Targeting the Clear Cell Sarcoma Oncogenic Driver Fusion Gene EWSR1::ATF1 by HDAC Inhibition

Abstract

Clear cell sarcoma (CCS), a rare but extremely aggressive malignancy with no effective therapy, is characterized by the expression of the oncogenic driver fusion gene EWSR1::ATF1. In this study, we performed a high-throughput drug screening, finding that the histone deacetylase inhibitor vorinostat exerted an antiproliferation effect with the reduced expression of EWSR1::ATF1. We expected the reduced expression of EWSR1::ATF1 to be due to the alteration of chromatin accessibility; however, assay for transposase-accessible chromatin using sequencing and a cleavage under targets and release using nuclease assay revealed that chromatin structure was only slightly altered, despite histone deacetylation at the EWSR1::ATF1 promoter region. Alternatively, we found that vorinostat treatment reduced the level of BRD4, a member of the bromodomain and extraterminal motif protein family, at the EWSR1::ATF1 promoter region. Furthermore, the BRD4 inhibitor JQ1 downregulated EWSR1::ATF1 according to Western blotting and qPCR analyses. In addition, motif analysis revealed that vorinostat treatment suppressed the transcriptional factor SOX10, which directly regulates EWSR1::ATF1 expression and is involved in CCS proliferation. Importantly, we demonstrate that a combination therapy of vorinostat and JQ1 synergistically enhances antiproliferation effect and EWSR1::ATF1 suppression. These results highlight a novel fusion gene suppression mechanism achieved using epigenetic modification agents and provide a potential therapeutic target for fusion gene–related tumors. Significance: This study reveals the epigenetic and transcriptional suppression mechanism of the fusion oncogene EWSR1::ATF1 in clear cell sarcoma by histone deacetylase inhibitor treatment as well as identifying SOX10 as a transcription factor that regulates EWSR1::ATF1 expression.