Abstract B15: Different structural features of EWS are required for distinct modes of EWS/FLI-mediated oncogenic gene regulation
Authors: Theisen Emily R. , Miller Kyle R. , Lessnick Stephen L. 
Published: February 2019

Objective: This study aims to define the biochemical characteristics of EWS required for distinct modes of EWS/FLI-mediated gene regulation. EWS/FLI is the fusion protein and oncogenic transcription factor that drives Ewing sarcoma. While EWS/FLI is well characterized as a transcriptional activator, both gene activation and repression are necessary for EWS/FLI-mediated oncogenesis. Whether EWS/FLI-mediated repression is active or passive is unknown. We previously demonstrated that recruitment of the nucleosome modeling and deacetylase (NuRD) and lysine specific demethylase 1 (LSD1) complex is important for repression, supporting an active model. Targeted inhibition of either the histone deacetylase (HDAC) activity of NuRD or LSD1 impairs repression. Interestingly, inhibition of LSD1, but not HDACs, also disrupts gene activation, suggesting different protein complexes dictate different modes of transcriptional regulation. We hypothesize that EWS/FLI-mediated activation and repression result from separate active molecular mechanisms driven by distinct elements within the EWS domain. Prior studies using a deletion mutant strategy failed to identify a mutant that activated target genes, but did not repress, or vice versa.

Methods: Exploring EWS structure/function relationships is limited by its disordered and repetitive nature. We used a strategy in which conserved tyrosine residues in repetitive regions are mutated to alanine. This mutant, DAF, was assayed for its ability to regulate key EWS/FLI targets and transformation using a knockdown-rescue approach with subsequent qRT-PCR, RNA-seq, and agar assays. We also evaluated the function of DAF in microsatellite response element in reporter assays.

Results: DAF is the first mutant profiled that partially rescues EWS/FLI function. We observe DAF is capable of activating microsatellite-driven target genes, including NR0B1, CAV1, and GSTM4, while failing to repress IGFBP3, LOX, and TGFBR2. RNA-seq studies largely confirm this. Rescue of EWS/FLI knockdown with DAF expression fails to rescue the growth of A673 cells in soft agar, demonstrating DAF is incapable of transformation. In luciferase assays, DAF activates gene expression from the NR0B1 microsatellite.

Conclusions: We conclude DAF separates microsatellite-driven gene activation from other modes of EWS/FLI-mediated gene regulation. Whether this difference is due to altered DNA binding or recruitment of protein interaction partners is unknown. Future studies are under way to investigate these questions. The DAF mutant thus represents an important tool to dissect the molecular requirements for distinct mechanisms of EWS/FLI function in order to identify novel therapeutic liabilities in Ewing sarcoma.

Citation Format: Emily R. Theisen, Kyle R. Miller, Stephen L. Lessnick. Different structural features of EWS are required for distinct modes of EWS/FLI-mediated oncogenic gene regulation [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B15.