NK cell tumor therapy modulated by UV-inactivated oncolytic herpes simplex virus type 2 and checkpoint inhibitors.

Oncolytic virotherapy is a new and safe therapeutic strategy for cancer treatment. In our previous study, a new type of oncolytic herpes simplex virus type 2 (oHSV2) was constructed. Following the completion of a preclinical study, oHSV2 has now entered into clinical trials for the treatment of melanoma and other solid tumors (NCT03866525). Oncolytic viruses (OVs) are generally able to directly destroy tumor cells and stimulate the immune system to fight tumors. Natural killer (NK) cells are important components of the innate immune system and critical players against tumor cells. But the detailed interactions between oncolytic viruses and NK cells and these interaction effects on the antitumor immune response remain to be elucidated. In particular, the functions of activating surface receptors and checkpoint inhibitors on oHSV2-treated NK cells and tumor cells are still unknown. In this study, we found that UV-oHSV2 potently activates human peripheral blood mononuclear cells, leading to increased antitumor activity in vitro and in vivo. Further investigation indicated that UV-oHSV2-stimulated NK cells release IFN-γ via Toll-like receptor 2 (TLR2)/NF-κB signaling pathway and exert antitumor activity via TLR2. We found for the first time that the expression of a pair of checkpoint molecules, NKG2A (on NK cells) and HLA-E (on tumor cells), is upregulated by UV-oHSV2 stimulation. Anti-NKG2A and anti-HLA-E treatment could further enhance the antitumor effects of UV-oHSV2-stimulated NK92 cells in vitro and in vivo. As our oHSV2 clinical trial is ongoing, we expect that the combination therapy of oncolytic virus oHSV2 and anti-NKG2A/anti-HLA-E antibodies may have synergistic antitumor effects in our future clinical trials.

Oncolytic Adenovirus H101 Synergizes with Radiation in Cervical Cancer Cells.

BACKGROUND: A major challenge in cervical cancer radiotherapy is tailoring the radiation doses efficiently to eliminate malignant cells and reduce the side effects in normal tissues. Oncolytic adenovirus drug H101 was recently tested and approved as a topical adjuvant treatment for several malignancies. OBJECTIVE: This study aimed to evaluate the potential neoadjuvant radiotherapy benefits of H101 by testing the inhibitory function of H101 in combination with radiation in different cervical cancer cells. METHODS: Human cervical cancer cell lines C33a, SiHa, CaSki, and HeLa were treated with varying concentrations of H101 alone or in combination with radiation (2 Gy or 4 Gy). Cell viability and apoptosis were measured at the indicated time intervals. HPV16 E6 and cellular p53 mRNA expression alteration was measured by qRT-PCR. In situ RNA scope was used to determine HPV E6 status. P53 protein alterations were detected by Western blot. RESULTS: Cell viability and apoptosis assays revealed that the combination of a high dose of H101 (MOI=1000, 10000) with radiation yielded a synergistic anticancer effect in all tested cervical cancer cell lines (P<0.05), with the greatest effect achieved in HPV-negative C33a cells (P<0.05). Low-HPV16-viral-load SiHa cells were more sensitive to the combination therapy than high-HPV16- viral-load CaSki cells (P<0.05). The combined treatment reduced HPV16 E6 expression and increased cellular P53 levels compared to those observed with radiation alone in SiHa and CaSki cells (P<0.05). CONCLUSION: Oncolytic adenovirus H101 effectively enhances the antitumor efficacy of radiation in cervical cancer cells and may serve as a novel combination therapy for cervical cancer.

Inactivated Sendai virus strain Tianjin induces apoptosis and autophagy through reactive oxygen species production in osteosarcoma MG-63 cells.

Sendai virus strain Tianjin, a novel genotype of Sendai virus, has been proven to possess potent antitumor effect on certain cancer cell types although inactivated by ultraviolet (UV). This study was carried out to investigate the in vitro anticancer properties of UV-inactivated Sendai virus strain Tianjin (UV-Tianjin) on human osteosarcoma cells and the underlying molecular mechanism. Our studies demonstrated UV-Tianjin significantly inhibited the viability of human osteosarcoma cell lines and triggered apoptosis through activation of both extrinsic and intrinsic pathways in MG-63 cells. Meanwhile, autophagy occurred in UV-Tianjin-treated cells. Blockade of autophagy with 3-methyladenine remarkably attenuated the inhibition of cell proliferation by UV-Tianjin, suggesting that UV-Tianjin-induced autophagy may be contributing to cell death. Furthermore, UV-Tianjin induced reactive oxygen species (ROS) production, which was involved in the execution of MG-63 cell apoptosis and autophagy, as evidenced by the result that treatment of N-acetyl-L-cysteine, a ROS scavenger, attenuated both apoptosis and autophagy. In addition, inhibition of apoptosis promoted autophagy, whereas suppression of autophagy attenuated apoptosis. Our results suggest that UV-Tianjin triggers apoptosis and autophagic cell death via generation of the ROS in MG-63 cells, which might provide important insights into the effectiveness of novel strategies for osteosarcoma therapy.

Adenovirus with DNA Packaging Gene Mutations Increased Virus Release.

Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses2016, 8, 6). In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis.

Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection.

Oncolytic adenoviruses (Ads) have been shown to be safe and have great potential for the treatment of solid tumors. However, the therapeutic efficacy of Ads is antagonized by limited spread within solid tumors. To develop Ads with enhanced spread, viral particles of an E1-wildtype Ad5 dl309 was repeatedly treated with UV type C irradiation and selected for the efficient replication and release from cancer cells. After 72 cycles of treatment and cancer selection, AdUV was isolated. This vector has displayed many favorable characteristics for oncolytic therapy. AdUV was shown to lyse cancer cells more effectively than both E1-deleted and E1-wildtype Ads. This enhanced cancer cell lysis appeared to be related to increased AdUV replication in and release from infected cancer cells. AdUV-treated A549 cells displayed greater expression of the autophagy marker LC3-II during oncolysis and formed larger viral plaques upon cancer cell monolayers, indicating increased virus spread among cancer cells. This study indicates the potential of this approach of irradiation of entire viral particles for the development of oncolytic viruses with designated therapeutic properties.

An oncolytic adenovirus regulated by a radiation-inducible promoter selectively mediates hSulf-1 gene expression and mutually reinforces antitumor activity of I131-metuximab in hepatocellular carcinoma.

Gene therapy and antibody approaches are crucial auxiliary strategies for hepatocellular carcinoma (HCC) treatment. Previously, we established a survivin promoter-regulated oncolytic adenovirus that has inhibitory effect on HCC growth. The human sulfatase-1 (hSulf-1) gene can suppress the growth factor signaling pathways, then inhibit the proliferation of cancer cells and enhance cellular sensitivity to radiotherapy and chemotherapy. I(131)-metuximab (I(131)-mab) is a monoclonal anti-HCC antibody that conjugated to I(131) and specifically recognizes the HAb18G/CD147 antigen on HCC cells. To integrate the oncolytic adenovirus-based gene therapy and the I(131)-mab-based radioimmunotherapy, this study combined the CArG element of early growth response-l (Egr-l) gene with the survivin promoter to construct a radiation-inducible enhanced promoter, which was used to recombine a radiation-inducible oncolytic adenovirus as hSulf-1 gene vector. When I(131)-mab was incorporated into the treatment regimen, not only could the antibody produce radioimmunotherapeutic effect, but the I(131) radiation was able to further boost adenoviral proliferation. We demonstrated that the CArG-enhanced survivin promoter markedly improved the proliferative activity of the oncolytic adenovirus in HCC cells, thereby augmenting hSulf-1 expression and inducing cancer cell apoptosis. This novel strategy that involved multiple, synergistic mechanisms, including oncolytic therapy, gene therapy and radioimmunotherapy, was demonstrated to exert an excellent anti-cancer outcome, which will be a promising approach in HCC treatment.

Increased oncolytic efficacy for high-grade gliomas by optimal integration of ionizing radiation into the replicative cycle of HSV-1.

Oncolytic viruses have been combined with standard cancer therapies to increase therapeutic efficacy. Given the sequential activation of herpes viral genes (herpes simplex virus-1, HSV-1) and the temporal cellular changes induced by ionizing radiation, we hypothesized an optimal temporal sequence existed in combining oncolytic HSV-1 with ionizing radiation. Murine U-87 glioma xenografts were injected with luciferase encoding HSV-1, and ionizing radiation (IR) was given at times before or after viral injection. HSV-1 replication and tumor-volume response were followed. Radiation given 6-9 h after HSV-1 injection resulted in maximal viral luciferase expression and infectious viral production in tumor xenografts. The greatest xenograft regression was also seen with radiation given 6 h after viral injection. We then tested if HSV-1 replication had a dose response to ionizing radiation. HSV-1 luciferase expression exhibited a dose response as xenografts were irradiated from 0 to 5 Gy. There was no difference in viral luciferase expression as IR dose increased from 5 Gy up to 20 Gy. These results suggest that the interaction of IR with the HSV-1 lytic cycle can be manipulated for therapeutic gain by delivering IR at a specific time within viral replicative cycle.

Improved killing of human high-grade glioma cells by combining ionizing radiation with oncolytic parvovirus H-1 infection.

PURPOSE: To elucidate the influence of ionizing radiation (IR) on the oncolytic activity of Parvovirus H-1 (H-1PV) in human high-grade glioma cells. METHODS: Short term cultures of human high-grade gliomas were irradiated at different doses and infected with H-1PV. Cell viability was assessed by determining relative numbers of surviving cells. Replication of H-1PV was measured by RT-PCR of viral RNA, fluorescence-activated cell sorter (FACS) analysis and the synthesis of infectious virus particles. To identify a possible mechanism for radiation induced change in the oncolytic activity of H-1PV we performed cell cycle analyses. RESULTS: Previous irradiation rendered glioma cells fully permissive to H-1PV infection. Irradiation 24 hours prior to H-1PV infection led to increased cell killing most notably in radioresistant glioma cells. Intracellular levels of NS-1, the main effector of H-1PV induced cytotoxicity, were elevated after irradiation. S-phase levels were increased one day after irradiation improving S-phase dependent viral replication and cytotoxicity. CONCLUSION: This study demonstrates intact susceptibility of previously irradiated glioma-cells for H-1PV induced oncolysis. The combination of ionizing radiation followed by H-1PV infection increased viral cytotoxicity, especially in radioresistant gliomas. These findings support the ongoing development of a clinical trial of H-1PV in patients with recurrent glioblastomas.

Impact of radiation therapy on the oncolytic adenovirus dl520: implications on the treatment of glioblastoma.

BACKGROUND AND PURPOSE: Viral oncolytic therapy is emerging as a new form of anticancer therapy and has shown promising preclinical results, especially in combination with radio- and chemotherapy. We recently reported that nuclear localization of the human transcription factor YB-1 in multidrug-resistant cells facilitates E1-independent adenoviral replication. The aim of this study was to evaluate the combined treatment of the conditionally-replicating adenovirus dl520 and radiotherapy in glioma cell lines in vitro and in human tumor xenografts. Furthermore, the dependency of YB-1 on dl520 replication was verified by shRNA directed down regulation of YB-1. METHODS AND MATERIAL: Localization of YB-1 was determined by immunostaining. Glioma cell lines LN-18, U373 and U87 were infected with dl520. Induction of cytopathic effect (CPE), viral replication, viral yield and viral release were determined after viral infection, radiation therapy and the combination of both treatment modalities. The capacity of treatments alone or combined to induce tumor growth inhibition of subcutaneous U373 tumors was tested also in nude mice. RESULTS: Quantitative real-time PCR demonstrated that the shRNA-mediated down regulation of YB-1 is leading to a dramatic decrease in adenoviral replication of dl520. Immunostaining analysis showed that the YB-1 protein was predominantly located in the cytoplasm in the perinuclear space and less abundant in the nucleus. After irradiation we found an increase of nuclear YB-1. The addition of radiotherapy increased the oncolytic effect of dl520 with enhanced viral replication, viral yield and viral release. The oncolytic activity of dl520 plus radiation inhibited the growth of subcutaneous U373 tumors in a xenograft mouse model. CONCLUSIONS: Radiation mediated increase of nuclear YB-1 in glioma cells enhanced the oncolytic potential of adenovirus dl520.

ReVOLT: radiation-enhanced viral oncolytic therapy.

Viral oncolytic therapy has been pursued with renewed interest as the molecular basis of carcinogenesis and viral replication has been elucidated. Genetically engineered, attenuated viruses have been rationally constructed to achieve a therapeutic index in tumor cells compared with surrounding normal tissue. Many of these attenuated mutant viruses have entered clinical trials. Here we review the preclinical literature demonstrating the interaction of oncolytic viruses with ionizing radiation and provides a basis for future clinical trials.