Oncolytic vesicular stomatitis virus alone or in combination with JAK inhibitors is effective against ovarian cancer.

Therapy-resistant ovarian cancers have a poor prognosis and novel effective treatment options are urgently needed. In this study, we evaluated the therapeutic efficacy of the oncolytic vesicular stomatitis virus (VSV) against a panel of patient-derived ovarian cancer cell lines of all epithelial subtypes. Notably, we found that most of the cell lines were sensitive to VSV virotherapy. With the objective of improving treatment efficacy for the oncolytic virus-resistant cell lines, we tested various combinations with ovarian cancer standard of care drugs: olaparib, carboplatin, paclitaxel, doxorubicin, cyclophosphamide, and gemcitabine. While none of these combinations revealed to be beneficial, further experiments demonstrated that the antiviral interferon pathway was functional in VSV-resistant cell lines. Given that interferons signal through Janus kinase (JAK)-STAT to mediate their antiviral function, we tested combinations of oncolytic VSV with clinically relevant JAK inhibitors. Our results show that combining VSV with various JAK inhibitors, including ruxolitinib, enhances VSV virotherapy and treatment efficacy. Altogether, we show that VSV, either as a stand-alone treatment or in combination with JAK inhibitors provides an effective therapeutic option for ovarian cancer patients.

PD-L1 promotes oncolytic virus infection via a metabolic shift that inhibits the type I IFN pathway.

While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced efficacy of oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 promoted a metabolic shift characterized by enhanced glycolysis rate that resulted in increased lactate production. In turn, lactate inhibited type I IFN responses. In addition to adding mechanistic insight into PD-L1 intrinsic function, our results will also help guide the numerous ongoing efforts to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials.

Increased expression of the immunoproteasome subunits PSMB8 and PSMB9 by cancer cells correlate with better outcomes for triple-negative breast cancers.

Proteasome dependency is a feature of many cancers that can be targeted by proteasome inhibitors. For some cancer types, notably breast cancer and triple-negative breast cancer (TNBC), high mRNA expression of a modified form of the proteasome, called the immunoproteasome (ImP), correlates with better outcomes and higher expression of one ImP subunit was associated with slower tumor growth in a small patient cohort. While these findings are in line with an anti-tumoral role of the ImP in breast cancer, studies investigating ImP expression at the protein level in large patient cohorts are lacking. Furthermore, while ImPs can be found in both immune and non-immune cells, the cellular source is often ignored in correlative studies. In order to determine the impact of ImP expression on breast cancer outcomes, we assessed the protein expression and cellular source of the ImP subunits PSMB8 and PSMB9 in a cohort of 2070 patients. Our data show a clear correlation between high ImP expression and better outcomes, most notably for TNBC patients and when tumor cells rather than stromal or immune cells express PSMB8 or PSMB9. Our results therefore suggest that ImP expression by tumor cells could be used as prognostic markers of TNBC outcomes.

High Levels of MFG-E8 Confer a Good Prognosis in Prostate and Renal Cancer Patients.

Milk fat globule-epidermal growth factor-8 (MFG-E8) is a glycoprotein secreted by different cell types, including apoptotic cells and activated macrophages. MFG-E8 is highly expressed in a variety of cancers and is classically associated with tumor growth and poor patient prognosis through reprogramming of macrophages into the pro-tumoral/pro-angiogenic M2 phenotype. To date, correlations between levels of MFG-E8 and patient survival in prostate and renal cancers remain unclear. Here, we quantified MFG-E8 and CD68/CD206 expression by immunofluorescence staining in tissue microarrays constructed from renal (n = 190) and prostate (n = 274) cancer patient specimens. Percentages of MFG-E8-positive surface area were assessed in each patient core and Kaplan-Meier analyses were performed accordingly. We found that MFG-E8 was expressed more abundantly in malignant regions of prostate tissue and papillary renal cell carcinoma but was also increased in the normal adjacent regions in clear cell renal carcinoma. In addition, M2 tumor-associated macrophage staining was increased in the normal adjacent tissues compared to the malignant areas in renal cancer patients. Overall, high tissue expression of MFG-E8 was associated with less disease progression and better survival in prostate and renal cancer patients. Our observations provide new insights into tumoral MFG-E8 content and macrophage reprogramming in cancer.

In silico trials predict that combination strategies for enhancing vesicular stomatitis oncolytic virus are determined by tumor aggressivity.

BACKGROUND: Immunotherapies, driven by immune-mediated antitumorigenicity, offer the potential for significant improvements to the treatment of multiple cancer types. Identifying therapeutic strategies that bolster antitumor immunity while limiting immune suppression is critical to selecting treatment combinations and schedules that offer durable therapeutic benefits. Combination oncolytic virus (OV) therapy, wherein complementary OVs are administered in succession, offer such promise, yet their translation from preclinical studies to clinical implementation is a major challenge. Overcoming this obstacle requires answering fundamental questions about how to effectively design and tailor schedules to provide the most benefit to patients. METHODS: We developed a computational biology model of combined oncolytic vaccinia (an enhancer virus) and vesicular stomatitis virus (VSV) calibrated to and validated against multiple data sources. We then optimized protocols in a cohort of heterogeneous virtual individuals by leveraging this model and our previously established in silico clinical trial platform. RESULTS: Enhancer multiplicity was shown to have little to no impact on the average response to therapy. However, the duration of the VSV injection lag was found to be determinant for survival outcomes. Importantly, through treatment individualization, we found that optimal combination schedules are closely linked to tumor aggressivity. We predicted that patients with aggressively growing tumors required a single enhancer followed by a VSV injection 1 day later, whereas a small subset of patients with the slowest growing tumors needed multiple enhancers followed by a longer VSV delay of 15 days, suggesting that intrinsic tumor growth rates could inform the segregation of patients into clinical trials and ultimately determine patient survival. These results were validated in entirely new cohorts of virtual individuals with aggressive or non-aggressive subtypes. CONCLUSIONS: Based on our results, improved therapeutic schedules for combinations with enhancer OVs can be studied and implemented. Our results further underline the impact of interdisciplinary approaches to preclinical planning and the importance of computational approaches to drug discovery and development.

The pros and cons of interferons for oncolytic virotherapy.

Interferons (IFN) are potent immune stimulators that play key roles in both innate and adaptive immune responses. They are considered the first line of defense against viral pathogens and can even be used as treatments to boost the immune system. While viruses are usually seen as a threat to the host, an emerging class of cancer therapeutics exploits the natural capacity of some viruses to directly infect and kill cancer cells. The cancer-specificity of these bio-therapeutics, called oncolytic viruses (OVs), often relies on defective IFN responses that are frequently observed in cancer cells, therefore increasing their vulnerability to viruses compared to healthy cells. To ensure the safety of the therapy, many OVs have been engineered to further activate the IFN response. As a consequence of this IFN over-stimulation, the virus is cleared faster by the immune system, which limits direct oncolysis. Importantly, the therapeutic activity of OVs also relies on their capacity to trigger anti-tumor immunity and IFNs are key players in this aspect. Here, we review the complex cancer-virus-anti-tumor immunity interplay and discuss the diverse functions of IFNs for each of these processes.

Magnetic targeting of oncolytic VSV-based therapies improves infection of tumor cells in the presence of virus-specific neutralizing antibodies in vitro.

Oncolytic viruses (OVs) are a class of biotherapeutics that are currently being explored for the treatment of cancer. While showing promise in several pre-clinical and clinical studies, systemic delivery of these anti-cancer agents is hampered by inefficient tumor targeting and a host immune system that is highly evolved to detect and neutralize pathogens. To shield the virus from immune recognition and destruction, the use of cells as delivery vehicles has been explored for the systemic delivery of OVs. Though several types of cell carriers are able to protect OVs during intravenous delivery, many still lack the ability to specifically home to or accumulate within the tumor microenvironment. Overall, OV-based therapeutics could benefit from tumor targeting strategies to maximize tumor-specific delivery and minimize infection of off-target tissues. In the current study, we examine magnetic targeting as a strategy to improve OV infection of tumor cells in vitro. We found that magnetic targeting of magnetically-labeled VSV particles or VSV-infected cell carriers resulted in increased infection and killing of tumor cells. Furthermore, this enhanced infection of target tumor cells was observed even in the presence of virus-specific neutralizing antibodies. Overall, our findings suggest that magnetic targeting strategies can improve the infection of tumor cells and may be a viable strategy to improve the tumor-targeted delivery of oncolytic VSV-based therapeutics.

Methionine Metabolism Shapes T Helper Cell Responses through Regulation of Epigenetic Reprogramming.

Epigenetic modifications on DNA and histones regulate gene expression by modulating chromatin accessibility to transcription machinery. Here we identify methionine as a key nutrient affecting epigenetic reprogramming in CD4+ T helper (Th) cells. Using metabolomics, we showed that methionine is rapidly taken up by activated T cells and serves as the major substrate for biosynthesis of the universal methyl donor S-adenosyl-L-methionine (SAM). Methionine was required to maintain intracellular SAM pools in T cells. Methionine restriction reduced histone H3K4 methylation (H3K4me3) at the promoter regions of key genes involved in Th17 cell proliferation and cytokine production. Applied to the mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis), dietary methionine restriction reduced the expansion of pathogenic Th17 cells in vivo, leading to reduced T cell-mediated neuroinflammation and disease onset. Our data identify methionine as a key nutritional factor shaping Th cell proliferation and function in part through regulation of histone methylation.

Pre-surgical oncolytic virotherapy improves breast cancer outcomes.

Oncolytic viruses (OVs) are a novel class of cancer biotherapeutics with the ability to kill cancers and trigger anti-tumor immunity. Using murine models of cancer in pre-clinical proof-of-concept studies, we found that neoadjuvant OV administration before surgery efficiently prevents relapse, controls metastases and sensitizes tumors to immune checkpoint inhibitors (ICIs).

Pre-surgical neoadjuvant oncolytic virotherapy confers protection against rechallenge in a murine model of breast cancer.

The use of oncolytic viruses (OVs) for cancer treatment is emerging as a successful strategy that combines the direct, targeted killing of the cancer with the induction of a long-lasting anti-tumor immune response. Using multiple aggressive murine models of triple-negative breast cancer, we have recently demonstrated that the early administration of oncolytic Maraba virus (MRB) prior to surgical resection of the primary tumor is sufficient to minimize the metastatic burden, protect against tumor rechallenge, cure a fraction of the mice and sensitize refractory tumors to immune checkpoint blockade without the need for further treatment. Here, we apply our surgical model to other OVs: Vesicular stomatitis virus (VSV), Adenovirus (Ad), Reovirus (Reo) and Herpes simplex virus (HSV) and show that all of the tested OVs could positively change the outcome of the treated animals. The growth of the primary and secondary tumors was differently affected by the various OVs and most of the viruses conferred survival benefits in this neoadjuvant setting despite the absence of direct treatment following rechallenge. This study establishes that OV-therapy confers long-term protection when administered in the pre-operative window of opportunity.

Contribution of NK cells to immunotherapy mediated by PD-1/PD-L1 blockade.

Checkpoint blockade immunotherapy targeting the PD-1/PD-L1 inhibitory axis has produced remarkable results in the treatment of several types of cancer. Whereas cytotoxic T cells are known to provide important antitumor effects during checkpoint blockade, certain cancers with low MHC expression are responsive to therapy, suggesting that other immune cell types may also play a role. Here, we employed several mouse models of cancer to investigate the effect of PD-1/PD-L1 blockade on NK cells, a population of cytotoxic innate lymphocytes that also mediate antitumor immunity. We discovered that PD-1 and PD-L1 blockade elicited a strong NK cell response that was indispensable for the full therapeutic effect of immunotherapy. PD-1 was expressed on NK cells within transplantable, spontaneous, and genetically induced mouse tumor models, and PD-L1 expression in cancer cells resulted in reduced NK cell responses and generation of more aggressive tumors in vivo. PD-1 expression was more abundant on NK cells with an activated and more responsive phenotype and did not mark NK cells with an exhausted phenotype. These results demonstrate the importance of the PD-1/PD-L1 axis in inhibiting NK cell responses in vivo and reveal that NK cells, in addition to T cells, mediate the effect of PD-1/PD-L1 blockade immunotherapy.

Enhanced susceptibility of cancer cells to oncolytic rhabdo-virotherapy by expression of Nodamura virus protein B2 as a suppressor of RNA interference.

Antiviral responses are barriers that must be overcome for efficacy of oncolytic virotherapy. In mammalian cells, antiviral responses involve the interferon pathway, a protein-signaling cascade that alerts the immune system and limits virus propagation. Tumour-specific defects in interferon signaling enhance viral infection and responses to oncolytic virotherapy, but many human cancers are still refractory to oncolytic viruses. Given that invertebrates, fungi and plants rely on RNA interference pathways for antiviral protection, we investigated the potential involvement of this alternative antiviral mechanism in cancer cells. Here, we detected viral genome-derived small RNAs, indicative of RNAi-mediated antiviral responses, in human cancer cells. As viruses may encode suppressors of the RNA interference pathways, we engineered an oncolytic vesicular stomatitis virus variant to encode the Nodamura virus protein B2, a known inhibitor of RNAi-mediated immune responses. B2-expressing oncolytic virus showed enhanced viral replication and cytotoxicity, impaired viral genome cleavage and altered microRNA processing in cancer cells. Our data establish the improved therapeutic potential of our novel virus which targets the RNAi-mediated antiviral defense of cancer cells.

Neoadjuvant oncolytic virotherapy before surgery sensitizes triple-negative breast cancer to immune checkpoint therapy.

Triple-negative breast cancer (TNBC) is an aggressive disease for which treatment options are limited and associated with severe toxicities. Immunotherapeutic approaches like immune checkpoint inhibitors (ICIs) are a potential strategy, but clinical trials have demonstrated limited success in this patient cohort. Clinical studies using ICIs have revealed that patients with preexisting anticancer immunity are the most responsive. Given that oncolytic viruses (OVs) induce antitumor immunity, we investigated their use as an ICI-sensitizing approach. Using a therapeutic model that mimics the course of treatment for women with newly diagnosed TNBC, we demonstrate that early OV treatment coupled with surgical resection provides long-term benefits. OV therapy sensitizes otherwise refractory TNBC to immune checkpoint blockade, preventing relapse in most of the treated animals. We suggest that OV therapy in combination with immune checkpoint blockade warrants testing as a neoadjuvant treatment option in the window of opportunity between TNBC diagnosis and surgical resection.

Brief Communication; A Heterologous Oncolytic Bacteria-Virus Prime-Boost Approach for Anticancer Vaccination in Mice.

Anticancer vaccination is becoming a popular therapeutic approach for patients with cancers expressing common tumor antigens. One variation on this strategy is a heterologous virus vaccine where 2 viruses encoding the same tumor antigen are administered sequentially to prime and boost antitumor immunity. This approach is currently undergoing clinical investigation using an adenovirus (Ad) and the oncolytic virus Maraba (MRB). In this study, we show that Listeria monocytogenes can be used in place of the Ad to obtain comparable immune priming efficiency before MRB boosting. Importantly, the therapeutic benefits provided by our heterologous L. monocytogenes-MRB prime-boost strategy are superior to those conferred by the Ad-MRB combination. Our study provides proof of concept for the heterologous oncolytic bacteria-virus prime-boost approach for anticancer vaccination and merits its consideration for clinical testing.

Amplification of Oncolytic Vaccinia Virus Widespread Tumor Cell Killing by Sunitinib through Multiple Mechanisms.

Oncolytic viruses pose many questions in their use in cancer therapy. In this study, we assessed the potential of mpJX-594 (mouse-prototype JX-594), a replication-competent vaccinia virus administered by intravenous injection, to target the tumor vasculature, produce immune activation and tumor cell killing more widespread than the infection, and suppress invasion and metastasis. These actions were examined in RIP-Tag2 transgenic mice with pancreatic neuroendocrine tumors that developed spontaneously and progressed as in humans. mpJX-594 initially infected tumor vascular endothelial cells, leading to vascular pruning and prolonged leakage in tumors but not in normal organs; parallel effects were observed in U87 gliomas. Viral infection spread to tumor cells, where tumor cell killing was much more widespread than the infection. Widespread tumor cell killing at 5 days was prevented by depletion of CD8+ T lymphocytes and did not require GM-CSF, as mpJX-594 variants that expressed human, mouse, or no GM-CSF produced equivalent amounts of killing. The antivascular, antitumor, and antimetastatic effects of mpJX-594 were amplified by concurrent or sequential administration of sunitinib, a multitargeted receptor tyrosine kinase inhibitor. These effects were not mimicked by selective inhibition of VEGFR2 despite equivalent vascular pruning, but were accompanied by suppression of regulatory T cells and greater influx of activated CD8+ T cells. Together, our results showed that mpJX-594 targets tumor blood vessels, spreads secondarily to tumor cells, and produces widespread CD8+ T-cell-dependent tumor cell killing in primary tumors and metastases, and that these effects can be amplified by coadministration of sunitinib.Significance: These findings reveal multiple unrecognized features of the antitumor properties of oncolytic vaccinia viruses, all of which can be amplified by the multitargeted kinase inhibitor sunitinib. Cancer Res; 78(4); 922-37. ©2017 AACR.

Oncolytic measles virus encoding interleukin-12 mediates potent antitumor effects through T cell activation.

Combination of oncolytic virotherapy with immunomodulators is emerging as a promising therapeutic strategy for numerous tumor entities. In this study, we developed measles Schwarz vaccine strain vectors encoding immunomodulators to support different phases in the establishment of antitumor immune responses. Therapeutic efficacy of the novel vectors was evaluated in the immunocompetent MC38cea tumor model. We identified vectors encoding an IL-12 fusion protein (MeVac FmIL-12) and an antibody against PD-L1 (MeVac anti-PD-L1), respectively, as the most effective. Treatment of established tumors with MeVac FmIL-12 achieved 90% complete remissions. Profiling of the tumor immune microenvironment revealed activation of a type 1 T helper cell-directed response, with MeVac FmIL-12 ensuring potent early natural killer and effector T cell activation as well as upregulation of the effector cytokines IFN-γ and TNF-α. CD8+ T cells were found to be essential for the therapeutic efficacy of MeVac FmIL-12. Results of this study present MeVac FmIL-12 as a novel approach for targeted IL-12 delivery and elucidate mechanisms of successful immunovirotherapy.

Taking a Stab at Cancer; Oncolytic Virus-Mediated Anti-Cancer Vaccination Strategies.

Vaccines have classically been used for disease prevention. Modern clinical vaccines are continuously being developed for both traditional use as well as for new applications. Typically thought of in terms of infectious disease control, vaccination approaches can alternatively be adapted as a cancer therapy. Vaccines targeting cancer antigens can be used to induce anti-tumour immunity and have demonstrated therapeutic efficacy both pre-clinically and clinically. Various approaches now exist and further establish the tremendous potential and adaptability of anti-cancer vaccination. Classical strategies include ex vivo-loaded immune cells, RNA- or DNA-based vaccines and tumour cell lysates. Recent oncolytic virus development has resulted in a surge of novel viruses engineered to induce powerful tumour-specific immune responses. In addition to their use as cancer vaccines, oncolytic viruses have the added benefit of being directly cytolytic to cancer cells and thus promote antigen recognition within a highly immune-stimulating tumour microenvironment. While oncolytic viruses are perfectly equipped for efficient immunization, this complicates their use upon previous exposure. Indeed, the host's anti-viral counter-attacks often impair multiple-dosing regimens. In this review we will focus on the use of oncolytic viruses for anti-tumour vaccination. We will explore different strategies as well as ways to circumvent some of their limitations.

MARCH1 E3 Ubiquitin Ligase Dampens the Innate Inflammatory Response by Modulating Monocyte Functions in Mice.

Ubiquitination was recently identified as a central process in the pathogenesis and development of numerous inflammatory diseases, such as obesity, atherosclerosis, and asthma. Treatment with proteasomal inhibitors led to severe side effects because ubiquitination is heavily involved in a plethora of cellular functions. Thus, new players regulating ubiquitination processes must be identified to improve therapies for inflammatory diseases. In addition to their role in adaptive immunity, endosomal MHC class II (MHCII) molecules were shown to modulate innate immune responses by fine tuning the TLR4 signaling pathway. However, the role of MHCII ubiquitination by membrane associated ring-CH-type finger 1 (MARCH1) E3 ubiquitin ligase in this process remains to be assessed. In this article, we demonstrate that MARCH1 is a key inhibitor of innate inflammation in response to bacterial endotoxins. The higher mortality of March1-/- mice challenged with a lethal dose of LPS was associated with significantly stronger systemic production of proinflammatory cytokines and splenic NK cell activation; however, we did not find evidence that MARCH1 modulates LPS or IL-10 signaling pathways. Instead, the mechanism by which MARCH1 protects against endotoxic shock rests on its capacity to promote the transition of monocytes from Ly6CHi to Ly6C+/- Moreover, in competitive bone marrow chimeras, March1-/- monocytes and polymorphonuclear neutrophils outcompeted wild-type cells with regard to bone marrow egress and homing to peripheral organs. We conclude that MARCH1 exerts MHCII-independent effects that regulate the innate arm of immunity. Thus, MARCH1 might represent a potential new target for emerging therapies based on ubiquitination reactions in inflammatory diseases.

Complement inhibition enables tumor delivery of LCMV glycoprotein pseudotyped viruses in the presence of antiviral antibodies.

The systemic delivery of therapeutic viruses, such as oncolytic viruses or vaccines, is limited by the generation of neutralizing antibodies. While pseudotyping of rhabdoviruses with the lymphocytic choriomeningitis virus glycoprotein has previously allowed for multiple rounds of delivery in mice, this strategy has not translated to other animal models. For the first time, we provide experimental evidence that antibodies generated against the lymphocytic choriomeningitis virus glycoprotein mediate robust complement-dependent viral neutralization via activation of the classical pathway. We show that this phenotype can be capitalized upon to deliver maraba virus pseudotyped with the lymphocytic choriomeningitis virus glycoprotein in a Fischer rat model in the face of neutralizing antibody through the use of complement modulators. This finding changes the understanding of the humoral immune response to arenaviruses, and also describes methodology to deliver viral vectors to their therapeutic sites of action without the interference of neutralizing antibody.

MARCH1 regulates insulin sensitivity by controlling cell surface insulin receptor levels.

Insulin resistance is a key driver of type 2 diabetes (T2D) and is characterized by defective insulin receptor (INSR) signalling. Although surface INSR downregulation is a well-established contributor to insulin resistance, the underlying molecular mechanisms remain obscure. Here we show that the E3 ubiquitin ligase MARCH1 impairs cellular insulin action by degrading cell surface INSR. Using a large-scale RNA interference screen, we identify MARCH1 as a negative regulator of INSR signalling. March1 loss-of-function enhances, and March1 overexpression impairs, hepatic insulin sensitivity in mice. MARCH1 ubiquitinates INSR to decrease cell surface INSR levels, but unlike other INSR ubiquitin ligases, MARCH1 acts in the basal state rather than after insulin stimulation. Thus, MARCH1 may help set the basal gain of insulin signalling. MARCH1 expression is increased in white adipose tissue of obese humans, suggesting that MARCH1 contributes to the pathophysiology of T2D and could be a new therapeutic target.