*3.3. Antagonizing Inhibitors of Apoptosis (IAPs)*

One major barrier to effective OV therapy is virus-induced expression of type I IFN and nuclear factor kappa B (NF-κB)-responsive cytokines, which can orchestrate an antiviral state in tumors. On the other hand, the subsequently produced cytokines (TNF-α, Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL), etc.) can also be exploited to induce tumor cell killing beyond the zone of initial infection, facilitated via co-treatment with a number of different pharmaceutical agents, such as SMAC-mimetic compounds (SMCs) [154,171,172] and B cell lymphoma-2 (BCL-2) homology domain 3 (BH3) mimetics [173,174]. Of note, tumor cells are often more sensitive to these chemical compounds than normal cells since NF-κB signaling is frequently constitutively activated [175], leading to elevated expression of proteins participating in cell death pathways [176].

The second mitochondria-derived activator of caspase (SMAC) is a pro-apoptotic factor released from the mitochondria during the process of cell death. Cytosolic SMAC can potentiate the activity of different caspases by inhibiting X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitors of apoptosis (cIAPs) (Figure 4B), which otherwise antagonize caspase cleavage [177]. SMAC mimetic compounds (SMCs) are small molecule mimetics of this cellular factor that can potentiate TRAIL- and TNF-α-mediated cell death (Figure 4A,B), especially in tumor cells where theses signaling pathways are aberrant [178]. Despite their potent effects on certain cell lines as a single agent due to the presence of endogenous TNF-α, SMAC mimetics are ineffective as a monotherapy in most tumor cell lines. In addition, drug resistance mechanisms include a SMC-induced upregulation of

cIAP2 [179] and LRIG1 [180]. As enhancers of pro-apoptotic stimuli, however, they act as strong enhancers of the cytotoxicity of many apoptosis-inducing therapies, such as OVs [181]. This synergy has been described for several SMCs and viruses (see Table 2) and is mainly facilitated by the cytokines produced in response to OV infection. The most important cytokines involved are TRAIL [178,182,183], IL-8 [183], IL-1A [183], IL-1β [184] and TNF-α [176,185]. To improve the synergy between SMC and OVs even further, OVs have been armed with exogenous tumor cell death enhancing (TCDE) cytokines, like TNF-α [186], which also addresses toxicity issues commonly associated with their systemic delivery. In an armed OV setting, production of these cytokines is largely limited to the tumor [187].


Apart from enhanced cytotoxic effects, SMC/OV combinations can also improve the antitumor response by modulating the adaptive immune response. Exhaustion of CD8<sup>+</sup> T-cells was reduced by an SMC-induced tumor macrophage M2 to M1 repolarization, an effect that could be further enhanced by PD-1 checkpoint blockade [190].

B cell lymphoma-2 (BCL-2) homology domain 3 (BH3) mimetics are antagonists that can bind with the hydrophobic Bcl-2 homology (BH) groove of Bcl-2 family proteins, thereby inhibiting these pro-survival proteins and restoring the apoptotic processes in tumor cells (Figure 4B) [192]. Several BH3 mimetics, namely GX15-070 (Obatoclax), EM20-25, BI-97D6 were shown to synergistically increase tumor cell death when combined with oncolytic vaccinia virus, VSV and AdV, respectively [173,174,193,194]. BH3 mimetics also could have a place in the cancer vaccine setting where treatment with GX15-070 (Obatoclax) increased intra-tumoral activated CD8<sup>+</sup> T-cells while reducing Treg activity [193].
