preclinical evaluation of nintedanib, a triple angiokinase ... · angiokinase inhibitor, in...

Small Molecule Therapeutics

Preclinical Evaluation of Nintedanib, a TripleAngiokinase Inhibitor, in Soft-tissue Sarcoma:Potential Therapeutic Implication for SynovialSarcomaParag P. Patwardhan1, Elgilda Musi1, and Gary K. Schwartz1,2

Abstract

Sarcomas are rare cancers that make up about 1% of allcancers in adults; however, they occurmore commonly amongchildren and young adolescents. Sarcomas are geneticallycomplex and are often difficult to treat given the lack of clinicalefficacy of any of the currently available therapies. Receptortyrosine kinases (RTK) such as c-Kit, c-Met, PDGFR, IGF-1R, aswell as FGFR have all been reported to be involved in drivingtumor development and progression in adult and pediatricsoft-tissue sarcoma. These driver kinases often act as criticaldeterminants of tumor cell proliferation and targeting thesesignal transduction pathways remains an attractive therapeuticapproach. Nintedanib, a potent triple angiokinase inhibitor,targets PDGFR, VEGFR, and FGFR pathways critical for tumorangiogenesis and vasculature. In this study, we evaluated the

preclinical efficacy of nintedanib in soft-tissue sarcoma celllines. Nintedanib treatment resulted in significant antiproli-ferative effect in vitro in cell lines with high expression of RTKdrug targets. Furthermore, treatment with nintedanib showedsignificant downregulation of downstream phosphorylatedAKT and ERK1/2. Finally, treatment with nintedanib resultedin significant tumor growth suppression in mouse xenograftmodel of synovial sarcoma. Notably, both the in vitro andin vivo efficacy of nintedanib was superior to that of imatinib,another multikinase inhibitor, previously tested with min-imal success in clinical trials in sarcoma. Overall, the datafrom this study provide a strong rationale to warrant furtherclinical exploration of this drug in patients with synovialsarcoma. Mol Cancer Ther; 17(11); 2329–40. �2018 AACR.

IntroductionSarcomas are rare cancers with subtypes that are often genet-

ically andbiologically distinct (1, 2). Sarcomasmakeupabout 1%of all cancers in adults with approximately 14,000 people diag-nosed with sarcoma per year in the United States. However, theyare relativelymore commonamong childrenmakingup to12%to15% of cancers in children under the age of 20 (3). Some of themore common andmolecularly heterogeneous sarcoma subtypesinclude liposarcoma, leiomyosarcoma, and synovial sarcomaamong others (4). Localized disease is usually surgically resectedfollowed by radiotherapy with or without adjuvant chemother-apy. However, lack of clinical efficacy coupled with toxicity oftenlimits the treatment options especially in metastatic cases of thedisease.

Receptor tyrosine kinases (RTK) act as key mediators of signaltransduction pathways that regulate cell proliferation, survival,

and migration (5). Role of RTKs such as IGF-1R, PDGFR, c-Met,AXL, and FGFR as well as nonreceptor tyrosine kinases (non-RTK) such as FAK and Src acting as tumor drivers has beenreported in various sarcoma subtypes over the past few years(6–11). However, the translation of these preclinical studiesinto effective molecularly targeted therapies has met with onlymodest clinical success (12, 13). Gastrointestinal stromaltumor (GIST) is one of the few sarcoma subtypes whereoncogenic mutations in PDGFRa and c-Kit (14, 15) have beentargeted successfully using tyrosine kinase inhibitors such asimatinib and crenolanib (16, 17).In addition, in patients withwell- and dedifferentiated liposarcomas carrying CDK4 ampli-fication have shown promising results including progression-free survival in clinical trials (18, 19).

Although targeted RTK therapy presents an attractive approachto treat this disease, lack of clinical success using traditionaltherapieswarrants exploration of novel agents that blockmultiplekinases such as PDGFR, FGFR, and VEGFR known to be critical fordriving sarcoma progression (8, 20–23).

In this study, we tested the in vitro and in vivo antiproliferativeefficacy of nintedanib (BIBF1120), a potent PDGFR, VEGFR, andFGFR inhibitor, in sarcoma. Nintedanib was recently approvedfor the treatment of non–small-cell lung cancer in the EuropianUnion (EU). Efficacy of other multikinase inhibitors in vitro andin clinical trials in sarcoma has been reported previously by ourlaboratory (24) as well as others (12). However, not manyeffective molecularly targeted therapies are currently availablefor patients with sarcoma including those with synovial sarcomawhich has a propensity to metastasize in many cases. This is thefirst report describing efficacy of nintedanib in this disease

1Department of Medicine, Columbia University Medical Center, New York,New York. 2Herbert Irving Comprehensive Cancer Center, Columbia UniversityCollege of Medicine, New York, New York.

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

Corresponding Author: Parag P. Patwardhan, Columbia University MedicalCenter, Herbert Irving Comprehensive Cancer Center, 1130 St. Nicholas Avenue,Room 207, New York, NY 10032. Phone: 212-851-4904; Fax: 212-851-4901;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-18-0319

�2018 American Association for Cancer Research.

MolecularCancerTherapeutics

www.aacrjournals.org 2329

on June 28, 2020. © 2018 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 30, 2018; DOI: 10.1158/1535-7163.MCT-18-0319

http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-18-0319&domain=pdf&date_stamp=2018-10-23

http://mct.aacrjournals.org/

setting and our results strongly warrant further clinical explora-tion of this drug in synovial sarcoma.

Materials and MethodsChemicals and drugs

Nintedanib (BIBF1120) was kindly provided by Boehringer-Ingelheim Pharmaceuticals. Imatinib was purchased from LCLaboratories. Selumetinib (MEKi) and MK-2206 (AKTi) werepurchased from Selleck Chemicals. Drugs were dissolved inDMSO and aliquots were stored at �20�C for long-term use.

Cell culture and reagentsCells were cultured inRPMI orDMEMwith 10%FBS, 100m/mL

penicillin, and 100 mg/mL streptomycin, maintained at 37�C in5% CO2, and passaged for no more than 3 months. Synovialsarcoma cell lines (SYO-1 and HSSY-II) were obtained from Dr.Marc Ladanyi (Memorial Sloan Kettering Cancer Center, NewYork, NY). Malignant peripheral nerve sheath tumor (MPNST)cell line was supplied by Dr. Jonathan Fletcher (Dana FarberCancer Institute, Boston,MA). ST8814 cell linewas a generous giftfrom Dr. Karen Cichowski (BWS Biomedical Research Institute,Boston, MA), Ewing sarcoma cell lines (CHP100, A673) wereobtained from Dr. Melinda S. Merchant (Center for CancerResearch, NCI/NIH, Bethesda, MD). Dedifferentiated liposar-coma (LS141, DDLS) were obtained from Dr. Samuel Singer(Memorial Sloan Kettering Cancer Center, New York, NY). Oste-osarcoma cell line (SaOS2) was obtained from ATCC. Cell linesCHP100 and A673 were authenticated using RT-PCR, and foundto have their expected characteristic chromosomal translocations.SYO-1 and HS-SY-II cell lines were authenticated by confirmingthe expression of the pathognomonic SYT-SSX fusion gene by RT-PCR. All cell lines were determined to beMycoplasma free by usingbiochemical assay MycoAlert.

Cell viability assaysCell viability assays were carried out using the Dojindo

Molecular Technologies (CCK-8) Kit as per the manufacturer'sinstructions. Briefly, 2,000 cells were plated in 96-well platesand then treated with the indicated drugs for 6 days unlessindicated otherwise. Media was replaced with 100 mL of mediawith 10% serum and 10% CCK-8 solution (Dojindo MolecularTechnologies Kit). After 1 hour, the optical density was read at450 nm using a Spectra Max 340 PC (Molecular DevicesCorporation) to determine viability. Background values fromnegative control wells without cells were subtracted for finalsample quantification. Cell viability is expressed as a percentageof untreated/DMSO-treated cells.

Western immunoblottingCells and tissues were lysed with radioimmunoprecipitation

assay (RIPA) buffer supplemented with protease inhibitorcocktail tablets (Roche Diagnostics) and 1 mmol/L Na3VO4.Equal amounts (20–30 mg) of protein were electrophoresedonto 4%–12% gradient gels (Life Technologies) and transferredonto polyvinylidene difluoride or 0.45-mmnitrocellulose mem-branes. Membranes were blocked with 5% nonfat driedmilk and probed with primary antibodies. A complete list ofantibodies that were used in this study is included in theSupplementary Table. Bound antibodies were detected withhorseradish peroxidase secondary antibodies (GE Healthcare)

and visualized by Enhanced Chemiluminescence Reagent (GEHealthcare).

Xenograft studiesApproximately 8-week-old athymic female mice were injected

with 10–15million cells in Matrigel of the cell line of interest andallowed to growuntil tumors reached100mm3 in volumeprior totreatment with vehicle, imatinib (30 mg/kg i.p. once daily), ornintedanib (50 mg/kg orally once daily). Tumors were measuredevery 2 to 3 days using calipers and volumes calculated using theformula p/6 x (large diameter)� (small diameter) where p standsfor "pi". Animal weights were measured every 2 to 3 days as asurrogate marker for overall toxicity. Animals were sacrificed 4hours after the final dose of treatment (on day 21 for vehiclecontrol or day 24 for imatinib and nintedanib treatment groups).Tumors were extracted from surrounding tissue and either flash-frozen in liquid nitrogen for Western immunoblotting or placedin formalin or paraformaldehyde for IHC studies. Flash-frozentissue was ground in RIPA lysis buffer and resin as per themanufacturer's instructions using Sample Grinding Kit (GEHealthcare) and analyzed via Western immunoblotting asdescribed above. Formalin-fixed tissues were sectioned andstained with the indicated antibodies following standard proto-cols using HistoWiz automated histology services.

Statistical analysisAll in vitro experiments were carried out at least three times

unless otherwise indicated. P valueswere calculated using Studentt test, with values of � 0.05 determined to be statistically signif-icant. SEwas calculated as the SDdivided by the square root of thenumber of samples tested.

ResultsTarget RTKs for nintedanib are expressed in multiple sarcomacell lines

Nintedanib is a small-molecule inhibitor that targets multipleRTKs involved in tumor angiogenesis such as VEGFR, PDGFR, andFGFR (25, 26). It is an indolinone derivative that has been shownto block RTKs, such as VEGFR and PDGFR families at lownanomolar concentrations, and also block downstream MAPKand AKT signaling in pericytes and endothelial cells (25). To testthe basal levels of expression for nintedanib drug targets, wecarried out a Western blot analysis using 9 different sarcoma celllines belonging to various subtypes such as Ewing sarcoma (A673,CHP100), liposarcoma (DDLS, LS141), malignant peripheralnerve sheath tumor (MPNST, ST8814), synovial sarcoma (SYO-1,HS-SY-II), and osteosarcoma (SaOS2). Figure 1A shows that thecell lines tested express variable levels of PDGFR, FGFR, andVEGFR family of RTKs with malignant peripheral nerve sheathtumor (MPNST, ST8814) and synovial sarcoma cell lines (SYO-1,HS-SY-II) showing expression of maximum number of ninteda-nib drug targets.

Nintedanib exhibits potent antiproliferative activity andinhibition of downstream signaling in sarcoma cell linesexpressing both PDGFR and FGFR

To test the antiproliferative efficacy of nintedanib in multiplesarcoma cell lines, we carried out a cell viability assay following 6days of exposure to increasing concentrations of the drug. Ninte-danib treatment resulted in inhibition of cell proliferation

Patwardhan et al.

Mol Cancer Ther; 17(11) November 2018 Molecular Cancer Therapeutics2330




especially in cell lines that express both PDGFR and FGFRfamily of RTKs (Fig. 1B). As shown in Fig. 1B, inhibition ofcell proliferation in the nanomolar range was observed forMPNST, ST8814, as well as both synovial sarcoma cell lines(SYO-1, HS-SY-II) compared with the two Ewing sarcoma celllines (A673 and CHP100). Modest activity was observed atmicromolar concentrations for liposarcoma cell lines (DDLS,LS141) and for osteosarcoma cell line, SaOS2. SYO-1 andST8814 cell lines showed IC50 values < 1 mmol/L, MPNST,HS-SY-II, and LS141 cell lines had IC50 values between 1–2mmol/L, whereas, SaOS2, DDLS, A673, and CHP100 cell linesshowed IC50 values > 5–10 mmol/L (Table 1). Approximate IC50

values for all the cell lines tested are summarized in Table 1. Totest for the inhibition of downstream signaling pathways, wecarried out Western blot analysis with increasing dose of

nintedanib in 4 cell lines (selected on the basis oftheir anti-proliferative efficacy). Figure 1C shows that treatment withincreasing doses of nintedanib results in significant inhibitionof p-AKT in both SYO-1 and ST8814 cell line. Inhibition ofp-ERK1/2 was also observed for the cell line SYO-1 but no suchinhibition of p-ERK1/2 was seen in ST8814 cell line. This couldpartly be due to the fact that ST8814 cell line has higher basallevels of p-ERK1/2, which are difficult to suppress owing to theNF1 (Neurofibromatosis Type 1) loss and increased Ras activityin this cell line (27). On the other hand, cell lines A673 andCHP100 showed no change in p-AKT and p-ERK1/2 levels afternintedanib treatment. This correlated with the observation thatboth these cell lines did not show any significant inhibition ofproliferation even at micromolar concentrations of the drug(Fig. 1B).

Figure 1.

Inhibition of cell proliferation and downstreamsignaling pathways by nintedanib in PDGFR andFGFR expressing sarcoma cell lines. A, Basal levelsof target RTK expression in 9 sarcoma cell lines.Cells were plated in 100 mm tissue culture plates(Costar) and grown for 24 hours in mediacontaining 10%FBS. Cell lysates were obtained byscraping in RIPA lysis buffer and 30 mg of lysateswere run on SDS/PAGE gels. Western blot analysiswas carried out using indicated antibodies. B,Indicated cell lines were plated in 96-well platesand treated in triplicate per condition withincreasing doses of nintedanib for 6 days. Cellviabilitywasmeasured usingDojindoCell CountingKit 8 according to the manufacturer's instructions.Dose response graphs were generated as apercentage of DMSO-treated control. Error barsrepresent SEM. Combined data from two replicatesfrom a single experiment representative of threeindependent experiments are shown. C, Cell lineswere grown to 60% confluency in serum-freemedia overnight. Next day, cells were treated withindicated concentrations of nintedanib in serum-free media for 3 hours. Following drug treatment,cells were stimulated in drug-free mediacontaining 10%FBS. 30 mg of RIPA lysates wereloaded on SDS/PAGE and immunoblotted usingindicated antibodies. Representative western blotsfrom two independent experiments are shown.

Triple Angiokinase Inhibition by Nintedanib in Sarcoma

www.aacrjournals.org Mol Cancer Ther; 17(11) November 2018 2331




To test whether the drug treatment results in inhibition of targetRTKs in various cell lines, we carried out phospho-RTK proteomeprofiler array analysis in the 4 cell lines tested above. Nintedanibtreatment (at 500 nmol/L) resulted in significant downregulationof RTK targets including PDGFRa and PDGFRb in all the cell linestested including A673 and CHP100 (Fig. 2). This suggests thateven though the phosphorylation of PDGFRb is blocked bynintedanib, the lack of antiproliferative efficacy in the two Ewingsarcoma cell lines A673 and CHP100 is possibly due to the factthat pathways other than PDGFR such as Ins-R and IGF-1R aredriving cell proliferation. Although we were able to observephosphorylated levels of RYK in SYO-1 and ST8814 cell lines, itis highly unlikely that it plays any significant role in drivingsarcoma cell growth especially because it lacks any catalyticactivity of its own (24).

In the SYO-1 cell line, which expressed high levels of VEGFR2(Fig. 1A), we were unable to detect any phosphorylated VEGFR2either by RTK array (Fig. 2) or by Western blot analysis (Supple-mentary Fig. S1A). AXL, a well-characterized RTK, has been shownto be induced as a result of resistance to imatinib in GISTs. To testwhether nintedanib treatment results in induction of AXL, wecarried out Western blot analysis. Our results indicate that SYO-1cells lacked total as well as phosphorylated levels of AXL (Sup-plementary Fig. S1A). CHP100 cell line showed presence of totalAXL receptor on the Western blot analysis; however, phosphor-ylated AXL was unable to be detected irrespective of whether thecells were treated with nintedanib (Supplementary Fig. S1A).Next, we wanted to test whether nintedanib treatment resultedin downregulation of non-RTKs such as SRC family kinases, aknown downstream target of nintedanib (25). Treatment with500 nmol/L nintedanib did not result in activation of SRC familykinases as determined by phospho-SRC Tyr416 Western blotanalysis (Supplementary Fig. S1A). No changes in total levels ofSRCwere observed after nintedanib treatment.We also carried outanalysis of non-RTK phosphorylation using non-RTK proteinprofiler array kit (R&D Systems). The majority of the non-RTKsthat were detected on the array in both SYO-1 and CHP100 celllines included JNK, GSK3b, c-Jun, PRAS40, and HSP60 kinases(Supplementary Fig. S1B). Phosphorylated FAKwas detected onlyin the SYO-1 cell line; however, no significant changes in phos-phorylation of FAK were observed post-nintedanib treatment. Inaddition, in both the cell lines, no major change in phosphory-lation of any of the non-RTKs was observed other than phosphor-ylated p53 in SYO-1 cell line (Supplementary Fig. S1B). We wereunable to detect any phosphorylated SRC on the non-RTK array.

Antiproliferative activity of nintedanib is a result of sustainedp-AKT and p-ERK1/2 inhibition

To determine whether the inhibitory effect of nintedanib is aresult of combined inhibitionof bothp-AKTandp-ERKpathways,

we tested the efficacy of MEK inhibitor (MEKi) selumetinib(28) and AKT inhibitor (AKTi) MK-2206 (29) alone or incombination and compared the inhibitory effects against nin-tedanib and imatinib, another well-known c-Kit and PDGFRinhibitor (30). Cell proliferation assay in Fig. 3A and B showsthat inhibitory effects of nintedanib in synovial sarcoma cellline SYO-1 were significantly (P < 0.01 and P < 0.05, respec-tively) better than imatinib alone or a combination of MEKiand AKTi. On the other hand, no significant inhibition ofproliferation was observed for the CHP100 (Ewing sarcoma)cell line (Fig. 3B). In ST8814 cell line, the inhibition of cellproliferation by a combination of MEKi and AKTi was signif-icantly better than nintedanib treatment alone (Fig. 3A). For theA673 cell line, nintedanib treatment did not result in anysignificant inhibition of cell proliferation compared with nodrug control; however, a combination of MEKi andAKTi treatment resulted in significant inhibition of prolifera-tion (Fig. 3B). Western blot analysis after 24 hours of drugtreatment (Fig. 3C) to test pathway inhibition shows that MEKialone or in combination with AKTi did not result in suppres-sion of p-ERK1/2. Nintedanib treatment, on the other hand,resulted in significant inhibition of p-AKT and p-ERK1/2 levelsin SYO-1 cell line. When nintedanib treatment was combinedwith MEKi, the combination treatment resulted in furtherinhibition of cell proliferation (Fig. 3A) compared with ninte-danib alone (P < 0.05) as well as further inhibition of p-ERK1/2

levels (Fig. 3C). No such downstream signaling pathway inhi-bition was observed in Ewing sarcoma cell lines, A673 andCHP100 (Fig. 3C). It must be noted though that when the cellviability was assayed after 3 days of drug treatment, the inhi-bition of cell proliferation for nintedanib versus MEKi and AKTicombination treatment seemed comparable in the SYO-1 cellline (Supplementary Fig. S2). However, when the assay wasperformed after 6 days of drug exposure, nintedanib treatmentresulted in further inhibition of cell proliferation (Supplemen-tary Fig. S2, viability at day 3 vs. day 6) compared with MEKiand AKTi combination.

Antiproliferative activity of nintedanib is a result of combinedinhibition of both PDGFR and FGFR pathways

To test whether the inhibitory effects of nintedanib are medi-ated through inhibition of PDGFR and/or FGFR pathways, wecarried out cell proliferation assay andWestern blot analysis afternintedanib or pan-FGFR inhibitor, PD173074 (31) treat-ment. Figure 4A shows that inhibition by pan-FGFR inhibitor,PD173074, resulted in significant (P < 0.001) inhibition of cellproliferation compared withDMSO control. However, inhibitionof cell proliferation by nintedanib was significantly better thanPD173074 (P < 0.01) especially at 500 and 1,000 nmol/L con-centrations (Fig. 4A). Western blot analysis (Fig. 4B) showedthat PD173074 treatment resulted in significant inhibition ofp-ERK1/2 signaling in SYO-1 cell line; however, no decrease inp-AKT signal was observed (Fig. 4B, lanes 4 and 5). Nintedanibtreatment, on the other hand, showed significant reduction inp-AKT as well as p-ERK1/2 signaling at 500 nmol/L concentration(Fig. 4B, lanes 2 and 3). Results of nintedanib versus pan-FGFRinhibition in SYO-1 cell line strongly suggest a role for combinedinhibition of PDGFR and FGFR in downregulating p-AKT signal-ing compared with inhibition of FGFR activity alone in down-regulating p-ERK1/2 signaling. No significant inhibition of cellproliferation or any decrease in downstream p-AKT signaling was

Table 1. Table showing approximate IC50 values for all the cell lines tested

Cell Line [IC50] mmol/L

A673 >10CHP100 >10DDLS >10LS141 1.266SaOS2 7.120SYO-1 0.525HS-SY-II 1.397MPNST 1.431ST8814 0.734

Patwardhan et al.





observed in CHP100 cell line (Fig. 4A and B). Interestingly, basallevels of p-ERK1/2 in CHP100 cell line were lower compared withSYO-1 cell line and showed only a modest decrease after ninte-danib or PD173074 treatment.

Pazopanib, another multitargeted kinase, was recentlyapproved for the treatment of soft-tissue sarcoma (32). To testwhether nintedanib treatment fares better than pazopanib treat-ment in SYO-1, we carried out a cell viability assay and Western

Figure 2.

Inhibition of phosphorylated RTK targets by nintedanib. Approximately, 106 cells were plated in 60 mm plates in serum-free media for 24 hours. Next day,DMSOor 500 nmol/L nintedanibwas added in serum-freemedia and treated for 3 hours. Cellswere then stimulated in drug-freemedia containing 10%FBS for 1 hour.Following stimulation, lysates were prepared according to the manufacturer's instructions (R&D Systems, ARY001B) and 200 mg of lysates were applied tophospho-RTK membranes overnight. Arrows indicate phosphorylated RTK spots in duplicate.






Figure 3.

Combined inhibition of p-AKT and p-ERK can mimic inhibitory effects of nintedanib. A and B, Cell lines were plated in 96-well plates and treated in triplicateper condition with indicated drugs at 1,000 nmol/L for 6 days. Cell viability was measured using Dojindo Cell Counting Kit 8 according to the manufacturer'sinstructions. MEKi ¼ selumetinib, AKTi ¼ MK-2206. Dose response graphs were generated as a percentage of DMSO-treated control. Error bars represent SEM.Combined data from two replicates from a single experiment representative of three independent experiments are shown. P values indicated as � , ¼ P < 0.05,�� ,¼P <0.01.C,Approximately, 5� 105 cellswere plated in 60mmplated in serum-freemedia for 24 hours. Next day, cellswere treatedwith 500 nmol/L of indicateddrugs in media containing 10%FBS for another 24 hours. Cell lysates were prepared using RIPA lysis buffer and 30 mg lysates were loaded on SDS/PAGE andimmunoblotted using indicated antibodies. Representative western blots from two independent experiments are shown.

Patwardhan et al.





Figure 4.

Inhibition of cell proliferation and downstream signaling by nintedanib is a result of combined inhibition of PDGFR and FGFR. A and C, Cell lines were platedin 96-well plates and treated in triplicate per condition with increasing doses of indicated drugs for 6 days. Cell viability was measured using Dojindo CellCounting Kit 8 according to the manufacturer's instructions. Dose response graphs were generated as a percentage of DMSO-treated control. Error bars representSEM. Combined data from two replicates from a single experiment representative of three independent experiments are shown. P values indicated as � ,¼ P < 0.05,�� , ¼ P < 0.01. B and D, Approximately, 5 �105 cells were plated in 60 mm plated in serum-free media for 24 hours. Next day, cells were treated with500 nmol/L of indicated drugs in media containing 10%FBS for another 24 hours. Cell lysates were prepared using RIPA lysis buffer and 30 mg lysates wereloaded on SDS/PAGE and immunoblotted using indicated antibodies. Representative western blots from two independent experiments are shown.






blot analysis to compare their efficacy. Figure 4C shows thatnintedanib treatment resulted in significantly better inhibition(P < 0.05) compared with pazopanib in the SYO-1 cell line. Asseen earlier, nintedanib treatment (at 1,000 nmol/L) resulted ininhibition of p-AKT as well as p-ERK1/2 (Fig. 4D), whereaspazopanib treatment (at 1,000 nmol/L) showed downregulationof p-AKT signaling but failed to show any downregulation of p-ERK1/2 signaling strongly suggesting that nintedanib treatment is

superior in the synovial sarcoma setting. Furthermore, it has beenshown recently that nintedanib has a more potent inhibitionprofile across the triple angiokinase panel when compared head-to-head with other in-class competitor molecules such as suniti-nib, pazopanib, and vandetanib (26).

To further confirm the role of both PDGFR and FGFR in drivingcell proliferation, we carried out siRNA-mediated analysis of cellproliferation in SYO-1 cell line. siRNA-mediated knockdown of

Figure 5.

siRNA-mediated knockdown of PDGFR and FGFR can mimic inhibition of cell proliferation and downstream signaling by nintedanib. A, Approximately,2.5� 105 cellswere plated and transfectedwith 100 nmol/L of indicated siRNAs (pooled siRNAs, GEDharmacon) after 24 hours. Twenty-four hours after transfection,media were changed and cells were transfected again with 100 nmol/L siRNA. After another 48 hours, cells were harvested and approximately 2,000 cellsper well were plated in 96-well plates in triplicates. Cell viability was measured after 6 days using Dojindo Cell Counting Kit 8. Error bars represent SEM. Combineddata from two replicates from a single experiment representative of three independent experiments are shown. P values indicated as � , ¼ P < 0.05. B, Cell lysateswere prepared using RIPA lysis buffer 72 hours post siRNA transfection and 30 mg lysates were loaded on SDS/PAGE and immunoblotted using indicatedantibodies. Representative western blots from two independent experiments are shown.

Patwardhan et al.





Figure 6.

Effect of nintedanib treatment in SYO-1 xenografts. A, Tumor growth of SYO-1 xenografts (n ¼ 7/group) treated with the indicated drugs is shown.Drug treatment was stopped on day 21 for Vehicle control group (when the tumors grew more than 2,000 mm3 in size) and on day 24 for the remaining twotreatment groups. P values indicated as � , ¼ P < 0.05, �� , ¼ P < 0.01, ��, ¼ P < 0.005. B, Thirty micrograms of RIPA lysates obtained using sample grinding kit(GE Healthcare) from xenograft tissues at the end of drug treatment were loaded on SDS/PAGE and immunoblotted using indicated antibodies.Representative blots from two animals sacrificed at the end of drug treatment are shown. C, Xenograft tissues obtained from mice at the end of drugtreatment with the indicated drugs were stained immuno-histochemically using CD31 (HistoWiz). Magnification of 40� is shown for each image. BrownCD31 staining is indicated with red arrows in the zoomed portion of the image. Representative images from two animals sacrificed at the end of drugtreatment are shown.






individual RTKs (PDGFRa, FGFR1, and FGFR2) resulted in sig-nificant inhibition of cell proliferation compared with controlsiRNA (P < 0.01; Fig. 5A). However, combined knockdown ofPDGFRa, FGFR1, and FGFR2 (thus, mimicking nintedanib treat-ment) resulted in further inhibitionof cell proliferation comparedwith any of the single or double siRNA-mediated RTK silencing(P < 0.05; Fig. 5A). Western blot in Fig. 5B shows that siRNAtreatment resulted in significant downregulation of RTKs. As seenwith pan-FGFR inhibitor PD173074 (Fig. 4B), siRNA-mediatedknockdown of FGFR1 and FGFR2 resulted in downregulation ofpERK1/2, whereas, only combined knockdown of PDGFRa andFGFR1, and FGFR2 resulted in downregulation of p-AKT signal(Fig. 5B).

Nintedanib treatment results in significant tumor growthsuppression compared with imatinib in vivo in SYO-1 xenograftmodel

To test the efficacy of nintedanib treatment in vivo, we carriedout mouse xenograft experiments using SYO-1 xenograft model.We compared the efficacy of nintedanib against imatinib in vivo astested earlier in vitro (Fig. 3A andB). Both imatinib andnintedanibtreatment resulted in significant suppression (P < 0.05 and P <0.005) of tumor growth compared with vehicle control (Fig. 6A).However, tumor suppression after nintedanib treatment wassignificantly better than imatinib treatment (P < 0.01). Westernblot analysis of tissue samples obtained at the end of 3 weeks ofnintedanib treatment showed induction of cleaved PARP, a sur-rogate marker for apoptosis, as well as greater inhibition ofphosphorylation of FGFR, p-AKT, and p-ERK1/2 (Fig. 6B) com-paredwith imatinib treatment. Because nintedanib blocks VEGFRin addition to PDGFR andFGFR,we tested the effect of nintedanibtreatment on tumor vasculature as determined by CD31 IHC. Inaccordance with previous reports (25), nintedanib treatmentresulted in inhibition of tumor vessel density seen as completeabsence of brown IHC staining compared with vehicle control(Fig. 6C, red arrows in the zoomed image pointing to the brownsignal for CD31 staining). Imatinib treatment, on the other hand,did not show any reduction in CD31 staining (Fig. 6C). In vivotumor growthdata aswell asWestern blot analysis and IHC resultsobtained in SYO-1 xenografts confirmed our in vitro findings thatnintedanib can be explored further as a potential therapy forsynovial sarcoma.

DiscussionIn this study, we report preclinical evaluation of nintedanib

(25), a triple angiokinase inhibitor, inmultiple sarcoma cell lines.Nintedanib, a potent inhibitor of PDGFR, VEGFR, and FGFRfamily of kinases that are involved in tumor angiogenesis, hasundergone considerable preclinical and clinical evaluation incancers such as non–small-cell lung cancer, ovarian cancer,as well as in diseases like idiopathic pulmonary fibrosis (33);however, it has largely been untested as a potential therapy fororphan diseases like sarcoma. Pazopanib, another multikinaseinhibitor that blocks VEGFR and PDGFR has been approved forthe treatment of soft-tissue sarcoma albeit with limited clinicalsuccess (32). Recent studies (24) from our laboratory have alsoevaluated another multikinase inhibitor, sitravatinib, for its anti-proliferative efficacy in sarcoma and is currently in clinical trialfor liposarcoma (ClinicalTrials.gov Identifier: NCT02978859).Although broad-spectrum RTK inhibition approach seems attrac-

tive, disease-specific treatment is often needed given the com-plexities of various subtypes.

Recent evidence has suggested involvement of RTKs such asPDGFR (8, 34), c-Met (11) as well as IGF1-R (35), and ErbB4(9) among others in various sarcoma subtypes. Phosphopro-teomic analysis using a mass spectrometry approach has furtherrevealed that RTKs such as PDGFR, IGF-1R, c-Met, and ALK playa role in driving sarcoma development and progression (6, 7).Previous studies from our laboratory (8) and others (20) havealso elucidated the role of PDGFR and FGFR signaling pathwaysin driving synovial sarcoma proliferation. Despite many ofthese preclinical and clinical advances in the treatment ofsarcomas, the survival rate for this disease remains poor andnovel therapeutic approaches are needed to treat this disease.

To determine the efficacy of nintedanib in various sarcoma celllines, we first analyzed the basal levels of expression of many ofthe RTK drug targets such as PDGFRa, b as well as FGFR andVEGFR family of kinases. Many sarcoma subtypes includingMPNST and synovial sarcoma cell lines expressed multiple drugtargets specific for nintedanib. When we determined the antipro-liferative efficacy of nintedanib in a cell proliferation assay, weobserved increased sensitivity with increasing doses of the drugmostly in the cell lines with high levels of target RTK expression(such as SYO-1, HS-SY-II). Western blot analysis confirmed inhi-bition of downstream signaling pathways, p-AKT andp-ERK1/2, insynovial andMPNST cell line (ST8814), but not in Ewing sarcomacell lines (A673, CHP100), which also showed the lowest expres-sion of drug targets. Although nintedanib treatment resulted insignificant inhibition of phosphorylated RTKs when tested in aphospho-RTK array experiment, little-to-no inhibition of cellproliferation was observed in the two Ewing sarcoma cell lines(A673, and CHP100). Interestingly, phosphorylated ErbB4 aswell as phosphorylated IGF-1R and insulin receptor were detectedon the phospho-RTK array. This strongly suggests that RTK path-ways other than PDGFRa, b such as IGF-1R (10), or ErbB4 (9) areinvolved in driving Ewing sarcoma progression.

Multikinase inhibitors such as imatinib, which block RTKsincluding c-Kit and PDGFR, have been used in clinical trials withmodest success (12). When we tested the efficacy of nintedanibagainst imatinib using in vitro cell proliferation assay and westernimmunoblotting, nintedanib treatment showed significantly bet-ter inhibition of cell proliferation as well as blockade of signalingpathways compared with imatinib. Nintedanib has been shownpreviously to inhibit MAP kinase as well as AKT signaling (25). Totest whether the observed inhibition of cell proliferation in thesynovial sarcoma cell line SYO-1 is indeed a result of p-ERKand p-AKT inhibition, we combined a MEK and AKT inhibitor(selumetinib and MK-2206, respectively) to mimic nintedanib-mediated inhibition of downstream signaling. As hypothesized,combined inhibition of p-ERKandp-AKT resulted in inhibition ofcell proliferation comparable with nintedanib treatment. Inter-estingly, combined treatment with nintedanib and selumetinibresulted in further inhibition of cell proliferation than nintedanibalone. This is probably the result of further suppression of MAPKsignaling (seen as p-ERK downregulation in Fig. 3C) after theaddition of selumetinib as confirmed by western immunoblot-ting. Notably, in the SYO-1 cell line, we were unable to observeany significant downregulation of p-ERK1/2 when treated withselumetinib alone or in combination with MK-2206. Treatmentwith selumetinib alone was also accompanied by an increase inp-AKT which could be a result of feedback inhibition after MAPK

Patwardhan et al.





signaling inhibition by selumetinib as has been reported inother cancers as a result of relief of negative feedback loop (36).Combined treatment with nintedanib and selumetinib, how-ever, prevented this feedback activation of p-AKT as well asresulted in almost complete downregulation of p-ERK1/2 signalon the Western blot analysis. It must also be noted thatnintedanib treatment did not result in any significant inhibi-tion of cell proliferation in A673 cell line which carries a BRAFmutation (V600E; ref. 37); however, combined inhibition ofMEKi and AKTi resulted in inhibition of cell proliferationwhich could be a result of greater suppression of p-ERK byMEKi treatment as seen on Western blot analysis.

Previous studies from our laboratory have reported that syno-vial sarcomas often show uniquely high levels of PDGFRa expres-sion and PDGFRa is primarily involved in rapamycin inducedfeedback activation of AKT (8). Expression of multiple FGF genesand growth stimulatory effect of FGFR-mediated p-ERK1/2 insynovial sarcomas has also been documented previously (20).Combined siRNA-mediated knockdown of PDGFRa and FGFR1,and 2 in the SYO-1 cell line resulted in inhibition of cell prolif-eration significantly higher than siRNA knockdown of any of theRTKs individually. This strongly supported our hypothesis thatnintedanib-mediated inhibition of PDGFR, FGFR pathways, anddownstream p-AKT and p-ERK1/2 pathways is indeed responsiblefor the observed antiproliferative effect in SYO-1 cell line. Finally,the SYO-1 xenograft model confirmed that nintedanib-mediatedinhibition of RTK signaling pathways results in significant sup-pression of tumor growth, decreased tumor vasculature which isfar better than the effects observed for imatinib treatment.

Taken together, data from this study strongly suggest thatnintedanib-mediated inhibition of PDGFR and FGFR in synovialsarcoma could have potential therapeutic implication given therole of these RTKs in this disease setting (8, 20). Downregulationof p-AKT and p-ERK1/2 by nintedanib results in decreased cell

proliferation and significant tumor growth suppression that issuperior to imatinib which has previously been tested in clinicaltrials in synovial sarcoma with no clinical benefit (ClinicalTrials.gov Identifier:NCT00031915; ref. 12).Data obtained in this studystrongly warrant further preclinical and clinical evaluation ofnintedanib in synovial sarcoma.

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

Authors' ContributionsConception and design: P.P. Patwardhan, G.K. SchwartzDevelopment of methodology: P.P. Patwardhan, G.K. SchwartzAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P.P. Patwardhan, E. Musi, G.K. SchwartzAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P.P. Patwardhan, E. Musi, G.K. SchwartzWriting, review, and/or revision of the manuscript: P.P. Patwardhan, E. Musi,G.K. SchwartzAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P.P. Patwardhan, G.K. SchwartzStudy supervision: P.P. Patwardhan

AcknowledgmentsThe authors would like to thank Dr. Frank Hilberg and Dr. Ulrike Weyer-

Czernilofsky from Boehringer Ingelheim for valuable feedback and helpfulscientific discussions. Funding for this work was provided by the Herbert IrvingComprehensive Cancer Center, Columbia University (to G.K. Schwartz).Finan-cial support for this work was provided by Herbert Irving ComprehensiveCancer Center, Columbia University College of Medicine (New York, NY).

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

Received March 27, 2018; revised July 25, 2018; accepted August 21, 2018;published first August 30, 2018.

References1. Borden EC, Baker LH, Bell RS, Bramwell V, Demetri GD, Eisenberg BL, et al.

Soft tissue sarcomas of adults: state of the translational science. Clin CancerRes 2003;9:1941–56.

2. Clark MA, Fisher C, Judson I, Thomas JM. Soft-tissue sarcomas in adults.N Engl J Med 2005;353:701–11.

3. Anderson JL, Denny CT, Tap WD, Federman N. Pediatric sarcomas: trans-lating molecular pathogenesis of disease to novel therapeutic possibilities.Pediatr Res 2012;72:112–21.

4. Daugaard S. Current soft-tissue sarcoma classifications. Eur J Cancer2004;40:543–8.

5. Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell 2010;141:1117–34.

6. Bai Y, Li J, Fang B, Edwards A, Zhang G, Bui M, et al. Phosphoproteomicsidentifies driver tyrosine kinases in sarcoma cell lines and tumors. CancerRes 2012;72:2501–11.

7. Fleuren EDG, Vlenterie M, van der Graaf WTA, Hillebrandt-Roeffen MHS,Blackburn J, Ma X, et al. Phosphoproteomic profiling reveals ALK andMETas novel actionable targets across synovial sarcoma subtypes. Cancer Res2017;77:4279–4292.

8. Ho AL, Vasudeva SD, La�e M, Saito T, Barbashina V, Antonescu CR, et al.PDGF receptor alpha is an alternative mediator of rapamycin-induced Aktactivation: implications for combination targeted therapy of synovialsarcoma. Cancer Res 2012;72:4515–25.

9. Mendoza-Naranjo A, El-Naggar A, Wai DH, Mistry P, Lazic N, Ayala FR,et al. ERBB4 confersmetastatic capacity in Ewing sarcoma. EMBOMolMed2013;5:1087–102.

10. O'Neill A, Shah N, Zitomersky N, Ladanyi M, Shukla N, Uren A, et al.Insulin-like growth factor 1 receptor as a therapeutic target in ewing

sarcoma: lack of consistent upregulation or recurrent mutation and areview of the clinical trial literature. Sarcoma 2013;2013:450478.

11. Torres KE, Zhu QS, Bill K, Lopez G, Ghadimi MP, Xie X, et al. ActivatedMET is a molecular prognosticator and potential therapeutic target formalignant peripheral nerve sheath tumors. Clin Cancer Res 2011;17:3943–55.

12. Chugh R, Wathen JK, Maki RG, Benjamin RS, Patel SR, Meyers PA, et al.Phase II multicenter trial of imatinib in 10 histologic subtypes of sarcomausing a bayesianhierarchical statisticalmodel. J ClinOncol 2009;27:3148–53.

13. Ho AL, Schwartz GK. Targeting of insulin-like growth factor type 1 receptorin Ewing sarcoma: unfulfilled promise or a promising beginning? J ClinOncol 2011;29:4581–3.

14. Heinrich MC, Corless CL, Duensing A, McGreevey L, Chen CJ, Joseph N,et al. PDGFRA activating mutations in gastrointestinal stromal tumors.Science 2003;299:708–10.

15. Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, Ishiguro S, et al.Gain-of-function mutations of c-kit in human gastrointestinal stromaltumors. Science 1998;279:577–80.

16. Demetri GD, vonMehrenM, Blanke CD, Van denAbbeele AD, Eisenberg B,Roberts PJ, et al. Efficacy and safety of imatinib mesylate in advancedgastrointestinal stromal tumors. N Engl J Med 2002;347:472–80.

17. Heinrich MC, Griffith D, McKinley A, Patterson J, Presnell A, Ramachan-dran A, et al. Crenolanib inhibits the drug-resistant PDGFRA D842Vmutation associated with imatinib-resistant gastrointestinal stromaltumors. Clin Cancer Res 2012;18:4375–84.

18. DicksonMA, Schwartz GK, KeohanML, D'Angelo SP, Gounder MM, Chi P,et al. Progression-Free survival among patients with well-differentiated or






dedifferentiated liposarcoma treated with CDK4 inhibitor palbociclib: aphase 2 clinical trial. JAMA Oncol 2016;2:937–40.

19. DicksonMA, TapWD, KeohanML, D'Angelo SP, GounderMM, AntonescuCR, et al. Phase II trial of the CDK4 inhibitor PD0332991 in patients withadvanced CDK4-amplified well-differentiated or dedifferentiated liposar-coma. J Clin Oncol 2013;31:2024–8.

20. Ishibe T, Nakayama T, Okamoto T, Aoyama T, Nishijo K, Shibata KR, et al.Disruption of fibroblast growth factor signal pathway inhibits the growthof synovial sarcomas: potential application of signal inhibitors to molec-ular target therapy. Clin Cancer Res 2005;11:2702–12.

21. Zhou WY, Zheng H, Du XL, Yang JL. Characterization of FGFR signalingpathway as therapeutic targets for sarcoma patients. Cancer Biol Med2016;13:260–8.

22. Zhang L, Hannay JA, Liu J, Das P, Zhan M, Nguyen T, et al. Vascularendothelial growth factor overexpression by soft tissue sarcoma cells:implications for tumor growth, metastasis, and chemoresistance. CancerRes 2006;66:8770–8.

23. Movva S, Wen W, Chen W, Millis SZ, Gatalica Z, Reddy S, et al. Multi-platformprofiling of over 2000 sarcomas: identification of biomarkers andnovel therapeutic targets. Oncotarget 2015;6:12234–47.

24. Patwardhan PP, Ivy KS, Musi E, de Stanchina E, Schwartz GK. Significantblockade of multiple receptor tyrosine kinases by MGCD516 (Sitravati-nib), a novel small molecule inhibitor, shows potent anti-tumor activity inpreclinical models of sarcoma. Oncotarget 2016;7:4093–109.

25. Hilberg F, Roth GJ, Krssak M, Kautschitsch S, Sommergruber W,Tontsch-Grunt U, et al. BIBF 1120: triple angiokinase inhibitor withsustained receptor blockade and good antitumor efficacy. Cancer Res2008;68:4774–82.

26. Hilberg F, Tontsch-GruntU, BaumA, LeAT,Doebele RC, Lieb S, et al. Tripleangiokinase inhibitor nintedanib directly inhibits tumor cell growth andinduces tumor shrinkage via blocking oncogenic receptor tyrosine kinases.J Pharmacol Exp Ther 2018;364:494–503.

27. Barkan B, Starinsky S, Friedman E, Stein R, Kloog Y. The Ras inhibitorfarnesylthiosalicylic acid as a potential therapy for neurofibromatosis type1. Clin Cancer Res 2006;12:5533–42.

28. CiomborKK, Bekaii-Saab T. Selumetinib for the treatment of cancer. ExpertOpin Investig Drugs 2015;24:111–123.

29. Pal SK, Reckamp K, Yu H, Figlin RA. Akt inhibitors in clinical developmentfor the treatment of cancer. Expert Opin Investig Drugs 2010;19:1355–66.

30. Jones RL, Judson IR. The development and application of imatinib. ExpertOpin Drug Saf 2005;4:183–91.

31. PardoOE, Latigo J, Jeffery RE, Nye E, PoulsomR, Spencer-Dene B, et al. Thefibroblast growth factor receptor inhibitor PD173074 blocks small celllung cancer growth in vitro and in vivo. Cancer Res 2009;69:8645–51.

32. Nguyen DT, Shayahi S. Pazopanib: approval for soft-tissue sarcoma. J AdvPract Oncol 2013;4:53–7.

33. Roth GJ, Binder R, Colbatzky F, Dallinger C, Schlenker-Herceg R, Hilberg F,et al. Nintedanib: from discovery to the clinic. J Med Chem 2015;58:1053–63.

34. Zietsch J, ZiegenhagenN,Heppner FL, ReussD, vonDeimling A,HoltkampN. The 4q12 amplicon in malignant peripheral nerve sheath tumors:consequences on gene expression and implications for sunitinib treatment.PLoS One 2010;5:e11858.

35. Yang J, Ylip€a€a A, Sun Y, Zheng H, Chen K, Nykter M, et al. Genomic andmolecular characterization of malignant peripheral nerve sheath tumoridentifies the IGF1R pathway as a primary target for treatment. Clin CancerRes 2011;17:7563–73.

36. Turke AB, Song Y, Costa C, Cook R, Arteaga CL, Asara JM, et al. MEKinhibition leads to PI3K/AKT activation by relieving a negative feedback onERBB receptors. Cancer Res 2012;72:3228–37.

37. Slotkin EK, Patwardhan PP, Vasudeva SD, de Stanchina E, Tap WD,Schwartz GK. MLN0128, an ATP-competitive mTOR kinase inhibitor withpotent in vitro and in vivo antitumor activity, as potential therapy for boneand soft-tissue sarcoma. Mol Cancer Ther 2015;14:395–406.


Patwardhan et al.




2018;17:2329-2340. Published OnlineFirst August 30, 2018.Mol Cancer Ther Parag P. Patwardhan, Elgilda Musi and Gary K. Schwartz Synovial Sarcomain Soft-tissue Sarcoma: Potential Therapeutic Implication for Preclinical Evaluation of Nintedanib, a Triple Angiokinase Inhibitor,

Updated version

10.1158/1535-7163.MCT-18-0319doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2018/08/30/1535-7163.MCT-18-0319.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/17/11/2329.full#ref-list-1

This article cites 37 articles, 19 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/17/11/2329To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-18-0319

http://mct.aacrjournals.org/content/suppl/2018/08/30/1535-7163.MCT-18-0319.DC1

http://mct.aacrjournals.org/content/17/11/2329.full#ref-list-1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/17/11/2329


preclinical evaluation of nintedanib, a triple angiokinase ... · angiokinase inhibitor, in...

Documents