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

基本信息

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
项目类别：
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

项目摘要

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 10-15kb 的 TTAGGG 序列组成，这些结构通过以下方式发挥基因组稳定性的守护者作用：不必要地限制染色体末端的 DNA 修复活性，并通过控制细胞的总次数可以分裂，从而限制活跃增殖细胞中基因组不稳定性的积累。端粒结构和功能本质上受损的细胞的生长可能会带来灾难性的后果后果，因为它促进染色体缠结，可能导致染色体碎裂（希腊语为 “染色体破碎”）或断裂-融合-桥循环，这些事件与癌症密切相关为了防止这种情况发生，端粒的缩短或自发去保护会激活细胞。周期检查点信号触发衰老，这是肿瘤形成的重要障碍。增殖并最终渗透组织和器官，癌细胞必须绕过复制衰老并激活端粒维持机制（TMM），大多数癌细胞会重新激活端粒的催化亚基。端粒酶，hTERT，已被广泛研究，然而，hTERT 在许多癌症中受到抑制。这些癌症通过端粒替代延长 (ALT) 途径来维持端粒长度。最近的数据表明，在染色质组装过程中，组蛋白动力学缺陷会激活 ALT 尽管 ALT 的许多细节还不清楚，但它会导致端粒复制叉的进展受到干扰。据了解，预计这些分叉的修复是通过断裂诱导复制（BIR）发生的，并且这些过程被认为发生在 ALT 终止的细胞结构内。相关的 PML 小体（APB）是 ALT 癌细胞所特有的。最近的观察强调了维持 ALT 途径的叉修复活动，其中治疗具有通用复制抑制剂的 ALT 细胞已被证明可以预防 APB 的组装和 ALT 癌症在蛋白质组纯化的后续研究中，细胞对 ATR 抑制剂表现出增强的敏感性。通过对 ALT+ 细胞端粒的研究，我们发现维持 ADP-核糖平衡是 ALT+ 细胞端粒的一个关键特征。 ALT 机制消耗一种独特的酶，即聚 ADP-核糖糖水解酶 (PARG)，该酶可降解聚 ADP-核糖糖水解酶 (PARG)。 ADP-核糖 (PAR) 会破坏 APB 的形成并对 ALT 活性产生负面影响。 DNA 修复调节因子，迄今为止尚未与端粒调节相关。研究 PARG 在利用 ALT 的癌细胞中的作用及其抑制作用在 AIM 1 中，我们将研究 PARG 以及抑制的细胞中的端粒结构。 AIM 2 的时空动力学被设计为我们初步数据的扩展，其中我们已经确定 PAR 直接干扰 RPA 与 ALT+ 细胞中端粒的结合。使用新型 PARG 抑制剂和蛋白质组学进行生化研究，以深入了解该机制通过干扰 PAR 降解来支持 ALT 抑制最后，在 AIM 3 中，我们将研究细胞。 PARG 耗竭的影响并研究 ALT 受到抑制的细胞的命运。