Abstract 1305: Structure-function mapping reveal FLI1 contributes more than the DNA-binding properties to drive Ewing sarcoma biology

Abstract

Objective: Ewing sarcoma (ES) is the second most common pediatric bone cancer. To address a blatant lack of advancement in treatment, it is of the utmost importance to understand the underlying biology driving disease and through these developments, we hope to better outcomes through bench-to-bedside projects. ES is driven by a chromosomal translocation, which produces a fusion oncoprotein known as EWS/FLI1. Though it is known that FLI1 contains a DNA-binding domain that is essential for protein function and disease formation, very little further research has been completed to understand precisely how it contributes to ES etiology and how we can disrupt these functions. Our goal is to complete structure-function mapping that allows an in-depth analysis of FLI1 domain contributions that ultimately may elicit new targets for treatment development. Methods: EWS/FLI1 cDNA was used to create several different versions of EWS/FLI1, including a full-length protein and two EWS/FLI1 proteins with truncated FLI1 domains. A ES cellular model was used to test functionality of these constructs. A variety of molecular biology and new sequencing techniques were employed to establish structure-function relationships of the FLI1 domain of EWS/FLI1, including luciferase reporter assays, soft agar assays, RNA-sequencing, CUT&RUN-sequencing, and ATAC-sequencing. Results: In vitro transcriptional activation assays revealed EWS/FLI1 containing only the DNA-binding domain of FLI1 (EWS/FLI1DBD) was sufficient to drive transcription, but this result did not translate to the ability to oncogenically transform ES cells in a soft agar assay. We used various sequencing techniques to understand the process that allows EWS/FLI1 to actually drive transformation. CUT&RUN-sequencing revealed that EWS/FLI1 binds various places throughout the genome and, surprisingly, that EWS/FLI1DBD was able to bind all the same places. RNA-sequencing showed that EWS/FLI1 was able to regulate gene expression at the majority of bound regions, but EWS/FLI1DBD starkly lacked this ability. These results caused us to question what role EWS/FLI1 has as a pioneer transcription factor and what happens between DNA-binding and gene regulation. Through ATAC-sequencing results, we expect to see that EWS/FLI1 has the ability to open closed chromatin regions, but that EWS/FLI1DBD will lack this ability. It is likely that EWS/FLI1 recruits various additional co-factors to open chromatin and through these constructs, we hope to identify more of these important proteins. Conclusions: We have revealed FLI1 has a novel role in EWS/FLI1 activity that goes beyond DNA-binding, as well as isolated the essential regions of FLI1 necessary for activity. Ultimately, we hope that these novel properties or interactions will provide new ways to target ES cells that can benefit patient outcomes. Citation Format: Megann A. Boone, Jesse C. Crow, Julia Selich-Anderson, Cenny Taslim, Emily R. Theisen, Stephen L. Lessnick. Structure-function mapping reveal FLI1 contributes more than the DNA-binding properties to drive Ewing sarcoma biology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1305.