ASP5878, a Novel Inhibitor of FGFR1, 2, 3, and 4, Inhibits ... · is under development. It inhibits...

Small Molecule Therapeutics

ASP5878, a Novel Inhibitor of FGFR1, 2, 3, and 4,Inhibits the Growth of FGF19-ExpressingHepatocellular CarcinomaTakashi Futami, Hidetsugu Okada, Rumi Kihara, Tatsuya Kawase, Ayako Nakayama,Tomoyuki Suzuki, Minoru Kameda, Nobuaki Shindoh, Tadashi Terasaka, Masaaki Hirano,and Sadao Kuromitsu

Abstract

Hepatocellular carcinoma is an aggressive cancer with poorprognosis. Fibroblast growth factor 19, a member of the fibro-blast growth factor family, is a ligand for fibroblast growthfactor receptor 4. Moreover, it plays a crucial role in theprogression of hepatocellular carcinoma. ASP5878 is a novelinhibitor of fibroblast growth factor receptors 1, 2, 3, and 4 thatis under development. It inhibits fibroblast growth factorreceptor 4 kinase activity with an IC50 of 3.5 nmol/L. ASP5878potently suppressed the growth of the fibroblast growth factor19–expressing hepatocellular carcinoma cell lines Hep3B2.1-7,HuH-7, and JHH-7. In the Hep3B2.1-7 cell line, ASP5878

inhibited the phosphorylation of fibroblast growth factorreceptor 4 and its downstream signaling molecules as well asinduced apoptosis. Oral administration of ASP5878 at 3 mg/kginduced sustained tumor regression in a subcutaneous xeno-graft mouse model using Hep3B2.1-7. In HuH-7, an orthotopicxenograft mouse model, ASP5878 induced complete tumorregression and dramatically extended the survival of the mice.These results suggest that ASP5878 is a potentially effectivetherapeutic agent for hepatocellular carcinoma patients withtumors expressing fibroblast growth factor 19. Mol Cancer Ther;16(1); 68–75. �2016 AACR.

IntroductionHepatocellular carcinoma (HCC) is an aggressive cancer with

poor prognosis and the third most common cause of cancer-related deaths worldwide (1). Surgical resection is the mostsuccessful treatment for early-stage HCC; however, 70% ofpatients have recurrence after 5 years (2, 3). Sorafenib, a broad-spectrum kinase inhibitor, has been approved as a molecular-targeteddrug for surgically unresectableHCCandhas been shownto improve the duration of survival to 10.7months in comparisonwith 7.9 months in patients receiving placebo in a phase 3 trial(4). However, more effective therapeutic approaches are urgentlyrequired for unresectable HCC.

The signaling pathway activated by FGFRs and their cognateligands, i.e., fibroblast growth factors (FGF), plays an importantrole in the course of development fromearly embryogenesis to theformation of various organs. There are 18 genes encoding FGFsand 4 genes encoding FGFR, which are expressed in various cells.Upon ligand binding to FGFR, the receptor is dimerized for theautophosphorylation and recruitment of adaptor proteins such asFGFR substrate 2 (FRS2), which activates several intracellularsignaling pathways involved in cell growth, differentiation, and

survival (5). FGF19 is a physiologic ligand for FGFR4, which isexpressed in and secreted from the small intestine and gallbladderand controls the catabolism of cholesterol in the liver throughFGFR4 and coreceptor, bKlotho (5). Furthermore, in recent years,it has been found that FGF19 is involved in several types of cancerssuch as HCC and colon cancer (6, 7). In addition, FGF19 geneamplification has been reported to be involved in 14% of HCCpatients, and FGF19 is overexpressed in approximately 50% ofHCC patients (6, 8, 9). FGF19 expression correlates with poorerprognosis, recurrence, and tumor progression in HCC patients(9, 10). FGF19-expressing transgenicmice spontaneously developHCC (11), and the knockout of FGFR4 shows that it is required fortumorigenesis in this FGF19-mediated HCCmodel (12). Further-more, the growth ofHCC cell lines with FGF19 gene amplificationis inhibited by the addition of FGF19-neutralizing antibodies, anRNAi that targets FGFR4, or FGFR4 inhibitors (6, 10, 13, 14).

Here, we describe the preclinical profile of ASP5878, which is anovel FGFR-selective inhibitor, targeting FGF19-expressing HCC.In addition, we demonstrated that ASP5878 suppressed thegrowth of FGF19-expressing HCC cell lines, accompanied by theinhibition of FGFR4 phosphorylation and its downstream sig-naling molecules, which led to the induction of apoptosis. Fur-thermore, we showed that the oral administration of ASP5878induced sustained tumor regression in FGF19-expressing HCCmouse models. Our data indicate that ASP5878 is a potentiallyeffective therapeutic agent for HCC patients with tumors expres-sing FGF19.

Materials and MethodsCompounds

ASP5878 (Astellas Pharma Inc.; ref. 15) and sorafenib weresynthesized in-house. ASP5878 was dissolved in dimethyl

Drug Discovery Research, Astellas Pharma Inc., Ibaraki, Japan.

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

Corresponding Author: Takashi Futami, Astellas Pharma Inc., 21 MiyukigaokaTsukuba-shi, Ibaraki 305-8585, Japan. Phone: 16504921488; Fax: 16509260740;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0188

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 16(1) January 201768

on August 14, 2020. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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sulfoxide for in vitro experiments or suspended in 0.5% methylcellulose (MC) for in vivo experiments. Sorafenib was sus-pended in 12.5% Cremophor EL/12.5% ethanol for in vivoexperiments.

Kinase assayInhibitory activities of 128 serine/threonine kinases were mea-

sured using the Mobility Shift Assay Kit (Carna Biosciences). IC50

values were determined for kinases that were inhibited by morethan 50% by 200 nmol/L of ASP5878.

Cell growth assayThe humanHCC cell lines HuH-7 (JCRB0403; ref. 16), HUH-6

Clone 5 (JCRB0401; ref. 17), HLF (JCRB0405; ref. 18), huH-1(JCRB0199; ref. 19), JHH-1 (JCRB1062; ref. 20), JHH-2(JCRB1028; ref. 20), JHH-5 (JCRB1029; ref. 20), JHH-6(JCRB1030; ref. 20), and JHH-7 (JCRB1031; ref. 20) wereobtained from the Health Science Research Resources Bank.Hep3B2.1-7 (HB-8064), SNU-423 (CRL-2238; ref. 21), SNU-449 (CRL-2234; ref. 21), SNU-182 (CRL-2235; ref. 21), SNU-387 (CRL-2237; ref. 21), SNU-398 (CRL-2233; ref. 21), HepG2(HB-8065), and MDA-MB-453 (HTB-131) were obtained fromthe American Type Culture Collection. Li-7 (RCB1941) wasobtained from RIKEN Bioresource Center. SNU-739 andSNU-368 were obtained from the Korean Cell Line Bank.PLC/PRF/5 was obtained from DS Pharma Biomedical. HCCcell lines were cultured according to each manufacturer's guide-line. The cell lines in this study were not authenticated by anytests in our laboratory but were purchased from the providers ofauthenticated cell lines and stored at early passages in a centralcell bank at Astellas Pharma Inc. The experiments were con-ducted using low-passage cultures of these stocks. The cells wereseeded in 96-well plates and incubated overnight. The cellswere treated with ASP5878 for 5 days. Cell viability wasmeasured using CellTiter-Glo (Promega).

Western blotting, ELISA, and pharmacodynamicsHCC cell lines were lysed with cell lysis buffer containing

phosphatase and protease inhibitors, and the protein levels ofFGF19, FGFR4, bKlotho, and actin (Clone AC-74) were deter-mined by immunoblotting. Hep3B2.1-7 cells were treated withASP5878 for 2 hours, and the protein levels of phospho-FRS2(Tyr436), FRS2, phospho-extracellular signal-regulated kinase(phospo-ERK), ERK, glyceraldehyde-3-phosphate dehydroge-nase (GAPDH), and actin (Clone AC-74) were detected byimmunoblotting. Phosphorylated FGFR4 and total FGFR4 weremeasured by immunoprecipitation using FGFR4 antibody(A10) or mouse IgG1 isotype control, followed by immuno-blotting using an antibody specific for phospho-FGFR (Tyr653/654) and FGFR4 (C16) or sandwich ELISA assay. PARP cleavageand actin (Clone AC-15) were detected by immunoblottingafter 48-hour treatment with ASP5878. FRS2, phospho-ERK,ERK, and GAPDH levels in Hep3B2.1-7 tumors were measuredby immunoblotting at 6 hours after single oral administrationof ASP5878. The following antibodies were purchased: FGFR4(A10) (sc-136988), FGFR4 (C16) (sc-124), FRS2 (sc-8318),and GAPDH (sc-20357) from Santa Cruz Biotechnology;bKlotho (AF5889) and mouse IgG1 isotype control (MAB002)from R&D systems; phospho-FGFR (Tyr653/654; #3471), PARP(#9542), ERK (#9102), phospho-ERK (Thr202/Tyr204; #9101),and phospho-FRS2 (Tyr436; #3861) from Cell Signaling Tech-

nology; and actin (Clone AC-15, A5441), actin (Clone AC-74,A5316), and FGF19 (HPA036082) from Sigma-Aldrich. Thefollowing materials were purchased: cell lysis buffer from CellSignaling Technology, phosphatase inhibitor cocktail fromThermo Scientific, protease inhibitor cocktail from Roche Diag-nostics, and human total FGFR4 ELISA and human phospho-FGFR4 ELISA kits from R&D systems.

Xenograft modelsAll animal experimental procedures were approved by the

Institutional Animal Care and Use Committee of AstellasPharma Inc. In addition, Astellas Pharma Inc., TsukubaResearch Center, has accreditation status awarded by The Asso-ciation for Assessment and Accreditation of Laboratory AnimalCare International. Four-week-old male nude mice (CAnN.Cg-Foxn1nu/CrlCrlj [nu/nu]) were obtained from Charles RiverJapan. Hep3B2.1-7 cells were subcutaneously inoculated intothe flank of mice at 3 � 106 cells/0.1 mL (Matrigel:PBS ¼ 1:1)/mouse and allowed to grow. ASP5878 was administered as aonce-daily oral dose initiated after confirming tumor growth ineach experiment. Tumor volume was determined using theformula length � width2 � 0.5. Matrigel was purchased fromCorning Incorporated (Life Sciences).

Xenograft models (sorafenib switch)Hep3B2.1-7 cells were subcutaneously inoculated into the

flank of mice at 3 � 106 cells/0.1 mL (Matrigel:PBS ¼ 1:1)/mouse and allowed to grow. After confirming tumor growth,mice were divided into two groups (n ¼ 10 and 20 for vehicleand sorafenib groups, respectively) on day 0 on the basisof tumor volume and body weight. Vehicle (CremophorEL/ethanol) or sorafenib (10 mg/kg) was administered as aonce-daily oral dose from days 0 to 13. On day 14, the vehiclegroup was further divided into two groups (five per group), andthe sorafenib group was further divided into four groups (fiveper group) on the basis of tumor volume and body weight.Vehicle (Cremophor EL/ethanol or 0.5% MC), sorafenib (10mg/kg), or ASP5878 (3 mg/kg) was administered as a once-daily dose from days 14 to 52. The other two groups were usedfor a different research purpose. Tumor volume was determinedusing the formula length � width2 � 0.5.

HCC orthotopic xenograft modelHuH-7–expressing luciferase (HuH-7-Luc) cells were

obtained by infecting HuH-7 cells with lentivirus-expressingluciferase in the presence of blasticidin S (10 mg/mL). HuH-7-Luc cells were inoculated into hepatic parenchyma at 3 � 105

cells/0.01 mL (Matrigel 100%)/mouse. One week after inocu-lation, the mice were divided into three groups (n ¼ 5 pergroup) on day 0 on the basis of bioluminescent imaging.Vehicle (Cremophor EL/ethanol or 0.5% MC), sorafenib (30mg/kg), or ASP5878 (3 mg/kg) was administered as a once-daily oral dose for 91 days. Tumor growth was monitored byin vivo bioluminescent imaging of the abdomen after intraper-itoneally injecting luciferin using IVIS-Lumina2 (PerkinElmerInc.). During the study period (181 days), the survival of micebearing hepatic tumors was recorded. The condition of the micewas monitored daily. The mice were scored as dead if any of thefollowing signs of suffering were observed: cachexia, weaken-ing, and difficulty in moving or eating. Mice that were scored asdead were euthanized.

ASP5878, a Novel FGFR1, 2, 3, and 4 Inhibitor for HCC

www.aacrjournals.org Mol Cancer Ther; 16(1) January 2017 69




ResultsKinase inhibition profile of ASP5878

ASP5878 (Fig. 1A) potently inhibited the tyrosine kinaseactivities of recombinant FGFR1, 2, 3, and 4 with IC50 values of0.47, 0.60, 0.74, and 3.5 nmol/L, respectively (Table 1). Kinaseselectivity was investigated against a panel of 128 human kinases,and the kinases that showedmore than50% inhibitionbyASP5878(200nmol/L)were FGFRs,VEGFR2, andFMS(Table1). In total, 117kinases showed less than50% inhibition (SupplementaryTable S1).

Antiproliferative effect of ASP5878 and FGF19 expression in 20HCC cell lines

A previous report suggested that an FGF19 antibody has thepotential to be effective in FGF19-amplifiedHCC cell lines (6). To

characterize the antiproliferative sensitivity of HCC cell lines toASP5878, we examined the antiproliferative effect of ASP5878 in20 human HCC cell lines (Fig. 1B). FGF19 protein expression inthese cell lines was detected byWestern blotting (Fig. 1C).HuH-7,Hep3B2.1-7, and JHH-7 cell lines exhibited potent sensitivity toASP5878, with IC50 values of 27, 8.5, and 21 nmol/L, respectively(Table 2). FGF19expressionwasobserved inHuH-7,Hep3B2.1-7,and JHH-7 but not in other cell lines (Fig. 1C). Furthermore, theexpression of FGFR4 and bKlotho was observed in HuH-7,Hep3B2.1-7, and JHH-7 (Fig. 1C). HUH-6 Clone 5, SNU-398,Li-7, andHLFwere also sensitive toASP5878 (Fig. 1B). The growthinhibition rate of HLF was 64% and those of other ASP5878-sensitive cell lines were higher than 95% at 1,000 nmol/L, highestconcentration examined (data not shown). The mechanisms of

Figure 1.

Antiproliferative effect of ASP5878 in FGF19-expressing HCC cell lines. A, Chemical structure of ASP5878. B, In vitro proliferation assay. C, Expression ofFGF19, FGFR4, bKlotho, and actin across a panel of 20 human HCC cell lines. D, Inhibition of activated FGFR4 phosphorylation after ASP5878 treatment for 2 hoursin Hep3B2.1-7 cells. E, Downstream signals after ASP5878 treatment for 2 hours in Hep3B2.1-7 cells. F, PARP cleavage induced by ASP5878. Hep3B2.1-7cells were incubated with ASP5878 at the indicated concentration for 48 hours. Each protein was detected by immunoblotting using specific antibodies.

Futami et al.

Mol Cancer Ther; 16(1) January 2017 Molecular Cancer Therapeutics70




inhibition are under investigation. Thus, ASP5878 inhibited thecell proliferation of HCC cell lines expressing FGF19.

Effect of ASP5878 on FGFR signalingTo assess the modulation of FGFR4 phosphorylation and the

downstream signaling pathway by ASP5878, Hep3B2.1-7 cellswere treated with ASP5878 and cell lysates were analyzed byWestern blotting. FGFR4 phosphorylation was constitutivelyobserved and inhibited by ASP5878 in a concentration-depen-dent manner (Fig. 1D). In addition, ASP5878 treatment resultedin the suppression of phosphorylation, mobility shift of FRS2,and suppression of ERK phosphorylation (Fig. 1E). Furthermore,concentration-dependent increases in PARP cleavage, an apopto-sis marker, were detected at 48 hours after ASP5878 treatment(Fig. 1F). The antiproliferative effects of Hep3B2.1-7 (IC50 ¼8.5 nmol/L) and ASP5878-induced PARP cleavage at 10 nmol/Lare comparable and have no gaps (Table 2). A similar effect wasconfirmed in HuH-7 and JHH-7 cell lines (data not shown).The suppression of phosphorylation and mobility shift of FRS2were shown by another FGFR inhibitor in several cell lines (22).These experiments suggest that the FRS2 mobility shift reflectsthe phosphorylation status of FRS2 through FGFR signaling.

ASP5878 induces shrinkage of FGF19-expressing HCCxenograft model

We next evaluated the antitumor activity of ASP5878 in aHep3B2.1-7 subcutaneous xenograft mouse model. In this mod-el, a once-daily oral administration of ASP5878 induced tumorregressionby 9%and88%at 1 and3mg/kg, respectively (Fig. 2A),without affecting the body weight for 14 days (Fig. 2B). To assessthe pharmacodynamic effects in vivo, we evaluated the inhibitoryeffect of ASP5878 on the constitutively activated FGFR down-stream signals in Hep3B2.1-7 tumors. The mobility shift of FRS2and suppression of ERK phosphorylation in Hep3B2.1-7 tumorswere observed following the administration of 1 and 3 mg/kgASP5878 (Fig. 2C). Because it was difficult to detect FGFR4phosphorylation in Hep3B2.1-7 tumors under xenograft condi-tions, we examined the effect of ASP5878 on FGFR4 phosphor-ylation in anMDA-MB-453 breast cancer xenograft model. MDA-

MB-453 harbors FGFR4-Y367C mutation, which elicits constitu-tive phosphorylation (23). Six hours after the administration of asingle dose, ASP5878 induced the suppression of FGFR4 phos-phorylation, mobility shift of FRS2, and suppression of ERKphosphorylation (Supplementary Fig. S1). This effect was similarto that detected in Hep3B2.1-7 tumors. These data indicated thatASP5878 suppresses FGFR4 downstream signaling through theinhibition of FGFR4 phosphorylation in Hep3B2.1-7 tumors.

Survival prolongation of ASP5878 in FGF19-expressing HCCorthotopic xenograft model

To further evaluate the effect of ASP5878 in another HCCmodel, we established an imageable FGF19-expressing HCCorthotopic xenograft model. HuH-7-Luc cells were directly inoc-ulated into the livers of mice, and the implanted cells weremonitored by bioluminescent imaging of the upper abdominalarea (Fig. 3A). The bioluminescent emissions of vehicle-treatedcontrol mice increased during the subsequent 24 days. Moreover,we evaluated the effect of sorafenib in this model. Althoughsorafenib treatment had a slight effect that reduced the emission,the emission was reduced to approximately one-fourth of thebaseline treatment levels by 3 mg/kg ASP5878 administered as aonce-daily oral dose for 24 days. The mean bioluminescentemission in the ASP5878-treated group was lower than that inthe vehicle-treated group. These results indicate that the ASP5878-treated mice exhibited a lower tumor burden than vehicle- andsorafenib-treated mice. The emission of ASP5878-treated micereached background levels on day 31. Continuous dosing ofASP5878 sustained the emissions at the background level, andno increase was observed by the end of the dosing at day 90 (Fig.3A and B). These data indicate that ASP5878 dosing inducedsustained tumor regression without tumor regrowth. To confirmcomplete tumor regression, tumor recurrencewasmonitored afterthe cessation of ASP5878 treatment from day 91. No increase inbioluminescent emissions was observed 90 days after the cessa-tion of administration (day 181) when the measurement wasstopped (Fig. 3B). These data indicate that ASP5878 inducedcomplete tumor regression at 90 days. Disease-related mortalitywas also measured (Fig. 3C). All the vehicle-treated control micedied by day 52, and all the sorafenib-treated mice died by day 90.In contrast, no mice treated with 3 mg/kg ASP5878 died duringthe 90-day experimental period. The effects on survival wereaccompanied by changes in the bioluminescent emissions, withvehicle-treated mice showing increased emission, whereas micetreated with 3 mg/kg ASP5878 showing reduced emissions.

Antitumor activity of ASP5878 in the Hep3B2.1-7 xenograftmodel after sorafenib treatment

We investigated the effect of ASP5878 on the Hep3B2.1-7 cellline after sorafenib treatment. According to previous reports, adose of 10 mg/kg/day sorafenib was orally administered(24, 25). In our experiments, sorafenib administration for 14days caused 40% tumor growth inhibition in the Hep3B2.1-7xenograft model. Even after continuous sorafenib treatment,the Hep3B2.1-7 tumor gradually enlarged, and 47% tumorgrowth inhibition was observed by day 31. In contrast, theswitch from sorafenib to ASP5878 on day 14 induced 83%tumor regression on day 52 relative to the tumor size observedon day 14 (Fig. 4). This indicates the therapeutic potential ofASP5878 for FGF19-overexpressing HCC patients who previ-ously received sorafenib treatment.

Table 1. Kinase inhibition profile of ASP5878

Kinase IC50 (nmol/L) 95% confidence interval

FGFR4 3.5 2.1–5.9FGFR1 0.47 0.28–0.78FGFR2 0.60 0.36–1.0FGFR3 0.74 0.3–1.8FGFR3 (K650E) 1.6 1.3–1.9FGFR3 (K650M) 4.2 3.1–5.6VEGFR2 25 19—32FGFR4 (N535K) 78 26—230FMS 150 100—220

NOTE: Other TKs, <50% inhibition at 200 nmol/L.

Table 2. Antiproliferative effect of ASP5878 in FGF19-expressing HCC cell lines

Cellline

FGF19status

IC50

(nmol/L)95% confidenceinterval

Hep3B2.1-7 HCC (overexpression) 8.5 6.7–11HuH-7 HCC (overexpression) 27 20–37JHH-7 HCC (overexpression) 21 18–24JHH-5 HCC (no expression) 410 270–620PLC/PRF/5 HCC (no expression) 730 660–800






DiscussionRecent clinical success in molecular-targeted therapies for can-

cer based on the identification of oncogenic gene alterations andtheir specific inhibitors is associated with dramatic antitumoreffect, reduced side effects, and improved patient survival. Themolecular-targeted therapies includeHerceptin forHER2/neu recep-tor amplification and overexpression in breast cancer patients(26), XALKORI for echinoderm microtubule-associated protein-like4–anaplastic lymphoma kinase fusion oncogene-positive non–small-cell lung cancer (27), and Tarceva for EGFR-mutated non–smallcell lungcancer (28).Although therehavebeennodriveroncogene-targeted therapies for HCC, recent data suggest that amplified oroverexpressed FGF19 is a "driver oncogene" in HCC and thatblockage of the FGF19–FGFR4 axis could be a novel therapeuticapproach for FGF19-overexpressing HCC (6, 10, 12, 29).

There have been reports on the importance of FGF signaling inthe development of various types of cancer, and this explains theinterest in the development of FGFR- or FGF-selective inhibitors(30). The most clinically advanced agents targeting HCC aremainly broad-spectrum kinase inhibitors with VEGFR2 inhibitoryactivity (31). The inhibitory activity of such kinase inhibitors onFGFR4 kinase activity is less effective than the inhibitory activityon VEGFR2 (31). On the other hand, FGFR-selective inhibitors(NPV-BGJ398, AZD4547, and JNJ-42756493) are being devel-oped for the treatment of lung/breast cancer with FGFR1 geneamplification, gastric cancer with FGFR2 gene amplification,cholangiocarcinoma with FGFR2 fusion, and urothelial cancerwith FGFR3 gene alternations but not for HCC (31–33). Recently,twoFGFR4-specific kinase inhibitors, BLU9931andFGF401, havebeen reported and tested in clinical trials including HCC patients(13, 14).

We developed ASP5878, an FGFR tyrosine kinase inhibitorwith potent FGFR4 inhibitory activity. Here, we demonstrated thepreclinical profile of ASP5878 in the FGF19-overexpressing HCCmodels. ASP5878 is a potent inhibitor of FGFR tyrosine kinases 1,2, 3, and 4 and is selective against a number of other kinases.Previous studies have shown that cell growth is inhibited by thesuppression of FGFR4 expression in FGF19-amplified HCC celllines with an FGFR4 RNAi or FGFR4-neutralizing antibody andFGFR-selective inhibitors (6, 10, 12–14, 29). These reports suggestthat ASP5878 has the potential to be effective in the treatment ofFGF19-expressing HCC. Of the 20 HCC cell lines, we observedabnormal FGF19 protein expression in HuH-7, Hep3B2.1-7, andJHH-7 cell lines. The proliferation of these cell lines was sensitiveto ASP5878 and effectively inhibited by ASP5878. Furthermore,ASP5878 treatment potently inhibited cellular FGFR4, FRS2, andERK activation and induced apoptosis in these HCC cell lines.Although the expression of FGFR4 and bKlothowas also observedinHuH-7,Hep3B2.1-7, and JHH-7 cell lines, someotherHCC celllines such as HepG2 and JHH-5 expressing FGFR4 and bKlothowithout abnormal FGF19 expression showed low sensitivity toASP5878. Thus, using ASP5878, we confirmed that FGF19 over-expression is an oncogenic driver of HCC and a response markerfor FGFR4 inhibition in HCC. Immunohistochemical analysis inprevious studies demonstrated that FGF19 expression is associ-ated with recurrence and poor prognosis in HCC patients, andserum FGF19 levels in HCC patients are higher than those inhealthy individuals (9, 10). These data indicate that the immu-nohistochemical staining of tumor FGF19 or measurement ofserum FGF19 could be a stratification method for FGF19-over-expressing HCC patients. In addition to FGF19-overexpressingHCC, HUH-6 Clone 5, HLF, SNU-398, and Li-7 were sensitive toASP5878; however, FGF19 expressionwasnot observed. ASP5878

Figure 2.

ASP5878 treatment leads to tumorregression in FGF19-expressingHep3B2.1-7 HCC subcutaneousxenograft model. ASP5878 was orallyadministrated once daily. Tumorvolume (A) and body weight (B) areexpressed as the mean� SEM (n ¼ 10).C, Tumor tissues from Hep3B2.1-7 HCCsubcutaneous xenograft model treatedwith 1 and 3 mg/kg ASP5878 wererecovered 6 hours after single dosingand analyzed for FRS2 and ERKactivation by Western blotting.

Futami et al.





sensitivity of these cell lines could be based on the geneticalterationof other FGF-signaling genes andnot the FGF19–FGFR4axis. For instance, the amplification of FGF3/FGF4 genes andFGF21 overexpression has recently been observed in an HCCpatient (34, 35). A subpopulation of HCC patients with FGFR1overexpression was thought to be addicted to FGFR1 (36).FGFR2 and FGFR3 overexpression contributed to advancedHCC tumorigenesis (37–39). For instance, the SNU398 cellline was found to be sensitive to ASP5878 and NPV-BGJ398 butnot to the FGFR4-selective inhibitor BLU9931 (14). FGFR1, 2,and 3, but not FGFR4, could be oncogenic drivers in theSNU398 cell line. We are presently conducting further studiesto validate those hypotheses.

Once-daily oral administration of ASP5878 resulted in tumorregression in the Hep3B2.1-7 xenograft model. The antitumoreffect was accompanied by the inhibition of FGFR downstreamsignaling in Hep3B2.1-7 xenograft tumors. These aspects weresupported by the results that oral administration of 3 mg/kgASP5878 inhibited phospho-FGFR4 and activated FRS2 and ERKin the MDA-MB-453 xenograft model. In the Hep3B2.1-7 xeno-graft model, the tumor volume decreased after treatment withASP5878 and was almost at an undetectable level at the end of3 mg/kg ASP5878 treatment following sorafenib treatment. Sor-afenib is the standard first-line systemic drug for advanced HCC,which can extend the duration of survival to 10.7 months in

comparison with 7.9 months in the placebo arm (4). In ourexperiments, sorafenib induced slight retardation of tumorgrowth in the HuH-7 orthotopic xenograft and Hep3B2.1-7xenograft models. On the other hand, oral administration of 3mg/kg ASP5878 induced tumor regression and downstream sig-nals declined to background levels in the Hep3B2.1-7 xenograftmodel. Furthermore, the survival was enhanced in associationwith the potent antitumor effect of ASP5878 in the HuH-7orthotopic xenograft model. Importantly, no tumor regrowthwas observed among the two different models during ASP5878administration. Furthermore, ASP5878 induced complete tumorregression, which was sustained for at least 90 days after thecessation of ASP5878 treatment in the HuH-7 orthotopic xeno-graft model. These data indicated that ASP5878 treatment ofFGF19-expressing HCC induced sustained complete regression.

Several FGFR inhibitors are being developed in clinical trials,but ASP5878 has a different profile than FGFR inhibitors (13).AZD4547 and NPV-BGJ398 show at least 30-fold greater selec-tivity toward FGFR1–3 than toward FGFR4 (22, 40). These inhi-bitors are categorized as pan-FGFR1, 2, and 3 inhibitors. It has notbeen demonstrated that those two inhibitors showed activity inHCC vivo model. In contrast, ASP5878 has potent FGFR4 inhib-itory activity. It showed remarkable tumor regression and extend-ed survival in an HCCmodel in vivo, without weight loss. Similarefficacy and tolerability may be achieved in HCC patients; this

Figure 3.

ASP5878 treatment leads to tumor regression in FGF19-expressingHuH-7HCCorthotropic graftmodel. HuH-7-Luc cellsweredirectly inoculated into the liver ofmice.Vehicle (Cremophor EL/Ethanol), ASP5878 (3 mg/kg), or sorafenib (30 mg/kg) was orally administered once daily at the indicated dose (n ¼ 5). ASP5878was orally administered once daily from days 1 to 90, and the administration was stopped from day 91 to end of the study, i.e., day 181. A, The implanted cells weremonitored using bioluminescent imaging (BLI). Each point represents the mean BLI � SEM. B, Representative BLI image of each group at the indicated day.C, The survival of mice was monitored daily.






hypothesis is currently being evaluated in clinical trials. BLU9931showed >50-fold greater inhibition of FGFR4 than of FGFR1, 2,and 3 (14). FGF401 has reported to be an FGFR4-selectiveinhibitor, but the kinase selectivity profile remains unknown(13). Similar to ASP5878, these inhibitors are expected toimprove the therapeutic outcome of HCC patients with FGF19overexpression. The advantage of ASP5878 over FGFR4-selectiveinhibitors is the ability toprovide therapeutic benefit to a subset ofpatients with HCC whose tumors are driven by FGFR1, 2, or 3. Inaddition to FGFR4, the overexpression of FGFR1, FGFR2, andFGFR3 contributes to advanced HCC tumorigenesis, metastasis,and poor prognosis (36–38). In particular, Paur and colleaguesobserved that FGFR3 and/or FGFR4 expression was elevated in68% of HCC patients (38). Moreover, the amplification ofFGF3/FGF4 genes and FGF21 overexpression has recently beenobserved in an HCC patient (34, 35). These ligands bind toFGFR1, 2, and/or FGFR3. ASP5878 may be beneficial for theseHCC subpopulations. In fact, the SNU398 cell line was sensitiveto ASP5878 and BGJ398 but not to BLU9931; SNU398 may beone of the representatives of these HCC subsets. JNJ-42756493was found to inhibit all members of the FGFR family at the lownanomolar IC50 range (41). This type of compound is categorized

as a "pan-FGFR1, 2, 3, and 4 inhibitor," including ASP5878. JNJ-42756493 is currently being evaluated in clinical trials. Hyper-phosphatemia has been observed with other FGFR inhibitors(e.g., AZD4547 and JNJ-42756493) and is likely based on mech-anismof action (32, 33). In linewith thefindings, increased serumphosphate was observed in rodents repeatedly administered withASP5878 (unpublished data). ASP5878 is currently being evalu-ated in phase I clinical trials to evaluate the safety, pharmacoki-netics, and pharmacodynamics and defines the recommendedphase II dose.

In conclusion, using a panel of HCC cell lines in vitro, we haveshown that FGF19 expression levels predict the antiproliferativesensitivity to the novel and potent FGFR-selective agent ASP5878.ASP5878 induced almost complete tumor regression in associa-tion with the pharmacodynamic modulation of FGFR down-stream signals in a Hep3B2.1-7 xenograft model. Furthermore,ASP5878 showed complete and sustained tumor regression andextended survival in a HuH-7 orthotopic xenograft model. Ourfindings demonstrate that ASP5878, a novel FGFR inhibitor withpotent FGFR4 inhibitory activity, is expected to improve thetherapeutic outcomes of HCC patients with FGF19 overexpres-sion. ASP5878 is currently being evaluated in phase I clinicaltrials.

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

Authors' ContributionsConception and design: T. Futami, R. Kihara, T. Suzuki, M. HiranoDevelopment of methodology: T. Futami, H. Okada, R. Kihara, T. SuzukiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Futami, H. Okada, R. Kihara, T. Kawase,A. Nakayama, M. KamedaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Futami, H. Okada, R. Kihara, A. Nakayama,T. Terasaka, M. HiranoWriting, review, and/or revision of the manuscript: T. Futami, H. Okada,R. Kihara, T. Kawase, A. Nakayama, T. Suzuki, M. Kameda, N. Shindoh,M. Hirano, S. KuromitsuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Kihara, A. NakayamaStudy supervision: T. Futami, T. Suzuk, T. Terasaka, M. Hirano, S. Kuromitsu

AcknowledgmentsThe authors thank Hiroshi Fushiki, Aya Kikuchi, Taisuke Nakazawa, Masa-

teru Iizuka, and Hideki Sakagami for their involvement in this study.The costs of publication of this article were defrayed in part by the

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

Received April 6, 2016; revisedOctober 10, 2016; acceptedOctober 11, 2016;published OnlineFirst November 11, 2016.

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2017;16:68-75. Published OnlineFirst November 11, 2016.Mol Cancer Ther Takashi Futami, Hidetsugu Okada, Rumi Kihara, et al. Growth of FGF19-Expressing Hepatocellular CarcinomaASP5878, a Novel Inhibitor of FGFR1, 2, 3, and 4, Inhibits the

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