BACKGROUND: Rhabdoid tumor (RT) is an aggressive pediatric malignancy defined by the genetic deletion of SMARCB1 (BAF47/SNF5/INI1), a subunit of the SWI/SNF chromatin remodeling complex. SMARCB1 loss redistributes the SWI/SNF complex to favor a proliferative non-differentiated cellular state. Nevertheless, while EZH2, the catalytic subunit of PRC2, is an established, synthetically lethal therapeutic target, other vulnerabilities that result from this imbalance between the SWI/SNF and PRC2 complexes are relatively unexplored.

METHODS: 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 SWI/SNF and PRC2 dynamics. Here, we describe an epigenetic mechanism of action for mithramycin in rhabdoid tumor. We characterize KDM6A, a H3K27me3 histone demethylase, as a novel therapeutic vulnerability in rhabdoid tumor with live cell imaging, western blot, small molecule inhibition, and chromatin immunoprecipitation (ChIP) sequencing. We identify SWI/SNF eviction from chromatin as a novel mechanism of action for mithramycin with biochemical fractionation, ChIP-seq, and ATAC-seq. Finally, we demonstrate these findings in vivo using RT xenograft models.

RESULTS: Mithramycin induced apoptosis and a striking increase in H3K27me3, the catalytic mark of PRC2, in rhabdoid tumor cell lines in a dose and time-dependent manner. 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, a H3K27me3 histone demethylase, potentiates the effects of MMA providing further evidence that KDM6A loss and H3K27me3 amplification lead to apoptosis. We have identified SP1 as the transcription factor driving KDM6A and mithramycin interferes with SP1-dependent activation of KDM6A by evicting SWI/SNF from chromatin. We are currently working on the mechanism of action for mithramycin in rhabdoid tumor cells, including H3K27me3 ChIP-seq and ATAC-seq to elucidate global transcription and chromatin changes. We have recapitulated these results in vivo with xenograft mouse models.

CONCLUSIONS: Mithramycin inhibits KDM6A, a histone demethylase, and SWI/SNF leading to apoptosis through the amplification of H3K27me3. Eviction of SWI/SNF from chromatin is a novel mechanism of action for mithramycin. Overall, this study indicates KDM6A and SWI/SNF loss are novel therapeutic vulnerabilities in rhabdoid tumor and mithramycin may be a promising clinical candidate for the treatment of rhabdoid tumor.

Citation Format: Maggie H. Chasse, Elissa Boguslawski, Katie Sorensen, Ian Beddows, Kristin Rybski, Zachary Madaj, Susan M. Kitchen-Goosen, Patrick J. Grohar. Mithramycin inhibits KDM6A (UTX) and SWI/SNF to induce apoptosis in malignant rhabdoid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2883.