Abstract 1499: Gene therapy in a mouse model for malignant melanoma: Combining the activities of the p53/Arf and interferon pathways

Abstract

Abstract Background: Malignant melanoma cells typically maintain wild-type p53 in a non-functional state, due in part to the loss of ARF (p14ARF in humans, p19ARF in mice). Data have shown that interferon-β (IFNβ) can activate the transcription of p53 and interact with the ARF pathway as well. By using a p53-driven adenovirus, we sought to combine the action and regulation of p53, p19ARF and IFNβ for the treatment of malignant melanoma in an immunocompetent mouse model. Methods: p53-responsive AdPG adenoviral vectors (Bajgelman and Strauss, 2008) containing p53, p19ARF and IFNβ were produced and expression verified by immunofluorescence and ELISA. In vitro evaluation of the antiproliferative effect of single or co-transduction of B16F10 cells (B16, p53 wild-type) was done by FACS after annexin/PI staining. C57/Bl6 mice were injected subcutaneously with B16 cells and tumors were allowed to develop (∼0.5 cm2). At this point, three consecutive intra-tumor injections (3 day interval between injections) of individual or combined adenoviral vectors were performed. Animals were then sacrificed and tumors collected and analyzed. Results: The combination of IFNβ and p19ARF proved to be more effective in inhibiting proliferation of B16 cells than either treatment alone, both in vitro and in vivo. In vitro we observed that neither AdPGp19 nor AdPGIFNβ alone altered the cell cycle, though co-transduction with these vectors resulted in extensive cell death. Annexin-V staining was also increased by the combined, but not individual, treatments. Cell death was observed only after 90-120h of incubation, yet prior to this event, indicators of senescence (positive SA-βGal staining, loss of histone H3 acetylatation at Lys9) were revealed in both single and combined vector treatments. In vivo, we observed that the combined gene transfer strategy led to reduced tumor volume, reduced incorporation of BrdU and increased staining by TUNEL in the primary tumor. Assays are in progress to determine if the combined treatment is effective in reducing metastases and prolonging survival. Conclusion: The use of p53-responsive vectors for the restoration of ARF expression in the presence of IFNβ represents a potential strategy for melanoma tumor suppression. While mechanistic studies are underway, we propose that the complementation of the p53/ARF and interferon pathways in the primary tumor may lead to reduction of metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1499.