Olaratumab Exerts Antitumor Activity in Preclinical Models ... · malignant rhabdoid tumor, and this activity was enhanced by combination with either doxorubicin or cisplatin. Conclusions:

Cancer Therapy: Preclinical

Olaratumab Exerts Antitumor Activity inPreclinical Models of Pediatric Bone and SoftTissue Tumors through Inhibition ofPlatelet-Derived Growth Factor Receptor aCaitlin D. Lowery1,Wayne Blosser1, Michele Dowless1, Shelby Knoche1, Jennifer Stephens1,Huiling Li1, David Surguladze1, Nick Loizos1, Debra Luffer-Atlas1, Gerard J. Oakley III1,Qianxu Guo1, Seema Iyer1, Brian P. Rubin2,3, and Louis Stancato1

Abstract

Purpose: Platelet-derived growth factor receptor a (PDGFRa)is implicated in several adult and pediatric malignancies, whereactivated signaling in tumor cells and/or cells within the micro-environment drive tumorigenesis and disease progression. Olar-atumab (LY3012207/IMC-3G3) is a humanmAb that exclusivelybinds to PDGFRa and recently received accelerated FDA approvaland conditional EMA approval for treatment of advanced adultsarcoma patients in combination with doxorubicin. In this study,we investigated olaratumab in preclinical models of pediatricbone and soft tissue tumors.

Experimental Design: PDGFRa expression was evaluated byqPCR and Western blot analysis. Olaratumab was investigated inin vitro cell proliferation and invasion assays using pediatricosteosarcoma and rhabdoid tumor cell lines. In vivo activity of

olaratumabwas assessed in preclinical mousemodels of pediatricosteosarcoma and malignant rhabdoid tumor.

Results: In vitro olaratumab treatment of osteosarcoma andrhabdoid tumor cell lines reduced proliferation and inhibitedinvasion driven by individual platelet-derived growth factors(PDGFs) or serum. Furthermore, olaratumab delayed primarytumor growth in mouse models of pediatric osteosarcoma andmalignant rhabdoid tumor, and this activity was enhanced bycombination with either doxorubicin or cisplatin.

Conclusions: Overall, these data indicate that olaratumab,alone and in combination with standard of care, blocks thegrowth of some preclinical PDGFRa-expressing pediatric boneand soft tissue tumor models. Clin Cancer Res; 24(4); 847–57.�2017 AACR.

IntroductionThe platelet-derived growth factor (PDGF) pathway is com-

posed of two receptors (PDGFRa and PDGFRb), four homodi-meric ligands (PDGF-AA, -BB, -CC, and -DD), and one hetero-dimeric ligand (PDGF-AB). After ligand binding, PDGFRa and/orPDGFRb form complexes as homo- or heterodimers, resulting inreceptor transphosphorylation and activation of downstreamsignaling pathways, which in turn regulate normal cellular pro-cesses, such as proliferation, migration, and survival (1). Aberrantactivation of the PDGF pathway through overexpression of key

nodes or receptor mutation often facilitates tumorigenesis anddisease progression across several cancer subtypes (2, 3). Inaddition, PDGF signaling in tumor-associated stromal cells pro-motes fibroblast activation and angiogenesis (4–7).

The PDGF pathway has been implicated in bone and soft tissuesarcoma, a collection of mesenchymal malignancies comprisedof nearly 80 distinct histologies. Olaratumab (LY3012207/IMC-3G3) is a fully human mAb that specifically binds to andinhibits PDGFRa (8). In a phase II clinical study, olaratumab incombination with doxorubicin improved advanced adult sarco-mapatient outcomewith amedian overall survival benefit of 11.8monthswhen comparedwith doxorubicin alone (9).On the basisof these data, olaratumab received accelerated approval from theFDA and conditional approval from the European MedicinesAgency for treatment of advanced soft tissue sarcoma in combi-nation with doxorubicin in adult patients.

Sarcoma subtypes occurring primarily in the pediatric popu-lation, including osteosarcoma, rhabdomyosarcoma, and Ewingsarcoma, account for approximately 15% of childhood cancers(10). Despite intensive multimodal therapy, which typicallyincludes a combination of chemotherapies, surgery, and/or radio-therapy, the current 5-year overall survival rate for pediatricsarcoma patients is approximately 60%; for those who experiencea relapse or havemetastatic disease, survival drops to only 20% to30%(11).Malignant rhabdoid tumor (MRT) is ahighly aggressivepediatric cancer typically occurring in the kidney and soft tissuesor the central nervous system [where it is referred to as atypicalteratoid/rhabdoid tumor (AT/RT)] and is characterized by a loss

1Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana. 2Depart-ment of Pathology, Robert J Tomsich Pathology and Laboratory MedicineInstitute and Cleveland Clinic, Cleveland, Ohio. 3Department of Cancer Biology,Robert J Tomsich Pathology and Laboratory Medicine Institute and ClevelandClinic, Cleveland, Ohio.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Current address for S. Knoche: 1) Fred & Pamela Buffett Cancer Center and 2)Eppley Institute for Research in Cancer &Allied Diseases, University of NebraskaMedical Center, Omaha, Nebraska.

Corresponding Author: Louis F. Stancato, Eli Lilly and Company, Lilly CorporateCenter, Indianapolis, IN 46285. Phone: 1-317-655-6910; Fax: 1-317-276-1414;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-1258

�2017 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 847

on October 7, 2020. © 2018 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst November 30, 2017; DOI: 10.1158/1078-0432.CCR-17-1258

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-17-1258&domain=pdf&date_stamp=2018-1-29

http://clincancerres.aacrjournals.org/


of SMARCB1 (12, 13). Currently, no standard of care exists for thispatient population, and overall survival remains poor (14–16).For pediatric cancer survivors, the possibility of debilitating long-term side effects, chronic health conditions, and even secondarycancers resulting from demanding therapeutic regimens remains(17, 18). Therefore, it is of the utmost importance to identify andevaluate targeted agents in the pediatric setting to improve patientoutcome.

Although the prevalence of PDGFRA genetic aberrations inpediatric cancer is reported to be only about 2% (19), membersof the PDGF pathway are highly expressed in several subtypes ofpediatric bone and soft tissue tumors, including rhabdomyosar-coma (20, 21), synovial sarcoma (22), osteogenic sarcoma (23),Ewing sarcoma (24), andMRT (25). In addition, PDGFRa expres-sion is linked to adverse outcomes in pediatric patients withrhabdomyosarcoma (26). In this study, we investigated olaratu-mab alone and in combination with standard-of-care (SOC)chemotherapy in preclinical models of pediatric bone and softtissue tumors.

Materials and MethodsTest compounds

Olaratumab (LY3012207/IMC-3G3, Eli Lilly and Company)was prepared in PBS for both in vitro and in vivo use. The mouseanti-PDGFRa antibody, 1E10, was also prepared in PBS for in vivoexperiments.

Cell cultureThe HuO9 osteosarcoma cell line was purchased from the

Japanese Collection of Research Bioresources (JCRB) Cell Bank.Rh18, Rh28, Rh36, and Rh41 rhabdomyosarcoma cell lines wereobtained from St. Jude Children's Research Hospital (Memphis,TN). Other pediatric cancer cell lines and WS-1 normal humanfibroblasts were ordered from ATCC. Cell lines were Mycoplasmanegative prior to freezing working stocks. The frozen workingstocks of each cell line were authenticated by STR-based DNAprofiling and multiplex PCR. The genetic profiles for the sampleswere identical to the genetic profiles reported for these cell lines.BT-12 and BT-16 AT/RT cell lines were a gift from Dr. PeterHoughton (Greehey Children's Cancer Research Institute, San

Antonio, TX). Cell line details are listed in Supplementary TableS1. All cells were maintained at 37�C and 5% CO2 in tissueculture–treated flasks. Cells were used for experiments within10 to 20 passages and then discarded.

Reagents and antibodiesSpecific details regarding recombinant human PDGFs (AA, BB,

CC, DD, and AB) are displayed in Supplementary Table S2.Antibodies against PDGFRa, PDGFRa Y754, PDGFRb, PDGFRb751, ERK1/2, ERK1/2 T202/Y204, AKT, and AKT S473 werepurchased from Cell Signaling Technology or Santa Cruz Bio-technology; details are included in Supplementary Table S2.

Western blot analysisTo determine receptor status and baseline pathway signaling in

a panel of pediatric bone and soft tissue tumor lines, cells wereserum starved overnight and harvested into 1% SDS lysis buffercontaining 1� HALT protease and phosphatase inhibitor(Thermo Fisher Scientific, cat #78441). To determine effects ofolaratumab treatment, cell lines were treated with either an IgGcontrol antibody or olaratumab for 15 minutes and then stim-ulated with individual PDGFs (final concentration, 5 nmol/L) foran additional 15 minutes. Cells were immediately washed withPBS and harvested into 1% SDS lysis buffer containing 1� HALTprotease and phosphatase inhibitor.

Protein expression and phosphorylation status was assessed bySDS-PAGE and immunoblotting as described previously (27).Briefly, whole-cell lysates (30–50 mg of protein/sample) wereseparated on gradient Tris-Glycine protein gels (Novex, ThermoFisher Scientific) and transferred to nitrocellulose via TransBlotTurbo (Bio-Rad, cat #170–4159). After blocking with 5%milk inTBST, membranes were probed with primary antibody overnightat 4�C, washed 3 times with TBST, and incubated with secondaryantibodies for 1 hour at room temperature. Following 3 washeswith TBST, membranes were developed with SuperSignal WestFemtoChemiluminescent Substrate (Thermo Fisher Scientific, cat#34095) and imaged with a Bio-Rad ChemiDoc XRS.

Cell proliferation assayTo determine antiproliferation EC50 values for olaratumab,

pediatric cancer cells were seeded in 96-well plates. The next day,cells were incubated with increasing concentrations of olaratu-mab or the IgG antibody control in serum-free media for 2 hoursat 37�C and then stimulated with individual PDGFs. Cell prolif-eration was assayed by CellTiter Glo Luminescent Cell ViabilityAssay (Promega, cat #G7571) 72 hours after stimulation. Datawere normalized to the unstimulated IgG-treated control. RelativeEC50 values were calculated using GraphPad Prism software.Experiments were repeated in triplicate and data from a repre-sentative experiment are displayed in Table 1.

To evaluate the effects of combination treatment on pediatriccancer cell proliferation, cells were plated in 96-well microtiterplates in normal media containing 10% FBS. The next morning,mediawere changed tomedia containing 0.5%FBS. After 4 hours,cells were costimulated with 5 nmol/L PDGF-AA and 5 nmol/LPDGF-CC and treated with 1 mmol/L olaratumab, 0.5 mmol/Ldoxorubicin, or 2mmol/L cisplatin or combination of olaratumaband chemotherapy. Cell proliferation was assayed after 72 hoursby CellTiter Glo. Luminescence was normalized to the average ofthe untreated control for each cell line. Results are presented as the

Translational Relevance

Olaratumab (LY3012207/IMC-3G3) is a human mAbagainst the platelet-derived growth factor receptor a(PDGFRa), which recently received accelerated approval fromthe FDA and conditional EMA approval for treatment of adultadvanced soft tissue sarcoma patients in combination withdoxorubicin. Pediatric patients with bone and soft tissuetumors are treated with intensive, multimodal therapeuticregimens that often result in debilitating long-term side effects.PDGFRa has been implicated in these pediatric malignancies.In this study, we demonstrate the antitumor activity of olar-atumab in combination with standard chemotherapy in pre-clinical models of pediatric bone and soft tissue tumors. Thesedata support clinical evaluation of olaratumab in combina-tion with chemotherapy in pediatric patients with solidtumors (NCT02677116).

Lowery et al.

Clin Cancer Res; 24(4) February 15, 2018 Clinical Cancer Research848




mean of duplicate or triplicate experiments (indicated in figurelegends) � SEM. Statistical significance was determined by theStudent t test.

Evaluation of cell invasionInvasion assays were performed using the CultreCoat LowBME

Cell Invasion Assay (Trevigen, cat #3481–096-K) per the manu-facturer's instructions. Briefly, 2.5�104 pediatric cancer cells wereseeded in the upper chamber of the well with 1 mmol/L IgGcontrol antibody or olaratumab. Serum-free media was added tothe lower chamber, and individual PDGFs were used as a che-moattractant. Media with 10% FBS as a chemoattractant served asa positive control. Final concentration of ligand was 5 nmol/L foreach,with the exceptionof PDGF-DD,whichwas20nmol/L. After48 hours, cells able to invade through themembrane to the lowerchamber were dissociated, and plates were read at 485 nmexcitation, 520 nm emission. Results were normalized to theserum-free media, IgG control for each cell line. Results arepresented as the average of duplicate or triplicate experiments(indicated in figure legends)� SEM. Significance was determinedby the Student t test.

Expression profiling of patient-derived xenograft mousemodels

Samples from pediatric sarcoma patient-derived xenografts(PDX) were obtained from Champions Oncology, START Dis-covery, andOncotest (Charles River Laboratories). Frozen tumorswere manually dissociated using a TissueLyser (Qiagen).RNA extraction was performed using the MagMAX-96 total RNAIsolation Kit (Thermo Fisher Scientific, cat#AM1830) per themanufacturer's instructions. The High-Capacity cDNA ReverseTranscription Kit (Thermo Fisher Scientific, cat #4374967) wasused to generate cDNA. TaqMan Gene Expression Master Mix(cat#4369016) and TaqMan probes for human PDGFRA,PDGFRB, PDGFA, PDGFC, and 18Swere purchased from ThermoFisher Scientific (cat #4331182). Probe IDs are as follows:PDGFRA – HS00998018_m1; PDGFRB – HS01019589_m1;

PDGFA – HS00964426_m1; PDGFC – HS01044219_m1;and 18S – HS03928985_g1. A standard curve was generated foreach gene using a plasmidDNA template, and data were extractedusing this curve. Values are expressed as copies of mRNA/ngof cDNA.

In vivo evaluation of olaratumabA colorimetric ELISA was used to quantify the concentration

of olaratumab in mouse serum and mouse plasma. Briefly, thePDGFRa extracellular domain was immobilized in coatingbuffer in 96-well microtiter plate. After washing the plates,wells were blocked with block/diluent buffer between 1 and 2hours. Plates were then washed and analytes (diluted 1:500 inblock/diluent buffer) were added to each well, incubated forapproximately 1 hour, and washed. Goat a-human IgG F(ab)2was added to each well and incubated for approximately 1hour. The plates were washed, followed by the addition oftetramethylbenzidine to each well and incubation for 15 min-utes. The reaction was terminated with the addition of a StopSolution. Plates were read at 450 nm for detection of olaratu-mab and at 620 nm for background signal. The concentrationof olaratumab was determined on a standard curve.

In vivo studies were performed in accordance with AmericanAssociation for Laboratory Animal Care institutional guide-lines. A-204 and HuO9 in vivo experiments were approved bythe Eli Lilly and Company Animal Care and Use Committee. Toevaluate the effects of olaratumab in HuO9 and A-204 cell line–derived xenografts, cells were harvested during log-phasegrowth and resuspended in Hank balanced salt solution. Cellsuspensions containing 10 � 106 (HuO9) or 5 � 106 (A-204)cells (0.2 mL total volume) were subcutaneously injected intothe right flank of female athymic nude mice and tumor growthmonitoring started 1 week postinjection. When tumor volumesaveraged 200 mm3, mice were randomized into treatmentgroups (n ¼ 6/group) based on tumor volume and bodyweight. In vivo experiments utilizing the osteosarcoma PDXmodel CTG-1095 were conducted at Champions Oncology.

Table 1. Receptor status and relative EC50 values for proliferation inhibition in a panel of pediatric bone and soft tissue tumor cell lines

Relative EC50 (nmol/L) - antiproliferationCell line Disease PDGFRaa PDGFRba þAA þBB þCC þDD þAB

A-204 MRT + � 45.74 170.7 71.42 NDb 123BT-12 AT/RT � þ Not evaluatedBT-16 AT/RT � � Not evaluatedG-401 MRT � + Not evaluatedG-402 MRT + þ 167.3 498.2 70.95 ND NDRD Embryonal RMS � � Not evaluatedRD-ES Ewing sarcoma � � Not evaluatedRh18 Alveolar RMS � þ Not evaluatedRh28 Alveolar RMS � � Not evaluatedRh36 Embryonal RMS � � Not evaluatedRh41 Alveolar RMS þ � Not evaluatedSJCRH30 Alveolar RMS � � Not evaluatedHuO9 Osteosarcoma + � 204.7 311.3 35.11 20.1 164KHOS/NP Osteosarcoma þ þ NDMG-63 Osteosarcoma + + 52.37 186 38.03 ND 101.1MNNG/Hos Osteosarcoma þ þ NDSaos-2 Osteosarcoma þ � Not evaluatedSJSA-1 Osteosarcoma þ + NDWS-1 Normal fibroblast + + Not evaluated

Abbreviations: ND, not detected; RMS, rhabdomyosarcoma.aExpression determined by Western blot analysis; boldface indicates strong signal observed.bND, not determined due to ambiguous or nonconverged curves.

Preclinical Activity of Olaratumab in Pediatric Tumor Types

www.aacrjournals.org Clin Cancer Res; 24(4) February 15, 2018 849




The CCSARC005 osteosarcoma PDX model was generated andevaluated at Covance, Inc.

Mice with A-204 xenografts were treated with 40 mg/kg IgG orolaratumab three times a week for 4 weeks. Animals bearingHuO9 or CTG-1095 tumors were treated with control (80 mg/kgIgG, i.p. þ vehicle), cisplatin (5 mg/kg once weekly, i.p.), doxo-rubicin (5 mg/kg once weekly, i.v.), olaratumab þ 1E10 (60 and20 mg/kg, respectively, twice weekly, i.p.), or combination ofolaratumab þ 1E10 and a SOC for 5 weeks. Mice withCCSARC005 xenograft tumors were treated with control(60 mg/kg IgG, i.p. þ vehicle), olaratumab (60 mg/kg, twiceweekly, i.p.), cisplatin (4 mg/kg once weekly, i.p.), or a combi-nation of the two. Tumor volume was monitored twice weekly.For all studies, experiments were terminated within 2 weeksfollowing the end of the treatment period.

ResultsExpression of PDGF pathway components is detected in severalpediatric sarcoma cell lines

Gene expression of PDGFRA and PDGFRB and of PDGFRa-activating ligands PDGFA and PDGFCwas evaluated in 7 pediatricbone and soft tissue tumor cell lines (Fig. 1). The osteosarcomacell line HuO9 and the MRT cell line A-204 exclusively expressedPDGFRA, while the transcripts of both receptors were detected inMG-63, KHOS/NP, andMNNG/Hos osteosarcoma cell lines (Fig.1A). A-204, HuO9, and MG-63 also expressed PDGFA and/orPDGFC, indicating the potential for autocrine activation ofPDGFRa (Fig. 1B). Gene expression correlated with PDGFRa andPDGFRb protein expression (Supplementary Fig. S1). Further-more, PDGFRa expression was detected in G-402 MRT cells butnot in G-401 MRT cells nor in BT-12 or BT-16 AT/RT cells(Supplementary Fig. S1A). Expression of PDGFRa and bwas alsonot observed in the embryonal rhabdomyosarcoma cell line RDor the Ewing's sarcoma cell line RD-ES (Supplemental Fig. S1B).Surprisingly, although previous studies reported robust PDGFRaexpression in human and murine rhabdomyosarcoma (20, 28),low levels of PDGFRA and PDGFRB transcript were detected in thealveolar rhabdomyosarcoma cell line SJCRH30 (Fig. 1A). Simi-

larly, PDGFRa was not detected at the protein level in 3 of 4rhabdomyosarcoma cell lines (Supplementary Fig. S1C). As olar-atumab is a selective anti-PDGFRa antibody, this study focusedon receptor-expressing pediatric models (Table 1; Fig. 1; andSupplementary Fig. S1). Receptor status for all evaluated cell linesis summarized in Table 1.

PDGFRa phosphorylation is blocked by olaratumab inpediatric osteosarcoma cell lines

Olaratumab specifically binds PDGFRa and prevents ligandbinding to the receptor and stimulating transphosphorylation(8). The influence of PDGFRb expression and potentialPDGFRa:PDGFRb heterodimers on olaratumab activity is cur-rently not well understood; therefore, in vitro evaluation ofolaratumab was conducted using cell lines with varying expres-sion levels of PDGFRa and/or PDGFRb. Furthermore, BT-16, anAT/RT cell line that does not express either receptor, wasinvestigated alongside PDGFRþ cell lines as a negative control.The WS-1 normal fibroblast cell line was used as a nontrans-formed cell control.

Phosphorylation status of the PDGF receptors and their down-stream effectors was assessed following stimulation by individualPDGFs (Fig. 2; Supplementary Figs. S2 and S3A). Phosphoryla-tion of PDGFRa at tyrosine 754 (Y754) was detected in PDGFRa-positive cell lines, albeit to varying degrees, and indicated receptoractivation. Stimulation with PDGF-BB or -DD elicited phosphor-ylation of PDGFRb at tyrosine 771 (Y771) in PDGFRb-expressingcell lines (Fig. 2B and C; Supplementary Fig. S2); however,phospho-PDGFRb was not detected in BT-12 AT/RT cells (Sup-plementary Fig. S3A). Stimulation of HuO9 and MG-63 OS cellswith PDGFs resulted in activation of downstream PI3K andMAPK pathway signaling [as measured by phosphorylation ofAKT at serine 473 (S473) and ERK1/2 threonine 202/tyrosine 204(T202/Y204); Fig. 2A and B]. Baseline AKT and ERK1/2 phos-phorylation was observed in all MRT and AT/RT cell lines eval-uated regardless of PDGF receptor status (Fig. 2; SupplementaryFig. S3A). Stimulation with any PDGF (in the case of A-204 andG-402; Fig. 2A and B) or PDGFRb-activating ligands (in G-401

Figure 1.

Pediatric bone and soft tissue tumor cell lines express platelet-derived growth factors and their receptors. A panel of pediatric bone and soft tissue tumor cell lineswere evaluated for receptor and ligand expression by qPCR. A, Results are plotted as expression of PDGFRA by PDGFRB on a log10 scale. Note: expressionof PDGFRB did not exceed 1,000 copies/ng cDNA. B, Expression of PDGFA and PDGFC are presented on a linear scale.

Lowery et al.





and BT-12 cells; Fig. 2C; Supplementary Fig. S3A) resulted in adetectable increase in pAKT and pERK1/2. Surprisingly, PDGF-CCstimulation elicited a marked increase in AKT and ERK1/2 phos-phorylation in BT-16 cells, suggesting that PDGFRa may beexpressed below the level that can be detected by Western blotanalysis (Supplementary Fig. S3A).

As expected, incubation of cells with olaratumab prior toligand stimulation blocked PDGFRa Y754 phosphorylation inPDGFRa-positive cell lines (Fig. 2; Supplementary Fig. S2).Interestingly, olaratumab blocked activation of AKT andERK1/2 resulting from stimulation with any ligand in HuO9cells (Fig. 2A). Similarly, phosphorylation of AKT was stronglyinhibited with olaratumab treatment in A-204 cells, whereasERK1/2 phosphorylation was reduced to baseline signal withtreatment (Fig. 2A). PDGFRb phosphorylation at Y771 wassustained following olaratumab treatment in PDGFRb-expres-sing MG-63 and G-401 as well as WS-1 fibroblasts (Fig. 2B andC; Supplementary Fig. S2). However, PDGF-BB and -AB-stim-ulated PDGFRb phosphorylation was noticeably reduced witholaratumab treatment in G-402 MRT cells (Fig. 2B). Reducedphosphorylation of AKT and ERK1/2 in PDGFRa/PDGFRbcoexpressing cells (Fig. 2B; Supplementary Fig. S2) wasobserved only when olaratumab prevented PDGFRa stimula-tion by PDGF-AA, -AB, or (with the exception of MG-63) PDGF-CC; furthermore, AKT and ERK1/2 phosphorylation in G-401cells did not change with olaratumab treatment (Supplemen-tary Fig. S2A), indicating that PDGFRb homodimeric signalingis not affected by olaratumab. Interestingly, olaratumab treat-ment slightly reduced pAKT and pERK1/2 in BT-16 cells stim-ulated with PDGF-AA or -CC (Supplementary Fig. S3A).

PDGF-stimulated cell proliferation is inhibited by olaratumabalone and in combination with chemotherapeutic agents

As the PDGF pathway supports tumor cell growth (1), thepotential antiproliferative effect of olaratumab was assessed ina panel of pediatric bone and soft tissue tumor cell lines(summarized in Table 1). In addition, olaratumab was eval-uated in combination with either doxorubicin or cisplatin inosteosarcoma and rhabdoid tumor cell lines (SupplementaryFigs. S3B, S3C, and S4; Supplementary Table S3). Stimulationwith PDGF-AA and PDGF-CC increased cell proliferation inPDGFRa-expressing cell lines when compared with unstimu-lated control cells (Supplementary Fig. S4A–S4D; P valuesreported in Supplementary Table S3). The increase in A-204and HuO9 proliferation in response to ligand stimulation wasmodest when compared with the unstimulated control; how-ever, these cell lines also express PDGFRa-activating ligandsand likely activate the receptor in an autocrine manner (Sup-plementary Fig. S4A and S4B). As expected, PDGFRa-nullrhabdoid cell lines G-401, BT-12, and BT-16 did not respondto PDGF-AA/-CC treatment with increased proliferation (Sup-plementary Figs. S3B, S3C and S4E).

Doxorubicin significantly reduced tumor cell proliferationregardless of receptor status or stimulationwith exogenous ligand(Supplementary Figs. S3B, S3C, and S4). Combination of doxo-rubicin with olaratumab in A-204 andHuO9modestly enhancedthis reduction by an additional 10% (Supplementary Fig. S4A andS4B). Cisplatin plus olaratumab resulted in a 20% to 30% furtherreduction in proliferation in PDGFRa-positive osteosarcoma celllines when compared with single-agent treatment (Supplemen-tary Fig. S4A–S4D). As expected, olaratumab treatment did not

Figure 2.

Olaratumab prevents ligand-induced phosphorylation of PDGFRa. A–C, A-204 and HuO9 cells (A), which only express PDGFRa, G-402 and MG-63 cells (B),which express both PDGF receptors, or G-401 cells (C), which are PDGFRb positive, were treated with 1 mmol/L IgG or olaratumab for 15 minutesand then stimulated with individual PDGFs (5 nmol/L). Whole-cell lysates were probed for phosphorylation of PDGF receptors and downstreameffectors AKT and ERK1/2.






affect proliferation in cells that do not express the receptor(Supplementary Figs. S3B, S3C, and S4E).

Olaratumab has varying effects on PDGF-driven tumor cellinvasion

The ability of olaratumab to modulate cell invasion throughPDGFRa inhibition was also evaluated in osteosarcoma andrhabdoid tumor cell lines (Fig. 3; Supplementary Fig. S3D and

S3E). Serum elicited tumor cell invasion when used as a chemoat-tractant regardless of PDGF receptor status. Interestingly, serum-stimulated A-204, HuO9, and G-402 cell invasion was reducedwith olaratumab treatment by approximately 50% when com-pared with the IgG control treatment group (Fig. 3A–C). Unex-pectedly, A-204 and HuO9 cell invasion did not increase sub-stantially when any of the individual PDGFs were used as thechemoattractant, which again suggests that PDGFRa is already

Figure 3.

Olaratumab reduces osteosarcoma andMRT cell invasion in vitro.A–E,A-204 (A), HuO9 (B), G-402 (C), MG-63 (D), or G-401 cells (E) were evaluated for cell invasionusing a simplified Boyden chamber assay. Cells were incubated with IgG or olaratumab; individual PDGFs (5 nmol/L each, with the exception of 25 nmol/LPDGF-DD) or FBS served as chemoattractants. After 48 hours, the number of invasive cells was quantified using a fluorescence-based method and normalizedto the serum-free media (SFM) IgG control. Results are presented as the summary of duplicate (G-402) or triplicate experiments � SEM. P values werecalculated using the Student t test. Significance indicators: � , P < 0.05; �� , P < 0.001.

Lowery et al.





activated via an autocrine loop (Fig. 3A and B). In contrast, allPDGFs (with the exception of PDGF-AB in the case of MG-63)promoted invasion of cells that coexpressed PDGFRa andPDGFRb (Fig. 3C and D). MG-63 cells were particularly respon-sive to the ligands when compared with the 10% FBS controlchemoattractant (Fig. 3D). Olaratumab treatment significantlyblockedHuO9,G-402, andMG-63 cell invasion stimulated by thedifferent ligands; however, although olaratumab treatmentreduced HuO9 invasion, these changes were less than 20% ofthe untreated control. Furthermore, olaratumab did not inhibitthe invasive potential of tumor cells that lacked PDGFRa expres-sion (G-401, BT-12, and B-16; Fig. 3E; Supplementary Fig. S3Dand S3E).

Olaratumab, alone or in combination with chemotherapy,delays xenograft growth

Single-agent olaratumab was first investigated in mice bearingA-204 MRT xenografts, as the A-204 cell line expresses bothPDGFRa and its activating ligand PDGFC. Tumor-bearing ani-

mals were given 40 mg/kg olaratumab or control antibody threetimes weekly for 4 weeks. Using this dosing schedule, olaratumabtreatment significantly delayed A-204 xenograft growth in alltreated animals,with stable disease observed in 55%of the treatedarm (Fig. 4A and B; Supplementary Table S4).

A dose-dependent increase in mean trough serum concentra-tion was measured following olaratumab dose escalation in twocell line–derived xenograft mouse models (Supplementary Fig.S5A). On the basis of these results, 60 mg/kg olaratumab twiceweekly was selected as the dosing schedule used in future in vivoexperiments. Trough mean serum concentrations of olaratumabwere determined in several cell line–derived and PDX mousemodels and were found to be relatively consistent across theevaluated models (Supplementary Fig. S5B).

Olaratumab was further investigated in mice bearingHuO9 osteosarcoma xenografts (Fig. 4C and D; SupplementaryTable S4). To interrogate and potentially disrupt the relation-ship between the human tumor and mouse stroma, the mouseanti-PDGFRa antibody 1E10 was given in conjunction with

Figure 4.

Olaratumab inhibits tumor growth in A-204 and HuO9 cell line–derived xenograft mouse models. A and B, Mice with A-204 subcutaneous xenografts were treatedwith either 40 mg/kg IgG control or 40 mg/kg olaratumab three times weekly for 4 weeks. A, Average tumor volume � SEM (n ¼ 11/group) during thetreatment period is shown.B,Waterfall plot of %Dtumor/control or %regression for individual animals at day 30. One animal in the treatment armwas found dead onday 17 andwas not included in downstream analysis. C andD,Mice bearing HuO9 subcutaneous xenografts were treated with olaratumab (60mg/kg)/1E10 (20mg/kg) twice weekly and/or cisplatin or doxorubicin (5 mg/kg) once weekly. Treatment began at day 44 (dotted line) and continued for 5 weeks. C, A single HuO9in vivo experiment is displayed in two separate graphs with the same control and olaratumab/1E10 arms but different chemotherapy-containing arms containingchemotherapy (left, cisplatin; right, doxorubicin). The graph displays average tumor volume � SEM (n ¼ 6/group). D, Waterfall plot of individual %Dtumor/control or %regression at day 60. P values for tumor growth curves are displayed in Supplementary Table S4. PD, progressive disease (>10% growth); SD, stabledisease (�50%–10% growth); PR, partial regression (��50% and >14 mm3).






olaratumab. In addition to single-agent olaratumab/1E10 treat-ment, combination with doxorubicin or cisplatin (two SOCagents used in the treatment of osteosarcoma patients) was alsoevaluated. Administration of olaratumab/1E10, cisplatin, ordoxorubicin alone delayed tumor growth when compared withthe control arm (Fig. 4C). Furthermore, stable disease wasachieved in 1 or 2 animals in each group (Fig. 4D). Thecombination of olaratumab/1E10 plus cisplatin (Fig. 4C, left)resulted in stable disease in 4 of 6 mice (Fig. 4D), whereascombination with doxorubicin (Fig. 4C, right) elicited a partialregression in one animal in addition to 3 of 6 animals withstable disease.

PDX models of pediatric bone and soft tissue sarcomawere profiled for expression of PDGFRA, PDGFRB, PDGFA, andPDGFC (Supplementary Table S5). Expression of each gene wasvariable, both within and between sarcoma subtypes. PDGFRAtranscriptwas detectable in 50%(11/22) ofmodels assayedwith acutoff of at least 500 copies mRNA/ng cDNA. OsteosarcomaPDXs made up more than half (6/11) of the PDGFRA-expressingmodels. Activity of olaratumab was evaluated in the CTG-1095model, an osteosarcoma PDX that expressed moderate levels ofPDGFRA, PDGFRB, and PDGFC (Supplementary Table S5). Olar-atumab/1E10 treatment impaired tumor growth, as evidenced bya reduction in tumor volume when compared with the control

Figure 5.

Olaratumab, alone or in combination with cisplatin, reduces tumor volume in two osteosarcoma PDXmousemodels. A and B,Mice bearing CTG-1095 osteosarcomaPDX tumors were treated with olaratumab/1E10 (60 mg/kg olaratumab/20 mg/kg 1E10) twice weekly and/or SOC [cisplatin or doxorubicin (5 mg/kg)] onceweekly (n ¼ 5/group). One animal in the olaratumab/1E10 group was sacrificed prior to experiment termination due to circumstances unrelated to treatment.A, Dosing began on day 0, and average tumor volume � SEM over the 5-week dosing period is shown. B, Waterfall plot of %Dtumor/control or %regressionfor individual animals at day 21. C and D, Animals with CCSARC005 osteosarcoma PDX tumors were treated with olaratumab (60 mg/kg) twice weeklyand/or 4 mg/kg cisplatin once weekly (n ¼ 5/group). C, Dosing period is indicated by the dotted vertical lines. Average tumor volume � SEM is shown.D, Waterfall plot of %Dtumor/control or %regression for individual mice at day 62. P values for tumor growth curves are displayed in SupplementaryTable S4. PD, progressive disease (>10% growth); SD, stable disease (�50%–10% growth); PR, partial regression (��50% and >14 mm3).

Lowery et al.





arm (Fig. 5A and B; Supplementary Table S4). Although doxo-rubicin did not demonstrate any activity in the xenograft tumors,single-agent cisplatin significantly delayed tumor growth. Inaddition, stable disease was achieved in 2 animals receivingolaratumab/1E10 in combination with cisplatin (Fig. 5B).

Olaratumab treatment, alone or in combination with cisplatin,was also investigated in the CCSARC005 osteosarcoma PDXmodel. IHC analysis of xenograft tumors revealed that a smallnumber of tumor cells express PDGFRa; however, PDGFRbwas readily detectable by Western blot analysis (SupplementaryFig. S6). Treatment with olaratumab or cisplatin alone had noeffect on tumor growth when compared with tumors from vehi-cle-treated animals (Fig. 5C). The combination of olaratumaband cisplatin significantly inhibited xenograft tumor growthwith tumors in 2 of 5 mice meeting the criteria for stable disease(Fig. 5C and D) and the remaining mice within the cohortresponding with strong tumor growth delay. Tumors from thecombination arm were significantly smaller when comparedwith vehicle- or single agent–treated tumors at study termination(Fig. 5C; Supplementary Table S4).

DiscussionAberrant expression and/or activation of PDGFRa is impli-

cated in several subtypes of adult and pediatric sarcoma andsupports protumorigenic processes in both the tumor cells andassociated stroma (1). Therefore, anti-PDGFRa therapies are ofhigh interest; several molecules targeting PDGFRa have beendeveloped and evaluated both preclinically and clinically inadult and pediatric malignancies (29–31). Olaratumab(LY3012207/IMC-3G3) is a fully human IgG1 mAb, whichspecifically binds to PDGFRa, thus inhibiting ligand-bindingand receptor activation (8). Following a pivotal randomizedphase II trial, olaratumab received accelerated FDA approvaland conditional EMA approval for treatment of advanced softtissue sarcoma in adult patients in combination with doxoru-bicin (9). In this study, we demonstrate olaratumab activity inpreclinical pediatric bone and soft tissue tumor models. Olar-atumab specifically blocked phosphorylation of PDGFRa andattenuated associated downstream MAPK and PI3K pathwaysignaling in PDGFRa-positive tumor cell lines. Furthermore, asa single agent, olaratumab significantly delayed osteosarcomaand rhabdoid xenograft growth; combination with doxorubicinor cisplatin resulted in stable disease in three mouse models ofhuman osteosarcoma.

Therapeutic targeting of PDGFRa with small-molecule inhi-bitors, such as sunitinib and imatinib, has been investigatedpreclinically and clinically in multiple pediatric cancer types,including osteosarcoma and MRT (25, 32–35). As many ofthese drugs are designed to bind the ATP site within the kinasedomain, target specificity is often lacking, resulting in inhibi-tion of multiple receptor tyrosine kinases and associated tox-icity (34, 36). This target promiscuity makes it difficult to assignthe direct contribution of PDGFRa to tumor growth and cancercell survival in preclinical studies using small-molecule inhi-bitors. In contrast, mAbs are engineered toward a specific target,and cross-reactivity has not been reported (36). Olaratumabblocks ligand binding and receptor dimerization by specificallybinding to PDGFRa and as such is able to suppress potentialkinase domain-independent signaling (37). Furthermore, olar-atumab-bound PDGFRa is internalized and degraded, which

attenuates downstream pathway activation and likely furthercontributes to drug activity (8, 37).

Although PDGFRA is not frequently altered in childhoodcancers (19, 38, 39), receptor expression is associated withprogressive disease and poor prognosis in some pediatricmalignancies of bone and soft tissue (11, 26, 40). Expressionof the receptor was readily detected in several osteosarcoma celllines and PDX models, which was expected, as 50% of osteo-sarcoma primary samples were previously found to be PDGFRapositive (23). In addition, PDGFRa-activating ligands (namely,PDGFA and/or PDGFC) were also expressed in these cells lines.Conversely, PDGFRa was not expressed at the gene or proteinlevel in the majority of rhabdomyosarcoma cell lines and PDXmodels in our study nor in a previous study that examinedPDGFRa in SJCRH30 and other rhabdomyosarcoma cell lines(41). These findings were particularly surprising, as PDGFRa isreadily detectable in rhabdomyosarcoma clinical specimens byqPCR or IHC and PDGFRA is a transcriptional target of thefusion protein driving alveolar rhabdomyosarcoma (PAX3-FOXO; refs. 20, 22, 26). Similarly, the Ewing sarcoma PDXmodels evaluated in this study were negative for PDGFCexpression, despite previous work identifying PDGFC as a targetgene of the EWS-FLI1 fusion transcription factor (42). Thesedata are suggestive of potential alterations in the tumor tran-scriptome when cultured or propagated outside of its nativeenvironment and are reminiscent of a previous report describ-ing changes in the epigenome and transcriptome of mamma-lian cells isolated from primary tissues upon exposure to cellculture (43). Furthermore, a recent study demonstrated that ananti-PDGFRa polyclonal antibody frequently used in previousIHC analyses cross-reacts with PDGFRb, further confoundingprevious reports regarding the prevalence of PDGFRa expres-sion in pediatric bone and soft tissue tumor patient samples(44). In light of this discordance in receptor expression betweenavailable preclinical bioresources and clinical specimens, it isdifficult to investigate the full range of olaratumab activity inpediatric sarcoma in a preclinical setting and to identify poten-tial predictive biomarkers. Furthermore, although PDGFRaexpression in cell lines and xenograft models was necessaryfor olaratumab to demonstrate some antitumor activity inpreclinical models, expression alone was insufficient to predictsensitivity, echoing the findings of the pivotal phase II study inadult STS patients (9). Further development of preclinicalmodels that better recapitulate human disease is necessary topredict the clinical activity of novel agents and to understandadditional factors that may influence sensitivity.

PDGFC has been reported to be highly expressed in A-204 cells,suggesting the potential for an autocrine manner of PDGFRaactivation (41); indeed, shRNA-mediated silencing of PDGFCsignificantly reduced A-204 proliferation, comparable with theknockdownofPDGFRA.We confirmed thatPDGFCwas expressedinA-204 cells and found thatHuO9 expressed both PDGF-AA and-CC, indicating another potential autocrine loop. Furthermore,we observed that exogenous PDGFswere not chemoattractive andcould not promote invasion of serum-starved HuO9 or A-204cells. Insensitivity of HuO9 and A-204 to exogenously suppliedPDGFs may be explained by saturation of PDGFRa via endoge-nously produced ligands. Interestingly, the addition of serumelicited an increase in A-204 and HuO9 osteosarcoma cellinvasion well above that observed when PDGFs were used aschemoattractants. Olaratumab subsequently blocked this






serum-induced invasion, suggesting that PDGFRa may accom-modate signaling initiated by non-PDGF ligands and/or throughnoncanonical receptor dimerization partners.

As expected, olaratumab blocked PDGF-stimulated PDGFRaphosphorylation in alpha-positive normal and pediatric tumorcell lines. PDGFRb phosphorylation and PDGFRb-activateddownstream pathways were not inhibited by treatment, furtherillustrating the specificity of olaratumab for the alpha receptor.Interestingly, varied effects on downstream MAPK and PI3Kpathway signaling were observed following ligand stimulationand olaratumab treatment in MG-63 and WS-1 cells, potentiallyinfluenced by coexpression of PDGFRa and PDGFRb. Dimeriza-tion and subsequent activation of PDGFRa is primarily driven bybinding PDGF-AA and PDGF-CC, whereas PDGFRb has a higheraffinity for PDGF-BB and PDGF-DD (1, 45). Heterodimerizationcan occur after binding any ligand except for PDGF-AA. Theexpression of PDGFRa alone in HuO9 OS cells and A-204 MRTcells seemingly heightens the affinity of the receptor for individualPDGFs, and olaratumab is therefore highly effective at blockingMAPK and PI3K pathway activation caused by PDGFRa signaling.In contrast, MG-63 and WS-1 cells express both PDGFRa andPDGFRb and downstream signaling persisted after PDGF-BB,-DD, and -AB stimulation, even in the presence of olaratumab.Furthermore, PDGFRb phosphorylation and downstream path-way signaling was unaffected by olaratumab in G-401 MRT cells,which only express the beta receptor; however, olaratumabreduced PDGF-BB or -AB-stimulated PDGFRb phosphorylationin G-402 MRT cells. The differences in signal propagation initi-ation and maintenance are most likely influenced by the patternsof homo- and heterodimerization. Furthermore, although it isgenerally accepted that specific PDGFs activate PDGFRa orPDGFRb, our data suggest that receptor affinity for ligand maybe flexible and dependent on relative expression levels ofPDGFRa and PDGFRb protein.

To our knowledge, this study is the first to demonstrate that theantitumor activity of olaratumab observed in adult sarcomas canextend to preclinical models of pediatric bone and soft tissuemalignancies. In the phase II clinical trial combining olaratumabwith doxorubicin, progression-free survival was improved by 2.5months, and overall survival was extended by a median gain of11.8 months with combination treatment when compared withsingle-agent doxorubicin (9). In this study, single-agent olaratu-mab delayed tumor growth in preclinical mouse models ofosteosarcoma and MRT, indicating that PDGFRa signaling isnecessary to drive some aspect of xenograft growth; however, a

recent report demonstrated that inhibition of PDGFRa alone isinsufficient for durable control of MRT growth (25), and combi-nation with other targeted agents or chemotherapy is most likelynecessary. Indeed, we observed enhanced antitumor activity withcoadministration of olaratumab and either doxorubicin or cis-platin in osteosarcoma mouse models. These data indicate thatthe improvement in activity observed upon combining SOCwitholaratumab is not limited to doxorubicin. Therefore, other che-motherapies commonly used in pediatric tumors, such as ifosfa-mide, cyclophosphamide, or vincristine (46, 47), could poten-tially combine with olaratumab to enhance antitumor activity inpreclinical models of pediatric bone and soft tissue tumors.

Disclosure of Potential Conflicts of InterestB.P. Rubin reports receiving speakers bureau honoraria from and is a

consultant/advisory board member for Eli Lilly. L.F. Stancato has ownershipinterests in Eli Lilly. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: C.D. Lowery, W. Blosser, M. Dowless, J. Stephens,H. Li, N. Loizos, G.J. Oakley III, L. StancatoDevelopment of methodology: W. Blosser, M. Dowless, J. Stephens, H. Li,G.J. Oakley III, Q. Guo, S. IyerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. Blosser, M. Dowless, S. Knoche, J. Stephens,H. Li, D. Surguladze, N. Loizos, D. Luffer-Atlas, G.J. Oakley III, Q. Guo,B.P. RubinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.D. Lowery, W. Blosser, S. Knoche, J. Stephens,H. Li,D. Surguladze,N. Loizos,D. Luffer-Atlas, G.J.Oakley III, S. Iyer, L. StancatoWriting, review, and/or revision of the manuscript: C.D. Lowery, D. Surgu-ladze, N. Loizos, D. Luffer-Atlas, G.J. Oakley III, L. StancatoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.D. Lowery, W. Blosser, S. IyerStudy supervision: N. Loizos, S. Iyer

AcknowledgmentsThe authorswould like to thankDr. PeterHoughton for his gift of the atypical

teratoid/rhabdoid tumor cell lines BT-12 and BT-16. This study was funded byEli Lilly and Company, Lilly Corporate Center.

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 May 1, 2017; revised September 7, 2017; accepted November 28,2017; published OnlineFirst November 30, 2017.

References1. HeldinCH. Targeting the PDGF signaling pathway in tumor treatment. Cell

Commun Signal 2013;11:97.2. Demoulin JB, Essaghir A. PDGF receptor signaling networks in normal and

cancer cells. Cytokine Growth Factor Rev 2014;25:273–83.3. Cao Y. Multifarious functions of PDGFs and PDGFRs in tumor growth and

metastasis. Trends Mol Med 2013;19:460–73.4. Pietras K, Pahler J, Bergers G, Hanahan D. Functions of paracrine PDGF

signaling in the proangiogenic tumor stroma revealed by pharmacologicaltargeting. PLoS Med 2008;5:e19.

5. Quail DF, Joyce JA. Microenvironmental regulation of tumor progressionand metastasis. Nat Med 2013;19:1423–37.

6. Gerber DE, Gupta P, Dellinger MT, Toombs JE, Peyton M, Duignan I, et al.Stromal platelet-derived growth factor receptor alpha (PDGFRalpha) pro-vides a therapeutic target independent of tumor cell PDGFRalpha expres-sion in lung cancer xenografts. Mol Cancer Ther 2012;11:2473–82.

7. Anderberg C, Li H, Fredriksson L, Andrae J, Betsholtz C, Li X, et al.Paracrine signaling by platelet-derived growth factor-CC promotestumor growth by recruitment of cancer-associated fibroblasts. CancerRes 2009;69:369–78.

8. LoizosN, Xu Y,Huber J, LiuM, LuD, Finnerty B, et al. Targeting the platelet-derived growth factor receptor alpha with a neutralizing human mono-clonal antibody inhibits the growth of tumor xenografts: implications as apotential therapeutic target. Mol Cancer Ther 2005;4:369–79.

9. Tap WD, Jones RL, Van Tine BA, Chmielowski B, Elias AD, Adkins D, et al.Olaratumab and doxorubicin versus doxorubicin alone for treatment ofsoft-tissue sarcoma: an open-label phase 1b and randomised phase 2 trial.Lancet 2016;388:488–97.

10. Horner M, Ries L, Krapcho M, Neyman N, Aminou R, Howlader N, et al.SEER Cancer Statistics Review, 1975–2006, National Cancer Institute.Bethesda, MD: NIH; 2009.

Lowery et al.





11. Anderson JL, Denny CT, Tap WD, Federman N. Pediatric sarcomas:translating molecular pathogenesis of disease to novel therapeutic possi-bilities. Pediatr Res 2012;72:112–21.

12. Roberts CW, Biegel JA. The role of SMARCB1/INI1 in development ofrhabdoid tumor. Cancer Biol Ther 2009;8:412–6.

13. Horazdovsky R, Manivel JC, Cheng EY. Surgery and actinomycinimprove survival in malignant rhabdoid tumor. Sarcoma 2013;2013:315170.

14. Farber BA, Shukla N, Lim II, Murphy JM, LaQuagliaMP. Prognostic factorsand survival in non-central nervous system rhabdoid tumors. J Pediatr Surg2017;52:373–6.

15. ReinhardH, Reinert J, Beier R, Furtwangler R, AlkasserM, Rutkowski S, et al.Rhabdoid tumors in children: prognostic factors in 70 patients diagnosedin Germany. Oncol Rep 2008;19:819–23.

16. Torchia J, Picard D, Lafay-Cousin L, Hawkins CE, Kim SK, Letourneau L,et al. Molecular subgroups of atypical teratoid rhabdoid tumours inchildren: an integrated genomic and clinicopathological analysis. LancetOncol 2015;16:569–82.

17. Robison LL, Hudson MM. Survivors of childhood and adolescent cancer:life-long risks and responsibilities. Nat Rev Cancer 2014;14:61–70.

18. Fernandez-Pineda I, Hudson MM, Pappo AS, Bishop MW, Klosky JL,Brinkman TM, et al. Long-term functional outcomes and quality of lifein adult survivors of childhood extremity sarcomas: a report from the St.Jude Lifetime Cohort Study. J Cancer Surviv 2017;11:1–12.

19. Chmielecki J, Bailey M, He J, Elvin J, Vergilio JA, Ramkissoon S, et al.Genomic profiling of a large set of diverse pediatric cancers identifiesknown and novel mutations across tumor spectra. Cancer Res 2017;77:509–19.

20. Taniguchi E, Nishijo K,McCleish AT,Michalek JE, GraysonMH, Infante AJ,et al. PDGFR-A is a therapeutic target in alveolar rhabdomyosarcoma.Oncogene 2008;27:6550–60.

21. EhnmanM,Missiaglia E, Folestad E, Selfe J, Strell C, Thway K, et al. Distincteffects of ligand-induced PDGFRalpha and PDGFRbeta signaling in thehuman rhabdomyosarcoma tumor cell and stroma cell compartments.Cancer Res 2013;73:2139–49.

22. Ho AL, Vasudeva SD, Lae 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.

23. Abdeen A, Chou AJ, Healey JH, Khanna C, Osborne TS, Hewitt SM, et al.Correlation between clinical outcome and growth factor pathway expres-sion in osteogenic sarcoma. Cancer 2009;115:5243–50.

24. Bozzi F, Tamborini E, Negri T, Pastore E, Ferrari A, Luksch R, et al. Evidencefor activation of KIT, PDGFRalpha, and PDGFRbeta receptors in the Ewingsarcoma family of tumors. Cancer 2007;109:1638–45.

25. Wong JP, Todd JR, Finetti MA, McCarthy F, Broncel M, Vyse S, et al. Dualtargeting of PDGFRalpha and FGFR1 displays synergistic efficacy in malig-nant rhabdoid tumors. Cell Rep 2016;17:1265–75.

26. Blandford MC, Barr FG, Lynch JC, Randall RL, Qualman SJ, Keller C.Rhabdomyosarcomas utilize developmental, myogenic growth factors fordisease advantage: a report from the Children's Oncology Group. PediatrBlood Cancer 2006;46:329–38.

27. Lowery CD, VanWye AB, Dowless M, Blosser W, Falcon BL, Stewart J,et al. The checkpoint kinase 1 inhibitor prexasertib induces regression ofpreclinical models of human neuroblastoma. Clin Cancer Res 2017;23:4354–63.

28. Armistead PM, Salganick J, Roh JS, Steinert DM, Patel S, Munsell M, et al.Expression of receptor tyrosine kinases and apoptotic molecules in rhab-domyosarcoma: correlation with overall survival in 105 patients. Cancer2007;110:2293–303.

29. Sethi TK, Keedy VL. Histology-specific uses of tyrosine kinase inhibitors innon-gastrointestinal stromal tumor sarcomas. Curr Treat Options Oncol2016;17:11.

30. Selt F, Deiss A, Korshunov A, Capper D, Witt H, van Tilburg CM, et al.Pediatric targeted therapy: clinical feasibility of personalized diagnostics inchildrenwith relapsed and progressive tumors. Brain Pathol 2016;26:506–16.

31. Heymann MF, Brown HK, Heymann D. Drugs in early clinical develop-ment for the treatment of osteosarcoma. Expert Opin Investig Drugs2016;25:1265–80.

32. Keir ST, Maris JM, Lock R, Kolb EA, Gorlick R, Carol H, et al. Initial testing(stage 1) of the multi-targeted kinase inhibitor sorafenib by the pediatricpreclinical testing program. Pediatr Blood Cancer 2010;55:1126–33.

33. Kubo T, Piperdi S, Rosenblum J, Antonescu CR, Chen W, Kim HS, et al.Platelet-derived growth factor receptor as a prognostic marker and atherapeutic target for imatinib mesylate therapy in osteosarcoma. Cancer2008;112:2119–29.

34. Gobin B, Moriceau G, Ory B, Charrier C, Brion R, Blanchard F, et al.Imatinib mesylate exerts anti-proliferative effects on osteosarcoma cellsand inhibits the tumour growth in immunocompetent murine models.PLoS One 2014;9:e90795.

35. Koos B, Jeibmann A, Lunenburger H, Mertsch S, Nupponen NN, RoselliA, et al. The tyrosine kinase c-Abl promotes proliferation and isexpressed in atypical teratoid and malignant rhabdoid tumors. Cancer2010;116:5075–81.

36. Imai K, Takaoka A. Comparing antibody and small-molecule therapies forcancer. Nat Rev Cancer 2006;6:714–27.

37. DolloffNG, RussellMR, LoizosN, Fatatis A.Humanbonemarrow activatesthe Akt pathway in metastatic prostate cells through transactivation of thealpha-platelet-derived growth factor receptor. Cancer Res 2007;67:555–62.

38. Sulzbacher I, Birner P, DominkusM, Pichlhofer B, Mazal PR. Expression ofplatelet-derived growth factor-alpha receptor in human osteosarcoma isnot a predictor of outcome. Pathology 2010;42:664–8.

39. Do I, Araujo ES, Kalil RK, Bacchini P, Bertoni F, Unni KK, et al. Proteinexpression of KIT and gene mutation of c-kit and PDGFRs in Ewingsarcomas. Pathol Res Pract 2007;203:127–34.

40. Kilvaer TK, ValkovA, Sorbye SW,DonnemT, Smeland E, Bremnes RM, et al.Platelet-derived growth factors in non-GIST soft-tissue sarcomas identify asubgroupof patients withwide resectionmargins and poor disease-specificsurvival. Sarcoma 2010;2010:751304.

41. McDermott U, Ames RY, Iafrate AJ, Maheswaran S, Stubbs H, Greninger P,et al. Ligand-dependent platelet-derived growth factor receptor (PDGFR)-alpha activation sensitizes rare lung cancer and sarcoma cells to PDGFRkinase inhibitors. Cancer Res 2009;69:3937–46.

42. Zwerner JP, MayWA. PDGF-C is an EWS/FLI induced transforming growthfactor in Ewing family tumors. Oncogene 2001;20:626–33.

43. Nestor CE, Ottaviano R, Reinhardt D, Cruickshanks HA, Mjoseng HK,McPherson RC, et al. Rapid reprogramming of epigenetic and transcrip-tional profiles in mammalian culture systems. Genome Biol 2015;16:11.

44. Holzer TR,O'Neill Reising L,Credille KM, SchadeAE,OakleyGJ. Variabilityin platelet-derived growth factor receptor alpha antibody specificity mayimpact clinical utility of immunohistochemistry assays. J HistochemCytochem 2016;64:785–810.

45. Andrae J, Gallini R, Betsholtz C. Role of platelet-derived growth factors inphysiology and medicine. Genes Dev 2008;22:1276–312.

46. Merchant MS, Mackall CL. Current approach to pediatric soft tissuesarcomas. Oncologist 2009;14:1139–53.

47. JacksonTM, BittmanM,Granowetter L. Pediatricmalignant bone tumors: areview and update on current challenges, and emerging drug targets. CurrProbl Pediatr Adolesc Health Care 2016;46:213–28.






2018;24:847-857. Published OnlineFirst November 30, 2017.Clin Cancer Res Caitlin D. Lowery, Wayne Blosser, Michele Dowless, et al.

αPlatelet-Derived Growth Factor Receptor Pediatric Bone and Soft Tissue Tumors through Inhibition of Olaratumab Exerts Antitumor Activity in Preclinical Models of

Updated version

10.1158/1078-0432.CCR-17-1258doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2017/11/30/1078-0432.CCR-17-1258.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/24/4/847.full#ref-list-1

This article cites 46 articles, 10 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://clincancerres.aacrjournals.org/content/24/4/847To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-17-1258

http://clincancerres.aacrjournals.org/content/suppl/2017/11/30/1078-0432.CCR-17-1258.DC1

http://clincancerres.aacrjournals.org/content/24/4/847.full#ref-list-1

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

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/24/4/847


Documents

Olaratumab Exerts Antitumor Activity in Preclinical Models ... · malignant rhabdoid tumor, and this activity was enhanced by combination with either doxorubicin or cisplatin. Conclusions: