External Beam Radiation Therapy and Enadenotucirev: Inhibition of the DDR and Mechanisms of Radiation-Mediated Virus Increase.

Tzveta D Pokrovska,William K Taverner,Richard Baugh,Janet Lei-Rossmann,Egon J Jacobus,Rathi Puliyadi,Geoff S Higgins,Gonzalo Rodriguez-Berriguete,Remko Prevo,Arthur Dyer,Kerry Fisher,Len W Seymour,Katharine Herbert,Giovanna Granata,Sally Frost,Brian R Champion

doi:10.3390/cancers12040798

Tzveta D Pokrovska, William K Taverner + Show 14 more

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https://doi.org/10.3390/cancers12040798

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Abstract

Ionising radiation causes cell death through the induction of DNA damage, particularly double-stranded DNA (dsDNA) breaks. Evidence suggests that adenoviruses inhibit proteins involved in the DNA damage response (DDR) to prevent recognition of double-stranded viral DNA genomes as cellular dsDNA breaks. We hypothesise that combining adenovirus treatment with radiotherapy has the potential for enhancing tumour-specific cytotoxicity through inhibition of the DDR and augmentation of virus production. We show that EnAd, an Ad3/Ad11p chimeric oncolytic adenovirus currently being trialled in colorectal and other cancers, targets the DDR pathway at a number of junctures. Infection is associated with a decrease in irradiation-induced 53BP1 and Rad51 foci formation, and in total DNA ligase IV levels. We also demonstrate a radiation-associated increase in EnAd production in vitro and in a pilot in vivo experiment. Given the current limitations of in vitro techniques in assessing for synergy between these treatments, we adapted the plaque assay to allow monitoring of viral plaque size and growth and utilised the xCELLigence cell adhesion assay to measure cytotoxicity. Our study provides further evidence on the interaction between adenovirus and radiation in vitro and in vivo and suggests these have at least an additive, and possibly a synergistic, impact on cytotoxicity.

Highlights

Radiotherapy, alongside chemotherapy, immunotherapy and resection, is one of the major treatment modalities in the management of cancer
DNA damage response (DDR) are mediated through early viral proteins, but we elected to assess the relationship between these two treatments by irradiating infected cells 24 h post infection by the virus
To understand the effect of virus infection on the DDR in both irradiated and non-irradiated individual cells, we explored the formation of DNA damage foci in infected cells, using GFP-expressing EnAd to exclude “noise” from non-infected cells. 53BP1 foci are indicative of double-stranded break repair, promoting non-homologous end-joining (NHEJ) [52], while Rad51 foci are associated with homologous recombination repair (HR) [53], and γH2AX foci denote double-stranded breaks [54]

Summary

Introduction

Radiotherapy, alongside chemotherapy, immunotherapy and resection, is one of the major treatment modalities in the management of cancer. Current radiotherapy techniques allow improved precision in the treatment of target cells, decreasing normal tissue toxicity without compromise in effective tumour eradication remains an ongoing challenge. Oncolytic viruses are viruses that replicate in and lyse cancer cells. A number of viruses, including adenoviruses, are currently being explored as oncolytic agents in the clinic [6,7]. Oncolytic viruses are being explored as potential radiosensitisers, those with DNA genomes that spend part of their replication cycle inside the host cell nucleus [8,9]. Nuclear-resident viruses, those that have linear genomes such as adenoviruses, are likely to have mechanisms to inhibit the cellular DNA damage response (DDR) that would otherwise allow the cell to detect and mount an antiviral response against the replicating virus

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