Abstract B25: HNRNPH1-dependent splicing of the fusion oncogene EWS-FLI1 reveals a targetable RNA G-quadruplex interaction

Abstract

Abstract The primary oncogenic event in ~85% of Ewing sarcomas is a chromosomal translocation that generates a fusion gene encoding an aberrant transcription factor EWS-FLI1. In a third of EWS-FLI1 driven tumors, translocations that retain exon 8 of EWSR1 must exclude this exon to synthesize a protein-encoding mRNA. We have previously demonstrated that the heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) binds to G-rich sequences within EWSR1 exon 8 and that this binding event is required to express an in-frame EWS-FLI1 mature mRNA. G-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 rare 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 HNRNPH1 preferentially binds these RNAs when in a folded state. Critically, we also demonstrate that transfection of a single-stranded RNA oligomer corresponding to one of these G-rich sequences (rG1) reduces the expression of EWS-FLI1 mRNA and EWS-FLI1 protein and decreases its transcriptional activity. 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 (rG1) at varying concentrations of a G4-binding molecule, pyridostatin (PDS). We found that PDS disrupts the formation of the HNRNPH1-rG1 complex (IC50 of 7 micromolar). Furthermore, treatment with PDS selectivity inhibits the growth of Ewing sarcoma cell lines 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 modulation of the alternative splicing 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 E. Boer, Nicholas Aboreden, Allison M. Cross, Robert L. Walker, Bong-Hyun Kim, Suntae Kim, John S. Schneekloth, Natasha J. Caplen. HNRNPH1-dependent splicing of the fusion oncogene EWS-FLI1 reveals a targetable RNA G-quadruplex interaction [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B25.