PROJECT SUMMARY Pediatric cancer is the leading cause of disease-related death in children, yet very few drugs are specifically labeled for pediatric malignancies, underscoring a need to identify novel molecular therapeutic targets to improve outcomes for children with cancer, which is our long-term goal. Specifically, diffuse pediatric high- grade gliomas (pHGGs) are resistant to multi-modal treatment and have had no new FDA-approved drugs in the past 20 years, thus patients with these tumors are in urgent need of novel, effective therapeutic strategies. Aberrant splicing contributes to neoepitope formation and represents a class of untapped targetable genetic alterations that are largely unexplored in pHGG. Our central hypothesis of this research plan is that aberrant splicing events can result in tumor-specific neoepitopes in pHGGs and these data can be rapidly harnessed and prioritized for therapeutic targeting. The proposed work will test this hypothesis with two integrated specific aims: 1) identify putative immunotherapeutic subtype-specific splice targets in pHGGs and 2) characterize aberrant splice variation in pHGG preclinical models and validate immunotherapeutic splice targets for preclinical testing. These studies will integrate transcriptional splice events with tumor tissue expression (PBTA and Kids First X01), normal tissue expression (GTEx and available pediatric matched tissue normals), peptide sequences (UniProt), and known extracellular domain annotations (UniProt) to identify and prioritize neoepitopes generated in pHGGs. This work will elucidate novel splice-driven immunotherapeutic targets through rigorous integrative computational analysis of splice variation in pHGG tumors, coupled with orthogonal molecular assays, to validate presence and expression of these targets. The successful completion of this project will generate significant new knowledge of aberrant splicing programs in pHGG and will identify potential immunotherapeutic targets. This work is critical to understanding the genetic contributions of aberrant splicing to pediatric cancer, will enable the research and clinical communities to rationally inform novel immunotherapeutic strategies for pHGG, and will serve as a roadmap for investigation of neoepitopes in other pediatric brain tumors. This work is highly relevant to the critical mission of the National Cancer Institute to advance scientific knowledge and identify novel strategies to improve overall survival of cancer patients.

Project Narrative Pediatric diffuse high-grade gliomas are the leading cause of death by central nervous system tumor and are in urgent need of novel therapies. The proposed research project is relevant to public health because it will generate significant new knowledge by identifying and prioritizing tumor-specific neoepitopes generated through aberrant splicing. This research is highly relevant to the NIH mission and the critical unmet need of accelerating time to identification of novel therapeutic targets and developing rational evidence-based strategies to reduce the health burden of pediatric cancer.