phospho- (), and total levels in the cell lysates are also shown. C FLAG-epitope-tagged WT was transfected into HEK293 cells. After 24 h, the cells were treated with 2DG (11 mm) and/or PD (1 lm) for 4 h. FLAG-tagged was immunoprecipitated (IP), and the immunocomplexes were Western-blotted for endogenous (left panel), endogenous (right panel), and FLAG. Endogenous (left panel), endogenous (right panel), and FLAG levels in the cell lysates are also shown. D -epitope-tagged WT, R665H, WT, or R718H were transfected into MelJuso cells. After 24 h, the cells were treated with 2DG (11 mm) for 4 h. - tagged WT, WT, and their respective mutants were immunoprecipitated (IP), and the immunocomplexes were Western-blotted for, phospho-mek1/2 (), and. E -epitope-tagged WT, R665H, WT, R718H, and FLAG-epitope-tagged WT or K375M were transfected into HEK293 cells. After 24 h, cells were treated with rotenone (Rot; 5 lm) for 4 h. Cell extracts were Western-blotted for -tagged WT, WT, FLAG, phospho- (), and total. Source data are available online for this figure. ª 2017 The Authors EMBO reports Vol 19 No

7 EMBO reports Metabolic stress controls KSR-RAF dimers Amandine Verlande et al by replacing WT with its kinase-dead (KD) version, K375M, and observed that the pathway was not activated when cells were treated with rotenone (Fig 3E, right panel). In fact, rotenone even decreased MEK phosphorylation when K375M was expressed, presumably because the overexpressed KD sequesters KSR from endogenous. These data indicated that the dimerization and transactivation of by KSR cause the hyperactivation of the ERK pathway after metabolic stress. NRAS- and BRAF V600E -mutant melanoma cells are differentially regulated by metabolic stressors The above-described experiments showed that metabolic perturbations enhance the activity of the ERK pathway by promoting the interactions between KSR and proteins. Next, we were interested in comparing the response to metabolic stressors of NRASmutant cells to that of the more common genomic melanoma subtype bearing the oncogenic BRAF V600E mutation. In BRAF V600E - mutant A375 melanoma cells, ERK activity was enhanced upon the treatment with 2DG, rotenone, and metformin albeit to a lesser extent than in RAS-mutant cells (Figs 4A and EV3A). The enhanced ERK activity after 2DG was dose-dependent as the pathway was downregulated at the highest concentration tested (11 mm) and this response was similar in RKO, another BRAF V600E -mutant cell line (Figs 4A and EV3A). Next, we examined the response of A375 to other metabolic stressors (Fig 4B), as we did in MelJuso cells (Fig 1B). An increased activity of the ERK pathway was observed in response to 5TG, 6AN, oligomycin A, and piericidin A but not to antimycin A (Fig 4B). However, similarly to 2DG, a decreased ERK pathway activity was detectable at the highest doses used (Fig 4B). As we did in the case of in NRAS-mutant cells, we tested whether oncogenic BRAF and KSR dimerize upon the treatment with our initial set of metabolic stressors in BRAF V600E -mutant cells. Interestingly, oncogenic BRAF associated with endogenous and especially in response to 2DG and rotenone in A375 cells (Fig 4C). HA-epitope-tagged BRAF V600E transiently transfected in HEK293 cells also dimerized with the KSR proteins after the treatment with 2DG (Fig 4D). HA-BRAF V600E bound more strongly to endogenous than in response to metabolic stress in HEK293 cells (Fig 4D), while the opposite was seen in A375 cells (Fig 4C). However, we do not know how cells regulate the distinct dimer formation between the two KSR isoforms and mutant BRAF. It is of note that 2DG could induce the dimerization when BRAF V600E was transfected into HEK293 cells but not when wild-type was overexpressed in the same cell line without the addition of PD (Fig 3B and C). We attributed this difference to the fact that oncogenic BRAF, but not wild-type, can bypass the inhibitory effect of the negative feedback regulation by ERK [34]. As we observed downregulation of the ERK pathway at the highest used concentrations of 2DG (11 mm) (Fig 4A) and other inhibitors (Fig 4B) in A375 cells, we also investigated the association of BRAF V600E with when the cells were treated with increasing concentrations of 2DG. As shown in Fig 4E, 2DG at 5.5 mm promoted binding to oncogenic BRAF and enhanced ERK pathway activation, while a higher dose (11 mm) did not induce heterodimerization of the two proteins. Thus, it appeared that low and high levels of metabolic stress differently impact the behavior of the ERK pathway in BRAF V600E -mutant cells while a uniform hyperactivation of the pathway by metabolic stress was observed in NRAS-mutant cells (Fig 1A C). In the next set of experiments, we investigated possible reasons why BRAF V600E -mutant cells exhibit a reduced ERK pathway activity when exposed to high metabolic stress. Apart from using high doses of 2DG and other metabolic drugs, we noticed that we could induce high metabolic stress by combining metabolic stressors hitting different pathways of cell energy metabolism. As shown in Fig 4F, the combination of 2DG with a mitochondrial complex I inhibitor, rotenone or metformin, downregulated MEK activity in three different BRAF V600E -mutant cell lines while it further enhanced it in NRASmutant cells. To ensure that the metabolic stress induced by the Figure 4. Under metabolic stress, KSR dimerizes with oncogenic BRAF stimulating ERK pathway activation, yet the pathway is downregulated when the stress is higher. A A375, BRAF V600E -mutant melanoma cells were treated with 2DG, rotenone, and metformin at the indicated concentrations for 14 h. RKO, BRAF V600E -mutant colorectal cells were treated with 2DG at the indicated concentrations for 14 h. Cell extracts were Western-blotted for phospho- () and total. B A375 cells were treated with 5TG, 6AN, oligomycin A, antimycin A, and piericidin A at the indicated concentrations for 14 h. Cell extracts were Western-blotted for phospho- () and total. C A375 cells were treated with 2DG (5.5 mm) or rotenone (Rot; 5 lm) for 4 h. Endogenous BRAF V600E was immunoprecipitated (IP), and the immunocomplexes were Western-blotted for endogenous BRAF V600E and endogenous and. Endogenous BRAF V600E,, and levels in the cell lysates are also shown. D HA-epitope-tagged BRAF V600E was transfected into HEK293 cells. After 24 h, cells were treated with 2DG (11 mm) for 4 h. HA-tagged BRAF V600E was immunoprecipitated (IP) with HA antibody, and the immunocomplexes were Western-blotted for HA and endogenous and. HA and endogenous and levels in the cell lysates are also shown. E A375 cells were treated with 2DG (5.5 and 11 mm) for 4 h. Endogenous BRAF V600E was immunoprecipitated (IP), and the immunocomplexes were Western-blotted for endogenous BRAF V600E and. Endogenous BRAF V600E,, phospho- (), total, phospho-ampka T172 (pampka), and AMPKa levels in the cell lysates are also shown. F MelJuso, IPC298, and SKMel30, NRAS-mutant melanoma cells and A375, RVH421, RKO, BRAF V600E -mutant cells were treated with 2DG (5.5 mm) and/or rotenone (Rot; 5 lm) and/or metformin (Met; 5 mm) for 14 h. Cell extracts were Western-blotted for phospho-mek1/2 () and total. G MelJuso and A375 cells were treated with 2DG (5.5 mm), rotenone (Rot; 5 lm), and metformin (Met; 5 mm) for 14 h. Cell extracts were Western-blotted for phospho- AMPKa T172 (pampka), total AMPKa, and a-tubulin. H ATP levels in MelJuso and A375 lysates were measured using a luciferase-based assay after 14 h of treatment with the indicated stressors. Bars show mean % SEM (n = 4). Differences between control and experimental groups were evaluated by Student s t-test: MelJuso cells [2DG (***P < ); rotenone (***P = ); 2DG + rotenone (***P < ); metformin (P = ); 2DG + metformin (**P = )]. A375 cells [2DG (P = ); rotenone (P = ); 2DG + rotenone (**P = ); metformin (P = ); 2DG + metformin (**P = )]. Source data are available online for this figure. 326

8 Amandine Verlande et al Metabolic stress controls KSR-RAF dimers EMBO reports A B 2DG (mm) Rotenone (μm) Metformin (mm) 5TG (mm) 6AN (μm) RKO A : perk/erk A :perk/erk Oligomycin A (μm) Antimycin A (μm) Piericidin A (μm) : perk/erk :perk/erk C D E - 2DG - Rot BRAF V600E IP BRAF V600E HA-BRAF V600E - - 2DG IP HA-BRAF V600E - 2DG 2DG 5.5mM 11mM BRAF V600E IP BRAF V600E HA BRAF V600E BRAF V600E HA p F NRAS-mutated cell lines G MelJuso H - 2DG Rot 2DG Met 2DG Rot Met - 2DG Rot 2DG 2DG Met Rot Met SKMel30 IPC298 MelJuso - 2DG Rot A375 2DG 2DG Met Rot Met p α-tubulin p BRAF V600E -mutated cell lines - 2DG Rot 2DG Met 2DG Rot Met α-tubulin A375 RVH421 RKO (colorectal) Figure 4. ª 2017 The Authors EMBO reports Vol 19 No

9 EMBO reports Metabolic stress controls KSR-RAF dimers Amandine Verlande et al combinations is higher than the stress caused by individual drugs, we measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of the cells after a 4-h treatment with the metabolic stressors. The drug combination decreased the basal levels of ECAR and OCR in both A375 and MelJuso cells (Fig EV3B and C). Furthermore, cells treated with the combinations of metabolic stressors responded only slightly to glucose, oligomycin, and 2DG when challenged using the Seahorse Glycolytic Stress Test Kit, suggesting that the metabolic capacity of these cells was low (Fig EV3B and C). We also evaluated the activity of the central energy sensor, AMP-activated protein kinase (AMPK) in both cell types. We found that the kinase was activated after the treatment with the metabolic stressors alone or in combination in NRASmutant cells but not in BRAF V600E -mutant cells. Here, AMPK was activated solely in response to a high concentration of 2DG (Fig 4E) or the combination of metabolic stressors (Fig 4G), leading to a significant ATP depletion (Fig 4H). The downregulation of the BRAF signaling correlated with AMPK activation in the cells. We, therefore, investigated whether AMPK could play a role in the reduced ERK signaling in BRAF V600E -mutant cells. Two-hour treatment of A375 cells with 2DG plus rotenone activated AMPK and induced the dissociation of from BRAF V600E (Fig 5A). The combination of 2DG and metformin did not activate AMPK, most probably because the 2-h treatment with the drugs was not long enough to activate the kinase and we did not observe the dissociation of from BRAF V600E (Fig 5A). Interestingly, in 2DG plus metformin-treated sample, AMPK was found to strongly bind to BRAF V600E, although the kinase was not activated (Fig 5A). A more prolonged treatment of the cells with 2DG and metformin led to AMPK activation and a partial unbinding of from BRAF V600E (Fig 5B). Under these conditions, we could hardly detect any AMPK in the immunoprecipitated fraction of the 2DG and metformin-treated sample (Fig 5B). Next, we wanted to determine whether AMPK plays a role in the dissociation of from mutant BRAF. HEK293 cells were transfected with HA-BRAF V600E, depleted of AMPKa by sirnas, and the mutant BRAF protein was immunoprecipitated and assessed for binding. As shown in Fig 5C, the binding of with BRAF V600E after 2DG plus rotenone could be rescued, even strengthened, when AMPKa was depleted by RNAi. Furthermore, the activity of the ERK pathway was downregulated in 2DG plus rotenone-treated sample but not in 2DG plus rotenone-treated cells with depleted AMPKa levels (Fig 5C), suggesting that AMPK plays a critical role in regulating the ERK pathway activity by modulating the dimerization of oncogenic BRAF with upon metabolic stress. Importantly, we confirmed these data in A375 cells where the depletion of AMPKa in metabolically highly stressed cells also preserved the dimer formation between endogenous and endogenous BRAF V600E (Fig 5D). Considering the role of AMPK in controlling the dimerization of oncogenic BRAF with KSR, we also investigated whether AMPK could be found at the -KSR complexes under high metabolic stress conditions in NRAS-mutant cells. Surprisingly, AMPK localized at the complexes in these conditions (Fig 5E), and in contrast to BRAF V600E -mutant cells, we did not observe any dissociation of from (Fig 5E). In fact, increased stress promoted AMPK activation, AMPK binding to, and association with and hence also the ERK pathway hyperactivation (Fig 5E). NRAS- and BRAF V600E -mutant cells regulate the ERK pathway to promote cell cycle arrest in response to metabolic stress The above-presented data demonstrated that NRAS- and BRAF V600E - mutant cells differently regulate the ERK pathway when facing high metabolic stress; while the ERK pathway was strongly hyperactivated in NRAS-mutant cells, it was inhibited in BRAF V600E -mutant cells. Thus, our next set of experiments was to investigate the possible reasons for this differential regulation. The RAF-ERK pathway is one of the key signal transduction pathways that participate in cell cycle control. We first analyzed the progression through the S phase of the cell cycle in MelJuso and A375 cells. Importantly, we confirmed that 2DG induced a comparable inhibition of the glycolysis in the two cell lines and that rotenone and metformin promoted a similar decrease in the oxygen consumption at the concentrations used (Fig 6B). 2DG as a single agent significantly diminished the number of cells in S phase in MelJuso but not in A375 cells (Fig 6A). To better understand this disparity between the two cell lines in response to 2DG, we analyzed the percentage of cells in each cell cycle phase using propidium iodide DNA staining. As shown in Fig 6C, MelJuso cells treated with 2DG massively accumulated in G0/G1 phase (65% vs. 43% control). It Figure 5. AMPK mediates the dissociation of KSR from mutant BRAF but not in high metabolic stress conditions. A HA-epitope-tagged BRAF V600E was transfected into HEK293 cells. After 24 h, cells were treated with 2DG (11 mm) and/or rotenone (Rot; 5 lm) and/or metformin (Met; 10 mm) for 2 h. HA-tagged BRAF V600E was immunoprecipitated (IP) with HA antibody, and the immunocomplexes were Western-blotted for HA, endogenous, and endogenous AMPKa. HA, endogenous, phospho-ampka T172 (pampka), and total AMPKa levels in the cell lysates are also shown. B HA-epitope-tagged BRAF V600E was transfected into HEK293 cells. After 24 h, cells were treated with 2DG (11 mm) and/or metformin (Met; 10 mm) for 3 h. HA-tagged BRAF V600E was immunoprecipitated (IP) with HA antibody, and the immunocomplexes were Western-blotted for HA, endogenous, and endogenous AMPKa. HA, endogenous, phospho-ampka T172 (pampka), and total AMPKa levels in the cell lysates are also shown. C HA-epitope-tagged BRAF V600E and -epitope-tagged WT were transfected into HEK293 cells; 6 h later, one of the transfected dishes was transfected with an AMPKa sirna. After 48 h, cells were treated with 2DG (11 mm) and rotenone (Rot; 5 lm) for 2 h. HA-tagged BRAF V600E was immunoprecipitated (IP) with HA antibody, and the immunocomplexes were Western-blotted for HA and. HA,, phospho- (), and total levels in the cell lysates are also shown. The efficiency of the AMPKa knockdown is shown in the lower panel. D One of the dishes of A375 cells was transfected with an AMPKa sirna. After 48 h, cells were treated with 2DG (5.5 mm) and rotenone (Rot; 5 lm) for 2 h. Endogenous BRAF V600E was immunoprecipitated (IP), and the immunocomplexes were Western-blotted for endogenous BRAF V600E and endogenous. Endogenous BRAF V600E and levels in the cell lysates are also shown. The efficiency of the AMPKa knockdown is presented in the lower panel. E MelJuso cells were treated with 2DG (11 mm) and/or rotenone (Rot; 5 lm) and/or metformin (Met; 10 mm) for 2 h. Endogenous was immunoprecipitated (IP), and the immunocomplexes were Western-blotted for endogenous,, and AMPKa. Endogenous,, phospho-ampka T172 (pampka), total AMPKa, phospho- (), and total levels in the cell lysates are also shown. Source data are available online for this figure. 328

10 Amandine Verlande et al Metabolic stress controls KSR-RAF dimers EMBO reports has been demonstrated that a sustained activation of the ERK pathway is necessary for G1 to S phase progression [35,36]. However, the duration and the magnitude of the activation both have to be controlled as not only the inhibition but also too strong activation of this pathway can lead to a reversible or permanent cell cycle arrest [37,38]. In this study, we showed that 2DG promoted a stronger activation of the ERK pathway in MelJuso than in A375 cells (Figs 1A and 4A, and EV3A) and the difference became even more prominent when the level of metabolic stress was higher (Fig 4F). These data suggested that in MelJuso cells, the enhanced ERK pathway activation after 2DG could be high enough to induce cell cycle arrest in G0/G1 phase, but not in A375 where the activation was A HA-BRAF V600E B HA-BRAF V600E - - 2DG 2DG 2DG Rot Met - - 2DG 2DG Met IP HA-BRAF V600E IP HA-BRAF V600E HA HA HA HA p p C D E HA-BRAF V600E - WT HA IP HA-BRAF V600E - 2DG 2DG Rot 2DG Rot + sirna BRAF V600E IP BRAF V600E - 2DG 2DG 2DG Rot Met IP HA BRAF V600E p α-tubulin α-tubulin Figure 5. ª 2017 The Authors EMBO reports Vol 19 No

11 EMBO reports Metabolic stress controls KSR-RAF dimers Amandine Verlande et al A B C D E A375-2DG MelJuso - 2DG p21 Cip1 α-tubulin F G H - 2DG Rot 2DG Rot Cleaved PARP PARP Cleaved Caspase 3 Caspase 3 Cleaved Caspase 9 Caspase 9 Cytosolic fraction Cytochrome c α-tubulin Figure 6. weaker. Indeed, it did not seem to be sufficient to promote cell cycle arrest or a significant increase in cell proliferation (Fig 6A and C). High intensity or long-term ERK activation can cause cell cycle arrest due to the induction and accumulation of the cell cycle inhibitor p21 [39,40], promoted by an ERK-driven transcriptional induction and increased protein stability [41,42]. As shown in Fig 6D, p21 expression is highly induced by 2DG in MelJuso but not in A375 cells. Together, these data indicated that NRAS-mutant cells hyperactivate the ERK pathway in response to metabolic stress to efficiently block cell cycle progression. This strategy was not adopted in BRAF V600E -mutant cells that still proliferated when subjected to mild metabolic stress (Fig 6A and C). However, when the metabolic stressors were combined, and the levels of stress were higher (Fig 4G and H), the ERK pathway was downregulated (Fig 4F) and the cell cycle progression arrested in BRAF V600E - mutated cells (Fig 6A). 330

12 Amandine Verlande et al Metabolic stress controls KSR-RAF dimers EMBO reports Figure 6. Metabolic targeting might not be an efficient therapeutic strategy for BRAFV600E -mutant cells. A A375 and MelJuso cells were treated with 2DG (5.5 mm) and/or rotenone (5 lm) and/or metformin (5 mm) for 24 h followed by incubation with Edu nucleotide analog for 30 min. EdU-positive cells were visualized using the Click-iT EdU Alexa Fluor 488 Imaging Kit and quantified by flow cytometry. Bars show mean % SEM of EdU-positive cells (n = 4). Differences between control and experimental groups were evaluated by Student s t-test: A375 cells (2DG (P = ); rotenone (P = ); 2DG + rotenone (***P = ); metformin (P = ); 2DG + metformin (***P = ). MelJuso cells (2DG (**P = ); rotenone (P = ); 2DG + rotenone (**P = ); metformin (P = ); 2DG + metformin (**P = ). B ECAR measurements representing the glycolytic flux in control and 2DG-treated cells and OCR measurements depicting the basal respiration in control, rotenone, and metformin-treated cells (n = 3). Cells were pre-treated for 4 h with 2DG (11 mm) and/or rotenone (5 lm) and/or metformin (10 mm). Bars show means SEM (n = 3). Differences between the groups were evaluated by Student s t-test: ECAR (A375 control vs. MelJuso control (*P = )); (A375 2DG vs. MelJuso 2DG (P = )) OCR (A375 control vs. MelJuso control (*P = )); (A375 rotenone vs. MelJuso rotenone (P = )); (A375 metformin vs. MelJuso metformin (P = )). C A375 and MelJuso cells were treated with 2DG (5.5 mm) for 24 h and processed as described in the Materials and Methods section. Bars show mean % SEM of number of cells in each cell cycle phases (G0/G1, S, and G2/M) (n = 3). Differences between control and 2DG were evaluated by Student s t-test: A375 (G0/G1: P = ; S:P = ; G2/M: P = ), MelJuso (G0/G1: ***P < ; S:**P = ; G2/M: P = ). D A375 and MelJuso cells were treated with 2DG (5.5 mm) for 24 h. Cell extracts were Western-blotted for p21 Cip1 and a-tubulin. E Percentage of apoptotic A375 cells after 48 h of treatment with 2DG (5.5 mm) and/or rotenone (5 lm) and/or metformin (5 mm). Bars show means SEM of PIpositive cells (n = 3). Student s t-test: control vs. rotenone (***P = ); rotenone vs. 2DG + rotenone (***P = ). F A375 cells were treated with 2DG (5.5 mm) and/or rotenone (Rot; 5 lm) for 14 h. Cell extracts were Western-blotted for cleaved PARP, PARP, cleaved caspase 3, caspase 3, cleaved caspase 9, and caspase 9. Below: Amount of cytochrome c released in the cytosolic fraction of A375 cells. G Quantification of intracellular glutamine. Before metabolite extraction, cells were treated with 2DG (5.5 mm), rotenone (5 lm), and/or metformin (5 mm) for 14 h. Bars show mean % SEM (n = 3). Differences between control and each group were evaluated by Student s t-test: 2DG (P = ); 2DG + rotenone (***P < ); 2DG + metformin (P = ). H Percentage of apoptotic A375 cells after 48 h of treatment with 2DG (5.5 mm), rotenone (5 lm), and/or metformin (5 mm) BPTES (10 lm). Bars show means SEM of PI-positive cells (n = 3). Differences between the groups were evaluated by Student s t-test: 2DG + rotenone vs. 2DG + rotenone + BPTES (***P < ); 2DG + metformin vs. 2DG + metformin + BPTES (**P = ). Source data are available online for this figure. Analysis of the therapeutic potential of metabolic targeting in malignant melanoma Considering the different strategies to block cell cycle progression in the two genomic melanoma subtypes, we decided to determine whether the metabolic stressors, used separately or combined, could constitute a potential therapeutic strategy for the treatment of NRAS- or BRAF V600E -mutant cells. Most classical small molecule inhibitors of cellular respiration, including rotenone, exhibit significant toxicity and are not suitable for therapeutic use. In contrast, metformin, while being capable of inhibiting complex I activity [43], is commonly used in the clinic as the treatment of type II diabetes [20]. Therefore, we were particularly interested in determining the effect of 2DG as a single treatment and in combination with metformin, but we also used the combination of 2DG plus rotenone for comparison. 2DG, metformin, and their combination did not display any acute toxicity toward A375 and MelJuso cells in vitro (Figs 6E and EV4A). Surprisingly, the concomitant treatment of 2DG with rotenone improved A375 and MelJuso viability compared to rotenone-treated cells (Figs 6E and EV4A). This effect was also observed with 5TG, another glucose analog and glycolysis inhibitor. However, we did not observe a similar protective effect when rotenone was combined with inhibitors of other essential carbon sources, for example, the glutaminase inhibitor BPTES or the inhibitor of fatty acid b-oxidation etomoxir (Fig EV4B), or when 2DG was replaced by glucose-free medium (Fig EV4C). The unexpected protective effect of 2DG against rotenone-induced toxicity was confirmed when we analyzed the activation of caspases, specific proteases that are required for the execution of apoptotic cell death. In A375 cells, rotenone induced the cleavage of procaspase-9 and procaspase-3 and of poly(adp-ribose) polymerase (PARP), a cellular target of caspase-3. Activation of caspases and PARP cleavage were reduced when rotenone was applied concomitantly with 2DG (Fig 6F). We also examined the release of cytochrome c into the cytosol, another apoptotic feature, and observed that its release was also reduced when 2DG was used together with rotenone, compared to rotenone alone-treated cells (Fig 6F). Next, we tested the possibility that autophagy, an important catabolic process involved in cell survival and death [44], might play a role in the 2DG-protective effect of rotenone-treated cells. For that, we used bafilomycin A1 (Baf A1), an inhibitor of the late phase of autophagy. bafilomycin A1 induced a robust increase in LC3-II, a specific marker of autophagosome formation, in A375 and MelJuso cells (Fig EV4D), indicating autophagy blockage. Interestingly, cells treated concomitantly with bafilomycin A1 and the combination of metabolic stressors accumulated less LC3-II than cells treated with bafilomycin A1 alone (Fig EV4D), suggesting that these cells did not turn on autophagy even though AMPK was activated (Fig EV4D). We also evaluated whether autophagy could have an impact on the improved cell viability after the treatment with the combination of metabolic drugs. As shown in Fig EV4E, bafilomycin A1 did not significantly increase cell death in any of the cell types when added to the mix of metabolic stressors. Therefore, we concluded that autophagy did not participate in the 2DG-induced protective effect of rotenone-treated cells. In the next step, we tested whether this pro-survival effect could be linked to the reduced glycolytic flux and substrate availability for the TCA cycle caused by 2DG. For that, we added the membrane-permeable pyruvate analog, methyl-pyruvate (MP) to 2DG and rotenone-treated cells. To confirm that the externally supplied MP is taken up by the cells, we monitored the levels of alanine, which can be produced from pyruvate, by NMR analysis. As this reaction is coupled to the conversion of glutamate to a-ketoglutarate (AKG), we also analyzed the level of AKG in the cells. Intracellular alanine increased to about 400% and intracellular AKG to approx. 1,500% of the control levels in MP-treated A375 cells, suggesting that the exogenously provided MP is indeed metabolized by the cells (Fig EA). MP effectively restored cell death, indicating that the glycolytic flux is probably contributing to the decreased viability of melanoma cells treated with rotenone ª 2017 The Authors EMBO reports Vol 19 No

13 EMBO reports Metabolic stress controls KSR-RAF dimers Amandine Verlande et al (Fig EB). The NMR analysis also revealed that cells treated with 2DG in combination with metformin or rotenone accumulated significantly more (approx. 300%) intracellular glutamine than control cells (Fig 6G). Glucose and glutamine are the two most important carbon sources for cell growth, and glutamine was shown to maintain TCA cycle and cell viability when mitochondrial pyruvate transport is inhibited [45]. Reductive carboxylation of glutamine-derived AKG is stimulated when TCA cycle function is altered [46 49]. To determine whether glutamine metabolism could be contributing to the maintenance of cell viability in 2DG and rotenone-treated A375 cells, we inhibited glutamine utilization with BPTES. The glutaminase inhibitor reverted the 2DG-induced protective effect (Fig 6H), suggesting that A375 cells facing metabolic perturbations rely on glutamine to preserve their viability. Importantly, the addition of BPTES strongly potentiated the cytotoxicity of 2DG and metformin combination. Discussion Fast-growing tumors are often subjected to periods of metabolic stress caused by factors such as hypoxia or lack of nutrients [50]. In our study, we induced metabolic perturbations in the two most common genomic melanoma subtypes, bearing mutated BRAF and NRAS genes, and examined the effects on RAF signaling and cellular functions. Our data suggest that metabolic stress promotes dimerization of the KSR proteins with RAF kinases; that is, in NRAS-mutant cells and oncogenic BRAF in BRAF V600E -mutant cells, stimulating ERK signaling. In NRAS-mutant cells, the formation of dimers positively regulated kinase activity. Critically, expression of transactivation-impaired forms of KSR, R665H and R718H, abrogated the enhancement of kinase activity induced by metabolic stressors. In BRAF V600E -mutant cells, the formation of dimers did not regulate the activity of oncogenic BRAF as the mutant kinase is constitutively activated. However, KSR invariably associates with the RAF substrate MEK [9,10] and hence, its heterodimerization with BRAF V600E could bring MEK and oncogenic BRAF into closer proximity. The mutant kinase has been described to signal primarily as a monomer which does not require dimerization for ERK pathway activation. Yet, it has been shown that the capacity of BRAF V600E to activate ERK is significantly compromised in the absence of [51], indicating that KSR function is necessary for the oncogenic potential of BRAF V600E. Additionally, we found that in NRAS-mutant cells, the activation of by metabolic stress was independent of its upstream regulator NRAS. Notably, mutant ( R89L ) that cannot bind to RAS [24] had an increased kinase activity when cells underwent metabolic stress. This result does not constitute the first example of RAS-independent activation of RAF. For example, it has been shown that RNA interference-mediated knockdown of RAS does not affect the ability of KSR to drive RAF activation in Drosophila Schneider S2 cells [12]. Leicht et al [52] also demonstrated that MEK1 could activate in a RAS-independent manner, an event which was mediated by the interaction of both kinases. The authors suggest that the association might promote a conformational change that enhances susceptibility to phosphorylation and activation or that the interaction could stabilize the phosphorylated active form of. Similar mechanisms could be responsible for the positive effect of the enhanced interaction between KSR and under metabolic stress on kinase activity. While the ERK pathway was hyperactivated in both genomic subtypes under metabolic stress, consequences on cell functions were distinct. The glycolysis inhibitor 2DG induced p21 expression and cell cycle arrest in G0/G1 phase in NRAS-mutant cells but not in BRAF V600E -mutant cells. This might be because the ERK pathway was more activated in NRAS-mutant cells than in BRAF V600E -mutant cells in response to 2DG. Too strong activation of the ERK pathway, in the duration or magnitude, can result in a reversible or permanent cell cycle arrest in G1 phase [39,40]. Interestingly, we showed that the cell cycle arrest could also be achieved in BRAF V600E -mutant cells, but a higher level of metabolic stress had to be reached. This state was induced by the combination of two metabolic stressors or by a high concentration of one metabolic stressor, both situations leading to the activation of AMPK. In NRAS-mutant cells, 2DG was already enough to activate AMPK, and therefore, it appears that BRAF V600E -mutant cells might be more resistant to metabolic stress than NRAS-mutant cells. They were also less sensitive to glucose starvation (Fig EV4C). This difference in sensitivity might come from the fact that the AMPK and BRAF V600E signaling cannot be activated simultaneously [53], but active AMPK and could co-exist in NRAS-mutant cells as we observed AMPK activation concomitantly to ERK pathway hyperactivation. Contrary to NRAS-mutant cells that strongly hyperactivated the ERK pathway to induce cell cycle arrest, BRAF V600E -mutant cells downregulated the ERK pathway when facing high metabolic stress. Both an excessive activation and the inhibition of the ERK signaling can result in cell cycle arrest [37,38]. Thus, even though the two genomic subtypes oppositely control ERK activity, both have a common goal to block the cell cycle under metabolic insult. The next question we wanted to answer was why the two cell types used a different strategy to modulate ERK activity when facing high metabolic stress. We showed that in NRAS-mutant cells, increased stress promoted the association of with AMPK and additionally with, enhancing ERK signaling. This scenario was different in BRAF V600E -mutant cells where non-activated AMPK bound to BRAF V600E and activation of the kinase correlated with the dissociation of oncogenic BRAF from KSR. Critically, RNA interference-mediated knockdown of AMPKa in metabolically stressed cells rescued the interaction of mutant BRAF with, demonstrating that AMPK plays a critical role in the dissociation of oncogenic BRAF from KSR. This negatively regulated ERK pathway activity, most probably due to mutant BRAF being diverted from its substrate MEK, which constitutively binds to the KSR proteins. These data suggested that the differential dependence of the two RAF isoforms on AMPK could be the cause of the opposite responses seen in the two genomic melanoma subtypes facing high metabolic stress. We are currently investigating this possibility further. A previous study found that KSR proteins can associate with AMPK and regulate its activity [54]. AMPK binds to the CA3 region of KSR and an additional portion located between the CA2 and CA3 regions for, while RAF and MEK bind to a more carboxy-terminal region of KSR [54]. While these binding sites are distinct, mutations affecting key residues involved in the interactions of with RAF and MEK often lead to a reduced affinity for AMPK compared to their wild-type counterpart [55]. These data strongly suggest that the association of KSR proteins with AMPK depends on their interaction with RAF kinases. 332