Abstract B014: Effects of chronic histone deacetylase inhibitor treatment on Ewing sarcoma cells highlight Ret as a potential therapeutic target

Abstract Histone deacetylase inhibitors (HDACi’s) have multiple effects on cancer cell lines, including inhibition of cell growth in vitro, and of human tumor xenografts in vivo, and have had modest therapeutic efficacy in a subset of human malignancies. Previously, we have shown that chronic treatment of colon cancer cells with HDACI’s can result in adaptation, associated with marked decreases in cell growth, colony formation, and tumorigenicity, and extensive changes in gene expression, suggesting an attenuation of malignant phenotype and a possible novel approach to using these agents. Ewing Sarcoma (ES), likely derived from a very primitive mesenchymal-related cell, is an aggressive malignancy of children often resistant to therapy. ES cells classically contain the characteristic EWS-FLI1 translocation and fusion gene, known to dramatically alter histone modifications and epigenetic regulation. We hypothesized that chronic treatment of ES cell lines with HDACi’s will major changes in the program of ES cells gene expression that may alter their malignant phenotype. We treated the ES cell line A673, which contains the EWS-FLI fusion gene, with either stepwise increasing amounts of Entinostat (selective class I HDACi, up to 1. 5µM) or single dose chronic treatment (1. 5µM). We observed treatment-associated dramatic reductions in growth rates, colony formation, anchorage-independent growth, and major changes in gene expression (RNA sequencing, RT-qPCR) and chromatin organization (ATAC-seq). Gene expression analysis revealed hundreds of differentially expressed genes including changes associated with differentiation along both neural and mesenchymal pathways, consistent with observed changes in lineage-determining transcription factors of the Nkx, Sox, and Pax families. Other genes with changed expression levels included potential novel therapeutic targets such as the tyrosine kinase, RET, whose expression was reduced at both the RNA and protein levels, in association with reduced chromatin accessibility. We hypothesized that Ret may play a role in the malignant phenotype of ES and were interested in whether HDACi-induced reduction in Ret expression might sensitize A673 cells to Ret inhibitors. Co-treatment of A673 cells with Entinostat resulted in over two-fold increase in sensitivity to Cabozantinib, a tyrosine kinase inhibitor that inhibits a select group of tyrosine kinases including Reg. We are continuing to characterize the role of Ret as a potential therapeutic target, and as a mediator of the ES cell malignant phenotype through CRISPR-mediated gene. Acknowledgement: This work was supported by funding from the NJ Pediatric Hematology and Oncology Center of Excellence of the Rutgers Cancer Institute. Citation Format: Hsin-Ching Lin, Erica Raj, Luis Sanchez-Gonzalez, Aisling Byrne, Shin-Heng Chiou, Arnold B Rabson. Effects of chronic histone deacetylase inhibitor treatment on Ewing sarcoma cells highlight Ret as a potential therapeutic target abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl₂): Abstract nr B014.