Published OnlineFirst October 5, 2015; DOI: 10.1158/1535-7163.MCT-15-0587 Cancer Biology and Signal Transduction Molecular Cancer Therapeutics PARP Inhibitors Sensitize Ewing Sarcoma Cells to Temozolomide-Induced Apoptosis via the Mitochondrial Pathway Florian Engert1, Cornelius Schneider1, Lilly Magdalena Weib1,2,3, Marie Probst1,and Simone Fulda1,2,3 Abstract Ewing sarcoma has recently been reported to be sensitive to that subsequent to DNA damage-imposed checkpoint activa- poly(ADP)-ribose polymerase (PARP) inhibitors. Searching for tion and G2 cell-cycle arrest, olaparib/TMZ cotreatment causes synergistic drug combinations, we tested several PARP inhibi- downregulation of the antiapoptotic protein MCL-1, followed by tors (talazoparib, niraparib, olaparib, veliparib) together with activation of the proapoptotic proteins BAX and BAK, mitochon- chemotherapeutics. Here, we report that PARP inhibitors syner- drial outer membrane permeabilization (MOMP), activation of gize with temozolomide (TMZ) or SN-38 to induce apoptosis caspases, and caspase-dependent cell death. Overexpression of a and also somewhat enhance the cytotoxicity of doxorubicin, nondegradable MCL-1 mutant or BCL-2, knockdown of NOXA or etoposide, or ifosfamide, whereas actinomycin D and vincris- BAX and BAK, or the caspase inhibitor N-benzyloxycarbonyl-Val- tine show little synergism. Furthermore, triple therapy of ola- Ala-Asp-fluoromethylketone (zVAD.fmk) all significantly reduce parib, TMZ, and SN-38 is significantly more effective compared olaparib/TMZ-mediated apoptosis. These findings emphasize with double or monotherapy. Mechanistic studies revealed that the role of PARP inhibitors for chemosensitization of Ewing the mitochondrial pathway of apoptosis plays a critical role in sarcoma with important implications for further (pre)clinical mediating the synergy of PARP inhibition and TMZ. We show studies. Mol Cancer Ther; 14(12); 2818–30. Ó2015 AACR. Introduction accompanied by the release of mitochondrial intermembrane space proteins such as cytochrome c into the cytosol, which in Ewing sarcoma is the second most common pediatric primary turn results in caspase activation and apoptosis (5). MOMP is bone tumor in children and young adults (1). State-of-the-art tightly controlled by various factors including proteins of the BCL- treatment options for Ewing sarcoma patients include chemo- 2 family. BCL-2 family proteins consist of both antiapoptotic therapy consisting of vincristine, ifosfamide, doxorubicin, temo- members, for example BCL-2, BCL-x , and MCL-1, and proapop- zolomide (TMZ), actinomycin D, irinotecan, etoposide, and L totic molecules such as the multidomain proteins BAK and BAX cyclophosphamide (2). Despite aggressive treatment protocols, and BH3-only domain proteins, for example, BIM, BMF, PUMA, patients especially with advanced or relapsed diseases still harbor and NOXA (6). an overall poor survival (2). This highlights the unmet medical Characteristic for Ewing sarcoma is the existence of a chimeric need to develop innovative treatment strategies. fusion protein which is in >85% of cases generated by the Tumor response to cytotoxic therapies including chemotherapy translocation of the EWS gene on chromosome 22 and the FLI1 critically relies on intact cell death programs in cancer cells, as gene on chromosome 11 (11;22)(q24;q12) (7). This fusion chemotherapeutics exert their anticancer effects to a large extent protein encodes the chimeric transcription factor EWS-FLI1 that by engaging programmed cell death (3). Apoptosis is mediated causes upregulation of a range of target genes including PARP1 via two well-defined pathways, that is the extrinsic (death recep- (7, 8), which promotes transcriptional activation by EWS-FLI1 in tor) pathway and the intrinsic (mitochondrial) pathway, which a positive feedback loop (9). Elevated PARP levels have been both eventually lead to activation of caspases as cell death effector detected in Ewing sarcoma specimens (8, 10), indicating that molecules (4). Engagement of the mitochondrial pathway results PARP may represent a promising target for therapeutic exploita- in mitochondrial outer membrane permeabilization (MOMP) tion in Ewing sarcoma. Indeed, Ewing sarcoma cells have been shown to be particularly sensitive to PARP inhibition (9, 10). 1Institute for Experimental Cancer Research in Pediatrics, Goethe- Several PARP inhibitors including talazoparib (BMN-673), nir- University, Frankfurt, Germany. 2German Cancer Consortium (DKTK), aparib (MK-4827), olaparib (AZD-2281), and veliparib (ABT- 3 Heidelberg, Germany. German Cancer Research Center (DKFZ), Hei- 888) are currently under investigation (11). delberg, Germany. Despite the sensitivity of Ewing sarcoma cells to PARP inhibi- Note: Supplementary data for this article are available at Molecular Cancer tors in vitro (9, 10, 12), PARP inhibitors as single therapy displayed Therapeutics Online (http://mct.aacrjournals.org/). limited efficacy in xenograft models of Ewing sarcoma and in Corresponding Author: Simone Fulda, Goethe-University, Komturstr. 3a, 60528 initial clinical trials (12–15), highlighting the need for combina- Frankfurt, Germany. Phone: 49-69-67866557; Fax: 49-69-6786659157; E-mail: tion therapies. PARP inhibitors have been documented to poten- [email protected] tiate DNA damage-mediated cytotoxicity caused in particular by doi: 10.1158/1535-7163.MCT-15-0587 topoisomerase I poisons, DNA methylating agents, or radiation in Ó2015 American Association for Cancer Research. several cancers including Ewing sarcoma, which has been related 2818 Mol Cancer Ther; 14(12) December 2015 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst October 5, 2015; DOI: 10.1158/1535-7163.MCT-15-0587 Synergistic Apoptosis by PARP Inhibitors and Temozolomide A A4573 SK-ES-1 Talazoparib Talazoparib Niraparib Niraparib Olaparib Olaparib Veliparib Veliparib 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 Cell viability (% (% of viability control) Cell 20 Cell viability (% (% of viability Cell control) 20 10 10 0 0 0.1 1 10 100 1,000 10,000 100,000 0.1 1 10 100 1,000 10,000 100,000 Drug concentration (nmol/L) Drug concentration (nmol/L) B Talazoparib (nmol/L) Niraparib (nmol/L) Olaparib (nmol/L) Veliparib (nmol/L) A4573 CI values 0.5 1 2.5 5 7.5 25 50 75 100 300 600 1,600 2,200 2,800 25 < 0.1 Very strong synergism TMZ (µmol/L) 50 0.1–0.3 Strong synergism 100 0.3–0.7 Synergism 0.2 0.7–0.85 Moderate synergism SN38 (nmol/L) 0.4 0.85–0.9 Slight synergism 0.6 0.9–1.1 Nearly additive 25 1.1–1.2 Slightly antagonistic Etoposide (ng/mL) 50 1.2–1.45 Moderate antagonistic 75 >1.45 Antagonism 0.5 Not calculated Ifosfamide (µmol/L) 1 2 1 Doxo (ng/µL) 2.5 4 0.1 Vincristine (nmol/L) 0.25 0.4 0.1 AMD 0.25 (ng/mL) 0.4 Talazoparib (nmol/L) Niraparib (nmol/L) Olaparib (nmol/L) Veliparib (nmol/L) SK-ES-1 CI values 0.5 1 2.5 5 7.5 25 50 75 100 300 600 1,600 2,200 2,800 25 < 0.1 Very strong synergism TMZ (µmol/L) 50 0.1–0.3 Strong synergism 100 0.3–0.7 Synergism 0.2 0.7–0.85 Moderate synergism SN38 (nmol/L) 0.4 0.85–0.9 Slight synergism 0.6 0.9–1.1 Nearly additive 25 1.1–1.2 Slightly antagonistic Etoposide (ng/mL) 50 1.2–1.45 Moderate antagonistic 75 >1.45 Antagonism 0.5 Not calculated Ifosfamide (µmol/L) 1 2 1 Doxo (ng/µL) 2.5 4 0.1 Vincristine (nmol/L) 0.25 0.4 0.1 AMD 0.25 (ng/mL) 0.4 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 indicated concentrations of talazoparib, niraparib, olaparib, or veliparib. Cell viability was assessed by crystal violet assay and is shown as percentage of untreated control. 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 with different cytostatic drugs (i.e., TMZ, SN-38, etoposide, ifosfamide, doxorubicin, vincristine, and actinomycin D). Cell viability was assessed by crystal violet assay. Synergistic, additive, or antagonistic drug interactions were calculated by combination index (CI). CI values were then used to generate a heatmap of drug interactions according to the subclassification provided by the software's manual. www.aacrjournals.org Mol Cancer Ther; 14(12) December 2015 2819 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst October 5, 2015; DOI: 10.1158/1535-7163.MCT-15-0587 Engert et al. A4573 SK-ES-1 A 100 100 *** 100 *** 100 *** * 80 80 80 80 60 60 60 60 40 40 40 40 20 20 20 20 DNA fragmentation (%) fragmentation DNA DNA fragmentation (%) fragmentation DNA DNA fragmentation (%) fragmentation DNA DNA fragmentation (%) fragmentation DNA 0 0 0 0 Olaparib – + – + Olaparib – + – + Olaparib – + – + Olaparib – + – + TMZ – – + + SN-38 – – + + TMZ – – + + SN-38 – – + + B ** ** * ** 100 100 100 100 80 80 80 80 60 60 60 60 40 40 40 40 20 20 20 20 Cell viability (% of Cell viability control) Cell viability (% of Cell viability control) Cell viability (% of Cell viability control) 0 0 0 (% of Cell viability control) 0 Olaparib – + – + Olaparib – + – + Olaparib – + – + Olaparib – + – + TMZ – – + + SN-38 – – + + TMZ – – + + SN-38 – – + + C ****** 120 120 120 100 100 100 100 80 80 80 80 60 60 60 60 40 40 40 40 20 20 20 20 Colonies (% Colonies (% of control) Colonies (% Colonies (% of control) Colonies (% Colonies (% of control) Colonies (% Colonies (% of control) 00 0 0 Olaparib – + – + Olaparib – + – + Olaparib – + – + Olaparib – + – + TMZ – – + + SN-38 – – + + TMZ – – + + SN-38 – – + + – Olaparib + Olaparib – Olaparib + Olaparib – Olaparib + Olaparib – Olaparib + Olaparib – TMZ – TMZ – SN-38 – SN-38 + TMZ + TMZ + SN-38 + SN-38 D *** *** 100 100 ** * 80 80 60 60 40 40 20 20 Cell viability (% Cell viability of control) Cell viability (% of Cell viability control) 0 0 Olaparib – + – – – + + + Olaparib – + – – – + + + TMZ – – + – + + – + TMZ – – + – + + – + SN-38 – – – + + – + + SN-38 – – – + + – + + 2820 Mol Cancer Ther; 14(12) December 2015 Molecular Cancer Therapeutics Downloaded from mct.aacrjournals.org on September 24, 2021.