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性的端粒酶，因此具有长期更新能力，但大多数人类体细胞缺乏端粒酶功能，因此更新能力有限。端粒酶缺乏导致骨髓衰竭、再生障碍性贫血和肺纤维化的患者加速了端粒缩短，这导致了这些组织衰竭。与此相反的效果 是端粒酶的重新激活，它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强烈的影响，但我们缺乏对人类细胞调节端粒酶活性以确保组织动态平衡的分子机制，以及端粒酶功能障碍如何导致肿瘤发生。人类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类细胞的影响只能在原代人类干细胞系统中进行研究。直到最近，技术上的限制，尤其是基因操作的低效，一直阻碍着人类干细胞作为研究工具的使用。我们已经克服了这一点，建立了使用位点特异性核酸酶来高效地设计人类多能干细胞(HPSCs)的基因工程。这项技术使我们第一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控事件：其转录调控和端粒酶向端粒的募集。Aim 1中描述的实验使用基因工程hPSC来阐明转录调控干细胞中的端粒酶的分子机制，以及在分化过程中端粒酶的表达是如何沉默的。在Aim 2中概述的实验将揭示控制端粒酶重新聚集到端粒的机制，以及端粒结合蛋白在这一招募步骤之后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系的增殖能力而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端粒酶非依赖性永生化的基因改变，该途径被少数未重新激活端粒酶表达的癌症所使用。综上所述，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受到严格调控的步骤，并从机制上理解该途径中的突变如何促进癌症的形成。这种完整的机制理解将开辟抑制端粒维持的新途径，作为不损害正常干细胞长期增殖的特定抗癌疗法。

 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.