Abstract A010: GGAA Repeat Binding by EWSR1/FLI1 Drives Chromatin Destabilization: A Mechanistic and Drug Discovery Platform

Abstract Ewing sarcoma is driven by the EWSR1–FLI1 fusion oncoprotein, a transcription factor created by chromosomal translocation in otherwise low-mutation tumors. EWSR1–FLI1 recruits chromatin-remodeling complexes, opens chromatin, and establishes broad H3K27ac/H3K4me1 domains. Uniquely, it binds normally silent GGAA microsatellites dispersed across the genome and converts them into de novo enhancers, elevating transcription of downstream targets. We hypothesize that EWSR1–FLI1 engagement of GGAA repeats globally destabilizes chromatin and enforces a differentiation block, and that direct inhibition of its DNA binding will restore transcriptional control. To test this, we established a propidium iodide (PI) uptake assay after exposure to the chromatin-damaging agent CBL0137 as a surrogate for chromatin accessibility, benchmarking ES against non-ES tumor and non-tumor cells, and in parallel engineered a DNA-binding–dependent reporter to screen small molecules that disrupt fusion–DNA interactions. ES cells showed greater PI uptake following CBL0137 than controls, consistent with globally relaxed chromatin. The reporter screen identified candidate compounds that decreased fusion-dependent activity in a dose-responsive manner; orthogonal luciferase assays supported potential on-target inhibition, and viability assays revealed selective toxicity in ES relative to fusion-negative sarcomas. These findings support a model in which fusion oncoproteins promote chromatin relaxation and establish a complementary screening platform that nominates direct fusion inhibitors, providing mechanistic insight and therapeutic starting points for pediatric sarcomas. Citation Format: Ayah Salameh. GGAA Repeat Binding by EWSR1/FLI1 Drives Chromatin Destabilization: A Mechanistic and Drug Discovery Platform [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr A010.