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.

PUBLIC HEALTH RELEVANCE: In order to survive, proliferate and eventually infiltrate tissues and organs, it is essential that cancer cells maintain their telomeres. This is achieved by activating one of two different telomere length maintenance mechanisms. One is common to both normal cells and cancerous cells, whereas the other less well understood mechanism, known as ALT, is only found in cancerous cells. Cancers that rely on ALT tend to be among the most aggressive and patients often have a poor prognosis. Therefore, understanding the molecular basis for the activation and maintenance of the ALT pathway and identifying the key regulators of ALT is critically important for improving the detection and treatment of this devastating disease. This is the focus of our study.