Abstract 4494: HNRNPH1-dependent splicing of a fusion oncogene reveals a targetable RNA G-quadruplex interaction

Ewing sarcoma (EWS) is the second most common bone tumor in children and adolescents. The primary oncogenic event in ~85% of EWS is a chromosomal translocation that generates a fusion gene encoding the aberrant transcription factor EWS-FLI1. A third of EWS-FLI1 driven tumors harbor translocations that retain exon 8 of the EWSR1 gene, and in these tumors, skipping of this exon is vital to express an in-frame EWS-FLI1 mRNA. We have shown previously that the heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) binds to G-rich sequences within EWSR1 exon 8 and facilitates the exclusion of this exon from distinct EWS-FLI1 pre-mRNAs. Guanine-rich nucleic acids may fold into stable tertiary structures, such as RNA guanine quadruplexes (rG4s). Our current studies are focused on the tertiary RNA structure of the G-rich regions within EWSR1 exon 8 and its role in HNRNPH1-dependent RNA processing. 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 wild-type EWSR1. We demonstrate that guanine-rich sequences within EWSR1 exon 8 can fold into RNA G-quadruplex structures and that this structure favors the recruitment of HNRNPH1. Bioinformatic analysis of transcriptome-wide profiles for RNA G-quadruplexes and RNA targets of HNRNPH1 revealed sequences containing two-quartet G-quadruplex configurations are central for HNRNPH1 binding to exonic elements. Critically, we also demonstrate that the glycine-tyrosine-arginine-rich domain of HNRNPH1 is the minimal domain required for G-quadruplex recognition. Small molecules that can displace HNRNPH1 binding may provide a therapeutic vulnerability in a subset of Ewing sarcoma. To evaluate if this protein-RNA interaction is amenable to small molecules, we performed displacement assays with HNRNPH1 protein and an EWSR1 exon 8 RNA oligomer at varying concentrations of the pan-quadruplex binding molecule, pyridostatin (PDS). PDS disrupts the HNRNPH1-RNA complex with an IC50 of 7 micromolar. Furthermore, treatment with PDS selectivity inhibits the growth of EWS cells harboring EWSR1 exon 8 fusions, decreases EWS-FLI1 activity in cell-based reporter assays, and restores mRNA expression of EWS-FLI1 deregulated transcriptional targets. Our findings illustrate that splicing modulation of EWS-FLI1 pre-mRNA is a promising strategy for future therapeutics against this fusion oncogene expressed in a third of Ewing sarcoma. Citation Format: Carla Neckles, Robert Boer, Nicholas Aboreden, Robert L. Walker, Bong-Hyun Kim, Suntae Kim, John S. Schneekloth, Natasha J. Caplen. HNRNPH1-dependent splicing of a fusion oncogene reveals a targetable RNA G-quadruplex interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4494.