Ewing's sarcoma family of tumors (ESFT) refers to aggressive malignancies which frequently harbor characteristic EWS-FLI1 or EWS-ERG genomic fusions. Here, we report that these fusion products interact with the DNA damage response protein and transcriptional coregulator PARP-1. ESFT cells, primary tumor xenografts, and tumor metastases were all highly sensitive to PARP1 inhibition. Addition of a PARP1 inhibitor to the second-line chemotherapeutic agent temozolamide resulted in complete responses of all treated tumors in an EWS-FLI1–driven mouse xenograft model of ESFT. Mechanistic investigations revealed that DNA damage induced by expression of EWS-FLI1 or EWS-ERG fusion genes was potentiated by PARP1 inhibition in ESFT cell lines. Notably, EWS-FLI1 fusion genes acted in a positive feedback loop to maintain the expression of PARP1, which was required for EWS-FLI–mediated transcription, thereby enforcing oncogene-dependent sensitivity to PARP-1 inhibition. Together, our findings offer a strong preclinical rationale to target the EWS-FLI1:PARP1 intersection as a therapeutic strategy to improve the treatment of ESFTs. Cancer Res; 72(7); 1608–13. ©2012 AACR.
The Ewing's sarcoma family of tumors (ESFT) refers to malignant neoplasms of bone and soft tissue which frequently harbor reciprocal chromosomal translocations that result in the production of chimeric proteins containing the transcriptional activation domain from the Ewing's sarcoma breakpoint region 1 gene (EWS) fused to the DNA-binding domain of an E26 transformation-specific sequence (ETS) transcription factor (FLI1 or ERG; refs. 1, 2). Dysregulation of these chimeric proteins have previously been implicated in abnormal proliferation, invasion, and tumorigenesis (3, 4). Although the EWS-FLI or EWS-ERG fusion proteins represent potential disease–specific molecular targets, transcription factors such as these have been notoriously difficult to inhibit therapeutically. However, given the poor outcomes of patients with metastatic EFST (5), there is a clear need to improve therapy for these patients.
We have recently shown that an alternative strategy to inhibit transcription factor activity is to target critical interacting enzymes (6). Similar to ESFTs, 50% of prostate cancers harbor genomic rearrangements of ETS transcription factors (7). However, unlike the EWS-FLI or EWS-ERG fusions which produce a multifunctional chimeric protein, prostate cancer rearrangements usually place an androgen-regulated promoter upstream of an ETS gene (ERG or ETV1), resulting in increased ETS transcription factor activity by overexpression (3). We have previously shown that PARP1 and DNA-dependent protein kinase (DNA-PKcs) are key ETS protein family cofactors in prostate cancer, and therapeutic inhibition of PARP1 disrupts the growth of ETS positive, but not ETS negative, prostate cancer xenografts (6). Importantly, we showed that this ETS protein interaction axis is mediated by the ETS DNA-binding domain (6). Although this interaction site is present in the EWS-FLI1 and EWS-ERG chimeras, these fusion proteins activate oncogenic pathways independent of their DNA-binding domain (8). As such, despite the presence of the ETS DNA-binding domain, the effectiveness of PARP inhibitors against these multifunctional gene fusion proteins is unclear.
Mechanistically, PARP1 is a functionally complex enzyme that has been shown to both drive transcription and to accelerate base excision repair (9–11). PARP1 inhibitors have shown promising activity in early clinical trials (12), particularly in BRCA-mutant cancers defective in homologous repair (HR), in which they may cause replication fork stalling and subsequent synthetic lethal cell death (13, 14). We now hypothesize that the EWS-FLI and EWS-ERG gene fusions in ESFTs depend on the activity of PARP1 and may be targeted with PARP1 inhibition.
Materials and Methods
Cell lines and inhibitors
PC3, RD-ES, SAOS-2, A-204 (ATCC), CADO-ES1 (DSMZ), COG10, and COG258 cell lines were grown in RPMI-1640 (Invitrogen) supplemented with 10% FBS (Invitrogen). The genetic identity of each cell line was confirmed by genotyping samples. Olaparib was purchased from Axon Biochem.
Gene-specific knockdown was accomplished with commercially available siRNA duplexes for EWS, ERG, FLI1, DNA-PKcs, or PARP-1 (Dharmacon). Two independent siRNA sequences were used for each gene for knockdown experiments.
In vitro assays
Soft agar colony formation, chemosensitivity, and basement membrane matrix invasion assays were conducted per standard protocols (see SOM). Approaches for immunofluorescence, immunoprecipitation, Western blot, COMET assays, luciferase reporter assays, and Oncomine analysis are also detailed in the SOM. Primer sequences available in Supplementary Table S1.
Severe combined immunodeficient mice were purchased from Charles River, Inc. (Charles River Laboratory) and implanted subcutaneously with RD-ES, PC3-luciferase-LACZ, or PC3-luciferase-EWS-FLI (1 × 106 cells per injection). After 2 weeks, mice with tumors were treated with Olaparib and/or Temozolamide. All procedures involving mice were approved by the University of Michigan, The University Committee on Use and Care of Animals (UCUCA) and conform to their relevant regulatory standards. Additional experimental details are available in the SOM.
Results and Discussion
To test the hypothesis that EWS fusions interact with PARP1, we carried out immunoprecipitation (IP)-immunoblot analysis on cell lines naturally harboring the rearrangements. Using an ERG or FLI1 antibody that recognizes the EWS-ERG and EWS-FLI1 fusion proteins, respectively, we pulled down both PARP1 and DNA-PKcs in a DNA-independent manner from 2 cell lines, CADO-ES1 (EWS-ERG) or RD-ES (EWS-FLI1; Fig. 1A). Reverse IP from RD-ES cells carried out using a PARP1 antibody enriched for EWS-FLI1 and DNA-PKcs in a DNA-independent manner (Fig. 1B), confirming the interaction between EWS fusions and PARP1.
We then tested the sensitivity of ESFT cells to the PARP1 inhibitor Olaparib. To analyze the sensitivity of ESFT cells overexpressing the EWS-FLI1 and EWS-ERG fusions, compared with the prostate cancer harboring the TMPRSS2-ERG fusion, we carried out soft agar colony formation assays on PC3 cells in which we had stably overexpressed these fusion products. Overexpression of any of these 3 ETS fusion genes caused sensitivity to Olaparib (P < 0.05 at 3 and 10 μmol/L), and the EWS-FLI1 and EWS-ERG fusions were more sensitive than the TMPRSS2-ERG fusion (P < 0.05 at 1 μmol/L; Fig. 1C). Soft agar assays conducted with CADO-ES1, RD-ES, or VCaP (TMPRSS2-ERG rearranged prostate cancer) cells confirmed that while all ETS-positive cell lines were more susceptible to Olaparib than the ETS-negative cell line PC3, ESFT cell lines were even more sensitive than VCaP cells (P < 0.05; Fig. 1D). Importantly, Olaparib did not have an effect on an osteosarcoma cell line, SAOS-2, or a rhabdomyosarcoma cell line, A-204, which were used as sarcoma model controls without ETS rearrangement (Fig. 1D). We also found that 2 Ewing's cell lines [COG10 and COG258 (both EWS-FLI1)] derived from front-line chemotherapy patient relapses (and thus heavily pretreated) were also extremely sensitive to PARP1 inhibition (Fig. 1D).
Given the sensitivity of ESFTs to PARP inhibition, we sought to explore the mechanism underlying this sensitivity. In prostate cancer, TMPRSS2-ERG overexpression causes DNA damage, which is then potentiated by PARP inhibition (6). Surprisingly, Ewing's cells showed significantly more γH2A.X foci (a marker of DNA double-strand breaks) per cell than TMPRSS2-ERG–positive VCaP cells (P < 0.05 for all EFST cells; Fig. 2A) and 1 μmol/L Olaparib caused massive induction of γH2A.X foci (Supplementary Fig. S1A). Neutral COMET assays confirmed that natural EFST cells have more DNA double-strand breaks per cell than VCaP cells (P < 0.05; Fig. 2A). Importantly, overexpression of EWS-FLI1 in PC3 cells led to increased DNA breaks (P < 0.05; Supplementary Fig. S1B), and knockdown of either EWS-FLI1 or EWS-ERG fusions caused a reduction in COMET tail moment, and significantly decreased the induction of DNA damage after Olaparib treatment (P < 0.05; Fig. 2B and Supplementary Figs. S2A, S2B, S3A, and S3B) suggesting that Olaparib-potentiated DNA damage is due to expression of the EWS-FLI1 and EWS-ERG fusion genes. Inhibition of nonhomologous end-joining (NHEJ) can synergistically potentiate DNA damage in the context of deficient HR. However, siRNA knockdown of XRCC4 (needed for NHEJ) had little effect (Supplementary Fig. S4), suggesting that EFST cells have functional HR. Although PARP1 inhibition dramatically accentuated DNA damage, it did not have an effect on short-term cell viability (Supplementary Fig. S5A and S5B).
We next assessed the effect of PARP1 inhibition on short-term cell invasion. Intriguingly, siRNA against EWS, PARP1, or DNA-PKcs reduced CADO-ES1 and RD-ES cell invasion (P < 0.05), while knockdown of key components of DNA repair pathways (XRCC1 for base excision repair, XRCC3 for HR, and XRCC4 for NHEJ) had no effect (Fig. 2C and Supplementary Figs. S2A and S2B), supporting a DNA repair–independent role of the enzymes. Importantly, Olaparib disrupted ESFT cell invasion (P < 0.05) at approximately 1 log-fold lower concentration than VCaP cell invasion (6) and did not affect ETS negative, control sarcoma cell invasion (Fig. 2D and Supplementary Figs. S2A and S2B).
Because PARP inhibition disrupts the long-term growth and invasion of ESFT cells, we assessed the ability of EWS-FLI1 to sensitize xenografts to Olaparib. Surprisingly, Olaparib significantly impeded PC3-EWS-FLI1 (P < 0.001), but not PC3-LACZ, xenograft growth (Fig. 3A and B), showing that EWS-FLI1 overexpression is alone sufficient to sensitize PC3 xenografts to Olaparib. Because PARP1 inhibitors are unlikely to be used clinically as a monotherapy, we also combined PARP1 inhibition with the DNA alkylating agent temozolomide (TMZ) to treat a highly aggressive ESFT xenograft model (RD-ES) harboring an endogenous EWS-FLI1 rearrangement. TMZ potentiates the effects of PARP1 inhibitors (15) and is currently being assessed in Ewing's sarcoma patients as second-line therapy (16). Surprisingly, TMZ alone only delayed RD-ES xenograft growth (P < 0.001; Fig. 3C). However, Olaparib also caused significant delay in RD-ES xenograft growth (P < 0.001), and, when administered in combination with TMZ, led to an immediate tumor regression and a highly significant sustained complete response. Mice were maintained without therapy for an additional 30 days without any observable recurrence (P < 0.001). Furthermore, we harvested lungs from RD-ES–xenografted mice, assessed metastasis and found only 1 of 5 mice treated with Olaparib developed lung metastasis as compared with 6 of 8 untreated mice (P = 0.058; Fig. 3D). This is the first mouse model showing that PARP1 activity is required for metastasis.
Given that PARP1 has roles in both DNA repair and transcriptional regulation (9, 10), we hypothesized that the sensitivity of ESFT xenograft growth and metastasis depends on both PARP1-mediated roles. Consistent with this, knockdown of PARP1 or DNA-PKcs, but not other DNA repair proteins, disrupted transcriptional activity to a similar extent as fusion targeting EWS siRNA as evidenced by disruption of consensus EWS-FLI1 target genes (ref. 17; Fig. 4A and Supplementary Figs. S2A, S2B).
In modulating EWS-FLI1 and EWS-ERG expression, we observed that both EWS fusions maintain PARP1 mRNA expression levels (P < 0.05; Supplementary Figs. S2A, S3A, S6A, and S6B). This was unexpected because the ETS gene fusions found in prostate cancer do not regulate PARP1 mRNA expression (6), but consistent with reports showing that ETS transcription factors drive PARP1 expression in ESFTs (18, 19). Likewise, in EFST cells, knockdown of the EWS-FLI1 fusion led to a decrease in PARP1 protein expression and promoter activity (P < 0.05), while ERG siRNA did not have these effects in VCaP cells (Fig. 4B). Conversely, EWS-FLI1 expression was not altered by Olaparib in RD-ES cells (Supplementary Figs. S6C and S6D). Finally, Oncomine tumor microarray compendium analysis showed that PARP1 mRNA expression correlates with FLI1 expression in EFST patient samples (P < 0.01, Spearman correlation shown in figure), but did not correlate with ERG expression in prostate cancer (Fig. 4C). Taken together, our data suggest that PARP1 is critical to a positive-feedback loop in EFST, in which the EWS-FLI fusion product drives expression of PARP1, which then further promotes transcriptional activation by EWS-FLI.
In conclusion, our data show that ESFT cells and xenografts are sensitive to PARP1 inhibition, to a significantly greater degree than their ETS fusion-positive prostate cancer counterparts. This increased sensitivity may stem from both greater potentiation of DNA damage by PARP1 inhibition, as well as a EWS-FLI:PARP1–positive feedback loop in transcriptional activation (Fig. 4D). Our findings suggest that PARP1 inhibition should be explored as a strategy for targeted therapy in Ewing's sarcoma, which may represent a tumor type that may be as profoundly affected by PARP inhibitors as BRCA1/2 mutated carcinomas (12–14).
Disclosure of Potential Conflicts of Interest
The University of Michigan has filed a patent linking ETS fusions in Ewing's sarcoma and prostate cancer as markers of sensitivity to PARP inhibitors and DNA-PKcs inhibitors. A.M. Chinnaiyan and J.C. Brenner are named as inventors. No potential conflicts of interest were disclosed by the other authors.
The authors thank Xuhong Cao and Steven Kronenberg for technical support.
This work was supported, in part, by the DOD National Functional Genomics Center (W81XWH-11-1-0520). A.M. Chinnaiyan is a Howard Hughes Medical Institute Investigator, is supported by the Doris Duke Foundation and the American Cancer Society, and is a University of Michigan Taubman Scholar. A.M. Chinnaiyan, J.C. Brenner, F.Y. Feng, and S.A. Tomlins are supported by the Prostate Cancer Foundation.