*2.1. JAK-STAT Signaling Inhibition*

Inhibitors of Janus kinases (JAK), such as JAK inhibitor I (a pan-JAK inhibitor) or ruxolitinib (a specific JAK1/2 inhibitor) (Figure 1), were able to rescue the replication of VSV in several human pancreatic ductal adenocarcinoma (PDA) cells that were otherwise resistant due to constitutive high-level expression of certain interferon stimulated genes (ISGs) [38,45,46]. This effect was improved even further when Polybrene or DEAE-dextran were additionally added, improving VSV attachment and entry and allowing more cells to be infected [47]. A similar effect was seen for refractory human head and neck squamous cell carcinoma (HNSCC) cell lines which owed their VSV resistance to the constitutive expression of a different set of ISGs. Here, JAK inhibitor I and ruxolitinib were also successful

in rescuing virus replication with a 100- to 1000-fold increase in yield. Interestingly, other innate immune small molecule compounds, such as histone deacetylase inhibitors (HDI; LBH589), phosphoinositide 3-kinase (PI3K) inhibitors (GDC-0941, LY294002), mammalian target of rapamycin complex 1 (mTORC1) inhibitors (rapamycin) or STAT3 inhibitor VII were not effective [48]. Combination therapy with ruxolitinib and VSV-IFNβ also enhanced viral replication and oncolysis in several non-small cell lung cancer (NSCLC) cell lines [49]. However, several of these compounds were effective in rescuing OV replication in other tumor cell types, as discussed in the sections below underlining the heterogeneity in mechanisms among different tumor cells by which synergy with OVs can occur. In melanoma, mutations in the JAK1/2 signaling pathway as well as JAK1/2 inhibition increase sensitivity to VSV-dM51 [50]. The dual inhibitor of JAK1 and IκB kinase (IKK), TPCA-1 was also shown to improve HSV replication of malignant peripheral nerve sheath tumor (MPNST) cells [51]. OVs that replicate in the cytoplasm, such as RNA viruses and poxviruses, can also trigger direct antiviral effector responses that can hamper their replication and subsequent oncolytic effects. Viral RNA activates the cytosolic PKR by inducing dimerization and subsequent auto-phosphorylation reactions. The protein kinase R (PKR) pathway leads to a stress response by activating other pathways such as the interconnected nuclear factor κ-light chain enhancer of activated B cells (NF-κB) & c-Jun N-terminal kinase (JNK) pathways (Figure 2) [52–54]. JNK are kinases involved in a diverse set of cellular functions, ranging from cell death, survival and proliferation to innate immunity [54]. Specifically, JNK are essential for the expression regulation of many immune mediator genes, such as cytokines (e.g., interleukins (ILs) IL-2, IL-4, IL-8, IL-18, IFN-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), C-C motif chemokine ligand 5 (CCL5), tumor necrosis factor α (TNF-α)) [55–59] and adhesion molecules (ICAM-1) [53]. While JNK inhibition has been reported to act antivirally on encephalomyocarditis virus, rotavirus and HSV [60–62], a virus promoting effect was seen for vaccinia virus. Here, murine embryonic fibroblasts devoid of JNK showed a significant increase in titer. In line with these results, an increase of apoptosis was seen when wildtype murine embryonic fibroblast cells were co-treated with the JNK-specific inhibitor SP600125 [55,63]. This suggests that JNK inhibition, at least under very specific conditions, can be beneficial for OV therapy [63].
