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: 9154553
• 负责人: Roderick O'Sullivan
• 金额: $ 36.17万
• 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/9154553
• 关键词: 3-Dimensional; Address; Adenosine Diphosphate Ribose; Affect; 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; Health; Histones; Length; Lesion; Link; Maintenance; Malignant Neoplasms; Metabolism; Methods; Molecular; 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; X-Linked Mental Retardation; abstracting; alpha-Thalassemia; alternative treatment; base; cancer cell; cancer initiation; cell growth; chromothripsis; design; event cycle; homologous recombination; improved; inhibitor/antagonist; innovation; insight; live cell imaging; novel; outcome forecast; poly ADP-ribose glycohydrolase; prevent; 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.

摘要 端粒是染色体的天然末端，由10- 15 kb的TTAGGG序列组成， 健康细胞生理学的关键调节器。这些结构作为基因组稳定性的监护人， 限制染色体末端不需要的DNA修复活性，并通过控制细胞 可以分裂，从而限制活跃增殖细胞中基因组不稳定性的积累。持续 具有固有受损的端粒结构和功能的细胞的生长可能具有灾难性的 结果，因为它促进了染色体的缠结，可能导致染色体断裂（希腊语， “染色体破碎”）或断裂-融合-桥周期，这些事件与癌症密切相关 入会仪式为了防止这种情况发生，端粒的缩短或自发去保护激活细胞 周期检查点信号传导触发衰老，这是肿瘤形成的重要屏障。为了生存， 由于癌细胞会增殖并最终浸润组织和器官，因此癌细胞必须绕过复制衰老， 启动端粒维持机制（TMM）。大多数癌细胞会重新激活 端粒酶，hTERT，被广泛研究。然而，hTERT在许多癌症中被抑制。 这些癌症通过参与端粒替代延长（ALT）途径来维持端粒长度。 最近的数据表明，ALT在染色质组装过程中被有缺陷的组蛋白动力学激活， 导致通过端粒的复制叉进程受到干扰。虽然ALT的许多细节都很差， 据了解，预计这些分叉的修复通过断裂诱导复制（BIR）发生， 同源重组这些过程被认为发生在细胞结构内，称为ALT 相关的PML小体，或APB，是ALT癌细胞所特有的。顶端参与复制 最近的观察结果强调了维持ALT途径的分叉修复活性， 具有通用复制抑制剂的ALT细胞已被证明可以防止APB和ALT癌症的组装 细胞对ATR抑制剂显示出增强的敏感性。在后续的几个命中从蛋白质组纯化的 从ALT+细胞的端粒，我们已经确定，维持ADP-核糖平衡是一个关键的特征， ALT机制。降解聚的独特酶聚ADP-核糖糖羟化酶（PARG）的耗尽 ADP-核糖（PAR）破坏APB形成并对ALT活性产生负面影响。PARG是重要的 到目前为止，它还没有与端粒调节相关的DNA修复调节因子。本研究 研究PARG在使用ALT的癌细胞中的作用，并分析其抑制对 癌细胞存活率在AIM 1中，我们将研究PARG抑制细胞中的端粒结构，以及 端粒的时空动态AIM 2被设计为我们初步数据的扩展， 我们已经确定PAR直接干扰RPA与ALT+细胞中端粒的结合。我们会委聘 使用新型PARG抑制剂和蛋白质组学进行生化研究，以深入了解其机制 通过干扰PAR降解来支持ALT抑制。最后，在AIM 3中，我们将研究细胞 研究PARG耗竭的影响，并研究其中ALT被抑制的细胞的命运。