Oncolytic virus-mediated manipulation of DNA damage responses: synergy with chemotherapy in killing glioblastoma stem cells

J Natl Cancer Inst. 2012 Jan 4;104(1):42-55. doi: 10.1093/jnci/djr509. Epub 2011 Dec 15.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antineoplastic Agents, Alkylating / pharmacology*
  • Ataxia Telangiectasia Mutated Proteins
  • Brain Neoplasms / drug therapy*
  • Brain Neoplasms / genetics*
  • Cell Cycle Proteins / metabolism
  • Cell Line, Tumor
  • Cell Survival
  • Chlorocebus aethiops
  • DNA Damage*
  • DNA Modification Methylases / metabolism
  • DNA Repair / drug effects
  • DNA Repair Enzymes / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Dacarbazine / analogs & derivatives*
  • Dacarbazine / pharmacology
  • Female
  • Gene Knockdown Techniques
  • Genetic Vectors
  • Glioblastoma / drug therapy*
  • Glioblastoma / genetics*
  • Humans
  • Immunoblotting
  • Immunohistochemistry
  • Methylation
  • Mice
  • Mice, Nude
  • Neoplastic Stem Cells / drug effects*
  • Oncolytic Virotherapy* / methods
  • Polymerase Chain Reaction
  • Protein-Serine-Threonine Kinases / metabolism
  • Simplexvirus* / genetics
  • Temozolomide
  • Tumor Suppressor Proteins / metabolism
  • Vero Cells
  • Virus Replication

Substances

  • Antineoplastic Agents, Alkylating
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • G-T mismatch-binding protein
  • Tumor Suppressor Proteins
  • Dacarbazine
  • DNA Modification Methylases
  • MGMT protein, human
  • ATM protein, human
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Atm protein, mouse
  • Protein-Serine-Threonine Kinases
  • DNA Repair Enzymes
  • Temozolomide