Abstract 2008: Ewing sarcoma cells harboring a translocation that retains EWSR1 exon 8 require HNRNPH1 to express the in-frame oncogenic fusion transcript EWS-FLI1
Authors: Kim Suntae , Olivero Christiane , Rangel Rivera Guillermo O. , Sen Nirmalya , Haddock Sara , Huppi Konrad , Helman Lee J. , Grohar Patrick J. , Caplen Natasha J. 
Published: July 2016

The primary oncogenic event in ∼85% of Ewing sarcomas is a t(11:22)(q24:q12) translocation. This translocation generates a fusion gene containing the 5’ end of the EWSR1 gene and the 3’ end of the FLI1 gene 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. 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. In this study, we demonstrate that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require HNRNPH1 to express an in-frame EWS-FLI1 mature mRNA.

Using a genome-wide RNAi screen, we identified several proteins involved in RNA processing as required for the activity of EWS-FLI1, including the heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1). The role of HNRNPH1 in alternative splicing led us to hypothesize that ES cells are dependent on HNRNPH1 for the expression of the EWS-FLI1 transcript. Analysis of the expression of EWS-FLI1 following HNRNPH1 silencing in Ewing sarcoma cell lines representing different translocation breakpoints and transcript isoforms showed that only Ewing sarcoma cell lines retaining EWSR1 exon 8 at a genomic level (TC32 and SKNMC cells) are dependent on HNRNPH1 expression. Silencing of HNRNPH1 in TC32 or SKNMC cells, results in the expression of an out-of-frame EWS-FLI1 transcript that cannot express the EWS-FLI1 oncogenic transcription factor. This leads to the reversal of expression of EWS-FLI1 gene targets and cell death. Ewing sarcoma cell lines that harbor a translocation upstream of EWSR1 exon 8 (TC71 and RD-ES) exhibit none of these molecular or phenotypic changes upon HNRNPH1 silencing.

We next employed an RNA pull-down and PCR strategy to identify putative binding sites for HNRNPH1 on the EWS-FLI1 pre-mRNA. This analysis showed enrichment for the binding of EWSR1 exon 8 by HNRNPH1. Towards the 3’ end of EWSR1 exon 8 we identified two G-rich sequences, a motif typically bound by HNRNPH1. To determine if HNRNPH1 binds one or both of these sites, we developed an in vitro protein-RNA-oligomer binding assay. This assay confirmed the binding of HNRNPH1 to both G-rich sites in EWSR1 exon 8. Current studies are focused on using the protein-RNA-oligomer binding assay to fully map the interaction of HNRNPH1 with sequences within EWSR1 exon 8 and understand the molecular mechanism to target it. These results demonstrate a sequence-specific, breakpoint-dependent vulnerability in Ewing sarcoma that has the potential to be exploited as a therapeutic target and suggests a novel strategy to target fusion oncogenes.

Citation Format: Suntae Kim, Christiane Olivero, Guillermo O. Rangel Rivera, Nirmalya Sen, Sara Haddock, Konrad Huppi, Lee J. Helman, Patrick J. Grohar, Natasha J. Caplen. Ewing sarcoma cells harboring a translocation that retains EWSR1 exon 8 require HNRNPH1 to express the in-frame oncogenic fusion transcript EWS-FLI1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2008.