Abstract Background: EWS-FLI1 requires the protein binding of RNA Helicase A (RHA) to enable oncogenic transformation. RHA is a DExH box RNA helicase family member. RHA has critical roles modulating transcription, splicing and translation. RHA also functions as a scaffolding protein in multi-protein complexes including BRCA1, DICER, EGFR, POL2R, TOPO2. Our previous work demonstrated that YK-4-279 inhibited the protein-protein interaction of RHA, with EWS-FLI1 and led to cellular apoptosis. The biochemical mechanism remained cryptic, so we investigated the effect of EWS-FLI1 upon the helicase activity of RHA. Methods: Full-length purified recombinant RHA from insect cells. Infrared-labeled double stranded RNA was used as a substrate to measure helicase activity of RHA. We included full-length recombinant EWS-FLI1 in to the helicase reaction to test the effect of EWS-FLI1. Results: RHA unwinds double stranded RNA in the presence of ATP with Km value of 5.322 nM. We also showed that RHA manifests reannealing activity of single stranded RNA to double stranded RNA in the absence of ATP. Unlike ADP, ATP and additionally the non-hyrolysable gamma-S-ATP prevent reannealing activity of RHA. A molecular modeling of RHA structure predicted the surface exposure of the region (between 823-832aa), which EWS-FLI1 interacts with. The function of this region is not well described but presumed to support helicase activity. EWS-FLI1 interferes RNA helicase activity of RHA in a dose dependent manner. Moreover, the reduction of RHA protein level exerted cytotoxic effect on Ewing Sarcoma cells. Decreased RHA protein level changed EWS-FLI1 and its target gene expression profile. IGFBP3, GRK5, p21, and PRR3 gene expression levels increased massively. RPLA1 and NR0B1 mRNA levels were also increased by moderately in these cells. The change in the profile may contribute to apoptosis. Conclusions: The purified recombinant RHA is very active in vitro helicase assays. We demonstrated that RHA's helicase activity might depend on ATP binding but not ATP hydrolysis. The protein partnering as well as posttranslational modification of RHA might modulate the helicase and/or reannealing activity of RHA, which may augment oncogenesis of EWS-FLI1. This is the first time that a protein partner reduces the helicase activity of RHA. Further experiments are ongoing that investigate whether YK-4-279 blocking of EWS-FLI1 binding to RHA will restore the helicase activity of RHA as part of it contribution to cell death. Citation Format: Hayriye Verda Erkizan, Kamal Sajwan, Maksymilian Chruszcz, Jeffrey Schneider, Sarah E. Gamble, Aykut Uren, Wladek Minor, Radhakrishnan Padmanabhan, Jeffrey Toretsky. EWS-FLI1 reduces RNA Helicase A activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2747. doi:10.1158/1538-7445.AM2013-2747