RNA Helicase A is a functional partner of EWS-FLI1 in Ewing’s sarcoma family of tumors

4327 RNA helicase A (RHA), a member of the DexD/H box helicase family of proteins, is an integral component of the human transcriptosome. RHA has not previously shown to interact with tumor specific oncogenes. Ewing’s sarcoma Family of Tumors (ESFT) are most often characterized by a chromosomal translocation t(11;22) that results in an aberrant transcription EWS-FLI1. Elimination of EWS-FLI1 protein from ESFT cells induces apoptosis and reduces xenograft tumor growth. We identified RHA as a novel partner for EWS-FL1 by unbiased phage display screening. We hypothesized that the interaction of RHA with the EWS-FLI1 results in a potent transcriptional activator/coactivator complex amplifying the functions of both proteins and together drive the malignant phenotype of ESFT. We produced and purified recombinant EWS-FLI1. EWS-FLI1 screening of a phage display library identified phage that bound to EWS-FLI1 and the tail proteins of those phage were sequenced. Co-immunoprecipitation experiments were performed with EWS and FLI1 antibodies. The direct binging of RHA and EWS-FLI1 was tested by ELISA. Chromatin immunoprecipitation (ChIP) assay was performed in crosslinked TC32 and TC71 cells with antibodies recognized either RHA or FLI1. Decrosslinked chromatin was then subject to PCR with primers amplifying genomic region in the PTPL1 promoter. Transformation assays were performed in primary mouse fibroblasts that stably expressed low levels of EWS-FLI1 and were subsequently transfected with RHA and selected with G418 prior to being placed in soft agar. We found RHA occurring in a complex with endogenous EWS-FLI1 in ESFT cells by co-immunoprecipitation using both EWS and FLI1 antibodies. ChIP using antibodies to both RHA and FLI1 followed by PCR demonstrated that both proteins exist on the genomic region of the PTPL1 promoter. EWS-FLI1 regulated promoter activity was increased an a dose dependent fashion using co-transfected RHA along with endogenous Id2 promoter (3-fold increase) and the synthetic GGAA promoter (15-fold increase) in ESFT cell lines. Three independent transfections of mouse embryonic fibroblasts, stably expressed low levels of EWS-FLI1, with RHA or empty vector control were performed followed by soft agar colony assays. A 33% increase in colony formation was reproducibly seen in cells expressing both EWS-FLI1 and RHA. RHA appears to interact with EWS-FLI1 directly. Our studies demonstrate enhanced EWS-FLI1 function when RHA levels are increased. Ongoing studies are evaluating the impact of EWS-FLI1 upon RHA helicase and ATPase function. Further characterization of these interactions might allow for development of novel targeted therapeutic agents for anti-tumor activity.