Role of POT1 mutations in glioma initiation

项目总结 弥漫性胶质瘤构成了大多数的恶性原发脑肿瘤，通常是致命的。在探索中 为了更好地了解它们的生物学，在过去的十年里已经发现了许多与胶质瘤相关的突变。 其中，我们已经证明POT1的胚系突变与神经胶质瘤的风险增加有关。 发展。已知POT1结合和保护端粒，其他癌症中的几个POT1突变是 已知会导致端粒延长。然而，目前尚不清楚胶质瘤相关的POT1是否 突变可以导致端粒延长，更根本的是，连接生殖系POT1的机制 突变导致胶质瘤的发生尚不清楚。该提案的总体目标是确定POT1如何 突变促进了胶质瘤的发生。我假设POT1突变通过(1)驱动细胞 增殖和(2)使端粒延长和绕过复制衰老。我们计划使用Glial 祖细胞(GPC)作为胶质瘤起始细胞的模型。我们将使用鼠标GPC来解决第一个问题 假设，因为小鼠允许活体建模，而人类GPC解决第二个假设，作为人 细胞更容易发生复制性衰老。我的第一个具体目标是量化 小鼠POT1突变表达或缺失在体内GPC增殖和命运中的作用 建立小鼠子宫电穿孔模型，以及体外实验，在不同时间点进入成年期。我们还将 使用选择性启动子驱动基因表达来评估这些对谱系受限的GPC的影响。我的 第二个特定的目标是通过以下方法确定POT1突变在复制衰老旁路中的作用 人类的GPC。我们将评估POT1突变表达或POT1缺失对端粒长度和 人类GPC的人口倍增能力。我们还将量化这些细胞在体内的肿瘤形成潜力 抑制P53和激活RAS通路的异种移植模型。这些研究将有助于揭示 POT1突变在脑胶质瘤发生中的作用。从长远来看，我打算利用这一知识进一步获得 洞察胶质瘤的端粒生物学机制并将其脆弱性转化为潜在的 治疗措施。我设想自己的职业生涯是一名研究生物学的独立内科医生兼科学家。 神经胶质瘤遗传学与活跃的脑肿瘤外科实践专注于治疗神经胶质瘤患者。 为了实现这一目标，我已经确定了我的培训中需要解决的几个不足之处。这些 包括端粒生物学和基因工程技术知识以及实验台和实验室 管理技能。我计划通过向我的导师和同事以及 参加课程和会议。贝勒医学院和神经外科提供 一个非常肥沃的研究环境，我享受着我的导师、顾问和部门的全力支持 椅子。这份计划书中概述的计划将为我提供成功完成我的 建议的研究以及作为独立调查者竞争研究资金的记录。

PROJECT SUMMARY Diffuse gliomas comprise the majority of malignant primary brain tumors and are commonly fatal. In a quest to better understand their biology, many glioma-associated mutations have been identified over the past decade. Among these, we have shown that germline mutations in POT1 are associated with increased risk of glioma development. POT1 is known to bind and protect telomeres, and several POT1 mutations in other cancers are known to result in telomere elongation. However, it is not known whether the glioma-associated POT1 mutations can cause telomere elongation, and more fundamentally, the mechanisms linking germline POT1 mutations to glioma initiation are not understood. The overall goal of this proposal is to determine how POT1 mutations facilitate glioma initiation. I hypothesize that POT1 mutations accomplish this by (1) driving cell proliferation and (2) enabling telomere elongation and bypass of replicative senescence. We plan to use glial progenitor cells (GPCs) as a model for glioma initiating cells. We will use mouse GPCs to address the first hypothesis, as mice allow in vivo modeling, and human GPCs to address the second hypothesis, as human cells are more readily capable of replicative senescence. My first Specific Aim seeks to quantify the effects of POT1 mutant expression or loss of mouse POT1 orthologs in GPC proliferation and fate in vivo, using an in utero electroporation mouse model, as well as in vitro, at various time points into adulthood. We will also assess these effects in lineage-restricted GPCs using selective promoters to drive gene expression. My second Specific Aim seeks to establish the role of POT1 mutations in replicative senescence bypass using human GPCs. We will assess the effects of POT1 mutant expression or POT1 loss on telomere length and population doubling capacity of human GPCs. We will also quantify the tumorigenesis potential of these cells in a xenograft model upon inhibition of p53 and activation of Ras pathways. These studies will help to reveal the role of POT1 mutations in glioma initiation. In the long term, I intend to leverage this knowledge to gain further insight into the mechanisms of telomere biology in glioma and translate its vulnerabilities into potential therapeutic measures. I envision a career for myself as an independent physician-scientist studying the biology and genetics of gliomas with an active brain tumor surgical practice focused on treating patients with glioma. To reach this goal, I have identified several areas of deficiency in my training that require addressing. These include knowledge of telomere biology and genetic engineering techniques as well as bench and laboratory management skills. I plan to address these deficiencies by learning from my mentor and colleagues as well as participation in courses and conferences. Baylor College of Medicine and Department of Neurosurgery provide a highly fertile environment for research, and I enjoy the full support of my mentor, advisors, and Department Chair. The program outlined in this proposal will provide me with the training to successfully complete my proposed studies as well as the track record to compete for research funding as an independent investigator.