In Vitro and In Vivo Biology of Telomere Stress Induced Senescence

项目摘要 衰老细胞被认为是衰老和年龄相关疾病的驱动因素。衰老细胞具有 持续某种形式的压力，导致不可逆的细胞周期停滞，这些细胞避免凋亡。这些 细胞具有高代谢表型并分泌导致衰老的细胞因子和趋化因子 相关分泌表型（SASP）。SASP含有多种炎性成分， 促进组织功能障碍。这些观察结果一直是开发衰老药物的动力。这些 药物被设计为引起衰老细胞的选择性凋亡， 已进入临床试验，可能用于特发性肺纤维化、骨关节炎、慢性糖尿病等。 许多这些senolytic药物已经通过候选方法开发，其中药物最初 用于治疗癌症的药物被重新用于清除衰老细胞。这些药 不幸的是，具有靶向和脱靶剂量限制性副作用，如血小板减少症， 骨小梁丢失。因此，需要开发靶向特定的潜在衰老抑制剂的衰老清除药物。 衰老细胞的脆弱性，而不影响健康细胞。在这个项目中，我们进行了一个公正的 基于CRISPR的全基因组合成致死性筛选，以确定基因敲除时，在 已经经历了端粒诱导衰老的细胞的设置，但当敲除端粒时， 一个健康的、不衰老的细胞。通过这个筛选，我们确定ER应激中的遗传改变 是衰老细胞特有的脆弱性具体来说，靶向ER驻留蛋白PARP16 和BIP，它们在CRISPR筛选中被鉴定，调节ER应激途径。所以我们 假设由于它们的高分泌负荷，衰老细胞特别容易受到以下改变的影响： ER应激通路。 我们提出了以下研究：（1）识别和表征的基因集，当 在衰老细胞中敲除，导致这些细胞的特异性死亡，而不影响健康细胞的功能。 （2）了解经历端粒诱导衰老的衰老细胞是否具有更高水平的 内质网应激以及这些途径的改变是否影响衰老细胞的活力和 （3）了解端粒诱导的衰老是否影响后叶细胞的功能， 有丝分裂细胞如心肌细胞，进而心肌细胞衰老是否影响心脏功能。 因此，这一建议将导致更好地了解独特的分子脆弱性， 衰老细胞，并澄清细胞如何维持衰老状态的分子基础。 该提案的成功完成将为开发senolytics治疗老年痴呆症提供基础。 各种与衰老相关的疾病。最后，这一建议将为我提供一个出色的平台，建立 我的基本科学和临床思维能力，使我可以追求作为一个医生科学家的职业生涯。

PROJECT SUMMARY Senescent cells have been implicated as drivers of aging and age-associated diseases. Senescent cells have sustained some form of stress that causes irreversible cell-cycle arrest, and these cells avoid apoptosis. These cells have a hyper-metabolic phenotype and secrete cytokines and chemokines resulting in the senescence associated secretory phenotype (SASP). The SASP contains multiple inflammatory components which promotes tissue dysfunction. These observations have been the impetus to develop senolytic drugs. These drugs are designed to cause the selective apoptosis of senescent cells and several senolytics have already entered clinical trials for potential use in idiopathic pulmonary fibrosis, osteoarthritis, chronic diabetes, etc. Many of these senolytic drugs have been developed via a candidate approach where drugs that were initially developed for the treatment of cancer are repurposed for the clearance of senescent cells. These drugs, unfortunately, have both on-target and off-target dose-limiting side effects such as thrombocytopenia and trabecular bone loss. Therefore, there is a need to develop senolytic drugs that target a specific underlying vulnerability of senescent cells without affecting healthy cells. In this project, we have performed an unbiased CRISPR-based, whole genome synthetic lethality screen to identify genes when knocked out, are lethal in the setting of a cell that has undergone telomere-induced senescence yet are well-tolerated when knockout in the context of a healthy, non-senescent cell. Through this screen, we identified that genetic alterations in ER stress pathways are specific vulnerabilities of senescent cells. Specifically, the targets ER-resident proteins, PARP16 and BIP, which were identified in the CRISPR screen, modulate ER stress pathways. Therefore, we hypothesize that due to their high secretory burden, senescent cells are particularly vulnerable to alterations in ER stress pathways. We propose to investigate the following: (1) Identify and characterize the set of genes which when knocked out in senescent cells cause the specific death of these cells without affecting the function of healthy cells; (2) Understand whether senescent cells that undergo telomere induced senescence have higher levels of ER stress and whether alterations in these pathways affect the viability of senescent cells and the production of SASP components; (3) Understand whether telomere-induced senescence affects the function of post- mitotic cells such as cardiomyocytes and in turn, whether cardiomyocyte senescence affect heart function. This proposal will therefore lead to a better understanding of the unique molecular vulnerabilities of senescent cells and clarify the molecular underpinnings of how cells maintain a state of senescence. Successful completion of this proposal will provide a foundation for the development of senolytics to treat a variety of aging-associated diseases. Finally, this proposal will provide me with an outstanding platform to build my basic science and clinical thinking skills so that I can pursue a career as a physician scientist.