Abstract 2356: The epigenomic characterization of clear cell sarcoma

Abstract

Abstract Clear cell sarcoma (CCS) is an aggressive malignancy that affects adolescents and young adults. CCS is a translocation-driven sarcoma subtype meaning a genetic fusion event defines and drives the malignancy. The chromosomal translocation occurring between EWSR1 on chromosome 22 and ATF1 on chromosome 12 results in the EWSR1-ATF1 fusion oncogene (hereafter abbreviated EA1) which drives more than 90% of CCS cases. The EA1 oncoprotein is a transcription factor that reprograms gene expression to drive CCS. However, the target genes of EA1 and how EA1 affects target gene expression levels are largely unknown. Elucidation of these effectors is critical in order to understand how the EA1 oncoprotein drives the epigenetic reprogramming in CCS. We utilized a robust mouse model that recapitulates CCS in which mice express TATCre-inducible EA1. Using CCS tumors that form in these mice, ChIP-sequencing identified where EA1 is binding along the genome. Analysis in combination with RNA-sequencing data revealed target genes that are bound by EA1 and either upregulated or downregulated compared to control tissue. Target gene expression levels were corroborated in a human context by RNA-sequencing of human tumors and a CCS human cell line. RNAi knockdown of EA1 in the human cell line confirmed functionality of the fusion protein at these target genes. We show that EA1 can bind at promoter regions to directly regulate transcription of target genes. These binding sites include the canonical ATF binding motif. The EA1 target genes overlap with known wildtype ATF1 target genes. Many upregulated genes are involved in cell cycle and proliferation as expected. Alternatively, EA1 can bind intergenic regions including H3K27ac-defined super enhancers to distally regulate target genes. These binding sites contain unique DNA motifs that are different from the canonical ATF binding motif by 1 base pair. One highly recurrent motif identified matches the motif of the AP1 complex, a putative co-factor that may be functioning with EA1. These so-called variant motifs appear to define enhancer regions where EA1 can bind along with AP1. In summary, EA1 binds the expected ATF motif at promoter regions to directly regulate target gene expression. Secondly, EA1 binds intergenic enhancer regions defined by variant motifs including the AP1 motif. Target genes can either be upregulated or downregulated by the fusion, suggesting that there are secondary epigenetic mechanisms. One such model involves differential co-factor composition, such as AP1 co-binding, that dictates where EA1 binds or subsequent transcriptional effects. This research sets up the foundation for how EA1 functions as a transcription factor including the distribution of EA1 across the genome, the motifs that EA1 binds, and the key genes that are regulated by EA1. These epigenetic findings are crucial to understanding how a powerful fusion oncoprotein drives this aggressive, under-studied malignancy. Citation Format: Benjamin B. Ozenberger, Li Li, Kevin B. Jones. The epigenomic characterization of clear cell sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2356.