Cancer Therapy: Preclinical Synergistic Effects of Oncolytic Reovirus and Cisplatin Chemotherapy in Murine Malignant Melanoma

Purpose: To test combination treatment schedules of reovirus and cisplatin chemotherapy in human and murine melanoma cell lines and murine models of melanoma and to investigate the possible mechanisms of synergistic antitumor effects. Experimental Design: The effects of reovirus ± chemotherapy on in vitro cytotoxicity and viral replication were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and plaque assay. Interactions between agents were assessed by combination index analysis. Mode of cell death was assessed by Annexin V/propidium iodide fluorescence-activated cell sorting–based assays; gene expression profiling of single versus combination treatments was completed using the Agilent microarray system. Single agent and combination therapy effects were tested in vivo in two immunocompetent models of murine melanoma. Results: Variable degrees of synergistic cytotoxicity between live reovirus and several chemotherapy agents were observed in B16.F10 mouse melanoma cells, most significantly with cisplatin (combination index of 0.42 ± 0.03 at ED50). Combination of cisplatin and reovirus exposure led to increased late apoptotic/necrotic cell populations. Cisplatin almost completely abrogated the inflammatory cytokine gene up-regulation induced by reovirus. Combination therapy led to significantly delayed tumor growth and improved survival in vivo (P < 0.0001 and P = 0.0003, respectively). Cisplatin had no effect on the humoral response to reovirus in mice. However, cisplatin treatment suppressed the cytokine and chemokine response to reovirus in vitro and in vivo. Conclusion: The combination of reovirus and several chemotherapeutic agents synergistically enhanced cytotoxicity in human and murine melanoma cell lines in vitro and murine tumors in vivo. The data support the current reovirus/chemotherapy combination phase I clinical studies currently ongoing in the clinic. (Clin Cancer Res 2009; 15(19):6158–66) There has been rapid preclinical and clinical development of viruses with oncolytic activity as anticancer therapeutics (1). As with other novel agents, the true potential of this approach to cancer treatment may only be realized in combination with other treatment modalities. Reoviruses (respiratory enteric orphan viruses) are ubiquitous, nonpathogenic viruses that have been isolated from the human respiratory and gastrointestinal tract. They are nonenveloped icosahedral viruses with a segmented genome composed of double-stranded RNA. To date, reovirus infection in humans has not been associated with any known disease. Reovirus has innate oncolytic potential in an extensive range of murine and human tumor cells, and this is, at least in part, dependent on the transformed state of the cell (2, 3). The precise mechanism of reoviral tropism and selective oncolysis in malignant cells is yet to be fully determined. In normal cells, the presence of an intact double-stranded RNA–activated protein kinase system prevents the establishment of a productive infection. In malignant cells with activated Ras pathway (or up-regulation of upstream or downstream components of the cell signaling pathway) through either Ras mutation or upregulated epidermal growth factor receptor (EGFR) signaling (4–6), this cellular antiviral response mechanism is perturbed, and viral replication and subsequent lysis of the host cell results. Reovirus has been shown to cause tumor regression after intralesional injections in immunodeficient mice and after systemic administration in immunocompetent mice (4, 5). In Authors' Affiliations: Oncology, Postgraduate Medical School, University of Surrey, Guildford, United Kingdom; Cancer Research UK Clinical Centre, St. James's University Hospital, Leeds, United Kingdom; Oncolytics Biotech, Inc., Calgary, Alberta, Canada; and Targeted Therapy Team, Institute for Cancer Research, Chester Beatty Laboratories, London, United Kingdom Received4/1/09; revised5/18/09; accepted5/19/09; publishedOnlineFirst 9/22/09. Grant support: Prostate Project (H.S. Pandha, L. Heinemann, andR.Morgan), University of Surrey (G.R. Simpson), Cancer Research UK (A. Melcher, R. Prestwich, F. Errington, and K.J. Harrington), and Oncolytic Biotech, Inc. (M. Coffey, K.J. Harrington, and A. Mecher). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Hardev S. Pandha, Oncology, Postgraduate Medical School, University of Surrey, Daphne Jackson Road, Manor Park, Guildford, Surrey GU2 7WG, United Kingdom. Phone: 44-1483-688550; Fax: 44-1483688558; E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-09-0796 6158 Clin Cancer Res 2009;15(19) October 1, 2009 www.aacrjournals.org Research. on April 14, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Published OnlineFirst September 22, 2009; DOI: 10.1158/1078-0432.CCR-09-0796