Abstract 2760: Identification of ET-743 analogs with improved selectivity and potency for EWS-FLI1 and Ewing sarcoma cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC BACKGROUND: Ewing sarcoma is a pediatric malignancy characterized by the fusion of the EWS and FLI1 genes, which creates a constitutively activated transcription factor, EWS/FLI1. The transcriptional program of EWS/FLI1 drives malignant transformation as well as high-risk features such as increased angiogenesis, resistance to apoptosis, and increased metastatic potential. It is well known that perturbing the transcriptional activity of EWS/FLI1 leads to cell death in vitro and should be considered as a possible site of therapeutic intervention in Ewing sarcoma patients. To that end, we identified ET-743 (trabectedin, Yondelis), a natural product, which inhibits EWS/FLI1 activity in vitro. Using ET-743 as a lead compound, we have characterized a number of structural analogs of ET-743 to identify more potent and/or more selective inhibitors of EWS/FLI1 activity. METHODS: We evaluated the potency of the ET-743 structural analogs using MTS viability assays using Ewing sarcoma, alveolar rhabdomyosarcoma, and osteosarcoma cell lines. The specific inhibition of EWS/FLI1 downstream transcriptional activity was measured using a luciferase-based approach and western blotting. We also evaluated each analog's ability to suppress a gene signature of EWS/FLI1, which included more than 20 genes known to be deregulated by EWS/FLI1 using a novel microfluidic based assay and the Fluidigm platform. RESULTS: Several analogs proved to be more potent than ET-743 and selective for transcriptional suppression of EWS/FLI1. All of the analogs induced varying degrees of DNA damage and cell death, which were detected by biochemical and metabolic assays. We also show that the analogs sensitize ES cells to treatment with camptothecins to a variable degree based on analog relative to the parent compound. CONCLUSIONS: In comparison to ET-743, we have shown that its structural analogs confer better pharmacological properties such as increased potency and specificity towards Ewing sarcoma cell lines. We are currently working to further characterize the mechanism of action for these analogs and evaluate the compounds in an in vivo model. The ultimate goal would be to translate these compounds to the clinic and improve patient outcomes with Ewing sarcoma. Citation Format: Matt Harlow, Nichole Maloney, Laura Segars, Girma Woldemichael, Lee J. Helman, Patrick J. Grohar. Identification of ET-743 analogs with improved selectivity and potency for EWS-FLI1 and Ewing sarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2760. doi:10.1158/1538-7445.AM2013-2760