Reestablishment of p53/Arf and interferon-\u03b2 pathways mediated by a novel adenoviral vector potentiates antiviral response and immunogenic cell death

Aline Hunger,Daniela B Zanatta,Thiago De Almeida Salles,Bryan E Strauss,Paulo R Del Valle,Silvina O Bustos,Renata De Freitas Saito,Christian A Merkel,Eugenia Costanzi-Strauss,Daniel G Ferrari,Tatiane K Furuya,Ruan Fv Medrano

doi:10.1038/cddiscovery.2017.17

Abstract

Late stage melanoma continues to be quite difficult to treat and new therapeutic approaches are needed. Since these tumors often retain wild-type p53 and have a strong immunogenic potential, we developed a gene transfer approach which targets these characteristics. Previously, we have shown that combined gene transfer of p19Arf and interferon-beta (IFNβ) results in higher levels of cell death and superior immune-mediated antitumor protection. However, these experiments were performed using B16 cells (p53wt) with forced expression of the adenovirus receptor and also the mechanism of death was largely unexplored. Here we take advantage of a novel adenoviral vector (AdRGD-PG), presenting an RGD-modified fiber as well as a p53-responsive promoter, in order to investigate further potential benefits and cell death mechanisms involved with the combined transfer of the p19Arf and IFNβ genes to the parental B16 cell line. Simultaneous p19Arf and IFNβ gene transfer is more effective for the induction of cell death than single gene treatment and we revealed that p19Arf can sensitize cells to the bystander effect mediated by secreted IFNβ. Strikingly, the levels of cell death induced upon activating the p53/p19Arf and interferon pathways were higher in the presence of the AdRGD-PG vectors as compared to approaches using pharmacological mimetics and this was accompanied by the upregulation of antiviral response genes. Only combined gene transfer conferred immunogenic cell death revealed by the detection of key markers both in vitro and in vivo. Finally, whole-genome transcriptome analysis revealed unique expression profiles depending on gene function, including immune activation, response to virus and p53 signaling. In this way, cooperation of p19Arf and IFNβ activates the p53 pathway in the presence of an antiviral response elicited by IFNβ, culminating in immunogenic cell death.

Highlights

Malignant melanoma is a type of cancer with high death rates due, in part, to the lack of efficient treatments once metastases have formed.[1]
These results indicate a strong bystander effect mediated by IFNβ that is further enhanced in the presence of p19Arf
The bystander effect of IFNβ would be interesting in vivo since we do not expect that all tumor cells are transduced upon gene therapy

Summary

Introduction

Malignant melanoma is a type of cancer with high death rates due, in part, to the lack of efficient treatments once metastases have formed.[1] The tumor suppressor protein p53 is found in its wild-type form in 90% of melanoma cases, though other components of this pathway may be altered. We showed that introduction of the p53 functional partner, p19Arf (p19Arf in mice, p14ARF in humans) into B16 melanoma cells restored the p53 pathway and promoted cell death in vitro and in vivo.[8] the impact of apoptotic cell death may be limited and is not likely to actively promote an antitumor immune response. We sought to develop a cancer gene therapy approach that would induce cell death by a mechanism that would have wide-spread anti-cancer effects that reach beyond the treated cell

Methods

Results

Conclusion