Inhibition of the ALT pathway by interfering with Poly-ADP-Ribose metabolism

通过干扰聚 ADP 核糖代谢来抑制 ALT 途径

• 批准号: 9280913
• 负责人: Roderick O'Sullivan
• 金额: $ 34.92万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9280913
• 关键词: 3-Dimensional; Address; Adenosine Diphosphate Ribose; Affect; Alpha Cell; Apical; BRCA1 gene; Binding; Biochemical; Bypass; Cancerous; Cell Aging; Cell Cycle Checkpoint; Cell Line; Cell Proliferation; Cell Survival; Cell model; Cell physiology; Cells; Cellular Structures; Chromatin; Chromatin Modeling; Chromosomes; Coupled; DNA; DNA Damage; DNA Repair; DNA replication fork; Data; Detection; Development; Disease; Enzymes; Equilibrium; Event; Excision; Exhibits; Functional disorder; Generic Drugs; Genetic Recombination; Genome Stability; Genomic Instability; Genomics; Glioma; Goals; Greek; Hand; Histones; Impairment; Length; Lesion; Link; Maintenance; Malignant Neoplasms; Mental Retardation; Metabolism; Methods; Molecular; Molecular Analysis; Molecular Cytogenetics; Mutation; Normal Cell; Organ; Outcome; Patients; Phenotype; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Polymerase; Polymers; Process; Proliferating; Proteins; Proteomics; Recruitment Activity; Regulation; Research; Residencies; Role; Signal Transduction; Stem cells; Stress; Structure; TERT gene; Telomerase; Telomere Length Maintenance; Telomere Maintenance; Telomere Pathway; Time; Tissues; alpha-Thalassemia; alternative treatment; base; cancer cell; cancer initiation; cell growth; chromothripsis; design; genotoxicity; homologous recombination; improved; inhibitor/antagonist; innovation; insight; live cell imaging; novel; outcome forecast; poly ADP-ribose glycohydrolase; prevent; public health relevance; repaired; response; senescence; small molecule inhibitor; spatiotemporal; targeted treatment; telomere; tumor

Abstract Telomeres, the natural termini of chromosomes, are composed of 10-15kb of the TTAGGG sequence and are critical regulators of healthy cellular physiology. These structures function as guardians of genome stability by limiting unwanted DNA repair activity at chromosome ends, and by controlling the total number of times a cell can divide thereby limiting the accumulation of genomic instability in actively proliferating cells. The sustained growth of cells with inherently compromised telomeric structure and function can have catastrophic consequences as it promotes the entanglement of chromosomes that may result in chromothripsis (Greek for “chromosome shattering”) or breakage-fusion-bridge cycles, events that are strongly linked with cancer initiation. To prevent this from occurring, shortening or spontaneous de-protection of telomeres activates cell cycle checkpoint signaling that triggers senescence, an essential barrier to tumor formation. In order to survive, proliferate and eventually infiltrate tissues and organs, cancer cells must bypass replicative senescence and activate a telomere maintenance mechanism (TMM). Most cancer cells reactivate the catalytic subunit of telomerase, hTERT, which is widely investigated. However, hTERT is suppressed in a number of cancers. These cancers maintain telomere length by engaging the alternative lengthening of telomeres (ALT) pathway. Recent data indicates that ALT is activated by defective histone dynamics during chromatin assembly that results in perturbed replication fork progression through telomeres. Though many details of ALT are poorly understood it is anticipated that the repair of these forks occurs via break-induced replication (BIR) and homologous recombination. These processes are thought to occur within cellular structures termed ALT associated PML bodies, or APBs, that are unique to ALT cancer cells. The apical involvement of replication fork repair activities in sustaining the ALT pathway is underscored by recent observations where treatment of ALT cells with generic replication inhibitors has been shown to prevent the assembly of APBs and ALT cancer cells display enhanced sensitivity to ATR inhibitors. In following-up several hits from a proteomic purification of telomeres from ALT+ cells we have identified that maintaining ADP-ribose equilibrium is a critical feature of the ALT mechanism. Depletion of a unique enzyme, poly ADP-ribose glycohyrolase (PARG), which degrades poly ADP-ribose (PAR), disrupts APB formation and negatively impacts ALT activity. PARG is an important regulator of DNA repair that, until now, has not been associated with telomere regulation. This study investigates the role of PARG in cancer cells that employ ALT and analyzes the effects of its inhibition on cancer cell survival. In AIM 1 we will investigate telomere structure in cells with suppressed PARG, as well as the spatiotemporal dynamics of telomeres. AIM 2 is designed as an extension of our preliminary data in which we have identified that PAR directly interferes with RPA binding to telomeres in ALT+ cells. We will employ biochemical studies with novel PARG inhibitors and proteomics to generate insights of the mechanism underpinning ALT inhibition by interfering with PAR degradation. Finally, in AIM 3 we will study the cellular effects of PARG depletion and investigate the fate of cells in which ALT in inhibited.

抽象的 端粒是染色体的天然末端，由ttaggg序列的10-15kb组成，是 健康细胞生理的关键调节剂。这些结构通过 限制染色体末端的不需要的DNA修复活动，并通过控制单元的总数 可以划分从而限制基因组不稳定性在主动增殖细胞中的积累。持续 具有固有损害的远程结构和功能的细胞的生长可能具有灾难性的 促进可能导致染色体的染色体纠缠的后果（希腊语 “染色体破碎”）或断裂融合桥周期，与癌症密切相关的事件 引发。为了防止这种情况发生，缩短或赞助端粒的去保护会激活细胞 循环检查点信号传达触发肿瘤形成的基本障碍。为了生存， 癌细胞增殖，有时甚至有时浸润组织和器官，必须绕过复制感应感， 激活端粒维护机制（TMM）。大多数癌细胞重新激活 端粒酶HTERT，已广泛研究。但是，HTERT被许多取消抑制。 这些癌症通过参与端粒（ALT）途径的替代延长来保持端粒长度。 最近的数据表明，ALT在染色质组装过程中被缺陷的组蛋白动力学激活 导致通过端粒的扰动复制叉进展。尽管ALT的许多细节很差 了解这些叉子的修复是通过突破引起的复制（BIR）和 同源重组。这些过程被认为发生在称为ALT的细胞结构中 ALT癌细胞独有的相关PML体或APB。重复的顶端参与 最近的观察结果强调了维持ALT途径中的叉子维修活动 已显示具有通用复制抑制剂的ALT细胞可防止APB和ALT癌的组装 细胞表现出对ATR抑制剂的敏感性增强。在跟踪蛋白质组学纯化的几次命中 来自Alt+细胞的端粒我们已经确定，维持ADP-核糖等效是该的关键特征 替代机制。独特的酶，多ADP-核糖糖醇（PARG）的耗竭，该酶降低了多个 ADP-ribose（PAR）破坏APB的形成，并对ALT活动产生负面影响。 parg是一个重要的 到目前为止，DNA修复的调节剂尚未与端粒调节有关。这项研究 研究PARG在采用ALT的癌细胞中的作用，并分析其抑制作用对 癌细胞存活。在AIM 1中，我们将研究抑制PARG的细胞中的端粒结构，以及 端粒的时空动力学。 AIM 2被设计为我们的初步数据的扩展 我们已经确定PAR直接干扰了Alt+细胞中端粒的RPA结合。我们将雇用 具有新型PARG抑制剂和蛋白质组学的生化研究，以产生有关该机制的见解 通过干扰PAR降解来支撑ALT抑制。最后，在AIM 3中，我们将研究细胞 PARG部署的影响并研究了抑制ALT的细胞的命运。