Abstract 4634: Mithramycin amplifies the imbalance between the BAF and PRC2 complexes to drive apoptosis in rhabdoid tumor
Authors: Maggie H. Chasse, Elissa Boguslawski, Courtney E. Wernette, Susan Kitchen-Goosen, Patrick J. Grohar
Journal: Cancer Research (2018)

Abstract BACKGROUND: Rhabdoid tumor (RT) is an aggressive pediatric malignancy defined by the genetic deletion of BAF47 (SMARCB1/SNF5/INI1), a subunit of the BAF chromatin remodeling complex. Loss of BAF47 leads to BAF complex redistribution and overexpression of EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2) to favor a proliferative non-differentiated cellular state. Nevertheless, while EZH2 itself is an established, synthetically lethal therapeutic target, other vulnerabilities that result from this imbalance between the BAF and PRC2 complexes are relatively unexplored. EXPERIMENTAL DESIGN: In a screen of multiple pediatric cancer cell lines, we identified a heightened sensitivity of RT cells to mithramycin (MMA), a small molecule transcription inhibitor. We hypothesized the hypersensitivity of rhabdoid tumor to mithramycin was due to the disruption of BAF and PRC2 dynamics. Here, we characterized the activity of mithramycin in three RT cell lines. To understand the effect of MMA, we performed in vitro qRT-PCR, immunoblot assays, and viability assays to characterize the expression and activity of the PRC2 complex, including a H3K27me3 histone demethylase. We further investigate the kinetics of cell viability and induction of apoptosis with live cell imaging. Finally, we demonstrate these findings in vivo using rhabdoid tumor xenograft models. RESULTS: Mithramycin inhibited mRNA and protein expression of all four PRC2 subunits in a dose-dependent manner in two different rhabdoid tumor cell lines. Even though EZH2 is suppressed with MMA treatment, we found a striking increase in H3K27me3 that correlated with EZH2 trafficking into the nucleus. This amplification of H3K27me3 preceded suppression of cellular proliferation and induction of apoptosis suggesting H3K27me3 amplification may drive the apoptotic phenotype. In addition, knockdown of KDM6A potentiates the effects of MMA providing further evidence that KDM6A loss and H3K27me3 amplification lead to apoptosis. We are currently working on the mechanism of action for mithramycin in RT cells, including H3K27me3 ChIP-seq and RNA-seq to elucidate global transcription and chromatin changes. We have recapitulated these results in vivo with xenograft mouse models. CONCLUSIONS: Mithramycin represses KDM6A, a histone demethylase, leading to apoptosis through the amplification of H3K27me3. Here, H3K27me3 amplification, in addition to depletion, is detrimental to rhabdoid tumor proliferation and progression. Overall, this study indicates KDM6A loss and H3K27me3 amplification are novel therapeutic vulnerabilities in rhabdoid tumor. Citation Format: Maggie H. Chasse, Elissa Boguslawski, Courtney E. Wernette, Susan Kitchen-Goosen, Patrick J. Grohar. Mithramycin amplifies the imbalance between the BAF and PRC2 complexes to drive apoptosis in rhabdoid tumor [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 4634.