pathway by fgf receptor 3 (fgfr3)/ras mediates resistance to

Reactivation of Mitogen-activated Protein Kinase (MAPK)Pathway by FGF Receptor 3 (FGFR3)/Ras Mediates Resistanceto Vemurafenib in Human B-RAF V600E Mutant Melanoma*□S

Received for publication, April 30, 2012, and in revised form, June 11, 2012 Published, JBC Papers in Press, June 22, 2012, DOI 10.1074/jbc.M112.377218

Vipin Yadav‡, Xiaoyi Zhang‡, Jiangang Liu§, Shawn Estrem§, Shuyu Li§, Xue-Qian Gong§, Sean Buchanan§,James R. Henry¶, James J. Starling‡, and Sheng-Bin Peng‡1

From ‡Oncology Discovery Research, §Translational Sciences, and ¶Discovery Chemistry, Lilly Research Laboratories, Eli Lilly andCompany, Indianapolis, Indiana 46285

Background: B-RAF V600E melanomas rapidly develop resistance to B-RAF inhibitors in the clinic.Results: FGFR3/Ras signaling is elevated and induces resistance to vemurafenib in vemurafenib-resistant cells.Conclusion: FGFR3/Ras confers resistance to B-RAF inhibition via MAPK pathway reactivation.Significance: A novel mechanism of resistance to B-RAF inhibitors is described and potential therapeutic strategies aresuggested.

Oncogenic B-RAF V600E mutation is found in 50% of mela-nomas and drives MEK/ERK pathway and cancer progression.Recently, a selective B-RAF inhibitor, vemurafenib (PLX4032),received clinical approval for treatment of melanoma withB-RAF V600E mutation. However, patients on vemurafenibeventually develop resistance to the drug and demonstratetumor progression within an average of 7 months. Recentreports indicated thatmultiple complex and context-dependentmechanisms may confer resistance to B-RAF inhibition. In thestudy described herein, we generated B-RAF V600E melanomacell lines of acquired-resistance to vemurafenib, and investi-gated the underlying mechanism(s) of resistance. Biochemicalanalysis revealed thatMEK/ERK reactivation through Ras is thekey resistancemechanism in these cells. Further analysis of totalgene expression by microarray confirmed a significant increaseof Ras and RTK gene signatures in the vemurafenib-resistantcells. Mechanistically, we found that the enhanced activation offibroblast growth factor receptor 3 (FGFR3) is linked to Ras andMAPK activation, therefore conferring vemurafenib resistance.Pharmacological or genetic inhibition of the FGFR3/Ras axisrestored the sensitivity of vemurafenib-resistant cells to vemu-rafenib. Additionally, activation of FGFR3 sufficiently reacti-vated Ras/MAPK signaling and conferred resistance to vemu-rafenib in the parental B-RAF V600E melanoma cells. Finally,we demonstrated that vemurafenib-resistant cells maintaintheir addiction to theMAPKpathway, and inhibition ofMEKorpan-RAF activities is an effective therapeutic strategy to over-come acquired-resistance to vemurafenib. Together, wedescribe a novel FGFR3/Rasmediatedmechanism for acquired-resistance to B-RAF inhibition. Our results have implicationsfor the development of new therapeutic strategies to improvethe outcome of patients with B-RAF V600E melanoma.

Melanoma is the sixth most common cancer in the UnitedStates, and the incidence continues to rise across the world (1).Metastatic melanoma has a poor prognosis with a five year sur-vival rate of less than 20% (2). The Ras-RAF-MEK-ERK MAPkinase signaling plays an important role in melanoma etiologyand progression, and is considered a key target for anti-mela-noma therapies (3, 4). Approximately 50% of melanomas har-bor an activating B-RAF V600E mutation that leads to consti-tutive activation of the B-RAF kinase and downstream MAPKsignaling (4, 5). B-RAF V600E melanoma cells are addicted tothe constitutive activation of the MAPK signaling and arehighly sensitive to B-RAF inhibition (6). As a result, B-RAF isconsidered an attractive target for treatment of melanoma.Vemurafenib/PLX4032 and PLX4720 (preclinical analog of

PLX4032) are potent small molecule inhibitors of mutantB-RAF V600E (7–9). Vemurafenib was recently approved byUnited States Food and Drug Administration for treatment ofmetastatic and unresectable melanomas that carry an activat-ing B-RAF V600E mutation. In recent clinical studies inpatients with metastatic melanomas carrying mutant B-RAF,single agent vemurafenib exhibited robust efficacy with aresponse rate of 50%, and improved overall survival as com-pared with the chemotherapeutic agent dacarbazine (10, 11).Although these patient responses were encouraging, they wererelatively short-lived. Almost all of the patients who initiallyresponded to vemurafenib therapy developed drug resistanceand eventually relapsed within an average of 7 months (10).Therefore, similar to many other targeted therapies, theacquired resistance to B-RAF inhibition presents a significanttherapeutic challenge to long-term survival benefit in thispatient population.To improve the clinical benefit of B-RAF inhibitors, it is

urgent and critical to identify the mechanisms which rendermutant B-RAF-expressing melanoma cells resistant to B-RAFinhibition. Recent studies have indicated that reactivation oftheMAPK pathway is an importantmechanism of resistance toB-RAF inhibition. Resistant mechanisms primarily involvereactivation of ERK signaling via bypass mechanisms that are

* This work was supported by Eli Lilly and Company.□S This article contains supplemental Tables S1 and S2 and Figs. S1–S6.1 To whom correspondence should be addressed: Oncology Discovery

Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis,IN 46285. Tel.: 317-433-4549; Fax: 317-276-1414; E-mail: [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 33, pp. 28087–28098, August 10, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

AUGUST 10, 2012 • VOLUME 287 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 28087

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either Ras/RAF dependent, such as Ras activation (12, 13) orC-RAF up-regulation (14, 15), or likely Ras/RAF independent(Tpl2/COT overexpression) (14). Recently an activating muta-tion in MEK was reported in the vemurafenib-resistant tumorin one patient (16). Thus,multiplemechanisms could attenuatethe effect of B-RAF inhibition on MAPK signaling in B-RAFmutant melanomas. Recent studies have also suggested thatactivation ofMAPK-redundant signaling pathways by receptortyrosine kinases (RTKs)2 such as IGF-1R or PDGFR� could playa role in acquired resistance to B-RAF inhibition (12, 17, 18).In the study described herein, we have generated vemu-

rafenib-resistant cell lines by chronic treatment of the humanmelanoma cell lines, A375 andM14 both harboring the B-RAFV600E mutation with increasing concentrations of vemu-rafenib or its close structural analog PLX4720. Using theseacquired-resistance cell models, we describe FGFR3/Ras sig-naling pathway as a novel mechanism for acquired-resistanceto B-RAF inhibition throughMAPK pathway reactivation. Ourresults shed new light on the complexity of the underlying sig-naling pathways responsible for resistance to B-RAF inhibitionandmay provide strategies to overcome vemurafenib resistancein the clinic.

EXPERIMENTAL PROCEDURES

Cell Culture, Reagents, and Transfections—A375 and M14human melanoma cells with B-RAF V600E mutation wereobtained from ATCC. All of the parental and resistant cellswere cultured inDulbecco’smodified Eagle’smedium (DMEM,Thermo Scientific) supplemented with 10% fetal bovine serum(FBS, Invitrogen) only. B-RAF inhibitors, PLX4720 and vemu-rafenib were obtained from Axon MedChem BV. MEK inhibi-tor AZD6244, pan-Raf inhibitor RAF265 and FGFR inhibitorLY2874455were synthesized by Eli Lilly and Company. siRNAsfor target gene knock downwere obtained fromThermo Scien-tific (OnTargetPlus SiRNA Pools, Dharmacon). siRNA trans-fections were carried out using LipofectamineTM RNAiMAXtransfection reagent (Invitrogen) as per the manufacturer’sinstructions. All plasmid transient transfections were carriedout using FuGENE�HDTransfection Reagent according to themanufacturer’s instructions (Promega).Generation of B-RAF V600E Melanoma Cell Lines Resistant

to B-RAF Inhibition—To generate resistant cells, we culturedA375 orM14 cells in growthmedium in the presence of vehicle(0.1% DMSO) or gradually increasing concentrations ofPLX4720 or vemurafenib from 0.02 to 2 �M through �4months and 30 passages to result in resistant cell lines desig-nated as A375-R1, A375-R3, and M14-R. A375-R1 was gener-ated by treatmentwith PLX4720 and, A375-R3 andM14-R cellswere generated by treatment with vemurafenib.Cell Proliferation Assay—Cells (5 � 103), maintained in

growth medium described above, were plated onto poly-D-ly-sine-coated well in 96-well plates (BD Biosciences) a day beforethe treatment. The cells were treated for 48–72 h, and thenanalyzed for viability using the CellTiterGlo Luminescent Cell

Viability Assay according to the manufacturer’s instructions(Promega) and a SpectraMax plate reader (Molecular Devices).Nonlinear regression and sigmoidal dose-response curves wereused to calculate the half maximal inhibitory concentration(IC50) with GraphPad Prism 4 software.Immunoblotting and Phospho-antibody Array—Western

blotting analysis was performed as described previously (19).Proteins were detected using the Odyssey Infrared ImagingSystem (Li-COR Biosciences). Antibodies against P-Tyr724-FGFR3 (sc-33041) and FGFR3 (c-15) were obtained from SantaCruz Biotechnology, Inc. Antibodies against ERK1/2, phospho-ERK1/2 (9101), phospho-Akt1 (S473) (4060), phospho-FGFR(3476), A-Raf (4432), B-Raf (9434), C-Raf (9422), phospho-MEK1/2 (9154), and Akt (9272), were obtained from Cell Sig-naling Technology. Anti-tubulin (ab7291, AbCam), bFGF (Mil-lipore), and anti-Ras (Upstate) antibodies were obtained fromthe indicated companies. To identify the relative phosphoryla-tion levels of human RTKs, we used human RTK SignalingAntibody Array Kit (7949, Cell Signaling Technologies) forphospho-protein analysis according to the manufacturer’sinstructions. Supplemental Table S2 describes the list of RTKsand the layout of the antibody array.Ras Activation Assay—Ras activation assay was performed

using Ras Activation Assay Kit from Millipore as per the man-ufacturer’s instructions. Briefly, 500 �l of cell lysates (proteinconcentration: 4 �g/�l) were incubated with 60 �l of Raf Ras-binding domain (RBD)-conjugated agarose beads for 1 h at 4 °C.Lysates loaded with GDP or with GTP�S (non hydrolyzableform of GTP) for 30 min were used as negative and positivecontrols, respectively. After three times washing in 1� PBS, thebeads were then boiled in reducing sample buffer. Levels ofactive Ras (Ras-GTP) were assessed by Western blotting usingmonoclonal anti-Ras antibody (1 �g/ml, Upstate) via OdysseyInfrared Imaging System as described above.Mutational and Sequencing Analysis—Genomic DNA was

purified from A375 and A375-R1 cells using Wizard GenomicDNA purification kit (Promega) as per manufacturer’s instruc-tions. Genotyping and bidirectional DNA sequencingwere per-formed by Agencourt Biosciences (Beverly, MA).Microarray Study—Total RNA was isolated from five inde-

pendent cultures of A375 andA375-R1 cells using RNAeasy Kitfrom Qiagen as per manufacturer’s instructions. Affymetrixgene expression profiling was performed by Asuragen Inc.(Austin, TX). Briefly, biotin-labeled targets (cRNA) were pre-pared using a MessageAmp™ II-based protocol (Ambion Inc.,Austin, TX). The cRNA yields were quantified by UV spectro-photometry and the distribution of transcript sizes wasassessed using the Agilent Bioanalyzer 2100 capillary electro-phoresis system. Labeled cRNA was fragmented and used forHG-U133 Plus 2.0 microarray hybridization and washing,according to the manufacturer’s protocol (Affymetrix, FosterCity, CA). Then the Affymetrix MAS5 algorithm and quantilenormalization across samples were applied, followed by log2transformation and mean-centered standardization. A total of13678 genes that were reliably detected on at least 80% of thearrays with a signal intensity of 64 or greater were used forfurther analysis.

2 The abbreviations used are: RTK, receptor tyrosine kinase; FGFR, fibroblastgrowth factor receptor; GTP�S, guanosine 5�-3-O-(thio)triphosphate;GSEA, gene set enrichment analysis.

Resistance to B-RAF Inhibition by MAPK Pathway Reactivation

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Gene Set Enrichment Analysis (GSEA)—GSEA was per-formed as previously described (20, 21), using a total of 3272gene sets from a Molecular Signature Data base called Mol-SigDB (21) (version c2.v3). The enrichment with GSEA wasassessed by: (a) ranking all genes on the HG-U133PLUS2 chipwith respect to the phenotype “resistant (A375-R1) versus no-resistant (A375 parental)”; (b) locating the represented mem-bers of a given gene set within the ranked genes; and (c) mea-suring the proximity of each gene set with respect to both endsof the ranked list using a Kolmogorov-Smirnoff (KS) non-para-metric rank statistic score (with a higher score correspondingto a higher proximity); (d) comparing the observed KS score tothe distribution of 1000permutedKS scores for all gene sets. Anempirical cutoff, FDR � .05, was used to define statisticalsignificance.Human FGF2 ELISA—Cells were cultured for 48 h in growth

medium described above, and the conditioned medium sam-ples (cell free culture supernatant) were analyzed for concen-trations of human FGF2 using human FGF2 Quantikine ELISAKit (R&D Systems). ELISA was performed as per the manufac-turer’s instructions.

RESULTS

Generation of Cell Line Models of Acquired-resistance toB-RAF V600E Inhibitors—To investigate the potential mecha-nisms of resistance to B-RAF inhibition in melanoma, we usedA375 and M14 human melanoma cell lines. These cells carrythe B-RAF V600E mutation and have been previously demon-strated to be highly sensitive to B-RAF inhibition (22). Afterconfirming the sensitivity of these cells to B-Raf inhibitors,PLX4720, and vemurafenib in cell proliferation assays, wetreated and cultured these cells in the presence of either vehicle(0.1% DMSO) or gradually increasing concentrations of vemu-rafenib or PLX4720 from 0.02 �M to 2 �M through �4 monthsand 30 passages (Fig. 1A). A375-R1 was generated usingPLX4720, and A375-R3 and M14-R cells were generated usingvemurafenib after the publication of its chemical structure (9).As demonstrated in Fig. 1B, vehicle-treated A375 parental cellsremained sensitive to vemurafenib inhibition in Cell Titer Gloassaywith IC50 of 85 nM,whileA375-R1 andA375-R3 cells wereonly sensitive at concentrations�2 orders ofmagnitude higher(IC50 of 4800 nM and 3000 nM, respectively). Similarly, M14parental cells were sensitive to vemurafenibwith IC50 of 180 nMwhile M14-R cells were resistant to vemurafenib with IC50 of9500 nM (Fig. 1C). The resistant cell lines were maintained inthe absence of B-Raf inhibition. Interestingly, late-passage(�20) resistant cells demonstrated increased sensitivity toB-Raf inhibition when compared with early-passage (�10)resistant cells (data not shown). Therefore, all of the experi-ments in this study were carried out using early passage-resis-tant cells only.MEK/ERK Signaling Is Reactivated and Resistant to B-RAF

Inhibition in Vemurafenib-resistant Cells—We characterizedthe A375-R1 cell line further. We initially examined the down-streamphospho-MEK and phospho-ERK activities inA375 andA375-R1 cells by immunoblotting. As revealed in Fig. 1D, thebasal levels of phospho-MEK and phospho-ERK were elevatedin the A375-R1 cells, while the changes in phospho-AKT levels

were minimal. As expected, treatment of parental cells withvemurafenib caused a robust dose dependent inhibition ofMEK and ERK activities, and 0.1 �M of vemurafenib signifi-cantly reduced phospho-MEK and phospho-ERK levels.However, phospho-MEK and phospho-ERK levels remainedrelatively elevated in the A375-R1 cells in presence of vemu-rafenib as high as 3 �M (Fig. 1D). Similar to A375-R1,A375-R3 and M14-R cells also displayed sustained phospho-ERK and phospho-MEK activity in the presence of vemu-rafenib as high as 10 �M (supplemental Fig. S1). The contin-ued activation of the MAPK pathway observed in thepresence of vemurafenib in A375-R1 cells is believed to be amajor reason of the resistance.Mutational and Microarray Analysis of A375 and A375-R1

Cells—To determine if any activating mutation associated withMAPK pathway is responsible for MAPK reactivation andresistance inA375-R1 cells, we isolated genomicDNAand con-ducted mutational analysis by direct bidirectional DNAsequencing of the following genes: RAF (A-RAF, B-RAF,C-RAF), Ras (H-Ras, K-Ras, N-Ras), RTK families (EGFRs,PDGFRs, cMet, FGFRs, RET, IGF-1R, VEGFRs, etc), MEK(MEK1 and MEK2), and ERK (ERK1 and ERK2), of both A375and A375-R1 cells as described under “Experimental Proce-dures.” Genotyping analysis confirmed that both A375 andA375-R1 cells have retained B-RAFV600Emutation. However,no additional mutation was observed in either cell line amongthe genes analyzed. To identify potential mechanisms involvedin resistance of A375-R1 cells, we also performed a microarraygene expression analysis utilizing the total RNA isolated fromA375 and A375-R1 cells. As shown in Fig. 2A, comparativeanalysis of gene expression patterns between A375 andA375-R1 cells revealed that the downstream gene signatures ofoncogenic Ras and RTK signaling are among the most consis-tent changes, indicating that RTK/Ras signaling is potentiallyinvolved in resistance of melanoma cells to B-RAF inhibition.The Ras and RTK gene signatures were defined based on pre-vious studies (23, 24).Ras Is Activated in Vemurafenib-resistant Cells and Required

for Resistance—RAF/MEK/ERK signaling is the key down-stream effector of Ras. Reactivation of MAPK signaling (Fig.1D) and increase of Ras gene signature by microarray study(Fig. 2A) trigged us to validate the activation status of Ras invemurafenib-resistant cells. For this purpose, we determinedthe levels of active Ras in the parental A375 and vemu-rafenib-resistant A375-R1 and A375-R3 cells by immuno-precipitating active Ras using beads coated with GST-boundRaf binding domain (RBD) as described under “ExperimentalProcedures.” As shown in Fig. 2B, we observed significantlyelevated levels of GTP bound Ras (Ras-GTP) in A375-R1 andA375-R3 cells when compared with parental A375 cells. Weverified the efficiency of this assay by pretreating the celllysates with GTP�S and immunoprecipitated similar levelsof Ras-GTP among A375 and A375-R1 cells.Furthermore, we investigated if active Ras is responsible for

the resistance of A375-R1 cells to the B-RAF inhibition. Weperformed specific knockdown of total Ras using siRNA pool(supplemental Fig. S2) and evaluated its effect on cell prolifer-ation in the presence of vemurafenib. Knockdown of Ras has no




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significant effect on the sensitivity of parental cells to vemu-rafenib (Fig. 2C). In contrast, knockdown of Ras restored thesensitivity of resistant A375-R1 and A375-R3 cells to vemu-rafenib inhibition (Fig. 2C). This indicates that Ras is requiredfor resistance to B-RAF inhibition in A375-R1 cells, althoughthe role of individual Ras isoform has not been defined in thisstudy.FGFR3 Signaling Is Elevated in Vemurafenib-resistant Cells—

Ras is generally activated in response to activation of RTKsby growth factors or activating mutation. Mutational analy-sis of RTKs and other components by direct DNA sequenc-ing revealed no additional activating mutations among thesegenes. To identify the mechanism of Ras activation in thevemurafenib-resistant cells, we used a phospho-antibodyarray and analyzed the tyrosine phosphorylation levels of 28different RTKs in parental and vemurafenib-resistant cells.Interestingly, we detected significantly elevated levels of

phospho-FGFR3 in all of the three resistant cells A375-R1,M14-R, and A375-R3 cells when compared with their paren-tal cells (Fig. 3A and supplemental Fig. S3A). To further val-idate the activation of FGFR3, we examined the levels ofphospho-Y724 FGFR3, the active form of FGFR3 (25) inparental and resistant cells by immunoblotting. As demon-strated in Fig. 3B, increased phospho-Y724-FGFR3 levelswere observed in the A375-R1 and M14-R cells. Similarly,increased phospho-FGFR3 levels were observed in A375-R3cells (supplemental Fig. S3B). In tumors and cell lines, FGFRsignaling is often enhanced by gene amplification and ele-vated autocrine or paracrine activation (26). Somatic gain-of-function mutations in FGFRs have also been reported incancers such as colorectal, urothelial endometrial and mel-anoma (27–29). However, we did not detect any mutation inthe members FGFR gene family in A375-R1 cells by DNAsequencing (data not shown). In addition, we did not detect

FIGURE 1. Vemurafenib-resistant cells display enhanced MAPK pathway activation. A, schematic representation of generation of B-RAF V600E melanomacells resistant to B-RAF inhibitors PLX4720 or PLX4032. B, sensitivity of A375, A375-R1, and A375-R3 cells to vemurafenib. C, sensitivity of M14 and M14-R cellsto vemurafenib. D, MAPK activation in vemurafenib resistance cells. A375 or A375-R1 cells were treated with indicated concentrations of vemurafenib for 1 h.Cell lysates were analyzed for activities of MAPK and AKT signaling by immunoblotting.




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any significant change in the total protein levels of FGFR3 inthe parental and the resistant cells by immunoblotting (Fig.3B). However, we detected increased levels of secreted FGF2,the major ligand of FGFRs, in the growth media of A375-R1and M14-R cells when compared with their respectiveparental cells by ELISA (Fig. 3C). We also measured the pro-tein levels of bFGF by Western blot analysis and detectedsignificant increase of bFGF protein in A375-R1 andA375-R3 cells compared with parental cells (supplementalFig. S4). Together, these results confirm the activation of

FGFR3 in the resistant cells possibly via an autocrine modeof activation.FGFR3 Activity Contributes to Resistance of A375-R1 Cells to

Vemurafenib—FGFR signaling has been previously implicatedin resistance to EGFR inhibitors and Her-2-targeted therapies.These suggest an important role of the FGF/FGFR axis in thedevelopment of resistance to targeted therapies in cancer (30,31). However, a role of FGFR signaling in resistance to B-RAFinhibition has not previously been reported. To verify our find-ing, we examined if FGFR3 activity is required for the resistance

FIGURE 2. Enhanced activation of Ras and downstream gene signatures in vemurafenib-resistant A375-R1 cells. A, heatmap of normalized Kolmogorov-Smirnov scoring for selected pathways. Affymetrix gene expression profiling was performed in A375 and A375-R1 cells. Gene expression data were obtainedfrom five independent cultures from each cell type. Gene signature pathway analysis showed the significant difference in “RTK_SIGNATURE” and “RAS_ONCOGENIC” pathway activities between the resistant cells (A375-R1) and parental cells (A375). The color represents the normalized KS score with each cellline, with red indicating strong pathway activity and blue indicating weak pathway activity. Statistical testing and the name of the genes involved in Ras and RTKgene signatures are provided in the supplementary information (supplemental Table S1) B, total Ras levels are shown (left). Ras-GTP levels were analyzed in theindicated cells lysates using Ras pulldown assay (right). Cell lysates were immunoprecipiated using GST-RBD beads. Lysates loaded with GTP�S (non-hydro-lyzable form of GTP) were used as positive control. C, A375 and A375-R1 cells were transfected with either control siRNA or siRNA pool targeting all the Rasisoforms (H-Ras, N-Ras, and K-Ras). After 24 h, cells were either treated with DMSO or vemurafenib (1 �M). Cell proliferation was assessed 48 h post-treatmentusing CellTIter-Glo. t test was performed on the indicated groups (* indicates P � 0.05).




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to vemurafenib. First, we investigated if FGFR3 is required forMAPK activation andmaintaining resistance to vemurafenib invemurafenib-resistant cells. We used LY2874455, an FGFRinhibitor described recently (32), and evaluated if targetingFGFR3 activity can re-sensitize A375-R1 cells to B-RAF inhibi-tion. Interestingly, LY2874455 blocked the activation of MEK/ERK signaling in presence of vemurafenib as low as 100 nM (Fig.4A). However, treatment with either LY2874455 or vemu-rafenib alone had no significant effect on the phospho-MEK orphospho-ERK levels in the A375-R1 cells (Fig. 4A). This indi-cates that combined inhibition of FGFR3 andB-RAF is requiredfor total inhibition of MEK/ERK signaling in the vemurafenib-resistant cells. Consistent with our results with LY2874455 inA375-R1 cells, knockdown of FGFR3 by siRNA reduced MEK/ERK signaling in response to vemurafenib (Fig. 4B). Interest-ingly in A375-R1 cells, knockdown of FGFR3 by siRNA orLY2874455 treatment hadminimal effect on pERK levels in theabsence of vemurafenib. This suggests that in the absence ofB-RAF inhibition, MEK/ERK signaling is driven primarily bymutant B-RAF in the A375-R1 cells as well (Fig. 4, A and B).Together, we show that FGFR3 is required for sustained activa-tion of MEK/ERK signaling in A375-R1 cells in the presence ofvemurafenib inhibition.Additionally, we tested if combined inhibition of B-RAF and

FGFR3 activities could have anti-proliferation effect in vemu-rafenib-resistant cells. Inhibition of FGFR3 by FGFR inhibitor,LY2874455 or FGFR3-specific siRNA pool restored the sensi-tivity of A375-R1 cells to vemurafenib with IC50 values of 385nM and 130 nM, respectively (Fig. 4, C and D). Together, our

results suggest that FGFR3 is required for resistance to vemu-rafenib inA375-R1 cells and targeting FGFR3 could be an effec-tive therapeutic strategy to overcome vemurafenib resistance.FGFR3 Activation Induces Resistance to Vemurafenib in

A375 Cells—To determine if FGFR3 activation can induceresistance to vemurafenib, we transfected the parental A375cells with either empty vector or constitutively active FGFR3K650Emutant expressing vector, and examinedMEK/ERK sig-naling in response to B-RAF inhibition. We found that activeFGFR3 K650E-expressing cells demonstrated up-regulatedERK activation in the presence of vemurafenib (Fig. 5A), buthadno significant effect on theAkt activation.We also exploredif bFGF stimulation is sufficient for inducing resistance tovemurafenib in the parental A375 cells. Interestingly, treatmentof parental cells with bFGF induced a dose-dependent reactiva-tion ofMEK/ERK signaling in the presence of vemurafenib (Fig.5B). FGFR3 was previously demonstrated to mediate its effectson MAPK pathway primarily via Ras activation (25). We thusanalyzed if bFGF stimulation leads to Ras activation in A375cells by immunoprecipitating Ras-GTP using GST-RBD beads.As demonstrated in Fig. 5C, stimulation of A375 cells withbFGF indeed induces Ras activation. Taken together, theseresults demonstrate that FGFR3 activity is sufficient for reacti-vation of Ras/Raf/MAPK signaling in B-RAF V600Emelanomacells by amechanism that is independent of B-RAFkinase activ-ity. Additionally, we evaluated the effect of FGFR3 activation onthe sensitivity of A375 cells to vemurafenib. Expression of con-stitutively active FGFR3 or stimulation with bFGF attenuatedthe inhibitory effect of vemurafenib on the parental cells corre-

FIGURE 3. Enhanced FGFR3 activation in vemurafenib-resistant B-RAF V600E melanoma cells. A, phospho-RTK antibody array analysis. Cell lysates fromA375, M14, A375-R1, and M14-R cell lines were incubated on RTK antibody array for 16 h and phosphorylation status was determined as described under“Experimental Procedures.” Each RTK antibody is spotted in duplicate. Supplemental Table S2 describes the list of RTKs and the layout of the antibody array.B, confirmation of phospho-FGFR3 levels by Western blot analysis. Protein levels of total and phosho-FGFR3 were assessed using immunoblotting. C, ELISAanalysis of secreted FGF2 in the conditioned media obtained from A375, A375-R1, M14, and M14-R cells. ELISA was performed as described in “ExperimentalProcedures.”




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sponding to increased IC50 values of 1480 nM and 1190 nM,respectively (Fig. 5, D and E). However, bFGF failed to signifi-cantly affect the sensitivity of A375 cells to pharmacologicinhibitors of pan-RAF or MEK kinases suggesting that mecha-nism of bFGF induced resistance to B-RAF inhibition requiresRAF/MEK activity (supplemental Fig. S5, A and B). Takentogether, our results indicate that FGFR3 activation inducesresistance to B-RAF inhibition in B-RAFV600Emelanoma cellsvia reactivation of MAPK signaling.A375-R1 Cells Are Sensitive toMEK and pan-RAF Inhibition—

B-RAF V600E melanomas are addicted to the RAF/MEK/ERKsignaling (6, 22). To investigate if vemurafenib-resistant cells

rely on MAPK signaling for growth and proliferation, we eval-uated the sensitivity of A375-R1 cells to pharmacologic inhibi-tion of RAF orMEK kinases.We used AZD6244, a highly selec-tive and potent inhibitor of MEK1/2, to test the effect of MEKinhibition on cell viability and ERK activity (33). AZD6244inhibited cell proliferation in the parental and resistant cellswith similar potency as indicated by IC50 values of 90 nM and 51nM, respectively (Fig. 6A). In addition, AZD6244 strongly inhib-ited ERK activity in parental A375 as well as resistant A375-R1cells (supplemental Fig. S6). Furthermore, we evaluated if RAFactivities are required for sustained activation ofMEK/ERK sig-naling in A375-R1 cells by using RAF265, a small molecule

FIGURE 4. FGFR3 activity is required for resistance to B-RAF Inhibition. A, A375 and A375-R1 cells were treated with indicated concentrations of eithervemurafenib or LY2874455 alone, or in combination for 1 h. Cell lysates were analyzed by immunoblotting using indicated antibodies. B, A375-R1 cells weretransfected with control or FGFR3 siRNA, and then A375-R1 cells were treated with indicated increasing concentrations of vemurafenib 48 h post-siRNAtransfection. Cell lysates were analyzed by immunoblotting using indicated antibodies. C, sensitivity of A375-R1 cells to either vemurafenib alone or incombination with FGFR inhibitor LY2874455 (300 nM) was assessed by CellTiter-Glo assay at 72 h post-treatment. D, A375 and A375-R1 cells were transientlytransfected with control or FGFR3 siRNA pool, and then treated with increasing concentrations of vemurafenib (0 –30 �M). Cell viability was assessed usingCellTiter-Glo. Data are represented as mean � S.D. from a representative experiment performed in triplicate.




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inhibitor of pan-RAF (34, 35). RAF265 inhibited the prolifera-tion of the parental A375 andA375-R1 cells with similar poten-cies with IC50 values of 458 nM and 235 nM, respectively (Fig.6B). Thus vemurafenib-resistant cells maintain their depen-dence on RAF/MEK/ERK signaling for cell proliferation. Previ-ous reports have shown that alternate RAF isoforms can regu-late the proliferation of vemurafenib-resistant melanomas viaactivation of MEK/ERK signaling (12, 17). Thus, we tested theRAF dependence of resistant cells by knockdown of all RAFisoforms via siRNA. Consistent with our results using pan-Rafinhibitor RAF265, treatment with siRNA targeting multipleRAF isoforms significantly reduced ERK activity and inhibited

proliferation inA375 andA375-R1 cells (Fig. 6,C andD). Takentogether, our data suggest that the vemurafenib-resistant cellsare still dependent on RAF/MEK/ERK activities and targetingthis pathway remains a promising therapeutic strategy againstB-RAF V600E melanomas, including ones resistant to B-RAFinhibition.

DISCUSSION

In this study, we generated three different resistant cell linesand showed that B-RAF V600E melanoma cells treated withB-RAF inhibitor developed resistance to B-RAF inhibitionthrough Ras activation and subsequent reactivation of MAPK

FIGURE 5. FGFR3 signaling induces resistance to B-RAF inhibition. A, A375 cells were transiently transfected with control pcDNA3 or constitutively activeFGFR3 K650E expressing vector (pcDNA3-FGFR3 K650E), and then treated with indicated concentrations of vemurafenib for 1 h. Cell lysates were analyzed byimmunoblotting using indicated antibodies. B, A375 parental cells were treated with indicated concentrations of bFGF in presence of 1% FBS, and then treatedwith vemurafenib (1 �M) for 1 h. Cell lysates were analyzed by immunoblotting using indicated antibodies. C, Ras-GTP levels were analyzed using the cellslysates from A375 cells untreated or treated with bFGF (100 ng/ml) by Ras pulldown assay as described under “Experimental Procedures.” Cell lysates treatedwith GDP or GTP�S (non-hydrolyzable form of GTP) were used as negative and positive controls, respectively. D, vemurafenib sensitivity of A375 cellstransfected with either control vector or FGFR3 K650E-expressing vector was assessed by CellTiter-Glo assay. E, sensitivity of A375 cells to vemurafenib with orwithout bFGF (100 ng/ml) was assessed by CellTiter-Glo assay.




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pathway. Additional molecular characterization revealed thatenhanced FGFR3 signaling is involved in Ras activation andacquired-resistance to vemurafenib. These resistant cellsmain-tain their addiction to the MAPK pathway as they are sensitiveto the MEK or pan-RAF inhibition.Consistent with the previous studies (12, 14, 17), we did not

detect any additional B-RAF mutation in the vemurafenib-re-sistant cells, confirming that a secondary B-RAF mutation isnot themechanismof resistance to B-RAF inhibition. Althoughmutation in N-Ras or MEK have been reported in low fre-quency from B-RAF V600E melanoma patients who failedvemurafenib therapy (12, 16), we did not identify any mutationin Ras isoforms, the downstream RAF/MEK/ERK componentsor the upstream RTKs in the A375-R1 cells (data not shown),indicating that a non-genetic mechanism is involved in resis-tance to B-RAF inhibition in our cell lines. We subsequentlyperformed a microarray-based gene expression study inattempt to reveal the underlyingmechanisms of acquired-resis-tance to vemurafenib. Interestingly, we found a consistent andsignificant up-regulation of Ras and RTKdownstream gene sig-natures in A375-R1 cells when compared with the parentalA375 cells, indicating the mechanism of acquired-resistance tovemurafenib involves activation of RTK and Ras signaling (Fig.2A). As demonstrated previously, mechanism of resistance to

B-RAF inhibition primarily involves either reactivation ofMEK/ERK signaling by switching of RAF isoforms, or enhancedactivation of PI3K/AKT signaling, ultimately resulting inreduced B-RAF dependence of the cells (36). Overall, theseresults indicate that activation of Ras, either by RTK signalingor via activating mutations, could be one of the primary mech-anisms of resistance to B-RAF inhibitors. In this study, weshowed that active Ras-GTP levels are elevated in the vemu-rafenib resistant B-RAF V600E melanoma cells (Fig. 2B). Inaddition, we showed that activation of FGFR3 induces Rasactivation in B-RAF V600E melanomas and reduces their sen-sitivity to B-RAF inhibition (Fig. 5). Our findings indicate Rasactivation as a critical node in mediating resistance to B-RAF-targeted therapies. Our data and others support this hypothesisas PDGFR� (12, 18), IGF-1R (17) andN-Rasmutation (12) wereidentified as a B-RAF resistant mechanism in different cellularbackgrounds. It is our speculation that Ras activation is a keymechanism involved in multiple resistance mechanisms toB-Raf inhibition.FGFR signaling has been previously implicated in resistance

to EGFR and Her-2 targeted therapies (30, 31). However, itsrole in resistance to B-RAF inhibition has never been reported.Herewe describe FGFR3 signaling as an important player in thedevelopment of acquired resistance to vemurafenib. However,

FIGURE 6. RAF and MEK activities are required for proliferation of vemurafenib-resistant A375-R1 cells. A, sensitivity of A375 and A375-R1 cells toAZD6244 was assessed by CellTiter-Glo assay. B, sensitivity of A375 and A375-R1 cells to RAF265 was assessed by CellTiter-Glo assay. C, A375 and A375-R1 cellswere treated with control siRNA or a combined siRNA pool directed against A-RAF, B-RAF, and C-RAF (pan-RAF). Levels of phospho-ERK, A-RAF, B-RAF, andC-RAF were examined by immunoblotting. D, cell proliferation was evaluated for A375 and A375-R1 cells transfected with control or pan-RAF siRNA usingCellTIter-Glo.




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the resistant mechanism involving other RTKs particularlyIGF-1R seems to require activation of alternate signaling path-ways (i.e. PI3K/Akt) that may reduce the dependence of B-RAFV600Emelanoma cells to RAF/MEK/ERK signaling (17). In thisstudy however, we show that phospho-FGFR3protein levels areup-regulated in the vemurafenib resistant B-RAF V600E mela-noma cells (Fig. 3, A and B). Although we detected significantincrease in the secretion of FGF2 ligand in the media by vemu-rafenib resistant B-RAF V600E melanoma cells, the precisemechanism of FGFR3 activation is currently under investiga-tion (Fig. 3C and supplemental Fig. S4). Furthermore, weshowed that FGFR3 signaling results in enhanced activation ofdownstream Ras/RAF/MEK/ERK signaling, thus conferringresistance to B-RAF inhibition (Figs. 3–5). In our resistant cells,no significant change in phospho-AKT status was observedwhen compared with the parental cells (Fig. 1D). In addition,activation of FGFR3 induced resistance to vemurafenib inB-RAF V600E cells without significantly affecting phospho-AKT levels (Fig. 5A), thus, MAPK reactivation is the dominantresistance mechanism in our resistant cells.We also show that vemurafenib-resistantmelanoma cells are

equally sensitive to pan-RAF inhibitor RAF265 or MEK selec-tive inhibitor AZD6244 when compared with parental cells,suggesting that vemurafenib-resistant cells indeed maintaintheir addiction toMAPK pathway (Fig. 6). Genetic depletion ofRaf isoforms by siRNA in B-Raf resistant cells further con-firmed these results (Fig. 6,C andD). Although, the precise roleof individual RAF isoforms in resistance to B-RAF inhibition isyet to be fully investigated, our data are consistent with theearlier findings that B-RAF V600E melanoma cells can escapeB-RAF kinase inhibition through MAPK reactivation by alter-native RAF isoforms (12, 14, 15, 17). Therefore, a selectiveMEKinhibitor or a pan-Raf inhibitor may provide clinical benefit tomelanoma patients who have failed or developed resistance tovemurafenib therapy.Finally, we propose the following model to illustrate the

mechanisms how B-RAF V600E melanoma cells develop resis-tance to vemurafenib treatment based on our results and otherpublished studies (Fig. 7).Whenmelanoma patients are treatedwith vemurafenib, two potential mechanisms of resistance candevelop; a compensatory mechanism and/or genetic mutation.The compensatory mechanism we believe is the most commonand dominantmechanism of resistance, and ismediated by oneor more RTKs or other cell signaling component, such as COT(14). The genetic mutations identified and responsible forvemurafenib resistance include N-Ras Q61K/R mutation (12),K-Ras K117N (13), or MEK C121S (16), and these mutationswere confirmed in few patients who have relapsed from B-RAFinhibitor therapy. Thus, both compensatory mechanism andgenetic mutations eventually lead to MAPK reactivation.Recently, dimerization of spliced form of BRAF V600E (p61)was also reported to induce MAPK pathway reactivation andresistance to vemurafenib (37). To date, activation of FGFR3,PDGFR�, or IGF-1R was observed in different resistant cells,and the RTK(s) to be activated is likely context dependent.Importantly, activation of RTK leads to Ras activation, subse-quent MAPK reactivation, and consequent drug resistance.Generally these resistant cells are still addicted toMAPK activ-

ity, and therefore, MAPK pathway inhibition by a pan RAFinhibitor or a MEK selective inhibitor could overcome theirresistance to B-RAF inhibition. In certain context, in additionto MAPK reactivation, enhanced PI3K/AKT activities due toRas activation or other cell signaling could contribute to theB-RAF resistance. Therefore, PI3K/AKT pathway inhibitioncould also be part of the strategy for overcoming resistance toB-RAF inhibitors.

Acknowledgments—We thank Dr. Philip J. Elbert for DNA sequenceand mutational analysis, and Dr. Genshi Zhao and Robert DanielVan Horn for helpful discussions.

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Sean Buchanan, James R. Henry, James J. Starling and Sheng-Bin PengVipin Yadav, Xiaoyi Zhang, Jiangang Liu, Shawn Estrem, Shuyu Li, Xue-Qian Gong,

V600E Mutant MelanomaReceptor 3 (FGFR3)/Ras Mediates Resistance to Vemurafenib in Human B-RAF

Reactivation of Mitogen-activated Protein Kinase (MAPK) Pathway by FGF

doi: 10.1074/jbc.M112.377218 originally published online June 22, 20122012, 287:28087-28098.J. Biol. Chem.

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http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=287/33/28087&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/287/33/28087

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2012/06/22/M112.377218.DC1

http://www.jbc.org/content/287/33/28087.full.html#ref-list-1

http://www.jbc.org/

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