loss of nf1 in cutaneous melanoma is associated with...

Therapeutics, Targets, and Chemical Biology

Loss of NF1 in Cutaneous Melanoma Is Associated with RASActivation and MEK Dependence

Moriah H. Nissan1,2, Christine A. Pratilas3,6, Alexis M. Jones2, Ricardo Ramirez2,7, Helen Won4, Cailian Liu8,Shakuntala Tiwari8, Li Kong2, Aphrothiti J. Hanrahan2, Zhan Yao6, Taha Merghoub8, Antoni Ribas9,Paul B. Chapman5, Rona Yaeger5,6, Barry S. Taylor10,11, Nikolaus Schultz7, Michael F. Berger2,4,Neal Rosen5,6, and David B. Solit2,5

AbstractMelanoma is a disease characterized by lesions that activate ERK. Although 70% of cutaneous melanomas

harbor activating mutations in the BRAF and NRAS genes, the alterations that drive tumor progression in theremaining 30% are largely undefined. Vemurafenib, a selective inhibitor of RAF kinases, has clinical utilityrestricted to BRAF-mutant tumors. MEK inhibitors, which have shown clinical activity in NRAS-mutantmelanoma,maybe effective in other ERKpathway-dependent settings. Here, we investigated a panel ofmelanomacell lines wild type for BRAF and NRAS to determine the genetic alteration driving their transformation and theirdependence on ERK signaling in order to elucidate a candidate set for MEK inhibitor treatment. A cohort of theBRAF/RAS wild type cell lines with high levels of RAS-GTP had loss of NF1, a RAS GTPase activating protein.In these cell lines, the MEK inhibitor PD0325901 inhibited ERK phosphorylation, but also relieved feedbackinhibition of RAS, resulting in induction of pMEK and a rapid rebound in ERK signaling. In contrast, the MEKinhibitor trametinib impaired the adaptive response of cells to ERK inhibition, leading to sustained suppressionof ERK signaling and significant antitumor effects. Notably, alterations in NF1 frequently co-occurred with RASand BRAF alterations in melanoma. In the setting of BRAF(V600E), NF1 loss abrogated negative feedback onRAS activation, resulting in elevated activation of RAS-GTP and resistance to RAF, but not MEK, inhibitors.We conclude that loss of NF1 is common in cutaneous melanoma and is associated with RAS activation,MEK-dependence, and resistance to RAF inhibition. Cancer Res; 74(8); 2340–50. �2014 AACR.

IntroductionThemitogen-activated protein kinase (MAPK) signaling path-

way is a critical regulator of cell growth and cell-cycle progres-sion. Receptor tyrosine kinases (RTK), activated by extracellularmitogens, stimulate the RAS (H-, N-, and KRAS) small GTPaseproteins. RAS proteins are activated when guanine exchangefactors facilitate binding to GTP and inactivated by GTPaseactivating proteins (GAP), such as NF1, that facilitate the hydro-lysis of GTP to GDP (1). Active RAS facilitates dimerization (2)and activation of RAF (A-, B-, and CRAF/RAF1) kinases, which in

turn activate the mitogen-activated protein kinase kinase 1/2(MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2)kinases. Activated, phosphorylated ERK regulates the transcrip-tion of proteins such as cyclin D1 that promote cell-cycle pro-gression, transcription factors that promote the transformedphenotype and a network of genes that negatively inhibit path-way output by regulating the activity of RTKs, RAS, and RAF (3).

Alterations resulting in aberrant activation of ERK can befound at almost every level of the MAPK pathway and arecommon in human tumors. BRAF mutations, particularly atcodon 600, are found inmany cancers, including approximately50% of cutaneous melanomas (4). Selective inhibitors of RAF(vemurafenib, dabrafenib) have unprecedented clinical activityin patients whosemelanomas harbor BRAF(V600E)mutations,and their use results in a prolongation of progression-free andoverall survival (5). NRASmutations, most commonly at codon61, have been identified in another 15% to 20% of melanomasand occur in a mutually exclusive pattern with BRAF(V600E)(4, 6, 7). Treatment options remain limited for these patientsand those whose tumors are wild type for BRAF and NRAS, andthe prognosis of such patients is particularly grim with amedian overall survival of less than 1 year (8).

Given the high prevalence of BRAF and NRAS alterations inmelanoma, we hypothesized that melanomas, including thosewild type for both genes (BRAFWT/NRASWT), may exhibit adependence on ERK pathway activation for maintenance of the

Authors' Affiliations: 1Louis V. Gerstner, Jr. Graduate School of Biomed-ical Science; 2Human Oncology and Pathogenesis Program; Departmentsof 3Pediatrics, 4Pathology, and 5Medicine; 6Programs in Molecular Phar-macology and Chemistry; 7Computational Biology; 8Ludwig CollaborativeLab, Memorial Sloan-Kettering Cancer Center, New York, New York;9Department of Medicine, Jonsson Comprehensive Cancer Center, Uni-versity of California, Los Angeles; Departments of 10Epidemiology andBiostatistics and 11Medicine, and the Helen Diller Family ComprehensiveCancer Center, University of California, San Francisco, California

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

Corresponding Author: David B. Solit, Memorial Sloan-Kettering CancerCenter, 1275 York Avenue, New York, NY 10065. Phone: 646-888-2641;Fax: 646-888-2595; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-2625

�2014 American Association for Cancer Research.

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transformed phenotype that may be exploited for therapeuticadvantage (9, 10). Here, we performed a functional and genomicanalysis of BRAFWT/NRASWT melanoma cell lines to determinewhether occult MAPK pathway alterations are present in suchcells. NF1 alterations that result in RAS activation and MEKdependence were identified in a subset of BRAFWT/NRASWT

melanoma cell lines. However, NF1 alterations were not mutu-ally exclusive with RAS and BRAF aberrations. Loss of NF1 incells co-mutated for BRAF was sufficient to overcome theupstream negative feedback that results in suppression of RASactivation in BRAF(V600E) cells and was sufficient to conferresistance to vemurafenib. Furthermore, NF1 loss and corre-sponding relief of upstream ERK-dependent negative feedbackattenuated the antiproliferative effects of the selective MEKinhibitor PD0325901 in NF1-null cells. In contrast, trametinib,an allostericMEK inhibitor recently approved for use in patientswith BRAF-mutant melanoma (11), attenuated the phosphor-ylation of MEK resulting from relief of upstream negativefeedback and exhibited greater potency than PD0325901 inNF1-null melanoma cells. In summary, loss of NF1 expressionin melanoma results in RAS activation and vemurafenib resis-tance even in the setting of BRAF mutation. Allosteric MEKinhibitors that impair the adaptive response of cells to ERKinhibition by blocking MEK phosphorylation should be studiedin patients with melanoma whose tumors harbor NF1 loss.

Materials and MethodsCell lines and culture conditions"SK-Mel" cell lines were provided by TahaMerghoub and Alan

Houghton (Memorial Sloan-Kettering Cancer Center, New York,NY). MeWo, Malme3M, A375, and SNF96.2 were purchasedfrom the American Type Culture Collection. M308 was providedby Antoni Ribas (University of California Los Angeles, Los Ange-les, CA) and WM3918 by Katherine Nathanson and MeenhardHerlyn (Wistar Institute, Philadelphia, PA). Other than A375 andSNF96.2 (grown in Dulbecco's Modified Eagle Medium), all celllines were grown in RPMI 1640 as previously described (12).

Genomic studiesCellular DNAwas extracted using the Qiagen DNeasy Tissue

Kit. DNA was analyzed using a mass spectrometry–basedfingerprinting assay to validate cell line identity as describedpreviously (13). NRAS (G12A, G12D, Q61K, Q61R, Q61L), BRAF(V600E, V600K, V600R, K601E), and c-KIT (D816V) mutationswere detected using a mass spectrometry–based assay (Seque-nom) and validated by Sanger sequencing (13). Selected celllineswere screened formutations and copy number alterationsin 279 cancer-associated genes using the integrated mutationprofiling of actionable cancer targets (IMPACT) assay as hasbeen described (14) and viewed in the integrated genomicsviewer (15). Genomic data from the TCGA melanoma projectwere derived from the cBioPortal for Cancer Genomics (http://cbioportal.org).

RNA-seqRNA was extracted from cells using the RNeasy Mini Kit

(Qiagen Inc.). Quality assessment, poly-A selection, andsequencing with an Illumina HiSeq 2000 were performed bythe Genomics Core Laboratory at Memorial Sloan-KetteringCancer Center. All samples had a minimum RNA integritynumber (RIN) of 7.0 (16). Sequencing produced 40 to 120million 75 bp reads per sample. FASTQ files were generatedusing CASAVA 1.8.2 software (Illumina). Low-quality bases andadapter sequences were removed with Cutadapt. Trimmedreads were aligned to human genome assembly GRCh37 usingTophat 2.0.8 (17, 18). Gene quantification and differentialexpression were calculated using Cufflinks 2.1.1 (19). Datavisualizations were created with the gplots package for R.

Western blottingCells were collected, lysed, and blotted as previously

described (12). Secondary antibodies were detected usingSuper Signal (Thermo) and chemiluminesence imaged usinga Fuji LAS-4000 imager (GE Lifesciences). Anti-NF1 (SC-67),cyclin D1 (SC-718), KRAS (SC-30), NRAS (SC-519), HRAS(SC-520), and actinin (SC-17829) were from Santa Cruz

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A BFigure 1. NF1-null melanoma celllines express high levels ofactivated RAS. A, BRAF andNRAS status of the melanoma cellline panel (n ¼ 191). B, activatedRAS protein (RAS-GTP) wasquantitated in select melanomacell lines via immunoprecipitationwith the RAS-binding domain ofRAF (RAF-1 RBD) followed byimmunoblot using pan-RAS andisoform selectiveNRAS andKRASantibodies. Expression of NF1,total RAS (pan-RAS), and actinin(as a loading control) wasmeasured by immunoblot fromwhole cell lysate (WCL). Alt,alteration; mut, mutant; WCL,whole cell lysate.

RAS Activation and MEK Dependence in NF1-Null Melanoma

www.aacrjournals.org Cancer Res; 74(8) April 15, 2014 2341




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Biotechnology; anti-Ras (#1862335) from Thermo Scientific;and anti-pERK (#9101), ERK (#9102), pMEK (#9121), MEK(#9122), and p-CRAF S338 (#9427) from Cell SignalingTechnology.

Proliferation assays/FACS analysisCell viability was measured by trypan blue incorporation

using a Vi-CELL XR 2.03 (Beckman Coulter) as previouslydescribed (20). Percent growth was calculated using the equa-tion 100 � ([Day 5 drug] � [Day 0])/([Day 5 DMSO] � [Day0]). FACS analysis was performed as previously described (21).

RAS-GTP assayGTP-bound RAS was isolated via immunoprecipitation

using recombinant RAS binding domain of RAF1 (RAF1-RBD;Active RAS Pull-down and Detection Kit; Thermo Scientific),according to themanufacturer's instructions. The product wasdetected using total (pan-RAS) or isoform specific (H-, K-, N-)RAS antibodies.

RNAi studiessiRNA studies usedON-TARGETplus siNF1 SMARTpool and

ON-TARGET plus nontargeting siRNA#2. shRNA studies wereaccomplished using GIPZ shNon-targeting RNA or TRIPZinducible shRNA against NF1. shRNAs (shNF1 #2: CloneIDV2THS-260806; shNF1 #4: CloneID V3THS-380114; shRNA#6:CloneID V3THS-380110) were induced with 2 mg/mL doxycy-cline daily for 1 week before vemurafenib studies.

ResultsNF1 expression is lost in a subset of RAS-activatedBRAFWT/RASWT cell linesTo identify a cohort of BRAFWT/NRASWT melanoma cell

lines for genomic and biologic characterization, 191 mela-noma cell lines were genotyped for BRAF and NRAS muta-tions using a mass spectrometry–based (Sequenom) assay(13, 22). This screen identified 66 cell lines that lackedhotspot mutations in BRAF or NRAS (Fig. 1A and Supple-mentary Table S1). As this assay was designed to detect onlythe most common BRAF and NRAS mutations, we furtherperformed Sanger sequencing of BRAF exons 11 and 15 andNRAS exons 2 and 3. This analysis identified BRAF mutationsnot present in the Sequenom assay in 2 cell lines (D594G inSK-Mel-264 and N581S in SK-Mel-215; Supplementary TableS2). Direct sequencing of KRAS and HRAS further identifiedactivating mutations in KRAS in 2 and HRAS in 1 cell line,respectively (Supplementary Table S2). In summary, 61 celllines were wild type for RAS and BRAF.Given the high prevalence of ERK activation in melanoma

(10), we hypothesized that a subset of the BRAFWT/RASWT

cohort likely harbored occult alterations within the MAPKpathway that cause RAS to become refractory to negativefeedback and thus confer activation of ERK.We thusmeasured

levels of activated, GTP-bound RAS in a subset of the BRAFWT/RASWT cell lines as a surrogate of pathway activation. Similarto human tumors, KRAS- and NRAS-mutant melanoma celllines exhibit high levels of RAS-GTP, whereas BRAF-mutantcell lines have low to undetectable levels of RAS-GTP (Fig. 1B;refs. 3, and 23). RAS was activated to varying levels in theBRAFWT/RASWT melanoma cells, with some expressing levelsof activated RAS similar to those present in RAS-mutant cells(Supplementary Fig. S1).

The NF1 gene encodes a protein that functions as thepredominant RASGTPase activating protein (RASGAP), whichsuppresses RAS activity and reduces RAS-GTP levels by pro-moting endogenous RAS GTPase activity. NF1 is inactivated indiverse human cancers (24–27) and would be predicted, if lost,to cause RAS to become refractory to negative feedback. Weperformed Western blot analysis to determine whether loss ofNF1 protein expression occurred in, and was correlated with,elevated RAS-GTP levels in BRAFWT/RASWT melanoma celllines. Complete loss of NF1 expression was noted in 5 of theBRAFWT/RASWT cell lines, all of which had high levels of RAS-GTP (Supplementary Fig. S1). Having previously performedhigh-resolution DNA copy number profiling (array CGH) on 92melanoma cell lines (22), we identified a sixth NF1-null cellline that harbored homozygous NF1 gene deletion and con-current NRAS(Q61R) mutation (SK-Mel-103).

NRASmutations are significantly more prevalent than otherRASmutations in melanoma even though KRASmutations arepredominant in most other cancers (4). To determine whichRAS isoforms were activated in NF1-null melanomas, weassayed activated KRAS, HRAS, and NRAS by performingimmunoprecipitation with the RAS binding domain of RAF1(RAF1-RBD; see Materials and Methods) followed by RASisoform-specific immunoblots. All 4 NF1-null cell lines exam-ined expressed high levels of total active RAS when comparedwith a BRAF(V600E) control cell line (Fig. 1B and Supplemen-tary Fig. S1). NRAS(Q61K) SK-Mel-30 cells expressed high levelsof GTP-bound NRAS, but no detectable levels of activatedKRAS, similar to the NRAS(Q61R)/NF1-null SK-Mel-103 line.GTP-bound NRAS was also highly expressed in the other NF1-null cell lines, whereas only a subset had concurrent activationof KRAS, including SK-Mel-217, which harbored KRAS geneamplification. Elevated levels of GTP-bound KRAS and NRASwere also detected in the KRAS (G12C)-mutant SK-Mel-285 cellline. Levels of activated HRAS were low or undetectable in allthe NF1-null melanoma cell lines (Supplementary Fig. S1).

To define the mechanistic basis for the loss of NF1 expres-sion in the melanoma cell lines, we performed next-generationsequencing of 279 genes commonly mutated in human cancerusing an exon capture-based approach (IMPACT assay; refs. 14and 28). Two cell lines were found to harbor nonsense muta-tions in NF1 (Fig. 2A): MeWo, a hemizygous Q1336� mutation,and Sk-Mel-266, L161� and Q282� mutations. The remaining 4cell lines had deletions involving the NF1 gene locus: SK-Mel-

Figure 2. The genomic basis of NF1 loss in melanoma cell lines. A, DNA from NF1-null cell lines was analyzed using a capture based, next-generationsequencingmethod (IMPACT). Shownare aligned sequencing reads highlighting selectNF1mutations. Percentages (left) are the ratio ofmutant readsover totalreads (right). B, homozygous deletions of NF1 in 4 melanoma cell lines. Exon-level copy number data are shown for target genes on chromosome17. C, summary of mutations and copy-number alterations in NF1-null cell lines by IMPACT and NF1 mutant melanoma tumors analyzed by TCGA.






113, focal homozygous loss of the N-terminal domain; SK-Mel-103 and WM3918, focal homozygous loss of the C-terminaldomain, and SK-Mel-217, broad monoallelic loss, as well as afocal, intragenic deletion in the second NF1 allele (Fig. 2B). Insum, genomic alterations sufficient to account for completeloss of NF1 protein expression were identified in all 6 NF1-nullmelanoma cell lines.

Although loss of NF1 was identified in the BRAFWT/RASWT

cohort, it was not mutually exclusive with RAS alterations.Notably, concurrent alterations in the NF1 and RAS genes havealso been noted in 2 recent whole-exome sequencing studiesof melanoma tumors, including Hodis and colleagues and themelanoma study performed by the Cancer Genome Atlas(TCGA) working group (Fig. 2C; ref. 29). CDKN2A and/orTP53 were among the genes most commonly co-altered in theNF1-null melanoma cell lines and tumors, suggesting thatthese genes may cooperate with NF1 loss in promoting mel-anomagenesis as has been reported in other cancer types,such as astrocytomas and malignant peripheral nerve sheathtumors (30).

NF1-null cell lines are sensitive to MEK inhibitors thatimpair the adaptive response of cells to ERK inhibition

The growth of NF1-null cell lines derived from humanmalignant peripheral nerve sheath tumors (MPNST) andMPNSTs that arise in NF1�/� mice have been shown to bedependent on mTORC1 signaling and exquisitely sensitive tothe mTORC1 inhibitor rapamycin (31, 32). To determinewhether NF1-null melanomas were also mTORC1 dependent,we treated the NF1-null melanoma cell lines with rapamycinand compared their sensitivity with that of SNF96.2, a repre-sentative human NF1-null MPNST cell line (Fig. 3A). The NF1-null melanomas as a group were significantly less sensitive torapamycin (IC50 ranging from 1 to 286 nmol/L) than SNF96.2cells (IC50 ¼ 0.05 nmol/L), suggesting that mTORC1 depen-dency is not a feature of all NF1-null cells.

To probe the MEK dependence of NF1-null melanomas, weused PD0325901, a selective allosteric inhibitor of MEK1/2(Ki

app of 1 nmol/L for MEK1 and MEK2; ref. 33). The effect ofPD0325901 on the proliferation and survival of 4 NF1-nullmelanoma cell lines was compared with that of a MEK inhib-itor-sensitive BRAF(V600E) melanoma cell line (SK-Mel-239)and to a MEK inhibitor–resistant ERBB2 amplified breastcancer cell line (BT-474). The proliferation of all 4 NF1-nullcell lines was inhibited by PD0325901, albeit with IC50s thatwere 6- to 20-fold greater than that of the BRAF(V600E) SK-Mel-239 cells (Fig. 3B).

To explore the basis for this differential sensitivity, weassessed the effects of drug exposure on downstream targetsof MEK/ERK signaling as a function of concentration andtime. Treatment of both BRAF(V600E) and NF1-null cellswith 50 nmol/L PD0325901 resulted in decreased activationof phosphorylated ERK1/2 (pERK) by 1 hour (Fig. 3C andSupplementary Fig. S2). In BRAF(V600E) cells, suppressionof pERK was durable and maintained at 6 and 24 hours andwas accompanied by loss of cyclin D1 expression, accumu-lation of cells in G1 phase, and induction of apoptosis (Fig.3C and D). In contrast, a partial rebound in pERK activation

was apparent by 6 hours in NF1-null cells and was accom-panied by a failure of the drug to potently suppress cyclin D1expression. This rebound in pERK in the NF1-null cellswas attenuated with use of a higher concentration of drug(500 nmol/L), leading to potent suppression of cyclin D1expression, maximal accumulation of cells in G1 phase, andinhibition of proliferation (Fig. 3B–D). Together, these datasuggest that cyclin D1 expression and cell-cycle progressionare MEK-dependent in NF1-null melanoma cells, but thatrapid rebound in ERK activity may account for the lowersensitivity of NF1-null cells to the MEK inhibitor PD0325901.Induction of cell death was not observed following treat-ment with the MEK inhibitor in any of the NF1-null cell lines(Fig. 3D). Furthermore, cotreatment with PD0325901 and thepan-AKT inhibitor MK2206 did not augment growth inhi-bition or induce apoptosis as has been shown in colorectalcells with RAS activation (Supplemental Fig. S3; ref. 34).

Resistance to allosteric MEK inhibitors can be induced byupstream pathway hyperactivation (35, 36). We have previous-ly shown that treatment of BRAFWT but not BRAF (V600E) cellswith PD0325901 leads to increased levels of phosphorylatedMEK (pMEK), which results from relief of upstream ERK-dependent negative feedback (3). Consistent with these priorobservations, treatment of NF1-null melanoma cells withPD0325901 resulted in increased pMEK (Fig. 3C).

As the induction of pMEK in the NF1-null melanomasparalleled the rebound in pERK activation, we further studiedthe effects of a second allosteric MEK inhibitor on MEKsignaling and cellular proliferation. Trametinib (GSK1120212)has a similar in vitro affinity for MEK1/2 as PD0325901 (IC50sfor MEK1 andMEK2 of 0.7 and 0.9 nmol/L, respectively), but incontrast to PD0325901, binding of trametinib toMEKblocks itsphosphorylation at serine 217 (11). To compare the relativepotencies of PD0325901 and trametinib in vivo, we firstexposed BRAF(V600E) SK-Mel-239, and NF1-null SK-Mel-113cells to increasing concentrations of both drugs and assessedthe effect of drug treatment on pERK activation at 1 hour. InBRAF(V600E) SK-Mel-239 cells, both drugs were equipotent intheir ability to suppress ERK activation at 1 hour (Fig. 4A). Incontrast, in NF1-null SK-Mel-113 cells, trametinib was consid-erably more potent in its ability to suppress pERK activationthan either PD0325901 or 2 additional allosteric MEK inhi-bitors currently in clinical testing (AZD6244 and MEK162;Fig. 4A and Supplementary Fig. S4; refs. 37–39). Treatment ofNF1-null melanoma cells with either PD0325901 or trametinibresulted in hyperactivation of RAS consistent with relief ofupstream negative feedback following inhibition of ERK (Fig.4B). However, relief of upstream feedback following MEKinhibition was accompanied by a significant increase inpMEK levels in PD0325901-treated NF1-null cells, which wasattenuated in cells treated with trametinib (Fig. 4B and Sup-plementary Fig. S4). This attenuation of MEK phosphorylationwas unique to trametinib and was not observed withPD0325901, AZD6244, or MEK162 (Supplementary Fig. S4).Furthermore, the resistance of MEK to upstream hyperactiva-tion by RAS in trametinib-treated cells was accompanied by amore durable downregulation of pERK and more potentinhibition of genes whose transcription is dependent upon

Nissan et al.





ERK, including CCND1 (cyclin D1), ETV1, MYC, and SPRY4, ascompared with PD0325901 (Fig. 4B and D). Consistent withthese biologic differences among the MEK inhibitors, theantiproliferative effects of trametinib were similar in BRAF(V600E) and NF1-null cells, whereas BRAF(V600E) cells exhib-ited greater sensitivity than NF1-null cells to PD0325901 (Fig.4C and Supplementary Fig. S4).

NF1 loss and resulting RAS activation inBRAF(V600E) melanoma cells confers resistanceto RAF inhibitionLevels of GTP-bound RAS are low in BRAF(V600E) mel-

anomas as a result of high levels of ERK-dependent negativefeedback (23). It was unclear whether loss of NF1 function inthis context would be sufficient to overcome the feedback-

mediated suppression of RAS activity in BRAF(V600E) cells.We therefore screened a panel of 10 BRAF(V600E) melano-ma cell lines for loss of NF1 expression and activation ofRAS-GTP. Nine of the 10 BRAF(V600E)–mutant melanomacell lines expressed low to undetectable levels of RAS-GTP(Fig. 5A). A single BRAF(V600E) cell line (M308) had highlevels of RAS-GTP similar to that of an NRAS(Q61K) cell line,and notably, M308 was devoid of NF1 expression by immu-noblot (Fig. 5A). Genomic analysis by IMPACT confirmed thepresence of a nonsense mutation in the NF1 gene (Q1070�) asthe basis for the loss of NF1 protein expression in M308 cells(Supplementary Fig. S5). To determine whether NF1 lossdesensitizes BRAF(V600E) cells to RAF inhibition, weassessed the sensitivity of M308 [BRAF(V600E)/NF1-null]cells to vemurafenib. Vemurafenib treatment of SK-Mel-

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Figure 3. NF1-null melanoma cell lines are MAPK pathway dependent. A, cells were treated with increasing concentrations of rapamycin for 5 days.Results are percent cell growth as a function of drug concentration (nmol/L). B, cells were treated with increasing concentrations of the MEK inhibitorPD0325901 for 5 days. Results as in A. C, cells were treated with 50 or 500 nmol/L PD0325901 for 0, 1, 6, and 24 hours. Phospho- and total levels of MAPKpathway components were determined by immunoblot. D, cells were treated for 24 hours with 50 or 500 nmol/L PD0325901 before undergoing FACSanalysis for cell-cycle distribution. Error bars, SEM; n ¼ 3.






239 [BRAF(V600E)/NF1WT] cells resulted in potent down-regulation of phosphorylated MEK and ERK and inhibitionof cell growth (Fig. 5B). In contrast, vemurafenib had littleeffect on levels of phosphorylated MEK and ERK in BRAF(V600E)/NF1-null M308 cells and no effect on cell prolifer-ation (Fig. 5B and C).

RAS activation is sufficient to induce vemurafenib resistancein BRAF(V600E) cells (Supplementary Fig. S6). To determinewhether NF1 loss activates RAS sufficiently to overcome ERK-dependent negative feedback and induce vemurafenib resis-tance, we knocked down NF1 expression in BRAF(V600E)-mutant A375 cells and assessed levels of RAS activation in thepresence and absence of vemurafenib. siRNA and shRNAmediated knockdown of NF1 resulted in induction of RAS-

GTP and decreased sensitivity to vemurafenib (SupplementaryFig. S6). These data suggest that loss of NF1 function in BRAF(V600E) mutant cells is sufficient to induce RAS-GTP activa-tion and, as a consequence, vemurafenib resistance (Fig. 5A–Cand Supplementary Fig. S6).

To assess theMEK dependence of theM308melanoma cells,we determined the effects of PD0325901 and trametinib treat-ment on ERK activation and cellular proliferation. Analogousto the results seen with these inhibitors in BRAFWT/NF1-nullmelanoma cells, exposure of M308 cells to 50 nmol/LPD0325901 was insufficient to durably suppress ERK signalingand cell proliferation (Fig. 5D and E). In contrast, treatment ofM308 cells with trametinib resulted in durable suppressionof pERK activation, potent downregulation of cyclin D1

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Figure 4. Trametinib durably inhibits ERK activation and proliferation in NF1-null melanoma cell lines. A, cells were treated with increasing concentrationsof PD0325901 or trametinib for 1 hour. pERK was measured by immunoblot. B, cells were treated with 50 nmol/L PD0325901 or 50 nmol/L trametinibfor 0, 1, 6, and 24 hours. RAS-GTP was quantitated as described in Materials and Methods. Phospho- and total levels of MAPK pathway components weredetermined by immunoblot. C, cells were treated with increasing doses of PD0325901 or trametinib for 5 days. Results are percent cell growth as afunction of drug concentration (nmol/L). D, NF1-null SK-Mel-113 cells were treated with dimethyl sulfoxide (DMSO) or 50 nmol/L PD0325901 or trametinib for1, 6, or 24 hours. RNA levels were quantitated by RNA-seq. Shown are relative mRNA expression levels as a function of duration of drug exposurefor selected ERK output genes.

Nissan et al.





expression, and potent inhibition of cellular proliferation (Fig.5D and E).

DiscussionNeurofibromatosis Type I is a hereditary disorder caused by

germline mutation of the NF1 gene (350 kb genomic DNA; 60exons; 2,818 amino acids). This syndrome is characterized bythe formation of benign neurofibromas as well as pigmentedcaf�e-au-lait spots and Lisch nodules. Individuals with neuro-fibromatosis are at risk of developingmalignant neoplasms at ayoung age, most commonly optic gliomas, malignant periph-eral nerve sheath tumors, and juvenile myelomonocytic leu-kemia (40). Only recently have somatic alterations in the NF1gene been implicated in malignancies, including gliomas,

breast cancers, and leukemias (24–27). The prior underesti-mation of the prevalence ofNF1 alterations in human tumors islikely attributable to the technical challenges previously inher-ent in sequencing large genes with tumor suppressive functionin which mutations throughout the coding region are poten-tially oncogenic. With the advent of massively parallel nextgeneration sequencing methods, many of these technicalchallenges have now been overcome.

Here, we report that a significant subset of melanoma celllines, including those wild type for BRAF and RAS, exhibittotal loss of NF1 protein expression. In all cases, a mutationand/or focal deletion of the NF1 gene, rather than posttran-scriptional regulation (41) could be identified as the basis forNF1 loss. In contrast to prior reports (42, 43), all the NF1-null

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Figure 5. NF1 loss in the context of BRAF(V600E) mutation results in elevated RAS-GTP levels and sensitivity to MEK but not RAF inhibition. A, RAS-GTPwas quantitated as described in Materials and Methods. Mut, mutant; WCL, whole cell lysate. B, cells were treated with 2 mmol/L vemurafenib for 0, 1, 6, and24 hours. Phospho- and total levels of MAPK pathway components were determined by immunoblot. C, cells were treated with increasing concentrationsof the RAF inhibitor vemurafenib for 3 or 5 days. Results are cell count as a function of drug concentration over time. Error bars, SEM; n ¼ 3. D, cellswere treated with 50 nmol/L of PD0325901 or trametinib for 0, 1, 6, and 24 hours. E, cells were treated with increasing concentrations of PD0325901 ortrametinib for 3 or 5 days. Results are cell count as a function of drug concentration over time. Error bars, SEM; n ¼ 3.






melanoma cell lines expressed levels of active GTP-boundRAS comparable with those found in RAS-mutant cells.Notably, NF1 loss was not mutually exclusive with RAS orBRAF mutations. In fact, mutations in NF1 were found to co-associate with additional activating alterations in the RAS/MAP kinase pathway, in particular with exon 11 BRAFmutations (Fig. 2C). Several of these BRAF mutations exhibitimpaired kinase activity but induce ERK signaling by dimer-izing with and activating CRAF (44). As induction of RASactivity through NF1 loss would be predicted to promote theformation of CRAF homo- and heterodimers, NF1 alterationsmay cooperate with low and impaired activity BRAFmutants to induce transformation by further enhancing RAFdimer formation.

Studies using genetically engineered mouse models withmelanocyte-targeted deletion of NF1 also suggest that NF1loss cooperates with BRAF(V600E) mutation to promotemelanomagenesis and suggest that this cooperativity results,at least in part, through abrogation of oncogene inducedsenescence (45). We observed that knockdown of NF1expression was sufficient to overcome the ERK-dependentfeedback suppression of RAS observed in BRAF(V600E) cells.However, loss of both copies of NF1 is likely required tomaximally elevate RAS-GTP expression, as a heterozygoussplice site mutation in NF1 in A375 cells (45) was notaccompanied by NF1 loss or increased RAS-GTP levels(Supplementary Fig. S7). These results suggest that loss ofNF1 in BRAF(V600E) melanoma cells may provide a selectiveadvantage, even in the absence of RAF inhibitor exposure, bydiminishing the oncogene-induced suppression of RASmediated by ERK-dependent negative feedback. A secondaryconsequence of this co-mutation pattern is that such tumorsexhibit intrinsic resistance to selective RAF inhibitors. Ourstudies suggest that partial loss of NF1 function may resultin a more pronounced and rapid restoration of RAS signalingfollowing RAF inhibitor therapy. This could result in anattenuation of drug response sufficient to promote theemergence of drug-resistant clones.

Efforts to develop clinically useful direct inhibitors of RAShave been unsuccessful to date (46). One alternative phar-macologic strategy for the treatment of tumors with con-stitutive RAS activation, including those with loss of NF1, isto target the pathways downstream of RAS responsible formaintenance of transformation (Supplementary Fig. S8). Weobserved that in contrast to NF1-null MPNSTs (31, 32), NF1-null melanomas were dependent on ERK pathway activationand not TORC1 for cell-cycle progression and cell prolifer-ation. This result indicates that the lineage context withinwhich NF1 is inactivated influences the downstream effectorpathways that facilitate RAS-mediated transformation andthus likely dictates the potential utility of targeted pathwayinhibitors.

Although NF1-null melanomas were dependent uponMEK-ERK activation for cell proliferation, we observed starkdifferences in the relative potency of allosteric, non–ATP-competitive MEK inhibitors in the NF1-null cohort (11).Specifically, trametinib, which attenuates phosphorylationof MEK by RAF at Serine 217, had greater antitumor effects

than PD0325901. Monophosphorylated MEK has only partialactivity (11) and the ability of trametinib but not PD0325901to abrogate the hyperphosphorylation of MEK resultingfrom relief of upstream negative feedback was associatedwith more durable inhibition of pERK and cyclin D1 expres-sion and greater antiproliferative effects. A similar lack ofpotency was also noted in NF1-null melanoma cells withAZD6244 (Supplementary Fig. S4), a second non–ATP-com-petitive MEK inhibitor incapable of abrogating RAF phos-phorylation of MEK. The inability of AZD6244 to block MEKphosphorylation likely accounts for the partial resistance toMEK inhibition observed following NF1 knockdown in aprior study (47). Our data are also consistent with a recentstudy suggesting that differences in the cellular potency ofMEK inhibitors in KRAS-mutant cells can result from dif-ferences in the strength of hydrogen bonding with S212 inMEK, which is critical for blocking feedback induced MEKphosphorylation by wild-type RAF (48). In sum, the dataimply that MEK inhibitors that block the phosphorylation ofMEK by RAF may have greater clinical activity in tumorswith activated RAS, including those with loss of NF1 func-tion. Such inhibitors may, however, have a narrow thera-peutic index in patients, as they would be predicted topotently inhibit RAS-dependent ERK signaling in normaltissues.

In summary, NF1 loss is common in cutaneous melano-mas. Loss of NF1 is associated with RAS activation, MEKdependence and, in the setting of concurrent BRAF muta-tion, vemurafenib resistance. Upstream hyperactivation ofRAS and RAF resulting from loss of negative feedbackfollowing ERK pathway inhibition can result in an attenu-ation of the antitumor activity of allosteric MEK inhibitors.Inhibitors that prevent RAF-mediated phosphorylation ofMEK abrogate this adaptive resistance to MEK inhibitionand have greater antitumor activity in NF1-null cells. Withthe recent approval of trametinib by the U.S. Food and DrugAdministration for the treatment of BRAF-mutant melano-mas, these findings have potential therapeutic implicationsfor patients with melanoma and others tumor types withNF1 alterations.

Disclosure of Potential Conflicts of InterestP.B. Chapman is a consultant/advisory board member of Glaxo-Smith-

Kline and Roche-Genentech. R. Yaeger is a consultant/advisory board mem-ber of GlaxoSmithKline Advisory Board—Combination therapies for BRAFmutant CRC. N. Rosen is a consultant/advisory board member of Astra-zeneca, Chugai, and Novartis. D.B Solit is a consultant/advisory boardmember of Pfizer.

Authors' ContributionsConception and design: M.H. Nissan, C.A. Pratilas, A.J. Hanrahan, R. Yaeger,N. Rosen, D.B. SolitDevelopment of methodology: M.H. Nissan, A.M. Jones, H. Won, L. Kong,N. Rosen, D.B. SolitAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.H. Nissan, C.A. Pratilas, A.M. Jones, R. Ramirez,S. Tiwari, L. Kong, T. Merghoub, A. Ribas, M.F. Berger, D.B. SolitAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.H. Nissan, C.A. Pratilas, A.M. Jones, R. Ramirez,H. Won, Z. Yao, T. Merghoub, P.B. Chapman, B.S. Taylor, N. Schultz, M.F. Berger,N. Rosen, D.B. Solit

Nissan et al.





Writing, review, and/or revision of the manuscript: M.H. Nissan,C.A. Pratilas, A.M. Jones, C. Liu, A.J. Hanrahan, Z. Yao, T. Merghoub, R. Yaeger,B.S. Taylor, M.F. Berger, N. Rosen, D.B. SolitAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M.H. Nissan, A.M. Jones, C. Liu,S. Tiwari, A.J. Hanrahan, D.B. SolitStudy supervision: P.B. Chapman, N. Rosen, D.B. Solit

AcknowledgmentsThe authors thank A. Houghton for his pioneering work developing the

"SK-Mel" cell lines; J. Wolchok for insightful comments and collaboration;K. Nathanson and M. Herlyn for the "WM" cell lines; the Geoffrey Beene Core,Agnes Viale, and the Genomics Core at MSKCC; The Cancer Genome Atlas forallowing pertinent TCGA NF1 results to be incorporated into our analyses; andthe patients for contributing tissue for research.

Grant SupportThis work was supported by the NIH; the Melanoma Research Alliance;

Mr. William H. Goodwin and Mrs. Alice Goodwin and the CommonwealthFoundation for Cancer Research, and the Experimental Therapeutics Center ofMemorial Sloan-Kettering Cancer Center; and the STARR Foundation; and aStand Up To Cancer - Melanoma Research Alliance Melanoma Dream TeamTranslational Research Grant (SU2C-AACR-DT0612). Stand Up To Cancer is aprogram of the Entertainment Industry Foundation administered by the Amer-ican Association for Cancer Research.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 11, 2013; revised January 30, 2014; accepted January 30,2014; published OnlineFirst February 27, 2014.

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2014;74:2340-2350. Published OnlineFirst February 27, 2014.Cancer Res Moriah H. Nissan, Christine A. Pratilas, Alexis M. Jones, et al. Activation and MEK DependenceLoss of NF1 in Cutaneous Melanoma Is Associated with RAS

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