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

摘要 端粒是染色体的自然末端，由10-15kb的TTAGGG序列组成，是 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守护者，通过 在染色体末端限制不需要的DNA修复活动，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的积累。持续的 具有固有的端粒结构和功能受损的细胞的生长可能具有灾难性的 后果，因为它促进了染色体的纠缠，可能会导致染色质增多(希腊语中的意思是 染色体断裂)或断裂-融合-桥周期，这些事件与癌症密切相关 入会仪式。为了防止这种情况的发生，端粒的缩短或自发的去保护会激活细胞 触发衰老的循环检查点信号，衰老是肿瘤形成的基本屏障。为了生存， 增殖并最终渗透到组织和器官，癌细胞必须绕过复制衰老和 激活端粒维持机制(TMM)。大多数癌细胞重新激活其催化亚单位 端粒酶，hTERT，被广泛研究。然而，端粒酶逆转录酶在一些癌症中被抑制。 这些癌症通过参与替代的端粒延长(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 通过端粒导致复制分叉进程受扰。尽管ALT的许多细节都很差 可以理解，预计这些分叉的修复是通过中断诱导复制(BIR)和 同源重组。这些过程被认为发生在被称为ALT的细胞结构中 相关的PML小体，或APB，是ALT癌细胞独有的。复制的顶端受累 最近的观察强调了维持ALT途径的分叉修复活动，其中治疗 含有通用复制抑制剂的ALT细胞已被证明可以防止APB的组装和ALT癌症 细胞对ATR抑制剂表现出更高的敏感性。在后续的几个命中从蛋白质组纯化 来自ALT+细胞的端粒我们已经证实，维持ADP-核糖平衡是ADP-核糖的一个重要特征 Alt机制。耗尽一种独特的酶，聚ADP-核糖糖水解酶(PARG)，它能降解多聚糖 ADP-核糖(PAR)，破坏APB的形成，对ALT活性产生负面影响。PARG是一个重要的 DNA修复的调节者，到目前为止，还没有与端粒调节有关。本研究 研究PARG在使用ALT的癌细胞中的作用，并分析其对 癌细胞存活。在目标1中，我们将研究PARG抑制的细胞的端粒结构，以及 端粒的时空动力学。AIM 2被设计为我们初步数据的扩展，其中 我们已经发现PAR直接干扰RPA与ALT+细胞中端粒的结合。我们将聘用 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的降解来支持ALT抑制。最后，在目标3中，我们将研究细胞 PARG耗竭的影响，并调查ALT被抑制的细胞的命运。

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.