Abstract 520: Functional genomic screens identify microRNA regulators of the oncogenic fusion transcription factor EWS-FLI1

Abstract

Abstract Ewing sarcoma (ES) is a highly aggressive malignant pediatric cancer of the bone and soft tissue with a poor prognosis. The genetic hallmark of ES is the t(11:22)(q24:q12) translocation that generates a fusion transcript containing the 5’ end of EWSR1 and the 3’ end of FLI1 referred to as EWS-FLI1. This fusion generates an oncogenic transcription factor, EWS-FLI1 that is responsible for malignant transformation and is necessary for ES cell survival. To enhance our understanding of the molecular mechanisms underlying ES, with the long-term aim of enhanced treatment for this cancer, we are applying RNAi functional genomic approaches. MicroRNAs (miRNAs) are critical regulators of gene expression. There is evidence that many transcription factors and miRNAs act in concert to regulate gene expression. To identify miRNAs that directly or indirectly modulate EWS-FLI1 activity we conducted parallel screens of mimics or inhibitors of the human miRome using a cell-based reporter assay. The reporter assay consists of two isogenic reporter cell lines generated in the EWS-FLI1 expressing TC32 cell line. One line harbors a luciferase reporter driven by the promoter of an established EWS-FLI1 target NR0B1, while the second line expresses luciferase from a constitutive CMV promoter. A comparison of the effects of a miRNA on these two reporter constructs can identify proteins that specifically modulate EWS-FLI1 activity. We first identified those miRNAs for which a differential effect was seen when the NR0B1- and CMV-luciferase signals were compared and prioritized those where the mimic and inhibitor corresponding to a specific miRNA exhibited opposite effects. Twenty-eight miRNAs were identified, including miR-145, an established regulator of EWS-FLI1. The screen also identified miRNAs that have not been linked to EWS biology. One such candidate is miR-200b, a known regulator of epithelial-mesenchymal transition and tumor progression. We have confirmed that biotinylated-miR-200b captures the EWS-FLI1 mRNA when transfected into TC32 and ∼300 other mRNAs that includes EWS-FLI1 target genes. This functional genomic approach gives us an opportunity to investigate the co-regulatory activity of an oncogenic transcription factor and miRNAs in a systematic fashion that could be extended to investigate the co-regulation of other transcription factors and miRNAs. Citation Format: Suntae Kim, Patrick J. Grohar, Carleen Klumpp, Ashish Lal, Scott E. Martin, Lee J. Helman, Natasha J. Caplen. Functional genomic screens identify microRNA regulators of the oncogenic fusion transcription factor EWS-FLI1. [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 520. doi:10.1158/1538-7445.AM2014-520