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. Interrogation of complete protein networks remains challenging because it is difficult to modify single interactions while preserving overall network architecture. We hypothesized that EWS-FLI1, Ewing sarcoma (ES) oncoprotein, modulates post-transcriptional gene regulation through novel protein interactions. EWS-FLI1 has multiple connections to the spliceosome and reduction of EWS-FLI1 alters significant numbers of exon skipping and intron inclusion events identified from RNA-seq. We validated the effect of EWS-FLI1 on alternative splicing (AS) using putative oncogenic genes including CLK1, PPFIBP1, CASP3 and TERT. In the presence of EWS-FLI1, the γ-isoform of hTERT is expressed and RNA immunoprecipitation reveals EWS-FLI1 binding TERT transcripts. We have validated a small molecule probe, YK-4-279, as an enantio-specific inhibitor of EWS-FLI1 that directly disrupts both RHA and p68 (DDX5). 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 (as shown by BruDRB-seq). We also determined that splicing patterns from 75 ES patients match splicing patterns for 10 genes in cell line models supporting the clinical relevance of our findings. Further, while overall gene expression levels did not stratify for overall survival, principal component isoform specific analyses did segregate survivors from those who died. These experiments establish oncogenic aspects of splicing which are specific to cancer cells and thereby illuminate potentially oncogenic splicing shifts as well as provide a potential stratification mechanism for ES patients. Assessment of aberrant splicing driven by EWS-FLI1 may inform oncogenesis and reciprocally, EWS-FLI1 activities may inform splicing mechanisms.
Dr. Jeffrey Toretsky received his MD in 1988 from the University of Minnesota. He completed his pediatric residency at the Medical College of Virginia in 1991, and his pediatric oncology fellowship at the National Cancer Institute Pediatric Branch in 1997. Dr. Toretsky joined the faculty in the Department of Pediatrics at the University of Maryland at Baltimore and served from 1997 until 2002. In 2002, Dr. Toretsky was recruited to Georgetown where he was granted tenure in 2005 and achieved the rank of professor in 2011. He was inducted into the American Society of Clinical Investigation in 2007 and received the Burroughs-Wellcome Clinical Scientist Award in Translational Research in 2008.
Dr. Toretsky actively pursues research that will lead to new and more specific therapies for a very rare cancer, Ewing sarcoma. His work focuses on Ewing sarcoma, since the tumors contain a unique target that is not found in non-tumor cells. This unique target offers an opportunity to create new medicines that will more specifically eliminate tumor growth while sparing normal cells. In 2009, Dr. Toretsky and his team revealed the molecule called YK-4-279 that targets Ewing sarcoma with an article in the prestigious journal Nature Medicine. YK-4-279 has the potential to be a potent new strategy in the fight against not only Ewing sarcoma, but also other cancers and diseases with similar characteristics. Given the small numbers of Ewing sarcoma patients, Big Pharma (and many not so big) was uninterested in pursuing clinical development of YK-4-279. Therefore, in 2010, Dr. Toretsky founded TDP Biotherapeutics, Inc. in order to facilitate the drug development. Dr. Toretsky continues to be the principal investigator of his NIH-funded laboratory group, co-leads the Molecular Oncology Program of the Lombardi Comprehensive Cancer Center, and maintains a clinical practice of pediatric oncology.