The Association of PARP1 Enzymatic Inhibition and Chromatin Complex Formation with Radiosensitization by PARP Inhibitors and Their Combination with ATR Inhibition in Pancreatic Cancer

Abstract

Pancreatic cancer has a five-year overall survival rate of less than 10%. Locoregional progression is responsible for death in up to a third of all patients, highlighting the need for local control. The use of chemotherapeutic agents in combination with radiation represents a current standard of care for locally advanced PDAC, but new therapies are urgently needed. PARP inhibitors have shown promise as radiosensitizing agents preclinically, both alone and in combination with chemotherapeutic or molecularly targeted agents. Work has demonstrated that their antitumor effect may be the product of their enzymatic inhibition or ability to form PARP1-DNA complexes at DNA damage sites known as PARP trapping. These complexes have been implicated as the cause for both therapeutic efficacy as well as many dose-limiting toxicities seen clinically. In our work, we investigated the radiosensitizing properties of PARP inhibitors in pancreatic cancer cell line models. Using olaparib, which inhibits PARP at both low and high concentrations but only traps at high concentrations, we demonstrated that pancreatic cancer cells proficient in homologous recombination were only radiosensitized by high concentrations of olaparib. While we demonstrated that olaparib possesses the ability to inhibit enzymatically at both high and low concentrations, it is known to trap only at high. The ability to form PARP1-DNA complexes correlated with increased DNA double strand breaks at higher olaparib concentrations. We discovered that the radiosensitizing potency of three PARP inhibitors, veliparib, olaparib, and talazoparib, corresponded to their described increasing potency as PARP trappers. Deletion of PARP1 failed to phenocopy the radiosensitizing effects of PARP inhibitors and protected cells from cytotoxicity from talazoparib. In order to more fully characterize PARP trapping, we adapted the proximity ligation assay to measure trapped PARP1 in situ by measuring the proximity of PARP1 and total histone H2AX in treated cells. To further potentiate the radiosensitizing effects of PARP inhibition, we leveraged the knowledge that PARP inhibition causes replication stress. ATR is known to regulate the cellular response to replication stress and fork stability. Thus, we sought to combine PARP inhibition with the ATR inhibitor AZD6738. The combinatorial effect of ATR and PARP inhibition in HR proficient cell lines to radiosensitize and damage DNA was most pronounced at trapping concentrations of olaparib. Further, this effect required presence of the PARP1 protein. Combined treatment in mice bearing pancreatic cancer xenografts with olaparib, AZD6738, and radiation substantially inhibited tumor growth relative to all other treatment groups while causing minimal toxicity. These findings strongly recommend the merits of clinical investigation into the efficacy of combined ATR and PARP inhibition with radiation for locally advanced disease and suggest that the PARP inhibitors with greater PARP trapping potency may be most efficacious.