Abstract 3180: Preclinical validation of EZH2 as a therapeutic target in pediatric Ewing's sarcoma

Abstract

Abstract Disease-free survival in relapsed Ewing's Sarcoma Family of Tumors (ESFT) has not improved over the past 25 years. Current standard-of-care (SOC) agents result in 70% survival in patients with localized ESFT; however, for relapsed patients the survival rates remain between 15-20%. Approximately 85% of ESFTs have the chromosomal translocation t(11;22)(q24;q12) which encodes for the oncogenic EWS/FL1 fusion protein. The EWS/FL1 functions as a potent transcription factor leading to the dysregulated expression of genes that promote and maintain tumorigenesis. A major epigenetic regulator that is a downstream target of EWS/FL1 is the enhancer of Zeste Homolog 2 (EZH2). EZH2 is the catalytic component of the polycomb repressor complex 2 (PRC2). Notably, it is overexpressed in ESFT and maintains tumor oncogenicity by tri-methylating histone 3 lysine 27 (H3K27me3) to modulate gene expression. Genome and transcriptome data obtained by the Pediatric Precision Genomics Program at Riley Hospital for Children at Indiana University Health (IUH) indicate that EZH2 is expressed at high levels in ESFT biopsies. Additionally, other groups have reported that high levels of EZH2 protein in ESFT and other cancers correlate with increased chemoresistance to SOC therapy. We are testing tested the hypothesis that EZH2 contributes to chemoresistance in ESFT by regulating critical growth and survival genes. In addition, we are investigating if pharmacological inhibition of EZH2 will enhance sensitivity to the cytotoxic effects of SOC agents. Pediatric primary and relapsed ESFT cell lines and ESFT xenografts were validated for the EWS/FLI, EZH2, and H3K27me3 signatures. In vitro- and in vivo-pharmacodynamic studies of EZH2 inhibition via tazemetostat were conducted to optimize dosing effect. In ESFT cell lines (TC71, A673, CHLA-9, and CHLA-10), tazemetostat dose-response experiments indicated a significant reduction of H3K27me3 by one day post-treatment which was either sustained or completely blocked by 7-days post-treatment compared to vehicle treated (p<0.001). EZH2-mediated gene regulation was confirmed via qPCR. Inhibition via siRNA or tazemetostat resulted in transcriptional up-regulation of p21 (p<0.05) and down-regulation of C-MYC (p<0.01) in all ESFT cell lines. In TC71 sarcoma xenografts, 200-400 mg/kg BID was well tolerated, and decreased H3K27me3 was evident by 5 and 10 days post-treatment (n=4-5 per cohort, vehicle vs treated, p<0.001). While decreased tumor growth was evident at day 5 post-treatment, growth had recovered by day 10. In vitro assays indicate that tazemetostat potentiates SOC agents, etoposide and irinotecan (SN-38), in mediating ESFT growth inhibition (p<0.001). These data provide rationale for systematic investigation of EZH2 inhibition in combination with SOC. New information will be obtained on how to reprogram the epigenome to mitigate therapeutic resistance in ESFT that overexpress EZH2. Citation Format: Pankita H. Pandya, Barbara Bailey, Adily E. Elmi, Heather R. Bates, Courtney N. Hemenway, Anthony L. Sinn, Khadijeh Bijangi-Vishehsaraei, M. Reza Saadatzadeh, Harlan E. Shannon, Jixin Ding, Mark S. Marshall, Michael J. Ferguson, Lijun Cheng, Lang Li, Mary E. Murray, Jamie L. Renbarger, Karen E. Pollok. Preclinical validation of EZH2 as a therapeutic target in pediatric Ewing's sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3180.