1. Introduction

Virotherapy is a new promising approach against different types of cancers through the use of oncolytic viruses (OVs). Virotherapy was born in the 19th century [22,23,24], and in the 1950s–1970s, the first clinical trials began and live viruses were deliberately injected into patients with cancer to promote tumor regression [22]. In the last decade, thanks to genetic engineering and the advent of in vitro experiments, the viral genome has been easily manipulated and modified to make viruses more selective for cancer cells and minimize their potential side effects [25,26,27], causing a great burst of oncolytic virotherapy. Since 2018, only three OVs have been approved for cancer therapy.

In fact, cancer cells have peculiar characteristics able to strengthen viral replication [124]: (i) they oppose apoptosis causing indefinite proliferation [125]; As a result, OVs replicate and lyse selectively tumor cells spreading viral progeny and other products of oncolysis. The release of infectious viral progeny allows oncolysis amplification also towards neighboring tumor cells. Potential adverse phenomena, such as viral encephalitis caused by HSV [134], require risk monitoring, which must be considered before treating patients.

Combinatorial treatments are required to improve the immune response and allow viral entry, replication, and diffusion between adjacent cells. In this review we discuss firstly the major viral families used in virotherapy and the clinical trials in which OVs are used; then we focus on the specific combinatorial therapies, including co-administered inhibitors of chromatin modifiers (combination strategies) and inserted target sites for miRNAs (recombination or arming strategies).

2. OVs and HDACs as Combinatorial Therapy

HDACs are epigenetic modulators that act on the epigenetic asset of the cellular system [201,202,203,204,205,206,207,208].

HDACi are anticancer agents that induce cell cycle arrest, and, additionally, they can inhibit the growth and differentiation of cancer cells [204,205,209,210,211]. , short-chain fatty acids (sodium butyrate or valproic acid), benzamide (MS-275 or entinostat), cyclic peptides (romidepsin or FR901228), and benzenesulfonamides (resminostat) [212]. In detail, SAHA is a wide HDAC class I and II inhibitor known to block the growth of cancer cells, including cutaneous T-cell lymphoma, breast, and prostate cancer. In addition, SAHA can induce cell cycle arrest in G1 phase in cancer cells through the up-regulation of cyclin-dependent kinase inhibitor p 21 [215].

To date, it has not yet received approval for clinical use, but the US FDA allowed its combinatorial treatment with exemestane for the management of advanced breast cancer [229]. It is also used in the treatment of prostate carcinoma as it is capable of preventing the development of metastases by inducing cell death [221,230] and transcriptional activation of specific genes [231]. Romidepsin (FK228 or FR90128) is a depsipeptide belonging to the group of cyclic peptides, approved by the FDA in 2009 for the anticancer treatment of cutaneous T-cell lymphoma (CTCL) and in prostatitis carcinoma [232,233]. It inhibits class I and IIb HDAC by preventing the growth of cancer cells and enhancing apoptotic processes [238,239].

To date, several oncolytic viruses have been associated with HDACi with the aim of increasing antitumor efficacy and, on the other hand, reducing the antiviral response. HSV, AdV, reovirus, VSV, vaccinia virus (VV), paramyxoviruses, and parvovirus are the most representative.

However, the effect of TSA decreased by using SN50, a NF-kB inhibitor, reducing the accumulation of p65 in the nucleus, and thus playing an important role in viral replication [241]. TSA can also up-regulate viral replication by increasing cytotoxicity [227,243]: it was able to upregulate the cyclin-dependent kinase inhibitor p21 and interrupt the cell cycle in the G0/G1 phase [138,240]. In co-treatment assay, glioma cells were treated with oHSV and VPA at the same time, meanwhile, in pre-treatment test, cellular monolayer was stimulated before with HDACi and, later, it was infected with the virus. GFP intensity resulted higher in pre-treatment than in co-treatment assay.

Kitazono et al. evaluated the transgenic expression of adenovirus in cancer cells subjected to treatment with the HDACi romidepsin (FR901228). The authors described the treatment of malignant cells with FR901228 and the subsequent infection with Ad5 CMV-LacZ, a replication-defective type 5 adenovirus, devoid of the E1 and E3 gene. Pre-treatment caused an increase in the expression of CAR and integrin-alpha, important for mediating the attack of adenovirus on cells [247]. Effects of oncolytic virotherapy with AdV have also been observed in cervical cancer cells.

However, many cancer cells present residual innate activity that can generate resistance to viral propagation [138]. On the other hand, in vivo experiments have analyzed the combination SAHA or MS-275 and rVSV M Delta 51 in prostate, ovarian, and breast cancer xenograft models, and showed enhanced survival [252,253]. Furthermore, Muscolini et al identified SIRT1 as a probable factor limiting viral infection in prostate cancer cells. Indeed, it has been considered a restriction factor known for its importance in prostate cancer, where it acted on the permissiveness of specific tumor cells.

Combinatorial therapy between HDACi and oncolytic reoviruses has also been evaluated in patients with multiple myeloma (MM). Life expectancy is reduced: in most cases, death occurs within 5 years of diagnosis, and, in cases where the tumor is aggressive, within 24 months [256,257,258]. By performing Western blot and flow cytometry analysis, lower expression of the reovirus receptor junctional adhesion molecule 1 (JAM-1) was observed in resistant tumor cells, infected with different amounts of virus, compared to sensitive ones. In addition, Jaime-Ramirez et al. assessed the impact of the combination of oncolytic reovirus and SAHA in head and neck squamous cell carcinomas (HNSCC), demonstrating an improvement in viral replication and immune-mediated anti-cancer responses both in vitro and in vivo [259].

Important results have been obtained in cervical cancer and pancreatic duct adenocarcinoma by the combination of HDACi. It has been reported that co-treating cancer cells with VPA and H1PV, as a result the onset of oxidative stress and apoptosis of cancer cells occurred [260]. The same effects have been observed by using H1PV and NaB at sub-lethal doses. There was an increase in viral oncotoxicity determining the eradication of neoplasm, but, on the other hand, there was the regression of carcinoma [138,185].

It has been shown that under optimal conditions, when cells were infected with the P/V-CPI mutant alone, it caused an increase in the production of IFN beta, while in cells infected with the oncolytic mutant virus and treated with scriptaid, there was a reduction in the production of INF and an increase in viral propagation in cancer cells [192]. Significant progress has also been assessed in the treatment of HCC in which the oncolytic measles vaccine virus (MeV) has been associated with the oral HDACi resminostat (Res) [238]. Res-MeV co-treatment increased viral replication and apoptosis, and improved primary infections. Furthermore, Res could exert a remarkable effect on innate cellular immunity, as it could prevent the activation of genes stimulated by IFN [238].

Currently, VV is under study and their activity can be enhanced by the use of HDACi [252,261]. Among the various HDACi, TSA represents the VV enhancer both in vitro and in vivo. Indeed, TSA caused a greater effect in vitro than other inhibitors, enhancing viral replication and the killing of infection-resistant tumor cells and, on the contrary, it was able to reduce toxicity to the mice [227]. Even in vivo studies with human colon carcinoma xenografts have shown that the combinatorial treatment resulted in improved survival [138,262].

3. OVs and DNMTi as Combinatorial Therapy

and DNMT3L are not canonical demethylating enzymes, as they do not contain the catalytic activity [263]. does not methylate genomic DNA but the anticodon loop of aspartic acid transfer RNA [267]. They perform different functions by acting in particular on the remodeling of chromatin and they are responsible for the up/down expression of proteins causing the onset of different pathologies [269]. Furthermore, the role of DNA methylation in common human pathologies has also been investigated, in particular in neurological disorders [275,276] and autoimmune diseases [277,278,279].

rQNestin34.5, remarkable results were obtained both in vivo and in vitro in the treatment of glioma [280]. By treating glioma cells with oHSV and 5-aza, there was an increase in the viral replication, as reported by the high expression of some viral genes and by the increase in the number and size of infected GFP-positive glioma cells [280]. Furthermore, Okemoto et al demonstrated that rQNestin34.5 and 5-aza can act synergistically causing apoptosis of glioma tumor cells. Monotherapy and combinatorial experiments were conducted in vitro, using cells derived from spontaneous breast fibrosarcomas (LCRT).

TMZ is mainly used for the treatment of malignant melanoma and glioma [284,285,286]; however, like the other drugs, prolonged use can induce resistance by producing the O 6- methylguanine mutagen and causing DNA damage. It has been observed that inhibition of MGMT improved the antitumor activity of the drug [284]. The combination between oncolytic adenoviruses and shRNA targeting MGMT activity could be an effective approach for fighting resistance to TMZ and for improving anticancer outcomes.

Therapeutic studies have also been conducted to enhance the treatment of onco-hematological diseases such as acute T-cell lymphocytic leukemia. Hastie et al used murine EL-4 cells from acute T-cell lymphocytic leukemia. before therapy with the DNMTi, caused tumor remission in 70% cases [289]. Cells which survived two consecutive treatments with the epigenetic modulator were more sensitive to oncolytic viral therapy, leading to durable remissions.

4. OVs and miRNA: Promising Combinatorial Treatment

In the past decade, the study of miRNAs has largely influenced the field of oncolytic virotherapy. Specific miRNAs target sequences can be integrated into the viral genome and can regulate viral proteins, improving the safety profile and strengthening the anticancer efficacy of oncolytic viruses (Table 3).

miRNAs are small non-coding RNA molecules approximately 22 nucleotides long that can negatively regulate gene expression at the post-transcriptional level [305]. In this scenario, synthetic target sequences complementary to specific miRNAs have been inserted in the UTRs of viral genes essential for replication. This approach promotes the degradation of the viral genome in healthy tissues, but not in cancer cells [131,310,311,312]. Its usefulness has been widely demonstrated and tissue specificity has been improved for many oncolytic viruses [304,311,313,314,315,316].

The results demonstrated that ICP27 protein level was higher in tumor cells than in healthy cells, indicating that this type of regulation could control HSV-1 by selectively killing NSCLC cells in vitro [292]. Generally, miRNA-21 was found to be upregulated in cancer cells [320]. An inverse miRNA control setup was created, in which miR-21 was used in cancer cells to induce, rather than repress, HSV replication. This study has shown that a viral gene under the control of miR-21 limited viral replication in healthy cells, where miR-21 was downregulated, and, at the same time, it induced a vigorous replication in cancer cells expressing miR-21 [290].

These results have prompted other studies that have combined miR-122 with miR-19, also specific for hepatocytes and downregulated in cancer cells. This modification effectively inhibited adenoviral infection in healthy pancreatic tissue and, on the contrary, it has improved the viral anti-tumor activity in pancreatic tumors [323]. The presence of viral proteins in normal tissues could create immunogenic reactions, as well as inflammation and cell death. However, other studies and clinical trials will need to be performed before the therapeutic potential of this innovative approach and its safety can be assessed in humans.

One of the first miRNA-regulated oncolytic viruses was the Coxsackievirus B3 (CVB3) characterized by the strong ability to lyse human cells of NSCLC [324,325]. The recombinant virus, called 53a-CVB, showed minimal levels of toxicity in healthy tissues and, furthermore, retained its full oncolytic activity in xenotransplant mice with human lung cancer [303]. [311] inserting target sequences complementary to miR-206 and miR-133a, specific to skeletal muscle tissue. On the contrary, the recombinant virus retained its replication ability in cancer cells, causing total regression of subcutaneous tumors, and did not replicate in healthy cells expressing complementary miRNAs, thereby reducing myotoxicity, and retaining the oncolytic potential [311].

It has shown oncolytic activity but its use can also cause side effects. In order to improve the safety profile and reduce toxicity, Ruiz et al. engineered the virus by inserting target sequences complementary to miR-124 (enriched in nerve tissue) in the 5′ UTR of the viral genome, and sequences complementary to miR-133 and miR-208 (enriched in heart tissue) were introduced in the 3′ UTR [304]. In vivo toxicity assays confirmed that miR-124, inserted within the 5′ UTR of the viral genome, suppressed viral replication in the central nervous system, while miR-133 and miR-208 inhibited viral replication in the heart tissue. This study has shown that the simultaneous use of multiple targets for miRNA reduces the saturation potential of a single miRNA.

This entry is adapted from the peer-reviewed paper 10.3390/cancers13112761