Abstract 479: Inhibition of the splicing of the EWS-FLI1 fusion transcript reverses EWS-FLI1 driven oncogenic expression in Ewing sarcoma
Authors: Grohar Patrick J. , Kim Suntae , Haddock Sara , Rangel Rivera Guillermo , Harlow Matt , Maloney Nichole K. , Huppi Konrad , Gehlhaus Kristen , Grandin Magdalena , Klumpp-Thomas Carleen , Buehler Eugen , Helman Lee J. , Martin Scott E. , Caplen Natasha J. 
Published: August 2015

Abstract
Ewing sarcoma (ES) is a highly aggressive cancer of the bone and soft tissue. In ∼85% of ES tumors the primary oncogenic event is a t(11:22)(q24:q12) translocation that generates a fusion of the 5′ end of EWSR1 and the 3′ end of FLI1 referred to as EWS-FLI1. The exact genomic breakpoints within the EWSR1 and FLI1 genes vary, but typically occur within introns and require the splicing machinery to generate an in-frame EWS-FLI1 transcript. The most common EWS-FLI1 transcripts fuse either exon 7 of EWSR1 to exon 6 of FLI1 (a type I or a 7/6 fusion), or fuse exon 7 of EWSR1 to exon 5 of FLI1 (a type II or 7/5 fusion). In an estimated 40% of EWS-FLI1 driven tumors the generation of an in-frame EWS-FLI1 fusion transcript requires alternative splicing. In particular, translocations that retain exon 8 of EWSR1 generate an out-of-frame transcript unless this exon is removed. Using an assay of EWS-FLI1 activity and genome-wide siRNA screening we have identified RNA processing as a therapeutic vulnerability in ES.
Parallel genome-wide siRNA-mediated RNAi screens were conducted in ES TC32 cell lines expressing a luciferase (luc) reporter protein driven by either the promoter of the EWS-FLI1 target gene NR0B1 (TC32-NR0B1-luc) or the CMV promoter (TC32-CMV-luc). The top gene ontology terms associated with the 28 priority candidate genes that when silenced induced a differential decrease in the TC32-NR0B1-luc signal versus the TC32-CMV-luc signal were mRNA splicing (p-value = 1.42E-08) and mRNA processing (p-value = 2.32E-07). To investigate the mechanistic basis for the identification of specific RNA processing proteins as required for the activity of EWS-FLI1 we focused on two lead candidate genes, the heterogeneous nuclear ribonucleoprotein H1, HNRNPH1, and the core splicing factor, SF3B1. Using PCR analysis we determined that HNRNPH1 is required for the splicing of EWS-FLI1 fusion transcripts expressed in ES cells in which the chromosome 22 breakpoint retains EWSR1 exon 8, specifically in TC32 and SKNMC ES cells. We also show ES cell lines harboring 7/ 6 (TC32, SKNMC, and TC71) or 7/ 5 (RD-ES) EWS-FLI1 fusions are all sensitive to the loss-of-function of SF3B1. Quantitative RT-PCR, immunoblot, and whole transcriptome analysis show that disrupted splicing of the EWS-FLI1 transcript alters its expression and reverses the expression of a significant proportion of genes that are targets of EWS-FLI1. These observations were confirmed in four ES cell lines using the splicing inhibitor Pladienolide B.
Our results provide the basis for a novel strategy to target fusion oncogenes by interfering with RNA processing. This study has implications for the treatment of ES through inhibition of proteins required for expression of the EWS-FLI1 transcript and identifies a candidate lead compound for further clinical development. Our findings may also open up strategies for treatment of other cancers driven by fusion oncogenes.
Citation Format: Patrick J. Grohar, Suntae Kim, Sara Haddock, Guillermo Rangel Rivera, Matt Harlow, Nichole K. Maloney, Konrad Huppi, Kristen Gehlhaus, Magdalena Grandin, Carleen Klumpp-Thomas, Eugen Buehler, Lee J. Helman, Scott E. Martin, Natasha J. Caplen. Inhibition of the splicing of the EWS-FLI1 fusion transcript reverses EWS-FLI1 driven oncogenic expression in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 479. doi:10.1158/1538-7445.AM2015-479