title: Oncolytic virus-mediated manipulation of DNA damage responses: synergy with chemotherapy in killing glioblastoma stem cells.
abstract: BACKGROUND
  Although both the alkylating agent temozolomide (TMZ) and oncolytic viruses hold promise for treating glioblastoma, which remains uniformly lethal, the effectiveness of combining the two treatments and the mechanism of their interaction on cancer stem cells are unknown.
  
  
  METHODS
  We investigated the efficacy of combining TMZ and the oncolytic herpes simplex virus (oHSV) G47Δ in killing glioblastoma stem cells (GSCs), using Chou-Talalay combination index analysis, immunocytochemistry and fluorescence microscopy, and neutral comet assay. The role of treatment-induced DNA double-strand breaks, activation of DNA damage responses, and virus replication in the cytotoxic interaction between G47Δ and TMZ was examined with a panel of pharmacological inhibitors and short-hairpin RNA (shRNA)-mediated knockdown of DNA repair pathways. Comparisons of cell survival and virus replication were performed using a two-sided t test (unpaired). The survival of athymic mice (n = 6-8 mice per group) bearing GSC-derived glioblastoma tumors treated with the combination of G47Δ and TMZ was analyzed by the Kaplan-Meier method and evaluated with a two-sided log-rank test.
  
  
  RESULTS
  The combination of G47Δ and TMZ acted synergistically in killing GSCs but not neurons, with associated robust induction of DNA damage. Pharmacological and shRNA-mediated knockdown studies suggested that activated ataxia telangiectasia mutated (ATM) is a crucial mediator of synergy. Activated ATM relocalized to HSV DNA replication compartments where it likely enhanced oHSV replication and could not participate in repairing TMZ-induced DNA damage. Sensitivity to TMZ and synergy with G47Δ decreased with O(6)-methylguanine-DNA-methyltransferase (MGMT) expression and MSH6 knockdown. Combined G47Δ and TMZ treatment extended survival of mice bearing GSC-derived intracranial tumors, achieving long-term remission in four of eight mice (median survival = 228 days; G47Δ alone vs G47Δ + TMZ, hazard ratio of survival = 7.1, 95% confidence interval = 1.9 to 26.1, P = .003) at TMZ doses attainable in patients.
  
  
  CONCLUSIONS
  The combination of G47Δ and TMZ acts synergistically in killing GSCs through oHSV-mediated manipulation of DNA damage responses. This strategy is highly efficacious in representative preclinical models and warrants clinical translation.
authors.affiliation: Brain Tumor Research Center, Department of Neurosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114 , USA.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Kanai
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Rabkin
authors.forename: Stephen
authors.initials: S
authors.lastname: Yip
authors.forename: Donatella
authors.initials: D
authors.lastname: Sgubin
authors.forename: Cecile M
authors.initials: CM
authors.lastname: Zaupa
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Hirose
authors.forename: David N
authors.initials: DN
authors.lastname: Louis
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Wakimoto
authors.forename: Robert L
authors.initials: RL
authors.lastname: Martuza
mesh_terms: D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D064007:Ataxia Telangiectasia Mutated Proteins; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival; D002522:Chlorocebus aethiops; D004249:DNA Damage; D015254:DNA Modification Methylases / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D045643:DNA Repair Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D005909:Glioblastoma / Q000188:drug therapy* / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008745:Methylation; D051379:Mice; D008819:Mice, Nude; D014411:Neoplastic Stem Cells / Q000187:drug effects*; D050130:Oncolytic Virotherapy / Q000379:methods; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D018139:Simplexvirus / Q000235:genetics; D000077204:Temozolomide; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014709:Vero Cells; D014779:Virus Replication
chemical_list: D018906:Antineoplastic Agents, Alkylating; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; D025521:Tumor Suppressor Proteins; D003606:Dacarbazine; D015254:DNA Modification Methylases; C515491:MGMT protein, human; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D045643:DNA Repair Enzymes; D000077204:Temozolomide