Combinatorial Therapy of Imipridones and Histone Deacetylase Inhibitors in Ewing Sarcoma Cell Lines Demonstrates Synergistic Cell Death.

Abstract Novel therapies are much needed in treating pediatric sarcomas, such as Ewing sarcoma. In the setting of metastatic Ewing sarcoma, survival expectancy ranges around 10%. A novel class of small molecules, imipridones, target G protein-coupled receptors and mitochondrial protease ClpP. Imipridones inactivate cell proliferation kinases Akt/ERK and induce cell death through the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor DR5. In pre-clinical and clinical trials, imipridone ONC201 shows increased activity against tumors with mutations in histones, specifically histone H3 at the location of lysine 27 (H3K27). Changes in histone acetylation and the epigenetic effects of imipridones remain under investigation. Most commonly caused by a fusion of the EWS-FLI1 genes, Ewing sarcoma exhibits epigenetic modifications with the EWS-FLI1 protein acting as a transcription factor that causes covalent modifications of histone H3, including acetylation and activation of H3K27. Ewing sarcomas have also shown in vitro response to ERK pathway inhibition, leading to apoptosis and inhibition of metastasis formation. We hypothesized that Ewing sarcoma cell lines, with increased H3K27 acetylation due to EWS-FLI1 acting as an activating transcription factor, would respond to the combination of imipridones with histone deacetylase inhibitors. We performed combinatorial drug treatment on Ewing sarcoma cell lines SK-N-MC and RD-ES with 3 imipridones (ONC201, ONC206, and ONC212) and 3 HDAC inhibitors (vorinostat, entinostat, and panobinostat). Cell viability was measured after drug treatment in vitro. We investigated markers of cell death in established pediatric sarcoma cell lines via protein analysis and flow cytometry analysis. Using protein quantification studies and downstream target analysis on combination drug-treated cells, we investigated the mechanisms of how these two targeted therapies interact synergistically to cause cell death. We show that imipridones inactivate cellular proliferation through the Akt/ERK pathway and induce cell death through the TRAIL pathway, with ONC212 exhibiting the highest cell killing potency. Protein quantification by Western blotting revealed treatment with imipridone ONC201 affected the mitochondrial Clp protease complex by decreasing the chaperone subunit ClpX. Cell viability studies and analysis with Compusyn demonstrate potent synergistic effects causing tumor cell death when imipridones are combined with HDAC inhibitors. Our work presents a novel therapeutic combination for the treatment of Ewing sarcoma. Citation Format: Wen-i Chang, Lanlan Zhou, Attila Seyhan, Varun V. Prabhu, Wafik S. El-Deiry. Combinatorial therapy of imipridones and histone deacetylase inhibitors in Ewing sarcoma cell lines demonstrates synergistic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1060.