Dissertation Defense: Garrett Graham
Candidate Name: Garrett Graham
Major: Tumor Biology
Advisor: Jeffrey A. Toretsky
Title: Cassette Exon RNA Splicing Altered in Ewing Sarcoma is Characterized by Higher Guanine-Cytosine Content and Lower Splice Site Strength Indicative of Intron-Definition Splicing
This dissertation details my work investigating RNA splicing in Ewing sarcoma (EwS). EwS is a pediatric cancer that typically presents at a mean of 15 years of age and is the second most common bone tumor in children and adolescents. While there have been substantial investigations into the mechanism of EwS oncogenesis, few of these findings have translated to improved therapeutics, and treatment of EwS, has largely remained static for the last 30 years. Patients that present with localized EwS have an approximately 70% 5-year survival rate whereas patients that present with metastatic EwS have only a 30% 5-year survival rate. New approaches to understanding the oncogenic process and potential vulnerabilities behind EwS are badly needed, as targeted therapeutics for EwS patients have the opportunity to drastically improve long-term outcomes and reduce systemic effects.
In this dissertation I present first the creation of a model to study EwS using bone marrow-derived human mesenchymal stem cells (hMSC-BM), as the lack of patient samples presents a substantial barrier to EwS research. I then describe my efforts to create a reference transcriptome using long-read sequencing that is more comprehensive than commonly used reference transcriptomes. I then use this transcriptome to quantify mRNA splicing in tumor samples, followed by a detailed analysis of sequence features associated with altered RNA splicing in EwS.
Here I also present an analysis of disrupted RNA splicing in EwS tumors, with a focus on the the sequence immediately surrounding splice sites. Motif enrichment of RNA-binding proteins indicates that splicing factors KHDRBS1, KHDRBS3, PCBP1, and RBMX may have a role in regulating RNA splicing observed in EwS. I further find that GC content, splice site strength, upstream intron length, and DNA methylation are identifying characteristics of altered splicing in EwS when compared to hMSC-BM. These features are indicative of intron-definition splicing and provides a framework for understanding the mechanism behind disrupted RNA splicing in EwS. This work presents a window into a vital component of EwS oncogenesis and may provide a path to targeted therapeutics in the future.