The Ewing Family of Tumors Relies on BCL-2 and BCL-X to ... · The Ewing family of tumors (EWFTs), consisting of primitive neuroectodermal tumor (PNET) and Ewing sarcoma, is a malig-nancy

Translational Cancer Mechanisms and Therapy

The Ewing Family of Tumors Relies on BCL-2and BCL-XL to Escape PARP Inhibitor ToxicityDaniel A.R. Heisey1, Timothy L. Lochmann1, Konstantinos V. Floros1,Colin M. Coon1, Krista M. Powell1, Sheeba Jacob1, Marissa L. Calbert1,Maninderjit S. Ghotra1, Giovanna T. Stein2, Yuki Kato Maves3, Steven C. Smith4,Cyril H. Benes2, Joel D. Leverson5, Andrew J. Souers5, Sosipatros A. Boikos6,and Anthony C. Faber1

Abstract

Purpose: It was recently demonstrated that the EWSR1-FLI1t(11;22)(q24;12) translocation contributes to the hypersensi-tivity of Ewing sarcoma to PARP inhibitors, prompting clinicalevaluation of olaparib in a cohort of heavily pretreated Ewingsarcoma tumors. Unfortunately, olaparib activity was disap-pointing, suggesting an underappreciated resistance mecha-nism to PARP inhibition in patients with Ewing sarcoma. Wesought to elucidate the resistance factors to PARP inhibitortherapy in Ewing sarcoma and identify a rational drug com-bination capable of rescuing PARP inhibitor activity.

Experimental Design: We employed a pair of cell linesderived from the same patient with Ewing sarcoma prior toand following chemotherapy, a panel of Ewing sarcoma celllines, and several patient-derived xenograft (PDX) and cell linexenograft models.

Results: We found olaparib sensitivity was diminishedfollowing chemotherapy. The matched cell line pair

revealed increased expression of the antiapoptotic proteinBCL-2 in the chemotherapy-resistant cells, conferring apo-ptotic resistance to olaparib. Resistance to olaparib wasmaintained in this chemotherapy-resistant model in vivo,whereas the addition of the BCL-2/XL inhibitor navitoclaxled to tumor growth inhibition. In 2 PDXs, olaparib andnavitoclax were minimally effective as monotherapy, yetinduced dramatic tumor growth inhibition when dosed incombination. We found that EWS-FLI1 increases BCL-2expression; however, inhibition of BCL-2 alone by veneto-clax is insufficient to sensitize Ewing sarcoma cells to ola-parib, revealing a dual necessity for BCL-2 and BCL-XL inEwing sarcoma survival.

Conclusions: These data reveal BCL-2 and BCL-XL acttogether to drive olaparib resistance in Ewing sarcoma andreveal a novel, rational combination therapy that may be putforward for clinical trial testing.

IntroductionThe Ewing family of tumors (EWFTs), consisting of primitive

neuroectodermal tumor (PNET) and Ewing sarcoma, is a malig-nancy of predominantly bone. These cancers are diagnosed mostoften in children and adolescents. Great strides have beenmade intreating localized disease by using intensive neoadjuvant andadjuvant chemotherapy regimens, increasing the 5-year survivalfrom about 10% to approximately 75%. However, there is a 30%

survival rate for patients with Ewing sarcoma presenting withmetastasis or that relapse following systematic chemotherapy (1).

The EWSR1-FLI1 t(11;22)(q24;q12) translocation event isfound in approximately 90% of EWFTs. Since the identificationof EWSR1-FLI1 in Ewing sarcoma, it has become clear that theresultant fusiononcogene is the vital driving event in these tumors(2–7). Themolecular consequence of juxtaposing the EWSR1 andFLI1 genes is a EWS-FLI1 fusion protein where EWS potentlyincreases the ability of transcription factor FLI1 to activate orsuppress target genes.

Unfortunately, FLI1 is currently undruggable, and effectivetargeted therapies for treating Ewing sarcoma remain elusive.Recent findings have highlighted the role of EWS-FLI1 at inducingawide range of changes throughout the epigenome, affecting bothhistone marks and enhancers (5–9), leading to simultaneousenhanced expression of tumor oncogenes and reduced expressionof tumor suppressors (6). However, these studies have yet toreveal specific, druggable targets with associated clinically avail-able therapies.

Brenner and colleagues (10) and the Genomics of Drug Sen-sitivity in Cancer (GDSC), a high-throughput drug screeningplatform (11), both demonstrated in 2012 that EWSR1-FLI1–translocated Ewing sarcoma displays hypersensitivity to PARPinhibition; this has since been replicated by several othergroups (12–14). These data have provided a promising

1VCU Philips Institute, School of Dentistry andMassey Cancer Center; Richmond,Virginia. 2Massachusetts General Hospital Cancer Center, Boston, Massachu-setts; Department of Medicine, Harvard Medical School, Boston, Massachusetts.3Crown Bioscience Inc., San Diego, California. 4Division of Anatomic Pathology,Virginia Commonwealth University, Richmond, Virginia. 5AbbVie, North Chi-cago, Illinois. 6Hematology, Oncology and Palliative Care, School of Medicineand Massey Cancer Center, Virginia Commonwealth University, Richmond,Virginia.

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

Corresponding Author: Anthony C. Faber, Virginia Commonwealth University,Perkinson Building, Room 4134, 1101 East Leigh Street, Richmond, VA 23298.Phone: 804-828-0841; Fax: 804-828-0150; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-0277

�2018 American Association for Cancer Research.

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drug target for EWFTs, with corresponding FDA-approved PARP1inhibitors (15).

However, in the initial clinical study of olaparib in Ewingsarcoma, no objective responses were observed in 12 evalu-able patients (15). Although there were no objectiveresponses, 4 of 12 patients achieved stable disease, with 2of the 4 achieving minor responses (tumor shrinkage of 9%and 12%), indicating a modest level of efficacy by PARPinhibition in these patients.

Based on the hypersensitivity of Ewing sarcoma to PARPinhibition in vitro and olaparib activity in patients with Ewingsarcoma, we sought to identify intrinsic resistance mechanismsto PARP inhibitor (PARPi) therapy as well as a rational drugcombination that could overcome these mechanisms. We andothers have shown that a low apoptotic response, even in thepresence of growth arrest, mitigates response to targeted therapies(16–21). We therefore hypothesized that mitigated responses ofPARP inhibition may be due to loss of apoptotic potential ofEWFTs, which could prove particularly true in the chemorefrac-tory population. This hypothesis was further supported by the factthat deficient DNA damage repair is thought to contribute to, ifnot define, PARPi sensitivity in Ewing sarcoma (12), as well as theestablished role of antiapoptotic BCL-2 family proteins in pro-tecting cancer cells from DNA damage–induced apoptosis(17, 22) and their inverse correlation of expression to cytotoxicagent sensitivity (23).

Materials and MethodsCell lines

A673 (ATCC CRL-1598) and HEK293T cells were cultured inDMEM (Gibco) with 10% FBS (Seradigm) and 1 mg/mL pen-icillin and streptomycin. CHLA9 and CHLA10 cells were grownin DMEM with 20% FBS, 1 mg/mL penicillin and streptomycin,and 1% Insulin-Transferrin-Selenium 100x (Gibco). SK-ES-1(ATCC HTB-86) was grown in DMEM/F12 (Corning) with 15%FBS and 1 mg/mL of penicillin and streptomycin. ES4 and EW16cells were grown in RPMI1640 (Lonza Group) with 10% FBSand 1 mg/mL of penicillin and streptomycin. Routine myco-plasma testing was performed on all cell lines. CHLA9 andCHLA10 were obtained from the Children's Oncology Group(COG) Cell Culture and Xenograft Repository, special thanks toDr. C. Pat Reynolds, Texas Tech University Health SciencesCenter. ES4, EW16, HEK293T, A673 (ATCC CRL-1598), andSK-ES-1 (ATCC HTB-86) were obtained from either the Molec-ular Center Therapeutics laboratory at Massachusetts GeneralHospital which performs routine testing of cell lines using shorttandem repeat and SNP analysis, or the American Type CultureCollection (ATCC).

Antibodies and reagentsPrimary antibodies used for Western blotting were as follows:

GAPDH (sc-3233) and FLI1 (sc-365294) from Santa Cruz Bio-technology; cleaved PARP1 (5625), BCL-2 (4223), BCL-XL

(2764), MCL-1 (5453), PARP1 (9532), gH2A.X (9781), andBIM (2933) from Cell Signaling Technology. Secondary anti-bodies used were mouse IgG (GE Healthcare Life Sciences;NXA931) and rabbit IgG (GE Healthcare Life Sciences; NA934).IgG (sc-2027) for immunoprecipitation was from Santa CruzBiotechnology. Olaparib (AZD-2281) was from Abmole, andA-1331852, navitoclax (ABT-263), and venetoclax (ABT-199)were kindly provided by AbbVie Inc.

Animal experimentsFor the SK-ES-1 xenograftmodel, 5� 106 cells were injected s.c.

into the flank of 6- to 8-week-old Nu/Nu mice. For the CHLA10xenograft model, 5 � 106 cells were injected s.c. into the flank of6- to 8-week-old Nod/SCID gamma (NSG) mice. The patient-derived xenograft (PDX) models were obtained from CrownBioscience, and 5 � 105 cells were injected into the flank of NSGmice s.c. Treatment began when tumors reached approximately150 to 200 mm3, and mice were randomized into treatmentcohorts. Tumor size and mouse weight were measured 3 daysper week with a digital scale and calipers, where tumor volumewas calculated as length�width2� 0.52.Navitoclax andolaparibwere administered by oral gavage. Navitoclax was dissolved in60% Phosal 50 PG, 30% PEG400, and 10% ethanol, for a finaldosage of 80 mg/kg of body weight. Olaparib was dissolvedin 10% hydroxylpropyl-b-cyclodextrin for a final dosage of100 mg/kg of body weight. All drugs were administered once perday, 5 days/week. For pharmacodynamics studies, tumor-bearingmicewere treatedwith drug for 3 days, and tumors were harvestedon the 3rd day 2 hours after the final treatment. Tumors wereflash frozen in liquid nitrogen. For the blood toxicity study,NSG mice were treated with no drug (no Rx), navitoclax,olaparib, or the combination, at the same doses as above. At3 and 7 days, mice were exsanguinated, and blood was sent toIDEXX BioResearch for testing. The recovery cohort was treatedfor 7 days and allowed 24 hours of recovery from treatmentbefore exsanguination. All animal experiments were approvedby the Virginia Commonwealth University Institutional AnimalCare and Use Committee (IACUC protocol #AD10001048).

Dataset analysisThe online database for publically accessible drug sensitivity

data (www.cancerRxgene.org) was used to generate Fig. 3C. Thecancer cell line encyclopedia (CCLE; ref. 24) was used to analyzeexpression between FLI1 and BCL-2 (Supplementary Fig. S7D).

Statistical considerationsFor gene expression analyses and the complete blood count

analyses, significance was determined using the nonparametricMann–Whitney U test. Differences were considered statisticallydifferent if P < 0.05. All other analyses were performed using theStudent t test and considered statistically different if P < 0.05.Asterisks indicate levels of significance: ns, P � 0.05; �, P < 0.05;��, P < 0.01; ��, P < 0.001; and ��, P < 0.0001.

Synergy assayCellswere seeded at 1�103 cells in a96-well plate. Twenty-four

hours after seeding, cells were treated with varied concentrations

Translational Relevance

Using matched patient samples prior to and followingchemotherapy, we have uncovered a drug combination thatis able to achieve substantial tumor regression in multiplemouse models including patient-derived xenografts andxenografts of chemotherapy-resistant cells. This warrants theclinical evaluation of olaparib in combination with navitoclaxin the EWS-FLI1–translocated Ewing family of tumors.

Navitoclax Sensitizes Ewing Sarcoma to Olaparib

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of navitoclax (0 to 2 mmol/L) and olaparib (0.1 to 10 mmol/L) for72 hours, followed by measurement of cell viability by CellTiter-Glo. Percent viability was constrained to a maximum of 100. Thepercentage over the bliss score was calculated as previouslydescribed (25).

ResultsA chemotherapy-na€�ve and chemotherapy-resistant cell linepair respond differently to olaparib

Olaparib performed poorly in patients with Ewing sarcoma(15) whose tumors were heavily pretreated and chemotherapy-resistant.We therefore utilized a pair of cell lines established fromthe same patient prior to and following chemotherapy treatment,at tumor relapse: CHLA9 cells were derived from the chemother-apy-na€�ve PNET, positive for the EWS-FLI1 translocation, whereasthe CHLA10 cell line was established after 4 cycles of inductionchemotherapy which included cisplatin, doxorubicin, cyclophos-phamide, and etoposide (26). We first assessed whether sensitiv-ity to olaparib was different in the 2 cell lines. We found that thechemotherapy-na€�ve cells were more sensitive to olaparib, asevidenced by a 5-day crystal violet viability assay (Fig. 1A, left),72-hour dose–response curve (Fig. 1A, right), and IC50 curve(Supplementary Fig. S2A) compared with the chemotherapy-resistant CHLA10 cells (Fig. 1A, left and right), despite both celllines reportedly expressing high levels of PARP1 (23, 27) andolaparib inducing similar growth arrest in both the CHLA9 andCHLA10 cells (Supplementary Fig. S2B). In addition, similarlevels of DNA damage were observed following olaparib treat-ment in both cell lines as evidenced by gH2A.X immunofluo-rescence staining (Supplementary Fig. S3A and S3B). Further-more, we confirmed the CHL10 cells were more resistant tochemotherapy compared with the CHLA9 cells (SupplementaryFig. S4A and S4B).

Chemotherapy-resistant CHLA10 cells do not undergo celldeath in response to olaparib

Because the lack of robust apoptotic responses can underlieresistance to both chemotherapy and targeted therapies, and theapoptotic response following many chemotherapies and targetedtherapies is largely governed by the BCL-2 family of proteins(16, 20, 28–30), we first explored the relationship betweenantiapoptotic BCL-2 family expression and olaparib response inthe CHLA9 and CHLA10 models. Expression of BCL-2 was upre-gulated (P < 0.05) in the CHLA10 cells compared with the CHLA9cells (Fig. 1B; Supplementary Fig. S4C), whereas expressions ofother key BCL-2 family members were not altered (Fig. 1B).

The increase in BCL-2 prompted us to evaluate BCL-2 expres-sion in pretreatment and postchemotherapy biopsy samples from2 patients with Ewing sarcoma treated at our cancer center.Interestingly, we did not detect an increase in BCL-2 expressionin these specimens, in contrast to the cell line pair; however, BCL-XL expression was markedly higher in chemotherapy-resistanttumors (Fig. 1C) relative to the matched chemotherapy-na€�vesamples. These data indicate that BCL-XL is overexpressed inEwing sarcoma patients' tumors that have undergone chemother-apy, and our findings in models of EWFTs implicate BCL-2 as acooperating partner with BCL-XL in resisting apoptosis. Together,these data indicated to us that both BCL-2 and BCL-XL may beimperative in Ewing sarcoma survival. We then moved to chem-ical interrogation of the cellswith specific BCL-2 family inhibitors.

Surprisingly, despite the increase in BCL-2, we found the BCL-2–specific inhibitor venetoclax (31) was unable to effectively sen-sitize CHLA10 cells to olaparib (Supplementary Fig. S5A).Because increased expression of BCL-XL is sufficient to induceresistance to venetoclax (32–34), we next tested the dual BCL-2/BCL-XL inhibitor navitoclax (35, 36) to determine if this agentsensitizes the CHLA10 cells to olaparib. Although venetoclaxshowed little potentiation of olaparib (Supplementary Fig. S5Aand S5B), navitoclax sensitized CHLA10 cells to olaparib treat-ment compared with venetoclax (P < 0.05), leading to a near-complete loss of cell viability (Fig. 1D), and mild synergy (Sup-plementary Fig. S5C). Impressively, at low doses of olaparib(1 mmol/L) where there was no single-drug efficacy in the CHLA9cells, the addition of navitoclax led to substantial loss of cellviability (Supplementary Fig. S5D). Similar to venetoclax, theBCL-XL–selective inhibitor A-1331852 (37) was not effective atsensitizing CHLA10 cells to olaparib (Supplementary Fig. S5E).Consistent with these findings, we found the CHLA9 cells under-went marked cell death in response to olaparib, as measured bycleaved PARP1 (Fig. 1E); in contrast, there was a near absence of acell death response in the olaparib-treated CHLA10 cells (Fig. 1E).However, the addition of navitoclax led to marked cleavage ofPARP1 in the presence of olaparib in the CHLA10 cells (Fig. 1E),despite the lack of modulation of BCL-2, BCL-XL, or the relatedMCL-1 (38) by olaparib (Fig. 1F). These data indicate that EWFTscan lose their sensitivity to olaparib following chemotherapytreatment and relapse, underscored by their inability to undergocell death, and can be rescued by the addition of navitoclax. Thiswas further supported by the observation that, at low concentra-tions of olaparib where sensitive CHLA9 cells do not yet respondto single-agent olaparib, navitoclax also sensitizes to olaparib(Supplementary Fig. S5D). Of note, the CHLA9 cells have func-tional p53, whereas the CHLA10 cells have nonfunctioningp53 (39). It is well established that functional p53 is capable ofbinding to and antagonizing the antiapoptotic functions of BH3proteins such as BCL-2 and BCL-XL (40, 41). In order to rule outp53 as the cause of inherent resistance to olaparib-inducedapoptosis in the CHLA10 cells when compared with the CHLA9cells, we used siRNA to knockdown TP53 in the CHLA9 cells andfound no difference in olaparib sensitivity (Supplementary Fig.S5F, left), consistent with a previous report on p53 and PARPisensitivity (2). To further support the role of BCL-2 and/or BCL-XL

overexpression in apoptotic resistance to olaparib treatment, weoverexpressed BCL-2 or BCL-XL in the CHLA9 cells. Here, we saw asignificant increase in resistance to olaparib treatment in cellsoverexpressing BCL-2 or BCL- XL compared with the GFP controls(P < 0.0001; Supplementary Fig. S6A). Together, these data reveala striking interplay between BCL-2/XL inhibition and PARP inhi-bition in the EWFTs.

Navitoclax and olaparib cooperate to inhibit tumor growth in aCHLA10 mouse model

We next grew CHLA10 tumors in NSG mice and evaluatedsingle-agent olaparib, navitoclax, and the combination of ola-parib and navitoclax to see if the in vitro results would translate invivo. Consistent with the cell culture experiments (Fig. 1D and E),we found the CHLA10 tumors were not sensitive to olaparib andminimally sensitive to navitoclax as a monotherapy comparedto untreated mice (Fig. 1G). However, the combination ofolaparib and navitoclax demonstrated robust inhibition of tumorgrowth (Fig. 1G). This contrasted with the venetoclax/olaparib

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Figure 1.

Chemotherapy-resistant CHLA10 cells are sensitized to olaparib with the inhibition of BCL-2 and BCL-XL. A, (left) Crystal violet staining of CHLA9 and CHLA10 cellsafter a 5-day treatment of 5 mmol/L olaparib compared to a no-treatment control (no Rx). Right, 72-hour CellTiter Glo of CHLA9 and CHLA10 using the indicatedconcentrations of olaparib. B, Western blot analysis with the indicated antibodies in chemotherapy-naive and chemotherapy-resistant paired lines. C, Twocases of Ewing sarcomawith available paired primary and recurrentmetastatic tissueswere immunostained for BCL-2 andBCL-XL. In both cases, similar expression ofBCL-2 was noted in primary. For BCL-XL, however, increased expression was noted in both recurrences compared with the primary tumors. Case 1: Primary: Archivalsections of the untreated biopsy of the primary tumor (patella), which was localized at presentation. Recurrence: Lung metastasis 5 years subsequent, aftersystemic chemotherapy (VAC-IE) and localized radiotherapy to the patellar primary site. Case 2: Primary: Archival sections of the biopsy of the untreated primarytumor (thoracic spine), which was metastatic (rib, lung, bone marrow) at presentation. Recurrence: Bone metastasis (right humerus) 8 months subsequent,after systemic chemotherapy (VAC-IE) and localized radiotherapy to the primary andmultiple metastatic sites. D, Crystal violet staining showing olaparib-resistantCHLA10 cells after 5-day treatment of 5 mmol/L olaparib, 1 mmol/L navitoclax, or the combination of 5 mmol/L olaparib þ 1 mmol/L navitoclax compared to ano-treatment control (no Rx). E,Western blot analysis of apoptosis indicated by an increase in cleaved PARP1 in CHLA9 and CHLA10 cells after 24-hour treatmentof 5 mmol/L olaparib, 1 mmol/L navitoclax, or 5 mmol/L olaparib þ 1 mmol/L navitoclax compared to a no-treatment control (no Rx). F, Western blot analysis ofthe indicated antibodies in the CHLA9 and CHLA10 cell lines after 24-hour treatment of 5 mmol/L olaparib, 1 mmol/L navitoclax, or 5 mmol/L olaparib þ 1 mmol/Lnavitoclax compared to a no-treatment control (no Rx).G,CHLA10 xenografts treated daily with olaparib (100mg/kg), navitoclax (80mg/kg), or the combination ofolaparib (100mg/kg)þ navitoclax (80mg/kg) for 28 days. Error bars areþSEM. Asterisks indicate a significant separation between the combination (olap/nav) andall other treatment cohorts using the Student t test (P < 0.05). H, CHLA10 xenografts treated daily with olaparib (100 mg/kg), venetoclax (100 mg/kg), or thecombination of olaparib (100 mg/kg) þ venetoclax (100 mg/kg) for 30 days. Error bars are þSEM.






Figure 2.

Combination of olaparib and navitoclax doesnot augment toxicity. NSG mice were treatedwith olaparib (100 mg/kg), navitoclax (80mg/kg), or the combination of olaparib (100mg/kg)þ navitoclax (80mg/kg) compared toa no-treatment control (no Rx). After theindicated 3- or 7-day treatment period, bloodwas collected and sent to IDEXX BioResearch(idexxbioresearch.com) for a complete bloodcount. The recovery cohort was treated for7 days with the combination of olaparib(100 mg/kg) þ navitoclax (80 mg/kg) andallowed 24 hours without treatment beforeblood was collected. Three-day treatmentwith navitoclax not significant compared with3-day treatment with the combinationolaparib þ navitoclax, nor was 7-daynavitoclax compared with 7-day olaparib þnavitoclax.

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combination, which was ineffective (Fig. 1H), consistent with thein vitro results (Supplementary Fig. S5A). The olaparib and navi-toclax combination did not induce substantial weight loss in themice or any overt signs of toxicity, suggesting the combination iswell tolerated (Supplementary Fig. S6B). To assess possible hema-tologic toxicity when using olaparib and navitoclax in combina-tion, we performed a complete blood count on NSGmice in vivo.Red blood cell and reticulocyte counts were not significantlyaffected by the combination.Wedid observe a significant decreasein platelet count as well as neutrophil count following navitoclaxtreatment that has previously been reported with its use (31);however, importantly, olaparib did not augment platelet loss ateither time point. Also of importance, there was no augmentationof neutropenia or other toxicity by the combination comparedwith any single-agent dosing at either time point (Fig. 2). Asthrombocytopenia is the major dose-limiting effect of navitoclax(42), we also assayed populations of cells following a 24-hourrecovery period after 7 days of treatment. Impressively, thesemicenearly fully recovered pretreatment platelet levels (Fig. 2). Thesedata demonstrate olaparib plus navitoclax may be both effectiveand tolerated as a novel combination therapy in EWFTs.

Most Ewing sarcoma cell lines do not undergo markedapoptosis following olaparib therapy

Followingourfindings in theCHLA9andCHLA10pair,wenextexpanded to a panel of Ewing sarcoma cell lines to determine theability of olaparib to induce apoptosis. Akin to CHLA10, thesecells had poor apoptotic responses to olaparib (Fig. 3A), incontrast to the CHLA9 cells. However, all Ewing sarcoma cellsunderwent G2–M accumulation, as previously reported (Supple-mentary Fig. S6C; ref. 43). Caspase 3 activity confirmed both the

differential apoptosis between the CHLA9 cells and other EWFTscell lines, as well as the apoptosis sensitization by navitoclax(Supplementary Fig. S7A). These data suggest our findings ofmitigated apoptotic responses to olaparib uncovered in theCHLA10 cells extend to other EWFTs models.

We next determined whether these other resistant EWFTsmodels had higher levels of BCL-2 or BCL-XL, as ourmodel wouldpredict. Indeed, in comparison with the CHLA9 cells, thesemodels had higher levels of BCL-2 and/or BCL-XL (Fig. 3B),associated with their poor apoptotic responses to olaparib (Fig.3A). We have uncovered an important role for both BCL-2 andBCL-XL in olaparib response in EWFTs (Figs. 1 and 3), and itwould strengthen the case of the importance of BCL-2 and BCL-XL

in EWFTs survival if these cancerswere sensitive to pharmaceuticaltargeting of these 2 proteins. We therefore examined in theupdated GDSC screen (www.cancerRxgene.org) whether Ewingsarcoma cells were more sensitive to navitoclax (Fig. 3C) com-pared with all other solid tumor cell lines grouped together. Infact, Ewing sarcoma cell lines were substantially more sensitive(P ¼ 8.69 � 10�5), with 8 of 21 cell lines demonstrating IC50s of700 nmol/L and below (Fig. 3C).

Interestingly, expression of EWS-FLI1 in HEK293T cells led tohigher BCL-2 transcript levels (P<0.05) comparedwith the emptyvector control, with consistent BCL-2 protein changes (Supple-mentary Fig. S7B and S7C); the FLI1 target genes EZH2 (44),STEAP1, and PRKCB (45–47) were all significantly upregulated aswell (Supplementary Fig. S7B). To further evaluate the relation-ship of FLI1 and BCL-2, we probed the CCLE (24) and found apositive correlation (P < 0.0001) between FLI1 and BCL-2 (Sup-plementary Fig. S7D); knockdown of EWS-FLI1 confirmeddecrease of BCL2 expression with the expected decrease in EZH2

Figure 3.

EWFTs are resistant to olaparib whichcorrelates with increased BCL-2 andnavitoclax sensitivity.A,FACSanalysisof apoptosis after 24-hour treatmentwith 5 mmol/L olaparib. Error bars areþSEM. Asterisks indicate a significantseparation between the olaparibtreatment groups of the CHLA9 andCHLA10 cells, using the Student t test(P < 0.01). B, Western blot analysis ofthe indicated antibodies in EWFT celllines. C, IC50 of navitoclax plottedfor solid tumor cell lines and 21 Ewingsarcoma cell lines from http://www.cancerrxgene.org/. A Mann–Whitneynonparametric test was performed(P ¼ 8.69 � 10�5).





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expression (Supplementary Fig. S7E), and increase in expressionof the normally EWS-FLI1 downregulated LOX, while EWS-FLI1upregulated targets STEAP1, NPY1R, and PRKCB expression werereduced following EWS-FLI1 downregulation, albeit not all sig-nificantly (45–47). Altogether, these data further demonstrate theimportance of BCL-2 and BCL-XL in EWFTs, which appear to playcomplimentary roles, constituting a critical survival signal forEWS-FLI1–driven EWFTs.

Navitoclax sensitizes a panel of Ewing sarcoma cells to olaparibWe moved to validate navitoclax as a sensitizing agent to

olaparib in EWFTs cells, as was determined in the CHLA9 andCHLA10 pair (Fig. 1). In fact, we noted marked sensitization bynavitoclax to olaparib-induced apoptosis in a panel of Ewingsarcoma cells as evidenced by cleaved PARP1 (Fig. 4A) and FACSmeasurement of Annexin-V–positive cells (Fig. 4B). Consistentwith the CHLA pair, apoptosis sensitization was sufficient fornavitoclax to markedly reduce total viable cells as determined byboth crystal violet assays (Fig. 4C) and 72-hour cell-viabilityassays (Fig. 4D and E). These data again indicate that olaparib-induced apoptosis can be rescued by the addition of navitoclax inEwing sarcoma.

Navitoclax sensitizes Ewing sarcoma cells to olaparib byaugmentation of DNA damage and disruption of BIMcomplexes

We next examined the DNA-damaging activity of the olaparib/navitoclax combination using gH2A.X immunofluorescencestaining as a marker for DNA damage. Interestingly, the DNAdamage observed following olaparib exposure was substantiallyincreased (P < 0.0001) with the addition of navitoclax, which byitself did not induce DNA damage (Fig. 5A; Supplementary Fig.S8A). These data are consistent with a direct role of BCL-2 andBCL-XL in the augmentation of DNA damage (48).

Navitoclax disrupts BIM:BCL-XL and BIM:BCL-2 complexes, toinduce apoptosis (20, 49–51), and can sensitize kinase inhibitorsin different cancers through modulation of the BCL-2 family(reviewed in ref. 7). We therefore asked whether reduction ofBIM protected from the olaparib/navitoclax combination. Asdemonstrated in Fig. 5B, reduction of BIM by siRNA led to aconcomitant loss in cleavage of PARP1. Immunoprecipitation ofBIM complexes verified navitoclax-disrupted BIM:BCL-2 com-plexes (Fig. 5C; Supplementary Fig. S8B and S8C). These dataindicate that olaparib/navitoclax induce apoptosis in EWFTsthrough disruption of BIM complexes, whereas this complexdisruption leads to BIM-mediated apoptosis (51, 52). Together,these data demonstratemultiplemechanisms inwhich navitoclaxsensitizes Ewing sarcoma to olaparib.

Mouse models of Ewing sarcoma are sensitive to olaparib plusnavitoclax

To robustly test this novel combination of olaparib andnavitoclax, we expanded our analyses to 3 models of Ewingsarcoma, the SK-ES-1 xenograft (ATCC HTB-86), and 2PDXs. Mice were treated daily with olaparib (100 mg/kg),navitoclax (80 mg/kg), or the combination of olaparib(100 mg/kg) þ navitoclax (80 mg/kg). In all 3 models, therewas limited activity of either agent when dosed as a mono-therapy. However, the combination of olaparib and navitoclaxmarkedly inhibited tumor growth in the SK-ES-1 model andPDX SA10233 and, impressively, almost completely shrank

tumors in the other PDX model, SA13542 (Fig. 6A and B).Again, the combination did not markedly affect mouse weightsor induce any overt signs of toxicity (Supplementary Fig. S6B).Assessment of the tumor lysates from the SA13542 PDXconfirmed marked apoptosis induction with the combination,but not single agents (Fig. 6C), and that both PDX modelsexpressed BCL-XL and with the SA10233 model expressinghigh levels of BCL-2 (Supplementary Fig. S8D). These data,along with the CHLA10 chemorefractory mouse model(Fig. 1G), demonstrate compelling activity of the combinationof olaparib and navitoclax in vivo.

DiscussionThrough an unbiased high-throughput drug screen, olaparib

was discovered to have marked in vitro activity in Ewing sarcoma(11).Despite several other reports demonstrating hypersensitivityof Ewing sarcoma to PARP inhibition (10, 12–14, 23, 53),subsequent clinical evaluation in a heavily pretreated cohort ofpatients with Ewing sarcoma with single-agent olaparib showedonly modest efficacy (15). Here, we demonstrated an importantrole for deficient apoptosis following olaparib therapy in Ewingsarcoma, with the antiapoptotic proteins BCL-2 and BCL-XL

playing key roles. We believe these experimental findings at leastin part explain the disappointing clinical data.

PARP inhibitorsprevent single-stranded (ss)DNAbreak repairs.This mechanism underlies PARPi activity in BRCA-deficientcancers, which are inherently deficient in double-stranded (ds)DNA break repair (54). In Ewing sarcoma, PARPi sensitivityhas been proposed to occur for several reasons: First, PARP1expression is higher in Ewing sarcoma (55), probably as a directresult of EWS-FLI1 (10, 55), and higher PARP1 expression is acause of enhanced PARPi sensitivity (56), most likely throughthe mechanism of PARP trapping at ssDNA breaks (57, 59).Second, Ewing sarcoma, like BRCA- deficient cancers, appearsto have a deficient dsDNA repair system (12). Third, FLI1 driveshigh SLFN11 expression (60), a gene tightly linked to DNA-damaging agent efficacy (24, 61). Fourth, EWS-FLI1 expressionis sufficient to increase dsDNA breaks (10). Fifth, EWS-FLI1causes R-loop accumulation, increases replication stress, andinterferes with BRCA1 function (62).

Although there are several factors that may have contributedto olaparib's lack of efficacy in patients with chemotherapy-resistant Ewing sarcoma, it is likely that a biological resistancemechanism served to rescue tumor cells from direct PARPinhibition. We propose that there is an inherent deficiency inmany Ewing sarcomas to undergo apoptosis following olaparibtreatment resulting from a protective effect of BCL-2 andBCL-XL (Figs. 1 and 3). Furthermore, exposure and resistanceto chemotherapy appear to contribute to this state of apoptoticresistance to olaparib, as evidenced by our results in theCHLA10 cell line (Figs. 1E and 3A) and observations inpatients' tumor specimens (Fig. 1C). It is likely that DNA-damaging agents used in induction chemotherapy lead toadditional pressure on the Ewing sarcoma tumor and, as aresult, the emergence of cells particularly reliant on BCL-2/BCL-XL for survival. Overall, further studies will be necessary toelucidate the precise relationship between these prosurvivalBCL-2 members and Ewing sarcoma tumorigenesis.

The strategy to sensitize Ewing sarcoma to PARP inhibition viaBCL-2/BCL-XL coinhibition is different from other explored

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Figure 4.

Combination of olaparib and navitoclax is effective in multiple Ewing sarcoma cell lines. A, Western blot analysis of apoptosis indicated by cleaved PARP1 after24-hour treatment with no-treatment control (no Rx), 5 mmol/L olaparib, 1 mmol/L navitoclax, or 5 mmol/L olaparib þ 1 mmol/L navitoclax. B, FACS analysis ofapoptosis after 24-hour treatment with 5 mmol/L olaparib, 1 mmol/L navitoclax, or 5 mmol/L olaparib þ 1 mmol/L navitoclax. The percentage of apoptosis inducedby drugs is normalized to the no-treatment control. Error bars areþSEM. C, Crystal violet staining after 5-day treatment with 5 mmol/L olaparib, 1 mmol/L navitoclax,or 5 mmol/L olaparib þ 1 mmol/L navitoclax. D, Dose–response curves in Ewing sarcoma cell lines after 72-hour treatment with increasing concentrations ofolaparib. Viability was determined using CellTiter-Glo. Data are graphed as percent viable cells from no-treatment control (no Rx), performed in quadruplicate. Errorbars are þSEM. E, Ewing sarcoma cell lines after 72-hour treatment with 1 mmol/L navitoclax or 1 mmol/L navitoclax in the presence of increasing concentrationsof olaparib, and viability was determined using CellTiter-Glo. Data are graphed as percent viable cells from no-treatment control (no Rx), performed inquadruplicate. Error bars are þSEM.






strategies to sensitize Ewing sarcoma to PARP inhibition; theseinclude the addition of DNA-damaging agents that intensifiesthe amount of active DNA damage in the cell, like irinotecanand temozolomide (13). Temozolomide has also been dem-onstrated to enhance PARP1 trapping (59) and, interestingly,the combination of temozolomide and PARP inhibition coop-eratively downregulates MCL-1, sensitizing to mitochondrial-mediated death (13). Although temozolomide–PARPi combi-nations are poorly tolerated in preclinical Ewing sarcomamouse models (12), irinotecan delivered to an orthotopicEwing sarcoma mouse model in dosing schedules consistentwith the pediatric population demonstrated marked activity(12). Consistent with these results, the combination of thePARPi veliparib and irinotecan was well tolerated in a recentphase I trial, including reaching a dose sufficient for PARPinhibition in adult cancers (63). Of note, BCL-XL blocks theability of irinotecan to induce apoptosis and BCL-XL inhibitionresults in a switch from irinotecan-induced senescence to apo-ptosis (64). Therefore, it is possible that PARPi/irinotecancombinations in other Ewing sarcoma models will face thesame issues we have found PARP inhibition monotherapy toface, namely a refractory apoptosis response. The PARPi/irino-tecan combination is currently being evaluated in pediatricpatients with solid tumors (NCT02392793).

The BCL-2 family of proteins monitors the integrity of themitochondria and integrates the signals of many pathways at themitochondria (65). Importantly, Javaheri and colleagues (66)elegantly demonstrated that EWS-FLI1 overexpression in mesen-chymal stem cells, the presumed cell of origin for Ewing sarcoma,was sufficient for blocking differentiation but led to high rates ofapoptosis; retrovirus containing BCL-2, BCL-XL, or MCL-1 expres-sion plasmids was able to rescue apoptosis and, importantly, led

to sarcoma formation, which was not accomplished in the par-allel, control-transduced cells. These data togetherwith the data inthis article present a compelling case where antiapoptotic activityof BCL-2 family members, particularly BCL-2 and BCL-XL, playsan intricate role in Ewing sarcoma tumorigenesis and affectsEwing sarcoma therapy.

It has been well known for several decades that BCL-2 has aprotective role against DNA damage–induced apoptosis (48, 67).In addition, BCL-XL expression has been reported to correlateinversely with the sensitivity of cancer cell lines to multipleantitumor agents, including those acting via a DNA-damagingmechanism (23). This becomes particularly relevant in the lightthat Ewing sarcoma have deficient DNA damage responses (12).Adding to the intrigue, Brohl and colleagues recently reported13% of patients with Ewing sarcoma have germline loss-of-function mutations in DNA repair genes (68). It is thereforetempting to speculate that, in order for EWS-FLI1–translocatedEwing sarcoma to develop and thrive, there must be an acquiredreliance on the antiapoptotic proteins BCL-2 and BCL-XL tomaintain survival. Consistent with this notion, our analyses ofHTS data revealed navitoclax has substantial single-agent activity(IC50 less than 700 nmol/L) across approximately 40% of Ewingsarcoma cell lines (Fig. 3C).

This notion is further supported by our findings in the CHLAcells derived from a patient prior to and following chemother-apy treatment. The postchemotherapy CHLA10 cells, derived atprogressive disease, had higher BCL-2 expression relative to thematched chemotherapy-na€�ve CHLA9 cells (Fig. 1B) and, unlikeCHLA9 cells, failed to undergo cell death following olaparibtherapy (Figs. 1E and 3A). It is important to note that we didnot account for other changes that occurred during the acqui-sition of chemotherapy resistance in this model, which could

Figure 5.

DNA damage is increased with thecombination of olaparib andnavitoclax. A, Quantification ofimmunofluorescence images probedfor gH2A.X foci intensity following24-hour treatment using no-treatment control (no Rx), 5 mmol/Lolaparib, 1 mmol/L navitoclax,5 mmol/L olaparib þ 1 mmol/Lnavitoclax, or 1 mmol/L etoposide.Error bars are þSEM. Asterisksindicate a significant separationbetween olaparib treatment and thecombination olaparib þ navitoclax.Significancewas determined using theMann–Whitney U test. B, siRNAknockdown of BIM or control(scramble sequence) in ES4 and A673cell lines followed by 24-hourtreatment with either no-treatmentcontrol (no Rx) or 5 mmol/L olaparibþ1 mmol/L navitoclax (Nav/Olap).C, An immunoprecipitation of lysatesfrom A673 cells using either aBCL-2 antibody or an IgG controlantibody (left) and the whole-celllysates (5% input; right), after 4-hourtreatment with 5 mmol/L olaparib,1 mmol/L navitoclax, or 5 mmol/Lolaparib þ 1 mmol/L navitoclax.

Heisey et al.





contribute to the resistance of these cells to olaparib. Forinstance, Mendoza-Naranjo and colleagues demonstrated theCHLA10 cells have enhanced flux through the PI3K pathwaycompared with the CHLA9 cells, which is a result of increasedErbB4 expression (69) and which may be contributing toolaparib resistance. Notwithstanding, the fact that navitoclaxwas sufficient to resensitize the cells to olaparib reflects theimportance of BCL-2 and BCL-XL. Interestingly, in the chemo-therapy-na€�ve CHLA9 cells, where olaparib was very effective(Fig. 1A), navitoclax fully sensitized these cells to a low dose ofolaparib (Supplementary Fig. S5D), which did not have markedsingle-agent anticancer activity. These data reveal an importantinterplay between PARP inhibition and BCL-2/XL inhibition,which likely contributes to the impressive activity of dual PARPand BCL-2/XL inhibition in Ewing sarcoma (Figs. 1, 3, and 4)and, again, supports the notion that BCL-2 and BCL-XL areimportant to counteract the intrinsic deficiencies in Ewing

sarcoma DNA damage repair, which are exacerbated by PARPinhibition. Indeed, BCL-2/BCL-XL inhibition causes accumula-tion of DNA damage following PARP inhibition (Fig. 5A;Supplementary Fig. S8A). Therefore, the robust activity of PARPinhibition and navitoclax is most likely due to both BCL-2 andBCL- XL inhibition, making these cells more vulnerable to DNAdamage–induced apoptosis, but also increasing the DNA dam-age itself. The result is a substantial increase in apoptosis(Fig. 4A–B), mediated by BIM (Fig. 5B and C), which translatesto impressive in vivo activity.

Overall, we demonstrate Ewing sarcoma tumors do notundergo a marked apoptotic response following olaparib ther-apy; however, cotargeting BCL-2 and BCL-XL dramatically sen-sitizes these tumors to olaparib in several mouse models ofEwing sarcoma, including chemotherapy-resistant Ewing sar-coma and 2 PDX models of Ewing sarcoma. As we foundneither drug augmented hematologic toxicity of the other

Figure 6.

Olaparib and navitoclax combinationis effective in mouse models of Ewingsarcoma. A, SK-ES-1 xenograftstreated daily for 26 days (top) andpatient-derived xenografts (bottom)treated daily for 31 days (PDXSA13542) or 27 days (PDX SA10233)with olaparib (100 mg/kg), navitoclax(80 mg/kg), or the combination ofolaparib (100 mg/kg) þ navitoclax(80 mg/kg). Error bars are þSEM.Asterisks indicate a significantseparation between the combination(olap/nav) and all other treatmentcohorts using the Student t test(P < 0.05). B, Fold change in tumorvolume, please note data are from theexperiment shown in Fig. 6A (PDXSA13542) after 31 days of treatment,and the x axis denotes individualxenografts. C, Western blot analysisof cleaved PARP1 from PDX tumorlysates after 3 daily treatments of no-treatment control (no Rx), olaparib(100 mg/kg), navitoclax (80 mg/kg),or the combination of olaparib(100 mg/kg) þ navitoclax(80 mg/kg).






(Fig. 2), and rational navitoclax-based combinations withother targeted therapies are ongoing in clinical trials (e.g.,NCT02520778), evaluation of PARP inhibitors and navitoclaxin Ewing sarcoma is warranted.

Disclosure of Potential Conflicts of InterestS.C. Smith received royalties for textbook authorship and consulting from

Elseveir/Amirsys. C.H. Benes reports receiving commercial research grants fromAmgen and Novartis. J.D. Leverson and A.J. Souers have ownership interests(including patents) at AbbVie. A.C. Faber is a consultant/advisory boardmember for AbbVie. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: D.A.R. Heisey, S.A. Boikos, A.C. FaberDevelopment of methodology: D.A.R. Heisey, S.C. Smith, A.C. FaberAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D.A.R. Heisey, T.L. Lochmann, K.V. Floros,C.M. Coon, K.M. Powell, S. Jacob, M.L. Calbert, M.S. Ghotra, G.T. Stein,S.C. SmithAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D.A.R. Heisey, T.L. Lochmann, K.V. Floros, S. Jacob,

M.L. Calbert, M.S. Ghotra, S.C. Smith, C.H. Benes, J.D. Leverson, S.A. Boikos,A.C. FaberWriting, review, and/or revision of the manuscript: D.A.R. Heisey,T.L. Lochmann, S.C. Smith, J.D. Leverson, A.J. Souers, A.C. FaberAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D.A.R. Heisey, Y.K. Maves, C.H. BenesStudy supervision: S.A. Boikos, A.C. Faber

AcknowledgmentsThis work was supported by an American Cancer Society Research Scholar

Grant (A.C. Faber) and the Sarcoma Foundation of America (A.C. Faber).A.C. Faber is supported by the George and Lavinia Blick Research Fund andis a Harrison Endowed Scholar in Cancer Research. Services and products insupport of the researchproject were generated by the VCUMassey Cancer CenterCancer Mouse Model Shared Resource, supported, in part, with funding fromNIH-NCI Cancer Center Support Grant P30 CA016059.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 25, 2018; revised July 11, 2018; accepted October 17, 2018;published first October 22, 2018.

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2019;25:1664-1675. Published OnlineFirst October 22, 2018.Clin Cancer Res Daniel A.R. Heisey, Timothy L. Lochmann, Konstantinos V. Floros, et al. Escape PARP Inhibitor Toxicity

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The Ewing Family of Tumors Relies on BCL-2 and BCL-X to ... · The Ewing family of tumors (EWFTs), consisting of primitive neuroectodermal tumor (PNET) and Ewing sarcoma, is a malig-nancy