*2.2. H-1PV + Histone Deacetylase Inhibitors (Valproic Acid)*

Preclinical proof-of-concept was also obtained for another treatment combining H-1PV with the histone deacetylase (HDAC) inhibitor (HDACi) valproic acid (VPA) [33]. HDACis hold significant promise in cancer therapy, due to their ability to cause malignant cell growth inhibition, re-differentiation and death [34]. Most interestingly, HDAC inhibition was also found to potentiate the oncotoxicity of various OVs, including vesicular stomatitis [35], herpes- [36], adeno- [37] and parvo [33]-viruses (for a review, see Reference [38]). The synergism between HDACi and H-1PV was first demonstrated by Li et al., who conducted preclinical testing of this combination in cervical carcinoma and PDAC models [33]. VPA proved to synergize with H-1PV in inducing DNA damage, oxidative stress and death in PDAC-derived cell lines. This cooperation was traced back, at least in part, to the ability of VPA to stimulate the acetylation and, in consequence, the oncotoxic activity of the viral protein NS1. Interestingly, VPA-induced hyperacetylation of NS1 was also associated with enhanced H-1PV DNA replication and viral gene transcription, ultimately boosting virus multiplication in tumor cells. The VPA-dependent increase in both H-1PV intrinsic oncotoxicity and multiplication was reflected in the potentiation of tumor suppression in animal models. In order to establish a clinically relevant animal model of PDAC, patient-derived material was xeno-transplanted in non-obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice. Alternatively, the human AsPC-1 cell line was implanted into nude rats. Tumors were subjected to mono versus combinatorial treatment and tumor growth parameters were comparatively evaluated. In line with the in vitro observations, H-1PV + VPA administration resulted in enhanced NS1 and H-1PV intra-tumoral accumulation, correlating with an increase in oxidative stress and subsequent apoptosis in co-treated tumors. The combination achieved complete AsPC-1 tumor eradication. Patient-derived xenografts were also responsive, yet to a somewhat

lesser extent, probably due to the characteristic PDAC intra-tumoral heterogeneity and prominent presence of stroma. tent, probably due to the characteristic PDAC intra-tumoral heterogeneity and prominent presence of stroma.

in vitro observations, H-1PV + VPA administration resulted in enhanced NS1 and H-1PV intra-tumoral accumulation, correlating with an increase in oxidative stress and subsequent apoptosis in co-treated tumors. The combination achieved complete AsPC-1 tumor eradication. Patient-derived xenografts were also responsive, yet to a somewhat lesser ex-

*Cancers* **2021**, *13*, x 6 of 15

It is noteworthy that besides its effects on tumor cell growth and OV oncotoxicity, VPA was reported to modulate the immune system, providing an additional possible interface for cooperation with OVs at the level of their intrinsic immuno-stimulating activity. VPA was indeed shown to: It is noteworthy that besides its effects on tumor cell growth and OV oncotoxicity, VPA was reported to modulate the immune system, providing an additional possible interface for cooperation with OVs at the level of their intrinsic immuno-stimulating activity. VPA was indeed shown to:


As a whole, the above data speak for the high translational relevance of VPA to the future development of PV-based combinatorial (immuno) therapies. As a whole, the above data speak for the high translational relevance of VPA to the future development of PV-based combinatorial (immuno) therapies.

In conclusion, two drugs that are available on the pharmaceutical market, i.e., gemcitabine (cytostatic) and VPA (antiepileptic), proved to be efficient in synergizing with H-1PV to suppress pancreatic cancer (Figure 2). In conclusion, two drugs that are available on the pharmaceutical market, i.e., gemcitabine (cytostatic) and VPA (antiepileptic), proved to be efficient in synergizing with H-1PV to suppress pancreatic cancer (Figure 2).

**Figure 2.** Parvovirus-based viro-immunotherapeutic combinations under development against pancreatic ducatl adenocarcinoma (PDAC). H-1PV-induced tumor cell lysis cooperates with gemcitabine-triggered programmed tumor cell death, valproic acid (VPA)-dependent epigenetic transcription regulation or interferon (IFN)-γ-induced immuno-stimulation to suppress PDAC. Preclinical data suggest that the immune system mediates, at least in part, this cooperation. H-1PV infection of tumor cells leads to the release of PAMPs/DAMPs, such as high-mobility group box 1 protein (HMGB1), which in turn alert the immune system to danger and mobilize an inflammatory antitumor immune response. Various aspects of H-1PV-, gemcitabine-, VPA- and IFN-γ-exerted immunomodulation may converge and synergize upon exposure of the host immune system to the respective combinations. The underlying mechanisms remain to be elucidated in detail by gathering extensive clinical experience. For details and references, see main text. **Figure 2.** Parvovirus-based viro-immunotherapeutic combinations under development against pancreatic ducatl adenocarcinoma (PDAC). H-1PV-induced tumor cell lysis cooperates with gemcitabine-triggered programmed tumor cell death, valproic acid (VPA)-dependent epigenetic transcription regulation or interferon (IFN)-γ-induced immuno-stimulation to suppress PDAC. Preclinical data suggest that the immune system mediates, at least in part, this cooperation. H-1PV infection of tumor cells leads to the release of PAMPs/DAMPs, such as high-mobility group box 1 protein (HMGB1), which in turn alert the immune system to danger and mobilize an inflammatory antitumor immune response. Various aspects of H-1PV-, gemcitabine-, VPA- and IFN-γ-exerted immunomodulation may converge and synergize upon exposure of the host immune system to the respective combinations. The underlying mechanisms remain to be elucidated in detail by gathering extensive clinical experience. For details and references, see main text.

#### Another combination with substantial potential for clinical development relies on the *2.3. H-1PV + Proinflammatory Cytokines (Interferon-Gamma)*

*2.3. H-1PV + Proinflammatory Cytokines (Interferon-Gamma)* 

mutual complementation of H-1PV- and IFN-γ-mediated immune stimulation. It was Another combination with substantial potential for clinical development relies on the mutual complementation of H-1PV- and IFN-γ-mediated immune stimulation. It was shown that IFN-γ improves the vaccination potential of the virus and diminishes the development of peritoneal carcinomatosis in preclinical PDAC models. Concomitant intraperitoneal administration of both H-1PV and IFN-γ in these models led to extended animal survival correlating with enhanced peritoneal macrophage and splenocyte responses against tumor cells [42].
