High-Throughput Chemical Screen for Inhibitors of EWS-FLI1 Using a Trangenic Zebrafish Model of Ewing Sarcoma

Abstract

Ewing Sarcoma is the second most common primary bone tumor of children and adolescents. With a lack of targeted therapies, survival remains poor despite remarkably intense chemotherapeutic regimens. 85% of Ewing cases are caused by a translocation between the EWS gene on chromosome 22 and the FLI1 gene on chromosome 11 [t(11;22)(q24;q12)]. The fusion EWS-FLI1 gene codes for a chimeric transcription factor which activates and suppresses expression of a diverse array of target genes, and is an enticing candidate for therapeutic investigation. EWS-FLI1 activity can be readily observed in a homozygous transgenic zebrafish model, in which the fusion gene is inserted downstream of the Microphthalmia-Associated Transcription Factor promoter, localizing EWS-FLI1 expression to melanocytes derived from the neural crest. These fish present with an increased melanocyte count on the dorsum of the head compared to wild type. We hypothesized that compounds which suppress EWS-FLI1 activity will prevent the formation of this phenotype in homozygous zebrafish larvae. A high-throughput chemical screen was performed using two libraries of FDA approved compounds. One compound was found to significantly reduce melanocyte count in EWS-FLI1 homozygous zebrafish compared to unexposed homozygous controls (p < 0.0001) in a dose-dependent manner. Furthermore, treated homozygous larvae displayed melanocyte phenotype similar to that of untreated wild type controls. From these results, we conclude that this compound interferes with EWS-FLI1 activity in the mitf:EWS-FLI1 homozygous zebrafish model. Further testing on Ewing Sarcoma cell lines will determine the potential significance of this compound as a specific inhibitor of tumor growth.