PROJECT SUMMARY Lineage-specific alternative splicing, where splicing occurs in a tissue-regulated manner involving evolutionary conserved alternative exons, has a determinative role in brain development. The role of tissue-specific alternative exons in malignant transformation and tumor development is undefined. The impact of lineage- specific splicing on glioblastoma (GBM) pathogenesis remains unclear but is conceptually attractive given the prevalence of this process in determining the fate of ancestral cells of potential GBM origin. We found that brain-enriched splice factor PTBP1 mediates lineage-specific alternative splicing of the ANXA7 tumor suppressor gene. PTBP1 is expressed in neural and glial precursor cells (NPCs/GPCs) and binds ANXA7 pre-mRNA to skip exon 6; this produces spliced ANXA7 isoform 2 (I2). During lineage specification, PTBP1 is downregulated and this allows the expression of unspliced ANXA7 I1 (I1), which includes exon 6. We determined that the patterned expression of the I2 splice variant in the brain is restricted to lineages that represent potential GBM cells of origin but that I1 is virtually absent in these cells. Our preliminary data illustrate that lineage-specific splicing can augment genetic mechanisms to deregulate oncogenic pathways. Specifically, we showed ANXA7 I1, but not I2, targets oncogenic receptor tyrosine kinases (RTKs) such as EGFR, MET and PDGFRA for endosomal degradation. In the adult brain, PTBP1 is nearly absent; in GBM, PTBP1 aberrantly persists and RTK levels are elevated. However, we demonstrate that upon PTBP1 knockdown and/or, ANXA7 I1 re-expression, RTK signaling is reduced, and GBM tumorigenicity is diminished. Therefore, we hypothesize that GBMs benefit from persistent PTBP1 expression as this suppresses ANXA7 I1, prevents RTK endosomal degradation, and sustains RTK signaling in GBM. Our Specific Aims are: 1. Define the Mechanism by which ANXA7 I1 Regulates Endosomal and Cellular Trafficking of EGFR. Herein, we will determine the detailed mechanism by which ANXA7 I1 interacts with and regulates endosomal and extracellular vesicle transport of EGFR as a prototype RTK. 2. Demonstrate ANXA7 I1 is a Signaling Modulator for Multiple RTKs and RTK-Targeted Therapies by Perpetuating Endosomal Degradation. Herein, we will demonstrate that ANXA7 I1 is competent to attenuate signaling through multiple pro-tumorigenic RTKs, modulate TKI response, and thus has a broad impact on GBM in vivo. These mechanistic and focused aims are needed to better define the role of lineage-specific splicing processes in tumorigenesis, which remains understudied. Our results will determine that ANXA7 I1 is sufficient to simultaneously inhibit signaling by multiple RTKs and thus holds potential to reveal a new therapeutic direction. This work will define the role of aberrant lineage-specific alternative splicing in GBM pathogenesis, offer a broader understanding of this process in malignant transformation, and provide a ...