(P 64) ENTINOSTAT ENHANCES PRO-INFLAMMATORY CHEMOKINE PRODUCTION AND INCREASES RADIATION-INDUCED APOPTOSIS IN EWING SARCOMA

Objective: Ewing sarcoma is an aggressive bone cancer most commonly diagnosed in adolescents and young adults. Histone deacetylase inhibitors (HDACi) are a class of drugs that target epigenetic modifications induced by tumor cells. Entinostat is an HDACi that is of specific interest in Ewing sarcoma based on promising pre-clinical data. However, trials of single-agent entinostat in patients with Ewing sarcoma have had limited efficacy. In other cancers, HDACi can enhance sensitivity to radiation, and can increase the expression of chemokines responsible for recruitment of anti-tumor immune cells into the tumor. However, the specific impact of entinostat on the Ewing sarcoma tumor immune microenvironment and sensitivity to radiation remains a gap-in-knowledge in the field. We hypothesize that entinostat will enhance the sensitivity of Ewing tumors to radiation and promote anti-tumor inflammation. The translational goal of this work is to better understand the impact of entinostat on Ewing tumor cells in detail in order to provide clues to new combination therapies for aggressive Ewing sarcoma.

Methods: To investigate the effect of entinostat on Ewing tumor sensitivity to radiation, Ewing sarcoma cell lines (A673, TC32, CHLA10) were treated with either entinostat (2mm or 3 mm dose) or DMSO control and either radiation (2 gy, 3 Gy, or 5 Gy) or no radiation. Cells were monitored for growth and apoptosis using a live-cell IncuCyte 3/7 caspase assay. To determine effect of entinostat on Ewing sarcoma production of pro-inflammatory chemokines, the Ewing sarcoma cell lines (TC32, CHLA10) were again treated with entinostat and radiation as above, and supernatant was collected. Supernatant was analyzed utilizing the Biolegend Legendplex Human Proinflammatory Chemokine Panel 1. RNA collected from Ewing cells treated with entinostat was also subjected to RNAseq analysis.

Results: We demonstrate that Ewing cells treated with the combination of entinostat and radiation in vitro demonstrate significantly increased apoptosis as compared to either treatment alone. Analysis of chemokine expression by Ewing cells following entinostat treatment demonstrates increased expression of multiple chemokines, including CXCL10 and IL8. Analysis of entinostat-induced transcriptional changes in Ewing cells in vitro is ongoing, as are in vivo effects on tumors in an immunocompetent model.

Conclusion: To date, single agent therapy with HDACi have not shown significant benefit in early phase clinical trials for Ewing sarcoma. However, we demonstrate that the HDACi entinostat enhances radiation induced apoptosis of Ewing tumor cell lines in vitro and increases tumor cell production of pro-inflammatory chemokines. These findings provide rationale to further investigate the role of entinostat in combinatorial therapies for Ewing sarcoma.