Histone deacetylase inhibitor boosts anticancer potential of fusogenic oncolytic vaccinia virus by enhancing cell–cell fusion

Abstract

Oncolytic viruses have two anticancer functions: direct oncolysis and elicitation of antitumor immunity. We previously developed a novel fusogenic oncolytic vaccinia virus (FUVAC) from a non‐fusogenic vaccinia virus (VV) and, by remodeling the tumor immune microenvironment, we demonstrated that FUVAC induced stronger oncolysis and antitumor immune responses compared with non‐fusogenic VV. These functions depend strongly on cell–cell fusion induction. However, FUVAC tends to have decreased fusion activity in cells with low virus replication efficacy. Therefore, another combination strategy was required to increase cell–cell fusion in these cells. Histone deacetylase (HDAC) inhibitors suppress the host virus defense response and promote viral replication. Therefore, in this study, we selected an HDAC inhibitor, trichostatin A (TSA), as the combination agent for FUVAC to enhance its fusion‐based antitumor potential. TSA was added prior to FUVAC treatment of murine tumor B16‐F10 and CT26 cells. TSA increased the replication of both FUVAC and parental non‐fusogenic VV. Moreover, TSA enhanced cell–cell fusion and FUVAC cytotoxicity in these tumor cells in a dose‐dependent manner. Transcriptome analysis revealed that TSA‐treated tumors showed altered expression of cellular component‐related genes, which may affect fusion tolerance. In a bilateral tumor‐bearing mouse model, combination treatment of TSA and FUVAC significantly prolonged mouse survival compared with either treatment alone or in combination with non‐fusogenic VV. Our findings demonstrate that TSA is a potent enhancer of cell–cell fusion efficacy of FUVAC.

Fusogenic oncolytic vaccinia virus combined with histone deacetylase inhibitor trichostatin A induced strong antitumor effects through enhancing cell–cell fusion induction.

Abbreviations

FUVAC

Fusogenic oncolytic vaccinia virus

HDAC

Histone deacetylase

OV

Oncolytic virus

TSA

Trichostatin A

VV

Vaccinia virus

1. INTRODUCTION

Oncolytic virotherapy is a novel cancer treatment that utilizes viruses as anticancer medicines. ¹ Oncolytic viruses show marked clinical benefits against refractory tumors. For example, the US FDA‐approved talimogene laherparepvec is used to treat melanoma, ² and the Japanese Pharmaceuticals and Medical Devices Agency‐approved teserpaturev is used to treat malignant glioma. ³ , ⁴ These OVs specifically replicate within the tumor and induce tumor cell lysis, leading to the activation of antitumor immunity. Some OVs have a cell–cell fusion phenotype. Cell–cell fusion induces rapid tumor cell lysis and tumor antigen release, causing immunogenic cell death. ⁵ , ⁶ Several OVs are armed with exogenous fusion proteins that confer the cell–cell fusion function. ⁵ , ⁶

We have previously developed a FUVAC from a non‐fusogenic VV. FUVAC was isolated from mitogen‐activated protein kinase‐dependent recombinant VV (MDRVV), whose tumor cell‐specific replication is attributed to the deletion of the virus growth factors, vaccinia growth factor, and O1L. ⁷ FUVAC has a nonsense mutation in the viral fusion inhibitor, K2L. ⁸ K2L conjugates with A56 and blocks the viral entry‐fusion complex, which initializes membrane fusion after the binding of viral attachment proteins. ⁹ , ¹⁰ The VV attachment proteins A26, A27, D8, and H3 bind to multiple surface molecules, such as laminin, heparan, and chondroitin. ⁹ , ¹¹ Thus, unlike the molecular mechanisms of entry and cell–cell fusion of other OVs, those of the VV are complicated.

Moreover, we recently found that FUVAC induced strong oncolysis in injected tumors, resulting in the remodeling of the tumor immune microenvironment by decreasing the tumor‐associated immune‐suppressive cell population locally and increasing that of cytotoxic T lymphocytes systematically. ¹² These functions are strongly dependent on cell–cell fusion induction. However, some tumor cells induce a small syncytium by FUVAC infection, causing the inhibition of the viral therapeutic effects.

Murine tumor cells tend to form smaller FUVAC syncytia than human tumor cells because of the low replication efficacy of the VV. Virus replication efficacy is commonly regulated by the host antivirus response represented by type 1 interferon (IFN) responses. ¹³ , ¹⁴ These IFN responses are involved in OV resistance by regulating virus replication within tumor cells. ¹⁵ OVs are combined with various type I IFN suppressors such as sunitinib, ¹⁶ ruxolitinib, ¹⁷ sulforaphane, ¹⁸ propranolol, ¹⁹ and anti‐interferon‐α/β receptor antibody. ²⁰ Hence, regulating the host antivirus response is critical for OV activity.

One of the host IFN suppressors, HDAC inhibitor, epigenetically regulates the expression of various genes by inhibiting deacetylation of histone or other target proteins. ²¹ , ²² HDACs control multiple cellular processes, such as cell cycle, differentiation, senescence, autophagy, apoptosis, and even immune processes, including the antivirus IFN response. ²³ , ²⁴ , ²⁵ Considering these features, HDAC inhibitors are often combined with oncolytic virotherapy to promote virus replication by inhibiting the virus defense mechanism. ²⁶ , ²⁷ , ²⁸ Non‐fusogenic oncolytic VV has been combined with the HDAC inhibitor TSA, resulting in the enhancement of virus growth, cell–cell spread, and in vivo anticancer effects while escaping the host IFN responses. ²⁶ However, the effect of TSA on the fusogenic oncolytic VV is unclear.

The details of VV cell–cell fusion induction have not been elucidated, and the cellular factors involved in cell–cell fusion sensitivity have not been identified. Therefore, it is important to determine whether TSA can change the tumor microenvironment to increase cell–cell fusion tolerance. This study aimed to examine whether TSA treatment could enhance the cell–cell fusion efficacy of FUVAC to improve its antitumor potential. Our study opened the possibility of a combination treatment with OVs and HDAC inhibitors to induce fusion‐based oncolytic activity.

2. MATERIALS AND METHODS

2.1. Virus preparation

Recombinant viruses MDRVV and FUVAC were constructed as described previously. ⁷ , ¹² Both viruses were purified before in vivo use. Briefly, the viruses were propagated in A549 cells. Subsequently, the infected cells were harvested and mixed with 200 units/mL benzonase (Merck, MA, USA) and incubated at 37°C for 1 h. Cell lysates were purified and dialyzed using OptiPrep™ (Axis‐Shield, Oslo, Norway) and Slide‐A‐Lyzer™ Dialysis Cassette (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturers' protocols.

2.2. Cell lines

Murine carcinoma cell lines, including melanoma B16‐F10 (cultured in DMEM), colon CT26.WT (in RPMI‐1640 medium), human lung carcinoma A549 (in Ham's F12K), and rabbit kidney‐derived RK13 were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in an appropriate medium (Wako, Osaka, Japan) with 10% fetal bovine serum (FBS; Corning, Oneonta, NY, USA), except for the RK13 (5% FBS) cells, at 37°C in a humidified atmosphere of 5% CO₂.

2.3. In vitro combination OV and TSA treatment

To determine the optimal TSA (Sigma‐Aldrich, MO, USA) dose, 1 × 10⁴ of murine melanoma B16‐F10 cells were treated with TSA at 0, 0.00375, 0.0375, 0.375, 3.75, or 37.5 μM and incubated at 37°C for 3 h. Subsequently, 48 h after infection, the cells were infected with MDRVV‐LG/DsRed or FUVAC‐LG/DsRed at a multiplicity of infection (MOI) of 5 and photographed under a fluorescence microscope (BZ‐X700; Keyence, Osaka, Japan). Cellular viability was examined using CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay (Promega, Madison, WI, USA) 72 h after infection.

Then, the TSA dose was selected (Figure 1), and B16‐F10 or CT26 cells were infected with MDRVV‐LG/DsRed or FUVAC‐LG/DsRed at an MOI of 0.1, 1, or 5 after treatment with dimethyl sulfoxide (DMSO) or 3.75 μM of TSA with incubation at 37°C for 24 h. The cells were photographed, and their viability was examined 72 h later, as described above.

FIGURE 1.

TSA dose‐escalation test. (A) B16‐F10 cells were treated with 0, 0.00375, 0.0375, 0.375, 3.75, or 37.5 μM of TSA and MDRVV‐LG/DsRed or FUVAC‐LG/DsRed at an MOI of 5, and the images were obtained 2 days after infection. Scale bars, 500 μm. (B) Viability of TSA‐treated and/or virus‐treated cells, as described in (A). The viability ratio was measured using the CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay (Promega) 72 h after infection. Data are expressed as the percentage of survival of mock‐infected cells and presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ****p < 0.0001 (two‐tailed unpaired t‐test). FUVAC, fusogenic oncolytic vaccinia virus; MDRVV, mitogen‐activated protein kinase‐dependent recombinant vaccinia virus; MOI, multiplicity of infection; SD, standard deviation; TSA, trichostatin A.

2.4. Virus growth analysis

Virus growth was examined by titrating progeny viruses from infected cell lysates. DMSO‐treated or TSA‐treated (3.75 μM, 24 h) and each line of virus‐infected (MOI of 1) tumor cells were harvested 12, 24, 36, 48, and 60 h after virus infection. Progeny viruses were extracted from the cell lysates by freeze‐thawing and sonication. Cellular debris was removed by centrifugation at 2000 g . Virus titration was performed in RK13 cells, virus plaques were counted 3 days after infection, and the plaque forming unit (PFU) was calculated as the plaque number relative to the fluid volume.

2.5. RNA‐sequencing analysis of TSA‐treated and TSA‐untreated tumor cells

In total, 3 × 10⁵ B16‐F10 or CT26 cells were treated with DMSO or 3.75 μM of TSA. After 24 h, cellular mRNA was isolated using TRIzol Reagent (Thermo Fisher Scientific) and the Pure Link RNA Mini Kit (Thermo Fisher Scientific), according to the manufacturer's protocols. RNA quality was assessed using Tapestation 4150 (Agilent Technologies, Waldbronn, Germany). The RNA integrity index was 10, and library construction and sequencing were conducted by Azenta Life Sciences (Tokyo, Japan). Changes in gene expression after TSA treatment were compared between DMSO‐treated and TSA‐treated cells.

2.6. In vivo OV and TSA combination therapy

Six‐week‐old female BALB/c mice (Charles River Laboratories, Yokohama, Japan) were subcutaneously transplanted with CT26 tumor cells (5 × 10⁵ cells) in their bilateral flanks. When the average tumor volume reached ~30 mm³, 5 μg of TSA was intraperitoneally injected on days −1, 1, and 3. Every 1 day after the TSA treatment (on days 0, 2, and 4), 2.5 × 10⁷ PFU of MDRVV‐Luc/LacZ or FUVAC‐Luc/LacZ was directly injected into the flank bearing the larger tumor. The average tumor volume exceeded 50 mm³ during the first virus treatment. Tumor volumes were measured using calipers, and the viral Fluc expression was monitored using an injection of VivoGlo Luciferin, In Vivo Grade (3 mg/mouse; Promega) on days 3, 5, and 7. The mice were anesthetized using isoflurane during bioimaging and visualized using NightSHADE LB985 (Berthold Technologies, Bad Wildbad, Germany). The luminescence intensity was quantified according to the manufacturer's protocol.

2.7. Immunohistochemical and immunological analyses

Mice with CT26 tumors were treated with TSA and FUVAC‐Luc/LacZ, as described above. Tumors were harvested on day 5 after the first virus treatment. Injected tumors were fixed in formalin and embedded in paraffin; then, the tissues were sliced and stained with hematoxylin and eosin or an anti‐β‐galactosidase polyclonal antibody (Thermo Fisher Scientific). Anti‐β‐galactosidase antibody reaction was detected with the SignalStain Boost IHC detection reagent and SignalStain DAB Substrate Kit (Cell Signaling Technology, MA, USA). The β‐galactosidase‐detected slides were counterstained with hematoxylin, and a coverslip was added with Soft Mount (Wako, Tokyo, Japan). Tissue sections were photographed using a BZ‐X700 fluorescence microscope (Keyence).

Non‐injected tumors were homogenized using a Tumor Dissociation Kit, mouse and gentleMACS Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. After Fc blocking (2.4G2: BD Bioscience, CA, USA), the cells were stained with antibodies against CD45 (30‐F11), CD3 (145‐2C11), and CD8 (53‐6.7: BioLegend, CA, USA). Furthermore, 7‐aminoactinomycin D (Beckman Coulter, CA, USA) was used for dead cell differentiation. After incubation (4°C for 25 min), the samples were suspended in fluorescence‐activated cell sorting buffer (PBS/2% FBS/0.02% sodium azide) and analyzed using a CytoFLEX flow cytometer and CytExpert Ver 2.0 software (Beckman Coulter).

2.8. Statistical analyses

Differences in cellular viability and the ratio of tumor‐infiltrating lymphocytes among the groups were evaluated using a two‐tailed unpaired t‐test. Virus growth, in vivo bioluminescence, and tumor volumes were analyzed using two‐way analysis of variance (ANOVA), followed by the Bonferroni test when the ANOVA showed an overall significance. Survival curves were generated using the Kaplan–Meier method and analyzed using the log‐rank test. A p‐value < 0.05 was considered to indicate statistical significance. All statistical analyses were performed using Prism version 9 (GraphPad, Inc., San Diego, CA, USA).

3. RESULTS

3.1. Combination effect of FUVAC and TSA

To determine the optimal condition for combining FUVAC and TSA, TSA dose escalation was performed in murine melanoma B16‐F10 cells. Treatment with TSA alone did not affect the tumor cells, except at the highest dose (37.5 μM; Figure 1A), which decreased cell viability by ~50% (Figure 1B) and strongly suppressed viral GFP expression. In addition, MDRVV increased virus spread in a dose‐dependent manner until 3.75 μM. FUVAC induced small syncytia in B16‐F10 cells without TSA treatment. In contrast, TSA increased syncytial formation in FUVAC‐infected cells in a dose‐dependent manner (Figure 1A). Cellular viability was not affected by TSA up to a dose of 3.75 μM. Importantly, FUVAC decreased cell viability more than MDRVV, depending on the enhancement of cell–cell fusion by TSA (Figure 1B).

The optimal TSA dose of 3.75 μM elicited the largest syncytium upon combination with FUVAC; the cell viability remained unchanged with TSA treatment alone. Combination treatment with TSA and oncolytic VVs was performed in B16‐F10 and murine colon carcinoma CT26 cells. Control DMSO‐treated and TSA‐treated cells were infected with MDRVV or FUVAC at an MOI of 0.1–5. Interestingly, MDRVV efficiently increased virus spread by TSA in B16‐F10 cells but not in CT26 cells. In contrast, combined FUVAC and TSA treatment clearly enhanced cell–cell fusion induction in both types of tumor cells (Figure 2A). The viability of B16‐F10 cells decreased more with MDRVV + TSA (up to a 50% decrease) than with MDRVV + DMSO (up to a 10% decrease), whereas that of CT26 cells was not affected by MDRVV + TSA. Nevertheless, FUVAC + TSA significantly decreased the viability of cells of both tumors compared with FUVAC + DMSO and MDRVV + DMSO or TSA (Figure 2B). The reduction in cell viability strongly corresponded to the cell–cell fusion efficacy.

FIGURE 2.

Combination effect of FUVAC and TSA in vitro. (A) B16‐F10 and CT26 cells were treated with DMSO or 3.75 μM of TSA and MDRVV‐LG/DsRed or FUVAC‐LG/DsRed at an MOI of 0.1, 1, or 5, and the images were obtained 3 days after infection. Scale bars, 1000 μm. (B) A CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay (Promega) was performed 72 h after DMSO or TSA and virus infection, as described in (A). Data are expressed as the percentage of survival of mock‐infected cells. Data are presented as mean ± SD (n = 3). ****p < 0.0001 (two‐tailed unpaired t‐test). FUVAC, fusogenic oncolytic vaccinia virus; MDRVV, mitogen‐activated protein kinase‐dependent recombinant vaccinia virus; MOI, multiplicity of infection; SD, standard deviation; TSA, trichostatin A.

3.2. Effect of TSA on virus replication and cell–cell fusion tolerance of tumor cells

Virus growth in DMSO‐treated or TSA‐treated tumor cells was measured 12–60 h after virus infection. In DMSO‐treated tumor cells, neither MDRVV nor FUVAC expanded the infection area after 24 h. However, in TSA‐treated tumor cells, FUVAC efficiently increased virus spread and induced a large syncytial formation over time, whereas the expansion of MDRVV increased slightly (Figure 3A). Virus growth curves showed that FUVAC tended to have higher titers than MDRVV in DMSO‐treated tumor cells. The replication capacity of both viruses was increased by TSA treatment (Figure 3B,C). Unexpectedly, the virus titer ratio of TSA:DMSO treatment indicated that virus growth escalation by TSA was comparable between MDRVV and FUVAC in both tumors (Figure S1a,b), regardless of the clear difference in cell–cell fusion induction. These results suggest that TSA increased the growth of both viruses and induced the cell–cell fusion of FUVAC.

FIGURE 3.

Viral growth in TSA‐treated tumor cells. (A) Virus replication images of B16‐F10 or CT26 cells treated with DMSO or 3.75 μM of TSA and MDRVV‐LG/DsRed or FUVAC‐LG/DsRed at an MOI of 1 obtained every 12 h. Scale bars, 500 μm. (B) Virus growth was titrated from the lysate of B16‐F10 cells treated with DMSO or TSA (3.75 μM) and MDRVV or FUVAC (MOI = 1), as described in (A). (C) Virus growth was titrated from the lysate of CT26 cells treated with DMSO or TSA (3.75 μM) and MDRVV or FUVAC (MOI = 1), as described in (A). Data in (B) and (C) are presented as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 (two‐way ANOVA, DMSO vs. TSA combined with MDRVV or FUVAC). ANOVA, analysis of variance; FUVAC, fusogenic oncolytic vaccinia virus; MDRVV, mitogen‐activated protein kinase‐dependent recombinant vaccinia virus; MOI, multiplicity of infection; SD, standard deviation; TSA, trichostatin A.

3.3. Effect of TSA on expression of cellular component‐related genes

To investigate changes in gene expression after TSA treatment, DMSO‐treated or TSA‐treated tumor cells were analyzed using RNA sequencing. Gene expression changes between DMSO‐treated and TSA‐treated cells were compared, and Gene Ontology (GO) analysis revealed the functions of TSA‐targeted genes. TSA treatment altered the expression of various genes in B16‐F10 and CT26 cells compared with DMSO treatment. GO analysis showed that the expression of the cellular component‐related genes (232 genes in B16‐F10 and 102 genes in CT26) fluctuated more than twice that of the molecular function‐related (83 genes in B16‐F10 and 34 genes in CT26) and biological process‐related genes (103 genes in B16‐F10 and 47 genes in CT26) in both B16‐F10 and CT26 cells (Figure 4A,B). However, the expression of a higher number of genes related to the host virus defense response differed in B16‐F10 cells (18 genes) than in CT26 cells (none). Cellular component‐related genes were involved in various extracellular and/or cell surface functions, such as those related to the extracellular space, region, and matrix. In total, 10 upregulated and seven downregulated genes were common to these cells (Figure 4C and Table S1). These results suggested that TSA altered the expression of cellular component genes, which may be related to cell–cell fusion tolerance in both types of tumor cells.

FIGURE 4.

Altered gene expression after TSA treatment. (A) Volcano plot shows the log10 (false discovery rate) and log fold change in cellular gene expression of B16‐F10 cells (DMSO‐treated vs. TSA‐treated). Bar plots show the number of significantly upregulated or downregulated genes after TSA treatment based on GO enrichment analysis. Highlighted genes in the volcano plot are cellular component‐related genes as detailed in (C) and Table S1. (B) Gene expression profiles and results of GO analysis of DMSO‐treated vs. TSA‐treated CT26 cells. (C) Venn diagrams showing the number of genes upregulated or downregulated by TSA related to the GO term of cellular components between B16‐F10 and CT26 cells. GO, Gene Ontology; TSA, trichostatin A.

3.4. FUVAC and TSA combination treatment in a bi‐flank tumor‐bearing mouse model

Combination treatment with oncolytic VVs and TSA was also attempted in an in vivo mouse model. BALB/c mice were bilaterally transplanted with CT26 tumor cells, and intraperitoneal TSA injection and unilateral MDRVV or FUVAC injection were administered alternately three times (Figure 5A). Bioimaging analysis showed that TSA treatment tended to increase the viral Fluc luminescence of FUVAC, although this was not significantly different compared with that observed for virus monotherapy (Figure S2). Without TSA treatment, FUVAC suppressed tumor growth more than MDRVV in both injected and non‐injected tumors (Figure 5B). In combination with TSA treatment, both MDRVV and FUVAC exhibited enhanced antitumor effects (Figure 5C). TSA increased the complete regression (CR) rate of the tumors injected with both viruses (4/9 of MDRVV and 6/10 of FUVAC) compared with that of the tumors injected with each virus alone (1/8 of MDRVV and 3/9 of FUVAC). Importantly, FUVAC combined with TSA led to CR of non‐injected tumors in 2/10 mice, although MDRVV could not eliminate virus‐untreated tumors, even with the TSA combination (Figure 5C). A single treatment with MDRVV or FUVAC significantly prolonged the survival of mice compared with that observed for PBS or TSA treatment alone. MDRVV combined with TSA treatment also prolonged survival but to the same extent as that of each virus monotherapy. Notably, mice treated with FUVAC and TSA showed longer survival than mice with the other single (PBS: ****p < 0.0001, MDRVV: **p < 0.0001, FUVAC: *p = 0.013) or combination treatments (PBS + TSA: ****p < 0.0001, MDRVV + TSA: *p = 0.0265, Figure 5D).

FIGURE 5.

Combination effect of FUVAC and TSA in vivo. (A) Schedule of virus and TSA treatment in a bilateral CT26 tumor‐bearing mouse model. CT26 tumors were bilaterally transplanted, and MDRVV‐Luc/LacZ or FUVAC‐Luc/LacZ was unilaterally injected on days 0, 2, and 4. TSA was intraperitoneally administered 1 day before virus treatment at each time point (days −1, 1, and 3). (B) Tumor growth curves after the first virus injection without TSA treatment. (C) Tumor growth curves after virus treatment combined with TSA. (B) and (C) are representative of two independent experiments comprising four or five mice per group (n = 8 for PBS and MDRVV; n = 9 for FUVAC, PBS + TSA, and MDRVV + TSA; n = 10 for FUVAC + TSA). Mean tumor volume (mm³) ± SD values are shown. **p < 0.01, ***p < 0.001, ****p < 0.0001 (two‐way ANOVA). (D) Kaplan–Meier survival curves associated with (B) and (C). MDRVV or FUVAC alone prolonged the survival of mice compared with PBS (**p = 0.0056 and ***p = 0.0002, respectively). FUVAC + TSA treatment prolonged survival compared with TSA alone, FUVAC alone, or MDRVV + TSA (****p = 0.0001, *p = 0.013, or *p = 0.0265, respectively, log‐rank test). ANOVA, analysis of variance; FUVAC, fusogenic oncolytic vaccinia virus; MDRVV, mitogen‐activated protein kinase‐dependent recombinant vaccinia virus; SD, standard deviation; TSA, trichostatin A.

Moreover, immunohistochemical analysis showed that TSA treatment increased β‐galactosidase expression of FUVAC in injected tumors (Figure S3a). Tumors treated with FUVAC + TSA showed a larger syncytial region, as represented by eosin dominance, than those treated with FUVAC alone (Figure S3b). Tumor‐infiltrating lymphocytes were simultaneously analyzed in non‐injected tumors. The combination treatment of FUVAC and TSA tended to increase the number of CD3⁺ and CD8⁺ T lymphocytes compared with FUVAC treatment alone (Figure S3c). These results corresponded to the enhancement of tumor regression of FUVAC combined with TSA. Thus, TSA increased the therapeutic potential of FUVAC compared with that of non‐fusogenic MDRVV.

4. DISCUSSION

In this study, the induction of cell–cell fusion of the oncolytic VV was enhanced by a combination treatment with the HDAC inhibitor TSA. HDAC inhibitors are utilized for cancer treatment due to their anticancer functions, such as induction of cell cycle arrest, antiangiogenesis, apoptosis induction, suppression of cell migration and invasion, and immune modulation. ²⁹ , ³⁰ , ³¹ In addition, HDAC inhibitors suppressed the type‐1 IFN response related to host virus defense. ²³ , ²⁴ , ²⁵ HDAC inhibition assists multiple processes of oncolytic virotherapy, such as enhancing virus entry, replication, propagation, and spread; decreasing antivirus response; promoting apoptosis; and induction of tumor antigen expression. ²⁷ , ²⁸ Among several HDAC inhibitors, TSA, the pan‐HDAC inhibitor, targets class I (HDAC1, HDAC2, HDAC3, and HDAC8) and class II (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10) HDACs and is frequently combined with several OVs, such as adenovirus, ³² , ³³ , ³⁴ , ³⁵ , ³⁶ bovine herpesvirus type 1, ³⁷ and herpes simplex virus. ³⁸ , ³⁹ , ⁴⁰ It is also the most potent enhancer of VV replication and spread among other HDAC inhibitors. ²⁶

B16‐F10 melanoma cells, which were previously used in combination with non‐fusogenic VV (TK‐B18R‐) and TSA, ²⁶ showed smaller cell–cell fusion formation with FUVAC treatment. In accordance with that report, in the present study, TSA pretreatment tended to increase the virus replication, spread, and cytotoxicity of non‐fusogenic MDRVV in these cells. However, TSA did not enhance the antitumor effect of MDRVV in colon carcinoma CT26 cells but increased virus replication compared with DMSO treatment. In contrast, TSA increased FUVAC cytotoxicity in both types of tumor cells, although the escalation in virus growth was comparable with that obtained with MDRVV. These results suggested that TSA efficiently enhanced the oncolytic effect of FUVAC, depending on the increment in virus replication and cell–cell fusion.

GO analysis revealed that the expression of genes related to the host virus defense response changed frequently in B16‐F10 cells after TSA treatment. Interestingly, TSA did not alter the host virus defense‐related gene expression in CT26 cells. TSA activated viral replication in CT26 cells in an antivirus IFN‐independent manner. Suppression of the IFN‐mediated antivirus response by TSA depends on the cell context, as reported previously. ³⁹ This might explain why both virus spread and cytotoxicity of non‐fusogenic MDRVV were observed only in B16‐F10 cells. In contrast, the expression of cellular component‐related genes fluctuated at similar levels in both types of tumor cells. HDAC modulates various cellular components to regulate cell adhesion, cell–cell interaction, and cellular differentiation, resulting in epithelial–mesenchymal transition (EMT) of tumor cells. ⁴¹ One of the TSA targets, HDAC6, is known as cytoskeleton‐associated deacetylase. ⁴² , ⁴³ HDAC6 deacetylates α‐tubulin and changes the microtubule dynamics, regulating cellular structure and motility. ⁴⁴ These changes affect tumor cell migration and metastasis by EMT. ⁴⁵ Moreover, HDAC6 regulates the process of macrophage infiltration by inducing filopodial formation. ⁴⁶ VV utilizes filopodia during viral entry and cell–cell spread. ⁴⁷ , ⁴⁸ , ⁴⁹ In addition, HDAC inhibition affects the expression of tight junction proteins, which play a role in cell–cell adhesion. ⁵⁰ , ⁵¹ Thus, HDAC inhibitors modulate multiple cellular components.

Although there are limitations to identifying the key factor in viral cell–cell fusion by current analyses, 17 TSA‐targeted cellular component‐related genes from both B16‐F10 and CT26 cells are strong candidates. In total, two of the 17 gene products directly compose the cell structure: Tnxb encodes extracellular matrix glycoprotein tenascin X, ⁵² and Prph encodes intermediate filament peripherin. ⁵³ Tnxb is involved in EMT transition, ⁵⁴ and Prph is involved in neurite growth and enterovirus‐A71 infection. ⁵⁵ The other candidates are also associated with virus infection and/or replication. For example, neuronal nitric oxide synthase 1 expression suppresses the replication of neurotropic viruses, such as vesicular stomatitis virus. ⁵⁶ Ppp1r15a, encoding the growth arrest and DNA damage‐inducible protein GADD34, dephosphorylates elf2a to promote viral protein synthesis, and several viruses utilize its ortholog. ⁵⁷ Slc7a11 encoding cystine/glutamic acid transporter is the target protein for Kaposi's sarcoma‐associated herpesvirus entry, ⁵⁸ and ddit3 suppresses cellular antiviral response to promote replication of several viruses, such as bovine alphaherpesvirus 1 and bovine viral diarrhea virus. ⁵⁹ , ⁶⁰ The genes encoding these proteins might also be responsible for viral growth escalation especially in CT26 cells. No genes were reported to be involved in cell–cell fusion induction. Detailed analyses of these candidates are currently underway.

Combination therapy with TSA and FUVAC also enhanced the oncolytic potential in the bilateral tumor‐bearing mouse model. CT26 syngeneic tumors are more resistant to non‐fusogenic VV treatment than B16‐F10 syngeneic tumors. ⁶¹ Although viral Fluc luminescence was only observed in virus‐treated CT26 tumors, the combination treatment with TSA and FUVAC clearly enhanced tumor suppression in injected and non‐injected tumors. The former showed the enhancement of local oncolysis, and the latter showed the induction of an antitumor immune response, as described in a previous report. ¹² MDRVV also showed enhanced local oncolysis on TSA treatment, although the antitumor immunity induced by this combination was insufficient to eliminate non‐injected tumors. FUVAC and TSA treatment achieved CR of 2/10 non‐injected tumors and prolonged the survival of mice. These results were corroborated by immunohistochemical and immunological analyses, showing that TSA treatment induced the systemic antitumor immune response of FUVAC by promoting local cell–cell fusion‐based oncolysis. TSA increased in vitro virus replication but did not significantly increase viral Fluc luminescence in vivo. However, histological analysis of the injected tumor revealed that TSA widely increased viral β‐gal expression along with enlarging the syncytial form of FUVAC. These results suggest that enhancement of the anticancer potential of oncolytic VVs is strongly dependent on the efficacy of virus replication and cell–cell fusion.

In conclusion, the oncolytic activity derived from viral replication and cell–cell fusion induction of the fusogenic VV was enhanced by the HDAC inhibitor TSA. TSA promoted the cell–cell fusion efficacy of FUVAC in a dose‐dependent manner by altering the expression of cellular component‐related genes. Combination treatment with FUVAC and TSA improved the therapeutic index, even in distant tumors. HDAC inhibitors, such as TSA, are excellent combination drugs for FUVAC.

AUTHOR CONTRIBUTIONS

Motomu Nakatake: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; resources; software; validation; visualization; writing – original draft; writing – review and editing. Hajime Kurosaki: Resources; writing – review and editing. Takafumi Nakamura: Conceptualization; data curation; formal analysis; funding acquisition; methodology; project administration; resources; software; supervision; validation; visualization; writing – original draft; writing – review and editing.

FUNDING INFORMATION

This study was funded by the Japan Society for the Promotion of Science KAKENHI (grant number 23K14618 and 21K20837 to M.N.) and the Japan Agency for Medical Research and Development (grant number JP22am0401017 to T.N.).

CONFLICT OF INTEREST STATEMENT

The authors have no conflict of interest.

ETHICS STATEMENT

Approval of the research protocol by an Institutional Review Board: N/A.

Informed Consent: N/A.

Registry and the Registration No. of the study/trial: N/A.

Animal Studies: All animal experiments were approved by the Animal Experiment Committee of Tottori University.

Supporting information

Figure S1.

Nakatake M, Kurosaki H, Nakamura T. Histone deacetylase inhibitor boosts anticancer potential of fusogenic oncolytic vaccinia virus by enhancing cell–cell fusion. Cancer Sci. 2024;115:600‐610. doi: 10.1111/cas.16032

DATA AVAILABILITY STATEMENT

The data reported in this study are available from the corresponding author (T.N.) upon reasonable request.