Background: NV1066, a replication-competent oncolytic herpes simplex virus type 1 (HSV-1) attenuated by a deletion in the gene γ 1 34.5, preferentially replicates in and kills malignant cells. γ 1 34.5 encodes ICP34.5, a viral protein essential for productive replication, which has homology with mammalian stress response induced GADD34 (growth arrest and DNA damage-inducible protein). We hypothesized that cisplatin upregulates GADD34 expression, which enhances NV1066 replication and oncolysis.Methods: Ten human malignant pleural mesothelioma (MPM) cell lines were infected with NV1066 at multiplicities of infection (MOI; ratio of viral particles per tumor cell) 0.005 to 0.8 in vitro, with and without cisplatin (1 to 4 µM). In the MPM cell line VAMT, viral replication was determined by plaque assay, cell kill by lactate dehydrogenase assay, and GADD34 induction by quantitative RT-PCR and Western blot. Synergistic efficacy was confirmed by the isobologram and combination index methods of Chou-Talalay. GADD34 upregulation by cisplatin was inhibited with GADD34 siRNA to further confirm the synergistic efficacy dependence with GADD34.Results: Combination therapy with NV1066 and cisplatin showed strong synergism in epithelioid (H-2452, H-Meso), sarcomatoid (H-2373, H-28), and biphasic (JMN, Meso-9, MSTO-211H) MPM cell lines, and an additive effect in others. In VAMT cells combination therapy enhanced viral replication 4 to11-fold (p < 0.01) and cell kill 2 to 3-fold (p < 0.01). Significant dose reductions for both agents (2 to 600-fold) were achieved over a wide range of therapeutic-effect levels (LD 50 -LD 99 ) without compromising cell kill. Synergistic cytotoxicity correlated with GADD34 upregulation (2 to 4-fold, p < 0.01) and was eliminated following transfection with GADD34 siRNA.Conclusion: Cisplatin-induced GADD34 expression selectively enhanced the cytotoxicity of the γ 1 34.5-deficient oncolytic virus, NV1066. This provides a cellular basis for combination therapy with cisplatin and NV1066 to treat MPM and achieve synergistic efficacy, while minimizing dosage and toxicity.

Background-Oncolytic herpes viruses are attenuated, replication-competent viruses that selectively infect, replicate within, and lyse cancer cells and are highly efficacious in the treatment of a wide variety experimental cancers. The current study seeks to define the pharmacologic interactions between chemotherapeutic drugs and the oncolytic herpes viral strain NV1066 in the treatment of pancreatic cancer cell lines.

Purpose: Although a variety of malignant tumors are susceptible to therapy with oncolytic herpes simplex viruses, the determinants of tumor sensitivity to these viruses are poorly understood. Nectin-1 is a cell surface adhesion molecule that is a component of intercellular adherens junctions and also functions as a herpes viral receptor. Because highly invasive cells may have decreased intercellular adhesion, we sought to determine if such cells might also have altered availability of cell surface nectin-1 to act as a herpes receptor. Experimental Design and Results: A series of squamous cell carcinoma lines of increasing migratory and invasive potential, termed MG1-MG14, were selected by serial passages of murine SCC7 through Matrigel invasion chambers. Available cell surface nectin-1 was enhanced on the MG11 and MG14 cell lines in comparison to SCC7 as measured by cellular ELISA and immunofluorescence microscopy. A replication-competent, oncolytic herpes virus (NV1023) showed an increased ability to enter MG11and MG14 cells as compared with SCC7 cells. Furthermore, MG11 and MG14 supported increased herpes viral replication and cytotoxicity over SCC7. For all three of the cell lines, viral entry assays revealed that the actively migrating cells were significantly more susceptible to herpes infection than the nonmigrating cells. Conclusions: These results show that malignant cells with highly migratory and invasive properties may exhibit increased cell surface nectin-1availability, which may serve as a herpes viral receptor to enhance the efficacy of herpes oncolytic therapy. This finding has implications regarding patient selection for future clinical trials using these promising therapeutic vectors.Attenuated, replication-competent oncolytic viruses based on herpes simplex type 1 (HSV-1) have potent therapeutic effects in treating a variety of human malignancies. Studies have shown therapeutic efficacy in animal models of brain, breast, colorectal, prostate, ovarian, lung, pancreatic, gastric, esophageal, and head and neck cancers (1 -9). Recent phase 1 clinical trials have shown encouraging early results and suggest that these viruses may be safe for clinical application (10 -12). Although many different malignancies can be effectively treated with oncolytic HSV in animal models, the specific determinants that define tumor sensitivity to herpes viral therapy remain poorly understood. We examined a series of attenuated, oncolytic HSV and have repeatedly observed that they possess a greater affinity for malignant cells over normal cells in vivo (13 -15). We have also noted a degree of variability in the sensitivity of different malignant cell lines to infection, replication, and lysis by these herpes viruses (8, 9). These observations suggest that there are tumor-related factors which determine sensitivity to herpes viral infection, replication, and oncolysis. In this study, we sought to examine the expression of a herpes viral receptor as a potential determinant of herpes viral sensitivity.In the natu...

Purpose-Herpes simplex virus-one (HSV-1) oncolytic therapy and gene therapy are promising treatment modalities against cancer. NV1066, one such HSV-1 virus carries a marker gene for enhanced green fluorescent protein (EGFP). The purpose of this study was to determine whether NV1066 is cytotoxic to lung cancer and whether EGFP is a detectable marker of viral infection in vitro and in vivo. We further investigated whether EGFP expression in infected cells can be used to localize the virus and to identify small metastatic tumor foci (< 1 mm.) in vivo by means of minimally invasive endoscopic systems equipped with fluorescent filters.Experimental Design-In A549 human lung cancer cells, in vitro viral replication was determined by plaque assay, cell kill by LDH release assay, and EGFP expression by flow cytometry. In vivo, A549 cells were injected into the pleural cavity of athymic mice. Mice were treated with intrapleural injection of NV1066 or saline and examined for EGFP expression in tumor deposits using a stereomicroscope or a fluorescent thoracoscopic system. Results-NV1066 replicated in, expressed EGFP in infected cells and killed tumor cells in vitro.In vivo, treatment with intrapleural NV1066 decreased pleural disease burden, as measured by chest wall nodule counts and organ weights. EGFP was easily visualized in tumor deposits, including microscopic foci, by fluorescent thoracoscopy.Conclusions-NV1066 has significant oncolytic activity against a human NSCLC cell line and is effective in limiting the progression of metastatic disease in an in vivo orthotopic model. By incorporating fluorescent filters into endoscopic systems, a minimally-invasive means for diagnosing small metastatic pleural deposits and localization of viral therapy for thoracic malignancies may be developed using the EGFP marker gene inserted in oncolytic herpes simplex viruses.

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