*2.2. Inhibition of NF-kB Signaling*

Nuclear factor (NF)-κB and inhibitor of NF-κB kinase (IKK) proteins regulate many cellular responses to stimuli, such as innate and adaptive immunity, cell death, and inflammation [64]. NF-κB and IKK therefore play key roles in regulating the innate immune response against OVs. Indeed, two types of compounds enhance OV replication through very distinct mechanisms at different stages of NF-κB-mediated transcription [65]. For instance, fumaric and maleic acid esters, such as dimethyl fumarate (DMF), block the nuclear translocation of NF-κB and have been shown to improve replication of several OVs and subsequent therapeutic outcomes by inhibiting type I IFN [66]. Another point of intervention is in the nucleus after NF-κB has already bound DNA [67]. At this point triptolide blocks transcription, leading to an increase of VSV replication in several VSVresistant tumor cell types (Figure 2) [68]. Before NF-κB can facilitate transcription of innate immune genes it has to be released from the IκB kinase β (IKKβ) complex. The activation of IKKβ, by the phosphorylation of IκBα and its subsequent proteasomal degradation, allows NF-κB to relocate to the nucleus [64]. Blocking IKKβ can be therapeutically exploited since NF-κB is overexpressed in many cancer types [69]. Consequently, inhibiting IKKβ shows much promise for synergizing with OVs (Figure 2). This would be especially advantageous for OVs, such as VSV and NDV, that rely on defective innate immunity for their onco-selectivity [70]. This was confirmed in studies on malignant peripheral nerve sheath tumor cells and some pancreatic ductal adenocarcinoma cell lines that showed resistance to oncolytic HSV and VSV infection, respectively. In combination with the IKKβ inhibitor TPCA-1, this resistance was overcome and productive infection was achieved [46,51].

**Figure 1.** JAK/STAT signaling inhibition for the improvement of OV efficacy. (**A**) IFN binding with its receptor can activate JAK1 and TYK2. This in turn facilitates the phosphorylation of the docking sites of STAT1 and STAT2. Following phosphorylation, both STATs associate with IRF6 to form the transcriptional regulation ISG3. ISG3 trans-locates to the nucleus where it mediates the transcription of ISG mRNAs. The appropriate DNA strains are made accessible for ISGF3 by different histone deacetylases. These mRNAs are in turn transported over microtubules in order to be translated. Targeting these pathways by means of different small molecule inhibitors (red annotated squares) allows OV replication to proceed for longer, resulting in increased viral spread and potentially efficacy. See the main text for more details. Created with biorender.com. (**B**) Selected chemical structures of compounds depicted in panel A. All structures throughout were drawn using MarvinSketch (ChemAxon) from publicly available information. Abbreviations: JAK, Janus kinase; STAT, signal transducers and activators of transcription; IRF9, Interferon regulatory factor 9; ISGF3, Interferon-stimulated gene factor 3; HDAC, histone deacetylase; ISRE, Interferon-sensitive response element; MDA, microtubule destabilizing agent; VPA, Valproate. **Figure 1.** JAK/STAT signaling inhibition for the improvement of OV efficacy. (**A**) IFN binding with its receptor can activate JAK1 and TYK2. This in turn facilitates the phosphorylation of the docking sites of STAT1 and STAT2. Following phosphorylation, both STATs associate with IRF6 to form the transcriptional regulation ISG3. ISG3 trans-locates to the nucleus where it mediates the transcription of ISG mRNAs. The appropriate DNA strains are made accessible for ISGF3 by different histone deacetylases. These mRNAs are in turn transported over microtubules in order to be translated. Targeting these pathways by means of different small molecule inhibitors (red annotated squares) allows OV replication to proceed for longer, resulting in increased viral spread and potentially efficacy. See the main text for more details. Created with biorender.com. (**B**) Selected chemical structures of compounds depicted in panel A. All structures throughout were drawn using MarvinSketch (ChemAxon) from publicly available information. Abbreviations: JAK, Janus kinase; STAT, signal transducers and activators of transcription; IRF9, Interferon regulatory factor 9; ISGF3, Interferon-stimulated gene factor 3; HDAC, histone deacetylase; ISRE, Interferon-sensitive response element; MDA, microtubule destabilizing agent; VPA, Valproate.

**Figure 2.** Compound classes that inhibit of NF-kB signaling and synergize with OV treatment. (**A**) Virus replication results in the production of cytosolic DNA and single- and double-stranded RNA. This triggers multiple signaling cascades, including the recruitment of RIG-I and Mda5 to the adaptor IPS-1 on the membrane of the mitochondria. This in turn leads to kinase activation through TRAF family members. More specifically, this activates the IKK complex, which phosphorylates IκB proteins. Phosphorylation of IκB leads to its ubiquitination and proteasomal degradation, freeing NF-κB complexes for transcription induction. TRAF6 signaling also leads to JNK activation. Activated JNK trans-locates to the nucleus and activates c-Jun and other target transcription factors. These transcription factors, such as cJun and NF-κB lead to the transcription of numerous proteins involved in innate immunity and cells death, including IFN-β. Interfering with the different steps of signaling pathways using different classes of compounds (red annotated red squares) have resulted in increased viral replication and subsequent efficacy. See the main text for more details. Created with biorender.com. (**B**) Selected chemical structures of compounds depicted in panel A. All structures throughout were drawn using MarvinSketch (ChemAxon) from publicly available information. Abbreviations: TRAF, TNF Receptor Associated Factor; JNK, c-Jun N-terminal kinase; Atf2, Activating transcription factor 2; IPS-1, interferon-β promoter stimulator 1; TSA, Trichostatin A; DMF, dimethyl fumarate; RigI, retinoic acid-inducible gene I; mda5, melanoma differentiation-associated protein 5; PRK, protein kinase R; Ubcl3, ubiquitin-conjugating enzyme 13; ubiquitin-conjugating enzyme E2 variant 1; Tak1, transforming growth factor-6-activated kinase 1; IKK, IκB kinase β. *2.2. Inhibition of NF-kB Signaling*  Nuclear factor (NF)-κB and inhibitor of NF-κB kinase (IKK) proteins regulate many cellular responses to stimuli, such as innate and adaptive immunity, cell death, and inflammation [64]. NF-κB and IKK therefore play key roles in regulating the innate immune **Figure 2.** Compound classes that inhibit of NF-kB signaling and synergize with OV treatment. (**A**) Virus replication results in the production of cytosolic DNA and single- and double-stranded RNA. This triggers multiple signaling cascades, including the recruitment of RIG-I and Mda5 to the adaptor IPS-1 on the membrane of the mitochondria. This in turn leads to kinase activation through TRAF family members. More specifically, this activates the IKK complex, which phosphorylates IκB proteins. Phosphorylation of IκB leads to its ubiquitination and proteasomal degradation, freeing NF-κB complexes for transcription induction. TRAF6 signaling also leads to JNK activation. Activated JNK trans-locates to the nucleus and activates c-Jun and other target transcription factors. These transcription factors, such as cJun and NF-κB lead to the transcription of numerous proteins involved in innate immunity and cells death, including IFN-β. Interfering with the different steps of signaling pathways using different classes of compounds (red annotated red squares) have resulted in increased viral replication and subsequent efficacy. See the main text for more details. Created with biorender.com. (**B**) Selected chemical structures of compounds depicted in panel A. All structures throughout were drawn using MarvinSketch (ChemAxon) from publicly available information. Abbreviations: TRAF, TNF Receptor Associated Factor; JNK, c-Jun N-terminal kinase; Atf2, Activating transcription factor 2; IPS-1, interferon-β promoter stimulator 1; TSA, Trichostatin A; DMF, dimethyl fumarate; RigI, retinoic acid-inducible gene I; mda5, melanoma differentiation-associated protein 5; PRK, protein kinase R; Ubcl3, ubiquitin-conjugating enzyme 13; ubiquitin-conjugating enzyme E2 variant 1; Tak1, transforming growth factor-6-activated kinase 1; IKK, IκB kinase β.

#### response against OVs. Indeed, two types of compounds enhance OV replication through *2.3. PI3K/AKT/mTOR Pathway Antagonists*

very distinct mechanisms at different stages of NF-κB-mediated transcription [65]. For instance, fumaric and maleic acid esters, such as dimethyl fumarate (DMF), block the nuclear translocation of NF-κB and have been shown to improve replication of several OVs and subsequent therapeutic outcomes by inhibiting type I IFN [66]. Another point of in-Important for cell survival and growth, the phosphoinositide 3-kinase (PI3K)/Ak strain transforming (AKT)/mTOR pathway is also crucially involved in the induction of type 1 interferons (Figure 3) [71]. It is commonly activated in numerous types of cancer [72] via mutations or amplification of genes encoding receptor tyrosine kinases, subunits of

PI3K, AKT or activating isoforms of rat sarcoma (Ras) [73]. The first agents, targeting the PI3K pathway with the specific purpose of treating cancer, were analogues of rapamycin, namely everolimus (RAD 001) and temsirolimus [73]. Hence, inhibition of mTOR is expected to augment the oncolytic activity particularly of those viruses depending on impaired antiviral responses within a tumor cell. The macrolide compound rapamycin is a prototypical inhibitor of the serine/threonine protein kinase mTOR. Combining rapamycin with the highly IFN-sensitive VSV-mutant strain (VSV∆M51) led to significant increase of the oncolytic effect [74]. In addition other oncolytic RNA viruses, such as NDV and reovirus, showed improved oncolytic effect in mice when co-treated with rapamycin [75,76]. Oncolytic DNA viruses also benefit from co-treatment with rapamycin. The yield and dissemination of an HSV-derived oncolytic virus was markedly increased in semi-permissive tumor cell lines [77]. An oncolytic vaccinia virus (VACV) only achieved complete remission in in vivo models when it was combined with rapamycin [78]. A key restriction factor for myxoma virus in human cells is its dependence on AKT activation [79]. By inhibiting mTORC1, AKT becomes hyperactivated through the release from the negative feedback loop between ribosomal protein S6 kinase beta-1 (S6K1) and insulin receptor substrate 1 (IRS-1) [80]. This subsequently enhances myxoma virus replication which also translates to increased survival in vivo [81–83]. mTOR inhibition can also lead to a decrease in phosphorylation of the effector proteins, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BPs) and S6Ks, which are essential for type I interferon (IFN) production (Figure 3) [84,85]. This inhibition of the type I interferon response also contributes to a more pronounced replication of myxoma virus in vitro and increased efficacy in vivo [86]. Everolimus was tested in combination with an oncolytic adenovirus. Even though, in vitro, RAD001 seemed to interfere with the viral replication, potent anti-glioma effects were seen in vivo. This was presumably due to the induction of autophagic cell death [87,88]. Increased efficacy through modulation of autophagy in similar settings is also described for other OVs [75,76]. The hyperactivation of AKT during mTORC1 inhibition might have benefits when combined with myxoma virus [81–83], but in other settings can have a negative effect on survival. In phosphate and tensin homolog (PTEN)-deficient glioblastoma patients, for instance, hyperactivation of AKT, following rapamycin treatment, was associated with more rapid onset of tumor progression [89]. The mTORC2 complex, which is insensitive to rapamycin and its analogues, activates AKT and has a distinct role in tumor maintenance and progression [90]. For OVs with a dependency on a weakened antiviral state within the tumor, mTORC2 antagonists that also inhibit mTORC1 would be a superior option. ATP-competitive mTOR kinase inhibitors (TKIs) achieve this by targeting the kinase domain of mTOR, thereby also blocking the activation feedback of PI3K/Akt signaling (Figure 3) [91]. Indeed, mTORC1/2 inhibitors, such as PP242, INK1341, INK128 or Torin1, were also able to increase HSV replication and oncolysis by altering eIF4E/4E-BPs expression [77]. Specific inhibitors, such as rapamycin and TKIs, are prone to trigger the development of secondary resistance after prolonged treatment [92]. Consequently, inhibitors were developed that target the same signaling pathway but at multiple sites. Dual PI3K/mTOR inhibitors, such as voxtalisib [93], target the p110α, β, and γ isoforms of PI3K as well as the ATP-binding sites of both mTORC1 and mTORC2, completely suppressing PI3K/Akt signaling [91]. Combinations with OVs have yet to be reported. BKM120, another pan-class PI3K inhibitor, targeting all four catalytic isoforms, in combination with oncolytic HSV-1, was effective in the treatment of Du145 prostate cancer sphere forming cells (PCSCs) [94]. Finally, the benefits of combining PI3K/Akt signaling blockade and OVs can also work in the opposite direction, demonstrated by the combination of an oncolytic HSV and PI3K/Akt inhibitors (LY294002, triciribine, GDC-0941, BEZ235). Here, treatment with the OV sensitized the tumor cells to the inhibitors through enhanced Akt activation [95,96]. Indirectly, PI3K inhibitors, more specifically PI3Kδ-selective inhibitors, could improve systemic OV delivery to tumors through attachment inhibition of systemic macrophages [97].

bition of systemic macrophages [97].

would be a superior option. ATP-competitive mTOR kinase inhibitors (TKIs) achieve this by targeting the kinase domain of mTOR, thereby also blocking the activation feedback of PI3K/Akt signaling (Figure 3) [91]. Indeed, mTORC1/2 inhibitors, such as PP242, INK1341, INK128 or Torin1, were also able to increase HSV replication and oncolysis by altering eIF4E/4E-BPs expression [77]. Specific inhibitors, such as rapamycin and TKIs, are prone to trigger the development of secondary resistance after prolonged treatment [92]. Consequently, inhibitors were developed that target the same signaling pathway but at multiple sites. Dual PI3K/mTOR inhibitors, such as voxtalisib [93], target the p110α, β, and γ isoforms of PI3K as well as the ATP-binding sites of both mTORC1 and mTORC2, completely suppressing PI3K/Akt signaling [91]. Combinations with OVs have yet to be reported. BKM120, another pan-class PI3K inhibitor, targeting all four catalytic isoforms, in combination with oncolytic HSV-1, was effective in the treatment of Du145 prostate cancer sphere forming cells (PCSCs) [94]. Finally, the benefits of combining PI3K/Akt signaling blockade and OVs can also work in the opposite direction, demonstrated by the combination of an oncolytic HSV and PI3K/Akt inhibitors (LY294002, triciribine, GDC-0941, BEZ235). Here, treatment with the OV sensitized the tumor cells to the inhibitors through enhanced Akt activation [95,96]. Indirectly, PI3K inhibitors, more specifically PI3Kδ-selective inhibitors, could improve systemic OV delivery to tumors through attachment inhi-

**Figure 3.** Overview of the PI3K(p85/p110)/AKT/mTOR pathway and small molecule compounds that target this pathway in synergy with OV therapy. (**A**) Activating (PI3K, AKT, PDK1, mTORC1 and mTORC2) and inhibiting proteins (PTEN, TSC1/2) of the signaling pathway are shown. PI3K consists of catalytic subunit p110 and the regulatory subunit p85. PI3K phosphorylates phosphatidylinositol bisphosphate, which in turn activates PDK1 and AKT. PTEN negatively regulates the activation of AKT, which can inhibit TSC1/2, a negative regulator of mTOR. Active mTOR phosphorylates S6K1 and 4EBP1 leading to increased translation and synthesis of, among others, ISGs [73]. Targeting this process by means of different small molecule inhibitors (red annotated squares) allows OV replication to proceed for longer, resulting in increased viral spread and efficacy. See the main text for more details. Created with biorender.com. (**B**) Selected chemical structures of compounds depicted in panel A. All structures throughout were drawn using MarvinSketch (ChemAxon) from publicly available information. Abbreviations: RTK, receptor tyrosine kinase; PDK1, phosphoinositide-dependent kinase 1; IRS1, insulin receptor substrate 1; PTEN, phosphatase and tensin homologue; mTOR, mammalian target of rapamycin. PKR, protein kinase R; Myx, GTP-binding protein MX; AOS, oligoadenylate synthetase; S6K, S6 kinase; 4E-BP1, Eukaryotic translation initiation factor 4E-binding protein 1; Rheb, Ras homolog enriched in brain; IRS1, insulin receptor substrate 1.
