NRAS-Mutated Rhabdomyosarcoma Cells Are Vulnerable to ... · NRAS-Mutated Rhabdomyosarcoma Cells Are Vulnerable to Mitochondrial Apoptosis Induced by Coinhibition of MEK and PI3Ka

Tumor Biology and Immunology

NRAS-Mutated Rhabdomyosarcoma Cells AreVulnerable to Mitochondrial Apoptosis Inducedby Coinhibition of MEK and PI3KaNadezda Dolgikh1, Manuela Hugle1, Meike Vogler1, and Simone Fulda1,2,3

Abstract

Sequencing studies have revealed recurrent mutations in theRAS pathway in rhabdomyosarcoma (RMS). However, RASeffector pathways in RMS are poorly defined. Here, we reportthat coinhibition of NRAS or MEK plus PI3Ka triggers wide-spread apoptosis in NRAS-mutated RMS cells. Subtoxic concen-trations of the MEK inhibitor MEK162 and the PI3Ka-specificinhibitor BYL719 synergized to trigger apoptosis inNRAS-mutat-ed RMS cells in vitro and in vivo. NRAS- or HRAS-mutated celllines were more vulnerable to MEK162/BYL719 cotreatmentthan RAS wild-type cell lines, and MEK162/BYL719 cotreatmentwas more effective to trigger apoptosis in NRAS-mutated thanRAS wild-type RMS tumors in vivo. We identified BCL-2–mod-ifying factor (BMF) as an inhibitory target of oncogenic NRAS,

with either NRAS silencing or MEK inhibition upregulating BMFmRNA and protein levels, which BYL719 further increased. BMFsilencing ablated MEK162/BYL719-induced apoptosis. Mecha-nistic investigations implicated a proapoptotic rebalancing ofBCL-2 family members and suppression of cap-dependent trans-lation in apoptotic sensitivity upon MEK162/BYL719 cotreat-ment. Our results offer a rationale for combining MEK- andPI3Ka-specific inhibitors in clinical treatment of RAS-mutatedRMS.

Significance: These findings offer a mechanistic rationale forcombining MEK- and PI3Ka-specific inhibitors in the clinicaltreatment of RAS-mutated forms of often untreatable rhabdo-myosarcomas. Cancer Res; 78(8); 2000–13. �2018 AACR.

IntroductionRAS proteins have extensively been studied due to their

essential roles in normal physiology as well as in humanmalignancies (1). RAS proteins are small GTPases, which trans-mit signals from receptors on the cell surface and activatemultiple downstream effector pathways, including the RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways, to regulatevarious cellular processes, for example, cell growth, survival,and suppression of apoptosis (1). Oncogenic mutation andactivation of RAS genes frequently occur in a variety of humancancers (1).

Recently, whole-genome sequencing studies have identified ahigh rate of recurrent mutations in the RAS pathway in PAX genefusion–negative rhabdomyosarcoma (RMS) samples (2–4),which correlated with intermediate- and high-risk disease (4).RMS is the most common malignant soft-tissue sarcoma inchildren and adolescence and comprises embryonal RMS (ERMS)and alveolar RMS (ARMS) as the two major subtypes based on

histologic and genetic features (5). Although ARMS is character-ized by chromosomal translocations resulting in a PAX3-FOXO1or PAX7-FOXO1 fusion gene with few other chromosomal altera-tions, ERMS often acquires multiple chromosomal alterations(6). Among RAS pathway genes, NRAS was found to be mostcommonly mutated in RMS, followed by KRAS and HRAS (2).Besides oncogenic mutations in RAS genes, additional geneticalterations in genes directly interacting with RAS or receptortyrosine kinases acting upstream of RAS have been identified inRMS, leading to mutational activation of the RAS pathway in atleast 45% of PAX gene fusion–negative RMS (2). This stressesthe clinical relevance of the RAS pathway in RMS and thehigh medical need for developing novel therapeutic strategies forRAS-mutated RMS.

Cancers harboring RAS mutations are often the most difficultto treat (7). Treatment resistance is often due to the evasion ofprogrammed cell death (8), a characteristic feature of cancer cells(9). Apoptosis is one of the most intensively studied formsof programmed cell death and can be initiated via activationof the extrinsic (death receptor) or the intrinsic (mitochondrial)signaling pathway (10). These two pathways converge uponactivation of caspases as effector proteins (11). The intrinsicpathway of apoptosis is characterized by mitochondrial outermembrane permeabilization (MOMP), followed by the releaseof proapoptotic proteins from the mitochondria into the cytosol,leading to caspase activation and cell death (12). MOMP istightly regulated by BCL-2 family proteins, which consist ofthree functionally different classes, i.e., proapoptotic multido-main proteins (such as BAX and BAK), proapoptotic BH3-onlyproteins (e.g., BIM and BMF), and antiapoptotic proteins (e.g.,BCL-2, BCL-XL, and MCL-1). BCL-2 family proteins are regulatedin multiple ways, including transcriptional or translational

1Institute for Experimental Cancer Research in Pediatrics, Goethe-UniversityFrankfurt, Frankfurt, Germany. 2German Cancer Consortium (DKTK), PartnerSite Frankfurt, Frankfurt, Germany. 3German Cancer Research Center (DKFZ),Heidelberg, Germany.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Simone Fulda, Goethe-University Hospital, Komturstrasse3a, 60528 Frankfurt, Germany. Phone: 49-69-67866557; Fax: 49-69-6786659157;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-1737

�2018 American Association for Cancer Research.

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regulation, posttranslational modifications, and oligomerizationwith other BCL-2 proteins (13). Overall, the balance betweenpro- and antiapoptotic BCL-2 family proteins is essential forregulating sensitivity to apoptosis (13).

Because direct targeting of constitutively active RAS remains achallenge, inhibition of downstream effector pathways that arealtered by oncogenic RASoffers a feasible alternative, for example,by blocking MEK (7). However, the response to MEK inhibitorsvaries among different RAS-mutated cancers (14), and the inef-ficiency of single-agent treatment has been attributed to variouscross-talks and feedback loops between RAF/MEK/ERK and PI3K/AKT/mTOR pathways (15). This provides the rationale forthe concurrent inhibition of both pathways. Indeed, parallelblockage of both PI3K/AKT/mTOR and RAF/MEK/ERK pathwayshas been shown to suppress tumor growth in some in vitro and inseveral in vivomodels of RAS-mutated cancers (16, 17). However,the transfer of these findings into clinical application is beingcomplicatedby the fact that theRAS signalingnetwork is regulatedin a context-dependent manner in different cancer entities, forexample, by alterations affecting additional pathways. This high-lights the need to study RAS-controlled effector mechanisms in agiven tumor.

Because RMS has recently been shown to frequently har-bor mutations in RAS genes (2–4), in the current study, weaimed at dissecting the RAS effector pathways that controlcell death in RMS cells in order to provide a mechanism-based rationale for therapeutic targeting of aberrant RASsignaling in RMS.

Materials and MethodsCell culture and chemicals

RMS cell lines were obtained in 2014 or 2015 from the ATCCor from DSMZ (German Collection of Microorganisms andCell Cultures GmbH) and frozen upon arrival after authenti-cation by short tandem repeat profiling. VJ cells were generatedfrom a tumor specimen derived from a patient diagnosedwith fusion-gene–negative ERMS. Cells were maintained upto 25 passages in RPMI 1640 or DMEM medium (Life Tech-nologies, Inc.), supplemented with 10% FCS (Biochrom),1 mmol/L sodium pyruvate (Invitrogen), and 1% penicillin/streptomycin (Invitrogen), and regularly tested for lack ofmycoplasma contamination. NRAS-overexpressing RMS13 cellshave been described previously (18). N-benzyl-oxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) was purchasedfrom Bachem; BYL179, BKM120, MEK162, and ABT-199 fromSelleck Chemical; A-1210477 from Active Biochem; PI103 fromMerck Millipore; and all chemicals from Sigma unless indicatedotherwise.

Determination of cell viability, DNA fragmentation, caspaseactivity, clonogenic growth, Western blot analysis, and BAX/BAK activation

Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay according to themanufacturer's instructions (Roche Diagnostics); apoptosis byanalysis of DNA fragmentation of propidium iodide (PI)–stained nuclei using flow cytometry (FACSCanto II, BD Bio-sciences) as described previously (19); and caspase activity byCellEvent Caspase-3/7 Green Detection Reagent according tothe manufacturer's instructions (ThermoFisher Scientific) using

ImageXpress Micro XLS system for detection (MolecularDevices). For determination of colony formation, 200 cellswere seeded in a 6-well plate, allowed to adhere overnight,and treated for 24 hours, followed by medium exchange.Colonies were stained after 12 days with crystal violet solution(0.5% crystal violet, 30% ethanol, and 3% formaldehyde).Colonies were counted, and the percentage of colonies relativeto solvent-treated controls was calculated. Western blot analysiswas performed as described previously (19), and antibodies arelisted in Supplementary Methods. BAX/BAK activation wasperformed as described previously (20).

Overexpression and RNA interferenceCells were transfected with murine stem cell virus vector

(pMSCV, Clontech) containing murine BCL-2 or empty vector(EV) using calcium-phosphate transfection as described (21),were transduced with pCMV-Tag3B plasmid (Genentech) con-taining MCL-1 or EV, followed by selection with Neomycin, orwere reversely transfected with 10 nmol/L SilencerSelect siRNA(Invitrogen): Control siRNA (4390844) or two or three inde-pendent targeting siRNAs to ensure on-target effects (s54, s55,s56 for NRAS; s10520, 10521 for PIK3CA; s195011, s195012,and s223065 for BIM; s40385, s40386, and s40387 for BMF;s1880 and s1881 for BAK; s1889 and s1890 for BAX) usingOpti-MEM medium (Life Technologies, Inc.) and Lipofecta-mine RNAi MAX reagent (Life Technologies, Inc.) according tothe manufacturer's instructions.

Quantitative real-time PCR and RAS sequencingExpression levels of the target genes were determined using

quantitative real-time (qRT)-PCRmethod as described (20). Datawere normalized on 28S-rRNA expression as reference gene.Primers are listed in Supplementary Table S3. Primers for NRAS,KRAS, and HRAS exons containing activating mutations, exons 2and 3, are listed in Supplementary Table S1.

Chicken chorioallantoic membrane assayAt day 8 of fertilization of chicken eggs, 1 � 106 of RD or

RH30 cells mixed 1:1 with Matrigel were implanted onto thechorioallantoic membrane (CAM) to form tumors, which werethen treated for 3 consecutive days with the amount of drug percell corresponding to 1 mmol/L BYL719 and/or 1 mmol/LMEK162 in cell culture experiments. The CAM was fixed in4% paraformaldehyde, embedded in paraffin, cut in 3 mmsections, and then analyzed by immunohistochemistry using1:1 hematoxylin and 0.5% eosin or rabbit polyclonal antic-leaved caspase-3 (Asp175) antibody (Cell Signaling Technol-ogy) and hematoxylin counterstain. Images were digitallyrecorded, and tumor area was analyzed with ImageJ digitalimaging software (NIH, Bethesda, MD). The number of activecaspase-3–positive cells per tumor area was counted manuallyby two investigators.

Statistical analysisStatistical significance was assessed by the Student t-test (two-

tailed distribution, two-sample, unequal variance) or ANOVAand Tukey multiple comparison test. Drug interactions wereanalyzed by calculation of CI using CalcuSyn software (Biosoft)based on the methods described by Chou (22). CI < 0.9 indicatessynergism, 0.9–1.1 additivity, and >1.1 antagonism.

Cotargeting of MEK and PI3Ka to Block RAS Signaling in RMS

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ResultsNRASdepletion reduces cell viability and clonogenic survival ofNRAS-mutated RMS cells without inducing cell death

To investigate the role of oncogenic RAS in RMS, we initiallyassessed the RAS mutational status in a panel of RMS cell lines.We found NRAS mutation (codon Q61H) in four of nine estab-lished RMS cell lines and in one patient-derived RMS cultureas well as HRAS mutation (codon Q61K) in one RMS cell line,whereas four RMS cell lines were wild-type for NRAS, KRAS, andHRAS (Supplementary Table S2).

Next, we investigated the effects of NRAS knockdown on cellsurvival and cell death in threeNRAS-mutated RMS cell lines. Theefficacy of siRNA-imposed silencing of NRAS was controlled atboth 48 hours (Fig. 1A) and 144 hours (Supplementary Fig. S1A).NRAS knockdown significantly decreased cell viability as assessedbyMTT and crystal violet assays (Fig. 1B; Supplementary Fig. S1B)as well as long-term clonogenic survival (Fig. 1C). In contrast,NRAS silencing failed to cause spontaneous cell death in theabsence of a cytotoxic stimulus (Fig. 1D). This shows that cellviability and long-term clonogenic growth ofNRAS-mutated RMSdepend on the presence of oncogenic RAS; however, NRASdepletion is not sufficient to induce spontaneous cell death. Thus,NRAS-mutated RMS cells can compensate for the loss of onco-genic NRAS to prevent cell death.

RAS-mutatedRMScells rely onPI3Ka toprevent cell deathuponNRAS depletion or MEK inhibition

To explore howNRAS-mutatedRMS cells evade cell death upondepletion of oncogenic NRAS, we tested their dependency onactive PI3K/AKT/mTOR signaling. To this end, we used threeinhibitors that block distinct pathway elements, i.e., the PI3Ka-specific inhibitor BYL719, the pan-PI3K inhibitor BKM120, andthe dual pan-PI3K/mTOR inhibitor PI103. Importantly, all threePI3K inhibitors significantly increased cell death in NRAS knock-down cells compared with control cells (Fig. 2A–C). Interestingly,the PI3Ka-specific inhibitor BYL719 turned out to be similarlyeffective as pan-PI3K inhibitors to induce cell death in NRASknockdown cells with no or little single-agent cytotoxicity in bothtested RMS cell lines, whereas BKM120 and PI103 already assingle agents induced cell death in a dose-dependent manner inone or both RMS cell lines (Fig. 2A–C). In sharp contrast to PI3Kinhibitors, addition of the MEK inhibitor MEK162 failed topreferentially trigger cell death in NRAS knockdown comparedwith control cells (Fig. 2D).

Next, we used a dual pharmacological approach to blockeffector pathways of oncogenic RAS using the MEK1/2 inhibitorMEK162 in combination with the three PI3K inhibitors. Impor-tantly, BYL719 turned out to be the most potent inhibitor toinduce cell death togetherwithMEK162 in all testedRMS cell linesas compared with BKM120 or PI103 (Fig. 2E; Supplementary Fig.S2A–S2C). Calculation of CI revealed that the interaction inparticular of MEK162 and BYL719 is highly synergistic (Supple-mentary Table S3).

To further investigate the specific relevance of PI3Ka to com-pensate for inhibition of RAF/MEK/ERK signaling, we silencedPI3Ka via siRNA. Western blotting confirmed that two distinctsiRNAs against p110a caused marked, although not completedownregulation of p110a protein (Fig. 2F). Importantly, p110aknockdown significantly enhanced the sensitivity of all NRAS-mutated RMS cells to MEK162-induced cell death (Fig. 2G).

Together, this set of experiments shows that parallel inhibitionof both, the RAF/MEK/ERK pathway and PI3Ka, is necessary toelicit cell death in NRAS-mutated RMS cells, whereas verticalinhibition of the RAF/MEK/ERK cascade is not sufficient. Thisindicates that RAS-mutated RMS cell lines rely on PI3Ka toprevent cell death upon NRAS depletion or MEK inhibition.

The RAS mutational status predicts the responsiveness of RMScells toward combined MEK and PI3Ka inhibition

Next, we asked whether the sensitivity of RMS cells towardMEK162/BYL719 correlates with their RAS mutational status. Toaddress this question, we tested subtoxic concentrations ofBYL719 andMEK162 that cause less than 20% cell death as singleagents against a broad panel of RMS cell lines. Strikingly,MEK162/BYL719 cotreatment significantly increased apoptosisin the six RAS-mutated RMS cell lines, whereas it failed in the fourRAS wild-type cell lines (Fig. 3A). Similarly, ectopic expression ofmutant NRAS rendered the ARMS cell line RMS13 responsive totheMEK162/BYL719 cotreatment, whereas no cooperative induc-tion of cell death by MEK162 and BYL719 was found in RMS13cells expressing EV control (Fig. 3B and C). Also, PI3Ka silencingfailed to sensitize RASwild-type RH30 cells toMEK162-mediatedcell death (Fig. 3D and E), in contrast toNRAS-mutated RMS celllines (Fig. 2G). To test whether the RASmutational status predictssensitivity of RMS to MEK162/BYL719 cotreatment in vivo, weused the CAMmodel, an established in vivomodel for anticancerdrug testing (21, 23). Of note, MEK162/BYL719 cotreatment wasmore effective in vivo to trigger caspase-3 activation as a marker ofapoptosis in NRAS-mutated than in RAS wild-type RMS tumors(Fig. 3F). BeyondRMS,MEK162 andBYL719 synergized to inducecell death in NRAS-mutated SK-N-AS neuroblastoma and HL60leukemia cells (Supplementary Fig. S3). These findings indicatethat the RASmutational status predicts the responsiveness of RMSand other malignancies toward combined MEK and PI3Kainhibition.

MEK162 and BYL719 cooperate to induce caspase-dependentapoptosis

Next,we aimed at unraveling themolecularmechanismsunder-lying the synergy of combinedMEK and PI3Ka inhibition. To thisend, we assessed the activation of caspases known to mediateapoptotic cell death. Treatment withMEK162/BYL719 significant-ly increased caspase-3/7 activation in all three RMS cell lines(Fig. 4A). To examine whether caspases are required for theexecution of cell death, we used the broad-range caspase inhibitorzVAD.fmk. Of note, the addition of zVAD.fmk significantlydecreased MEK162/BYL719-induced apoptosis (Fig. 4B). Kineticanalysis showed that cotreatment with BYL719 and MEK162induced apoptosis in a time-dependentmanner (Fig. 4C) and thatzVAD.fmk-conferred protection from cell death was more pro-nounced at earlier time points (Supplementary Fig. S4A and S4B).Monitoring of long-termclonogenic survival revealed that BYL719and MEK162 cooperated to significantly suppress colony forma-tion compared with each agent alone or to control cells (Fig. 4D).

This set of experiments emphasizes that MEK162/BYL719cotreatment triggers caspase-dependent apoptosis.

MEK162 and BYL719 cooperate to suppress cap-dependenttranslation

To investigate how treatment with MEK162 alone or in com-binationwith BYL719 alters RAF/MEK/ERK andPI3K/AKT/mTOR

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Figure 1.

NRAS depletion reduces cell viability and clonogenic survival of NRAS-mutated RMS cells without inducing cell death. A–D, RD, VJ, and T174 cells were transientlytransfected with siRNA against NRAS or nonsilencing siRNA (siCtrl). A, Expression of NRAS and pan-RAS was determined after 48 hours by Western blotting.b-Actin served as loading control. B, Cell viability was analyzed by MTT assay at 144 hours. C, Colony formation was assessed after 12 days. The number ofcolonies is expressed as percentage of control (top), and representative images are shown (bottom). D, Apoptosis was determined by DNA fragmentation ofPI-stained nuclei using flow cytometry at 144 hours. Mean and SD (error bars) of three independent experiments performed in triplicate are shown. TheStudent t-test was used to calculate two-sided P values (� , P < 0.05; �� , P < 0.01; and �� , P < 0.001).






signaling, we assessed phosphorylation of AKT as surrogate read-out for PI3K activity, phosphorylation of ERK for MEK activity,and phosphorylation of S6 ribosomal protein and 4E-BP1 asreadouts for mTORC1 activity and cap-dependent translation.Constitutively, all three RMS cell lines exhibited phosphoryla-tion of AKT, ERK, S6 ribosomal protein, and 4E-BP1 (Fig. 5A).As expected, BYL719 as single agent reduced phosphorylationof AKT, whereas MEK162 alone suppressed phosphorylation ofERK (Fig. 5A). We also observed that BYL719 slightly reducedERK1/2 phosphorylation in RD cells (Fig. 5A), in line with aprevious study reporting that PI3Ka might regulate ERK phos-phorylation via RAF/MEK signaling (24). By comparison,knockdown of NRAS had a minor effect on ERK phosphoryla-tion (Supplementary Fig. S5), which might be due to differ-ences between acute pharmacological inhibition and siRNA-imposed knockdown of gene expression. Importantly, MEK162and BYL719 cooperated to reduce phosphorylation of S6 ribo-somal protein and 4E-BP1 in all three NRAS-mutated RMS celllines (Fig. 5A), whereas it failed to reduce 4E-BP1 phosphor-ylation in RAS wild-type RH30 cells (Supplementary Fig. S6A).

This indicates that combined MEK and PI3Ka inhibition isnecessary to potently suppress cap-dependent translation, acommon downstream process regulated by both PI3K/AKT/mTOR and RAF/MEK/ERK signaling.

MEK162 andBYL719 cooperate to increase BMF andBIMand tosuppress MCL-1, while oncogenic NRAS suppresses BMF andBIM with little effect on MCL-1

Next, we analyzed expression levels of BCL-2 family proteins askey regulators of apoptosis. Interestingly, we found that MEK162and BYL719 cooperated to upregulate BMF mRNA as well asprotein levels (Fig. 5B and C). Also, MEK inhibition causeddephosphorylation and increased expression of BIM protein, andthe addition of BYL719 further increasedmRNA levels of BIMand,to a lesser extent, its protein expression (Fig. 5B and C). Further-more, MEK162 especially in combination with BYL719 sup-pressed MCL-1 mRNA and protein levels in all three NRAS-mutated RMS cell lines (Fig. 5B and C), in line with the cooper-ative inhibition of 4E-BP1 phosphorylation by MEK162/BYL719cotreatment (Fig. 5A). By comparison, MEK162/BYL719

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Figure 2.

RAS-mutated RMS cells rely on PI3Ka to prevent cell death upon NRAS depletion or MEK inhibition. A–D, RD and VJ cells were transiently transfected with siRNAagainst NRAS or nonsilencing siRNA (siCtrl), and after 48 hours, cells were treated with BYL719, BKM120, PI103, and MEK162 at the indicated concentrations, andapoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry at 72 hours. E, RD, VJ, and T174 cells were treated with indicatedconcentrations of MEK162 in combination with 10 mmol/L BYL719, 1 mmol/L BKM120, and 2 mmol/L PI103, and apoptosis was determined by DNA fragmentationof PI-stained nuclei using flow cytometry at 72 hours. F and G, RD, VJ, and T174 cells were transiently transfected with siRNA against p110a or nonsilencing siRNA(siCtrl) and treatedwith 10mmol/LMEK162. F, Expressions of p110a and p110bwere determined after 48 hours byWestern blotting. b-Actin served as loading control.G, Apoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry at 72 hours. Mean and SD (error bars) of three independentexperiments performed in triplicate are shown. The Student t-test was used to calculate two-sided P values (� , P < 0.05; �� , P < 0.01; and �� , P < 0.001).

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cotreatment had little effect on MCL-1 levels in RAS wild-typeRH30 cells (Supplementary Fig. S6B), in line with its failure toinhibit 4E-BP1 phosphorylation in these cells (SupplementaryFig. S6A), whereas it increased BMF and BIM expression (Sup-plementary Fig. S6B). Consistently, genetic silencing of NRASresulted in upregulation of BMF and BIM mRNA and proteinexpression, whereas it had little effect on MCL-1 protein levels(Fig. 5D; Supplementary Fig. S7). Therefore, we investigatedwhetherNRAS silencing is synthetic lethal withMCL-1 inhibition.Intriguingly, the MCL-1 inhibitor A-1210477 cooperated withNRAS silencing to trigger cell death in NRAS-mutated RMS cells(Supplementary Fig. S8).

To further investigate whether oncogenic NRAS regulatesexpression of BCL-2 family proteins, we used the ARMS cell lineRMS13 with ectopic expression of mutant NRAS (18). Consis-tently, ectopic expression of mutant NRAS resulted in markedsuppression of BMF and BIM protein levels compared withRMS13 cells expressing EV control, whereas MCL-1 proteinexpression remained largely unchanged (Fig. 5E). By comparison,screening of a panel of RMS cell lines showed no obviousassociation between RASmutational status and expression levelsof BMForBIM(Supplementary Fig. S9), indicating that additionalfactors besides RAS mutation regulate BIM and BMF expression.This set of experiments shows that MEK162 and BYL719 coop-erate to upregulate BIM and BMF and to suppress MCL-1 levels,whereas oncogenic NRAS suppresses BMF and BIM with littlealterations of MCL-1 levels.

BMF, BIM, and MCL-1 regulate the sensitivity of RAS-mutatedRMS cells to MEK162/BYL719-induced apoptosis

To test if the elevated expression levels of BIM and BMF uponMEK/PI3K coinhibition result in their enhanced interactionwith antiapoptotic BCL-2 proteins, we immunoprecipitatedBCL-2, BCL-XL, and MCL-1 and analyzed their binding to BIMand BMF. Interestingly, treatment with MEK162 alone and incombination with BYL719 increased the binding of BIM toBCL-2, BCL-XL, and MCL-1, and BMF mainly to BCL-2 (Sup-plementary Fig. S10). This shows that elevated expression levelsof BIM and BMF result in their enhanced interaction withantiapoptotic BCL-2 proteins and thus increase the priming ofthese cells, rendering them more susceptible to undergoapoptosis.

To investigate the functional relevance of BMF and BIM forMEK162/BYL719-induced apoptosis, we genetically silencedthese proteins using three distinct siRNA sequences for each gene.Importantly, silencing of BMF or BIM significantly reducedMEK162/BYL719-induced apoptosis in all three RMS cell lines(Fig. 6A–D). To test the relevance of MCL-1, we generated RMScells with overexpression of MCL-1. Indeed, MCL-1 overexpres-sion significantly decreased MEK162/BYL719-induced apoptosis(Fig. 6E and F). These results indicate that MEK162/BYL719-stimulated changes in expression levels of BIM, BMF, andMCL-1 contribute to the induction of apoptosis by thiscombination.

MEK162/BYL719-induced apoptosis is mediated via themitochondrial pathway

Next, we investigated the question as to whether or not theobserved changes in the ratio of pro- and antiapoptotic BCL-2proteins promote activation of BAX and BAK. To this end, we

immunoprecipitated BAX and BAK using conformation-specificantibodies, which specifically bind to their activated forms.BYL719 andMEK162 cooperated to trigger activation of BAX andBAK (Supplementary Fig. S11). Notably, BAK and BAX doubleknockdown significantly reduced cell death upon MEK162/BYL719 cotreatment (Fig. 7A and B), demonstrating that BAXand BAK activation contributes to MEK162/BYL719-inducedapoptosis.

To further test the requirement of mitochondrial apoptosis,we overexpressed BCL-2, which is known to interfere withmitochondrial apoptosis. Notably, overexpression of BCL-2prevented BAX/BAK activation and significantly rescued cellsfrom MEK162/BYL719-induced apoptosis as well as loss ofclonogenic growth (Fig. 7C–F). To test whether BCL-2 mightimpede MEK162/BYL719 cotreatment, we used the BCL-2-selective inhibitor ABT-199 to neutralize BCL-2. Intriguingly,addition of ABT-199 completely reversed the BCL-2-imposedresistance to MEK162/BYL719 cotreatment (Fig. 7D). Togeth-er, this set of experiments underscores the relevance of anintact mitochondrial signaling pathway for MEK162/BYL719-induced apoptosis.

DiscussionRecent sequencing studies have revealed recurrent mutations

in the RAS pathway in primary RMS samples with NRAS as themost commonly mutated RAS gene (2–4). The signaling networkof oncogenic RAS is regulated in a context-dependent manner.In RMS, however, the RAS effector pathways are still poorlyunderstood.

Parallel NRAS and PI3K pathway inhibition is required toinduce cell death in NRAS-mutated RMS cells

Here, we report thatNRAS-mutated RMS cells can compensatefor the depletion of oncogenic NRAS or pharmacological inhibi-tion of MEK to prevent cell death. Importantly, subtoxic concen-trations of the MEK inhibitor MEK162 and the PI3Ka-specificinhibitor BYL719 synergize to trigger apoptosis inNRAS-mutatedRMS cells, demonstrating that parallel inhibition of both the RAF/MEK/ERK and the PI3K pathways is necessary to elicit cell death inNRAS-mutated RMS cells. This suggests that oncogenic NRASsignaling occurs via these two pathways in RMS cells. In addition,aberrant receptor tyrosine kinase signaling, a frequent event inRMS (2, 3, 25), likely contributes to PI3K/AKT/mTOR pathwayactivation. In contrast to parallel blockade of RAF/MEK/ERK andPI3K signaling, vertical inhibition of NRAS andMEK fails to elicitcell death in NRAS-mutated RMS cells.

NRAS-mutated RMS cells require PI3Ka to prevent cell deathupon NRAS depletion or MEK inhibition

Interestingly, we discover thatNRAS-mutated RMS cells rely inparticular on PI3Ka to prevent cell death uponNRAS silencing orMEK inhibition. This conclusion is supported by our datashowing that specific knockdown of PI3Ka is sufficient tocooperatively trigger cell death together with pharmacologicalMEK inhibition. In addition, pharmacological inhibitors of MEKor NRAS knockdown synergize in particular with the PI3Ka-specific inhibitor BYL719 to trigger cell death in NRAS-mutatedRMS cells. Our study is the first to demonstrate that PI3Ka playsa critical role in activating the PI3K/AKT/mTOR pathway in






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Dolgikh et al.





NRAS-mutated RMS cells. This finding has important implica-tions, because it provides a rationale for the combined use ofPI3Ka-specific inhibitors together with MEK inhibitors in RMSwith NRAS mutation. Although the four isoforms of PI3K

(p110a, p110b, p110g , and p110d) might exhibit functionalredundancy in retaining cell survival, one of the isoforms pref-erentially mediates signal transmission depending on the tumortype and genetic aberration of the cell (26). Some RAS-mutated

Figure 4.

MEK162 and BYL719 cooperate to induce caspase-dependent apoptosis. A, RD, VJ, and T174 cells were treated with 5 mmol/L MEK162 and/or 10 mmol/L BYl719 (RDand VJ), or with 10 mmol/L MEK162 and/or 20 mmol/L BYL719 (T174). Caspase activity was determined at 24 hours (RD and VJ) and 72 hours for T174. B, RD,VJ, andT174 cellswere treatedwith 5mmol/LMEK162 and/or 10mmol/LBYl719 (RDandVJ), orwith 10mmol/LMEK162 and/or 20mmol/LBYL719 (T174) for 72hours inthe presence or absence of 50 mmol/L of zVAD.fmk, and apoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry. C, RD,VJ, and T174 cells were treated with 5 mmol/L MEK162 and/or 10 mmol/L BYl719 (RD and VJ), or with 10 mmol/L MEK162 and/or 20 mmol/L BYL719 (T174), andapoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry at indicated time points. D, Cells were treated with 5 mmol/LMEK162 and/or 10 mmol/L BYl719 (RD and VJ), or with 10 mmol/L MEK162 and/or 20 mmol/L BYL719 (T174) for 24 hours, and colony formation was assessed after 12days. The number of colonies is expressed as percentage of control (top), and representative images are shown (bottom). Mean and SD (error bars) of threeindependent experiments performed in triplicate are shown. The Student t-test was used to calculate two-sided P values (� , P < 0.05; �� , P < 0.01; and�� , P < 0.001).

Figure 3.TheRASmutational status predicts the responsiveness of RMS cells toward combinedMEK and PI3Ka inhibition.A, The panels of RMS cells were treated for 72 hourswith 5 mmol/L MEK162 and/or BYL719 (10 mmol/L for RD, TE671, RH36, and VJ; 20 mmol/L for T174; 2.5 mmol/L for RH30 and TE381.T; 0.5 mmol/L for TE441.T,Kym1, and RH41). Apoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry. Mean and SD (error bars) of three independentexperiments performed in triplicate are shown. The Student t-test was used to calculate two-sided P values; �� , P < 0.001, comparing apoptosis upon MEK162/BYL719 cotreatment with that of single treatments or untreated control for each cell line. B and C, RMS13 cells with ectopic expression of mutant NRAS or EVcontrol were treatedwith 5 mmol/LMEK162 and/or 10 mmol/L BYL719 for 72 hours. NRAS protein expressionwas determined byWestern blotting, and b-actin servedas loading control (B). Apoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry (C). Mean and SD (error bars) of threeindependent experimentsperformed in triplicate are shown. The Student t-testwas used to calculate two-sidedP values; �� ,P<0.01; ns, not significant.D andE,RH30cells were transiently transfected with siRNA against p110a or nonsilencing siRNA (siCtrl). Expressions of p110a and p110b were determined after 48 hours byWestern blotting, and b-actin served as loading control (D). Apoptosiswas determined after treatment with 10 mmol/LMEK162 for 72 hours by DNA fragmentation ofPI-stained nuclei using flow cytometry (E). Mean and SD (error bars) of three independent experiments performed in triplicate are shown. The Studentt-testwas used to calculate two-sidedP values. � ,P<0.05; �� ,P<0.01; and �� ,P<0.001.F,RDandRH30 tumor xenografts on theCAMof fertilized chicken eggsweretreated with 1 mmol/L BYL719 and/or 1 mmol/L MEK162 or solvent for three consecutive days. Tumor sections were stained for active caspase-3 byimmunohistochemistry, and the number of active caspase-3–positive cells per tumor area was determined. Mean and SEM (error bars) of three independentexperiments are shown. ANOVA and Tukey multiple comparison test were used to calculate two-sided P values. �, P < 0.05; ��, P < 0.01; and �� , P < 0.001; ns, notsignificant.






cancers, for example, myeloid leukemia, non–small cell lungcancer, and pancreatic cancer, have previously been reported todepend in particular on PI3Ka in addition to RAF/MEK/ERKsignaling (27–30). This dependency has been attributed to thedirect interaction of oncogenic RAS with PI3Ka (26, 27, 31).However, KRAS depletion on its own has also been reported toinduce apoptosis in some KRAS-mutated cancer cell lines (32),emphasizing the context dependency of RAS signaling.

The RAS mutational status predicts the responsiveness of RMScells toward combined inhibition of MEK and PI3Ka

We identify the RAS mutational status as a marker predictingthe sensitivity of RMS toward combined inhibition of MEK andPI3Ka by screening a panel of RMS cell lines including apatient-derived RMS culture. This conclusion is underscoredby genetic evidence showing that (i) PI3Ka silencing cooperates

with MEK inhibition to trigger cell death in NRAS-mutated butnot in RAS wild-type RMS cells and that (ii) ectopic expressionof NRAS in RAS wild-type ARMS confers sensitivity to MEK162/BYL719 cotreatment. In addition, MEK162/BYL719 cotreat-ment proved to be more effective to trigger apoptosis inNRAS-mutated than in RAS wild-type RMS tumors in vivo. RASmutations were reported to predominantly occur in ERMS,and only some rare cases were found in fusion-negative ARMS(2, 3). This implies that the RAS mutational status may help toselect RMS patients that are particularly susceptible to MEK162/BYL719 cotreatment. Nevertheless, RMS cell lines harboringwild-type RAS genes were also found to respond to parallelinhibition of RAF/MEK/ERK and PI3K/AKT/mTOR pathwaysunder certain circumstances, i.e., under cellular stress caused byserum starvation or when MEK inhibition was combined withinhibition of downstream elements of the PI3K/AKT/mTOR

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MEK162 and BYL719 cooperate to suppress cap-dependent translation, to increase BMF and BIM, and to suppress MCL-1, whereas oncogenic NRAS suppresses BMFand BIM with little effect on MCL-1. A–C, RD and VJ cells were treated with 5 mmol/L MEK162 and/or 10 mmol/L BYl719, T174 cells were treated with 10 mmol/LMEK162 and/or 20 mmol/L BYL719, and mRNA expression was determined at 6 hours; protein expression was determined at 9 hours (RD), 20 hours (VJ), and24 hours (T174), corresponding to their different kinetics of cell death (Fig. 4C). A, Protein expression of phospho-AKT (S473), AKT, phospho-ERK1/2, ERK1/2,phospho-4E-BP1, 4E-BP1, phospho-S6, and S6 was determined byWestern blotting. b-Actin served as loading control. B, Protein expression of BMF, BIM, andMCL-1was determined by Western blotting. b-Actin served as loading control. C, mRNA expression of BIM, BMF, and MCL-1 was determined by qRT-PCR, normalizedto 28S expression, and is shown as fold change of mRNA expression compared with the control. D, RD, VJ, and T174 cells were transiently transfected withsiRNA against NRAS or nonsilencing siRNA (siCtrl). Protein expression of BMF, BIM, MCL-1, and NRAS was determined after 48 hours by Western blotting.E,Protein expressionofBMF, BIM, andMCL-1wasdeterminedbyWesternblotting in untreatedRMS13 cellswith ectopic expression ofmutantNRASor EVcontrol, andb-actin served as loading control. Mean and SD (error bars) of three independent experiments performed in triplicate are shown. The Student t-test wasused to calculate two-sided P values (� , P < 0.05; �� , P < 0.01; and �� , P < 0.001). Representative blots of at least two independent experiments are shown.

Dolgikh et al.





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BMF, BIM, andMCL-1 regulate the sensitivity ofRAS-mutated RMS cell toMEK162/BYL719-induced apoptosis.A–D,RD, VJ, and T174 cellswere transiently transfectedwith siRNA against BIM or BMF or nonsilencing siRNA (siCtrl) and were treated with 5 mmol/L MEK162 and/or 10 mmol/L BYL719 (RD and VJ) and 10 mmol/L MEK162and/or 20 mmol/L BYL719 (T174). Expression of BMF and BIM was assessed after 24 hours by Western blotting. b-Actin served as loading control. Apoptosis wasdetermined by DNA fragmentation of PI-stained nuclei using flow cytometry at 72 hours. E and F, RD cells engineered to overexpress MCL-1 were treatedwith 5mmol/LMEK162 and/or 10 mmol/L BYL719. E, Expression of MCL-1 in transfected RD cells was verified byWestern blotting. b-Actin served as loading control. F,Apoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry at 72 hours. Mean and SD (error bars) of three independentexperiments performed in triplicate are shown. The Student t-test was used to calculate two-sided P values (� , P < 0.05; �� , P < 0.01; and �� , P < 0.001).






pathway using a catalytical mTOR inhibitor or a dual pan-PI3K/mTOR inhibitor (33, 34).

Changes in pro- and antiapoptotic BCL-2 proteins andsuppression of cap-dependent translation mediate the synergyof combined MEK and PI3Ka inhibition

Our study provides novel insights into the molecular mechan-isms underlying the synergy of combined MEK and PI3Ka inhi-bition inNRAS-mutated RMS cells, i.e., (i) changes in the ratio of

pro- and antiapoptotic BCL-2proteins leading to apoptosis via themitochondrial pathway and (ii) suppression of cap-dependenttranslation (Fig. 7G).

Importantly, we identify BMF as a target that is suppressed byoncogenic NRAS and required for MEK162/BYL719-inducedapoptosis. So far, BMF has not yet been implied in mediatingapoptosis upon combined inhibition of RAF/MEK/ERK andPI3K/AKT/mTOR in RAS-mutated cancer cells. We show thatNRAS depletion or MEK inhibition upregulates mRNA and

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Figure 7.

MEK162/BYL719-induced apoptosis is mediated via the mitochondrial pathway. A and B, RD, VJ, and T174 cells were transiently transfected with siRNA againstBAK and BAX or nonsilencing siRNA (siCtrl) and treated with 5 mmol/L MEK162 and 10 mmol/L BYL719 (RD and VJ), or 10 mmol/L MEK162 and 20 mmol/LBYL719 (T174). A, Expression levels of BAX and BAK at 48 hours after double knockdown were analyzed by Western blotting. b-Actin served as loading control.B, Apoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry at 72 hours. C–F, RD cells were transfected with murine BCL-2or EV. C, Expression of BCL-2 was determined byWestern blotting. b-Actin served as loading control.D, BCL-2–overexpressing RD cells were treated with 5 mmol/LMEK162 and 10mmol/LBYL719, and/or 5mmol/LABT-199, and apoptosiswasdeterminedbyDNA fragmentation of PI-stainednuclei usingflowcytometry at 72hours.E, BCL-2–overexpressing RD cells were treated with 5 mmol/L MEK162 and 10 mmol/L BYL719 for 9 hours. BAX and BAK activations were assessed byimmunoprecipitation using active conformation-specific anti-BAX and anti-BAK antibody, and protein expression of BAX and BAK was analyzed by Westernblotting. b-Actin served as loading control. F, BCL-2–overexpressing RD cells were treated with 5 mmol/L MEK162 and/or 10 mmol/L for 24 hours, and colonyformation was assessed after 12 days. The number of colonies is expressed as percentage of control (right), and representative images are shown (left). Meanand SD (error bars) of three independent experiments performed in triplicate are shown. The Student t-test was used to calculate two-sided P values(� , P < 0.05; �� , P < 0.01; and �� , P < 0.001). G, Scheme of the proposed mechanism of BYL719/MEK162-induced mitochondrial apoptosis. NRAS mutationactivates RAF/MEK/ERK and PI3K/AKT/mTOR pathways; aberrant receptor tyrosine kinase (RTK) signaling additionally contributes to PI3K/AKT/mTORpathway activation. Inhibition of downstream RAS effector pathways using MEK inhibitor MEK162 and PI3Ka-specific inhibitor BYL719 cooperativelysuppresses cap-dependent translation via mTORC1, which in turn can suppress the antiapoptotic protein MCL-1. MEK162/BYL719 cotreatment changes thebalance of BCL-2 proteins toward apoptosis by upregulating BMF and BIM and by downregulating MCL-1, leading to BAX/BAK activation and caspase-dependent apoptosis.


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protein levels of BMF, which are further increased upon theaddition of BYL719. This increase in BMF expression is criticalfor MEK162/BYL719-stimulated apoptosis, because knock-down of BMF significantly rescues cell death. BMF has recentlybeen identified as a direct transcriptional target of FOXO3a(35), indicating that dephosphorylation of FOXO3a upon MEKand PI3K inhibition might contribute to transcriptional upre-gulation of BMF. In addition, BMF has been reported to beupregulated via internal ribosome entry sites (IRES)–mediatedtranslation upon inhibition of cap-dependent translation (36).This could explain why we detected upregulation of BMFprotein even under conditions when general translation wasblocked. Upregulation of BMF has previously been observed inBRAF-mutated melanoma cells treated with the BRAF inhibitorvemurafenib or the MEK inhibitor CI-1040 (37, 38), andphosphorylation of BMF by ERK2 has been reported to reduceits proapoptotic activity without changing its protein turnover(39). In mammary epithelial cells, oncogenic transformationby RASV12 has been described to suppress upregulation of BMFduring anoikis (40).

Besides BMF, we show that also other BCL-2 family proteinscontribute to MEK162/BYL719-stimulated apoptosis, as (i)MEK162/BYL719 cotreatment cooperates to upregulate BIM andto downregulate MCL-1 and as (ii) BIM silencing or MCL-1overexpression significantly protects RMS cells from MEK162/BYL719-induced cell death. Both AKT and ERK have beenreported to phosphorylate and inactivate the transcription factorFOXO3a that regulates BIM expression (41). In addition, ERKdirectly phosphorylates BIM, which enhances its proteasomaldegradation (42). MCL-1 expression is tightly controlled by bothRAF/MEK/ERK and PI3K/AKT/mTOR pathways (13, 43), involv-ing transcription factors such as ELK-1 (44) or CREB (45),mTORC1-stimulated translation of MCL-1 (46), or posttransla-tional modifications, such as phosphorylation, that control thestability of MCL-1 (47).

Together, these MEK162/BYL719-stimulated changes in theratio of pro- and antiapoptotic BCL-2 proteins toward a proa-poptotic state act in concert to promote activation of BAXand BAK and caspase-dependent mitochondrial apoptosis,as individual silencing of one of these proteins providespartial protection from MEK162/BYL719-triggered apoptosis(Fig. 7G). Our findings showing that MEK162/BYL719 cotreat-ment triggers upregulation of BMF and BIM as well as MCL-1downregulation, whereas NRAS knockdown upregulates BMFand BIM but has little effect on MCL-1 levels may explain whyNRAS knockdown on its own is not sufficient to induce apo-ptosis. The crucial role of the mitochondrial pathway in medi-ating MEK162/BYL719-induced apoptosis is emphasized byour data showing that BAX/BAK silencing, overexpression ofMCL-1 or BCL-2 as well as caspase inhibition rescue cell death.Intriguingly, the BCL-2–specific inhibitor ABT-199 completelyreversed the BCL-2–conferred resistance to MEK162/BYL719-induced apoptosis. This implies that therapeutic modulation ofmitochondrial apoptosis may offer new opportunities toenhance the antitumor activity of combined RAF/MEK/ERKand PI3K/AKT/mTOR inhibition. It also implicates that mar-kers of an intact mitochondrial apoptotic pathway, for exampleusing BH3 profiling, may help to predict the sensitivity of RMScells toward MEK162/BYL719 cotreatment, e.g., for patientstratification.

Besides changes in BCL-2 family proteins, we show thatcombined inhibition of RAF/MEK/ERK and PI3K/AKT/mTORpathways is necessary in NRAS-mutated RMS cells to potentlysuppress cap-dependent translation. Both 4E-BP1 and S6 areimportant regulators of cap-dependent translation (48), and4E-BP1 has been identified as a key common downstreameffector of active RAF/MEK/ERK and PI3K/AKT/mTOR signal-ing in RAS- and PIK3CA-mutated cancer cells (49). Interesting-ly, we show that MEK162/BYL719-conferred dephosphoryla-tion of 4E-BP1 and S6 is accompanied by downregulation ofMCL-1 and upregulation of BMF levels. MCL-1 is a short-livedprotein that for its translation depends on active mTORC1(46), and IRES-mediated upregulation of BMF has beendescribed as a cellular stress response to inhibition of cap-dependent translation (36). Thus, MEK162/BYL719-imposedsuppression of cap-dependent translation may well lead todownregulation of MCL-1 and upregulation of BMF levels. Inaddition, reduced proliferation upon blockage of cap-depen-dent translation is expected to facilitate the induction of pro-grammed cell death.

Our study has important implications. First, it might inspirefuture clinical trials in RMS by providing a rationale for thecombined use of MEK- and PI3Ka-specific inhibitors in NRAS-mutated RMS. Because sequencing of RMS tumor samples prior totreatment is being more and more introduced into clinical prac-tice and because several therapeutics targeting RAS effector net-works have already been approved or are in late-stage develop-ment, it is in principle feasible to translate our approach intoclinical application in the future. The relevance of concomitantinhibition of RAS and PI3K signaling in RMS is further empha-sized by documented simultaneous mutations of RAS andPIK3CA in some PAX gene fusion–negative RMS (2–4).

Second, specific targeting of PI3Ka may offer therapeuticadvantages, as it reduces the side effects of pan-PI3K inhibitors.BYL719 has been reported to have an improved safety profile withrespect to glucose metabolism and proved to be well-toleratedwith manageable side effects (50). Currently, the combination ofBYL719 and MEK162 is being evaluated in an ongoing clinicaltrial for adult solid cancers with documented RAS of BRAFmutations (NCT01449058).

Third, our findings are not restricted to NRAS-mutated RMSbut are likely of broader relevance also for RMS-harboring muta-tions in other RAS genes, because we show that HRAS-mutatedRMS cells similarly respond to MEK162/BYL719 cotreatment.In summary, by elucidating the RAS effector pathways in RMScells, our study provides a rationale for the combined use ofMEK-and PI3Ka-specific inhibitors in RAS-mutated RMS that warrantsfurther investigation.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: N. Dolgikh, S. FuldaDevelopment of methodology: N. Dolgikh, M. Hugle, M. VoglerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. DolgikhAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N. Dolgikh, M. Hugle, M. Vogler, S. FuldaWriting, review, and/or revision of the manuscript: N. Dolgikh, M. Hugle,M. Vogler, S. Fulda






Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. FuldaStudy supervision: S. Fulda

AcknowledgmentsThis work has partially been supported by grants from the BMBF (to

S. Fulda).We thankD. Br€ucher for expert technical assistance and C. Hugenbergfor expert secretarial assistance.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 12, 2017; revised December 6, 2017; accepted January 30,2018; published first February 6, 2018.

References1. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a

tumorigenic web. Nat Rev Cancer 2011;11:761–74.2. Shern JF, Chen L, Chmielecki J, Wei JS, Patidar R, Rosenberg M, et al.

Comprehensive genomic analysis of rhabdomyosarcoma reveals a land-scape of alterations affecting a common genetic axis in fusion-positive andfusion-negative tumors. Cancer Discov 2014;4:216–31.

3. Seki M, Nishimura R, Yoshida K, Shimamura T, Shiraishi Y, Sato Y, et al.Integrated genetic and epigenetic analysis defines novel molecular sub-groups in rhabdomyosarcoma. Nat Commun 2015;6:7557.

4. Chen X, Stewart E, Shelat AA, Qu C, Bahrami A, Hatley M, et al. Targetingoxidative stress in embryonal rhabdomyosarcoma. Cancer Cell 2013;24:710–24.

5. El Demellawy D, McGowan-Jordan J, de Nanassy J, Chernetsova E, Nasr A.Update onmolecular findings in rhabdomyosarcoma. Pathology 2017;49:238–46.

6. Arnold MA, Barr FG. Molecular diagnostics in the management of rhab-domyosarcoma. Expert Rev Mol Diagn 2017:1–6.

7. Gysin S, Salt M, Young A,McCormick F. Therapeutic strategies for targetingras proteins. Genes Cancer 2011;2:359–72.

8. Fulda S. Tumor resistance to apoptosis. Int J Cancer 2009;124:511–5.9. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell

2011;144:646–74.10. Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in

anticancer chemotherapy. Oncogene 2006;25:4798–811.11. Taylor RC, Cullen SP, Martin SJ. Apoptosis: controlled demolition at the

cellular level. Nat Rev Mol Cell Biol 2008;9:231–41.12. Fulda S, Galluzzi L, Kroemer G. Targetingmitochondria for cancer therapy.

Nat Rev Drug Discov 2010;9:447–64.13. Czabotar PE, Lessene G, Strasser A, Adams JM. Control of apoptosis by the

BCL-2 protein family: implications for physiology and therapy. Nat RevMol Cell Biol 2014;15:49–63.

14. Solit DB, Garraway LA, Pratilas CA, Sawai A, Getz G, Basso A, et al.BRAF mutation predicts sensitivity to MEK inhibition. Nature 2006;439:358–62.

15. Mendoza MC, Er EE, Blenis J. The Ras-ERK and PI3K-mTOR pathways:cross-talk and compensation. Trends Biochem Sci 2011;36:320–8.

16. Engelman JA, Chen L, Tan X, Crosby K, Guimaraes AR, Upadhyay R, et al.Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D andPIK3CA H1047R murine lung cancers. Nat Med 2008;14:1351–6.

17. PoschC,Moslehi H, Feeney L, Green GA, Ebaee A, Feichtenschlager V, et al.Combined targeting of MEK and PI3K/mTOR effector pathways is neces-sary to effectively inhibitNRASmutantmelanoma in vitro and in vivo. ProcNatl Acad Sci U S A 2013;110:4015–20.

18. Schott C, Graab U, Cuvelier N, Hahn H, Fulda S. Oncogenic RAS mutantsconfer resistance of RMS13 rhabdomyosarcoma cells to oxidative stress-induced ferroptotic cell death. Front Oncol 2015;5:131.

19. Fulda S, Sieverts H, Friesen C, Herr I, Debatin KM. The CD95 (APO-1/Fas)system mediates drug-induced apoptosis in neuroblastoma cells. CancerRes 1997;57:3823–9.

20. Heinicke U, Kupka J, Fichter I, Fulda S. Critical role of mitochondria-mediated apoptosis for JNJ-26481585-induced antitumor activity in rhab-domyosarcoma. Oncogene 2016;35:3729–41.

21. Hugle M, Belz K, Fulda S. Identification of synthetic lethality of PLK1inhibition and microtubule-destabilizing drugs. Cell Death Differ 2015;22:1946–56.

22. Chou TC.Drug combination studies and their synergy quantification usingthe Chou-Talalay method. Cancer Res 2010;70:440–6.

23. Vogler M, Walczak H, Stadel D, Haas TL, Genze F, Jovanovic M, et al.Targeting XIAP bypasses Bcl-2-mediated resistance to TRAIL and coop-

erates with TRAIL to suppress pancreatic cancer growth in vitro and in vivo.Cancer Res 2008;68:7956–65.

24. Xu YC, Wang X, Chen Y, Chen SM, Yang XY, Sun YM, et al. Integration ofreceptor tyrosine kinases determines sensitivity to PI3Kalpha-selectiveinhibitors in breast cancer. Theranostics 2017;7:974–86.

25. Crose LE, Linardic CM. Receptor tyrosine kinases as therapeutic targets inrhabdomyosarcoma. Sarcoma 2011;2011:756982.

26. Castellano E, Downward J. RAS interaction with PI3K: more than justanother effector pathway. Genes Cancer 2011;2:261–74.

27. Gupta S, Ramjaun AR, Haiko P, Wang Y, Warne PH, Nicke B, et al. Bindingof ras to phosphoinositide 3-kinase p110alpha is required for ras-driventumorigenesis in mice. Cell 2007;129:957–68.

28. Gritsman K, Yuzugullu H, Von T, Yan H, Clayton L, Fritsch C, et al.Hematopoiesis and RAS-driven myeloid leukemia differentially requirePI3K isoform p110alpha. J Clin Invest 2014;124:1794–809.

29. Ku BM, Jho EH, Bae YH, Sun JM, Ahn JS, Park K, et al. BYL719, a selectiveinhibitor of phosphoinositide 3-Kinase alpha, enhances the effect ofselumetinib (AZD6244, ARRY-142886) in KRAS-mutant non-small celllung cancer. Invest New Drugs 2015;33:12–21.

30. Baer R, Cintas C, DufresneM, Cassant-Sourdy S, Schonhuber N, Planque L,et al. Pancreatic cell plasticity and cancer initiation induced by oncogenicKras is completely dependent on wild-type PI 3-kinase p110alpha. GenesDev 2014;28:2621–35.

31. MurilloMM, Zelenay S, Nye E, Castellano E, Lassailly F, StampG, et al. RASinteraction with PI3K p110alpha is required for tumor-induced angiogen-esis. J Clin Invest 2014;124:3601–11.

32. Singh A, Greninger P, Rhodes D, Koopman L, Violette S, Bardeesy N,et al. A gene expression signature associated with "K-Ras addiction"reveals regulators of EMT and tumor cell survival. Cancer Cell 2009;15:489–500.

33. GuentherMK,GraabU, Fulda S. Synthetic lethal interaction between PI3K/Akt/mTOR and Ras/MEK/ERK pathway inhibition in rhabdomyosarcoma.Cancer Lett 2013;337:200–9.

34. Renshaw J, Taylor KR, Bishop R, Valenti M, De Haven Brandon A,Gowan S, et al. Dual blockade of the PI3K/AKT/mTOR (AZD8055) andRAS/MEK/ERK (AZD6244) pathways synergistically inhibits rhabdo-myosarcoma cell growth in vitro and in vivo. Clin Cancer Res 2013;19:5940–51.

35. Hornsveld M, Tenhagen M, van de Ven RA, Smits AM, van Triest MH, vanAmersfoort M, et al. Restraining FOXO3-dependent transcriptional BMFactivationunderpins tumour growth andmetastasis of E-cadherin-negativebreast cancer. Cell Death Differ 2016;23:1483–92.

36. Grespi F, Soratroi C, Krumschnabel G, Sohm B, Ploner C, Geley S, et al.BH3-only protein Bmfmediates apoptosis upon inhibition of CAP-depen-dent protein synthesis. Cell Death Differ 2010;17:1672–83.

37. VanBrocklin MW, Verhaegen M, Soengas MS, Holmen SL. Mitogen-acti-vated protein kinase inhibition induces translocation of Bmf to promoteapoptosis in melanoma. Cancer Res 2009;69:1985–94.

38. Boussemart L, Malka-Mahieu H, Girault I, Allard D, Hemmingsson O,Tomasic G, et al. eIF4F is a nexus of resistance to anti-BRAF and anti-MEKcancer therapies. Nature 2014;513:105–9.

39. Shao Y, Aplin AE. ERK2 phosphorylation of serine 77 regulates Bmf pro-apoptotic activity. Cell Death Dis 2012;3:e253.

40. Schmelzle T,Mailleux AA,OverholtzerM, Carroll JS, Solimini NL, LightcapES, et al. Functional role and oncogene-regulated expression of the BH3-only factor Bmf in mammary epithelial anoikis and morphogenesis. ProcNatl Acad Sci U S A 2007;104:3787–92.

41. Balmanno K, Cook SJ. Tumour cell survival signalling by the ERK1/2pathway. Cell Death Differ 2009;16:368–77.


Dolgikh et al.




42. Yang JY, Zong CS, XiaW, Yamaguchi H, Ding Q, Xie X, et al. ERK promotestumorigenesis by inhibiting FOXO3a via MDM2-mediated degradation.Nat Cell Biol 2008;10:138–48.

43. WarrMR, Shore GC.Unique biology ofMcl-1: therapeutic opportunities incancer. Curr Mol Med 2008;8:138–47.

44. Booy EP, Henson ES, Gibson SB. Epidermal growth factor regulates Mcl-1expression through theMAPK-Elk-1 signalling pathway contributing to cellsurvival in breast cancer. Oncogene 2011;30:2367–78.

45. Wang JM, Chao JR, Chen W, Kuo ML, Yen JJ, Yang-Yen HF. The anti-apoptotic genemcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containingCREB. Mol Cell Biol 1999;19:6195–206.

46. Mills JR, Hippo Y, Robert F, Chen SM, Malina A, Lin CJ, et al. mTORC1promotes survival through translational control of Mcl-1. Proc Natl AcadSci U S A 2008;105:10853–8.

47. Domina AM, Vrana JA, Gregory MA, Hann SR, Craig RW. MCL1 isphosphorylated in the PEST region and stabilized upon ERK activationin viable cells, and at additional sites with cytotoxic okadaic acid or taxol.Oncogene 2004;23:5301–15.

48. Bhat M, Robichaud N, Hulea L, Sonenberg N, Pelletier J, Topisirovic I.Targeting the translation machinery in cancer. Nat Rev Drug Discov2015;14:261–78.

49. She QB, Halilovic E, Ye Q, Zhen W, Shirasawa S, Sasazuki T, et al. 4E-BP1 is a key effector of the oncogenic activation of the AKT and ERKsignaling pathways that integrates their function in tumors. Cancer Cell2010;18:39–51.

50. Juric D, Rodon J, Gonzalez-Angulo A, Burris H, Bendell J, Berlin J, et al.BYL719, a next generation PI3K alpha specific inhibitor: preliminarysafety, PK, and efficacy results from the first-in-human study. Cancer Res2012;72.






2018;78:2000-2013. Published OnlineFirst February 6, 2018.Cancer Res Nadezda Dolgikh, Manuela Hugle, Meike Vogler, et al. αMitochondrial Apoptosis Induced by Coinhibition of MEK and PI3K

-Mutated Rhabdomyosarcoma Cells Are Vulnerable toNRAS

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NRAS-Mutated Rhabdomyosarcoma Cells Are Vulnerable to ... · NRAS-Mutated Rhabdomyosarcoma Cells Are Vulnerable to Mitochondrial Apoptosis Induced by Coinhibition of MEK and PI3Ka