Effect of chemotherapy-induced DNA repair on oncolytic herpes simplex viral replication.
BACKGROUND
Gliomas treated with the alkylating agent temozolomide have incomplete responses in part because of tumoral repair of chemotherapy-induced DNA damage. Data from phase I trials suggest that G207, an oncolytic herpes simplex virus (HSV) with mutated ribonucleotide reductase (RR) and gamma34.5 genes, is safe but needs greater viral oncolysis to be effective. We hypothesized that temozolomide and G207 treatment limitations could be jointly addressed using temozolomide-induced tumor-protective DNA repair pathways to enhance viral replication.


METHODS
Human glioblastoma cells (U87, T98, and U373) and U87 cells transfected with the gene for the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) were treated with G207 and/or temozolomide. Drug interactions, expression of the growth arrest DNA damage 34 (GADD34) and RR transcripts before and after their knockdown with short interfering RNAs, DNA strand breaks, and apoptosis were measured using Chou-Talalay analysis, real-time reverse transcription-polymerase chain reaction, the comet assay, and flow cytometry, respectively. Survival of mice (groups of ten) with intracranial U87 xenograft tumors treated with temozolomide and/or G207 was analyzed using Kaplan-Meier analysis.


RESULTS
Temozolomide exhibited strong synergy with G207 in both MGMT-negative and the MGMT inhibitor O6-benzylguanine-treated MGMT-expressing gliomas (Chou-Talalay combination indices = 0.005 to 0.39) and induced GADD34 expression primarily in nonapoptotic MGMT-negative U87 glioma cells (fold difference = 16, 95% confidence interval [CI] = 12.6 to 20.4, compared with untreated cells). MGMT-expressing T98 and U87/MGMT cells treated with temozolomide plus O6-benzylguanine had higher RR expression than untreated cells (fold difference =14.9, 95% CI = 10.1 to 22.0 [T98]; 9.9, 95% CI = 7.0 to 13.8 [U87/MGMT]). GADD34 and RR knockdown increased temozolomide-induced DNA damage and inhibited the synergy of G207 and temozolomide in U87 and O6-benzylguanine-treated U87/MGMT cells. Mice bearing intracranial U87 tumors survived longer after combination therapy (100% survival at 90 days) than after single-agent therapy (median survival = 46 and 48 days with G207 and temozolomide treatment, respectively).


CONCLUSIONS
Temozolomide-induced DNA repair pathways vary with MGMT expression and enhance HSV-mediated oncolysis in glioma cells. These findings unveil the potential of HSV to target cells surviving temozolomide treatment.
Aghi
Manish
M
Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. maghi@partners.org
Rabkin
Samuel
S
Martuza
Robert L
RL
D000818:Animals; D000943:Antigens, Differentiation / Q000187:drug effects / Q000378:metabolism; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D020552:Comet Assay; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects*; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004357:Drug Synergism; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005909:Glioblastoma / Q000188:drug therapy* / Q000201:enzymology; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism*; D050504:Oncolytic Viruses / Q000254:growth & development*; D010957:Plasmids; D054645:Protein Phosphatase 1; D034741:RNA, Small Interfering / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012264:Ribonucleotide Reductases / Q000235:genetics; D018139:Simplexvirus / Q000254:growth & development*; D000077204:Temozolomide; D014158:Transcription, Genetic; D014162:Transfection; D014183:Transplantation, Heterologous
D000943:Antigens, Differentiation; D018906:Antineoplastic Agents, Alkylating; D018797:Cell Cycle Proteins; D034741:RNA, Small Interfering; D006147:Guanine; D003606:Dacarbazine; C008449:O-(6)-methylguanine; D012264:Ribonucleotide Reductases; D019853:O(6)-Methylguanine-DNA Methyltransferase; C087519:PPP1R15A protein, human; C513256:Ppp1r15a protein, mouse; D054645:Protein Phosphatase 1; D000077204:Temozolomide