The Ewing Family of Tumors Relies on BCL-2 and BCL-XL to Escape PARP Inhibitor Toxicity Daniel A.R

Published OnlineFirst October 22, 2018; DOI: 10.1158/1078-0432.CCR-18-0277

Cancer Therapy: Preclinical Clinical Cancer Research The Ewing Family of Tumors Relies on BCL-2 and BCL-XL to Escape PARP Inhibitor Toxicity Daniel 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-FLI1 revealed increased expression of the antiapoptotic protein t(11;22)(q24;12) translocation contributes to the hypersensi- BCL-2 in the chemotherapy-resistant cells, conferring apo- tivity of Ewing sarcoma to PARP inhibitors, prompting clinical ptotic resistance to olaparib. Resistance to olaparib was evaluation of olaparib in a cohort of heavily pretreated Ewing maintained in this chemotherapy-resistant model in vivo, sarcoma tumors. Unfortunately, olaparib activity was disap- whereas the addition of the BCL-2/XL inhibitor navitoclax pointing, suggesting an underappreciated resistance mecha- led to tumor growth inhibition. In 2 PDXs, olaparib and nism to PARP inhibition in patients with Ewing sarcoma. We navitoclax were minimally effective as monotherapy, yet sought to elucidate the resistance factors to PARP inhibitor induced dramatic tumor growth inhibition when dosed in therapy in Ewing sarcoma and identify a rational drug com- combination. We found that EWS-FLI1 increases BCL-2 bination capable of rescuing PARP inhibitor activity. expression; however, inhibition of BCL-2 alone by veneto- Experimental Design: We employed a pair of cell lines clax is insufﬁcient to sensitize Ewing sarcoma cells to ola- derived from the same patient with Ewing sarcoma prior to parib, revealing a dual necessity for BCL-2 and BCL-XL in and following chemotherapy, a panel of Ewing sarcoma cell Ewing sarcoma survival. lines, and several patient-derived xenograft (PDX) and cell line Conclusions: These data reveal BCL-2 and BCL-XL act xenograft models. together to drive olaparib resistance in Ewing sarcoma and Results: We found olaparib sensitivity was diminished reveal a novel, rational combination therapy that may be put following chemotherapy. The matched cell line pair forward for clinical trial testing.

Introduction survival rate for patients with Ewing sarcoma presenting with metastasis or that relapse following systematic chemotherapy (1). The Ewing family of tumors (EWFTs), consisting of primitive The EWSR1-FLI1 t(11;22)(q24;q12) translocation event is neuroectodermal tumor (PNET) and Ewing sarcoma, is a malig- found in approximately 90% of EWFTs. Since the identification nancy of predominantly bone. These cancers are diagnosed most of EWSR1-FLI1 in Ewing sarcoma, it has become clear that the often in children and adolescents. Great strides have been made in resultant fusion oncogene is the vital driving event in these tumors treating localized disease by using intensive neoadjuvant and (2–7). The molecular consequence of juxtaposing the EWSR1 and adjuvant chemotherapy regimens, increasing the 5-year survival FLI1 genes is a EWS-FLI1 fusion protein where EWS potently from about 10% to approximately 75%. However, there is a 30% increases the ability of transcription factor FLI1 to activate or suppress target genes. Unfortunately, FLI1 is currently undruggable, and effective 1VCU Philips Institute, School of Dentistry and Massey Cancer Center; Richmond, Virginia. 2Massachusetts General Hospital Cancer Center, Boston, Massachu- targeted therapies for treating Ewing sarcoma remain elusive. fi setts; Department of Medicine, Harvard Medical School, Boston, Massachusetts. Recent ndings have highlighted the role of EWS-FLI1 at inducing 3Crown Bioscience Inc., San Diego, California. 4Division of Anatomic Pathology, a wide range of changes throughout the epigenome, affecting both Virginia Commonwealth University, Richmond, Virginia. 5AbbVie, North Chi- histone marks and enhancers (5–9), leading to simultaneous 6 cago, Illinois. Hematology, Oncology and Palliative Care, School of Medicine enhanced expression of tumor oncogenes and reduced expression and Massey Cancer Center, Virginia Commonwealth University, Richmond, of tumor suppressors (6). However, these studies have yet to Virginia. reveal specific, druggable targets with associated clinically avail- Note: Supplementary data for this article are available at Clinical Cancer able therapies. Research Online (http://clincancerres.aacrjournals.org/). Brenner and colleagues (10) and the Genomics of Drug Sen- Corresponding Author: Anthony C. Faber, Virginia Commonwealth University, sitivity in Cancer (GDSC), a high-throughput drug screening Perkinson Building, Room 4134, 1101 East Leigh Street, Richmond, VA 23298. platform (11), both demonstrated in 2012 that EWSR1-FLI1– Phone: 804-828-0841; Fax: 804-828-0150; E-mail: [email protected] translocated Ewing sarcoma displays hypersensitivity to PARP doi: 10.1158/1078-0432.CCR-18-0277 inhibition; this has since been replicated by several other 2018 American Association for Cancer Research. groups (12–14). These data have provided a promising

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Antibodies and reagents Translational Relevance Primary antibodies used for Western blotting were as follows: Using matched patient samples prior to and following GAPDH (sc-3233) and FLI1 (sc-365294) from Santa Cruz Bio- chemotherapy, we have uncovered a drug combination that technology; cleaved PARP1 (5625), BCL-2 (4223), BCL-XL is able to achieve substantial tumor regression in multiple (2764), MCL-1 (5453), PARP1 (9532), gH2A.X (9781), and mouse models including patient-derived xenografts and BIM (2933) from Cell Signaling Technology. Secondary anti- xenografts of chemotherapy-resistant cells. This warrants the bodies used were mouse IgG (GE Healthcare Life Sciences; clinical evaluation of olaparib in combination with navitoclax NXA931) and rabbit IgG (GE Healthcare Life Sciences; NA934). in the EWS-FLI1–translocated Ewing family of tumors. IgG (sc-2027) for immunoprecipitation was from Santa Cruz Biotechnology. Olaparib (AZD-2281) was from Abmole, and A-1331852, navitoclax (ABT-263), and venetoclax (ABT-199) were kindly provided by AbbVie Inc. drug target for EWFTs, with corresponding FDA-approved PARP1 inhibitors (15). Animal experiments 6 However, in the initial clinical study of olaparib in Ewing For the SK-ES-1 xenograft model, 5 10 cells were injected s.c. fl sarcoma, no objective responses were observed in 12 evalu- into the ank of 6- to 8-week-old Nu/Nu mice. For the CHLA10 6 fl able patients (15). Although there were no objective xenograft model, 5 10 cells were injected s.c. into the ank of responses, 4 of 12 patients achieved stable disease, with 2 6- to 8-week-old Nod/SCID gamma (NSG) mice. The patient- of the 4 achieving minor responses (tumor shrinkage of 9% derived xenograft (PDX) models were obtained from Crown 5 fl and 12%), indicating a modest level of efficacy by PARP Bioscience, and 5 10 cells were injected into the ank of NSG mice s.c. Treatment began when tumors reached approximately inhibition in these patients. 3 Based on the hypersensitivity of Ewing sarcoma to PARP 150 to 200 mm , and mice were randomized into treatment inhibition in vitro and olaparib activity in patients with Ewing cohorts. Tumor size and mouse weight were measured 3 days sarcoma, we sought to identify intrinsic resistance mechanisms per week with a digital scale and calipers, where tumor volume 2 to PARP inhibitor (PARPi) therapy as well as a rational drug was calculated as length width 0.52. Navitoclax and olaparib combination that could overcome these mechanisms. We and were administered by oral gavage. Navitoclax was dissolved in fi others have shown that a low apoptotic response, even in the 60% Phosal 50 PG, 30% PEG400, and 10% ethanol, for a nal presence of growth arrest, mitigates response to targeted therapies dosage of 80 mg/kg of body weight. Olaparib was dissolved fi (16–21). We therefore hypothesized that mitigated responses of in 10% hydroxylpropyl-b-cyclodextrin for a nal dosage of PARP inhibition may be due to loss of apoptotic potential of 100 mg/kg of body weight. All drugs were administered once per EWFTs, which could prove particularly true in the chemorefrac- day, 5 days/week. For pharmacodynamics studies, tumor-bearing tory population. This hypothesis was further supported by the fact mice were treated with drug for 3 days, and tumors were harvested fi that deficient DNA damage repair is thought to contribute to, if on the 3rd day 2 hours after the nal treatment. Tumors were fl not define, PARPi sensitivity in Ewing sarcoma (12), as well as the ashfrozeninliquidnitrogen.Forthebloodtoxicitystudy, established role of antiapoptotic BCL-2 family proteins in pro- NSG mice were treated with no drug (no Rx), navitoclax, tecting cancer cells from DNA damage–induced apoptosis olaparib, or the combination, at the same doses as above. At (17, 22) and their inverse correlation of expression to cytotoxic 3 and 7 days, mice were exsanguinated, and blood was sent to agent sensitivity (23). IDEXX BioResearch for testing. The recovery cohort was treated for 7 days and allowed 24 hours of recovery from treatment before exsanguination. All animal experiments were approved Materials and Methods by the Virginia Commonwealth University Institutional Animal Cell lines Care and Use Committee (IACUC protocol #AD10001048). A673 (ATCC CRL-1598) and HEK293T cells were cultured in DMEM (Gibco) with 10% FBS (Seradigm) and 1 mg/mL pen- Dataset analysis icillin and streptomycin. CHLA9 and CHLA10 cells were grown The online database for publically accessible drug sensitivity in DMEM with 20% FBS, 1 mg/mL penicillin and streptomycin, data (www.cancerRxgene.org) was used to generate Fig. 3C. The and 1% Insulin-Transferrin-Selenium 100x (Gibco). SK-ES-1 cancer cell line encyclopedia (CCLE; ref. 24) was used to analyze (ATCC HTB-86) was grown in DMEM/F12 (Corning) with 15% expression between FLI1 and BCL-2 (Supplementary Fig. S7D). FBS and 1 mg/mL of penicillin and streptomycin. ES4 and EW16 cells were grown in RPMI1640 (Lonza Group) with 10% FBS Statistical considerations and 1 mg/mL of penicillin and streptomycin. Routine myco- For gene expression analyses and the complete blood count plasma testing was performed on all cell lines. CHLA9 and analyses, significance was determined using the nonparametric CHLA10 were obtained from the Children's Oncology Group Mann–Whitney U test. Differences were considered statistically (COG) Cell Culture and Xenograft Repository, special thanks to different if P < 0.05. All other analyses were performed using the Dr. C. Pat Reynolds, Texas Tech University Health Sciences Student t test and considered statistically different if P < 0.05. Center. ES4, EW16, HEK293T, A673 (ATCC CRL-1598), and Asterisks indicate levels of significance: ns, P 0.05; , P < 0.05; SK-ES-1 (ATCC HTB-86) were obtained from either the Molec- , P < 0.01; , P < 0.001; and , P < 0.0001. ular Center Therapeutics laboratory at Massachusetts General Hospital which performs routine testing of cell lines using short Synergy assay tandem repeat and SNP analysis, or the American Type Culture Cells were seeded at 1 103 cells in a 96-well plate. Twenty-four Collection (ATCC). hours after seeding, cells were treated with varied concentrations

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Navitoclax Sensitizes Ewing Sarcoma to Olaparib

of navitoclax (0 to 2 mmol/L) and olaparib (0.1 to 10 mmol/L) for Surprisingly, despite the increase in BCL-2, we found the BCL-2– 72 hours, followed by measurement of cell viability by CellTiter- specific inhibitor venetoclax (31) was unable to effectively sen- Glo. Percent viability was constrained to a maximum of 100. The sitize CHLA10 cells to olaparib (Supplementary Fig. S5A). percentage over the bliss score was calculated as previously Because increased expression of BCL-XL is sufficient to induce described (25). resistance to venetoclax (32–34), we next tested the dual BCL-2/ BCL-XL inhibitor navitoclax (35, 36) to determine if this agent Results sensitizes the CHLA10 cells to olaparib. Although venetoclax showed little potentiation of olaparib (Supplementary Fig. S5A € A chemotherapy-na ve and chemotherapy-resistant cell line and S5B), navitoclax sensitized CHLA10 cells to olaparib treat- pair respond differently to olaparib ment compared with venetoclax (P < 0.05), leading to a near- Olaparib performed poorly in patients with Ewing sarcoma complete loss of cell viability (Fig. 1D), and mild synergy (Sup- (15) whose tumors were heavily pretreated and chemotherapy- plementary Fig. S5C). Impressively, at low doses of olaparib resistant. We therefore utilized a pair of cell lines established from (1 mmol/L) where there was no single-drug efficacy in the CHLA9 the same patient prior to and following chemotherapy treatment, cells, the addition of navitoclax led to substantial loss of cell at tumor relapse: CHLA9 cells were derived from the chemother- viability (Supplementary Fig. S5D). Similar to venetoclax, the apy-na€ve PNET, positive for the EWS-FLI1 translocation, whereas BCL-XL–selective inhibitor A-1331852 (37) was not effective at the CHLA10 cell line was established after 4 cycles of induction sensitizing CHLA10 cells to olaparib (Supplementary Fig. S5E). chemotherapy which included cisplatin, doxorubicin, cyclophos- Consistent with these findings, we found the CHLA9 cells under- fi phamide, and etoposide (26). We rst assessed whether sensitiv- went marked cell death in response to olaparib, as measured by ity to olaparib was different in the 2 cell lines. We found that the cleaved PARP1 (Fig. 1E); in contrast, there was a near absence of a € chemotherapy-na ve cells were more sensitive to olaparib, as cell death response in the olaparib-treated CHLA10 cells (Fig. 1E). evidenced by a 5-day crystal violet viability assay (Fig. 1A, left), However, the addition of navitoclax led to marked cleavage of – 72-hour dose response curve (Fig. 1A, right), and IC50 curve PARP1 in the presence of olaparib in the CHLA10 cells (Fig. 1E), (Supplementary Fig. S2A) compared with the chemotherapy- despite the lack of modulation of BCL-2, BCL-XL, or the related resistant CHLA10 cells (Fig. 1A, left and right), despite both cell MCL-1 (38) by olaparib (Fig. 1F). These data indicate that EWFTs PARP1 lines reportedly expressing high levels of (23, 27) and can lose their sensitivity to olaparib following chemotherapy olaparib inducing similar growth arrest in both the CHLA9 and treatment and relapse, underscored by their inability to undergo CHLA10 cells (Supplementary Fig. S2B). In addition, similar cell death, and can be rescued by the addition of navitoclax. This levels of DNA damage were observed following olaparib treat- was further supported by the observation that, at low concentra- fl ment in both cell lines as evidenced by gH2A.X immuno uo- tions of olaparib where sensitive CHLA9 cells do not yet respond rescence staining (Supplementary Fig. S3A and S3B). Further- to single-agent olaparib, navitoclax also sensitizes to olaparib fi more, we con rmed the CHL10 cells were more resistant to (Supplementary Fig. S5D). Of note, the CHLA9 cells have func- chemotherapy compared with the CHLA9 cells (Supplementary tional p53, whereas the CHLA10 cells have nonfunctioning Fig. S4A and S4B). p53 (39). It is well established that functional p53 is capable of binding to and antagonizing the antiapoptotic functions of BH3 Chemotherapy-resistant CHLA10 cells do not undergo cell proteins such as BCL-2 and BCL-XL (40, 41). In order to rule out death in response to olaparib p53 as the cause of inherent resistance to olaparib-induced Because the lack of robust apoptotic responses can underlie apoptosis in the CHLA10 cells when compared with the CHLA9 resistance to both chemotherapy and targeted therapies, and the cells, we used siRNA to knockdown TP53 in the CHLA9 cells and apoptotic response following many chemotherapies and targeted found no difference in olaparib sensitivity (Supplementary Fig. therapies is largely governed by the BCL-2 family of proteins S5F, left), consistent with a previous report on p53 and PARPi (16, 20, 28–30), we first explored the relationship between sensitivity (2). To further support the role of BCL-2 and/or BCL-XL antiapoptotic BCL-2 family expression and olaparib response in overexpression in apoptotic resistance to olaparib treatment, we the CHLA9 and CHLA10 models. Expression of BCL-2 was upre- overexpressed BCL-2 or BCL-XL in the CHLA9 cells. Here, we saw a gulated (P < 0.05) in the CHLA10 cells compared with the CHLA9 significant increase in resistance to olaparib treatment in cells cells (Fig. 1B; Supplementary Fig. S4C), whereas expressions of overexpressing BCL-2 or BCL- XL compared with the GFP controls other key BCL-2 family members were not altered (Fig. 1B). (P < 0.0001; Supplementary Fig. S6A). Together, these data reveal The increase in BCL-2 prompted us to evaluate BCL-2 expres- a striking interplay between BCL-2/XL inhibition and PARP inhi- sion in pretreatment and postchemotherapy biopsy samples from bition in the EWFTs. 2 patients with Ewing sarcoma treated at our cancer center. Interestingly, we did not detect an increase in BCL-2 expression Navitoclax and olaparib cooperate to inhibit tumor growth in a in these specimens, in contrast to the cell line pair; however, BCL- CHLA10 mouse model XL expression was markedly higher in chemotherapy-resistant We next grew CHLA10 tumors in NSG mice and evaluated tumors (Fig. 1C) relative to the matched chemotherapy-na€ve single-agent olaparib, navitoclax, and the combination of ola- samples. These data indicate that BCL-XL is overexpressed in parib and navitoclax to see if the in vitro results would translate in Ewing sarcoma patients' tumors that have undergone chemother- vivo. Consistent with the cell culture experiments (Fig. 1D and E), apy, and our findings in models of EWFTs implicate BCL-2 as a we found the CHLA10 tumors were not sensitive to olaparib and cooperating partner with BCL-XL in resisting apoptosis. Together, minimally sensitive to navitoclax as a monotherapy compared these data indicated to us that both BCL-2 and BCL-XL may be to untreated mice (Fig. 1G). However, the combination of imperative in Ewing sarcoma survival. We then moved to chem- olaparib and navitoclax demonstrated robust inhibition of tumor ical interrogation of the cells with specific BCL-2 family inhibitors. growth (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 cells after 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 indicated concentrations of olaparib. B, Western blot analysis with the indicated antibodies in chemotherapy-naive and chemotherapy-resistant paired lines. C, Two

cases of Ewing sarcoma with available paired primary and recurrent metastatic tissues were immunostained for BCL-2 and BCL-XL. In both cases, similar expression of BCL-2 was noted in primary. For BCL-XL, however, increased expression was noted in both recurrences compared with the primary tumors. Case 1: Primary: Archival sections of the untreated biopsy of the primary tumor (patella), which was localized at presentation. Recurrence: Lung metastasis 5 years subsequent, after systemic chemotherapy (VAC-IE) and localized radiotherapy to the patellar primary site. Case 2: Primary: Archival sections of the biopsy of the untreated primary tumor (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 and multiple metastatic sites. D, Crystal violet staining showing olaparib-resistant CHLA10 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 a no-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 treatment of 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 of the 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/L navitoclax compared to a no-treatment control (no Rx). G, CHLA10 xenografts treated daily with olaparib (100 mg/kg), navitoclax (80 mg/kg), or the combination of olaparib (100 mg/kg) þ navitoclax (80 mg/kg) for 28 days. Error bars are þSEM. Asterisks indicate a signiﬁcant separation between the combination (olap/nav) and all 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 the combination of olaparib (100 mg/kg) þ venetoclax (100 mg/kg) for 30 days. Error bars are þSEM.

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Figure 2. Combination of olaparib and navitoclax does not augment toxicity. A, NSG mice were treated with olaparib (100 mg/kg), navitoclax (80 mg/kg), or the combination of olaparib (100 mg/kg) þ navitoclax (80 mg/kg) compared to a no-treatment control (no Rx). After the indicated 3- or 7-day treatment period, blood was collected and sent to IDEXX BioResearch (idexxbioresearch.com) for a complete blood count. The recovery cohort was treated for 7 days with the combination of olaparib (100 mg/kg) þ navitoclax (80 mg/kg) and allowed 24 hours without treatment before blood was collected. Three- day treatment with navitoclax not signiﬁcant compared with 3-day treatment with the combination olaparib þ navitoclax, nor was 7-day navitoclax compared with 7-day olaparib þ navitoclax.

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Figure 3. EWFTs are resistant to olaparib which correlates with increased BCL-2 and navitoclax sensitivity. A, FACS analysis of apoptosis after 24-hour treatment with 5 mmol/L olaparib. Error bars are þSEM. Asterisks indicate a signiﬁcant separation between the olaparib treatment groups of the CHLA9 and CHLA10 cells, using the Student t test (P < 0.01). B, Western blot analysis of the indicated antibodies in EWFT cell

lines. C, IC50 of navitoclax plotted for solid tumor cell lines and 21 Ewing sarcoma cell lines from http://www. cancerrxgene.org/. A Mann–Whitney nonparametric test was performed (P ¼ 8.69 105).

combination, which was ineffective (Fig. 1H), consistent with the differential apoptosis between the CHLA9 cells and other EWFTs in vitro results (Supplementary Fig. S5A). The olaparib and navi- cell lines, as well as the apoptosis sensitization by navitoclax toclax combination did not induce substantial weight loss in the (Supplementary Fig. S7A). These data suggest our findings of mice or any overt signs of toxicity, suggesting the combination is mitigated apoptotic responses to olaparib uncovered in the well tolerated (Supplementary Fig. S6B). To assess possible hema- CHLA10 cells extend to other EWFTs models. tologic toxicity when using olaparib and navitoclax in combina- We next determined whether these other resistant EWFTs tion, we performed a complete blood count on NSG mice in vivo. models had higher levels of BCL-2 or BCL-XL, as our model would Red blood cell and reticulocyte counts were not significantly predict. Indeed, in comparison with the CHLA9 cells, these affected by the combination. We did observe a significant decrease models had higher levels of BCL-2 and/or BCL-XL (Fig. 3B), in platelet count as well as neutrophil count following navitoclax associated with their poor apoptotic responses to olaparib (Fig. treatment that has previously been reported with its use (31); 3A). We have uncovered an important role for both BCL-2 and however, importantly, olaparib did not augment platelet loss at BCL-XL in olaparib response in EWFTs (Figs. 1 and 3), and it either time point. Also of importance, there was no augmentation would strengthen the case of the importance of BCL-2 and BCL-XL of neutropenia or other toxicity by the combination compared in EWFTs survival if these cancers were sensitive to pharmaceutical with any single-agent dosing at either time point (Fig. 2). As targeting of these 2 proteins. We therefore examined in the thrombocytopenia is the major dose-limiting effect of navitoclax updated GDSC screen (www.cancerRxgene.org) whether Ewing (42), we also assayed populations of cells following a 24-hour sarcoma cells were more sensitive to navitoclax (Fig. 3C) com- recovery period after 7 days of treatment. Impressively, these mice pared with all other solid tumor cell lines grouped together. In nearly fully recovered pretreatment platelet levels (Fig. 2). These fact, Ewing sarcoma cell lines were substantially more sensitive 5 data demonstrate olaparib plus navitoclax may be both effective (P ¼ 8.69 10 ), with 8 of 21 cell lines demonstrating IC50sof and tolerated as a novel combination therapy in EWFTs. 700 nmol/L and below (Fig. 3C). Interestingly, expression of EWS-FLI1 in HEK293T cells led to Most Ewing sarcoma cell lines do not undergo marked higher BCL-2 transcript levels (P < 0.05) compared with the empty apoptosis following olaparib therapy vector control, with consistent BCL-2 protein changes (Supple- Following our findings in the CHLA9 and CHLA10 pair, we next mentary Fig. S7B and S7C); the FLI1 target genes EZH2 (44), expanded to a panel of Ewing sarcoma cell lines to determine the STEAP1, and PRKCB (45–47) were all significantly upregulated as ability of olaparib to induce apoptosis. Akin to CHLA10, these well (Supplementary Fig. S7B). To further evaluate the relation- cells had poor apoptotic responses to olaparib (Fig. 3A), in ship of FLI1 and BCL-2, we probed the CCLE (24) and found a contrast to the CHLA9 cells. However, all Ewing sarcoma cells positive correlation (P < 0.0001) between FLI1 and BCL-2 (Sup- underwent G2–M accumulation, as previously reported (Supple- plementary Fig. S7D); knockdown of EWS-FLI1 confirmed mentary Fig. S6C; ref. 43). Caspase 3 activity confirmed both the decrease of BCL2 expression with the expected decrease in EZH2

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expression (Supplementary Fig. S7E), and increase in expression tumors in the other PDX model, SA13542 (Fig. 6A and B). of the normally EWS-FLI1 downregulated LOX, while EWS-FLI1 Again, the combination did not markedly affect mouse weights upregulated targets STEAP1, NPY1R, and PRKCB expression were or induce any overt signs of toxicity (Supplementary Fig. S6B). reduced following EWS-FLI1 downregulation, albeit not all sig- Assessment of the tumor lysates from the SA13542 PDX nificantly (45–47). Altogether, these data further demonstrate the confirmed marked apoptosis induction with the combination, importance of BCL-2 and BCL-XL in EWFTs, which appear to play but not single agents (Fig. 6C), and that both PDX models complimentary roles, constituting a critical survival signal for expressed BCL-XL and with the SA10233 model expressing EWS-FLI1–driven EWFTs. high levels of BCL-2 (Supplementary Fig. S8D). These data, along with the CHLA10 chemorefractory mouse model Navitoclax sensitizes a panel of Ewing sarcoma cells to olaparib (Fig. 1G), demonstrate compelling activity of the combination We moved to validate navitoclax as a sensitizing agent to of olaparib and navitoclax in vivo. olaparib in EWFTs cells, as was determined in the CHLA9 and CHLA10 pair (Fig. 1). In fact, we noted marked sensitization by navitoclax to olaparib-induced apoptosis in a panel of Ewing Discussion sarcoma cells as evidenced by cleaved PARP1 (Fig. 4A) and FACS Through an unbiased high-throughput drug screen, olaparib measurement of Annexin-V–positive cells (Fig. 4B). Consistent was discovered to have marked in vitro activity in Ewing sarcoma with the CHLA pair, apoptosis sensitization was sufficient for (11). Despite several other reports demonstrating hypersensitivity navitoclax to markedly reduce total viable cells as determined by of Ewing sarcoma to PARP inhibition (10, 12–14, 23, 53), both crystal violet assays (Fig. 4C) and 72-hour cell-viability subsequent clinical evaluation in a heavily pretreated cohort of assays (Fig. 4D and E). These data again indicate that olaparib- patients with Ewing sarcoma with single-agent olaparib showed induced apoptosis can be rescued by the addition of navitoclax in only modest efficacy (15). Here, we demonstrated an important Ewing sarcoma. role for deficient apoptosis following olaparib therapy in Ewing sarcoma, with the antiapoptotic proteins BCL-2 and BCL-XL Navitoclax sensitizes Ewing sarcoma cells to olaparib by playing key roles. We believe these experimental findings at least augmentation of DNA damage and disruption of BIM in part explain the disappointing clinical data. complexes PARP inhibitors prevent single-stranded (ss) DNA break repairs. We next examined the DNA-damaging activity of the olaparib/ This mechanism underlies PARPi activity in BRCA-deficient navitoclax combination using gH2A.X immunofluorescence cancers, which are inherently deficient in double-stranded (ds) staining as a marker for DNA damage. Interestingly, the DNA DNA break repair (54). In Ewing sarcoma, PARPi sensitivity damage observed following olaparib exposure was substantially has been proposed to occur for several reasons: First, PARP1 increased (P < 0.0001) with the addition of navitoclax, which by expression is higher in Ewing sarcoma (55), probably as a direct itself did not induce DNA damage (Fig. 5A; Supplementary Fig. result of EWS-FLI1 (10, 55), and higher PARP1 expression is a S8A). These data are consistent with a direct role of BCL-2 and cause of enhanced PARPi sensitivity (56), most likely through BCL-XL in the augmentation of DNA damage (48). the mechanism of PARP trapping at ssDNA breaks (57, 59). Navitoclax disrupts BIM:BCL-XL and BIM:BCL-2 complexes, to Second, Ewing sarcoma, like BRCA- deficient cancers, appears induce apoptosis (20, 49–51), and can sensitize kinase inhibitors to have a deficient dsDNA repair system (12). Third, FLI1 drives in different cancers through modulation of the BCL-2 family high SLFN11 expression (60), a gene tightly linked to DNA- (reviewed in ref. 7). We therefore asked whether reduction of damaging agent efficacy (24, 61). Fourth, EWS-FLI1 expression BIM protected from the olaparib/navitoclax combination. As is sufficient to increase dsDNA breaks (10). Fifth, EWS-FLI1 demonstrated in Fig. 5B, reduction of BIM by siRNA led to a causes R-loop accumulation, increases replication stress, and concomitant loss in cleavage of PARP1. Immunoprecipitation of interferes with BRCA1 function (62). BIM complexes verified navitoclax-disrupted BIM:BCL-2 com- Although there are several factors that may have contributed plexes (Fig. 5C; Supplementary Fig. S8B and S8C). These data to olaparib's lack of efficacy in patients with chemotherapy- indicate that olaparib/navitoclax induce apoptosis in EWFTs resistant Ewing sarcoma, it is likely that a biological resistance through disruption of BIM complexes, whereas this complex mechanism served to rescue tumor cells from direct PARP disruption leads to BIM-mediated apoptosis (51, 52). Together, inhibition. We propose that there is an inherent deficiency in these data demonstrate multiple mechanisms in which navitoclax many Ewing sarcomas to undergo apoptosis following olaparib sensitizes Ewing sarcoma to olaparib. treatment resulting from a protective effect of BCL-2 and BCL-XL (Figs. 1 and 3). Furthermore, exposure and resistance Mouse models of Ewing sarcoma are sensitive to olaparib plus to chemotherapy appear to contribute to this state of apoptotic navitoclax resistance to olaparib, as evidenced by our results in the To robustly test this novel combination of olaparib and CHLA10 cell line (Figs. 1E and 3A) and observations in navitoclax, we expanded our analyses to 3 models of Ewing patients' tumor specimens (Fig. 1C). It is likely that DNA- sarcoma, the SK-ES-1 xenograft (ATCC HTB-86), and 2 damaging agents used in induction chemotherapy lead to PDXs. Mice were treated daily with olaparib (100 mg/kg), additional pressure on the Ewing sarcoma tumor and, as a navitoclax (80 mg/kg), or the combination of olaparib result, the emergence of cells particularly reliant on BCL-2/ (100 mg/kg) þ navitoclax (80 mg/kg). In all 3 models, there BCL-XL for survival. Overall, further studies will be necessary to was limited activity of either agent when dosed as a mono- elucidate the precise relationship between these prosurvival therapy. However, the combination of olaparib and navitoclax BCL-2 members and Ewing sarcoma tumorigenesis. markedly inhibited tumor growth in the SK-ES-1 model and The strategy to sensitize Ewing sarcoma to PARP inhibition via PDX SA10233 and, impressively, almost completely shrank BCL-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 after 24-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 of apoptosis 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 induced by drugs is normalized to the no-treatment control. Error bars are þSEM. 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 of olaparib. Viability was determined using CellTiter-Glo. Data are graphed as percent viable cells from no-treatment control (no Rx), performed in quadruplicate. Error bars 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 concentrations of olaparib, and viability was determined using CellTiter-Glo. Data are graphed as percent viable cells from no-treatment control (no Rx), performedin quadruplicate. Error bars are þSEM.

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Figure 5. DNA damage is increased with the combination of olaparib and navitoclax. A, Quantification of immunofluorescence images probed for gH2A.X foci intensity following 24-hour treatment using no- treatment control (no Rx), 5 mmol/L olaparib, 1 mmol/L navitoclax, 5 mmol/L olaparib þ 1 mmol/L navitoclax, or 1 mmol/L etoposide. Error bars are þSEM. Asterisks indicate a significant separation between olaparib treatment and the combination olaparib þ navitoclax. Significance was determined using the Mann–Whitney U test. B, siRNA knockdown of BIM or control (scramble sequence) in ES4 and A673 cell lines followed by 24-hour treatment with either no-treatment control (no Rx) or 5 mmol/L olaparib þ 1 mmol/L navitoclax (Nav/Olap). C, An immunoprecipitation of lysates from A673 cells using either a BCL-2 antibody or an IgG control antibody (left) and the whole-cell lysates (5% input; right), after 4-hour treatment with 5 mmol/L olaparib, 1 mmol/L navitoclax, or 5 mmol/L olaparib þ 1 mmol/L navitoclax. strategies to sensitize Ewing sarcoma to PARP inhibition; these to sarcoma formation, which was not accomplished in the par- include the addition of DNA-damaging agents that intensifies allel, control-transduced cells. These data together with the data in the amount of active DNA damage in the cell, like irinotecan this article present a compelling case where antiapoptotic activity and temozolomide (13). Temozolomide has also been dem- of BCL-2 family members, particularly BCL-2 and BCL-XL, plays onstrated to enhance PARP1 trapping (59) and, interestingly, an intricate role in Ewing sarcoma tumorigenesis and affects the combination of temozolomide and PARP inhibition coop- Ewing sarcoma therapy. eratively downregulates MCL-1, sensitizing to mitochondrial- It has been well known for several decades that BCL-2 has a mediated death (13). Although temozolomide–PARPi combi- protective role against DNA damage–induced apoptosis (48, 67). nations are poorly tolerated in preclinical Ewing sarcoma In addition, BCL-XL expression has been reported to correlate mouse models (12), irinotecan delivered to an orthotopic inversely with the sensitivity of cancer cell lines to multiple Ewing sarcoma mouse model in dosing schedules consistent antitumor agents, including those acting via a DNA-damaging with the pediatric population demonstrated marked activity mechanism (23). This becomes particularly relevant in the light (12). Consistent with these results, the combination of the that Ewing sarcoma have deficient DNA damage responses (12). PARPi veliparib and irinotecan was well tolerated in a recent Adding to the intrigue, Brohl and colleagues recently reported phase I trial, including reaching a dose sufficient for PARP 13% of patients with Ewing sarcoma have germline loss-of- inhibition in adult cancers (63). Of note, BCL-XL blocks the function mutations in DNA repair genes (68). It is therefore ability of irinotecan to induce apoptosis and BCL-XL inhibition tempting to speculate that, in order for EWS-FLI1–translocated results in a switch from irinotecan-induced senescence to apo- Ewing sarcoma to develop and thrive, there must be an acquired ptosis (64). Therefore, it is possible that PARPi/irinotecan reliance on the antiapoptotic proteins BCL-2 and BCL-XL to combinations in other Ewing sarcoma models will face the maintain survival. Consistent with this notion, our analyses of same issues we have found PARP inhibition monotherapy to HTS data revealed navitoclax has substantial single-agent activity face, namely a refractory apoptosis response. The PARPi/irino- (IC50 less than 700 nmol/L) across approximately 40% of Ewing tecan combination is currently being evaluated in pediatric sarcoma cell lines (Fig. 3C). patients with solid tumors (NCT02392793). This notion is further supported by our findings in the CHLA The BCL-2 family of proteins monitors the integrity of the cells derived from a patient prior to and following chemother- mitochondria and integrates the signals of many pathways at the apy treatment. The postchemotherapy CHLA10 cells, derived at mitochondria (65). Importantly, Javaheri and colleagues (66) progressive disease, had higher BCL-2 expression relative to the elegantly demonstrated that EWS-FLI1 overexpression in mesen- matched chemotherapy-na€ve CHLA9 cells (Fig. 1B) and, unlike chymal stem cells, the presumed cell of origin for Ewing sarcoma, CHLA9 cells, failed to undergo cell death following olaparib was sufficient for blocking differentiation but led to high rates of therapy (Figs. 1E and 3A). It is important to note that we did apoptosis; retrovirus containing BCL-2, BCL-XL, or MCL-1 expres- not account for other changes that occurred during the acqui- sion plasmids was able to rescue apoptosis and, importantly, led sition of chemotherapy resistance in this model, which could

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Figure 6. Olaparib and navitoclax combination is effective in mouse models of Ewing sarcoma. A, SK-ES-1 xenografts treated daily for 26 days (top) and patient-derived xenografts (bottom) treated daily for 31 days (PDX SA13542) or 27 days (PDX SA10233) with olaparib (100 mg/kg), navitoclax (80 mg/kg), or the combination of olaparib (100 mg/kg) þ navitoclax (80 mg/kg). Error bars are þSEM. Asterisks indicate a signiﬁcant separation between the combination (olap/nav) and all other treatment cohorts using the Student t test (P < 0.05). B, Fold change in tumor volume, please note data are from the experiment shown in Fig. 6A (PDX SA13542) after 31 days of treatment, and the x axis denotes individual xenografts. C, Western blot analysis of cleaved PARP1 from PDX tumor lysates 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).

contribute to the resistance of these cells to olaparib. For sarcoma DNA damage repair, which are exacerbated by PARP instance, Mendoza-Naranjo and colleagues demonstrated the inhibition. Indeed, BCL-2/BCL-XL inhibition causes accumula- CHLA10 cells have enhanced flux through the PI3K pathway tion of DNA damage following PARP inhibition (Fig. 5A; compared with the CHLA9 cells, whichisaresultofincreased Supplementary Fig. S8A). Therefore, the robust activity of PARP ErbB4 expression (69) and which may be contributing to inhibition and navitoclax is most likely due to both BCL-2 and olaparib resistance. Notwithstanding, the fact that navitoclax BCL- XL inhibition, making these cells more vulnerable to DNA was sufficient to resensitize the cells to olaparib reflects the damage–induced apoptosis, but also increasing the DNA dam- importance of BCL-2 and BCL-XL. Interestingly, in the chemo- age itself. The result is a substantial increase in apoptosis therapy-na€ve CHLA9 cells, where olaparib was very effective (Fig. 4A–B), mediated by BIM (Fig. 5B and C), which translates (Fig. 1A), navitoclax fully sensitized these cells to a low dose of to impressive in vivo activity. olaparib (Supplementary Fig. S5D), which did not have marked Overall, we demonstrate Ewing sarcoma tumors do not single-agent anticancer activity. These data reveal an important undergo a marked apoptotic response following olaparib ther- interplay between PARP inhibition and BCL-2/XL inhibition, apy; however, cotargeting BCL-2 and BCL-XL dramatically sen- which likely contributes to the impressive activity of dual PARP sitizes these tumors to olaparib in several mouse models of and BCL-2/XL inhibition in Ewing sarcoma (Figs. 1, 3, and 4) Ewing sarcoma, including chemotherapy-resistant Ewing sar- and, again, supports the notion that BCL-2 and BCL-XL are coma and 2 PDX models of Ewing sarcoma. As we found important to counteract the intrinsic deficiencies in Ewing neither drug augmented hematologic toxicity of the other

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(Fig. 2), and rational navitoclax-based combinations with M.L. Calbert, M.S. Ghotra, S.C. Smith, C.H. Benes, J.D. Leverson, S.A. Boikos, other targeted therapies are ongoing in clinical trials (e.g., A.C. Faber NCT02520778), evaluation of PARP inhibitors and navitoclax Writing, 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. Faber in Ewing sarcoma is warranted. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.A.R. Heisey, Y.K. Maves, C.H. Benes Disclosure of Potential Conflicts of Interest Study supervision: S.A. Boikos, A.C. Faber S.C. Smith received royalties for textbook authorship and consulting from Elseveir/Amirsys. C.H. Benes reports receiving commercial research grants from Acknowledgments Amgen and Novartis. J.D. Leverson and A.J. Souers have ownership interests This work was supported by an American Cancer Society Research Scholar (including patents) at AbbVie. A.C. Faber is a consultant/advisory board Grant (A.C. Faber) and the Sarcoma Foundation of America (A.C. Faber). member for AbbVie. No potential conflicts of interest were disclosed by the A.C. Faber is supported by the George and Lavinia Blick Research Fund and other authors. is a Harrison Endowed Scholar in Cancer Research. Services and products in support of the research project were generated by the VCU Massey Cancer Center Authors' Contributions Cancer Mouse Model Shared Resource, supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059. Conception and design: D.A.R. Heisey, S.A. Boikos, A.C. Faber Development of methodology: D.A.R. Heisey, S.C. Smith, A.C. Faber Acquisition of data (provided animals, acquired and managed patients, The costs of publication of this article were defrayed in part by the payment advertisement provided facilities, etc.): D.A.R. Heisey, T.L. Lochmann, K.V. Floros, of page charges. This article must therefore be hereby marked C.M. Coon, K.M. Powell, S. Jacob, M.L. Calbert, M.S. Ghotra, G.T. Stein, in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. S.C. Smith Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Received January 25, 2018; revised July 11, 2018; accepted October 17, 2018; computational analysis): D.A.R. Heisey, T.L. Lochmann, K.V. Floros, S. Jacob, published first October 22, 2018.

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OF12 Clin Cancer Res; 2018 Clinical Cancer Research

The Ewing Family of Tumors Relies on BCL-2 and BCL-XL to Escape PARP Inhibitor Toxicity

Daniel A.R. Heisey, Timothy L. Lochmann, Konstantinos V. Floros, et al.

Clin Cancer Res Published OnlineFirst October 22, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-0277

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