Identifying genetic vulnerabilities in KIAA1549-BRAF mutant pediatric low-grade gliomas

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Project Summary: Pediatric low-grade gliomas (pLGGs) typically harbor only a single oncogenic driver event which almost universally result in MAPK pathway activation. The most frequent of these alterations is a rearrangement that fuses the C-terminus of BRAF to the large transmembrane protein KIAA1549. Many patients whose tumors harbor this fusion respond to BRAF inhibitors, but challenges remain in extending clinical benefit to all patients. A key challenge is that patients require chronic treatment with these inhibitors throughout childhood because tumors often rebound upon cessation of treatment. These inhibitors are associated with significant dose-limiting toxicity issues, likely because the MAPK pathway is essential for brain development. These observations suggest that we need to identify new therapeutic targets for pLGG. The two core members of the POMT complex and two complex cofactors emerged as the top four dependencies in a genome-scale loss of function CRISPR/Cas9 screen. This dependency was specific for cells harboring the KIAA1549-BRAF fusion and were not dependencies in cells expressing BRAFV600E. The POMT complex is necessary for O-mannosylation of secreted and transmembrane proteins. This is striking because across all proteins in the human proteome, KIAA1549 one of the proteins most affected by this post-translational modification. It was originally hypothesized that the KIAA1549-BRAF fusion activates BRAF by truncating its negative regulatory domain, but this observation suggests that the KIAA1549 portion of the fusion may also be necessary for activating BRAF. The primary goals of this proposal are to understand why the POMT complex is a vulnerability in KIAA1549- BRAF mutant cells, and to understand how the KIAA1549 portion of KIAA1549-BRAF regulates the activity of the fusion. In Aim 1 I will test the hypothesis that POMT complex activity is necessary for the survival of KIAA1549-BRAF-dependent cells. In Aim 2 I will test the hypothesis that the POMT complex is required for O- mannosylation of KIAA1549-BRAF, and that O-mannosylation of KIAA1549-BRAF is necessary for oncogenic signaling. In Aim 3 I will test the hypothesis that the KIAA1549 portion of the KIAA1549-BRAF fusion tethers BRAF to the plasma membrane and enhances its ability to activate the MAPK pathway.