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Cancer Biology and Signal Transduction PARP Inhibitors Sensitize Ewing Sarcoma Cells to Temozolomide-Induced Apoptosis via the Mitochondrial Pathway Florian Engert 1 , Cornelius Schneider 1 , Lilly Magdalena Weib 1,2,3 , Marie Probst 1 , and Simone Fulda 1,2,3 Abstract Ewing sarcoma has recently been reported to be sensitive to poly(ADP)-ribose polymerase (PARP) inhibitors. Searching for synergistic drug combinations, we tested several PARP inhibi- tors (talazoparib, niraparib, olaparib, veliparib) together with chemotherapeutics. Here, we report that PARP inhibitors syner- gize with temozolomide (TMZ) or SN-38 to induce apoptosis and also somewhat enhance the cytotoxicity of doxorubicin, etoposide, or ifosfamide, whereas actinomycin D and vincris- tine show little synergism. Furthermore, triple therapy of ola- parib, TMZ, and SN-38 is signicantly more effective compared with double or monotherapy. Mechanistic studies revealed that the mitochondrial pathway of apoptosis plays a critical role in mediating the synergy of PARP inhibition and TMZ. We show that subsequent to DNA damage-imposed checkpoint activa- tion and G 2 cell-cycle arrest, olaparib/TMZ cotreatment causes downregulation of the antiapoptotic protein MCL-1, followed by activation of the proapoptotic proteins BAX and BAK, mitochon- drial outer membrane permeabilization (MOMP), activation of caspases, and caspase-dependent cell death. Overexpression of a nondegradable MCL-1 mutant or BCL-2, knockdown of NOXA or BAX and BAK, or the caspase inhibitor N-benzyloxycarbonyl-Val- Ala-Asp-uoromethylketone (zVAD.fmk) all signicantly reduce olaparib/TMZ-mediated apoptosis. These ndings emphasize the role of PARP inhibitors for chemosensitization of Ewing sarcoma with important implications for further (pre)clinical studies. Mol Cancer Ther; 14(12); 281830. Ó2015 AACR. Introduction Ewing sarcoma is the second most common pediatric primary bone tumor in children and young adults (1). State-of-the-art treatment options for Ewing sarcoma patients include chemo- therapy consisting of vincristine, ifosfamide, doxorubicin, temo- zolomide (TMZ), actinomycin D, irinotecan, etoposide, and cyclophosphamide (2). Despite aggressive treatment protocols, patients especially with advanced or relapsed diseases still harbor an overall poor survival (2). This highlights the unmet medical need to develop innovative treatment strategies. Tumor response to cytotoxic therapies including chemotherapy critically relies on intact cell death programs in cancer cells, as chemotherapeutics exert their anticancer effects to a large extent by engaging programmed cell death (3). Apoptosis is mediated via two well-dened pathways, that is the extrinsic (death recep- tor) pathway and the intrinsic (mitochondrial) pathway, which both eventually lead to activation of caspases as cell death effector molecules (4). Engagement of the mitochondrial pathway results in mitochondrial outer membrane permeabilization (MOMP) accompanied by the release of mitochondrial intermembrane space proteins such as cytochrome c into the cytosol, which in turn results in caspase activation and apoptosis (5). MOMP is tightly controlled by various factors including proteins of the BCL- 2 family. BCL-2 family proteins consist of both antiapoptotic members, for example BCL-2, BCL-x L , and MCL-1, and proapop- totic molecules such as the multidomain proteins BAK and BAX and BH3-only domain proteins, for example, BIM, BMF, PUMA, and NOXA (6). Characteristic for Ewing sarcoma is the existence of a chimeric fusion protein which is in >85% of cases generated by the translocation of the EWS gene on chromosome 22 and the FLI1 gene on chromosome 11 (11;22)(q24;q12) (7). This fusion protein encodes the chimeric transcription factor EWS-FLI1 that causes upregulation of a range of target genes including PARP1 (7, 8), which promotes transcriptional activation by EWS-FLI1 in a positive feedback loop (9). Elevated PARP levels have been detected in Ewing sarcoma specimens (8, 10), indicating that PARP may represent a promising target for therapeutic exploita- tion in Ewing sarcoma. Indeed, Ewing sarcoma cells have been shown to be particularly sensitive to PARP inhibition (9, 10). Several PARP inhibitors including talazoparib (BMN-673), nir- aparib (MK-4827), olaparib (AZD-2281), and veliparib (ABT- 888) are currently under investigation (11). Despite the sensitivity of Ewing sarcoma cells to PARP inhibi- tors in vitro (9, 10, 12), PARP inhibitors as single therapy displayed limited efcacy in xenograft models of Ewing sarcoma and in initial clinical trials (1215), highlighting the need for combina- tion therapies. PARP inhibitors have been documented to poten- tiate DNA damage-mediated cytotoxicity caused in particular by topoisomerase I poisons, DNA methylating agents, or radiation in several cancers including Ewing sarcoma, which has been related 1 Institute for Experimental Cancer Research in Pediatrics, Goethe- University, Frankfurt, Germany. 2 German Cancer Consortium (DKTK), Heidelberg, Germany. 3 German Cancer Research Center (DKFZ), Hei- delberg, Germany. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Simone Fulda, Goethe-University, Komturstr. 3a, 60528 Frankfurt, Germany. Phone: 49-69-67866557; Fax: 49-69-6786659157; E-mail: [email protected] doi: 10.1158/1535-7163.MCT-15-0587 Ó2015 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 14(12) December 2015 2818 on April 25, 2019. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst October 5, 2015; DOI: 10.1158/1535-7163.MCT-15-0587

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Page 1: PARP Inhibitors Sensitize Ewing Sarcoma Cells to ...mct.aacrjournals.org/content/molcanther/14/12/2818.full.pdfCancer Biology and Signal Transduction PARP Inhibitors Sensitize Ewing

Cancer Biology and Signal Transduction

PARP Inhibitors Sensitize Ewing Sarcoma Cellsto Temozolomide-Induced Apoptosis via theMitochondrial PathwayFlorian Engert1, Cornelius Schneider1, Lilly Magdalena Weib1,2,3, Marie Probst1, andSimone Fulda1,2,3

Abstract

Ewing sarcoma has recently been reported to be sensitive topoly(ADP)-ribose polymerase (PARP) inhibitors. Searching forsynergistic drug combinations, we tested several PARP inhibi-tors (talazoparib, niraparib, olaparib, veliparib) together withchemotherapeutics. Here, we report that PARP inhibitors syner-gize with temozolomide (TMZ) or SN-38 to induce apoptosisand also somewhat enhance the cytotoxicity of doxorubicin,etoposide, or ifosfamide, whereas actinomycin D and vincris-tine show little synergism. Furthermore, triple therapy of ola-parib, TMZ, and SN-38 is significantly more effective comparedwith double or monotherapy. Mechanistic studies revealed thatthe mitochondrial pathway of apoptosis plays a critical role inmediating the synergy of PARP inhibition and TMZ. We show

that subsequent to DNA damage-imposed checkpoint activa-tion and G2 cell-cycle arrest, olaparib/TMZ cotreatment causesdownregulation of the antiapoptotic protein MCL-1, followed byactivation of the proapoptotic proteins BAX and BAK, mitochon-drial outer membrane permeabilization (MOMP), activation ofcaspases, and caspase-dependent cell death. Overexpression of anondegradableMCL-1mutant or BCL-2, knockdown of NOXA orBAX and BAK, or the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) all significantly reduceolaparib/TMZ-mediated apoptosis. These findings emphasizethe role of PARP inhibitors for chemosensitization of Ewingsarcoma with important implications for further (pre)clinicalstudies. Mol Cancer Ther; 14(12); 2818–30. �2015 AACR.

IntroductionEwing sarcoma is the second most common pediatric primary

bone tumor in children and young adults (1). State-of-the-arttreatment options for Ewing sarcoma patients include chemo-therapy consisting of vincristine, ifosfamide, doxorubicin, temo-zolomide (TMZ), actinomycin D, irinotecan, etoposide, andcyclophosphamide (2). Despite aggressive treatment protocols,patients especially with advanced or relapsed diseases still harboran overall poor survival (2). This highlights the unmet medicalneed to develop innovative treatment strategies.

Tumor response to cytotoxic therapies including chemotherapycritically relies on intact cell death programs in cancer cells, aschemotherapeutics exert their anticancer effects to a large extentby engaging programmed cell death (3). Apoptosis is mediatedvia two well-defined pathways, that is the extrinsic (death recep-tor) pathway and the intrinsic (mitochondrial) pathway, whichboth eventually lead to activation of caspases as cell death effectormolecules (4). Engagement of the mitochondrial pathway resultsin mitochondrial outer membrane permeabilization (MOMP)

accompanied by the release of mitochondrial intermembranespace proteins such as cytochrome c into the cytosol, which inturn results in caspase activation and apoptosis (5). MOMP istightly controlled by various factors including proteins of the BCL-2 family. BCL-2 family proteins consist of both antiapoptoticmembers, for example BCL-2, BCL-xL, and MCL-1, and proapop-totic molecules such as the multidomain proteins BAK and BAXand BH3-only domain proteins, for example, BIM, BMF, PUMA,and NOXA (6).

Characteristic for Ewing sarcoma is the existence of a chimericfusion protein which is in >85% of cases generated by thetranslocation of the EWS gene on chromosome 22 and the FLI1gene on chromosome 11 (11;22)(q24;q12) (7). This fusionprotein encodes the chimeric transcription factor EWS-FLI1 thatcauses upregulation of a range of target genes including PARP1(7, 8), which promotes transcriptional activation by EWS-FLI1 ina positive feedback loop (9). Elevated PARP levels have beendetected in Ewing sarcoma specimens (8, 10), indicating thatPARP may represent a promising target for therapeutic exploita-tion in Ewing sarcoma. Indeed, Ewing sarcoma cells have beenshown to be particularly sensitive to PARP inhibition (9, 10).Several PARP inhibitors including talazoparib (BMN-673), nir-aparib (MK-4827), olaparib (AZD-2281), and veliparib (ABT-888) are currently under investigation (11).

Despite the sensitivity of Ewing sarcoma cells to PARP inhibi-tors in vitro (9, 10, 12), PARP inhibitors as single therapy displayedlimited efficacy in xenograft models of Ewing sarcoma and ininitial clinical trials (12–15), highlighting the need for combina-tion therapies. PARP inhibitors have been documented to poten-tiate DNA damage-mediated cytotoxicity caused in particular bytopoisomerase I poisons,DNAmethylating agents, or radiation inseveral cancers including Ewing sarcoma, which has been related

1Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany. 2German Cancer Consortium (DKTK),Heidelberg, Germany. 3German Cancer Research Center (DKFZ), Hei-delberg, Germany.

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

CorrespondingAuthor: Simone Fulda, Goethe-University, Komturstr. 3a, 60528Frankfurt, Germany. Phone: 49-69-67866557; Fax: 49-69-6786659157; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0587

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 14(12) December 20152818

on April 25, 2019. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst October 5, 2015; DOI: 10.1158/1535-7163.MCT-15-0587

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A4573 Veliparib (nmol/L)Olaparib (nmol/L)Niraparib (nmol/L)Talazoparib (nmol/L) CI values2,8002,2001,6006003001007550257.552.510.5

TMZ (µmol/L)

25 Very strong synergism< 0.150 Strong synergism0.1–0.3100 Synergism0.3–0.7

SN38 (nmol/L)

0.2 Moderate synergism0.7–0.850.4 Slight synergism0.85–0.90.6 Nearly additive0.9–1.1

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25 Slightly antagonistic1.1–1.250 Moderate antagonistic1.2–1.4575 Antagonism>1.45

Ifosfamide (µmol/L)

0.5 Not calculated12

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12.54

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0.10.250.4

SK-ES-1 Veliparib (nmol/L)Olaparib (nmol/L)Niraparib (nmol/L)Talazoparib (nmol/L) CI values2,8002,2001,6006003001007550257.552.510.5

TMZ (µmol/L)

25 Very strong synergism< 0.150 Strong synergism0.1–0.3100 Synergism0.3–0.7

SN38 (nmol/L)

0.2 Moderate synergism0.7–0.850.4 Slight synergism0.85–0.90.6 Nearly additive0.9–1.1

Etoposide (ng/mL)

25 Slightly antagonistic1.1–1.250 Moderate antagonistic1.2–1.4575 Antagonism>1.45

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0.5 Not calculated12

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12.54

Vincristine (nmol/L)

0.10.250.4

AMD (ng/mL)

0.10.250.4

Figure 1.Screening for synergistic drug interactions of PARP inhibitors and chemotherapeutic drugs. A, A4573 and SK-ES-1 cells were treated for 72 hours with indicatedconcentrations of talazoparib, niraparib, olaparib, or veliparib. Cell viability was assessed by crystal violet assay and is shown as percentage of untreatedcontrol. B, A4573 and SK-ES-1 cells were treated for 72 hours with different PARP inhibitors (i.e., talazoparib, niraparib, olaparib, and veliparib) in combination withdifferent cytostatic drugs (i.e., TMZ, SN-38, etoposide, ifosfamide, doxorubicin, vincristine, and actinomycin D). Cell viability was assessed by crystal violetassay. Synergistic, additive, or antagonistic drug interactions were calculated by combination index (CI). CI values were then used to generate a heatmap of druginteractions according to the subclassification provided by the software's manual.

Synergistic Apoptosis by PARP Inhibitors and Temozolomide

www.aacrjournals.org Mol Cancer Ther; 14(12) December 2015 2819

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Engert et al.

Mol Cancer Ther; 14(12) December 2015 Molecular Cancer Therapeutics2820

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to the role of PARP in the repair of theDNA lesions causedby thesecytotoxic therapies (9, 13, 16, 17). In addition to impairing single-strand break (SSB) repair, the anticancer effects of PARP inhibitorshave recently also been attributed to their ability to trap PARP–DNA complexes, thereby generating cytotoxic lesions (18).

Although different PARP inhibitors were reported to vary intheir potency of PARP-DNA trapping (18, 19), they have not yetbeen systematically tested in combination with a range of anti-cancer drugs in Ewing sarcoma. In addition, little is yet known oncell death signaling pathways that mediate the synergistic inter-action of PARP inhibitors and anticancer drugs. Therefore, in thisstudy, we aimed (i) at examining the effects of different PARPinhibitors in combinationwith clinically used chemotherapeuticsin Ewing sarcoma cells to identify themost potent synergistic drugcombinations and (ii) at elucidating the molecular mechanismsof synergy with a specific focus on cell death pathways.

Materials and MethodsCell culture and chemicals

Ewing sarcoma cell lines were kindly provided by C. Roessig(Muenster, Germany) or obtained from German Collection ofMicroorganisms and Cell Cultures (Braunschweig, Germany) orAmerican Type Culture Collection (Manassas, VA, USA) in 2013.Cells were maintained in DMEM GlutaMAX-l or RPMI medium(Life Technologies, Inc.), supplementedwith 10% fetal calf serum(FCS), 1% penicillin/streptomycin, 1 mmol/L sodium pyruvate(all from Life Technologies, Inc.). Cell lines were authenticated bySTR profiles and regularly tested for mycoplasma contamination.Talazoparib, olaparib, and veliparib were obtained from Sell-eckchem and niraparib from ChemieTek. actinomycin D, doxo-rubicin, etoposide, TMZ, SN-38, and vincristine were purchasedfrom Sigma, zVAD.fmk from Bachem, bortezomib from Jansen-Cilag. 4-Hydroperoxy-ifosfamide (active metabolite of ifosfa-mide and further designated as ifosfamide) was kindly providedby Baxter and TRAIL receptor-2 agonistic antibody lexatumumab(ETR2) Human Genome Sciences. Chemicals were purchasedfrom Sigma or Carl Roth unless otherwise indicated.

Determination of apoptosis, cell viability, colony formation,and mitochondrial membrane potential (MMP)

Apoptosis was determined by flow cytometric analysis (FACS-Canto II; BD Biosciences) of DNA fragmentation of propidiumiodide (PI)-stained nuclei as described previously (20). Cellviability was assessed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphe-nyltetrazolium bromide (MTT) assay according to the manufac-turer's instructions (Roche Diagnostics) or by crystal violet assayusing crystal violet solution (0.5% crystal violet, 30% ethanol, 3%formaldehyde). Plates were then rinsed with water and crystal

violet incorporated by the cells was resolubilized in a solutioncontaining 1% SDS. Absorbance at 550 nmwasmeasured using amicroplate reader (Infinite M100; Tecan). Results are expressed aspercentage of cell viability relative to untreated controls. Forcolony formation assay, cells were treated as indicated for 24hours. Subsequently, living cells were counted, 100 cells werereseeded, and cultured in drug-free medium for additional 12days before fixation and staining with 0.5% crystal violet, 30%ethanol, and 3% formaldehyde. Colonies were countedmacroscopically.

Western blot analysisWestern blot analysis was performed as described previously

(20) using the following antibodies: BAK, BCL-2, BCL-xL (BDBiosciences), pChk1, pChk2, Chk1, Chk2, caspase-3, caspase-9,PARP, BIM, PUMA (Cell Signaling), BAX NT, pH 3, a-tubulin(Millipore), BMF (Novus Biologicals) MCL-1, NOXA, caspase-8(Enzo Life Science), GAPDH (HyTest), murine BCL-2 (Life Tech-nologies, Inc.). Goat antimouse IgG and goat anti-rabbit IgGconjugated tohorseradish peroxidase (SantaCruzBiotechnology)were used as secondary antibodies and enhanced chemilumines-cence (Amersham Bioscience) or infrared dye-labeled secondaryantibodies and infrared imagingwere used for detection (OdysseyImaging System; LI-COR Bioscience). Representative blots of atleast two independent experiments are shown.

Cell-cycle analysisCells were stained with PI as described previously (20). Cell-

cycle analysis was performed using FlowJo Software (TreeStarInc.) following the manufacturer's instructions.

RNA interference and overexpressionCells were reversely transfected with 10 nmol/L Silencer Select

(Life Technologies, Inc.) control siRNA (10 nmol/L, 4390844),NOXA siRNA (10 nmol/L s10708 and s10709), or a combinationof targeting siRNAs (5 nmol/L for BAK, s1880 and s1881; 5 nmol/Lfor BAX, s1889 and s1890) using Lipofectamine RNAiMAX reagent(Life Technologies, Inc.) and Opti-MEM medium (Life Technolo-gies, Inc.). After 6 hours of incubation with transfection solution,the medium was changed and cells recovered for 48 hours beforedrug treatment. For transient overexpression, Ewing sarcoma cellswere transfectedwith 4 mg of pCMV-Tag 3B plasmid [empty vector;MCL-1 4A (S64A/S121A/S159A/T163A)], supplied with Lipofec-tamine 2000 (Life Technologies, Inc.) and selectedwith 500 mg/mLG418 (Carl Roth).Murine BCL-2was stably overexpressedbyusinglentiviral vectors. Briefly, Phoenix cells were transfected with 20 mgofpMSCVplasmid (emptyvector, BCL-2) using calciumphosphatetransfection. Virus-containing supernatant was collected, sterile-filtered, and used for spin transduction at 37�C in the presence of 8

Figure 2.Olaparib synergizes with TMZ and SN-38 to induce cell death in Ewing sarcoma cells. A and B, A4573 and SK-ES-1 cells were treated for 48 hours with0.3 mmol/L olaparib and/or 50 mmol/L TMZ and/or 0.6 nmol/L SN-38. Apoptosis was determined by analysis of DNA fragmentation of PI-stained nuclei using flowcytometry (A). Cell viability was measured by MTT assay and is expressed as percentage of untreated control (B). Data are shown as mean � SD of threeindependent experiments performed in triplicate; � , P < 0.05; �� , P < 0.01; ��� , P < 0.001. C, A4573 and SK-ES-1 cells were treated with 0.3 mmol/L olaparib and/or 50mmol/L TMZ and/or 0.6 nmol/L SN-38 for 24 hours, living cells were counted and subsequently 100 cells/well were reseeded in drug-free medium in asix-well plate for additional 12 days. Colony formation was assessed by crystal violet staining and colonies were counted macroscopically. The number of colonies isexpressed as percentage of untreated control (top) and representative images are shown (bottom). Data are shown as mean � SD of three independentexperiments performed in triplicate; � , P < 0.05; �� , P < 0.01. D, A4573 and SK-ES-1 cells were treated for 72 hours with 0.3 mmol/L olaparib and/or 10 mmol/LTMZ and/or 0.2 nmol/L SN-38. Cell viability was measured by crystal violet staining and is expressed as percentage of untreated control. Data are shownas mean � SD of three independent experiments performed in triplicate; � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.

Synergistic Apoptosis by PARP Inhibitors and Temozolomide

www.aacrjournals.org Mol Cancer Ther; 14(12) December 2015 2821

on April 25, 2019. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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Engert et al.

Mol Cancer Ther; 14(12) December 2015 Molecular Cancer Therapeutics2822

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mg/mL polybrene. Transduced Ewing sarcoma cells were selectedwith 10 mg/mL blasticidin (Carl Roth).

Determination of BAK and BAX activation or MMPBAK and BAX activation was determined by immunoprecipi-

tation of active conformation by specific antibodies. Briefly, cellswere lysed in CHAPS lysis buffer (10mmol/LHEPES, pH 7.4; 150mmol/L NaCl; 1% CHAPS). Five hundred micrograms of proteinwere incubated overnight at 4�C with 8 mg mouse anti-BAXantibody (clone 6A7; Sigma) or 0.5 mg mouse anti-BAK antibody(AB-1; Calbiochem) and 10 mL pan mouse IgG Dynabeads(Dako), washedwithCHAPS lysis buffer and analyzed byWesternblotting using rabbit anti-BAX NT antibody (Millipore) or rabbitanti-BAK antibody (BD Biosciences). Loss of MMP was assessedby tetramethylrhodamine methyl ester (TMRMþ) stainingaccording to the manufacturer's instructions (Life TechnologiesInc.).

Statistical analysisStatistical significancewas assessed by Student t-test (two-tailed

distribution, two-sample, equal variance) using Microsoft Excel(Microsoft Deutschland GmbH); �, P < 0.05; ��, P < 0.01; ���, P <0.001. Drug interactions were analyzed by the combination index(CI) method based on that described by Chou (21) using Calcu-Syn software (Biosoft). Subclassification of CI values according toCalcuSyn'smanual was used (CI < 0.1 very strong synergism, 0.1–0.3 strong synergism, 0.3–0.7 synergism, 0.7–0.85 moderatesynergism, 0.85–0.9 slight synergism, 0.9–1.1 nearly additive,1.1–1.2 slightly antagonistic, 1.2–1.45 moderate antagonistic,and CI > 1.45 antagonism.

ResultsScreening for synergistic drug interactions of PARP inhibitorsand chemotherapeutic drugs

To investigate the sensitivity of Ewing sarcoma against PARPinhibitors, we initially tested the dose response to four differentPARP inhibitors using the Ewing sarcoma cell lines A4573 and SK-ES-1 and subsequently extended our studies also to two addi-tional Ewing sarcoma cell lines. Talazoparib, niraparib, olaparib,and veliparib showed a differential ability to reduce cell viabilityof Ewing sarcoma cells with talazoparib being themost active andveliparib being the least active compound (talazoparib IC50� 10nmol/L > niraparib IC50 � 300 nmol/L > olaparib IC50 � 1000nmol/L > veliparib IC50� 10000 nmol/L; Fig. 1A, SupplementaryTable S1).

To investigate the question whether PARP inhibitors modulatechemosensitivity of Ewing sarcoma cells, we screened the efficacyof several anticancer drugs that are used in the clinic for thetreatment of Ewing sarcoma (11) in the presence and absence ofPARP inhibitors. We used suboptimal drug concentrations of

PARP inhibitors and anticancer drugs that caused up to � 20%reduction of cell viability when used as single agents comparedwith untreated control (Supplementary Fig. S1). Synergistic,additive, or antagonistic drug interactions were calculated by CI(Supplementary Table S2). CI values were then used to generate aheatmap of drug interactions according to the subclassificationprovided by the software's manual (Fig. 1B). The strongest syn-ergism was observed for PARP inhibitors in combination withTMZ; the secondbest synergistic interactionwas found for cotreat-ment with SN-38 (Fig. 1B, Supplementary Fig. S2). We also notedthat, among the tested anticancer drugs, talazoparib preferentiallysynergized with TMZ (Fig. 1B, Supplementary Fig. S2). In addi-tion, PARP inhibitors enhanced doxorubicin-, etoposide-, orifosfamide-induced cytotoxicity in Ewing sarcoma cells, althoughless consistently (Fig. 1B, Supplementary Fig. S2). By comparison,the DNA-binding drug actinomycin D and the vinca alkaloidvincristine exerted little synergistic effects together with PARPinhibitors (Fig. 1B, Supplementary Fig. S2).

Olaparib synergizeswith TMZ and SN-38 to induce cell death inEwing sarcoma cells

Because in our screening approachwe identified cotreatment ofolaparib with TMZ or SN-38 as themost potent combinations, wefocused our validation experiments on these combinations. ola-parib acted together with TMZ or SN-38 to increase DNA frag-mentation, a typical marker of apoptosis, and to reduce cellviability compared to treatment with either agent alone (Fig.2A and B). Moreover, we extended our studies to two additionalEwing sarcomacell lines. Similarly, olaparib cooperatedwithTMZor SN-38 to induce apoptosis and to decrease cell viability in TC-32 and TC-71 cells (Supplementary Fig. S3). To explore whethercombined treatment with olaparib and TMZ or SN-38 also affectslong-term survival of Ewing sarcoma cells, we performed colonyassays. Of note, cotreatment of olaparib together with TMZ or SN-38 acted in concert to significantly suppress colony formation ofEwing sarcoma cells compared to treatment with olaparib, TMZ,or SN-38 alone (Fig. 2C). Because TMZ and SN-38 are adminis-tered together in clinical protocols as second-line treatment ofEwing sarcoma (22), we also tested whether the addition ofolaparib further potentiates the antitumor activity of this chemo-therapy regimen.Ofnote, triple therapyof olaparib, TMZ, andSN-38was significantlymore effective to reduce cell viability of Ewingsarcoma cells compared to treatment with single agents or tocotreatment with TMZ/SN-38 (Fig. 2D). Together, this set ofexperiments shows that olaparib cooperates together with TMZor SN-38 to induce apoptosis, to reduce cell viability, and tosuppress long-term clonogenic survival of Ewing sarcoma cells. Toinvestigate in more detail the molecular mechanisms underlyingthe synergism of PARP inhibitors and chemotherapeutic drugs inEwing sarcoma, we focused the subsequent experiments on TMZ,

Figure 3.Olaparib/TMZ cotreatment causesG2 arrest prior to cell death. A, A4573 and SK-ES-1 cellswere treatedwith 0.3mmol/L olaparib and/or 50mmol/L TMZ for indicatedtime. Apoptosis was determined by quantification of DNA fragmentation of PI-stained nuclei using flow cytometry. Data are shown as mean � SD of threeindependent experiments performed in triplicate; � , P < 0.05; �� , P < 0.01; ��� , P < 0.001. B, A4573 and SK-ES-1 cells were treated with 0.3 mmol/L olaparib and/or50 mmol/L TMZ for 18 hours. Phosphorylation of checkpoint kinaseswas assessed byWestern blotting. Expression of GAPDH ora-tubulin served as loading controls.Representative blots of two independent experiments are shown. C, A4573 and SK-ES-1 cells were treated for 18 hours with 0.3 mmol/L olaparib and/or50 mmol/L TMZ. DNA was stained with PI and cell-cycle analysis was performed using FlowJo software. Data are shown as mean � SD of three independentexperiments performed in triplicate; � , P < 0.05; �� , P < 0.01 comparing olaparib/TMZ cotreated to single treated or untreated cells in G2–M phase. D, Ewingsarcoma cells were treated with 0.3 mmol/L olaparib and/or 50 mmol/L TMZ or 2.5 nmol/L vincristine for 18 hours. Expression ofmitotic marker pH3was analyzed byWestern blotting. Expression of GAPDH served as loading control. Representative blots of two independent experiments are shown.

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because this drug yielded the most pronounced synergisticinteraction.

Olaparib/TMZ cotreatment causes G2 arrest prior to cell deathTo define the onset of olaparib/TMZ-induced apoptosis we

performed a kinetic analysis. Olaparib and TMZ cooperated totrigger apoptosis in a time-dependentmanner starting at about 18hours (Fig. 3A).

Next, we investigated whether the combination treatmentleads to a DNA damage-related stress response. Olaparib andTMZ acted together to trigger phosphorylation of the check-point kinases Chk1 and Chk2 at 18 hours pointing to activationof DNA damage response pathways prior to cell death (Fig. 3B).Analysis of cell-cycle distribution using flow cytometry revealedthat olaparib/TMZ cotreatment caused a significant increase ofcells in G2–M phase compared with cells treated with eitherdrug alone or untreated cells (Fig. 3C, Supplementary Fig. S4).As flow cytometric analysis of cell-cycle distribution does notallow to distinguish between G2 or M phase arrest, we addi-tionally analyzed phosphorylation of histone H3 (pH3) as aspecific M-phase marker. Olaparib/TMZ cotreatment did notcause phosphorylation of histone H3 in contrast to the micro-tubule-interfering drug vincristine that was used as a positivecontrol for the induction of M-phase arrest (Fig. 3D). Together,these experiments show that olaparib and TMZ induce cell-cycle arrest in the G2 phase prior to induction of apoptosis inEwing sarcoma cells.

Olaparib/TMZ-induced apoptosis is executed via caspase-dependent effector pathways

Toexplorewhether inductionofapoptosis involvedactivationofcaspases, we performedWestern blotting. Olaparib and TMZ actedtogether to trigger cleavage of caspase-9 into active p37/p35 frag-ments, cleavage of caspase-3 into active p17/p12 fragments, andcleavage of PARP into p89 fragment. By comparison, little cleavageof caspase-8 was observed upon olaparib/TMZ combination treat-ment in contrast to treatmentwith the tumornecrosis factor-relatedapoptosis-inducing ligand (TRAIL) receptor 2 agonistic antibodylexatumumab that served as positive control (Fig. 4A). Expressionlevels of caspase-8 were very low in A4573 cells consistent withprevious studies demonstrating that caspase-8 is frequentlysilenced by epigenetic mechanisms in Ewing sarcoma (23, 24).

To test whether caspase activity is required for the induction ofapoptosis, we used the broad-range caspase inhibitor zVAD.fmk.Addition of zVAD.fmk significantly diminished olaparib/TMZ-induced apoptosis compared with cells that were treated witholaparib/TMZ in the absence of zVAD.fmk (Fig. 4B). These experi-ments show that olaparib and TMZ cooperate to trigger caspase

activation and caspase-dependent apoptosis in Ewing sarcomacells.

Olaparib/TMZ cotreatment downregulates MCL-1 levelsTo investigate whether olaparib/TMZ combination treatment

engages the mitochondrial pathway of apoptosis, we assessedthe MMP. Olaparib and TMZ cooperated to trigger loss of MMPin a time-dependent manner at the onset of apoptosis (Fig. 4C).Because BCL-2 family proteins are key regulators of the mito-chondrial pathway, we then asked whether olaparib/TMZcotreatment causes changes in their expression levels. Of note,cotreatment with olaparib and TMZ resulted in downregulationof MCL-1, whereas expression levels of BCL-2, BCL-xL, BIM,BMF, and PUMA remained largely unchanged (Fig. 4D). Wealso noted that NOXA levels decreased upon olaparib/TMZcotreatment (Fig. 4D).

Olaparib/TMZ cotreatment promotes proteasomal degradationof MCL-1

To investigate whether the observed downregulation of MCL-1upon olaparib/TMZ cotreatment is due to proteasomal degrada-tion and/or caspase-mediated cleavage, we tested the effects of theproteasome inhibitor bortezomib and/or the pan-caspase inhib-itor zVAD.fmk. Although addition of bortezomib significantlyrescued the olaparib/TMZ-imposed downregulation of MCL-1protein levels, addition of zVAD.fmk largely failed to significantlyprotect against MCL-1 downregulation upon olaparib/TMZcotreatment (Fig. 5A). This indicates that MCL-1 is degraded viathe proteasome upon treatment with olaparib/TMZ.

To elucidate the role of MCL-1 in olaparib/TMZ-mediated celldeath we overexpressed a phosphomutant variant of MCL-1(MCL-1 4A), which is resistant to phosphorylation of a phos-pho-degron and subsequent proteasomal degradation (Fig. 5B).Notably, ectopic expression of nondegradable MCL-1 mutantsignificantly decreased olaparib/TMZ-induced apoptosis (Fig.5C). Because NOXA is known to specifically antagonize MCL-1, we also knocked downNOXAbyRNAi to further investigate theinvolvement of the MCL-1/NOXA axis. NOXA silencing signifi-cantly reduced olaparib/TMZ-induced apoptosis in Ewing sarco-ma cells (Fig. 5D and E). Together, these results indicate thatolaparib/TMZ-triggered degradation of MCL-1 contributes toolaparib/TMZ-induced apoptosis.

Olaparib/TMZ cotreatment promotes BAX/BAK activation andMOMP

Next, we explored whether degradation of MCL-1 leads toactivation of BAK and BAX, two multidomain proapoptoticBCL-2 proteins that control MOMP. Since activation of BAK and

Figure 4.Olaparib and TMZ cooperate to trigger caspase activation andmitochondrial perturbations. A, A4573 andSK-ES-1 cellswere treatedwith 0.3 mmol/L olaparib and/or50 mmol/L TMZ for 24 hours. Cleavage of caspase-9, caspase-3, caspase-8, and PARP was analyzed by Western blotting. Arrowheads indicate activecleavage fragments, expression of GAPDH served as loading control; asterisk denotes unspecified protein bands. SK-ES-1 cells treated for 2 hours with 2 mg/mLlexatumumab (ETR-2) served as positive control for caspase activation. Representative blots of two independent experiments are shown. B, A4573 andSK-ES-1 cells were treated for 48 hours with 0.3 mmol/L olaparib and/or 50 mmol/L TMZ in the presence or absence of 50 mmol/L zVAD.fmk. Apoptosis wasdetermined by quantification of DNA fragmentation of PI-stained nuclei using flow cytometry. Data are shown as mean � SD of three independentexperiments performed in triplicate; ���, P < 0.001. C, A4573 and SK-ES-1 cells were treated with 0.3 mmol/L olaparib and/or 50 mmol/L TMZ for indicated timesand loss of MMP in the living cell population was determined by flow cytometry using TMRM fluorescent dye. Data are shown as mean � SD of threeindependent experiments performed in triplicate; � , P<0.05; ��, P<0.01. D, A4573 and SK-ES-1 cellswere treatedwith 0.3mmol/L olaparib and/or 50mmol/L TMZ for18 hours. Expression of BCL-2 family proteins was analyzed by Western blot, and expression of GAPDH served as loading control. Representative blots of twoindependent experiments are shown.

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BAX is accompanied by a conformational change that can bedetected by specific antibodies, we immunoprecipitated BAK andBAX using conformation-specific antibodies. Combination treat-ment with olaparib and TMZ stimulated activation of BAK andBAX (Fig. 6A). To explore the functional relevance of BAK andBAXin olaparib/TMZ-induced apoptosis we used a combined siRNAapproach to concomitantly silence BAK and BAX (Fig. 6B).Knockdown of BAK and BAX significantly rescued olaparib/TMZ-mediated apoptosis in all different siRNA construct combi-nations we used (Fig. 6C), emphasizing the importance of BAKand BAX in olaparib/TMZ-induced apoptosis.

Overexpression of BCL-2 protects against Olaparib/TMZ-induced apoptosis

To further examine the requirement of the mitochondrialpathway for the synergistic induction of apoptosis by olapariband TMZweoverexpressed the antiapoptotic protein BCL-2 that isknown to block mitochondrial apoptosis (Fig. 6D). Importantly,BCL-2 overexpression significantly decreased olaparib/TMZ-induced apoptosis (Fig. 6E). This underscores that the synergisticinduction of apoptosis by olaparib and TMZ is mediated via themitochondrial pathway of apoptosis.

DiscussionPARP is currently considered as a promising target for thera-

peutic exploitation in Ewing sarcoma. Therefore, the two aims ofthis study were (i) to examine the effects of different PARPinhibitors in combination with a range of commonly used che-motherapeutics to identify the best synergistic combinations inEwing sarcoma cells and (ii) to elucidate the molecular mechan-isms of synergy with a specific focus on cell death pathways.

Here, we report that different PARP inhibitors synergized inparticular together with TMZ and also with SN-38 to reduce cellviability and to trigger cell death in Ewing sarcoma cells. Calcu-lation of CI values confirmed that the combinations of PARPinhibitors together with TMZ or SN-38 were the best and secondbest combinations among the tested chemotherapeutics. Further-more, triple therapy of olaparib, TMZ, and SN-38 proved to besignificantly more effective to reduce cell viability of Ewingsarcoma cells compared to cotreatment with TMZ/SN-38 orsingle-agent treatment. These findings may have important clin-ical implications, because topoisomerase I poisons and DNAmethylating agents such as TMZ are used in second-line chemo-therapy regimens for Ewing sarcoma (22). However, increasedhost toxicitymay limit the therapeutic window for such combina-tions, because there is preclinical and clinical evidence for

enhanced normal tissue toxicity upon cotreatment with PARPinhibitors and TMZ or topoisomerase I inhibitors (12, 16, 25).PARP inhibitors also enhanced doxorubicin-, etoposide-, or ifos-famide-induced cytotoxicity in Ewing sarcoma cells, although lessconsistently. By comparison, the cytotoxic antibiotic actinomycinD and the vinca alkaloid vincristine did hardly act in a synergisticor additive manner together with PARP inhibitors and alsoshowed some antagonistic effects, depending on the drug con-centrations. Antagonistic interactions of olaparib together withvincristine have previously been described in Ewing sarcoma cellsand have been linked to vincristine's mechanism of action whichis independent ofDNAdamage (13). In linewith the results of oursystematic testing approach that identified TMZ and SN-38 as themost suitable anticancer drugs for combinations with PARPinhibitors in Ewing sarcoma, cooperative interaction of PARPinhibitors together with TMZ or SN-38 has previously beendescribed for Ewing sarcoma cells in vitro and in preclinical Ewingsarcoma models in vivo (9, 12, 13, 16, 26).

The differential ability of chemotherapeutic drugs to synergizewith PARP inhibitors has been attributed to the type of DNAlesions generated by the anticancer agents that differentiallyrequire PARP1 for DNA repair or affect binding of PARP1 to theDNA (27). In combination with PARP-trapping PARP inhibitorssuch as talazoparib, the DNA-methylating agent TMZ elicits itscytotoxicity through N3 and N7 methyl adducts that induce baseexcision repair. Single-strand breaks generated during base exci-sion repair are cytotoxic via PARP trapping, as PARP1binds to andis activated by these base excision repair intermediates, and poly(ADP-ribos)ylation is important for efficient repair (27). Consis-tently, we show that, among the tested anticancer drugs, talazo-parib, a potent PARP-trapping agent, preferentially synergizeswith TMZ, confirming that PARP inhibitors with high PARP-DNA-trapping activity are especially effective together with TMZ(28). By comparison, TMZ as single agent induces cytotoxicitymainly through O6 methylation followed by futile cycles ofmismatch repair, pointing to differentmechanisms of cytotoxicityfor TMZ monotherapy or in combination with PARP inhibitors.Topoisomerase I inhibitors such as SN-38 cause DNA SSBs andcovalent topoisomerase I–DNA complexes, which trigger PARPactivation and require PARP1 for repair (29). Accordingly, cata-lytic PARP inhibitors have shown highly synergistic effects incombination with topoisomerase I inhibitors (28). Consistently,we demonstrate that potent catalytic PARP inhibitors such asniraparib and olaparib cause synergistic cytotoxicity together withSN-38. Olaparib has previously been reported to act in concertwith doxorubicin, melphalan, and carboplatin in Ewing sarcoma

Figure 5.Olaparib/TMZ cotreatment promotes proteasomal degradation of MCL-1. A, A4573 and SK-ES-1 cells were treated for 18 hours with 0.3 mmol/L olaparib and/or 50mmol/L TMZ and/or 50 mmol/L zVAD.fmk and/or 5 ng/mL bortezomib. Expression of MCL-1 was analyzed by Western blotting, and expression of GAPDHserved as loading control. For further evaluation,Western blotswere quantifiedusing ImageJ software and changes inMCL-1 protein levels are given as fold change incomparison to untreated control. Data are shown asmean�SDof three independentWestern blots; � ,P<0.05; �� ,P<0.01; ��� ,P<0.001; n.s., not significant. B andC,A4573 and SK-ES-1 cells were transfected with nondegradable phospho-defective mutant of MCL-1 (MCL-1 4A) or empty vector (EV). Expression of MCL-1 wasanalyzed by Western blotting, and expression of GAPDH served as loading control; arrow indicates exogenously expressed MCL-1 (B; representative blotsof two independent experiments are shown). Cells were treated for 48 hours with 0.3 mmol/L olaparib and 50 mmol/L TMZ and apoptosis was determined byquantification of DNA fragmentation of PI-stained nuclei using flow cytometry (C). Data are shown as mean � SD of three independent experiments performed intriplicate; � , P < 0.05; �� , P < 0.01. D and E, A4573 and SK-ES-1 cells were transiently transfected with 10 nmol/L nonsilencing siRNA or two different constructstargeting NOXA. Expression of NOXA was analyzed by Western blotting, and a-tubulin served as loading control; representative blots of two independentexperiments are shown (D). Transiently transfected Ewing sarcoma cells were treated for 24 hours with 0.3 mmol/L olaparib and 50 mmol/L TMZ and apoptosis wasdetermined by quantification of DNA fragmentation of PI-stained nuclei using flow cytometry. Data are shown as mean � SD of three independent experimentsperformed in triplicate; �� , P < 0.01.

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cells (13), whereas no cooperative interaction was reported in thepast for the combination of etoposide with the first-generationPARP inhibitor NU1025 (30).

In addition to the identification of synergistic PARP inhibitor-based combination therapies, our study demonstrates markeddifferences in the single-agent cytotoxicity of the four PARP inhi-bitors in Ewing sarcoma cells with decreasing potency in the orderof talazoparib (IC50� 10 nmol/L) > niraparib (IC50� 300 nmol/L) > olaparib (IC50� 1,000 nmol/L) > veliparib (IC50� 10,000nmol/L). These results are in line with previous studies showingdifferential antitumor activity of PARP inhibitors as single agentsin various tumors including Ewing sarcoma (18, 28). The antitu-mor activity of PARP inhibitorshasbeen shown todependonbothcatalytic PARP inhibition and PARP-DNA trapping (18). Consis-tent with our findings showing that talazoparib exerts the highestsingle-agent cytotoxicity of the tested PARP inhibitors, the potencyof talazoparib has been attributed to its high efficiency at trappingPARP–DNA complexes (19).

Our molecular studies revealed that the mitochondrial path-way of apoptosis plays a critical role in mediating the synergyof concomitant PARP inhibition (using olaparib as a prototypicPARP inhibitor) and TMZ used as a prototypic DNA-damagingagent. This is of particular importance, as to our knowledgethe downstream signaling events mediating synergistic celldeath induction by PARP inhibition together with TMZ haveso far remained largely elusive. We show that subsequent toDNA damage-imposed checkpoint activation and G2 cell-cyclearrest, olaparib/TMZ cotreatment causes downregulation of theantiapoptotic protein MCL-1, activation of the proapoptoticproteins BAX and BAK, MOMP, proteolytic activation of cas-pases, and caspase-dependent cell death. This conclusion isunderscored by the following independent pieces of experi-mental evidence.

First, olaparib/TMZ cotreatment triggers activation of the DNAdamage response, indicated by increased phosphorylation of thecheckpoint kinases Chk1 and Chk2. In response to DNA damage,ATM and ATR phosphorylate and thereby activate Chk1 andChk2, which control the G2–M checkpoint (31). Consistently,olaparib/TMZ cotreatment results in cell-cycle arrest inG2 prior tocell death induction. Second, our rescue experiments showing thataddition of the proteasome inhibitor bortezomib rescues cellsfrom olaparib/TMZ-imposed downregulation of MCL-1 proteinindicate that MCL-1 is degraded via the proteasome upon treat-ment with olaparib/TMZ.MCL-1 expression is tightly regulated atmultiple levels, involving ubiquitination of MCL-1 that targets itfor proteasomal degradation (32). Four different E3 ubiquitin-ligases that mediate MCL-1 ubiquitination have been identified,

i.e., Mule, SCF/b-TrCP, SCF/Fbw7, and Trim17 (32). Mule isconsidered to be responsible for constitutive MCL-1 degradation,whereas ubiquitination ofMCL-1 by SCF/b-TrCP, SCF/Fbw7, andTrim17 is mediated via phosphorylation of a phosphodegron siteby several kinases, for example by JNK and GSK3 in interphase orpostmitotic cells (32). Furthermore, binding of NOXA to MCL-1has been reported to induce MCL-1 degradation by the protea-some (33), whichmay also explain the downregulation of NOXAthat we observed upon prolonged treatment with olaparib/TMZ.During the initial induction phase of apoptosis, constitutiveexpression of NOXA turned out to be necessary for olaparib/TMZ-induced cell death, because knockdown of NOXA signifi-cantly reduced olaparib/TMZ-mediated cell death. Moreover, weshow that expression of a nondegradable phosphomutant variantof MCL-1 significantly reduces olaparib/TMZ-mediated apopto-sis, emphasizing that degradation of MCL-1 contributes to ola-parib/TMZ-induced apoptosis.

Third, rescue experiments demonstrate that activation of BAXand BAK upon olaparib/TMZ cotreatment is required for thesynergistic induction of apoptosis, because genetic silencing ofBAX and BAK significantly protects Ewing sarcoma cells fromolaparib/TMZ-induced apoptosis. Fourth, the importance of themitochondrial pathway of apoptosis is underscored by overex-pression of the antiapoptotic protein BCL-2, which is known toblock mitochondria-mediated apoptosis, because BCL-2 over-expression inhibits olaparib/TMZ-induced apoptosis. Fifth, ola-parib and TMZ act together to trigger cleavage of caspases intotheir active fragments. The requirement of caspases for the induc-tion of cell death is demonstrated by using the broad-rangecaspase inhibitor zVAD.fmk, which significantly rescues ola-parib/TMZ-induced apoptosis.

Our study has important implications for the development ofPARP inhibitor-based therapies in Ewing sarcoma. Despite the factthat PARP inhibitors as single agents exertedpromising cytotoxicityagainst Ewing sarcoma cells in vitro (10), they exerted limitedefficacy in xenograft models of Ewing sarcoma and did not elicitobjective responses in patientswith Ewing sarcoma in early clinicaltrials (12–15). This underscores the need for combination strate-gies with PARP inhibitors, for example together with chemo-or radiotherapy. Phase I clinical trials combining PARP inhibitors(i.e., olaparib, niraparib, or talazoparib) together with TMZ forthe treatment of Ewing sarcoma are currently under way (i.e.,NCT01858168, NCT02044120, NCT02116777). Our presentstudy emphasizes the role of PARP inhibitors for chemosensitiza-tion of Ewing sarcoma. By showing that PARP inhibitors synergis-tically induce apoptosis together with several DNA-damagingagents, most notably TMZ and also with SN38, it provides new

Figure 6.Olaparib/TMZ cotreatment promotes BAX/BAK activation and MOMP. A, A4573 and SK-ES-1 cells were treated with 0.3 mmol/L olaparib and/or 50 mmol/LTMZ for 21 hours. Active conformations of BAK or BAX were immunoprecipitated using active conformation-specific antibodies and were analyzed by Westernblotting. Expression of total BAK or BAX and GAPDH served as loading controls. Representative blots of two independent experiments are shown. B andC, A4573 and SK-ES-1 cellswere transiently transfectedwith 10 nmol/L nonsilencing siRNAor 5 nmol/L each of different combinations of constructs targetingBAKorBAX and expression of BAK and BAX was analyzed by Western blotting (B). GAPDH served as loading control. Representative blots of two independentexperiments are shown. Transiently transfected Ewing sarcoma cells were treated for 24 hours with 0.3 mmol/L olaparib and/or 50 mmol/L TMZ and apoptosis wasdetermined by quantification of DNA fragmentation of PI-stained nuclei using flow cytometry (C). Data are shown as mean � SD of three independentexperiments performed in triplicate; � , P < 0.05. D and E, A4573 and SK-ES-1 cells were transfected with a murine BCL-2 construct or empty vector and BCL-2expression was analyzed by Western blotting (D). Expression of GAPDH or a-tubulin served as loading controls. Representative blots of two independentexperiments are shown. BCL-2-overexpressing Ewing sarcoma cells were treated for 48 hours with 0.3 mmol/L olaparib and 50 mmol/L TMZ and apoptosis wasdetermined by quantification of DNA fragmentation of PI-stained nuclei using flow cytometry (E). Data are shown as mean � SD of three independentexperiments performed in triplicate; ��� , P < 0.001.

www.aacrjournals.org Mol Cancer Ther; 14(12) December 2015 2829

Synergistic Apoptosis by PARP Inhibitors and Temozolomide

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insights into cotreatment rationales for distinct PARP inhibitorsand chemotherapeutic drugs and sets the ground for further (pre)clinical evaluationof PARP inhibitor-based combination regimenswith cytotoxic chemotherapy.

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

Authors' ContributionsConception and design: F. Engert, C. Schneider, S. FuldaDevelopment of methodology: F. Engert, L.M. WeibAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Engert, C. Schneider, M. ProbstAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Engert, C. Schneider, M. Probst, S. Fulda

Writing, review, and/or revision of the manuscript: F. Engert, C. Schneider,L.M. Weib, S. FuldaStudy supervision: S. Fulda

AcknowledgmentsThe authors thank Human Genome Sciences (Rockville, MD) for kindly

providing TRAIL-R2 agonistic antibody and C. Hugenberg for expert secretarialassistance.

Grant SupportThis work has been partially supported by a grant from the BMBF (to S.

Fulda).

Received July 17, 2015; revised September 22, 2015; accepted September 23,2015; published OnlineFirst October 5, 2015.

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