The E1B19K-deleted oncolytic adenovirus mutant AdΔ19K sensitizes pancreatic cancer cells to drug-induced DNA-damage by down-regulating Claspin and Mre11

Abstract

Adenovirus-mediated sensitization of cancer cells to cytotoxic drugs depends on simultaneous interactions of early viral genes with cell death and survival pathways. It is unclear what cellular factors mediate these interactions in the presence of DNA-damaging drugs. We found that adenovirus prevents Chk1-mediated checkpoint activation through inactivation of Mre11 and downregulation of the pChk1 adaptor-protein, Claspin, in cells with high levels of DNA-damage induced by the cytotoxic drugs gemcitabine and irinotecan. The mechanisms for Claspin downregulation involve decreased transcription and increased degradation, further attenuating pChk1-mediated signalling. Live cell imaging demonstrated that low doses of gemcitabine caused multiple mitotic aberrations including multipolar spindles, micro- and multi-nucleation and cytokinesis failure. A mutant virus with the anti-apoptotic E1B19K-gene deleted (AdΔ19K) further enhanced cell killing, Claspin downregulation, and potentiated drug-induced DNA damage and mitotic aberrations. Decreased Claspin expression and inactivation of Mre11 contributed to the enhanced cell killing in combination with DNA-damaging drugs. These results reveal novel mechanisms that are utilised by adenovirus to ensure completion of its life cycle in the presence of cellular DNA damage. Taken together, our findings reveal novel cellular targets that may be exploited when developing improved anti-cancer therapeutics.

The Novel Oncolytic Adenoviral Mutant Ad5-3Δ-A20T Retargeted to αvβ6 Integrins Efficiently Eliminates Pancreatic Cancer Cells

“…Our data demonstrate that viral efficacy was retained in the 3D cultures when Ad5-3D-A20T was combined with low doses of gemcitabine. The deletion of the E1B19K gene causes enhancement of gemcitabine-dependent apoptosis, previously demonstrated for AdDD in PDAC cells (6,11,12,14). We propose that the 3D cocultures are suitable model systems for investigating oncolytic virus replication and spread in the presence of the multiple cell types that constitute the tumor microenvironment in patients.…”

“…S4). We previously demonstrated that both AdDD and AdD19K synergized with low doses of gemcitabine in pancreatic cancer cells in 2D monocultures and in vivo, including Suit-2 models, by enhancing apoptotic death through mitotic aberrations (11,14). We found that the 3D coculture system was a suitable model for screening the efficacy of adenoviral mutants and for confirming compatibility with current therapeutic cytotoxic drugs.…”

“…The cell suspensions were pelleted, snap-frozen, and stored at À80 C. DNA was extracted using the QIAamp DNA Blood Mini Kit, according to the manufacturer's instructions (Qiagen) and used for quantitative PCR (qPCR) analysis as described previously (11,14).…”

“…Ad5 wild-type virus of known activity was included in every assay as internal control as described previously (14).…”

“…E1B19K is a functional Bcl-2 homolog that binds Bax and Bak and inhibits mitochondrial pore formation and apoptosis in response to death receptor activation and intrinsically induced apoptosis (13). AdDD synergized with the current clinical standard of care for pancreatic cancer, gemcitabine and irinotecan, without toxicity to normal cells (6,11,14). However, to efficiently target pancreatic cancer lesions in patients by systemic administration required further modifications.…”