Active-site mTOR inhibitors augment HSV1-dICP0 infection in cancer cells via dysregulated eIF4E/4E-BP axis.

Herpes Simplex Virus 1 (HSV1) is amongst the most clinically advanced oncolytic virus platforms. However, efficient and sustained viral replication within tumours is limiting. Rapamycin can stimulate HSV1 replication in cancer cells, but active-site dual mTORC1 and mTORC2 (mammalian target of rapamycin complex 1 and 2) inhibitors (asTORi) were shown to suppress the virus in normal cells. Surprisingly, using the infected cell protein 0 (ICP0)-deleted HSV1 (HSV1-dICP0), we found that asTORi markedly augment infection in cancer cells and a mouse mammary cancer xenograft. Mechanistically, asTORi repressed mRNA translation in normal cells, resulting in defective antiviral response but also inhibition of HSV1-dICP0 replication. asTORi also reduced antiviral response in cancer cells, however in contrast to normal cells, transformed cells and cells transduced to elevate the expression of eukaryotic initiation factor 4E (eIF4E) or to silence the repressors eIF4E binding proteins (4E-BPs), selectively maintained HSV1-dICP0 protein synthesis during asTORi treatment, ultimately supporting increased viral replication. Our data show that altered eIF4E/4E-BPs expression can act to promote HSV1-dICP0 infection under prolonged mTOR inhibition. Thus, pharmacoviral combination of asTORi and HSV1 can target cancer cells displaying dysregulated eIF4E/4E-BPs axis.

Multi-modal Potentiation of Oncolytic Virotherapy by Vanadium Compounds.

Oncolytic viruses (OV) are an emerging class of anticancer bio-therapeutics that induce antitumor immunity through selective replication in tumor cells. However, the efficacy of OVs as single agents remains limited. We introduce a strategy that boosts the therapeutic efficacy of OVs by combining their activity with immuno-modulating, small molecule protein tyrosine phosphatase inhibitors. We report that vanadium-based phosphatase inhibitors enhance OV infection in vitro and ex vivo, in resistant tumor cell lines. Furthermore, vanadium compounds increase antitumor efficacy in combination with OV in several syngeneic tumor models, leading to systemic and durable responses, even in models otherwise refractory to OV and drug alone. Mechanistically, this involves subverting the antiviral type I IFN response toward a death-inducing and pro-inflammatory type II IFN response, leading to improved OV spread, increased bystander killing of cancer cells, and enhanced antitumor immune stimulation. Overall, we showcase a new ability of vanadium compounds to simultaneously maximize viral oncolysis and systemic anticancer immunity, offering new avenues for the development of improved immunotherapy strategies.

Oncolytic Maraba Virus MG1 as a Treatment for Sarcoma.

The poor prognosis of patients with advanced bone and soft-tissue sarcoma has not changed in the past several decades, highlighting the necessity for new therapeutic approaches. Immunotherapies, including oncolytic viral (OV) therapy, have shown great promise in a number of clinical trials for a variety of tumor types. However, the effective application of OV in treating sarcoma still remains to be demonstrated. Although few pre-clinical studies using distinct OVs have been performed and demonstrated therapeutic benefit in sarcoma models, a side-by-side comparison of clinically relevant OV platforms has not been performed. Four clinically relevant OV platforms (Reovirus, Vaccinia virus, Herpes-simplex virus and Rhabdovirus) were screened for their ability to infect and kill human and canine sarcoma cell lines in vitro, and human sarcoma specimens ex vivo. In vivo treatment efficacy was tested in a murine model. The rhabdovirus MG1 demonstrated the highest potency in vitro. Ex vivo, MG1 productively infected more than 80% of human sarcoma tissues tested, and treatment in vivo led to a significant increase in long-lasting cures in sarcoma-bearing mice. Importantly, MG1 treatment induced the generation of memory immune response that provided protection against a subsequent tumor challenge. This study opens the door for the use of MG1-based oncolytic immunotherapy strategies as treatment for sarcoma or as a component of a combined therapy.

Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans.

The efficacy and safety of biological molecules in cancer therapy, such as peptides and small interfering RNAs (siRNAs), could be markedly increased if high concentrations could be achieved and amplified selectively in tumour tissues versus normal tissues after intravenous administration. This has not been achievable so far in humans. We hypothesized that a poxvirus, which evolved for blood-borne systemic spread in mammals, could be engineered for cancer-selective replication and used as a vehicle for the intravenous delivery and expression of transgenes in tumours. JX-594 is an oncolytic poxvirus engineered for replication, transgene expression and amplification in cancer cells harbouring activation of the epidermal growth factor receptor (EGFR)/Ras pathway, followed by cell lysis and anticancer immunity. Here we show in a clinical trial that JX-594 selectively infects, replicates and expresses transgene products in cancer tissue after intravenous infusion, in a dose-related fashion. Normal tissues were not affected clinically. This platform technology opens up the possibility of multifunctional products that selectively express high concentrations of several complementary therapeutic and imaging molecules in metastatic solid tumours in humans.

Bacterial-mediated knockdown of tumor resistance to an oncolytic virus enhances therapy.

Oncolytic viruses (OVs) and bacteria share the property of tumor-selective replication following systemic administration. In the case of nonpathogenic bacteria, tumor selectivity relates to their ability to grow extracellularly within tumor stroma and is therefore ideally suited to restricting the production of bacterially produced therapeutic agents to tumors. We have previously shown the ability of the type 1 interferon antagonist B18R to enhance the replication and spread of vesicular stomatitis virus (VSV) by overcoming related cellular innate immunity. In this study, we utilized nonpathogenic bacteria (E. coli) expressing B18R to facilitate tumor-specific production of B18R, resulting in a microenvironment depleted of bioactive antiviral cytokine, thus "preconditioning" the tumor to enhance subsequent tumor destruction by the OV. Both in vitro and in vivo infection by VSVΔ51 was greatly enhanced by B18R produced from E. coli. Moreover, a significant increase in therapeutic efficacy resulted from intravenous (i.v.) injection of bacteria to tumor-bearing mice 5 days prior to i.v. VSVΔ51 administration, as evidenced by a significant reduction in tumor growth and increased survival in mice. Our strategy is the first example where two such diverse microorganisms are rationally combined and demonstrates the feasibility of combining complementary microorganisms to improve therapeutic outcome.

Resistance to two heterologous neurotropic oncolytic viruses, Semliki Forest virus and vaccinia virus, in experimental glioma.

Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses. Also, vaccinia virus appears to be sensitive to SFV-induced antiviral interference.

Oncolytic Vaccinia virus safely and effectively treats skin tumors in mouse models of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is an orphan autosomal recessive disorder of DNA repair. When exposed to genotoxic stress, XP patients have reduced capacity to remove bulky adducts by nucleotide excision repair and are thus greatly predisposed to cancer. Unfortunately, given the nature of their underlying genetic defect, tumor-bearing XP patients cannot be treated with conventional DNA damaging therapies. Engineered strains of the poxvirus Vaccinia have been shown to cure cancer in numerous preclinical models, and based on promising Phase I/II clinical trials have recently been approved for late phase evaluation in humans. As poxviruses are nongenotoxic, we investigated whether clinical-candidate strains of Vaccinia can safely and effectively treat cancers arising from XP. In vitro, Vaccinia virus was highly cytotoxic against tumor-derived cells from XP patients, on average 10- to 100-fold more so than on nontumor derived control cells from similar patients. In vivo, local or systemic administration of Vaccinia virus led to durable tumor resolution in both xenograft and genetic models of XP. Importantly, Vaccinia virus was well tolerated in the genetic models, which are each null for a critical component of the DNA repair process. Taken together, our data suggest that oncolytic Vaccinia virus may be a safe and effective therapy for cancers arising from XP, and raise the possibility of similar therapeutic potential against tumors that arise in patients with other DNA repair disorders.

The oncolytic poxvirus JX-594 selectively replicates in and destroys cancer cells driven by genetic pathways commonly activated in cancers.

Oncolytic viruses are generally designed to be cancer selective on the basis of a single genetic mutation. JX-594 is a thymidine kinase (TK) gene-inactivated oncolytic vaccinia virus expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and lac-Z transgenes that is designed to destroy cancer cells through replication-dependent cell lysis and stimulation of antitumoral immunity. JX-594 has demonstrated a favorable safety profile and reproducible tumor necrosis in a variety of solid cancer types in clinical trials. However, the mechanism(s) responsible for its cancer-selectivity have not yet been well described. We analyzed the replication of JX-594 in three model systems: primary normal and cancer cells, surgical explants, and murine tumor models. JX-594 replication, transgene expression, and cytopathic effects were highly cancer-selective, and broad spectrum activity was demonstrated. JX-594 cancer-selectivity was multi-mechanistic; replication was activated by epidermal growth factor receptor (EGFR)/Ras pathway signaling, cellular TK levels, and cancer cell resistance to type-I interferons (IFNs). These findings confirm a large therapeutic index for JX-594 that is driven by common genetic abnormalities in human solid tumors. This appears to be the first description of multiple selectivity mechanisms, both inherent and engineered, for an oncolytic virus. These findings have implications for oncolytic viruses in general, and suggest that their cancer targeting is a complex and multifactorial process.

Harnessing oncolytic virus-mediated antitumor immunity in an infected cell vaccine.

Treatment of permissive tumors with the oncolytic virus (OV) VSV-Δ51 leads to a robust antitumor T-cell response, which contributes to efficacy; however, many tumors are not permissive to in vivo treatment with VSV-Δ51. In an attempt to channel the immune stimulatory properties of VSV-Δ51 and broaden the scope of tumors that can be treated by an OV, we have developed a potent oncolytic vaccine platform, consisting of tumor cells infected with VSV-Δ51. We demonstrate that prophylactic immunization with this infected cell vaccine (ICV) protected mice from subsequent tumor challenge, and expression of granulocyte-monocyte colony stimulating factor (GM-CSF) by the virus (VSVgm-ICV) increased efficacy. Immunization with VSVgm-ICV in the VSV-resistant B16-F10 model induced maturation of dendritic and natural killer (NK) cell populations. The challenge tumor is rapidly infiltrated by a large number of interferon γ (IFNγ)-producing T and NK cells. Finally, we demonstrate that this approach is robust enough to control the growth of established tumors. This strategy is broadly applicable because of VSV's extremely broad tropism, allowing nearly all cell types to be infected at high multiplicities of infection in vitro, where the virus replication kinetics outpace the cellular IFN response. It is also personalized to the unique tumor antigen(s) displayed by the cancer cell.

Sensitivity of cervical carcinoma cells to vesicular stomatitis virus-induced oncolysis: potential role of human papilloma virus infection.

High-risk carcinogenic subtypes of human papilloma virus (HPV) are associated with the development of squamous cell carcinomas of the cervix (CC) and a subset of head and neck (HNSCC). Recurrent metastatic diseases of these sites display a dismal prognosis. Therefore, there is an urgent need to uncover innovative therapeutic strategies in this clinical setting. Oncolytic viruses, including vesicular stomatitis virus (VSV), were identified due to their ability to specifically target tumor cells that generally display defects in interferon (IFN) signaling. HPV expressed proteins can inhibit IFN signaling; therefore, HPV-infected cells may be particularly sensitive to VSV oncolysis. In this study, we evaluated the sensitivity of four CC (HPV+) and four HNSCC (HPV-) derived cell lines to VSV oncolysis. Interestingly, the CC cell lines were consistently more sensitive to VSV cytotoxicity than the HNSCC cell lines tested. Exogenous IFN addition or infection with two attenuated VSV variants that are more susceptible to IFN inhibition failed to attenuate VSV oncolysis in hypersensitive CC cell lines. Furthermore, the expression of HPV-E6, that inhibits IFN receptor signaling, in the VSV-resistant HNSCC cell line SCC25 attenuated VSV-induced IFN response and significantly enhanced VSV cytotoxicity. Finally, differential VSV infection and replication was confirmed in xenograft murine tumor models and explant tumor tissues from two patients with CC. Taken together, these results demonstrate that HPV-infected cells are susceptible to oncolytic virus therapy and that this approach may represent a novel therapeutic approach in HPV positive CC and HNSCC patients.

Antiangiogenic arming of an oncolytic vaccinia virus enhances antitumor efficacy in renal cell cancer models.

Oncolytic vaccinia viruses have shown compelling results in preclinical cancer models and promising preliminary safety and antitumor activity in early clinical trials. However, to facilitate systemic application it would be useful to improve tumor targeting and antitumor efficacy further. Here we report the generation of vvdd-VEGFR-1-Ig, a targeted and armed oncolytic vaccinia virus. Tumor targeting was achieved by deletion of genes for thymidine kinase and vaccinia virus growth factor, which are necessary for replication in normal but not in cancer cells. Given the high vascularization typical of kidney cancers, we armed the virus with the soluble vascular endothelial growth factor (VEGF) receptor 1 protein for an antiangiogenic effect. Systemic application of high doses of vvdd-VEGFR-1-Ig resulted in cytokine induction in an immunocompromised mouse model. Upon histopathological analysis, splenic extramedullary hematopoiesis was seen in all virus-injected mice and was more pronounced in the vvdd-VEGFR-1-Ig group. Analysis of the innate immune response after intravenous virus injection revealed high transient and dose-dependent cytokine elevations. When medium and low doses were used for intratumoral or intravenous injection, vvdd-VEGFR-1-Ig exhibited a stronger antitumor effect than the unarmed control. Furthermore, expression of VEGFR-1-Ig was confirmed, and a concurrent antiangiogenic effect was seen. In an immunocompetent model, systemic vvdd-VEGFR-1-Ig exhibited superior antitumor efficacy compared to the unarmed control virus. In conclusion, the targeted and armed vvdd-VEGFR-1-Ig has promising anticancer activity in renal cell cancer models. Extramedullary hematopoiesis may be a sensitive indicator of vaccinia virus effects in mice.

Potent oncolytic activity of raccoonpox virus in the absence of natural pathogenicity.

A number of oncolytic virus (OV) candidates currently in clinical trials are human viruses that have been engineered to be safer for patient administration by limiting normal cell targeting and replication. The newest OVs include viruses that cause no disease in humans, yet still have natural tumor tropism. Raccoonpox virus (RCNV) is a member of the Orthopoxvirus genus of Poxviridae and closely related to vaccinia virus, yet has no known pathogenicity in any mammalian species. A screen of cells from the NCI-60 cancer cell panel using growth curves demonstrated greater than a log increase in replication of RCNV in nearly 74% of the cell lines tested, similar to other tested OV poxviruses. In normal cell lines, pretreatment with interferon (IFN)-alpha/beta resulted in significant inhibition of RCNV replication. In both xenograft and syngeneic models of solid tumors, injection of RCNV resulted in significantly slower tumor progression and increased survival of mice. RCNV treatment also prolonged survival in treatment-resistant models of brain tumors and decreased tumor burden by systemic administration in models of lung metastasis.

Synergistic interaction between oncolytic viruses augments tumor killing.

A major barrier to all oncolytic viruses (OVs) in clinical development is cellular innate immunity, which is variably active in a spectrum of human malignancies. To overcome the heterogeneity of tumor response, we combined complementary OVs that attack cancers in distinct ways to improve therapeutic outcome. Two genetically distinct viruses, vesicular stomatitis virus (VSV) and vaccinia virus (VV), were used to eliminate the risk of recombination. The combination was tested in a variety of tumor types in vitro, in immunodeficient and immunocompetent mouse tumor models, and ex vivo, in a panel of primary human cancer samples. We found that VV synergistically enhanced VSV antitumor activity, dependent in large part on the activity of the VV B18R gene product. A recombinant version of VSV expressing the fusion-associated small-transmembrane (p14FAST) protein also further enhanced the ability of VV to spread through an infected monolayer, resulting in a "ping pong" oncolytic effect wherein each virus enhanced the ability of the other to replicate and/or spread in tumor cells. Our strategy is the first example where OVs are rationally combined to utilize attributes of different OVs to overcome the heterogeneity of malignancies and demonstrates the feasibility of combining complementary OVs to improve therapeutic outcome.

A high-throughput pharmacoviral approach identifies novel oncolytic virus sensitizers.

Oncolytic viruses (OVs) are promising anticancer agents but like other cancer monotherapies, the genetic heterogeneity of human malignancies can lead to treatment resistance. We used a virus/cell-based assay to screen diverse chemical libraries to identify small molecules that could act in synergy with OVs to destroy tumor cells that resist viral infection. Several molecules were identified that aid in viral oncolysis, enhancing virus replication and spread as much as 1,000-fold in tumor cells. One of these molecules we named virus-sensitizers 1 (VSe1), was found to target tumor innate immune response and could enhance OV efficacy in animal tumor models and within primary human tumor explants while remaining benign to normal tissues. We believe this is the first example of a virus/cell-based "pharmacoviral" screen aimed to identify small molecules that modulate cellular response to virus infection and enhance oncolytic virotherapy.

Src-mediated phosphorylation of Hsp90 in response to vascular endothelial growth factor (VEGF) is required for VEGF receptor-2 signaling to endothelial NO synthase.

Nitric oxide (NO) release from endothelial cells, via endothelial NO synthase (eNOS) activation, is central to the proangiogenic actions of vascular endothelial growth factor (VEGF). VEGF signaling to eNOS is principally mediated by an Akt-dependent phosphorylation of eNOS and by increased association of eNOS to the molecular chaperone, heat-shock protein 90 kDa (Hsp90). Herein, we report that VEGFR-2 activation induces tyrosine phosphorylation of VEGF receptor 2 (VEGFR-2)-associated Hsp90beta. Tyrosine phosphorylation of Hsp90beta in response to VEGF is dependent on internalization of the VEGFR-2 and on Src kinase activation. Furthermore, we demonstrate that c-Src directly phosphorylates Hsp90 on tyrosine 300 residue and that this event is essential for VEGF-stimulated eNOS association to Hsp90 and thus NO release from endothelial cells. Our work identifies Y300 phosphorylation of Hsp90 as a novel regulated posttranslational modification of the chaperone and demonstrates its importance in the proangiogenic actions of VEGF, namely by regulating NO release from endothelial cells.

Viral Delivery of CAR Targets to Solid Tumors Enables Effective Cell Therapy.

Chimeric antigen receptor (CAR) T cell therapy has had limited efficacy for solid tumors, largely due to a lack of selectively and highly expressed surface antigens. To avoid reliance on a tumor's endogenous antigens, here we describe a method of tumor-selective delivery of surface antigens using an oncolytic virus to enable a generalizable CAR T cell therapy. Using CD19 as our proof of concept, we engineered a thymidine kinase-disrupted vaccinia virus to selectively deliver CD19 to malignant cells, and thus demonstrated potentiation of CD19 CAR T cell activity against two tumor types in vitro. In an immunocompetent model of B16 melanoma, this combination markedly delayed tumor growth and improved median survival compared with antigen-mismatched combinations. We also found that CD19 delivery could improve CAR T cell activity against tumor cells that express low levels of cognate antigen, suggesting a potential application in counteracting antigen-low escape. This approach highlights the potential of engineering tumors for effective adoptive cell therapy.

Endothelial cell migration during angiogenesis.

Endothelial cell migration is essential to angiogenesis. This motile process is directionally regulated by chemotactic, haptotactic, and mechanotactic stimuli and further involves degradation of the extracellular matrix to enable progression of the migrating cells. It requires the activation of several signaling pathways that converge on cytoskeletal remodeling. Then, it follows a series of events in which the endothelial cells extend, contract, and throw their rear toward the front and progress forward. The aim of this review is to give an integrative view of the signaling mechanisms that govern endothelial cell migration in the context of angiogenesis.

Phosphorylation of focal adhesion kinase (FAK) on Ser732 is induced by rho-dependent kinase and is essential for proline-rich tyrosine kinase-2-mediated phosphorylation of FAK on Tyr407 in response to vascular endothelial growth factor.

Focal adhesion kinase (FAK) is phosphorylated on tyrosine and serine residues after cell activation. In the present work, we investigated the relationship between tyrosine and serine phosphorylation of FAK in promoting endothelial cell migration in response to vascular endothelial growth factor (VEGF). We found that VEGF induces the activation of the Rho-dependent kinase (ROCK) downstream from vascular endothelial growth factor receptor (VEGFR) 2. In turn, activated ROCK directly phosphorylates FAK on Ser732. Proline-rich tyrosine kinase-2 (Pyk2) is also activated in response to VEGF. Its activation requires the clustering of integrin alphavbeta3 and triggers directly the phosphorylation of Tyr407 within FAK, an event necessary for cell migration. Interestingly, ROCK-mediated phosphorylation of Ser732 is essential for Pyk2-dependent phosphorylation of Tyr407, because the latter is abrogated in cells expressing a FAK mutant that is nonphosphorylatable on Ser732. We suggest that VEGF elicits the activation of the VEGFR2-ROCK pathway, leading to phosphorylation of Ser732 within FAK. In turn, phosphorylation of Ser732 would change the conformation of FAK, making it accessible to Pyk2 activated in response to its association with integrin beta3. Then, activated Pyk2 triggers the phosphorylation of FAK on Tyr407, promoting cell migration.

Deletion of Apoptosis Inhibitor F1L in Vaccinia Virus Increases Safety and Oncolysis for Cancer Therapy.

Vaccinia virus (VACV) possesses a great safety record as a smallpox vaccine and has been intensively used as an oncolytic virus against various types of cancer over the past decade. Different strategies were developed to make VACV safe and selective to cancer cells. Leading clinical candidates, such as Pexa-Vec, are attenuated through deletion of the viral thymidine kinase (TK) gene, which limits virus growth to replicate in cancer tissue. However, tumors are not the only tissues whose metabolic activity can overcome the lack of viral TK. In this study, we sought to further increase the tumor-specific replication and oncolytic potential of Copenhagen strain VACV ΔTK. We show that deletion of the anti-apoptosis viral gene F1L not only increases the safety of the Copenhagen ΔTK virus but also improves its oncolytic activity in an aggressive glioblastoma model. The additional loss of F1L does not affect VACV replication capacity, yet its ability to induce cancer cell death is significantly increased. Our results also indicate that cell death induced by the Copenhagen ΔTK/F1L mutant releases more immunogenic signals, as indicated by increased levels of IL-1ß production. A cytotoxicity screen in an NCI-60 panel shows that the ΔTK/F1L virus induces faster tumor cell death in different cancer types. Most importantly, we show that, compared to the TK-deleted virus, the ΔTK/F1L virus is attenuated in human normal cells and causes fewer pox lesions in murine models. Collectively, our findings describe a new oncolytic vaccinia deletion strain that improves safety and increases tumor cell killing.

Trial Watch: Oncolytic viro-immunotherapy of hematologic and solid tumors.

Oncolytic viruses selectively target and kill cancer cells in an immunogenic fashion, thus supporting the establishment of therapeutically relevant tumor-specific immune responses. In 2015, the US Food and Drug Administration (FDA) approved the oncolytic herpes simplex virus T-VEC for use in advanced melanoma patients. Since then, a plethora of trials has been initiated to assess the safety and efficacy of multiple oncolytic viruses in patients affected with various malignancies. Here, we summarize recent preclinical and clinical progress in the field of oncolytic virotherapy.