PARP-1 inhibition as a targeted strategy to treat Ewing's sarcoma

Abstract

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.

Figure 1. Ewing’s sarcoma gene fusion products (EWS-FLI1 and EWS-ERG) interact with PARP1 and DNA-PKcs, and confer sensitivity to PARP1 inhibition

A, Immunoprecipitation (IP)-immunoblot analysis performed on CADO-ES1 (EWS-ERG) cells using an ERG antibody and RD-ES (EWS-FLI1) cells using a FLI1 antibody. IPs were treated +/-100μg/mL ethidium bromide. B, As in A, except using a PARP1 antibody on RD-ES cells. C, Soft agar colony formation assays were performed using PC3 cells, overexpressing ERG, EWS-FLI1, EWS-ERG, or LACZ. Colonies counted after 3 weeks in culture. Representative photomicrographs shown. Scale bar= 50μM. D, As in C, except in ETS-rearranged cells (VCaP, RD-ES, CADO-ES1, COG10 and COG258) and ETS-negative sarcoma controls (SAOS-2 and A-204). Error bars indicate S.E.M. of three replicates. *p<0.05.

Figure 2. Ewing sarcoma gene fusion products confer increased DNA damage and cell invasion, which can be modulated by genetic or pharmacologic approaches

A, Staining and quantification of γH2A.X foci in prostate and EFST cells shown on left axis. On right axis, average tail moments from neutral COMET assays are shown. Representative COMET tails are shown, with calculated tail moments depicted numerically. B, Neutral COMET assays performed on cells treated with siRNA and/or 1μM Olaparib as indicated. C, Boyden chamber transwell migration assays were performed 48 hours after siRNA transfection or Olaparib treatment. For siRNA experiments, cells were plated in serum-free media and incubated for another 48 hours before fixing and staining. Representative photomicrographs are shown. D, As in C, except sarcoma control cell lines that do not harbor EFST gene fusions were used. Error bars indicate S.E.M. of three replicates. *p<0.05.

Figure 3. PARP inhibition prevents EWS-FLI1 positive xenograft growth and metastasis

A, Two weeks after engraftment, PC3-LACZ xenografts were treated with 100mg/kg Olaparib BID. Tumor volume measurements were recorded daily. B, As in A, except with PC3-EWS-FLI1 xenografts. C, As in A, except RD-ES were treated with Olaparib (100mg/kg BID) alone, TMZ (50mg/kg QD) alone or in combination. D, At the RD-ES xenograft assay endpoint, lungs were analyzed for metastases. Representative images were taken with a 4x objective (inset at 40x). In the plot, each dot represents an individual lung metastasis for different treatments as indicated along the x-axis. Metastasis diameter was measured and depicted along the y-axis.

Figure 4. EWS-FLI1 maintains a feed forward loop that drives PARP1 expression

A, CADO-ES1 or RD-ES cells were treated with siRNA for 48 hours. qPCR was then performed for several EWS-ETS target genes. B, Immunoblot analysis of cells treated with control or EWS siRNA. Bar graphs are PARP1 promoter reporter luciferase assays were performed on cells treated with siRNA for 24 hours and then transfected with the promoter reporter for an additional 24 hours. C, Oncomine scatter plots of publically available gene expression data sets. Each point represents gene expression values from an individual patient. First author of each published study is indicated and referenced in the S.O.M.. D, Model depicting proposed mechanism of increased PARP sensitivity of ESFTs as compared to ETS positive prostate cancers. Error bars indicate S.E.M. of three replicates. *p<0.05.