Robust Radiosensitization by Combined Treatment of Cancer Cells with Talazoparib and Polθ Inhibitors

Abstract

The PARP inhibitor talazoparib is synthetically lethal with HR-defective tumors and functions as a potent radiosensitizer specifically of cancer cells. Talazoparib exerts this unique radiosensitizing property by shifting ionizing radiation (IR)-induced DNA double strand break (DSB) repair towards error-prone alternative end-joining (alt-EJ). DNA polymerase theta (Polθ, encoded by POLQ) is a key component of alt-EJ. Here, we tested the hypothesis that inhibition of alt-EJ with Polθ ablation or using specific small molecule inhibitors can further increase talazoparib-induced radiosensitization. U2OS, A549, and their corresponding POLQ ablated/mutant cell lines were treated with talazoparib and/or Polθ inhibitors ART558/novobiocin prior to irradiation. siRNAs against CtIP, MRE11, EXO1; and a specific inhibitor of DNA2 were employed to suppress DNA end resection. Radiosensitization was assessed by clonogenic survival. Olaparib, rucaparib, and veliparib were also tested under similar conditions. DSB repair and end resection were measured by scoring γH2AX and RPA nuclear foci, respectively. Chromosomal abnormalities were assessed using G2-specific cytogenetics analysis. Genetic ablation or pharmacological inhibition of Polθ robustly enhanced talazoparib mediated radiosensitization by ∼40-70%. Notably, Polθ inhibition had a much lower effect (by ∼7-17%) when combined with other clinically used PARP inhibitors, olaparib, rucaparib, and veliparib. Polθ inhibition significantly suppressed talazoparib-induced translocation formation in irradiated cells. In addition, combined treatment with Polθ inhibitor and talazoparib attenuated DSB repair, resulting in ∼60% unresolved γH2AX foci and ∼40% unresolved chromatid breaks at 5h post IR. Talazoparib promoted resection of DNA ends as demonstrated by an increase in RPA foci. The resection process requires the activities of CtIP and MRE11, but not of DNA2 or EXO1. Finally, CtIP and MRE11 knockdown impaired radiosensitization following a combined talazoparib/Polθ inhibition treatment. Talazoparib increases the reliance of irradiated cancer cells on Polθ-mediated alt-EJ owing to the increased CtIP/MRE11-dependent resection it produces. Combining talazoparib with Polθ inhibitors has therefore great potential in improving radiotherapy of human tumors.