Oncolytic herpes simplex virus mutants exhibit enhanced replication in glioma cells evading temozolomide chemotherapy through deoxyribonucleic acid repair.

INTRODUCTION Engineered oncolytic viruses take advantage of cancer cell mutations and induce selective destruction.1–3 We hypothesized that chemotherapy-induced tumor-protective deoxyribonucleic acid (DNA) repair proteins promote oncolytic herpes simplex virus (HSV) replication. Specifically, in this manuscript, we demonstrate that human glioma cells respond to glioma chemotherapeutic temozolomide by expressing DNA repair genes that can be targeted to improve oncolytic HSV therapy. Glioblastomas are aggressive neoplasms resistant to current treatments, with surgery, radiation, and chemotherapy minimally altering the median survival of the disease (12–15 months) during the past decade.4 G207, an oncolytic HSV,3 has deletions of both copies of neurovirulence gene 34.5 and an inactivating mutation of UL39, encoding ICP6, the HSV ribonucleotide reductase (RR) large subunit. These mutations ensure that G207 selectively replicates in and lyses dividing cells, possibly because dividing cells express mammalian RR and growth-arrest DNA damage 34 (GADD34), genes whose products are not yet fully characterized but may regulate the cell cycle5,6 and complement G207 mutations. Mammalian RR generates deoxyribonucleotides in place of HSV RR and the GADD34 carboxyl terminus substitutes for the homologous 34.5 region.7 Intratumoral G207 inoculation has efficacy in glioma animal models and a phase I clinical trial demonstrated safety of intracranial G207 inoculation in glioma patients, with partial radiological responses.3,8,9 Temozolomide, an oral alkylating agent with slight phase II clinical trial efficacy against glioblastoma,10 spontaneously converts, at nonacidic pH, into its active metabolite, 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC), a DNA-alkylating agent that methylates guanines at O6 and N7 positions.11 O6-methylguanine is not itself lethal to cells, but pairs with thymine, triggering mismatch DNA repair, a threestep process involving: 1) mismatched base removal by N-methylpurine-DNA glycosylase (MPG)12; 2) strand cleavage by apurinic/apyrimidic endonuclease; and 3) strand breaks recruit poly(adenosine diphosphate [ADP]-ribose) polymerase (PADPRP), a nick sensor targeting the DNA repair synthetic machinery to damaged DNA.12 However, if repair fails to keep pace with DNA damage, repetitive futile rounds of mismatch repair create DNA strand breaks, activating the serine/threonine kinase, ataxia-telangectasia mutated (ATM) and Rad3-related (ATR) during S-phase.13 If ATR activation fails to cause cell cycle arrest, subsequent S-phases convert single-strand breaks into double-strand breaks, which activate serine/threonine kinase ATM. Activated ATM promotes cell cycle arrest and apoptosis.11 Unfortunately, many gliomas lack temozolomide sensitivity, primarily because of expression of DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). MGMT, expressed by 20% of gliomas, facilitates temozolomide resistance by removing the alkyl adduct from the O6 position of guanine before mismatch repair begins.14,15 MGMT-mediated temozolomide resistance can be partially overcome with O6-benzylguanine (O6BG), an MGMT inactivator proven safe and capable of enhancing temozolomide responsiveness of MGMT-expressing gliomas in phase I clinical trials.16 Because these new therapies generate only partial responses, glioblastoma treatment requires multimodal therapy. We hypothesized that limitations in temozolomide and G207 glioma treatment could be jointly addressed by using temozlomide-induced DNA repair genes to enhance HSV replication. Although some studies found synergy between oncolytic HSV and chemotherapy,17–21 the interaction was usually not quantified or mechanistically explained. Herein, we used Chou-Talalay multiple drugeffect analysis22 to quantify the interaction between temozolomide and oncolytic HSV. We demonstrate: 1) profound synergy between temozolomide and certain oncolytic HSVs in culture; 2) that glioma p53 expression influences MGMT expression; 3) that MGMT expression determines the HSV mutation required for synergy to Copyright © 2006 by Lippincott Williams & Wilkins 0148-703/06/5301-0065