Abstract A39: Alternative splicing in Ewing sarcoma may be driven by phase separation of spliceosome proteins
Authors: Toretsky Jeffrey , Graham Garrett , Dirksen Uta , Erkizan Hayriye Verda , Lawlor Elizabeth , Uren Aykut , Selvanathan Saravana 
Published: May 2016

Abstract
Alternative splicing has been implicated as an oncogenic process and provides both a categorization of cancer as well as an opportunity for more effective targeted treatments. Spliceosomal network interactions, including proteins that recognize splice enhancer and silencer regions, are critical for the regulation of alternative splicing, leading to oncogenic protein isoforms. Many proteins that participate in altering splicing have regions of low complexity amino acids, such as serine-arginine (SR) repeats. These SR repeats cause these proteins to have unique biophysical properties leading to phase separation and putatively formation of speckles in the nucleoplasm. EWS-FLI1 has low complexity regions and multiple validated direct interactions with spliceosome components. In addition, our work shows that EWS-FLI1 has novel direct binding to mRNA. We hypothesize that EWS-FLI1, the Ewing sarcoma (ES) oncoprotein, modulates post-transcriptional gene regulation through both novel protein interactions (trans-acting factors) and by directly binding to transcribed mRNA (cis-elements). Our initial results led us to build a novel EWS-FLI1 protein interaction model that identifies both direct and indirect binding throughout the spliceosome. Specific protein interactions of EWS-FLI1 include a newly discovered direct binding partner, DDX5, which is part of the U1 subunit. In addition, direct interactions with both U2 and U5 were identified. We have validated a small molecule probe, YK-4-279, as an enantio-specific inhibitor of EWS-FLI1 that directly blocks its binding to both RHA and p68 (DDX5). Reduction of EWS-FLI1 and YK-4-279 treatment alters exon skipping and intron inclusion events identified from RNA-seq. We then identified splicing events that were similarly reverted by both EWS-FLI1 reduction and YK-4-279, including CLK1, PPFIBP1, and CASP3. In addition, YK-4-279 reverts alternative splicing changes seen in the presence of EWS-FLI1, which was not an effect of altering RNA pol II activity. Our recent unpublished work explores the biophysical interactions of EWS-FLI1 in nucleoplasm. Inhibition of EWS-FLI1 protein interactions by YK-4-279 appear to alter nuclear speckle formation and may, through this mechanism, lead to altered spliceosomal activity. To establish that EWS-FLI1 alternative splicing changes the function of a protein through expression of a different isoform, we selected TERT as an enzyme with an isoform shift in the presence of EWS-FLI1 . When EWS-FLI1 is reduced, a γ-isoform of hTERT is synthesized that lacks exon 11. The γ-isoform of hTERT is reported by others to have increased hTERT activity and we confirm this in cell-based assays following EWS-FLI1 reduction. The cellular and oncogenic implications of this enzymatic activity change is under investigation. To confirm the relevance of splicing modulated by EWS-FLI1 occurring in human tumors, we determined splicing patterns from 75 ES patients and matched splicing patterns for 10 genes in cell line models. Exon-specific expression loci in this comparison were statistically significant for their overlap. In addition, overall gene expression levels did not stratify for overall survival, however, principal component isoform specific analyses did segregate survivors. In metastatic patients, this was significant (p=0.05) while in localized patients it approached significance (p=0.1). This work illuminates potentially critical oncogenic splicing shifts as well as provide a potential stratification mechanism for ES patients. A splicing map specific to ES will define and inform potential functional aspects of alternative isoforms. The biophysical role of EWS-FLI1 in creating or maintaining speckles is an ongoing investigation. Assessment of aberrant splicing driven by EWS-FLI1 may inform oncogenesis and reciprocally, EWS-FLI1 activities may inform splicing mechanisms.
Citation Format: Jeffrey Toretsky, Garrett Graham, Uta Dirksen, Hayriye Verda Erkizan, Elizabeth Lawlor, Aykut Uren, Saravana Selvanathan. Alternative splicing in Ewing sarcoma may be driven by phase separation of spliceosome proteins. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A39.