INVESTIGATION OF THE PROCESSING OF THE EWS-FLI1 FUSION TRANSCRIPT
Guillermo Rangel Rivera, Natasha Caplen.
Center for Cancer Research, National Cancer Institute, Bethesda, MD.
Ewing sarcomas (ES) are bone and soft tissue tumors affecting children. Most ES tumors (approximately 85%) harbor a translocation between chromosomes 22 and 11 [t(11:22) (q24;q12)] which results in the generation of the fusion oncogene EWS-FLI1. EWS-FLI1 codes for the oncoprotein EWS-FLI1, an aberrant transcription factor required for ES tumorigenesis. Recently, a genome-wide loss-of-function (LOF) RNAi screen identified several genes involved in RNA processing as required for EWS-FLI1 activity. Due to the complexity of EWS-FLI1 gene organization, we hypothesized that constitutive expression of specific RNA processing proteins are required for the expression of EWS-FLI1 and hence EWS-FLI1 activity. Understanding the mechanism by which RNA processing regulates EWS-FLI1 expression may reveal new druggable targets for ES. To this end, we validated genes from the primary RNAi screen using a combination of luciferase reporter assays, cell viability, and quantitative and qualitative PCR-based techniques in 4 different ES cell lines. Using a reporter assay of EWS-FLI1 activity and siRNA-mediated LOF, we identified XPA binding protein 2 (XAB2) as a lead candidate. In this preliminary study, silencing of XAB2 by RNAi leads to a 3.3-fold decrease in EWS-FLI1 mRNA and a decrease in EWS-FLI1 protein levels. ES cell viability decreased in a time-dependent manner by approximately 40 and 65%, 48 and 72 h, respectively, following XAB2 LOF. This data suggests silencing of XAB2 down regulates EWS-FLI1 expression, and thus decreases cell viability. However, the exact mechanism by which XAB2 regulates EWS-FLI1 expression is unknown and remains the subject of ongoing investigation.