Abstract A34: EWS/FLI regulates transcriptional activation in Ewing sarcoma via length dependent GGAA microsatellites

Abstract

Abstract The purpose of this study is to investigate how EWS/FLI transcriptionally activates gene targets via polymorphic GGAA microsatellites. Ewing Sarcoma is a pediatric bone malignancy initiated by a t(11;22) chromosomal translocation that produces the EWS/FLI oncoprotein. EWS/FLI transcriptionally activates and represses its target genes to mediate oncogenic reprogramming. Expression of its up-regulated targets correlates with EWS/FLI binding to associated GGAA microsatellites. These microsatellites show length polymorphisms, suggesting that microsatellite polymorphisms may have critical effects on EWS/FLI-responsiveness of key gene targets. For example, NR0B1 is necessary for EWS/FLI mediated oncogenic transformation, and we found a “sweet-spot” of 24-25 repeat length as optimal for EWS/FLI mediated transcriptional activity at NR0B1. The mechanism underlying this optimal length is unknown. We therefore explored the stoichiometry and binding affinity of EWS/FLI for different repeat lengths through biochemical studies combined with molecular modeling simulation. Our data demonstrate a complex relationship between microsatellite length and transcriptional activity. Fluorescence anisotropy studies demonstrate that FLI binding affinity is independent of GGAA microsatellite length. In contrast, the stoichiometry of protein to DNA binding increases in specific incremental patterns with increasing microsatellite repeats. Gel shift binding assays elucidate the minimal microsatellite length critical for binding. Overall our data suggests a model in which the DNA binding domain of multiple monomers of FLI function as independent binding units to facilitate transcriptional activity in a length-dependent fashion. We propose that GGAA microsatellites are necessary and sufficient for EWS/FLI-mediated oncogenic transformation and that repeat length affects optimal DNA binding stoichiometry and transcriptional activity. Citation Format: Kirsten M. Johnson, Kunal Gangwal, James Robertson, Thomas E. Cheatham, Stephen L. Lessnick. EWS/FLI regulates transcriptional activation in Ewing sarcoma via length dependent GGAA microsatellites. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A34.