E1B19K--deleted oncolytic adenoviruses enhancee the cytotoxicity of DNA--damaging drugs in pancreatic cancer through deregulation of cell--cycle mechanisms

Abstract

Pancreatic cancer is an aggressive disease with poor prognosis and a high fatality rate. Gemcitabine, the standard first-line chemotherapy for advanced disease, has negligible effects, necessitating the development of new therapies. We previously demonstrated that deletion of the anti-apoptotic gene E1B19K (AdΔ19K) in a replication-selective adenoviral mutant, caused synergistically-enhanced cell-killing when combined with low-dose DNA-damaging drugs in pancreatic cancer xenograft models. To delineate the cellular pathways targeted by the combination treatment we employed AdΔ19K and gemcitabine or irinotecan, with the goal of identifying cellular factors that are essential for the synergistic cell-killing. We hypothesised that AdΔ19K and DNA-damaging drugs act synergistically to deregulate cell-cycle mechanisms. Pancreatic cancer cell death induced by AdΔ19K and DNA-damaging drugs is apoptotic and time-dependent. AdΔ19K could not block DNA-damage responses (DDR) elicited by the drugs, despite virus-mediated degradation of the DDR factor Mre11. Mre11 siRNA-mediated knockdown augmented the synergistic cell death. Mitotic-index analysis in synchronised cells and immunofluorescence microscopy suggested that AdΔ19K promotes mitotic entry of gemcitabine-treated DNA-damaged cells. Moreover, AdΔ19K inhibited drug-induced accumulation of Claspin, a DDR protein whose degradation is required for checkpoint recovery. Treatment with AdΔ19K and gemcitabine accelerated Claspin degradation, and siRNA-mediated Claspin knockdown enhanced the synergistic cell death. Time-lapse microscopy in histoneH2B mCherry-expressing cells showed that AdΔ19K enhanced gemcitabine-induced mitotic catastrophe, characterised by prolonged mitosis, chromosome missegregation errors, cytokinesis failure and formation of multinucleated cells. Moreover, live-cell imaging revealed that the majority of cells treated with AdΔ19K and gemcitabine die before mitotic entry. 5 These findings suggest that E1B19K-deleted adenoviruses cannot prevent cell-cycle checkpoint responses elicited by DNA-damaging drugs, but enhance drug-induced cell death by downregulating DDR factors, such as Mre11 and Claspin. Additionally, the virus enhances mitotic catastrophe of DNA-damaged cells escaping cell-cycle checkpoints, eventually leading to increased apoptosis. Through these studies cellular pathways and factors involved in the synergistic cell killing were identified, that could be explored in the future to develop improved targeted therapies for pancreatic cancer.