Telomerase function in human tumor and stem cell biology

 描述（由申请人提供）：端粒酶在干细胞维持和肿瘤发生中的关键作用早已被认识到。人类多能干细胞具有活跃的端粒酶，因此具有长期的更新能力，但大多数人类体细胞缺乏端粒酶功能，因此更新能力有限。端粒酶缺陷导致骨髓衰竭、再生障碍性贫血和肺纤维化的患者会加速端粒缩短，从而导致这些组织衰竭。与此效果相反 端粒酶重新激活是大多数人类癌症增殖永生的基础。尽管对人类健康有这些重大影响，但我们对人类细胞调节端粒酶活性以确保组织稳态的分子机制及其功能障碍如何导致肿瘤发生缺乏了解。人体组织中端粒酶活性的自然调节以及端粒缩短对未转化的人体细胞的影响只能在原代人体干细胞系统中进行研究。直到最近，技术限制，特别是基因操作的低效，一直阻碍着人类干细胞作为研究工具的使用。我们通过使用位点特异性核酸酶来有效地对人类多能干细胞（hPSC）进行基因工程，从而克服了这一问题。这项技术使我们首次能够在基因定义的人类干细胞系统中研究人类端粒酶功能的两个关键调节事件：转录调节和端粒酶向端粒的募集。目标 1 中描述的实验使用基因工程 hPSC 来阐明干细胞中转录调节端粒酶的分子机制，以及端粒酶的表达如何在分化时被沉默。目标 2 中概述的实验将揭示控制端粒酶募集到端粒的机制，以及端粒结合蛋白在募集步骤后用于调节端粒酶活性的机制。这些实验将揭示人类干细胞如何建立端粒长度设定点，为人类组织再生提供足够的端粒储备，同时通过最终限制分化细胞谱系的增殖能力发挥肿瘤抑制机制的作用。在目标 3 中，我们将通过诱导替代端粒维持途径来鉴定导致不依赖端粒酶的永生化的遗传改变，少数未重新激活端粒酶表达的癌症使用该途径。 总而言之，这里描述的实验将使用基因定义的人类干细胞模型系统来阐明端粒维持途径中严格调控的步骤，并从机制上理解该途径中的突变如何促进癌症形成。这种完整的机制理解将为端粒维持抑制开辟新途径，作为不损害正常干细胞长期增殖的特异性抗癌疗法。

 DESCRIPTION (provided by applicant): The crucial role of telomerase in stem cell maintenance and tumorigenesis has long been recognized. Human pluripotent stem cells have active telomerase and therefore long-term renewal capacity, but most human somatic cells lack telomerase function and therefore have a limited capacity for renewal. Patients with telomerase deficiencies that cause bone marrow failure, aplastic anemia and pulmonary fibrosis have accelerated telomere shortening, which gives rise to these tissue failures. Opposite to this effect is the telomerase reactivation that underlies the proliferative immortality of most human cancers. Despite these strong implications for human health, we lack understanding of the molecular mechanisms by which human cells regulate telomerase activity to ensure tissue homeostasis and how its dysfunction can lead to tumorigenesis. The natural regulation of telomerase activity in human tissue and the impact of telomere shortening on untransformed human cells can only be studied in a primary human stem cell system. Until recently, technical limitations, especially the inefficiency of genetic manipulation, have impeded the use of human stem cells as research tools. We have overcome this by establishing the use of site-specific nucleases to efficiently genetically engineer human pluripotent stem cells (hPSCs). This technology allows us, for the first time, to investigate two key regulatory events of human telomerase function in a genetically defined human stem cell system: its transcriptional regulation and the recruitment of telomerase to telomeres. The experiments described in Aim 1 use genetically engineered hPSCs to elucidate the molecular mechanisms that transcriptionally regulate telomerase in stem cells and how the expression of telomerase is silenced upon differentiation. The experiments outlined in Aim 2 will reveal the mechanisms that control telomerase recruitment to telomeres and the mechanisms utilized by telomere-binding proteins to regulate telomerase activity after this recruitment step. These experiments will uncover how human stem cells establish a telomere length set-point that provides a sufficient telomere reserve for human tissue regeneration while also functioning as a tumor suppressor mechanism by ultimately restricting the proliferative capacity of differentiated cell lineages. In Aim 3 we will identify the genetic alterations that led to telomerase-independent immortalization by the induction of the alternative telomere maintenance pathway, which is used by the minority of cancers that have not reactivated telomerase expression. Taken together, the experiments described here will use a genetically defined human stem cell model system to elucidate the tightly regulated steps in the telomere maintenance pathway and to mechanistically understand how mutations in this pathway promote cancer formation. Such a complete mechanistic understanding will open novel avenues of telomere maintenance inhibition as specific anti-cancer therapeutics that do not compromise the long-term proliferation of normal stem cells.