Abstract LT001: Exploiting the dependency on DDX19A to induce transcriptional catastrophe in Ewing sarcoma

Abstract Ewing sarcoma (ES) is an aggressive pediatric malignancy driven by the EWS/FLI1 oncofusion, which reprograms enhancers, enforces transcriptional reprogramming, and imposes high levels of replication stress. Multimodal therapy yields ∼70% survival in localized ES, but <30% in relapsed/metastatic disease, underscoring the need for new targets. As part of the Pediatric Dependency Map Accelerator (PedDep), a collaboration between St. Jude Children’s Research Hospital, Dana-Farber Cancer Institute, and the Broad Institute, we analyzed CRISPR/Cas9 screens across 27 ES models to prioritize tumor-selective dependencies, identifying the DEAD-box helicase DDX19A as a top specific dependency in EWS/FLI-driven tumors. DDX19A is a conserved RNA helicase with roles in mRNA export and RNA remodeling, but its role in cancer remains unexplored. Using CRISPR knockout and degron-based systems, we validated DDX19A as essential for ES survival. Acute DDX19A loss caused rapid cytotoxicity, with near-complete cell death by 96 hours. Mechanistic studies showed within 24 hours there is a global increase in chromatin accessibility (ATACseq), gain of poised enhancers (H3K27ac-ChIPseq), along with a widespread transcriptional upregulation (RNAseq). These changes are consistent with a background already marked by elevated R-loops and replication stress driven by EWS/FLI1. DDX19A depletion further increased R-loop accumulation, triggering DNA damage, checkpoint activation, G2/M arrest by 72 hours and apoptosis. These data suggest that DDX19A acts as a transcriptional gatekeeper, constraining oncogenic overload and maintaining transcriptional homeostasis. Loss of this function destabilizes ES cells, producing transcriptional catastrophe and cell death. Importantly, DDX19A represents a druggable helicase, opening translational opportunities. In collaboration with the Cupido laboratory in Chemical Biology and Therapeutics at St. Jude, we are testing first-in-class small-molecule inhibitors of DDX19A in vitro and in ES xenograft models in vivo to assess efficacy. Ongoing studies focus on mapping EWS/FLI1 occupancy relative to DDX19A-dependent chromatin changes, dissecting cell death and replication stress pathways, and evaluating DDX19A inhibitors as single agents and in combination with DNA damage response modulators. In conclusion, our findings establish DDX19A as a selective and druggable vulnerability in ES. Targeting this transcriptional gatekeeper highlights a novel way to exploit transcriptional balance in cancer, leading to a new paradigm of therapeutic transcriptional overload in transcription factor-driven malignancies such as ES. Citation Format: Saikat Chakraborty, Victoria Akingbehin, Daniel Estevez. Prado, John Harper, Abigail Fish2, Tommaso Cupido, Lillian M. Guenther. Exploiting the dependency on DDX19A to induce transcriptional catastrophe in Ewing sarcoma [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 LT001.