Tumor cells exhibiting the Warburg effect rely on aerobic glycolysis for ATP production and have a notable addiction to anaplerotic use of glutamine for macromolecular synthesis. This strategy maximizes cellular biosynthetic potential while avoiding excessive depletion of NAD þ and provides an attractive anabolic environment for viral infection. Here, we evaluate infection of highly permissive and poorly permissive cancer cells with wild-type adenoviruses and the oncolytic chimeric adenovirus enadenotucirev (EnAd). All adenoviruses caused an increase in glucose and glutamine uptake along with increased lactic acid secretion. Counterintuitively, restricting glycolysis using 2-deoxyglucose or by limiting glucose supply strongly improved virus activity in both cell types. Antagonism of glycolysis also boosted EnAd replication and transgene expression within human tumor biopsies and in xenografted tumors in vivo. In contrast, the virus life cycle was critically dependent on exogenous glutamine. Virus activity in glutamine-free cells was rescued with exogenous membranepermeable a-ketoglutarate, but not pyruvate or oxaloacetate, suggesting an important role for reductive carboxylation in glutamine usage, perhaps for production of biosynthetic intermediates. This overlap between the metabolic phenotypes of adenovirus infection and transformed tumor cells may provide insight into how oncolytic adenoviruses exploit metabolic transformation to augment their selectivity for cancer cells. Significance: This study describes changes in glucose and glutamine metabolism induced by oncolytic and wild-type adenoviruses in cancer cells, which will be important to consider in the preclinical evaluation of oncolytic viruses.

BackgroundTumour-associated macrophages (TAMs) are often implicated in cancer progression but can also exert anti-tumour activities. Selective eradication of cancer-promoting (M2-like) TAM subsets is a highly sought-after goal. Here, we have devised a novel strategy to achieve selective TAM depletion, involving the use of T cell engagers to direct endogenous T cell cytotoxicity towards specific M2-like TAMs. To avoid “on-target off-tumour” toxicities, we have explored localising expression of the T cell engagers to the tumour with enadenotucirev (EnAd), an oncolytic adenovirus in Phase I/II clinical trials.MethodA panel of bi- and tri-valent T cell engagers (BiTEs/TriTEs) was constructed, recognising CD3ε on T cells and CD206 or folate receptor β (FRβ) on M2-like macrophages. Initial characterisation of BiTE/TriTE activity and specificity was performed with M1- and M2-polarised monocyte-derived macrophages and autologous lymphocytes from healthy human peripheral blood donors. T cell engagers were inserted into the genome of EnAd, and oncolytic activity and BiTE secretion assessed with DLD-1 tumour cells. Clinically-relevant ex vivo models (whole malignant ascites from cancer patients) were employed to assess the efficacies of the free- and virally-encoded T cell engagers.ResultsT cells activated by the CD206- and FRβ-targeting BiTEs/TriTEs preferentially killed M2- over M1-polarised autologous macrophages, with EC50 values in the nanomolar range. A TriTE with bivalent CD3ε binding – the first of its kind – demonstrated enhanced potency whilst retaining target cell selectivity, whereas a CD28-containing TriTE elicited non-specific T cell activation. In immunosuppressive malignant ascites, both free and EnAd-encoded T cell engagers triggered endogenous T cell activation and IFN-γ production, leading to increased T cell numbers and depletion of CD11b+CD64+ ascites macrophages. Strikingly, surviving macrophages exhibited a general increase in M1 marker expression, suggesting microenvironmental repolarisation towards a pro-inflammatory state.ConclusionsThis study is the first to achieve selective depletion of specific M2-like macrophage subsets, opening the possibility of eradicating cancer-supporting TAMs whilst sparing those with anti-tumour potential. Targeted TAM depletion with T cell engager-armed EnAd offers a powerful therapeutic approach combining direct cancer cell cytotoxicity with reversal of immune suppression.

Recent advances in cancer therapeutics clearly demonstrate the need for innovative multiplex therapies that attack the tumour on multiple fronts. Oncolytic or “cancer-killing” viruses (OVs) represent up-and-coming multi-mechanistic immunotherapeutic drugs for the treatment of cancer. In this study, we perform an in-vitro screen based on virus-encoded artificial microRNAs (amiRNAs) and find that a unique amiRNA, herein termed amiR-4, confers a replicative advantage to the VSVΔ51 OV platform. Target validation of amiR-4 reveals ARID1A, a protein involved in chromatin remodelling, as an important player in resistance to OV replication. Virus-directed targeting of ARID1A coupled with small-molecule inhibition of the methyltransferase EZH2 leads to the synthetic lethal killing of both infected and uninfected tumour cells. The bystander killing of uninfected cells is mediated by intercellular transfer of extracellular vesicles carrying amiR-4 cargo. Altogether, our findings establish that OVs can serve as replicating vehicles for amiRNA therapeutics with the potential for combination with small molecule and immune checkpoint inhibitor therapy.

Transplantation of progenitors from induced pluripotent stem cells reprogrammed by lentiviral vectors led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Combined transgene-free reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo.

High-fat diet (HFD)-fed mouse models have been widely used to study early type 2 diabetes. Decreased β-cell glucokinase (GCK) expression has been observed in HFD-induced diabetes. However, owing to its crucial roles in glucose metabolism in the liver and in islet β-cells, the contribution of decreased GCK expression to the development of HFD-induced diabetes is unclear. Here, we employed a β-cell-targeted gene transfer vector and determined the impact of β-cell-specific increase in GCK expression on β-cell function and glucose handling in vitro and in vivo. Overexpression of GCK enhanced glycolytic flux, ATP-sensitive potassium channel activation and membrane depolarization, and increased proliferation in Min6 cells. β-cell-targeted GCK transduction did not change glucose handling in chow-fed C57BL/6 mice. Although adult mice fed a HFD showed reduced islet GCK expression, impaired glucose tolerance and decreased glucose-stimulated insulin secretion (GSIS), β-cell-targeted GCK transduction improved glucose tolerance and restored GSIS. Islet perifusion experiments verified restored GSIS in isolated HFD islets by GCK transduction. Thus, our data identify impaired β-cell GCK expression as an underlying mechanism for dysregulated β-cell function and glycemic control in HFD-induced diabetes. Our data also imply an etiological role of GCK in diet-induced diabetes.

The 11th International Oncolytic Virus Conference (IOVC) was held from April 9-12, 2018 in Oxford, UK. This is part of the high-profile academic-led series of meetings that was started back in 2002 by Steve Russell and John Bell, with most of the previous meetings being held in North America (often in Banff). The conference brought together many of the major players in oncolytic virotherapy from all over the world, addressing all stages of research and development-from aspects of basic science and cellular immunology all the way through to early- and late-phase clinical trials. The meeting welcomed 352 delegates from 24 countries. The top seven delegate countries, namely, the UK, US, Canada, The Netherlands, Germany, Japan and South Korea, contributed 291 delegates while smaller numbers coming from Australia, Austria, Bulgaria, China, Finland, France, Iraq, Ireland, Israel, Italy, Latvia, Malaysia, Poland, Slovenia, Spain, Sweden and Switzerland. Academics comprised about half of the attendees, industry 30% and students 20%. The next IOVC is scheduled to be held on Vancouver Island in autumn 2019. Here we share brief summaries of the oral presentations from invited speakers and proffered papers in the different subtopics presented at IOVC 2018.

Ionising radiation causes cell death through the induction of DNA damage, particularly double-stranded DNA (dsDNA) breaks. Evidence suggests that adenoviruses inhibit proteins involved in the DNA damage response (DDR) to prevent recognition of double-stranded viral DNA genomes as cellular dsDNA breaks. We hypothesise that combining adenovirus treatment with radiotherapy has the potential for enhancing tumour-specific cytotoxicity through inhibition of the DDR and augmentation of virus production. We show that EnAd, an Ad3/Ad11p chimeric oncolytic adenovirus currently being trialled in colorectal and other cancers, targets the DDR pathway at a number of junctures. Infection is associated with a decrease in irradiation-induced 53BP1 and Rad51 foci formation, and in total DNA ligase IV levels. We also demonstrate a radiation-associated increase in EnAd production in vitro and in a pilot in vivo experiment. Given the current limitations of in vitro techniques in assessing for synergy between these treatments, we adapted the plaque assay to allow monitoring of viral plaque size and growth and utilised the xCELLigence cell adhesion assay to measure cytotoxicity. Our study provides further evidence on the interaction between adenovirus and radiation in vitro and in vivo and suggests these have at least an additive, and possibly a synergistic, impact on cytotoxicity.

BackgroundOncolytic viruses are currently experiencing accelerated development in several laboratories worldwide, with some forty-seven clinical trials currently recruiting. Many oncolytic viruses combine targeted cytotoxicity to cancer cells with a proinflammatory cell lysis. Due to their additional potential to express immunomodulatory transgenes, they are also often known as oncolytic viral vaccines. However, several types of oncolytic viruses are human-specific and the lack of suitable immune-competent animal models complicates biologically relevant evaluation of their vaccine potential. This is a particular challenge for group B adenoviruses, which fail to infect even those immunocompetent animal model systems identified as semi-permissive for type 5 adenovirus. Here, we aim to develop a murine cell line capable of supporting replication of a group B oncolytic adenovirus, enadenotucirev (EnAd), for incorporation into a syngeneic immunocompetent animal model to explore the oncolytic vaccine potential of group B oncolytic viruses.MethodsTransgenic murine cell lines were infected with EnAd expressing GFP transgene under replication-independent or -dependent promoters. Virus mRNA expression, genome replication, and late protein expression were determined by qRT-PCR, qPCR, and immunoblotting, respectively. We also use Balb/c immune-competent mice to determine the tumourogenicity and infectivity of transgenic murine cell lines.ResultsOur results show that a broad range of human carcinoma cells will support EnAd replication, but not murine carcinoma cells. Murine cells can be readily modified to express surface human CD46, one of the receptors for group B adenoviruses, allowing receptor-mediated uptake of EnAd particles into the murine cells and expression of CMV promoter-driven transgenes. Although the early E1A mRNA was expressed in murine cells at levels similar to human cells, adenovirus E2B and Fibre mRNA expression levels were hampered and few virus genomes were produced. Unlike previous reports on group C adenoviruses, trans-complementation of group B adenoviruses by co-infection with mouse adenovirus 1 did not rescue replication. A panel of group B adenoviruses expressing individual mouse adenovirus 1 genes were also unable to rescue EnAd replication.ConclusionTogether, these results indicate that there may be major differences in the early stages of replication of group C and B adenoviruses in murine cells, and that the block to the life cycle of B adenoviruses in murine cells occurs in the early stage of virus replication, perhaps reflecting poor activity of Ad11p E1A in murine cells.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0350-x) contains supplementary material, which is available to authorized users.