Small Molecule Therapeutics

The Fibroblast Growth Factor Receptor Genetic Status as aPotential Predictor of the Sensitivity to CH5183284/Debio1347, a Novel Selective FGFR Inhibitor

Yoshito Nakanishi1, Nukinori Akiyama1, Toshiyuki Tsukaguchi1, Toshihiko Fujii1, Kiyoaki Sakata1,Hitoshi Sase1, Takehito Isobe2, Kenji Morikami2, Hidetoshi Shindoh1, Toshiyuki Mio, Hirosato Ebiike1,Naoki Taka2, Yuko Aoki1, and Nobuya Ishii1

AbstractThe FGF receptors (FGFR) are tyrosine kinases that are constitutively activated in a subset of tumors by

genetic alterations such as gene amplifications, point mutations, or chromosomal translocations/rearrange-

ments. Recently, small-molecule inhibitors that can inhibit the FGFR family as well as the VEGF receptor

(VEGFR) or platelet-derived growth factor receptor (PDGFR) family displayed clinical benefits in cohorts of

patients with FGFR genetic alterations. However, to achieve more potent and prolonged activity in such

populations, a selective FGFR inhibitor is still needed. Here, we report the identification of CH5183284/Debio

1347, a selective and orally available FGFR1, FGFR2, and FGFR3 inhibitor that has a unique chemical scaffold.

By interacting with unique residues in the ATP-binding site of FGFR1, FGFR2, or FGFR3, CH5183284/Debio

1347 selectively inhibits FGFR1, FGFR2, andFGFR3but does not inhibit kinase insert domain receptor (KDR) or

other kinases. Consistent with its high selectivity for FGFR enzymes, CH5183284/Debio 1347 displayed

preferential antitumor activity against cancer cells with various FGFR genetic alterations in a panel of 327

cancer cell lines and in xenograft models. Because of its unique binding mode, CH5183284/Debio 1347 can

inhibit FGFR2 harboring one type of the gatekeeper mutation that causes resistance to other FGFR inhibitors

and block FGFR2 V564F–driven tumor growth. CH5183284/Debio 1347 is under clinical investigation for the

treatment of patients harboring FGFR genetic alterations. Mol Cancer Ther; 13(11); 2547–58. �2014 AACR.

IntroductionIn recent years, the use of molecular-targeted agents is

increasingly adopted in basic and clinical cancer researchwith some of these drugs, improving patient survivaltimes. In this context, a number of agents that targettyrosine kinases have been launched, such as the EGFreceptor (EGFR) inhibitor erlotinib, the anti-HER2 anti-body trastuzumab, theBCR-ABL inhibitor imatinib, the B-RAF inhibitor vemurafenib, and the ALK inhibitor crizo-tinib. Eachof these agents hasdemonstrable efficacywhenused inpatient cohorts that are stratifiedon the basis of thegenetic status of their respective molecular targets. How-ever, such agents cannot cover all tumors, thus a medical

need remains for novel agents against tumors harboringnovel genetic alterations.

Many tumors have genetic alterations in membersof the receptor tyrosine kinase (RTK) family, whichincludes FGF receptors (FGFR). The FGFR family con-sists of FGFR1, FGFR2, FGFR3, and FGFR4, and eachmember is bound by a subset of 22 FGF ligands. TheMAPK and PI3K/AKT pathways are critical down-stream mediators of FGFR signaling. In cancer, consti-tutive FGFR signaling is activated by gene amplifica-tion, point mutations, or chromosomal translocations/rearrangements in several tumor types and is known tobe involved in cell growth, angiogenesis, cell migration,invasion, and metastasis (1). Amplification of FGFR1 isone of the key genetic alterations in squamous cell lungcarcinoma and hormone receptor–positive breast cancer(2–4). In addition, FGFR2 is also amplified in gastric andbreast cancers (5, 6). Point mutations of FGFR2 andFGFR3 are mainly observed in endometrial cancer andbladder cancer, respectively (7–9). Furthermore, theexplosion in next-generation sequencing technology hasrevealed several chromosomal translocations/rearran-gements of FGFR1, FGFR2, and FGFR3 in glioblastoma,bladder cancer, and breast cancer as well as in othertumor types (10–12). Thus, there is a clear clinical needfor FGFR-selective inhibitors.

1Research Division, Chugai Pharmaceutical Co., Ltd., Kamakura, Kana-gawa, Japan. 2Research Division, Chugai Pharmaceutical Co., Ltd.,Gotemba, Shizuoka, Japan.

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

Corresponding Author: Yoshito Nakanishi, Research Division, ChugaiPharmaceutical Co. Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530,Japan. Phone: 81-467-47-6262; Fax: 81-467-46-5320; E-mail:nakanishiyst@chugai-pharm.co.jp

doi: 10.1158/1535-7163.MCT-14-0248

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

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Several multitargeted kinase inhibitors that inhibitFGFRs as well as VEGF receptors (VEGFR) and platelet-derived growth factor receptors (PDGFR), such as cedir-anib (13), dovitinib (14), and E-3810 (15), are currentlyunder clinical trials and show some clinical benefits inFGFR genetic alteration–positive patients. For instance,dovitinib showed three unconfirmed partial responses(PR) in hormone receptor–positive breast cancer (16).However, the drugs are also potent inhibitors of kinaseinsert domain receptor (KDR), which likely underlies thegrade 3/4 hypertension and dose-limiting toxicityobserved during clinical trials of these inhibitors (17,18). Because these inhibitors are unlikely to achieve therequired exposure to sufficiently inhibit FGFR in tumorsin a clinical setting, more selective FGFR inhibitors arerequired.

Currently, several FGFR-selective inhibitors, such asAZD4547 (19) and NVP-BGJ398 (20), are under clinicaltrials targeting patientswho have FGFR genetic alterations.These inhibitors, as well as the FGFR-selective inhibitorPD173074, share a common 3,5-dimethoxy-phenyl moiety.Thus, it is possible that a common single mutation wouldconfer resistance to these inhibitors. A potential strategy tomitigate this issue is the rational design of a novel inhibitorwith a different chemical scaffold. Proof of principle is thatresistance to the anaplastic lymphoma kinase (ALK) inhib-itor crizotinib was overcome by the design of alectinib/CH5424802, a highly selective ALK inhibitor with a differ-ent chemical scaffold (21).

In this study, we generated a highly selective FGFR1,FGFR2, and FGFR3 inhibitor with a new chemical scaf-fold and investigated its activity in a cell growth inhi-bition assay against a large panel of tumors with FGFRgenetic alterations and in several xenograft models.We also evaluated the potential of this novel agent toinhibit the activity of V564F, a gatekeeper mutant ofFGFR2, which is resistant to previously developedFGFR inhibitors.

Materials and MethodsReagents

CH5183284/Debio 1347 andAZD4547were synthesizedat Chugai Pharmaceutical Co. Ltd., according to the pro-cedure described in patent publications WO2011016528and WO2008075068. NVP-BGJ398, PD173074, cediranib,and dovitinib were purchased from Active Biochem andSigma-Aldrich.

Protein kinase assayProtein kinases (listed in Supplementary Materials and

Methods) were purchased from Carna Biosciences orMillipore Corporation. The inhibitory activity againsteach kinase was evaluated as described previously (22).

Cell proliferation assayCell lines were obtained from the ATCC, Deutsche

Sammlung von Mikroorganismen und ZellkulturenGmbH (DSMZ), Health Science Research Resources Bank

(HSRRB), Asterand Inc., and Health Protection AgencyCulture Collections (HPACC). All cell lines were authen-ticated by the cell banks with cytogenic analysis, DNAprofiling, or growth properties and were propagated forless than 6 months after resuscitation. Also, all cell lineswere cultured according to supplier instructions. The celllines were added to the wells of 96-well plates containing0.076 to 10,000 nmol/L CH5183284/Debio 1347 and incu-bated at 37�C. After 4 days of incubation, Cell CountingKit-8 solution (Dojindo Laboratories) was added and,after incubation for several more hours, absorbance at450 nm was measured with the iMark Microplate-Reader(Bio-Rad Laboratories). The antiproliferative activity wascalculated using the formula (1� T/C)� 100 (%), whereTand C represent absorbance at 450 nm of the cells treatedwith drugs (T) and that of untreated control cells (C). TheIC50 values were calculated using Microsoft Excel 2007.

Western blot analysisCells were treated with 0.1% DMSO or CH5183284/

Debio 1347 for 2 hours and were lysed with Cell LysisBuffer (Cell Signaling Technology) containing proteaseand phosphatase inhibitors. The grafted tumors werehomogenized using a BioMasher (K.K. Ashisuto) beforelysis. The lysates were denatured with sample buffersolution with reducing reagent for SDS-PAGE (Life Tech-nologies) and were then subjected to SDS-PAGE. Afterelectroblotting, Western blot analysis was performed asdescribed previously (23). The antibodies used for thisstudy are available in Supplementary Materials andMethods.

Telemetry study in ratsAll in vivo studies were approved by the Chugai Insti-

tutional Animal Care and Use Committee. Male Wistarrats (340–390 g; Japan Slc Inc.) implantedwith a telemetrytransmitter were used for the assessment of effects onblood pressure (BP; ref. 24). Vehicle (0.5% carmellosesodium, 0.5% polysorbate 20, and 0.9% benzyl alcohol inpurifiedwater) or CH5183284/Debio 1347 (10 and 30mg/kg) were administered by oral gavage once a day for4 consecutive days. Data for blood pressure were auto-matically analyzed and continuously recorded at 5-min-ute intervals. Baseline blood pressure was determined bythe 24-hour mean of blood pressure before administra-tion, and change in bloodpressure from the baseline value(DBP) is represented as mean � SD. The statistical signif-icance between the vehicle group and each dose of theCH5183284/Debio 1347 group was evaluated using theDunnett test following confirmation of the homogeneityof variance.

Mouse xenograft studyAll in vivo studies were approved by the Chugai Insti-

tutional Animal Care and Use Committee. Female BALB-nu/nu mice (CAnN.Cg-Foxn1<nu>/CrlCrlj nu/nu)were obtained from Charles River Laboratories and keptunder specified pathogen-free conditions. Cells (4� 106 to

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1.1 � 107) were suspended in 100 to 200 mL serum-freeculturemediumand injected subcutaneously into the rightflank of themice. Tumor size wasmeasured using a gaugetwice per week, and tumor volume (TV) was calculatedusing the following formula: TV ¼ ab2/2, where a is thelength of the tumor and b is the width. Once the tumorsreached a volume of approximately 200 to 300 mm3, ani-mals were randomized into groups (n ¼ 3, 4, or 5 in eachgroup), and treatment was initiated. CH5183284/Debio1347 or AZD4547 were orally administered once a day.

ImmunohistochemistryXenograft tumors were extracted, fixed in formalin,

and embedded in paraffin. Sections were stained withantibodies against phospho-FRS (ab78195, Abcam), phos-pho-FGFR (#3471), phospho-ERK (#4780), and phospho-S6 (#4858) purchased from Cell Signaling Technology.Immunohistochemistry was performed using the DIS-COVERYXT automated staining platform (VentanaMed-ical Systems).

Crystallization and structural determination of theFGFR1–CH5183284 complexProtein crystallography was performed by Roche. A

crystal structure of the kinase domain of human FGFR1(residues 462–763) in complex with CH5183284/Debio1347 was determined at 2.2 A resolution. Details aredescribed in Supplementary Materials and Methods. Forcrystallographic data and refinement statistics, see Sup-plementary Table S1.

Stable Ba/F3 transfectantsThe TEL-FGFR2 wild-type (WT), which is a fusion

protein of the dimerization domain of the TEL transcrip-tion factor and cytosolic domain of FGFR2, and TEL-FGFR2V564Fwere inserted into apCXND3vector (Kaket-suken). TEL-FGFR2 V564F was generated by PCR-basedsite-directedmutagenesis. Ba/F3-TEL-FGFR2WTand theV564F cell lines were generated by transfecting Ba/F3cells with pCXND3 TEL-FGFR2 WT and the V564Fmutant using the NucleoFector device (Amaxa); stabletransfectants were then isolated from the cultured medi-um without IL3.

ResultsSelective inhibition of FGFR1, FGFR2, and FGFR3kinase activity by CH5183284/Debio 1347To obtain an FGFR-selective inhibitor, we performed

high-throughput screening against a chemical library ofChugai Pharmaceutical Co. Ltd. and identified a leadcompound that inhibited a relatively broad range ofkinases but had a different chemical scaffold from otherknown FGFR inhibitors. We then structurally modifiedand improved the pharmacokinetics profile and selec-tivity against FGFR1, FGFR2, and FGFR3. Finally, wegenerated a 1-(1H-benzimidazol-5-yl)-5-aminopyrazolederivative, CH5183284/Debio 1347, as a potent, selec-tive, and orally available FGFR1, FGFR2, and FGFR3

inhibitor (Fig. 1A). Our analysis of the FGFR inhibitionkinetics of CH5183284/Debio 1347 in biochemicalenzyme assays revealed an ATP-competitive inhibitoryprofile against FGFR1 (Supplementary Fig. S1). Toinvestigate the selectivity of CH5183284/Debio 1347against FGFR1, FGFR2, and FGFR3, we screenedCH5183284/Debio 1347 for activity against the FGFRfamily members and its selectivity among 20 tyrosinekinases and 14 serine/threonine kinases in a cell-freesystem (Table 1). The IC50 values of CH5183284/Debio1347 on the enzyme activity of FGFR1, FGFR2, FGFR3,and FGFR4 were 9.3, 7.6, 22, and 290 nmol/L, respectiv-ely. Because the IC50 for KDR kinase was 2,100 nmol/L,these data indicate that CH5183284/Debio 1347 coulddistinguish FGFR1, FGFR2, and FGFR3 from FGFR4 andKDR at the enzyme level. As for the other 33 kinases,CH5183284/Debio 1347 did not inhibit them at compa-rable degrees to the inhibition observed for FGFR1,FGFR2, or FGFR3. To evaluate kinase selectivity further,we used a KINOMEscan panel (DiscoveRx) consistingof 442 WT and mutant kinases. At 100 nmol/L,CH5183284/Debio 1347 only bound to five kinases inthe panel, including FGFR1, FGFR2, and FGFR3 (morethan 80% inhibition to an ATP analog binding; Supple-mentary Table S2). We also confirmed the selectivity forFGFR1, FGFR2, and FGFR3 with this ATP analog in acompetitive binding assay. The binding affinity (Kd) ofCH5183284/Debio 1347 for FGFR1, FGFR2, FGFR3,FGFR4, and KDR was 20, 20, 25, 740, and 960 nmol/L,respectively (using the DiscoveRx panel).

To evaluate theFGFR1, FGFR2, andFGFR3 selectivity incells, we tested the effects of the compound on autopho-sphorylation of several RTKs, including FGFRs.As shownin Fig. 1B, CH5183284/Debio 1347 prevented autopho-sphorylation of FGFR1, FGFR2, and FGFR3 at 100 to 300nmol/L in the DMS114 (FGFR1 amplification), SNU-16(FGFR2 amplification), and KMS11 [t(4;14) translocationand FGFR3 Y373C mutation] cell lines. In contrast,CH5183284/Debio 1347 could not suppress autopho-sphorylation of FGFR4, KDR, PDGFRa, or c-KIT at thesame concentration in MDA-MB-453 (FGFR4 mutation),human umbilical vein endothelial cells (HUVEC), NCI-H1703 (PDGFRa amplification), and Kasumi-1 (c-KITN822K mutation).

To further demonstrate the selectivity of CH5183284/Debio 1347 for FGFR versus KDR, we conducted FGF- orVEGF-dependent proliferation assay in HUVECs (25, 26).The IC50 of CH5183284/Debio 1347 was 29 nmol/Lfor FGF-dependent proliferation and 780 nmol/L forVEGF-dependent proliferation (Fig. 1C). In addition,CH5183284/Debio 1347 did not inhibit VEGF-inducedtube formation of HUVECs (Supplementary Fig. S2).Because inhibition of VEGF signaling leads to hyperten-sion (17, 18), we also evaluated the effect of CH5183284/Debio 1347 on blood pressure in telemetry-instrumentedrats. Four consecutive daily oral administrations of 10 or30 mg/kg of CH5183824/Debio 1347 did not lead tosignificant changes in blood pressure compared with

Efficacy of CH5183284 against Tumors with FGFR Aberrations

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vehicle-dosed animals (Fig. 1D). Of note, at a dose of 30mg/kg, the CH5183284/Debio 1347 plasma exposure inrats (AUC, 102 mg/h mL) was 4-fold higher than theexposure inducing a greater than 100% tumor growthinhibition in the SNU-16 mouse xenograft model (AUC,26 mg/h mL). In the same model, we have confirmed theelevation of blood pressure by cediranib treatment, multi-targeted KDR inhibitor (24). Therefore, CH5183284/Debio 1347 has no biologically significant effect on bloodpressure atmultiples of the efficacious dose.We also inferthat Debio 1347 does not target KDR to a significant extentin vivo, as no increase in blood pressure (a feature of otherFGFR inhibitors that also target KDR)was observed in ourexperiments. These results indicate that CH5183284/Debio 1347 selectively inhibits FGFR1, FGFR2, and FGFR3kinase activity.

Unique interactions between the FGFR1 and theCH5183284/Debio 1347

To understand the mechanism of selectivity ofCH5183284/Debio 1347 for FGFR1, FGFR2, and FGFR3over other kinases,we solved the three-dimensional struc-ture of a complex of CH5183284/Debio 1347 and thebacterially expressed protein kinase domain of humanFGFR1 (residues 462–763).This revealed thatCH5183284/Debio 1347 binds to the ATP-binding site of FGFR1 in theDFG-in mode through five hydrogen bonds. Two of thesefive hydrogen bonds occur between the benzimidazole

moiety of CH5183284/Debio 1347 and a backbone nitro-gen atom of Asp641 and a side chain oxygen atom ofGlu531 of FGFR1. The remaining three hydrogen bondswere identified between the hinge binder of CH5183284/Debio 1347 and the hinge region of FGFR1 at Glu562,Tyr563, and Ala564 (Fig. 2A). Although AZD4547, NVP-BGJ398, and PD173074 have a common structure, 3,5-dimethoxy-phenyl moiety, CH5183284/Debio 1347 doesnot have it but a unique benzimidazole moiety to interactwith the FGFR1 at the back pocket, so several uniqueinteractions were suggested (Fig. 2B). One of the uniqueinteractions is an interaction between Phe642 in FGFR1and the methyl group of the benzimidazole moiety ofCH5183284/Debio 1347, only CH5183284/Debio 1347nicely interacted with Phe642, which exists at the mostinnerpart of theATP-bindingpocket.During the chemicalmodification of CH5183284/Debio 1347 derivatives, weevaluated the compound that does not have the methylgroup in the benzimidazole moiety. And the derivativehad less selective and less potent inhibitory activity thanCH5183284/Debio 1347. This observation also supportsthat the methyl group of the benzimidazole moiety ofCH5183284/Debio 1347 would contribute to exert itsselectivity against FGFR. The second is an interactionbetween sulfur atom ofMet535 in FGFR1 and the nitrogenatom and the methyl group of the benzimidazole moietyof CH5183284/Debio 1347. In the case of KDR, a leucine isused at the same position and a reasonable interaction is

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Figure 1. Selective inhibitoryactivity against FGFR1, FGFR2,and FGFR3. A, chemical structureof CH5183284/Debio 1347. B,inhibition of cellular pY-FGFR1in DMS-114 cells, pY-FGFR2 inSNU-16, pY-FGFR3 in KMS11,pY-FGFR4 in MDA-MB453,pY-KDR in HUVECs, pY-PDGFRain NCI-H1703, and pY-cKIT inKasumi-1 by CH5183284/Debio1347 at the indicatedconcentration for 2 hours. C, cellgrowth inhibition of HUVECs byCH5183284/Debio 1347. HUVECcells were seeded in mediumcontaining VEGF (20 ng/mL) orECGF (30 mg/mL) and treated withvarious concentrations ofCH5183284/Debio 1347 for 4days. Then, viable cells werecounted with WST-8. Data areshown as mean � SD (n ¼ 3). D,effects of CH5183284/Debio 1347on blood pressure in rats. Animalswere treated and effects on bloodpressure were assessed. Meandiastolic blood pressure wasmeasured in rats treatedwith 0, 10, or 30 mg/kg/d ofCH5183284/Debio 1347.

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not suggested between the leucine andCH5183284/Debio1347 (Fig. 2C). It would be one of the reasons whyCH5183284/Debio 1347 has selectivity to KDR. The thirdis an interaction between Val561, a gatekeeper residue ofFGFR1, and aromatic ring of the benzimidazole moiety ofCH5183284/Debio 1347. Although this interaction is con-served among FGFRs and NVP-BGJ398 or PD173074,CH5183284/Debio 1347 has wider space between FGFR1and the aromatic ring because CH5183284/Debio 1347

does not have bulky methoxy moiety common to otherFGFR selective inhibitors, so CH5183284/Debio 1347might be able to accept the mutation at a gatekeeperresidue which is insensitive to other FGFR inhibitors.Only 15 of 490 human protein kinases use valine as agatekeeper. Among them, only 7 kinases, including theFGFR family, use methionine and phenylalanine at theparticular part of pocket. By interacting with these threeunique residues, CH5183284/Debio 1347 obtains FGFRselective kinase inhibitory activity, and this interactionmode is different from other FGFR inhibitors.

FGFR4 has a cysteine residue in themiddle of the hingeregion (Cys552),whereas FGFR1, FGFR2, andFGFR3havea tyrosine residue at the same position (Tyr563 in FGFR1,Tyr566 in FGFR2, and Tyr557 in FGFR3). This tyrosineresidue in FGFR1 makes parallel-displaced p-p interac-tionswith the indolemoiety of CH5183284/Debio 1347. Incontrast, CH5183284/Debio 1347 would not be able tomake p-p interactions with FGFR4 at this position. This isconsistent with the lower FGFR4 enzymatic inhibition byCH5183284/Debio 1347 as comparedwith the other FGFRfamily members.

The selective FGFR inhibition leads the selectiveantiproliferative activity against the cancer cellsharboring the FGFR genetic alterations

Comparison of an encyclopedia of cell lines and theirsusceptibilities to drugs facilitates the identification ofbiomarkers, such as gene amplifications, point mutations,and chromosomal translocation/rearrangements, thatcan predict the efficacy of drugs (27, 28). Following thisrationale, we predicted that a selectivity of CH5183284/Debio 1347 for FGFR1, FGFR2, andFGFR3wouldmanifestas a selective activity to cell lines with activating altera-tions in these FGFRs. To test this, the antiproliferativeactivity of CH5183284/Debio 1347 was assessed against alarge panel of 327 human tumor cell lines that weregenetically profiled (Fig. 3; Supplementary Table S3).CH5183284/Debio 1347–sensitive cancer cell lines har-boring genetic alterations in FGFR accounted for 20 of24 (83%) of the lines examined. Among these lines, fourhave copy number variations (CNV) of FGFR1 (>2.2-fold),two have a chromosomal translocation of FGFR1(FGFR1OP-FGFR1), six have CNVs of FGFR2 (>2.2-fold),three have point mutations in FGFR2 (S252W, K310R,N549K), three have chromosomal translocation/rearran-gements of FGFR3 (FGFR3-TACC3, FGFR3-BAIAP2L1),and two have point mutations in FGFR3 (S249C,Y373C). Together, these data indicate that the FGFR-selective inhibitor CH5183284/Debio 1347 has selectiveantiproliferative activity against cancer cell lines harbor-ing genetic alterations in FGFR.

FGFR-selective antitumor activity of CH5183284/Debio 1347 in vivo

To confirm the selective antitumor activity ofCH5183284/Debio 1347 against cancers harboring FGFRgenetic alterations in vivo aswell as in vitro,weevaluated its

Table 1. Kinase inhibitory activity ofCH5183284/Debio 1347

Class Enzyme IC50, nmol/L

Tyrosine kinase FGFR1 9.3FGFR2 7.6FGFR3 22FGFR4 290PDGFRb 560FLT1 1,000KDR 2,100ABL 4,900EPHA2 5,000KIT 5,500SRC 5,900FLT3 6,400TIE2 >10,000EGFR >10,000MET >10,000INSR >10,000ALK >10,000JAK2 >10,000HER2 >10,000EPHB2 >10,000RON >10,000AXL >10,000LTK >10,000FAK >10,000

Serine/threonine kinase Raf-1 >10,000MEK1 >10,000PKA >10,000AKT1 >10,000PKACa >10,000PKACb1 >10,000PKACb2 >10,000p38a >10,000AurA >10,000CDC2/CycB1 >10,000CHK1 >10,000CHK2 >10,000TBK1 >10,000

NOTE: Detailed description of in vitro kinase inhibitoryassays in the presence of CH5183284/Debio 1347 is pre-sented in Materials and Methods.

Efficacy of CH5183284 against Tumors with FGFR Aberrations

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in vivo efficacy in xenograft mouse models. CH5183284/Debio 1347 showed significant antitumor activity againstxenografts with FGFR genetic alterations such as KG1[leukemia, FGFR1OP-FGFR1 fusion; maximum tumorgrowth inhibition (TGI), 134%], SNU-16 (gastric cancer,

FGFR2 amplification; maximum TGI, 147%), MFE-280(endometrial cancer, FGFR2 S252W mutation; maximumTGI, 100%), UM-UC-14 (bladder cancer, FGFR3 S249Cmutation; maximum TGI, 116%), and RT112/84 (bladdercancer, FGFR3-TACC3 fusion; maximum TGI, 125%). In

E562

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Figure 2. X-Ray structure of the FGFR1 and CH5183284/Debio 1347 complex. A, interaction of CH5183284/Debio 1347 with FGFR1 at the ATP-binding site(PDB ID 3WJ6). CH5183284/Debio 1347 is shown as a ball-and-stickmodel, colored by element type (C in purple, O in red, andN in blue). Hydrogen bonds areindicated by red dashes. B, superposition of CH5183284/Debio 1347-hFGFR1, NVP-BGJ398-hFGFR1 (PDB code: 3TT0), and PD173074-hFGFR1(PDB code: 2FGI; ref. 48). The distance between the centroid of aromatic ring of Phe642 and the methyl group attached to benzimidazole moiety is 0.37 nm,between Cg2 atom of Val561 and X-position carbon atom of benzimidazole moiety is 0.37 nm, and between sulfur atom of Met535 and Y-position nitrogenatom of benzimidazole moiety is 0.38 nm. The ball-and-stick model of NVP-BGJ398 (cyan) and PD173074 (green) are overlaid on CH5183284/Debio 1347(pink). C, superposition of CH5183284/Debio 1347-hFGFR1 (pink) and the binding model of CH5183284/Debio 1347 to KDR (green). Side chain ofPhe564 residue is shown as a stick model, colored in cyan.

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Figure 3. Selective antiproliferativeactivity of CH5183284/Debio 1347against cancer cell lines harboringgenetic alterations in FGFR. Cellswere treated with variousconcentrations of CH5183284 for4 days. The viable cells werecounted with WST-8. IC50 valueswere arranged from lowest tohighest and divided into tumortypes. Red, genetic alterations inFGFR1; orange, genetic alterationsin FGFR2; and green, geneticalterations in FGFR3. See alsoSupplementary Table S3.

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contrast, MKN-45 (gastric cancer, WT FGFRs,MET ampli-fication)was not sensitive toCH5183284/Debio 1347 (max-imum TGI 8% at MTD; Fig. 4A). These data are consistentwith the in vitro observations. We then investigated thesuppression of FGFR signaling in tumor tissues by con-ductingWestern blotting and immunohistochemistry aftersingle administration of the drug. CH5183284/Debio 1347suppressed phospho-FGFR for at least 7 hours in SNU-16xenograft tissue (Fig. 4B), as well as the downstreamsignaling, as indicated by a reduction in phospho-FRS,phospho-ERK, and phospho-S6 (Fig. 4C). These resultssuggest that CH5183284/Debio 1347 has selective antitu-mor activity against cancers harboringFGFR genetic altera-tions both in vitro and in vivo through suppression of theFGFR signaling pathway.

Effects of CH5183284/Debio 1347 on FGFR2 withgatekeeper mutationsGatekeeper mutations in kinases, such as T790M in

EGFR, T315I in ABL, and L1196M inALK, are responsiblefor acquired resistance to small-molecule inhibitors (29–31). We therefore investigated the inhibitory activity ofCH5183284/Debio 1347 against several gatekeepermutants of FGFR2. First, we compared the kinase activityof FGFR2 WT with three gatekeeper mutants of FGFR2(V564F, V564I, and V564L), which could be generatedwith a single base substitution at the codon correspondingto the gatekeeper residue. Also, these residues are fre-quently used in other kinases. When these mutants wereexpressed in HEK293 cells, they exhibited higher FGFR2kinase activities than FGFR2WT (Supplementary Fig. S3).We then examined the capability of the compound toinhibit the three FGFR2 gatekeeper mutants and foundthat inhibition was sustained against the FGFR2 V564Fmutant, but not against FGFR2 V564I and V564L. Incontrast, AZD4547 was ineffective against all three gate-keeper mutants (Supplementary Fig. S4). To expand theinvestigation to other FGFR inhibitors, we establishedTEL-FGFR2 WT- and TEL-FGFR2 V564F–driven Ba/F3cell lines. Although the efficacy of AZD4547, NVP-BGJ398, PD173074, or cediranib against the FGFR2V564F mutant–harboring Ba/F3 cells was significantlydiminished (IC50 for FGFR2 V564F vs. IC50 for FGFR2WT: �216- to 8,460-fold), the efficacy of CH5183284/Debio 1347 was maintained (IC50 for FGFR2 V564F vs.IC50 for FGFR2 WT: 7.3-fold; Fig. 5A; Supplementary Fig.S5). Suppression of phosphorylation of TEL-FGFR2 andTEL-FGFR2 V564F in the cells by CH5183284/Debio 1347was also confirmed by Western blotting (SupplementaryFig. S5B). In addition, CH5183284/Debio 1347 was effec-tive against the TEL-FGFR2 V564F–driven Ba/F3 in vivo;in contrast, AZD4547 failed to inhibit tumor growth (Fig.5B). Consistent with this TGI profile, suppression of TEL-FGFR2 V564F phosphorylation by CH5183284/Debio1347, but not by AZD4547, was observed in the xenografttissues (Fig. 5C). In contrast, the in vivo efficacy of bothdrugs in the TEL-FGFR2 WT–driven Ba/F3 xenograftmodel and their ability to suppress FGFR2 activation

in the tumor tissues were comparable (SupplementaryFig. S6).

To clarify the mechanism by which CH5183284/Debio1347 was able to inhibit the FGFR2 V564F mutant, wegenerated an in silico model of the FGFR2-CH5183284/Debio 1347 complex. Models of CH5183284/Debio 1347,AZD4547, and NVP-BGJ398 binding to FGFR2 V564Fwere constructed from the crystal structural informationof FGFR1 with CH5183284/Debio 1347 (PDB code 3WJ6),FGFR1with the AZD4547 analog (PDB code 4F65; ref. 32),and FGFR1 with NVP-BGJ398 (PDB code 3TT0; ref. 20),respectively. This modeling suggested that there is closevan der Waals contact between Phe564 and the 7 positionof the benzimidazole moiety of CH5183284/Debio 1347.In contrast, the 3,5-dimethoxy-phenyl moiety present inboth AZD4547 and NVP-BGJ398 would result in stericclashes with Phe564. These differences may explain whyCH5183284/Debio 1347, but not AZD4547 or NVP-BGJ398, can inhibit V564F (Fig. 5D).

We also examined the inhibitory activity in an enzy-matic assay against FGFR2 N549H, which has beenreported as a dovitinib-resistant mutation (33). All FGFRinhibitors that we tested were comparably less effectiveagainst FGFR2N549Hsimilar to dovitinib; however, thesestill kept some inhibitory activity (Supplementary Fig. S7).

DiscussionThe FGFR family is frequently activated by diverse

genetic alterations in cancer, and therefore, FGFR inhi-bitors are likely to be effective in patients stratified onthe basis of the presence of FGFR genetic alterations.Several multitargeted kinase inhibitors that inhibitFGFRs, VEGFRs, and PDGFRs have shown some clinicalbenefits in patients with FGFR aberrations (16). How-ever, dosage of these inhibitors is limited by adverseeffects, rendering the "safe" dose range suboptimal forFGFR inhibition and suppression of tumor growth. Thiscreates a need for FGFR-selective inhibitors that withthe potential to inhibit FGFR more significantly intumors.

To overcome this issue, several FGFR selective inhibitorshave been developed. But these inhibitors share a common3,5-dimethoxy-phenyl moiety. Thus, it is possible that acommon single mutation would confer resistance to theseinhibitors. For instance, erlotinib and gefitinib have thesame moiety and it causes the resistance to EGFR T790M(34).Apotential strategy tomitigate this issue is the rationaldesign of a novel inhibitor with a different chemical scaf-fold. Then, we generated CH5183284/Debio 1347, anFGFR1-, FGFR2-, and FGFR3-selective inhibitor. The keyresidues in the CH5183284/Debio 1347 binding site areVal561 at the gatekeeper residue and Phe642 andMet535 in a back pocket. Only 15 of 490 human proteinkinases use valine as a gatekeeper, which partly explainsthe relative selectivity of CH5183284/Debio 1347. Interest-ingly, although KDR uses valine as a gatekeeper, it is notinhibited by CH5183284/Debio 1347. We speculate that amethionine in the back pocket, such as Met535 in FGFR1,

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also contributes to the selectivity to KDR. KDR has aleucine at the same position as Met535 of FGFR1. Byinteracting with Val561, Met535, and Phe642, whichinteracts with methyl group of benzimidazole moiety,CH5183284/Debio 1347 could achieve selectivity forbinding to FGFR1, FGFR2, and FGFR3. This bindingmode is different from other FGFR selective inhibitors,so CH5183284/Debio 1347 would overcome the resis-tance of other FGFR selective inhibitors. Furthermore,the selectivity among the FGFR family may be explainedby the structure at the middle of the hinge region, likelybecause only FGFR4 contains a cysteine residue in thisregion. In fact, our in silico modeling suggests thatCH5183284/Debio 1347 cannot participate in a produc-

tive interaction with this cysteine, leading to weakerkinase-inhibitory activity in this case.

For our investigation into genetic alterations that mightprove useful as predictive biomarkers, we used the pub-licly available databases of genetic information (35), aswell as our own encyclopedia of cell lines and a largeantiproliferative assay system. This confirmed that FGFRgenetic alterations act as predictive biomarkers ofCH5183284/Debio 1347 efficacy. The mechanism under-lying the sensitivity of 4 of 24 cell lines has not beenclarified yet, but we consider that it might involve novelgenetic alterations in FGFR, such as an intragenic dupli-cation of the FGFR kinase domain (36) or constitutiveFGFR activation via epigenetic mechanisms.

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Figure 4. Selective antitumoractivity of CH5183284/Debio 1347in mouse models of cell linesharboring genetic alterations inFGFR. A, antitumor activity ofCH5183284/Debio 1347. Micebearing KG1, SNU-16, MFE280,UM-UC-14, RT112/84, orMKN-45cells were treated withCH5183284/Debio 1347 orallyonce daily for 11 days at theindicated doses. Tumor volumeand body weight change for eachdose group were measured. Dataare shown asmean�SD (n¼4–5).B, time course of phospho-FGFRinhibition in xenograft tissue. Micebearing SNU-16 cells were orallytreated with a single dose of 30mg/kg of CH5183284/Debio 1347,and xenograft tumors wereextracted at 0, 2, 4, 7, and 24 hoursafter dosing and analyzed byWestern blotting. C, vehicle or 100mg/kg of CH5183284/Debio 1347were administered, and SNU-16xenograft tissues were resected 4hours after administration andembedded in paraffin. Treatedsamples were immunostained.

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In contrast, few CH5183284/Debio 1347–insensitivecancer cell lines have genetic alterations in FGFR (4.6%:14 of 303), and six of 14 cell lines have point mutations,such as FGFR1 (S125L), FGFR2 (V12M, R612T, E636K), orFGFR3 (F384L, K650E). However, except for FGFR3K650E, these mutations are not associated with activationof FGFRs; rather, the FGFR3 F384L mutant was demon-strated to be devoid of transforming activity in NIH-3T3cells (37) and, although the J82 cell line has an FGFR3K650E mutation, FGFR3 is not detectable at the mRNAand protein levels (38). In breast cancer, approximately10% of patients have amplification of FGFR1 (3), anddovitinib showed some clinical benefits in these patients(16). We have tested six breast cancer cell lines with

amplification of FGFR1, but these cell lines were insensi-tive to CH5183284/Debio 1347. One possible explanationfor this resistance mechanism is a genetic alteration inanother signaling pathway. In fact, the MDA-MB-134IVcell line has a K-RAS G12R mutation, JIMT-1 has HER2amplification and a PIK3CA C420R mutation, andCAMA-1 and ZR-75-1 have PTEN D92H and L108Rmutations, respectively. Because these genetic altera-tions could activate downstream pathways and bypassthe FGFR signal blockade, these cell lines would exhibitresistance to an FGFR inhibitor (4, 39, 40). Recently,everolimus combined with an aromatase inhibitorimproved progression-free survival in patients withhormone receptor–positive advanced breast cancer who

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Figure 5. Efficacy of CH5183284/Debio 1347 against gatekeepermutants of FGFR2. A,antiproliferative activity ofCH5183284/Debio 1347,AZD4547, and NVP-BGJ398against TEL-FGFR2 WT–drivenBa/F3 cells and TEL-FGFR2V564F–driven Ba/F3 cells. Threeclones were treated with variousconcentrations of the indicatedinhibitors for 4 days. The viablecells were counted with WST-8. B,antitumor activity of CH5183284/Debio 1347 and AZD4547. Micebearing Ba/F3 TEL-FGFR2 V564Fcells were treated withCH5183284/Debio 1347 orAZD4547 orally once daily for11 days at the indicated doses.Tumor volume and body weightchange for each dose group weremeasured. Data are shown asmean�SD (n¼ 5). C, suppressionof phospho-FGFR in xenografttissue. Mice bearing Ba/F3 TEL-FGFR2 V564F cells were treatedfor 11 days at 50 and 100mg/kg ofCH5183284/Debio 1347 or 25 and50 mg/kg of AZD4547, andxenograft tumors were extracted 4hours after dosingandanalyzedbyWestern blotting. (n ¼ 3). D,binding models of CH5183284/Debio 1347, AZD4547, and NVP-BGJ398 to FGFR2 (V564F)homology models. The moleculesare shown as a ball-and-stickmodel, colored by element type (Cin green, O in red, N in blue, and Clin dark green). Side chain ofPhe564 residue is shown as a stickmodel, colored in cyan.

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relapsed from nonsteroidal aromatase inhibitors (41).FGFR1 amplification is enriched in this population, andsynergism in the combination of an anti-estrogen antag-onist with an FGFR inhibitor has been suggested (4). Wetherefore suggest that a clinical study of an FGFRinhibitor combined with an aromatase inhibitor in hor-mone receptor and FGFR1 gene amplification double-positive patients is warranted.

One of the most frequent causes of resistance to proteinkinase inhibitors is a gatekeeper mutation (29–31). Toovercome this, several small-molecule kinase inhibitorsthat can inhibit gatekeepermutant kinases are now underclinical development, such as neratinib and afatinib forEGFR T790M and alectinib/CH5424802 for ALK L1196M(21, 42). Although a gatekeeper mutation of FGFR has notbeen identified in patients yet, the gatekeeper mutantV555M of FGFR3 or V564I of FGFR2 were identified asmutations resistant to AZD4547 or dovitinib in preclinicalexperiments (33, 43). Therefore, we evaluated the activityofCH5183284/Debio 1347 against the gatekeepermutantsof FGFR2. With a single base substitution at the codoncorresponding to the gatekeeper residue, phenylalanine,isoleucine leucine, alanine, glycine, and aspartic acidcould be generated. However, only 2 kinases use alanineor glycine, andnokinaseuses aspartic acid as a gatekeeperresidue in 490 kinases. In addition, it is known that thesubstitution of alanine or glycine for a gatekeeper residueresults in diminished kinase activity andATP affinity (44–46). Therefore, we only selected Phe, Ile, and Leu for ourinvestigation. We demonstrated that gatekeeper mutantsof FGFR2 have higher kinase activities than WT FGFR2(Supplementary Fig. S2). Taken together, these data sug-gest that resistance conferred by a gatekeeper mutant ofFGFRmayappear in patients in the near future. Therefore,an inhibitor capable of blocking FGFRs with gatekeepermutations will be required. Existing FGFR selective inhi-bitors, such as AZD4547, NVP-BGJ398, and PD173074,have the common 3,5-dimethoxy-phenyl moieties thatinteract with a back pocket of the ATP-binding site.According to our study, these bulky substituents stericallyclash with Phe564 in the FGFR2 gatekeeper mutant, pre-venting them from blocking the kinase activity of thismutant. In contrast, CH5183284/Debio 1347doesnot havethe methoxy moieties and would thus be able to produc-tively interactwithPhe564.Consistentwith this,we foundthat CH5183284/Debio 1347 had sustained inhibitoryactivity against the FGFR2 V564F gatekeeper mutant.

Because the binding site of FGFR inhibitors and theposition of FGFR2 N549 are not close, we cannot explainwith three-dimensional structure analysis why thismutation would affect the inhibitory activity in theenzymatic assay. Also, FGFR inhibitors, including

CH5183284/Debio 1347, are effective on cancer cellsharboring FGFR2 N549K mutation, such as AN3 CA orMFE-296 (Fig. 3; Supplementary Table S3). Therefore, itis still unclear whether this mutation would be a resis-tant mechanism, and this should be proved in the futureclinical studies.

In summary, we suggest that CH5183284/Debio 1347will provide therapeutic opportunities for patients whohave FGFR genetic alterations and patients with acquiredresistance to existing FGFR selective inhibitors that con-tain the commonmethoxymoieties. Recently, many kindsof FGFR genetic alterations have been identified usingnext-generation sequencing technologies (47). Thus, strat-ifying patients based on FGFR status using next-genera-tion sequencing may improve the power of clinical trials,allowing a clinical response to be detected even in an earlycohort. CH5183284/Debio 1347 is currently under phase Iclinical investigation by Debiopharm International S.A.in selected patients harboring FGFR genetic alterations(NCT01948297).

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

Authors' ContributionsConception and design: Y. Nakanishi, N. Taka, Y. Aoki, N. IshiiDevelopment of methodology: Y. Nakanishi, N. Akiyama, T. Isobe,Y. AokiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Nakanishi, N. Akiyama, T. Tsukaguchi,H. Sase, T. Isobe, H. ShindohAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): Y. Nakanishi, N. Akiyama, T. Fujii,K. Sakata, T. Isobe, K. MorikamiWriting, review, and/or revision of the manuscript: Y. Nakanishi,T. Isobe, K. Morikami, T. Mio, N. Taka, N. IshiiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y. Nakanishi, Y. AokiStudy supervision: Y. Nakanishi, T. Mio, Y. Aoki, N. IshiiOther (design and synthesis of the inhibitor): H. Ebiike

AcknowledgmentsThe authors thankDrs. TakaakiMiura,Mitsuyasu Tabo, and Toshikazu

Yamazaki for helpful discussion; Yasuko Satoh, Yukako Tachibana-Kondo, Yasue Nagata, and Tsutomu Takahashi for pharmacologicalassays; Santina Russo and Joachim Diez at Expose GmbH for proteincrystallography; and Takaaki Fukami for submission of 3D structure datato PDB. They also thank Anne Vaslin Chessex, H�el�ene Maby-El Hajjami,and Corinne Moulon of Debiopharm International S.A. for their helpfuldiscussion and technical support.

Grant SupportThis study was funded by the Chugai Pharmaceutical Co. Ltd.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked asadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 10, 2014; revised June 25, 2014; accepted August 4, 2014;published OnlineFirst August 28, 2014.

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2014;13:2547-2558. Published OnlineFirst August 28, 2014.Mol Cancer Ther   Yoshito Nakanishi, Nukinori Akiyama, Toshiyuki Tsukaguchi, et al.   Novel Selective FGFR InhibitorPotential Predictor of the Sensitivity to CH5183284/Debio 1347, a The Fibroblast Growth Factor Receptor Genetic Status as a

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