HNRNPH1-dependent splicing of a fusion oncogene reveals a targetable RNA G-quadruplex interaction

The primary oncogenic event in ∼85% of Ewing sarcomas is a chromosomal translocation that generates a fusion oncogene encoding an aberrant transcription factor. The exact genomic breakpoints within the translocated genes, EWSR1 and FLI1, vary; however, in EWSR1, breakpoints typically occur within introns 7 or 8. We previously found that in Ewing sarcoma cells harboring EWSR1 intron 8 breakpoints, the RNA-binding protein HNRNPH1 facilitates a splicing event that excludes EWSR1 exon 8 from the EWS–FLI1 pre-mRNA to generate an in-frame mRNA. Here, we show that the processing of distinct EWS–FLI1 pre-mRNAs by HNRNPH1, but not other homologous family members, resembles alternative splicing of transcript variants of EWSR1. We demonstrate that HNRNPH1 recruitment is driven by guanine-rich sequences within EWSR1 exon 8 that have the potential to fold into RNA G-quadruplex structures. Critically, we demonstrate that an RNA mimetic of one of these G-quadruplexes modulates HNRNPH1 binding and induces a decrease in the growth of an EWSR1 exon 8 fusion-positive Ewing sarcoma cell line. Finally, we show that EWSR1 exon 8 fusion-positive cell lines are more sensitive to treatment with the pan-quadruplex binding molecule, pyridostatin (PDS), than EWSR1 exon 8 fusion-negative lines. Also, the treatment of EWSR1 exon 8 fusion-positive cells with PDS decreases EWS–FLI1 transcriptional activity, reversing the transcriptional deregulation driven by EWS–FLI1. Our findings illustrate that modulation of the alternative splicing of EWS–FLI1 pre-mRNA is a novel strategy for future therapeutics against the EWSR1 exon 8 containing fusion oncogenes present in a third of Ewing sarcoma.