Abstract Ewing sarcoma (ES) is the second most frequent pediatric bone tumor and still remains of poor prognosis especially for metastatic patients. Genetically, ES is characterized by a chromosomal translocation between EWSR1 and ETS family members (FLI1 in 85% of cases). This leads to the expression of EWS-FLI1 chimeric oncogene transcription factor. Aiming at identifying EWS-FLI1 regulated genes with potential therapeutic targets, a genome wide method was developed to rank these potential hits by combining Ewing sarcoma transcriptome and ChIPSeq data. Accordingly, 273 selected genes were further investigated using a siRNA approach for their impact on cell cycle phases and apoptosis using high throughput imaging methods. 133 of these genes displayed a phenotype and bioinformatics tools were used to connect these genes. NFkB turned out to be a major hub in this network and upstream activation pathways were further investigated. Interleukin-1 pathway may account for this observed effect and in vitro and in vivo experiments are currently on going to validate this hypothesis. Interestingly, preliminary results indicate that both tumor and tumor microenvironment are of prime importance for the activation of this pathway. Citation Format: Didier Surdez, Gautier Stoll, Franck Tirode, Karine Laud, Emmanuel Barillot, Olivier Delattre. High throughput screening highlights NFkB signaling in Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3989. doi:10.1158/1538-7445.AM2014-3989