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Up-regulation of GADD34 mediates the synergistic anticancer activity of mitomycin C and a ₁34.5 deleted oncolytic herpes virus (G207) ¹

JOSEPH J. BENNETT^*, PRASAD ADUSUMILLI^*, HENRIK PETROWSKY^*, BRYAN M. BURT^*, GRETCHEN ROBERTS^*, KEITH A DELMAN^*, JONATHAN S. ZAGER^*, TING-CHAO CHOU and YUMAN FONG^*,2

^* Department of Surgery and ²Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

² Correspondence: Hepatobiliary Division, Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021, USA. E-mail: fongy{at}mskcc.org

SPECIFIC AIMS

1. Determine whether oncolytic herpes viral therapy is synergistic with chemotherapy in treatment of gastric cancer
Oncolytic herpes simplex viral therapy is a promising experimental cancer treatment that exploits the ability of genetically engineered HSV to specifically infect, replicate within, and kill tumor cells. The current study seeks to determine in vitro and in vivo whether the oncolytic virus G207 is synergistic with the alkylating agent mitomycin C in treatment of human gastric cancer cells, a tumor highly resistant to standard therapies.

2. Examine the mechanistic basis of such synergy
One strategy for genetically engineering herpes viruses to be less neurovirulent and thereby to be safer cancer-killing agents is deletion of the viral ₁34.5 gene, which bears great homology to the mammalian GADD34 gene. The current experiments aim to determine whether use of the chemotherapy agent mitomycin C will selectively up-regulate the DNA repair gene GADD34 in tumor and whether this would complement the ₁34.5 gene deletion and augment viral antitumor efficacy.

PRINCIPAL FINDINGS

1. Using the isobologram method and a combination index method of Chou-Talalay, significant synergism was demonstrated in vitro between mitomycin C (MMC) and G207
G207 and MMC each demonstrated dose-dependent cytotoxicity against OCUM-2MD3 and MKN-45-P gastric cancer cells, and combination therapy showed greater efficacy than the expected additive effect. A combination index method and the isobologram method were used to demonstrate synergy between G207 and MMC in treating these two cell lines (Fig. 1 ).

View larger version (10K): [in this window] [in a new window]   Figure 1. Combination therapy using mitomycin C and G207 demonstrates a synergistic interaction over the range of doses evaluated. The Chou-Talaley combination index method of evaluating synergy was performed. The Fa-CI plot was constructed using experimental data points (dark circles) and by simulating CI values over the entire range of Fa values from 5 to 95% (solid line) using CalcuSyn software. The additive effect of G207 and MMC is represented at CI = 1 (dotted line). CI < 1 indicates synergism. G207 and MMC combination therapy results in strong synergy for the MKN-45-P cell line at all levels.

2. As a result of such synergism, a dose reduction for each agent can be accomplished over a wide range of drug effect levels without sacrificing tumor cell kill
The dose reduction index was calculated. For the OCUM-2MD3 cell line, MMC and G207 doses could be lowered by 2- to 3-fold when given as combination therapy. For the MKN-45-P cell line, MMC doses could be lowered 2- to 9-fold and G207 doses could be lowered 2- to 4-fold when administered as combination therapy (Fig. 2 ).

View larger version (18K): [in this window] [in a new window]   Figure 2. Combination chemotherapy and oncolytic viral therapy to kill gastric cancer cells demonstrate enhanced efficacy vs. single agent therapy alone. MKN-45-P gastric cancer cells were treated with different doses of mitomycin C (µg/cc) or G207 (MOI). Combination therapy was performed to keep the ratio of MMC:G207 constant at 1:25 for MKN-45-P cells. A standard MTT assay was used to assess cytotoxicity for each treatment group, with results presented as % survival compared with control.

3. Mitomycin increased viral production by tumor cells
The synergy is likely due to increased viral production. In the presence of 0.01 µg/cc MMC, a 24-fold increase in viral titers over titers for G207 infection alone was observed over 5 days postinfection. In the presence of 0.02 and 0.04 µg/cc MMC, there was an 8- and 2-fold increase in viral yields respectively.

4. MMC treatment increased expression of GADD34
Northern blot analysis confirmed that expression of GADD34 mRNA was increased by mitomycin C treatment. At 24 h after treatment, a 2.49-fold increase in the intensity of the GADD34 band was measured compared with the negative control. At 48 h, a 3.21-fold increase in intensity was noted.

5. Inhibition of GADD34 expression decreased viral proliferation and decreased synergistic effects of MMC with G207
Using siRNA directed at GADD34, the synergistic cytotoxicity of MMC with oncolytic viral therapy was inhibited. This was associated with a significant decrease in viral proliferation.

6. Gastric carcinomatosis was more effectively treated with a combination of G207 and MMC
In a murine model of peritoneal dissemination of gastric cancer, carcinomatosis was treated with i.p. injection of G207, MMC, or a combination of these agents. Mean peritoneal tumor burden (±SE) was 2470 (±330) mg for control mice, 1210 (±300) mg for mice treated with 1 x 10⁶ pfu of G207 (P=0.02 vs. controls), 1490 (±310) mg for mice treated with 0.1 mg/kg MMC (P=0.06 vs. controls), and 350 (±150) mg for combined G207 (1x10⁶ pfu) MMC (0.1 mg/kg) treatment (P<0.001 vs. controls, P=0.03 vs. G207, and P=0.01 vs. MMC). High-dose viral therapy with G207 (5x10⁶ pfu) resulted in a mean tumor burden of 990 (±320) mg (P<0.01 vs. controls). Combination therapy using 5 x 10⁶ pfu of G207 and 0.1 mg/kg MMC resulted in a mean tumor burden of 100 (±60) mg (P<0.01 vs. controls, P=0.04 vs. 5x10⁶ pfu of G207 alone, P<0.01 vs. MMC alone).

CONCLUSIONS AND SIGNIFICANCE

1. G207 is an attenuated, multimutated HSV-1 that has demonstrated cytotoxic activity against a wide variety of tumor cell types. In vitro and in vivo data presented in this experiment demonstrate a synergistic interaction between MMC and G207 in gastric cancer, a tumor type particularly resistant to conventional therapies. In the current study, the addition of only 0.1 mg/kg MMC to 1 x 10⁶ pfu of G207 resulted in a major improvement in tumor kill. Based on these data, a significant dose reduction can be accomplished by combination therapy without reduction in efficacy. These data should be considered when designing future clinical trials and should encourage evaluation of such combined therapy against other tumor cell types.

2. The ₁34.5 gene was deleted from many herpes oncolytic viruses in order to decrease toxicity but this deletion also reduces anti-tumor efficacy. Some groups have therefore attempted to selectively restore ₁34.5 function in tumor cells. The ₁34.5 genes have been inserted under control of transcriptionally regulated promoter to facilitate selective gene expression in rapidly dividing cells. Although this approach is promising, insertion of the entire ₁34.5 gene may theoretically restore neurovirulence in addition to providing any improvements in tumor cell kill. GADD34 function, however, is separate from the virulence phenotype. Mitomycin C therapy may therefore provide a selective means of restoring the GADD34 phenotype in tumor cells without the potential risk of increasing neurovirulence. The current study demonstrates that by pharmacologic induction of GADD34 expression, replication and antitumor efficacy of this ₁34.5-deficient mutant virus are greatly enhanced. These data indicate that induction of GADD34 selectively restores the virulent phenotype of the deleted gene in G207, providing a cellular basis for the combined use of DNA-damaging agents and ₁34.5 HSV mutants in the treatment of cancer.

3. A series of five growth arrest and DNA damage-inducible (gadd) genes has been identified in mammalian cells. Our data demonstrate that DNA damage from mitomycin C resulted in high expression of GADD34, which can functionally restore the ₁34.5 phenotype. Other stimuli of cellular injury including ionizing radiation, medium-depletion, and other chemotherapies can also induce GADD expression. Treatment of mammalian cells with hydroxyurea and methyl methanesulfonate results in elevated levels of mRNA for GADD34. These data encourage further study of these agents for potential synergistic actions with herpes oncolytic viruses.

4. This study was performed in an experimental model of gastric carcinomatosis because few effective therapies exist for this disease. A significant number of patients presenting with gastric cancer already have gross peritoneal disease, and those who undergo resection without obvious peritoneal disease will likely recur in the peritoneum. The peritoneum is therefore a clinically relevant target if survival is to be affected in those patients with gastric cancer. The diffuse nature of carcinomatosis precludes surgery as an option for cure in most patients. Although regional chemotherapy has been used for peritoneally disseminated gastric cancer, significant morbidity has been demonstrated that may outweigh any potential benefits. A model of gastric carcinomatosis is a paradigm for therapy of widely disseminated malignancy. Current preclinical studies demonstrate that combining chemotherapy with oncolytic viral therapy is a promising strategy for treating this common site of cancer failure.

View larger version (12K): [in this window] [in a new window]   Figure 3. Schematic diagram. Possible mechanism of synergy between MMC and G207.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1080fje;

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