PROJECT ABSTRACT Glioblastoma (GBM) is the most malignant type of brain tumor, with a median survival time of 15 months. Despite advances in cancer survival for many malignancies, GBM survival rates still remain low and have not significantly changed over the last 30 years, emphasizing the urgent need for new treatment options. One of the common hallmarks of cancer is splicing perturbations and alternatively spliced genes are an interesting new source for potential diagnostic biomarkers and therapeutic targets. Current methodologies to characterize splicing in tumors using mRNA sequencing can only indicate that alternative exons are transcribed, but additional validation is needed to verify that these spliceforms produce functional proteins. Our preliminary results comparing paired tumor/normal samples indicate that alternative splicing in GBM affects many more genes than previously expected, both at the transcript and protein levels. Based on these data, we hypothesize that a set of alternatively spliced genes are involved in tumor initiation and GBM pathogenesis. In this proposal, we will use a combination of precision medicine approaches to detect and quantify tumor-specific alternative splicing events in GBMs. We will verify that these events produce distinct proteoforms using splicing-aware proteogenomics experiments. Finally, we will functionally characterize differentially expressed splice isoform candidates for their effects on growth, apoptosis, and invasion in patient-derived glioma stem cells. If successful, this work will lead to new understanding of the biological impact of novel GBM proteoforms, and potentially lead to novel approaches to treatment of GBM based on unique tumor antigens and molecular pathways.