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

项目总结： 儿童低级别胶质瘤(PLGG)通常只有一个致癌驱动事件，几乎 普遍导致MAPK通路激活。这些变化中最频繁的是重新排列， 将BRAF的C末端与大的跨膜蛋白KIAA1549融合。许多患者的肿瘤 这种融合对BRAF抑制剂有反应，但在将临床益处扩大到所有患者方面仍然存在挑战。 一个关键的挑战是，患者需要在整个童年期间使用这些抑制剂进行长期治疗，因为 停止治疗后，肿瘤通常会反弹。这些抑制剂与显著的剂量限制有关。 毒性问题，可能是因为MAPK通路对大脑发育至关重要。这些观察结果表明 我们需要为pLGG寻找新的治疗靶点。 POMT复合体的两个核心成员和两个复合协因子成为四大依赖项 在基因组规模的功能丧失CRISPR/Cas9筛查中。此依赖项特定于包含 KIAA1549-BRAF融合，在表达BRAFV600E的细胞中不依赖。POMT综合体是 分泌和跨膜蛋白O-甘露糖化所必需的。这很令人震惊，因为在所有 人类蛋白质组中的蛋白质，KIAA1549是受这种翻译后反应影响最大的蛋白质之一 修改。最初的假设是KIAA1549-BRAF融合通过截断其 负调控结构域，但这一观察表明融合的KIAA1549部分也可能是 激活BRAF所必需的。 这项提案的主要目标是了解为什么POMT复合体是KIAA1549- BRAF突变细胞，并了解KIAA1549-BRAF的KIAA1549部分如何调节 核聚变。在目标1中，我将检验POMT复合体活性对于人类生存所必需的假设 KIAA1549-BRAF依赖细胞。在目标2中，我将检验POMT络合物是O-所必需的假设 KIAA1549-BRAF的甘露糖化和KIAA1549-BRAF的O-甘露糖化是致癌所必需的 发信号。在目标3中，我将测试KIAA1549-BRAF融合系链的KIAA1549部分的假设 BRAF与质膜结合，增强其激活MAPK途径的能力。

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.