Abstract A32: Therapeutically targeting PRC2 expression and dynamics in rhabdoid tumor

Background: Rhabdoid tumor (RT) is a rare pediatric cancer characterized by the genetic silencing of SMARCB1 (SNF5/INI1), a critical subunit of the SWI/SNF chromatin remodeling complex. SMARCB1 deletion drives oncogenic transformation through redistribution of SWI/SNF and overexpression of EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2). EZH2 catalyzes H3K27me3-modified heterochromatin to repress transcription of critical tumor suppressor genes, such as cell cycle inhibitors and differentiation programs. Thus, inhibition of PRC2 catalytic activity is established to be synthetically lethal in rhabdoid tumors. Experimental Design: The objective of this study is to therapeutically target PRC2 expression in rhabdoid tumor (RT). We have previously screened multiple pediatric cancer cell lines and identified a heightened sensitivity of RT cell lines to mithramycin, a compound that suppresses EZH2 expression in other cell lines. We hypothesized this unique hypersensitivity of rhabdoid tumor to mithramycin is due to the disruption of SWI/SNF and PRC2 dynamics in this tumor. Here, we characterized the effect of mithramycin treatment on the expression and activity of the entire PRC2 complex in vitro using quantitative reverse transcription-polymerase chain reaction, immunoblot analyses, and viability assays. To elucidate the role of EZH2 catalytic activity in the mithramycin mechanism of action, we assayed cellular viability with mithramycin treatment in the presence and absence of tazemetostat (EPZ-6438), an EZH2 small-molecule inhibitor. Results: Mithramycin inhibited expression of all four PRC2 subunits in a dose-dependent manner in two different rhabdoid tumor (RT) cell lines. These effects were also observed at the protein level for two of the obligate PRC2 subunits, EZH2 and SUZ12. Strikingly, we found a marked increase in global H3K27me3 that correlated with EZH2 trafficking into the nucleus. Further, we found suppression of KDM6A expression preceded EZH2 trafficking, suggesting KDM6A loss drives the amplification of H3K27me3. These effects correlated with a marked suppression of cellular proliferation and irreversible induction of apoptosis. In addition, suppression of EZH2 catalytic activity was antagonistic to the effects on cell viability, suggesting an important scaffolding function of EZH2, independent of catalytic activity. We are currently working on the mechanism of action for mithramycin in RT cells, including identifying the transcription factor driving KDM6A and EZH2 expression. Results from these studies will be confirmed in vivo with orthotropic xenograft mouse models. Conclusions: Mithramycin treatment represses PRC2 expression and induces cell death by amplifying H3K27me3, driven by KDM6A loss. These data establish that, in addition to depletion, amplification of H3K27me3 is detrimental to rhabdoid tumor proliferation, and KDM6A is a novel therapeutic vulnerability in rhabdoid tumor. Citation Format: Maggie H. Chasse, Elissa A. Boguslawski, Katie M. Sorensen, Courtney E. Wernette, Susan M. Goosen, Patrick J. Grohar. Therapeutically targeting PRC2 expression and dynamics in rhabdoid tumor [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A32.