(P 246) PANOBINOSTAT DECREASES DNA DAMAGE REPAIR AND INCREASES SENSITIVITY TO CHEMOTHERAPY IN EWING SARCOMA

Objective: Ewing sarcoma (EWS) is the second most common malignant bone sarcoma in children and young adults. Current treatment for EWS includes a combination of cytotoxic chemotherapeutic agents and local control with radiation and/or surgery. The survival rate of those with EWS has seen very little improvement over the past several decades and remains poor for those with recurrent or metastatic disease. The purpose of this study is to identify novel agents currently available for use in EWS.

Methods: Using RNA sequencing performed on 10 EWS patient tumors with bioinformatic analysis of gene expression we identified possible targeted agents for the treatment of EWS. Panobinostat was selected and tested on patient cell lines at escalating doses. At 48 hours cell viability was assessed with CellTiter-Glo 2.0 and analyzed with GraphPad Prism to determine the IC50. At 24- and 48-hours western blot analysis was performed to evaluate the effect on cell cycle and apoptosis . Combination studies with standard upfront and relapsed chemotherapy were performed to evaluate for synergy using SynergyFinder 2.0.

Results: RNA analysis identified HDAC2, ALK, CDK4 and JAK1 as potential therapeutic targets. The pan-HDAC inhibitor, Panobinostat, was cytotoxic to all Ewing sarcoma cell lines tested with clinically relevant IC50s (< 200nM) and HDAC expression levels correlate to cell sensitivity. Panobinostat also synergizes with Doxorubicin and etoposide, and significantly decreases cell viability when added to standard chemotherapy combinations at clinically relevant doses. Panobinostat represses expression of Cyclin D1, pRB, CHEK1 andCHEK2; increases cleavage of Caspase 3; and increases expression of pH2AX especially when used in combination with doxorubicin.

Conclusion: This work highlights that RNA expression may identify potential therapeutic agents which could be effective in treating Ewing sarcoma or potentially improve the efficacy of standard of care drugs. HDAC inhibition shows a decrease in cell viability in EWS cell line models and when combined with doxorubicin results in increased DNA double strand breaks, less DNA repair and increased apoptosis. Future studies combining HDAC inhibitors with standard chemotherapy will aim to determine efficacy in xenograft models.

CTOS Heatmap.pdf

CTOS IC50s.pdf

CTOS Synergy.pdf

CTOS Western Blot.pdf