Overcoming resistance to oncolytic virus M1 by targeting PI3K-γ in tumor-associated myeloid cells.

Oncolytic viruses (OVs) have become a category of promising anticancer immunotherapeutic agents over the last decade. However, the fact that many individuals fail to respond to OVs highlights the importance of defining the barely known immunosuppressive mechanisms that lead to treatment resistance. Here we found that the immunosuppression mediated by tumor-associated myeloid cells (TAMCs) directly quenches the antitumor effect of oncolytic virus M1 (OVM). OVM induces myeloid cells to migrate into tumors and strengthens their immunosuppressive phenotypes. Mechanically, tumor cells treated with OVM secrete interleukin-6 (IL-6) to activate the phosphatidylinositol 3-kinase (PI3K)-γ/Akt axis in TAMCs, promoting infiltration of TAMCs and aggravating their inhibition on cytotoxic CD8+ T lymphocytes. Pharmacologically targeting PI3K-γ relieves TAMC-mediated immunosuppression and enhances the efficacy of OVM. Additional treatment with immune checkpoint antibodies eradicates multiple refractory solid tumors and induces potent long-term antitumor immune memory. Our findings indicate that OVM functions as a double-edged sword in antitumor immunity and provide insights into the rationale for liberating T cell-mediated antitumor activity by abolishing TAMC-mediated immunosuppression.

Lonidamine potentiates the oncolytic efficiency of M1 virus independent of hexokinase 2 but via inhibition of antiviral immunity.

BACKGROUND: Viruses are obligate parasites that depend on host cells to provide the energy and molecular precursors necessary for successful infection. The main component of virus-induced metabolic reprogramming is the activation of glycolysis, which provides biomolecular resources for viral replication. However, little is known about the crosstalk between oncolytic viruses and host glycolytic processes. METHODS: A MTT assay was used to detect M1 virus-induced cell killing. Flow cytometry was used to monitor infection of M1 virus expressing the GFP reporter gene. qPCR and western blotting were used to detect gene expression. RNA sequencing was performed to evaluate gene expression under different drug treatments. Scanning electron microscopy was performed to visualize the endoplasmic reticulum (ER). Caspase activity was detected. Last, a mouse xenograft model was established to evaluate the antitumor effect in vivo. Most data were analyzed with a two-tailed Student's t test or one-way ANOVA with Dunnett's test for pairwise comparisons. Tumor volumes were analyzed by repeated measures of ANOVA. The Wilcoxon signed-rank test was used to compare nonnormally distributed data. RESULTS: Here, we showed that the glucose analog 2-deoxy-D-glucose (2-DG) inhibited infection by M1 virus, which we identified as a novel type of oncolytic virus, and decreased its oncolytic effect, indicating the dependence of M1 replication on glycolysis. In contrast, lonidamine, a reported hexokinase 2 (HK2) inhibitor, enhanced the infection and oncolytic effect of M1 virus independent of HK2. Further transcriptomic analysis revealed that downregulation of the antiviral immune response contributes to the lonidamine-mediated potentiation of the infection and oncolytic effect of M1 virus, and that MYC is the key factor in the pool of antiviral immune response factors inhibited by lonidamine. Moreover, lonidamine potentiated the irreversible ER stress-mediated apoptosis induced by M1 virus. Enhancement of M1's oncolytic effect by lonidamine was also identified in vivo. CONCLUSIONS: This research demonstrated the dependence of M1 virus on glycolysis and identified a candidate synergist for M1 virotherapy.

Selective replication of oncolytic virus M1 results in a bystander killing effect that is potentiated by Smac mimetics.

Oncolytic virotherapy is a treatment modality that uses native or genetically modified viruses that selectively replicate in and kill tumor cells. Viruses represent a type of pathogen-associated molecular pattern and thereby induce the up-regulation of dozens of cytokines via activating the host innate immune system. Second mitochondria-derived activator of caspases (Smac) mimetic compounds (SMCs), which antagonize the function of inhibitor of apoptosis proteins (IAPs) and induce apoptosis, sensitize tumor cells to multiple cytokines. Therefore, we sought to determine whether SMCs sensitize tumor cells to cytokines induced by the oncolytic M1 virus, thus enhancing a bystander killing effect. Here, we report that SMCs potentiate the oncolytic effect of M1 in vitro, in vivo, and ex vivo. This strengthened oncolytic efficacy resulted from the enhanced bystander killing effect caused by the M1 virus via cytokine induction. Through a microarray analysis and subsequent validation using recombinant cytokines, we identified IL-8, IL-1A, and TRAIL as the key cytokines in the bystander killing effect. Furthermore, SMCs increased the replication of M1, and the accumulation of virus protein induced irreversible endoplasmic reticulum stress- and c-Jun N-terminal kinase-mediated apoptosis. Nevertheless, the combined treatment with M1 and SMCs had little effect on normal and human primary cells. Because SMCs selectively and significantly enhance the bystander killing effect and the replication of oncolytic virus M1 specifically in cancer cells, this combined treatment may represent a promising therapeutic strategy.

Deficiency of the IRE1α-Autophagy Axis Enhances the Antitumor Effects of the Oncolytic Virus M1.

Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancer cells. M1 is a naturally occurring alphavirus (Togaviridae) which shows potent oncolytic activities against many cancers. Accumulation of unfolded proteins during virus replication leads to a transcriptional/translational response known as the unfolded protein response (UPR), which might counteract the antitumor effect of the oncolytic virus. In this report, we show that either pharmacological or biological inhibition of IRE1α or PERK, but not ATF6, substantially increases the oncolytic effects of the M1 virus. Moreover, inhibition of IRE1α blocks M1 virus-induced autophagy, which restricts the antitumor effects of the M1 virus through degradation of viral protein, in glioma cells. In addition, IRE1α suppression significantly increases the oncolytic effect of M1 virus in an orthotopic glioma model. From a molecular pathology study, we found that IRE1α is expressed at lower levels in higher-grade gliomas, suggesting greater antitumor efficacy of the oncolytic virus M1. Taken together, these findings illustrate a defensive mechanism of glioma cells against the oncolytic virus M1 and identify possible approaches to enhance the oncolytic viral protein accumulation and the subsequent lysis of tumor cells.IMPORTANCE Although oncolytic virotherapy is showing great promise in clinical applications, not all patients are benefiting. Identifying inhibitory signals in refractory cancer cells for each oncolytic virus would provide a good chance to increase the therapeutic effect. Here we describe that infection with the oncolytic virus M1 triggers the unfolded protein response (UPR) and subsequent autophagy, while blocking the UPR-autophagy axis significantly potentiates the antitumor efficacy of M1 in vitro and in vivo A survey of cancer tissue banks revealed that IRE1α, a key element in the UPR pathway, is commonly downregulated in higher-grade human gliomas, suggesting favorable prospects for the application of M1. Our work provides a potential predictor and target for enhancement of the therapeutic effectiveness of the M1 virus. We predict that the mechanism-based combination therapy will promote cancer virotherapy in the future.

Association of Low Zinc Finger Antiviral Protein Expression with Progression and Poor Survival of Patients with Hepatocellular Carcinoma.

BACKGROUND/AIMS: Zinc finger antiviral protein (ZAP) has been reported to be expressed in hepatocellular carcinoma (HCC), and ZAP expression is associated with apoptotic signaling in cancer cells. This study aimed at investigating the expression of ZAP in HCC cells and its significance in clinical pathology. METHODS: Real-time quantitative PCR and western blot assays were employed to detect ZAP RNA and protein expression in normal human hepatocytes, HCC cells, and five primary HCC cell lines. Immunohistochemistry was performed to detect ZAP expression in 147 paraffin-embedded HCC tissues and adjacent normal tissues. The clinical significance of ZAP expression was analyzed in tissue samples from patients with or without infection by hepatitis B virus (HBV). RESULTS: ZAP expression in HCC cells and human primary HCC cell lines was significantly lower than that of normal human hepatocytes. Among 147 HCC samples, ZAP expression was lower in HCC tissues than in adjacent normal tissues for 107 (77.0%) samples. In patients with HCC and HBV infection, ZAP expression was related to pathological grade (P < 0.05); in HBV-negative patients with HCC, ZAP expression was associated with tumor size (P < 0.05) and clinical stage (P < 0.05). The overall survival time in patients with low ZAP expression was significantly shorter than survival times of those with high ZAP expression (P < 0.05), especially for patients with moderately to well-differentiated HCC (Grade 1-2) and HCC at stage T1 and T2 (P < 0.05). Cox multivariate analysis showed that ZAP expression was an independent predictor of survival of patients with HCC (P < 0.01). CONCLUSION: Low ZAP expression is closely associated with disease progression and poor prognosis for patients with HCC.

Activation of Cyclic Adenosine Monophosphate Pathway Increases the Sensitivity of Cancer Cells to the Oncolytic Virus M1.

Oncolytic virotherapy is a novel and emerging treatment modality that uses replication-competent viruses to destroy cancer cells. Although diverse cancer cell types are sensitive to oncolytic viruses, one of the major challenges of oncolytic virotherapy is that the sensitivity to oncolysis ranges among different cancer cell types. Furthermore, the underlying mechanism of action is not fully understood. Here, we report that activation of cyclic adenosine monophosphate (cAMP) signaling significantly sensitizes refractory cancer cells to alphavirus M1 in vitro, in vivo, and ex vivo. We find that activation of the cAMP signaling pathway inhibits M1-induced expression of antiviral factors in refractory cancer cells, leading to prolonged and severe endoplasmic reticulum (ER) stress, and cell apoptosis. We also demonstrate that M1-mediated oncolysis, which is enhanced by cAMP signaling, involves the factor, exchange protein directly activated by cAMP 1 (Epac1), but not the classical cAMP-dependent protein kinase A (PKA). Taken together, cAMP/Epac1 signaling pathway activation inhibits antiviral factors and improves responsiveness of refractory cancer cells to M1-mediated virotherapy.

Identification and characterization of alphavirus M1 as a selective oncolytic virus targeting ZAP-defective human cancers.

Oncolytic virotherapy is a growing treatment modality that uses replicating viruses as selective antineoplastic agents. Safety and efficacy considerations dictate that an ideal oncolytic agent would discriminate between normal and cancer cells on the basis of common genetic abnormalities in human cancers. Here, we identify a naturally occurring alphavirus (M1) as a novel selective killer targeting zinc-finger antiviral protein (ZAP)-deficient cancer cells. In vitro, in vivo, and ex vivo studies showed potent oncolytic efficacy and high tumor tropism of M1. We showed that the selectivity depends on ZAP deficiency by systematic identification. A large-scale multicenter pathology study using tissue microarrays reveals that ZAP is commonly deficient in human cancers, suggesting extensive application prospects for M1. Additionally, M1 killed cancer cells by inducing endoplasmic reticulum stress-mediated apoptosis. Our report provides novel insights into potentially personalized cancer therapy using oncolytic viruses.

Enhancing antitumor efficacy of oncolytic virus M1 via albendazole-sustained CD8+ T cell activation.

The immune response plays a crucial role in the functionality of oncolytic viruses. In this study, Albendazole, an antihelminthic drug known to modulate the immune checkpoint PD-L1, was combined with the oncolytic virus M1 (OVM1) to treat mice with either prostate cancer (RM-1) or glioma (GL261) tumors. This combination therapy enhanced anti-tumor effects in immunocompetent mice, but not in immunodeficient ones, without increasing OVM1 replication. Instead, it led to an increase in the number of CD8+ T cells within the tumor, downregulated the expression of PD1 on CD8+ T cells, and upregulated activation markers such as Ki67, CD44, and CD69 and the secretion of cytotoxic factors including interferon (IFN)-γ, granzyme B, and tumor necrosis factor (TNF)-α. Consistently, it enhanced the in vitro tumor-killing activity of lymphocytes from tumor-draining lymph nodes or spleens. The synergistic effect of Albendazole on OVM1 was abolished by depleting CD8+ T cells, suggesting a CD8+ T cell-dependent mechanism. In addition, Albendazole and OVM1 therapy increased CTLA4 expression in the spleen, and the addition of CTLA4 antibodies further enhanced the anti-tumor efficacy in vivo. In summary, Albendazole can act synergistically with oncolytic viruses via CD8+ T cell activation, and the Albendazole/OVM1 combination can overcome resistance to CTLA4-based immune checkpoint blockade therapy.

Pharmacokinetic enhancement of oncolytic virus M1 by inhibiting JAK‒STAT pathway.

Oncolytic viruses (OVs), a group of replication-competent viruses that can selectively infect and kill cancer cells while leaving healthy cells intact, are emerging as promising living anticancer agents. Unlike traditional drugs composed of non-replicating compounds or biomolecules, the replicative nature of viruses confer unique pharmacokinetic properties that require further studies. Despite some pharmacokinetics studies of OVs, mechanistic insights into the connection between OV pharmacokinetics and antitumor efficacy remain vague. Here, we characterized the pharmacokinetic profile of oncolytic virus M1 (OVM) in immunocompetent mouse tumor models and identified the JAK‒STAT pathway as a key modulator of OVM pharmacokinetics. By suppressing the JAK‒STAT pathway, early OVM pharmacokinetics are ameliorated, leading to enhanced tumor-specific viral accumulation, increased AUC and Cmax, and improved antitumor efficacy. Rather than compromising antitumor immunity after JAK‒STAT inhibition, the improved pharmacokinetics of OVM promotes T cell recruitment and activation in the tumor microenvironment, providing an optimal opportunity for the therapeutic outcome of immune checkpoint blockade, such as anti-PD-L1. Taken together, this study advances our understanding of the pharmacokinetic-pharmacodynamic relationship in OV therapy.

STT3A-mediated viral N-glycosylation underlies the tumor selectivity of oncolytic virus M1.

Oncolytic viruses are emerging as promising anticancer agents. Although the essential biological function of N-glycosylation on viruses are widely accepted, roles of N-glycan and glycan-processing enzyme in oncolytic viral therapy are remain elusive. Here, via cryo-EM analysis, we identified three distinct N-glycans on the envelope of oncolytic virus M1 (OVM) as being necessary for efficient receptor binding. E1-N141-glycan has immediate impact on the binding of MXRA8 receptor, E2-N200-glycan mediates the maturation of E2 from its precursor PE2 which is unable to bind with MXRA8, and E2-N262-glycan slightly promotes receptor binding. The necessity of OVM N-glycans in receptor binding make them indispensable for oncolysis in vitro and in vivo. Further investigations identified STT3A, a key catalytic subunit of oligosaccharyltransferase (OST), as the determinant of OVM N-glycosylation, and STT3A expression in tumor cells is positively correlated with OVM-induced oncolysis. Increased STT3A expression was observed in various solid tumors, pointing to a broad-spectrum anticancer potential of OVM. Collectively, our research supports the importance of STT3A-mediated N-glycosylation in receptor binding and oncolysis of OVM, thus providing a novel predictive biomarker for OVM.

Oncolytic virus M1 functions as a bifunctional checkpoint inhibitor to enhance the antitumor activity of DC vaccine.

Although promising, dendritic cell (DC) vaccines still provide limited clinical benefits, mainly due to the immunosuppressive tumor microenvironment (TME) and the lack of tumor-associated antigens (TAAs). Oncolytic virus therapy is an ideal strategy to overcome immunosuppression and expose TAAs; therefore, they may work synergistically with DC vaccines. In this study, we demonstrate that oncolytic virus M1 (OVM) can enhance the antitumor effects of DC vaccines across diverse syngeneic mouse tumor models by increasing the infiltration of CD8+ effector T cells in the TME. Mechanically, we show that tumor cells counteract DC vaccines through the SIRPα-CD47 immune checkpoint, while OVM can downregulate SIRPα in DCs and CD47 in tumor cells. Since OVM upregulates PD-L1 in DCs, combining PD-L1 blockade with DC vaccines and OVM further enhances antitumor activity. Overall, OVM strengthens the antitumor efficacy of DC vaccines by targeting the SIRPα-CD47 axis, which exerts dominant immunosuppressive effects on DC vaccines.

Directed natural evolution generates a next-generation oncolytic virus with a high potency and safety profile.

Oncolytic viruses (OVs) represent a type of encouraging multi-mechanistic drug for the treatment of cancer. However, attenuation of virulence, which is generally required for the development of OVs based on pathogenic viral backbones, is frequently accompanied by a compromised killing effect on tumor cells. By exploiting the property of viruses to evolve and adapt in cancer cells, we perform directed natural evolution on refractory colorectal cancer cell HCT-116 and generate a next-generation oncolytic virus M1 (NGOVM) with an increase in the oncolytic effect of up to 9690-fold. The NGOVM has a broader antitumor spectrum and a more robust oncolytic effect in a range of solid tumors. Mechanistically, two critical mutations are identified in the E2 and nsP3 genes, which accelerate the entry of M1 virus by increasing its binding to the Mxra8 receptor and antagonize antiviral responses by inhibiting the activation of PKR and STAT1 in tumor cells, respectively. Importantly, the NGOVM is well tolerated in both rodents and nonhuman primates. This study implies that directed natural evolution is a generalizable approach for developing next-generation OVs with an expanded scope of application and high safety.

Differential regulation of H3K9/H3K14 acetylation by small molecules drives neuron-fate-induction of glioma cell.

Differentiation therapy using small molecules is a promising strategy for improving the prognosis of glioblastoma (GBM). Histone acetylation plays an important role in cell fate determination. Nevertheless, whether histone acetylation in specific sites determines GBM cells fate remains to be explored. Through screening from a 349 small molecule-library, we identified that histone deacetylase inhibitor (HDACi) MS-275 synergized with 8-CPT-cAMP was able to transdifferentiate U87MG GBM cells into neuron-like cells, which were characterized by cell cycle arrest, rich neuron biomarkers, and typical neuron electrophysiology. Intriguingly, acetylation tags of histone 3 at lysine 9 (H3K9ac) were decreased in the promoter of multiple oncogenes and cell cycle genes, while ones of H3K9ac and histone 3 at lysine 14 (H3K14ac) were increased in the promoter of neuron-specific genes. We then compiled a list of genes controlled by H3K9ac and H3K14ac, and proved that it is a good predictive power for pathologic grading and survival prediction. Moreover, cAMP agonist combined with HDACi also induced glioma stem cells (GSCs) to differentiate into neuron-like cells through the regulation of H3K9ac/K14ac, indicating that combined induction has the potential for recurrence-preventive application. Furthermore, the combination of cAMP activator plus HDACi significantly repressed the tumor growth in a subcutaneous GSC-derived tumor model, and temozolomide cooperated with the differentiation-inducing combination to prolong the survival in an orthotopic GSC-derived tumor model. These findings highlight epigenetic reprogramming through H3K9ac and H3K14ac as a novel approach for driving neuron-fate-induction of GBM cells.

Identification of the receptor of oncolytic virus M1 as a therapeutic predictor for multiple solid tumors.

Over the last decade, oncolytic virus (OV) therapy has shown its promising potential in tumor treatment. The fact that not every patient can benefit from it highlights the importance for defining biomarkers that help predict patients' responses. As particular self-amplifying biotherapeutics, the anti-tumor effects of OVs are highly dependent on the host factors for viral infection and replication. By using weighted gene co-expression network analysis (WGCNA), we found matrix remodeling associated 8 (MXRA8) is positively correlated with the oncolysis induced by oncolytic virus M1 (OVM). Consistently, MXRA8 promotes the oncolytic efficacy of OVM in vitro and in vivo. Moreover, the interaction of MXRA8 and OVM studied by single-particle cryo-electron microscopy (cryo-EM) showed that MXRA8 directly binds to this virus. Therefore, MXRA8 acts as the entry receptor of OVM. Pan-cancer analysis showed that MXRA8 is abundant in most solid tumors and is highly expressed in tumor tissues compared with adjacent normal ones. Further study in cancer cell lines and patient-derived tumor tissues revealed that the tumor selectivity of OVM is predominantly determined by a combinational effect of the cell membrane receptor MXRA8 and the intracellular factor, zinc-finger antiviral protein (ZAP). Taken together, our study may provide a novel dual-biomarker for precision medicine in OVM therapy.

Visualization of the Oncolytic Alphavirus M1 Life Cycle in Cancer Cells.

Oncolytic alphavirus M1 has been shown to selectively target and kill cancer cells, but cytopathic morphologies induced by M1 virus and the life cycle of the M1 strain in cancer cells remain unclear. Here, we study the key stages of M1 virus infection and replication in the M1 virus-sensitive HepG2 liver cancer cell line by transmission electron microscopy, specifically examining viral entry, assembly, maturation and release. We found that M1 virus induces vacuolization of cancer cells during infection and ultimately nuclear marginalization, a typical indicator of apoptosis. Specifically, our results suggest that the endoplasmic reticulum participates in the assembly of nucleocapsids. In the early and late stage of infection, three kinds of special cytopathic vacuoles are formed and appear to be involved in the replication, maturation and release of the virus. Taken together, our data displayed the process of M1 virus infection of tumor cells and provide the structural basis for the study of M1 virus-host interactions.

Real-Time Visualization and Quantification of Oncolytic M1 Virus In Vitro and In Vivo.

Alphavirus M1 is a promising oncolytic virus for cancer therapy. Here, we constructed a fluorescent reporter virus for real-time visualization and quantification of M1 virus both in vitro and in vivo. The reporter-encoding M1 virus maintained the characteristics of parental virus in the aspects of structure, replication capacity, the feature to induce cytopathic cell death, and the property of tumor targeting. The fluorescence is positively correlated with virus replication both in vitro and in vivo. More importantly, the reporter can be stably expressed for at least 10 generations in a serial passage assay. In summary, we successfully constructed stable and authentic reporter viruses for studying M1 virus and provided a feasible technical route for gene modification of oncolytic virus M1.

Suppression of CCDC6 sensitizes tumor to oncolytic virus M1.

Oncolytic virus is an effective therapeutic strategy for cancer treatment, which exploits natural or manipulated viruses to selectively target and kill cancer cells. However, the innate antiviral system of cancer cells may resistant to the treatment of oncolytic virus. M1 virus is a newly identified oncolytic virus belonging to alphavirus species, but the molecular mechanisms underlying its anticancer activity are largely unknown. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. RNA seq analysis was used to analyze the gene alternation after M1 virus infection. Small interfering RNAs transfection for gene knockdown was used for gene functional tests. Caspase-3/7 activity was detected by Caspase-Glo Assay Systems. A mice model of orthotopic bladder tumor was established to determine the oncolytic effectiveness of the M1 virus. The expression of cleaved-Caspase 3 as well as Ki-67 in tumor cells were detected by immunohistochemical analysis. To further define the molecular factors involved in M1 virus-mediated biological function, we knocked down genes related to alphavirus' activity and found that CCDC6 plays an important role in the oncolytic activity of M1 virus. Moreover, knocked down of CCDC6 augments the reproduction of M1 virus and resulted in endoplasmic reticulum (ER) stress-induced cell apoptosis in vitro as well as in vivo orthotopic bladder cancer model. Our research provides a rational new target for developing new compounds to promote the efficacy of oncolytic virus therapy.

Necroptotic virotherapy of oncolytic alphavirus M1 cooperated with Doxorubicin displays promising therapeutic efficacy in TNBC.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype among breast tumors and remains a challenge even for the most current therapeutic regimes. Here, we demonstrate that oncolytic alphavirus M1 effectively kills both TNBC and non-TNBC. ER-stress and apoptosis pathways are responsible for the cell death in non-TNBC as reported in other cancer types, yet the cell death in TNBC does not depend on these pathways. Transcriptomic analysis reveals that the M1 virus activates necroptosis in TNBC, which can be pharmacologically blocked by necroptosis inhibitors. By screening a library of clinically available compounds commonly used for breast cancer treatment, we find that Doxorubicin enhances the oncolytic effect of the M1 virus by up to 100-fold specifically in TNBC in vitro, and significantly stalls the tumor growth of TNBC in vivo, through promoting intratumoral virus replication and further triggering apoptosis in addition to necroptosis. These findings reveal a novel antitumor mechanism and a new combination regimen of the M1 oncolytic virus in TNBC, and highlight a need to bridge molecular diagnosis with virotherapy.

Systematic Characterization of the Biodistribution of the Oncolytic Virus M1.

Oncolytic viruses are emerging as important tools for immunotherapy for cancer treatment; however, most of the clinically tested oncolytic candidates are still administered by intratumoral injection, and new viruses capable of intravenous injection are urgently needed. The M1 virus is a positive-sense single-stranded RNA virus that belongs to the alphavirus family, and it was identified as an oncolytic virus that can selectively replicate in and kill tumor cells after intravenous injection. To further develop M1 for clinical research through intravenous injection, we systematically investigated the biodistribution characteristics of the M1 virus in normal rats, cynomolgus monkeys, and tumor-bearing immunocompromised mice. The data showed that the M1 virus was eliminated gradually from normal tissue but replicated and increased rapidly in tumor tissue. More importantly, the virus also infiltrated the blood-brain barrier and specifically replicated in and killed malignant glioma in immunocompetent mice. Our data proved the tumor selectivity and safety of the M1 virus, supporting its further clinical development.

Intravenous injection of the oncolytic virus M1 awakens antitumor T cells and overcomes resistance to checkpoint blockade.

Reversing the highly immunosuppressive tumor microenvironment (TME) is essential to achieve long-term efficacy with cancer immunotherapy. Despite the impressive clinical response to checkpoint blockade in multiple types of cancer, only a minority of patients benefit from this approach. Here, we report that the oncolytic virus M1 induces immunogenic tumor cell death and subsequently restores the ability of dendritic cells to prime antitumor T cells. Intravenous injection of M1 disrupts immune tolerance in the privileged TME, reprogramming immune-silent (cold) tumors into immune-inflamed (hot) tumors. M1 elicits potent CD8+ T cell-dependent therapeutic effects and establishes long-term antitumor immune memory in poorly immunogenic tumor models. Pretreatment with M1 sensitizes refractory tumors to subsequent checkpoint blockade by boosting T-cell recruitment and upregulating the expression of PD-L1. These findings reveal the antitumor immunological mechanism of the M1 virus and indicated that oncolytic viruses are ideal cotreatments for checkpoint blockade immunotherapy.