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

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.

Keywords: oncolytic viruses; radiotherapy; virotherapy; combination therapy; DNA damage repair; ionizing radiation

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