*2.6. Receptor Tyrosine Kinase Inhibitor*

In the antiviral context, direct inhibition of PKR and Rnase was also achieved by another class of small molecule compounds. The ATP-competitive inhibitor of vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptors, sunitinib, was reported also to be a strong inhibitor for both PKR and RnaseL [107] (Figure 2). These compounds also have more direct impact on tumor growth through their negative regulation of tumor vascularization. Due to their broader mode of action this group of inhibitors can be referred to in more general terms as receptor tyrosine kinase inhibitors (RTKIs). These compounds proved to be very beneficial when combined with oncolytic VSV, leading to the elimination of prostate, breast, and kidney malignant tumors in mice [108]. Synergistic effects with RTKIs were also shown for vaccinia and reovirus in pancreatic neuroendocrine tumors and renal cell carcinoma, respectively [109,110], as well as for the combination with HSV in glioblastoma [111]. However, vaccine virus is also connected to the activation of the epidermal growth factor receptor (EGFR) pathway for their replication and spread. Here, simultaneous administration of RTKIs, such as imatinib and sorafenib, resulted in the inhibition of vaccinia virus replication [112,113]. Nonetheless, oncolytic vaccinia virotherapy, followed by sorafenib treatment, showed enhanced efficacy compared to either monotherapy. This is most likely due to OV-mediated sensitization of the tumor cells and tumor vasculature to VEGF/VEGFR inhibitors [112]. Part of these reported benefits are also achieved through modulation of the tumor microenvironment. When MC38 tumor bearing mice were pretreated with sunitinib, the anti-tumor response, induced by a tumor associated antigen (TAA)-armed virus, was markedly improved through a decrease in inhibitory regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) after sunitinib treatment [114]. This adaptive immune modulation is achieved by interacting with RTKs expressed on regulatory immune cell populations, such as c-KIT and VEGFR-1 [115,116]. In a similar setting, the more broad-range RTK inhibitor cabozantinib also showed a more diverse and potent effect and immunomodulatory effects with additional expression of MHC-I molecules, ICAM-1, Fas, and calreticulin on tumor cells. Modulation of antigen expression is most likely to be facilitated by its hepatocyte growth factor receptor (MET) inhibition [117]. Another more specific EGFR inhibitor, erlotinib, also seems to enhance the oncolytic effect in some human pancreatic cancer cells through a similar mechanism for oncolytic HSV. Here, prolonged viral presence was reported [118]. On the other hand, in tumors, characterized by upregulated EGFR signaling, the synergism seemed predominantly driven by a concerted antiangiogenic effect [119].

*Cancers* **2021**, *13*, x 10 of 33

**Figure 4.** Increasing bystander killing of tumor cells by small molecules after OV treatment. (**A**) Cytokines produced in response to OV treatment of the tumor can activate the extrinsic pathway for apoptosis through binding with death receptors such as Fas and TNF-α receptor. Oligomerization of these receptors in turn facilitates the recruitment of adaptor proteins, for example, binding of Fas ligand with Fas recruits caspase-8 through the adaptor protein FADD. Cleaved **Figure 4.** Increasing bystander killing of tumor cells by small molecules after OV treatment. (**A**) Cytokines produced in response to OV treatment of the tumor can activate the extrinsic pathway for apoptosis through binding with death receptors such as Fas and TNF-α receptor. Oligomerization of these receptors in turn facilitates the recruitment of adaptor proteins, for example, binding of Fas ligand with Fas recruits caspase-8 through the adaptor protein FADD. Cleaved caspase-8 can directly

activate caspase-3 and result in cell death. (**B**) Additionally, cleaved caspase-8 connects to the pathways of intrinsic apoptosis. This occurs when it cleaves Bid. Truncated Bid subsequently translocates to the mitochondria where it induces cytochrome release leading to activation of caspase-9 and caspase-3. This cytochrome c release is facilitated by the oligomerization of the pro-apoptotic Bax and Bak proteins at the outer mitochondria membrane. This process stands under the control of several proteins including Bcl-2, Bcl-xL and MCL-1. These pro-survival proteins in turn are inhibited by "BH3 only" proteins. (**C**) Intrinsic apoptosis can also be additionally stimulated through compounds that induce DNA damage, since this leads to p53 upregulation, resulting in indirectly Bax/Bak activation. ER stress signaling, caused by the accumulation of misfolded protein in the ER, can also facilitate this effect through ASK1 with the activation and subsequent translocation of JNK to the mitochondrial membrane. In addition, ER stress can also promote cell death through the activation of MAPK-mediated activation of eIF2α and ATF4 leading to the nuclear translocation of CHOP where it promotes transcription of pro-apoptotic genes. Apart from promoting cell death, eIF2α and TRAF2 also attenuates protein translation when misfolded protein accumulate in the ER. Since this is often the case during OV replication, the inhibition of these mechanisms can improve the efficacy of OV treatment. (**D**) Also the stabilizing or destabilizing of microtubules can trigger apoptosis. More specially, when cells are arrested G2/M phase, this can lead to the activation of intrinsic apoptosis. Targeting these pathways can improve oncolysis, tumor immunogenicity and viral replication depending on what aspect of cell death is targeted. Small molecule compounds targeting different stages of this process are presented by red annotated squares. See the main text for more information. Created with biorender.com. (**E**) Selected chemical structures of compounds depicted in panels A-D. All structures throughout were drawn using MarvinSketch (ChemAxon) from publicly available information. Abbreviations: TRADD, TNFR1-associated death domain protein; TRAF2, TNF receptor-associated factor 2; cIAP, cellular inhibitor of apoptosis; RIP, receptor interacting protein; FADD, fas-associated death domain; BH3, BCL-2 homology domain 3; SMC, Second mitochondria-derived activator of caspase mimetic compounds; Ub, ubiquitin; MCL-1; myeloid cell leukemia 1; XIAP, X-linked inhibitor of apoptosis protein; BID, BH3 interacting-domain death agonist; tBID, truncated Bid; AdV, adenovirus; JNK, c-Jun NH2-terminal kinase; BCL-xL, B-cell lymphoma, extra-large; BCL-2, B-cell lymphoma 2; BAX, BCL2 associated X; BAK, Bcl-2 homologous antagonist killer; Apaf-1, apoptotic protease activating factor-1; ASK1, Apoptosis signal-regulating kinase 1; CHOP, CCAAT-enhancer-binding protein homologous protein; DOX, downstream of CHOP; DR5, death receptor 5 (DR5); MDA, microtubule-destabilizing agents; MSA, microtubule-stabilizing agent; ATF4, Activating transcription factor 4; PERK, PRKR-like endoplasmic reticulum kinase; IRE1, inositol-requiring enzyme; CP, cyclophosphamide; Gem, gemcitabine; 5-Fu, fluorouracil; GBF-1, Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1; GCA-2, GBF-1 inhibitor golgicide A; P, phosphorylated; VCP, valosin-containing protein; eIF2α, eukaryotic translation initiation factor 2α.
