Constitutive Impaired Expression of Antiviral Effectors Sensitizes Melanoma Cells Viral Oncolysis

Introduction:

Immunoediting of malignant cells commonly dampens the function of interferon (IFN) antiviral responses and the expression of IFN stimulated genes (ISGs), making cancer cells potentially susceptible for viral attacks. Oncolytic virotherapy exploits such characteristics to specifically infect and kill cancer cells and stimulate anti-tumor immunity. In recent years, the Ehrlich and Bacharach labs at the Tel Aviv University have developed a novel oncolytic virus, the Epizootic Hemorrhagic Disease Virus-Tel Aviv University (EHDV-TAU). Starting from the Ibaraki strain of EHDV2, and employing an in vitro evolution process in IFN-defective human prostate cancer cells (LNCaP), we have selected the EHDV-TAU clone. Initial characterization of EHDV-TAU showed a million-fold increased replication efficiency in LNCaP cells, selectivity towards cancer cells (vs. normal cells), and the ability to induce different forms of cancer cell death (including apoptosis and necroptosis).^{1, 2} In the present study, we expand on published studies and probe for the efficiency and safety of EHDV-TAU with the B16F10 murine melanoma in immunocompetent C57BL6 mice.

Methods:

EHDV-TAU-mediated oncolysis of B16F10 was characterized in vitro and in vivo. The former line of experimentation, which employed immortalized skin fibroblasts (ISFs) as control, comprised measurements of viral RNA and proteins, production of infectious virions, and assessments of cell death. Moreover we also assessed the expression and function of mediators of innate immune antiviral responses, prior to, and following infection with EHDV-TAU. In vivo oncolysis assessed EHDV-TAU-mediated effects on tumor growth, mouse survival, and immune activation.

Results and Discussion:

The susceptibility of B16F10 melanoma cells to EHDV-TAU infection and oncolysis was markedly higher than that of ISF cells, with high cell death rate and increased formation of infectious particles. The increased susceptibility correlated with near-absent constitutive expression of viral sensors and ISGs, and with the inability to mount JAK/STAT-based antiviral responses upon exposure to EHDV-TAU. Preliminary analysis with inhibitors of DNA methylation and histone deacetylation suggest the role of epigenetic silencing of antiviral genes in B16F10 cells. Notably, infected B16F10 cells retained the ability to secrete immune-attractants such as CXCL1, CXCL2 and CXCL10, suggesting their ability to activate immune cells. Indeed, in vivo, EHDV-TAU markedly reduced tumor volume, enhanced survival and promoted intratumoral infiltration of effector immune cells.

Conclusions:

Our results suggest that EHDV-TAU may function as a novel and effective anti-tumor agent. Moreover, we propose that epigenetic silencing of antiviral genes may serve as a molecular mechanism for the acquired Achilles’ Heel of cancer cells when challenged with oncolytic viruses.

1. Danziger O, Pupko T, Bacharach E, Ehrlich M. Interleukin-6 and Interferon-alpha Signaling via JAK1-STAT Differentially Regulate Oncolytic versus Cytoprotective Antiviral States. Front Immunol 2018;9: 94.
2. Danziger O, Shai B, Sabo Y, Bacharach E, Ehrlich M. Combined genetic and epigenetic interferences with interferon signaling expose prostate cancer cells to viral infection. Oncotarget 2016;7: 52115-34.