erk signal suppression and sensitivity to ch5183284/debio 1347

Cancer Biology and Signal Transduction

ERK Signal Suppression and Sensitivity toCH5183284/Debio 1347, a Selective FGFRInhibitorYoshito Nakanishi, Hideaki Mizuno, Hitoshi Sase, Toshihiko Fujii, Kiyoaki Sakata,Nukinori Akiyama, Yuko Aoki, Masahiro Aoki, and Nobuya Ishii

Abstract

Drugs that target specific gene alterations have proven ben-eficial in the treatment of cancer. Because cancer cells havemultiple resistance mechanisms, it is important to understandthe downstream pathways of the target genes and monitor thepharmacodynamic markers associated with therapeutic effica-cy. We performed a transcriptome analysis to characterize theresponse of various cancer cell lines to a selective fibroblastgrowth factor receptor (FGFR) inhibitor (CH5183284/Debio1347), a mitogen-activated protein kinase kinase (MEK) inhib-itor, or a phosphoinositide 3-kinase (PI3K) inhibitor. FGFRand MEK inhibition produced similar expression patterns, andthe extracellular signal–regulated kinase (ERK) gene signaturewas altered in several FGFR inhibitor–sensitive cell lines.Consistent with these findings, CH5183284/Debio 1347

suppressed phospho-ERK in every tested FGFR inhibitor–sen-sitive cell line. Because the mitogen-activated protein kinase(MAPK) pathway functions downstream of FGFR, we searchedfor a pharmacodynamic marker of FGFR inhibitor efficacy in acollection of cell lines with the ERK signature and identifieddual-specificity phosphatase 6 (DUSP6) as a candidate marker.Although a MEK inhibitor suppressed the MAPK pathway, mostFGFR inhibitor–sensitive cell lines are insensitive to MEKinhibitors and we found potent feedback activation of severalpathways via FGFR. We therefore suggest that FGFR inhibitorsexert their effect by suppressing ERK signaling without feedbackactivation. In addition, DUSP6 may be a pharmacodynamicmarker of FGFR inhibitor efficacy in FGFR-addicted cancers.MolCancer Ther; 14(12); 2831–9. �2015 AACR.

IntroductionSeveral tyrosine kinase-targeting agents have recently been

developed. Each of these agents has demonstrable efficacywhen used in patient cohorts that are stratified based on thegenetic status of their respective molecular targets. The fibro-blast growth factor receptors (FGFR) are tyrosine kinases thatare constitutively activated in a subset of tumors by geneticalterations such as gene amplification, point mutation, orchromosomal translocation/rearrangement (1, 2). Geneticalterations of FGFR may also be predictive indicators of patientresponse to FGFR inhibitors (2, 3). For instance, dovitinib, amultitargeted kinase inhibitor that inhibits FGFRs, producedthree unconfirmed partial responses in breast cancer harboringFGFR1 gene amplification (4). Although genetic alterationscould predict drug efficacy, acquired genetic alterations conferresistance to molecular-targeted drugs. Acquired mutations intarget genes or downstream components are major mechanisms

of resistance (5–13). Although acquired FGFR mutations havenot yet been identified in patients, several FGFR mutations thatconfer resistance to FGFR inhibitors have been reported (7, 14).Because cancer cells continue to utilize the pathway to whichthey are originally addicted, they acquire some genetic altera-tions to reactivate the pathway. Therefore, monitoring ofchanges in the pathways utilized by cancer cells could be usedto predict the efficacy of an inhibitor in tumors.

The FGFR family consists of FGFR1, FGFR2, FGFR3, andFGFR4,each of which is bound by a subset of 22 fibroblast growth factor(FGF) ligands. FGFRs are activatedby ligand-dependent or ligand-independent dimerization that leads to intermolecular phosphor-ylation. FGFR substrate 2 (FRS2) is a key adaptor protein that isphosphorylated by FGFR. Phosphorylated FRS2 recruits severalother adaptor proteins and activates the mitogen-associated pro-tein kinase (MAPK) or PI3K/AKT pathways (1). However, thepathway associated with effective FGFR suppression in FGFR-addicted cancers has not yet been identified. Gaining an under-standing of the FGFR pathway by studying its function in thepresence of FGFR inhibitors will enable identification of candi-date pathways utilized by FGFR-addicted cancers and the phar-macodynamic markers of these pathways.

In a previous study, we analyzed the signaling pathway of anFGFR fusion kinases, FGFR3-BAIAP2L1 (15). A Rat-2 cell linestably expressing FGFR3-BAIAP2L1 exhibited potent tumorigenicactivity. Gene expression analysis revealed strong upregulation ofgenes downstreamof theMAPKpathway, and upregulation of theMAPKpathwaywas validated byWestern blotting; in contrast, thePI3K/AKT pathwaywas not activated by FGFR3-BAIAP2L1. There-fore, we suggested that MAPK pathway activation is essential to

Research Division, Chugai Pharmaceutical Co., Ltd., Kamakura, Kana-gawa, Japan.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Yoshito Nakanishi, Research Division, Chugai Pharma-ceutical Co. Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan. Phone:81-467-47-6262; Fax: 81-467-46-5320; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0497

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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on March 30, 2018. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst October 5, 2015; DOI: 10.1158/1535-7163.MCT-15-0497

http://mct.aacrjournals.org/

the tumorigenic activity of FGFR3-BAIAP2L1. To generalize theMAPK pathway dependency of FGFR, we characterized the path-way modulation by CH5183284/Debio 1347, a selective FGFRinhibitor, in several FGFR genetically altered cancer cell lines. Weanalyzed differential transcript expression by microarray analysisand found that MAPK pathway modulation was associated withthe efficacy of CH5183284/Debio 1347. We identified the dualspecificity phosphatase 6 (DUSP6) gene, which lies downstreamof MAPK, as a candidate pharmacodynamic marker of FGFRinhibitor efficacy. Finally, we applied a MEK inhibitor to FGFRgenetically altered cancer cells to validate the significanceofMAPKpathway modulation by FGFR inhibition.

Materials and MethodsReagents and cell lines

CH5183284/Debio 1347 (FGFR inhibitor), CH4987655 (MEKinhibitor), CH5126766 (RAF-MEK inhibitor), and CH5132799(PI3K inhibitor) were synthesized at Chugai Pharmaceutical Co.Ltd., as previously described (7, 16–18). AZD4547 was synthe-sized at Chugai Pharmaceutical Co. Ltd. (patent publicationWO2008075068). PD173074, PD0325901, and selumetinibwere purchased from Sigma-Aldrich and Selleck Chemicals. TheDMS-114, NCI-H520, NCI-H1581, NCI-H716, KATO-III, SNU-16, AN3CA, RT-4, NCI-N87, ZR-75-1, andHCT116 cell lines werepurchased from theAmerican TypeCultureCollection (Manassas,VA, USA). The KMS-11 cell line was purchased from the HealthScience Research Resources Bank. MFE-296 and UM-UC-14 celllines were purchased from Health Protection Agency CultureCollections. The MFE-280 cell line was purchased from DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH. TheHSC-39 and MKN-45 cell lines were purchased from Immuno-Biological Laboratories, and the SUM-52PE cell line was pur-chased from Asterand. All cell lines were obtained more than oneyear prior to the experiments and were propagated for less than6 months after thawing and cultured according to supplierinstructions.

MicroarrayCells were treated with 1 mmol/L inhibitor doses and incubated

for 24 hours at 37�C. Total RNA was purified with the RNeasy Kit(Qiagen). Total RNAwas reverse transcribed, labeled, and hybrid-ized to Human Genome U133 Plus 2.0 arrays (Affymetrix)according to the manufacturer's instructions. Themicroarray datawere deposited in the GEO database (GEO number: GSE73024).The expression value for each probe was calculated using theguanine-cytosine robustmultiarray analysis (GC-RMA) algorithm(19). To reduce noise from the low-signal range, probes with amedian signal of <10 across samples were filtered out. The logratio of expression relative to dimethyl sulfoxide (DMSO)-treatedsamples was calculated to identify downregulated genes (<50%expression) and upregulated genes (>200% expression). Cluster3.0 was used for clustering and Java Treeview was used forheatmap construction (20).

Signature analysisThe microarray dataset from human keratinocytes treated with

extracellular signal–regulated kinase 1/2 (ERK1/2) siRNA(GSE15417; ref. 21) was obtained from the Gene ExpressionOmnibus. This dataset consists of profiles from two independentsiRNA experiments (set A and B). Both profile sets and the log-

transformed expression data for each probe were analyzed by a ttest comparison against the control. Then, significantly down-regulated probes with t test P < 0.01 in both comparisons wereselected. We used 672 of 22,277 probes as an ERK1/2-induciblegene signature.

For the datasets in the "Microarray" section, the log ratios forthe replicated experiments were averaged. Then, distributions ofthe log ratios for probes in the signature and not in the signaturewere plotted as a histogram. Differences in their distributionpatterns indicate a coordinated change in expression for the genesignature. R (http://www.r-project.org/) was used for calculation.

Cell-proliferation assayAll cell lines were cultured according to the supplier's instruc-

tions. The cells were seeded in 96-well plates and incubated at37�C with inhibitors. After 4 days, Cell Counting Kit-8 solution(Dojindo Laboratories) was added, and, after incubation forseveral more hours, the absorbance at 450 nm was measuredwith the iMark Microplate-Reader (Bio-Rad Laboratories). Anti-proliferative activity was calculated using the formula (1� T/C)�100 (%), where T and C represent the absorbance at 450 nm oftreated (T) and untreated controls (C). The IC50 values werecalculated using Microsoft Excel 2007.

Western blot analysisCells were treated with inhibitors or a solvent control (0.1%

DMSO) for 2 or 24 hours and lysed in Cell Lysis Buffer(Cell Signaling Technology) containing protease and phospha-tase inhibitors. For animal studies, xenograft tumors wereobtained 4 hours after the 11th treatment of 100 mg/kgCH5183284/Debio 1347 and homogenized using a BioMasher(K.K. Ashisuto) before lysis. All in vivo studies were approved bythe Chugai Institutional Animal Care and Use Committee. Celllysates were denatured with Sample Buffer Solution with Reduc-ing Reagent for SDS-PAGE (Life Technologies) and resolvedon precast 10% or 5–20% SDS-PAGE gels (Wako Pure ChemicalIndustries). After electroblotting, Western blot analysis was per-formed as described previously (22). The following primaryantibodies (Cell Signaling Technology) were used: anti-phos-pho-FGF receptor (pFGFR-Tyr653/654, #3471), anti-phospho-ERK1/2 (pERK-The202/Tyr204; #9101), anti-ERK1/2 (ERK;#9102), anti-phospho-AKT (pAKT-Ser473; #9271), anti-AKT(AKT; #9272), anti-phospho-STAT3 (pSTAT3-Tyr705; #9145),anti-STAT3 (STAT3; #9139), anti-phospho-MET (pMET-Tyr1234/1235; #3126), anti-MET (Met; #3127), anti-phospho-EGFR (pEGFR-Tyr1068; #2234), anti-phospho-Src (pSrc-Tyr416;#2101), anti-DUSP6 (MKP3; #3058), and anti-Cyclin D1 (CyclinD1; #2926). The primary antibody against FGFR2 (F0300) wasobtained from Sigma-Aldrich, the primary antibody against Src(OP07) was obtained from EMD Millipore, and the primaryantibody against EGFR (sc-03) was obtained from Santa CruzBiotechnology. Horseradish peroxidase–conjugated secondaryantibodies against rabbit IgG (NA934V) and mouse IgG(NA931V) were obtained from GE Healthcare Life Sciences.

ResultsFGFR inhibition affects gene expression via the MAPK pathwayin an FGFR1 gene-amplified cancer

The MAPK and PI3K/AKT pathways are the main down-stream effectors of FGFR (1). We investigated the effects of

Nakanishi et al.

Mol Cancer Ther; 14(12) December 2015 Molecular Cancer Therapeutics2832




CH5183284/Debio1347 and AZD4547 (both FGFR inhibi-tors), CH4987655 (MEK inhibitor), and CH5132799 (PI3Kinhibitor) on the transcriptome of the NCI-H1581 lung cancercell line harboring FGFR1 gene amplification. We determinedthe effects of a 24-hour exposure on gene expression. Expres-sion values are presented relative to the DMSO controland clustered by similarity (Fig. 1A). We identified 2850probes that were modulated by CH5183284/Debio 1347.Of these, 2273, 1675, and 158 were also affected by AZD4547,CH4987655, and CH5132799, respectively. Because FGFRinhibition and MEK inhibition produced similar differentialexpression patterns, we analyzed our data in the context of thegene set associated with ERK siRNA-mediated changes(GSE15417). To analyze the coordinated change in expressionof the gene signature, the log ratio to the control was calculatedand distributions of log ratios for probes in the signature(blue) and not in the signature (red) were plotted as ahistogram. The FGFR and MEK inhibitors, but not the PI3Kinhibitor, showed differential gene expression patterns regu-lated by ERK (Fig. 1B). These results suggest that the FGFRinhibitor mainly modulates the MAPK pathway and not thePI3K pathway.

ERK signal suppression is associated with sensitivity to FGFRinhibition

We investigated the effect of CH5183284/Debio 1347 onthe MAPK pathway in 14 cancer cell lines harboring otherFGFR alterations. Thirteen of the cell lines were sensitive toCH5183284/Debio 1347 and harbored FGFR genetic alterationssuch as NCI-H716, KATO-III, HSC-39, SNU-16, MFE-280, MFE-296, AN3CA, DMS-114, NCI-H520, NCI-H1581, KMS-11, UM-UC-14, and RT-4; the other cell line, NCI-N87, was insensitive toCH5183284/Debio 1347 and had no FGFR alterations. Data ongenetic status and sensitivity to CH5183284/Debio 1347 areavailable elsewhere (7) or in Supplementary Table S1. We deter-mined the effects of a 24-hour exposure to 1 mmol/LCH5183284/Debio 1347 on gene expression relative to the DMSO control. Weconfirmed that the ERK (GSE15417) and MEK signatures (23)were suppressed by CH5183284/Debio 1347 only in sensitivecell lines harboring FGFR alterations (Fig. 2A and SupplementaryFig. S1). Consistentwith this, suppression of phospho-ERKby a 2-hour treatment with CH5183284/Debio 1347 was observedin seven cancer cell lines harboring FGFR alterations (Fig. 2B).However, phospho-AKT was suppressed only in four cell lines(NCI-H1581, SNU-16, KATO-III, andSUM-52PE), suggesting that

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Figure 1.Differential expression in the presence of several pathway inhibitors in NCI-H1581, an FGFR1-amplified cancer cell line. A, heatmap showing expression log ratio tocontrol (DMSO) for each probe after a 24-hour treatment with 1 mmol/L CH5183284/Debio 1347, AZD4547 (FGFR inhibitor), CH4987655 (MEK inhibitor), orCH5132799 (PI3K inhibitor) inNCI-H1581 harboringFGFR1gene amplification (n¼ 2). Probeswere clustered by similarity at the left side of the panel. B, distributions oflog ratio to control (geometric mean of DMSO-treated samples)were plotted as a histogram. The x-axis represents the log ratio; the y-axis represents the frequency.The distribution pattern for probes in the ERK1/2 signature is blue; distributions of other probes are red (n ¼ 2)

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FGFRs likely depend on the MAPK pathway and that ERK signalsuppression is associated with sensitivity to FGFR inhibition.

Differential expression of DUSP6 and sensitivity to FGFRinhibition

Because FGFRs likely depend on the MAPK pathway, wesearched for genes that could be modulated by and associatedwith sensitivity to CH5183284/Debio 1347 among the 672 genesof the ERK signature (GSE15417). We calculated the ratio of the

geometric mean of each probe in 13 cell lines treated with DMSOand CH5183284/Debio 1347 (NCI-H716, KATO-III, HSC-39,SNU-16, MFE-280, MFE-296, AN3 CA, DMS-114, NCI-H520,NCI-H1581, KMS-11, UM-UC-14, and RT-4). We identified 47genes (51 probes) that were significantly modulated (P < 0.05; 2-fold change) by treatment (Fig. 3A and Supplementary Table S2).The most significantly modulated gene was the DUSP6 gene.Expression of DUSP6 was suppressed by CH5183284/Debio1347 in every sensitive cancer cell line that we tested but not in

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Figure 2.MAPK pathway suppression by CH5183284/Debio 1347 in FGFR-altered cancer cell lines. A, distributions of expression log ratio to control (geometric mean ofDMSO-treated samples) were plotted as a histogram. The x-axis represents the log ratio; the y-axis represents the frequency. Distribution patterns for probes in theERK1/2 signature are blue; other probes are red. Cells were treated with 1 mmol/L CH5183284/Debio 1347 or DMSO for 24 hours. Total RNA was analyzed bymicroarray. IC50 concentrations of CH5183284/Debio 1347 in antiproliferative assay are provided. B, inhibition of ERK or AKT phosphorylation. After a 2-hourincubation with CH5183284/Debio 1347, cultures of DMS-114, NCI-H1581, SNU-16, KATO-III, SUM-52PE, UM-UC-14, and KMS-11 cells were lysed and analyzed byWestern blotting. Ampli, amplification; mut, mutation

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NCI-N87, which is insensitive to CH5183284/Debio 1347 (Fig.3B and Supplementary Fig. S2). PD173074, another FGFR inhib-itor, also suppressed phospho-ERK, resulting in decreasedDUSP6protein expression levels in the FGFR inhibitor–sensitive cell linesafter a 24-hour treatment (Fig. 3C). As expected, phospho-AKTlevel was not constantly suppressed. Notably, one FGFR1 gene-amplified but FGFR inhibitor–insensitive cell line, ZR-75-1, didnot show suppression of phospho-ERK and DUSP6 expression.PD0325901, aMEK inhibitor, reduced phospho-ERK andDUSP6protein levels in the FGFR inhibitor–sensitive and -insensitive celllines. To show the usefulness of DUSP6 as a pharmacodynamicmarker of CH5183284/Debio 1347 in in vivomodel, we obtained

the tumor tissues 4 hours after the 11th daily administration of100 mg/kg CH5183284/Debio 1347. We utilized SNU-16 andDMS-114 as a CH5183284/Debio 1347-sensitive model andNCI-N87 andMKN-45 as an insensitivemodel. The tumor growthinhibitory activities of CH5183284/Debio 1347 against thosemodels are available (Supplementary Table S3). Consistent withthe in vitro findings, CH5183284/Debio 1347 suppressed phos-pho-ERK and decreased DUSP6 protein expression levels in theFGFR inhibitor–sensitive xenograft models but not in the insen-sitivemodels (Fig. 3D). These data suggest that DUSP6 is themostreliable pharmacodynamic marker associated with the efficacy ofFGFR inhibitors in FGFR genetically altered cancers.

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Figure 3.Modulation of DUSP6 expression in FGFR inhibitor–sensitive cancer cell lines. A, a volcano plot representation showing the magnitude (log2 ratio, x-axis) andsignificance (log10 P, y-axis) of all genes in the ERK signature. The red circles represent the significantly modulated probes and the white circles represent others.B, bar graphs show the ratio of probes that recognize DUSP6 vs. DMSO in 14 cancer cell lines. The black bars denote cell lines harboring FGFR alterations,and gray bars denote the FGFR wild-type cell line. C, inhibition of DUSP6 expression or ERK phosphorylation. After a 24-hour treatment with PD173074 (FGFRinhibitor) at 3 mmol/L or with PD0325901 (MEK inhibitor) at 1 mmol/L, cultures of NCI-H1581, DMS-114, AN3 CA, SNU-16, KMS-11, ZR-75-1, NCI-N87, and HCT116cells were lysed and analyzed byWestern blotting. D, pharmacodynamic response in in vivomodels. Mice-bearing SNU-16, DMS114, NCI-N87, or MKN-45 cells wereorally administered CH5183284/Debio 1347 at 100 mg/kg, and the tumors were collected and lysed at 4-hour postdosing (n ¼ 2). Lysates were then analyzed byWestern blotting. Ampli, amplification; Mut, mutation






MAPK pathway suppression without feedback activation ofbypass pathways could be important for FGFR inhibitor activityin FGFR genetically altered cancers

To investigate the significance of MAPK pathway suppressionby FGFR inhibitors, we tested the sensitivity of FGFR-addictedcancer cell lines to MEK inhibitors, such as CH4987655,CH5126766, or selumetinib. Although MEK inhibition sup-pressed DUSP6 protein expression in FGFR genetically alteredcancer cell lines, these cell lines were not sensitive to the MEKinhibitor, whereas the B-RAF-mutated cell line (SK-MEL-1), K-RAS-mutated cell line (HCT116), andNF-1-null cell line (MeWo)were sensitive to MEK inhibitors (Fig. 4). To clarify the mechan-ismsof innate resistance toMEK inhibitors,we treated the SNU-16cell line harboring FGFR2 gene amplification with CH5183284/Debio 1347, CH5126766 (RAF-MEK inhibitor), or CH5132799(PI3K inhibitor), either alone or in combination (Fig. 5).CH5183284/Debio 1347 suppressed phospho-FGFR, phospho-AKT, phospho-ERK, and DUSP6. In the presence of CH5126766-mediated suppression of phospho-ERK and DUSP6, we observedelevated phosphorylation of FGFR, MET proto-oncogene, recep-tor tyrosine kinase (MET), epidermal growth factor receptor(EGFR), AKT, SRC proto-oncogene, nonreceptor tyrosine kinase(SRC), and signal transducer and activator of transcription 3(STAT3). Feedback activation by MEK inhibition was abrogatedby CH5183284/Debio 1347. Thus, MAPK pathway suppressionalone reactivated FGFR and induced potent activation of otherpathways. CH5132799 did not produce this result. DUSP6 andCyclin D1 suppression, and thus MAPK pathway inhibition, wasstronger with CH5183284/Debio 1347 than with CH5126766.These results show that the FGFR inhibitor suppressed the MAPKpathway more effectively than the MEK inhibitor alone withoutfeedback activation of other signals, thus leading to the efficacy ofFGFR inhibitors in FGFR-addicted cancers.

DiscussionInvestigations of the signaling pathways associated with

oncogenes provide important information leading to combi-

nation therapies or increased understanding of resistancemechanisms. For instance, in an endocrine therapy–resistanthormone receptor–positive breast cancer, the PI3K/AKT path-way is aberrantly activated and a downstream kinase, S6 kinase,independently phosphorylates, and activates the estrogenreceptor ligand (24). Therefore, combination of endocrinetherapy with letrozole and PI3K pathway inhibition by ever-olimus exhibits great clinical efficacy (25). The MAPK andPI3K/AKT pathways are the main downstream effectors of FGFR(1). Every FGFR-altered cancer cell line that was tested in thisstudy was dependent on the MAPK pathway but not the PI3K/AKT pathway (Fig. 1, 2). This is consistent with several geneticprofiling studies. FGFR and RAS mutations are mutually exclu-sive in bladder cancer and endometrial cancer (26, 27); incontrast, FGFR mutations are frequently associated with muta-tions in the PI3K/AKT pathway (26, 27). Conversely, when anFGFR-altered cancer also carries a KRAS mutation, FGFR inhi-bitors are ineffective (28), suggesting that FGFR and RAS likelyactivate the same pathway. Although MAPK is considered themain downstream effector of FGFR, we have shown that MEKinhibitors are not always effective in FGFR-altered cancerbecause of feedback activation (Fig. 5). Similarly, PI3K inhibi-tion enhanced HER2 signaling in HER2 overexpressing breastcancer. In that context, the combined administration of PI3Kand HER2 inhibitors produced superior antitumor activity incomparison to monotherapy (29). Therefore, the combinationof FGFR andMEK inhibition could be another approach to treatpatients harboring FGFR alterations.

DUSP6 is amember of the dual-specificity protein phosphatasesubfamily that inactivates target kinases, such as ERK, by dephos-phorylation. Overexpression of DUSP6 was observed in responseto activated RAS or BRAF (30–34), representing an increase innegative feedback of the MAPK pathway. Therefore, DUSP6expression could reflect activity of the MAPK pathway. In addi-tion, a role of DUSP6 during early mouse development inresponse to FGF signaling has been suggested (35, 36). In mouseembryos, DUSP6 contributes as a negative feedback regulator ofFGF-stimulated ERK signaling. The DUSP6 loss-of-function

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Figure 4.Sensitivity of FGFR-altered cancer cell lines to MEK inhibitors. Cells were seeded in 96-well plates and treated for 4 dayswith various concentrations of CH4987655(MEK inhibitor), CH5126766 (RAF-MEK inhibitor), or selumetinib (MEK inhibitor) prior to viability assays.

Nakanishi et al.





mutant causes several symptoms, such as variably penetrant,dominant postnatal lethality, skeletal dwarfism, coronal cranio-synostosis, and hearing loss. These phenotypes are also charac-teristic of FGFR active mutations. Therefore, we suggest thatDUSP6 could be the most reliable pharmacodynamic marker ofFGFR inhibition; the responsiveness of DUSP6 expression couldpredict the efficacy of FGFR inhibitors.

Studying pharmacodynamic biomarkers will help acceleratethe development ofmolecular-targeted agents for the treatment ofcancer (37, 38). In general, the gold-standard pharmacodynamicassay is immunohistochemistry on a tumor sample. Severalclinical studies evaluated the phosphorylation status of the drugtarget or molecules on the downstream pathway (39, 40).Although the evaluation of differential phosphorylation couldbe used to assess inhibitor activity in the tumor, there are limita-tions to phospho-related assays. The loss of immunoreactivephospho-AKT and phospho-ERK during sample procurement hasbeen reported (41). Therefore, we must consider multiple pre-analytical factors that influence the stability of phosphorylatedproteins during procurement and preservation of clinical samples(42). Noninvasive samples such as circulating tumor cells andcirculating tumor DNA obtained from a liquid biopsy could bethe next-generation source of pharmacodynamicmarkers (43). Insolid tumors, for instance, monitoring for EGFR-activating muta-tions enables the prediction of anti-EGFR therapeutic efficacy inpatients; identification of acquired EGFRmutations in circulatingDNA is also a way to guide therapeutic decision-making (44, 45).The FGFR family incurs many types of genetic alterations inmultiple tumor types; therefore, assays must be developed tocapture all genetic alterations in circulatingDNA. The detection ofDUSP6 could overcome these issues, as assays would not need tobe developed for each FGFR alteration and the stability of phos-phorylation would not be an issue. Alternatively, DUSP6 mRNAcould also bemonitored in circulating tumor cells or the exosomein liquid biopsy samples.

Monitoring of downstreammolecules enables prediction of theefficacy ofmolecular-targeted drugs. Sensitivity to BYL719, a PI3Kp110a selective inhibitor, in breast cancer carrying a mutation inphosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic sub-unit alpha (PIK3CA) was associated with full inhibition of sig-

naling through the CREB-regulated transcription coactivator 1(TORC1) pathway. Conversely, cancer cells that were not phos-pho-S6-suppressed were resistant to BYL719, although AKT phos-phorylation was inhibited (46). CH5126766, a RAF-MEK inhib-itor, showed superior antitumor efficacy in comparison to theMEK inhibitor alone (17). Although both inhibitors suppressedphospho-ERK in tumors, CH5126766 more strongly suppresseddownstream signaling targets such as DUSPs and SPREADs (17).In FGFR-altered cancer cells, DUSP6 was more significantly sup-pressed byCH5183284/Debio 1347 thanbyCH5126766 (Fig. 5).CH5126766 suppressed phospho-ERK but not the downstreamERKeffectorDUSP6, suggesting that CH5126766does not strong-ly suppress MAPK, resulting in the resistance of FGFR-alteredcancer cells to MEK inhibition. Therefore, monitoring of DUSP6could be more beneficial than that of phospho-ERK.

In summary, measurement of the status of downstreamsignaling could be used to predict a molecule's therapeuticefficacy. FGFR likely depends on the MAPK pathway, and ERKsignal suppression is associated with sensitivity to FGFR inhi-bition. Therefore, DUSP6, which functions downstream of theERK signal, is one of the most reliable pharmacodynamicmarkers of the efficacy of an FGFR inhibitor. CH5183284/Debio 1347 is currently in phase I clinical trials by DebiopharmInternational S.A. in patients harboring FGFR alterations(NCT01948297).

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

Authors' ContributionsConception and design: Y. Nakanishi, Y. AokiDevelopment of methodology: Y. Nakanishi, K. SakataAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Nakanishi, H. Sase, N. AkiyamaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Nakanishi, H. Mizuno, T. FujiiWriting, review, and/or revision of the manuscript: Y. Nakanishi, H. Mizuno,N. IshiiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. NakanishiStudy supervision: Y. Nakanishi, Y. Aoki, M. Aoki, N. Ishii

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Figure 5.Pathway biology upon MEK inhibitortreatment in FGFR2-amplified cancercells. After a 24-hour treatment withCH5183284/Debio 1347, CH5132799(PI3K inhibitor), CH5126766 (RAF-MEKinhibitor), or combinations thereof,SNU-16 cancer cells harboring FGFR2gene amplification were lysed andanalyzed byWestern blotting. The drugconcentrations were 0.3 or 1 mmol/L.






AcknowledgmentsThe authors thank Yasue Nagata for performing the pharmacological assays.

The authors also thank Anne Vaslin and Corinne Moulon from DebiopharmInternational S.A. for their helpful discussions.

Grant SupportThis study was funded by the Chugai Pharmaceutical Co., Ltd.

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, 2015; revised September 15, 2015; accepted September 23,2015; published OnlineFirst October 5, 2015.

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2015;14:2831-2839. Published OnlineFirst October 5, 2015.Mol Cancer Ther Yoshito Nakanishi, Hideaki Mizuno, Hitoshi Sase, et al. a Selective FGFR InhibitorERK Signal Suppression and Sensitivity to CH5183284/Debio 1347,

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