Translational Cancer Mechanisms and Therapy The Ewing Family of Tumors Relies on BCL-2 and BCL-X L to Escape PARP Inhibitor Toxicity Daniel A.R. Heisey 1 , Timothy L. Lochmann 1 , Konstantinos V. Floros 1 , Colin M. Coon 1 , Krista M. Powell 1 , Sheeba Jacob 1 , Marissa L. Calbert 1 , Maninderjit S. Ghotra 1 , Giovanna T. Stein 2 , Yuki Kato Maves 3 , Steven C. Smith 4 , Cyril H. Benes 2 , Joel D. Leverson 5 , Andrew J. Souers 5 , Sosipatros A. Boikos 6 , and Anthony C. Faber 1 Abstract Purpose: It was recently demonstrated that the EWSR1-FLI1 t(11;22)(q24;12) translocation contributes to the hypersensi- tivity of Ewing sarcoma to PARP inhibitors, prompting clinical evaluation of olaparib in a cohort of heavily pretreated Ewing sarcoma tumors. Unfortunately, olaparib activity was disap- pointing, suggesting an underappreciated resistance mecha- nism to PARP inhibition in patients with Ewing sarcoma. We sought to elucidate the resistance factors to PARP inhibitor therapy in Ewing sarcoma and identify a rational drug com- bination capable of rescuing PARP inhibitor activity. Experimental Design: We employed a pair of cell lines derived from the same patient with Ewing sarcoma prior to and following chemotherapy, a panel of Ewing sarcoma cell lines, and several patient-derived xenograft (PDX) and cell line xenograft models. Results: We found olaparib sensitivity was diminished following chemotherapy. The matched cell line pair revealed increased expression of the antiapoptotic protein BCL-2 in the chemotherapy-resistant cells, conferring apo- ptotic resistance to olaparib. Resistance to olaparib was maintained in this chemotherapy-resistant model in vivo, whereas the addition of the BCL-2/X L inhibitor navitoclax led to tumor growth inhibition. In 2 PDXs, olaparib and navitoclax were minimally effective as monotherapy, yet induced dramatic tumor growth inhibition when dosed in combination. We found that EWS-FLI1 increases BCL-2 expression; 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-X L in Ewing sarcoma survival. Conclusions: These data reveal BCL-2 and BCL-X L act together to drive olaparib resistance in Ewing sarcoma and reveal a novel, rational combination therapy that may be put forward for clinical trial testing. Introduction The 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 most often in children and adolescents. Great strides have been made in treating localized disease by using intensive neoadjuvant and adjuvant chemotherapy regimens, increasing the 5-year survival from about 10% to approximately 75%. However, there is a 30% survival rate for patients with Ewing sarcoma presenting with metastasis or that relapse following systematic chemotherapy (1). The EWSR1-FLI1 t(11;22)(q24;q12) translocation event is found in approximately 90% of EWFTs. Since the identification of EWSR1-FLI1 in Ewing sarcoma, it has become clear that the resultant fusion oncogene is the vital driving event in these tumors (2–7). The molecular consequence of juxtaposing the EWSR1 and FLI1 genes is a EWS-FLI1 fusion protein where EWS potently increases the ability of transcription factor FLI1 to activate or suppress target genes. Unfortunately, FLI1 is currently undruggable, and effective targeted therapies for treating Ewing sarcoma remain elusive. Recent findings have highlighted the role of EWS-FLI1 at inducing a wide range of changes throughout the epigenome, affecting both histone marks and enhancers (5–9), leading to simultaneous enhanced expression of tumor oncogenes and reduced expression of tumor suppressors (6). However, these studies have yet to reveal 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 screening platform (11), both demonstrated in 2012 that EWSR1-FLI1– translocated Ewing sarcoma displays hypersensitivity to PARP inhibition; this has since been replicated by several other groups (12–14). These data have provided a promising 1 VCU Philips Institute, School of Dentistry and Massey Cancer Center; Richmond, Virginia. 2 Massachusetts General Hospital Cancer Center, Boston, Massachu- setts; Department of Medicine, Harvard Medical School, Boston, Massachusetts. 3 Crown Bioscience Inc., San Diego, California. 4 Division of Anatomic Pathology, Virginia Commonwealth University, Richmond, Virginia. 5 AbbVie, North Chi- cago, Illinois. 6 Hematology, Oncology and Palliative Care, School of Medicine and Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia. Note: Supplementary data for this article are available at Clinical Cancer Research 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: acfaber@vcu.edu doi: 10.1158/1078-0432.CCR-18-0277 Ó2018 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 25(5) March 1, 2019 1664 on July 7, 2020. © 2019 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst October 22, 2018; DOI: 10.1158/1078-0432.CCR-18-0277