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MicroRNAs play an important role in the regulation of mRNA translation and have therapeutic potential in cancer and other diseases. To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen in adult and pediatric GBM cells using a synthetic oligonucleotide library representing all known human microRNAs. Bioinformatics analysis was used to refine this list and the top seven microRNAs were validated in a larger panel of GBM cells using state-of-the-art in vitro assays. The cytotoxic effect of our most relevant candidate was assessed in a preclinical model. Our screen identified ~100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 transfected cells due to cytokinesis failure followed by apoptosis. This was also observed in two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In GBM cells, where miR-1300 is normally not expressed, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target. ECT2 siRNA phenocopied the effects of miR-1300, and ECT2 overexpression led to rescue of miR-1300 induced binucleation. We showed that ectopic expression of miR-1300 led to decreased tumor growth in an orthotopic GBM model. Our screen provides a resource for the neuro-oncology community and identified miR-1300 as a novel regulator of endomitosis with translatable potential for therapeutic application.

T cell receptor (TCR) triggering by antigen results in metabolic reprogramming that, in turn, facilitates the exit of T cells from quiescence. The increased nutrient requirements of activated lymphocytes are met, in part, by upregulation of cell surface transporters and enhanced uptake of amino acids, fatty acids, and glucose from the environment. However, the role of intracellular pathways of amino acid biosynthesis in T cell activation is relatively unexplored. Asparagine is a nonessential amino acid that can be synthesized intracellularly through the glutamine-hydrolyzing enzyme asparagine synthetase (ASNS). We set out to define the requirements for uptake of extracellular asparagine and ASNS activity in CD8+ T cell activation. At early time points of activation in vitro, CD8+ T cells expressed little or no ASNS, and, as a consequence, viability and TCR-stimulated growth, activation, and metabolic reprogramming were substantially impaired under conditions of asparagine deprivation. At later time points (more than 24 hours of activation), TCR-induced mTOR-dependent signals resulted in ASNS upregulation that endowed CD8+ T cells with the capacity to function independently of extracellular asparagine. Thus, our data suggest that the coordinated upregulation of ASNS expression and uptake of extracellular asparagine is involved in optimal T cell effector responses.

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T cell receptor triggering by antigen results in metabolic reprogramming that, in turn, facilitates T cells’ exit from quiescence. The increased nutrient requirements of activated lymphocytes are met in part by upregulation of cell surface transporters and enhanced uptake of amino acids, fatty acids and glucose from the environment. However, the role of intracellular pathways of amino acid biosynthesis in T cell activation is relatively unexplored. Asparagine (Asn) is a non-essential amino acid that can be synthesized intracellularly through the glutamine-hydrolyzing enzyme asparagine synthetase (ASNS). We set out to define the requirements for uptake of extracellular Asn and ASNS activity in CD8+ T cell activation. At early timepoints of activation, T cells expressed little or no ASNS and, as a consequence, viability and TCR-stimulated growth, activation and metabolic reprogramming were substantially impaired under conditions of Asn deprivation. At later timepoints (>48h of activation), TCR-induced mTOR-dependent signals resulted in upregulation of ASNS, that endowed T cells with the capacity to function independently of extracellular Asn. Thus, we have determined that the coordinated upregulation of ASNS expression and uptake of extracellular Asn is required for optimal T cell effector responses.

Immunotherapy is showing promise for otherwise incurable cancers. Oncolytic viruses (OVs), developed as direct cytotoxic agents, mediate their antitumor effects via activation of the immune system. However, OVs also stimulate antiviral immune responses, including the induction of OV-neutralizing antibodies. Current dogma suggests that the presence of preexisting antiviral neutralizing antibodies in patients, or their development during viral therapy, is a barrier to systemic OV delivery, rendering repeat systemic treatments ineffective. However, we have found that human monocytes loaded with preformed reovirus–antibody complexes, in which the reovirus is fully neutralized, deliver functional replicative reovirus to tumor cells, resulting in tumor cell infection and lysis. This delivery mechanism is mediated, at least in part, by antibody receptors (in particular FcγRIII) that mediate uptake and internalization of the reovirus/antibody complexes by the monocytes. This finding has implications for oncolytic virotherapy and for the design of clinical OV treatment strategies. Cancer Immunol Res; 6(10); 1161–73. ©2018 AACR.

Simple Summary High-grade gliomas remain difficult-to-treat cancers. Novel treatment options include targeting glycogen synthase kinase 3 (GSK-3) to induce cell death but the mode of drug activity remains unknown and combination with conventional treatment including radiotherapy has not been explored. Here, we describe the effect of targeting GSK-3 with the inhibitor AZD2858 in in vitro and in vivo models of glioma. We established that AZD2858 exposure induces mitotic defects leading to cell death in patient-derived glioma cell lines and tumor growth delay in glioma xenografts. Co-administration also enhanced the effect of radiotherapy. We therefore propose AZD2858 as an adjuvant to radiotherapy in high-grade glioma. Abstract Background: Previous data on glycogen synthase kinase 3 (GSK-3) inhibition in cancer models support a cytotoxic effect with selectivity for tumor cells compared to normal tissue but the effect of these inhibitors in glioma has not been widely studied. Here, we investigate their potential as cytotoxics in glioma. Methods: We assessed the effect of pharmacologic GSK-3 inhibition on established (U87, U251) and patient-derived (GBM1, GBM4) glioblastoma (GBM) cell lines using cytotoxicity assays as well as undertaking a detailed investigation of the effect on cell cycle, mitosis, and centrosome biology. We also assessed drug uptake and efficacy of GSK-3 inhibition alone and in combination with radiation in xenograft models. Results: Using the selective GSK-3 inhibitor AZD2858, we demonstrated single agent cytotoxicity in two patient-derived glioma cell lines (GBM1, GBM4) and two established cell lines (U251 and U87) with IC50 in the low micromolar range promoting centrosome disruption, failed mitosis, and S-phase arrest. Glioma xenografts exposed to AZD2858 also showed growth delay compared to untreated controls. Combined treatment with radiation increased the cytotoxic effect of clinical radiation doses in vitro and in orthotopic glioma xenografts. Conclusions: These data suggest that GSK-3 inhibition promotes cell death in glioma through disrupting centrosome function and promoting mitotic failure and that AZD2858 is an effective adjuvant to radiation at clinical doses.

Background CNS tumors, including medulloblastoma and pediatric glioblastoma (pGBM) account for the majority of solid pediatric malignancies. There remains an unmet need to identify novel treatment approaches in poor prognosis and relapsed pediatric brain tumors, where therapeutic options are limited. Small-molecule B-cell lymphoma 2 (BCL-2) family inhibitors may enhance tumor cell killing when combined with conventional and targeted chemotherapeutic agents. We investigated the effect of disrupting BCL-2 and B cell lymphoma-extra large (BCL-XL) protein function using ABT-263, ABT-199 and WEHI-539 in medulloblastoma and pGBM cells following treatment with MLN8237, an Aurora kinase inhibitor under investigation as a novel agent for the treatment of malignant brain tumors. Methods Tumor cell growth and viability were determined by MTT/WST-1 assays and flow cytometry. Effects on cell phenotype, cell cycle progression, and ploidy were determined by live cell imaging and DNA content analysis. Apoptosis was determined by annexin V/propidium iodide staining and time-lapse microscopy and confirmed by measuring caspase-3/7 activity and western blotting and by short interfering RNA (siRNA) knockdown of BCL-2 associated X protein/BCL-2 antagonist killer (BAX/BAK). Results ABT-263, in combination with MLN8237, reduced mitotic slippage and polyploidy and promoted the elimination of mitotically defective cells via a BAX/BAK-dependent, caspase-mediated apoptotic pathway. The BCL-XL antagonist, WEHI-539, significantly augmented tumor cell killing when used in combination with MLN8237, as well as sensitized resistant brain tumor cells to a novel BAX activator, SMBA1. In addition, siRNA-mediated knockdown of BCL-XL sensitized pGBM and medulloblastoma cells to MLN8237 and mimicked the effect of combination drug treatment. Conclusion Selective small-molecule inhibitors of BCL-XL may enhance the efficacy of MLN8237 and other targeted chemotherapeutic agents.

BackgroundMicroRNAs play an important role in the regulation of mRNA translation, and have therapeutic potential in cancer and other diseases.MethodsTo profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen using a synthetic oligonucleotide library representing all known human microRNAs in adult and pediatric GBM cells. Bio-informatics analysis were used to refine this list and the top seven microRNAs were validated in a larger panel of cells by flow-cytometry, and RTqPCR. The downstream mechanism of the strongest and most consistent candidate was investigated by siRNAs, 3’UTR luciferase assays and Western Blotting.ResultsOur screen identified ∼100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 expressing cells and characterized the mechanism of action as cytokinesis failure followed by apoptosis, which was observed in an extended GBM cell panel including two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In glioblastoma cells, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target of miR-1300. ECT2 siRNA phenocopied the effects of miR-1300, and its overexpression led to a significant rescue of miR-1300 induced binucleation.ConclusionMiR-1300 was identified as a novel regulator of endomitosis with translatable potential for therapeutic application. The datatasets will be a resource for the neuro-oncology community.Key points (2 or 3 key points 85 characters plus spaces each)70% of cytotoxic microRNAs were shared between adult and pediatric glioblastoma cellsMiR-1300 expression is restricted to endomitosis within megakaryocyte differentiationMiR-1300’s ectopic expression is a potent and promising therapeutic tool in cancerImportance of StudyPrevious functional studies of microRNAs involved in the regulation of glioblastoma cell proliferation and/or survival have focused on adult glioblastoma alone and are restricted to only a few microRNAs at a time. Our study provides the first encompassing landscape of potent cytotoxic microRNAs in pediatric and adult glioblastoma.Not only, does our data provide an invaluable resource for the research community but it also revealed that 70% of microRNAs with significant cytotoxicity were shared by adult and pediatric cells. Finally, we identified and characterized the previously undescribed role of microRNA-1300 in the tight regulation of megakaryocyte differentiation into platelets and how, when expressed outside of this context, miR-1300 consistently causes cytokinesis failure followed by apoptosis, and thus represents a powerful cytotoxic tool with potential for translation towards therapeutic applications.

BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

MicroRNAs are proving to be potent tumour suppressors in cancer. Many microRNAs are in pre-clinical and clinical validation.

We have assessed the ability of bispecific fusion proteins to improve adenovirus-mediated transfer of therapeutic and marker transgenes. We constructed an expression vector that can be easily modified to synthesize a variety of fusion proteins for retargeting adenoviral gene therapy vectors to cell surface markers, which are differentially expressed between normal and cancer cells. Adenoviral transduction can be improved in a number of tumour cell lines which overexpress EGFR (epidermal growth factor receptor) or uPAR (urokinase-type plasminogen activator receptor), but which have only low levels of endogenous hCAR (human coxsackie B and adenovirus receptor) expression. Up to 40-fold improvement in beta-galactosidase transgene expression was seen using an EGFR retargeting protein, and up to 16-fold using a second fusion protein targeting uPAR. In vitro, our uPAR retargeting fusion protein improved the sensitivity to adenoviral herpes simplex virus thymidine kinase/ganciclovir by an order of magnitude, whereas in vivo, our EGFR retargeting protein is able to significantly delay tumour growth in rodent animal models in a dose-dependent manner. The 'cassette' design of our fusion protein constructs offers a flexible method for the straightforward synthesis of multiple adenoviral retargeting proteins, directed against a variety of tumour-associated antigens, for use in clinical trials.

In vivo, dendritic cells (DC) are programmed to orchestrate innate and adaptive immunity in response to pathogen-derived “danger” signals. Under particular circumstances, DC can also be directly cytotoxic against tumor cells, potentially allowing them to release tumor associated Ags from dying cells and then prime antitumor immunity against them. In this study, we describe the innate characteristics of DC (OK-DC) generated in vitro after exposure of immature human myeloid-derived DC to OK432, a penicillin-inactivated and lyophilized preparation of Streptococcus pyrogenes. OK-DC produced proinflammatory cytokines, stimulated autologous T cell proliferation and IFN-γ secretion, expressed CCR7, and migrated in response to MIP-3β. Moreover, OK-DC displayed strong, specific cytotoxicity toward tumor cell targets. This cytotoxicity was associated with novel, OK432-induced up-regulation of CD40L on the cell surface of OK-DC, and was absolutely dependent on expression of CD40 on the tumor targets. These data demonstrate that maturation of human DC with OK432, an adjuvant suitable for clinical use, induces direct tumor cell killing by DC, and describes a novel CD40/CD40L-mediated mechanism for specific DC antitumor cytotoxicity.

Oncolytic viruses can exert their antitumor activity via direct oncolysis or activation of antitumor immunity. Although reovirus is currently under clinical investigation for the treatment of localized or disseminated cancer, any potential immune contribution to its efficacy has not been addressed. This is the first study to investigate the ability of reovirus to activate human dendritic cells (DC), key regulators of both innate and adaptive immune responses. Reovirus induced DC maturation and stimulated the production of the proinflammatory cytokines IFN-α, TNF-α, IL-12p70, and IL-6. Activation of DC by reovirus was not dependent on viral replication, while cytokine production (but not phenotypic maturation) was inhibited by blockade of PKR and NF-κB signaling. Upon coculture with autologous NK cells, reovirus-activated DC up-regulated IFN-γ production and increased NK cytolytic activity. Moreover, short-term coculture of reovirus-activated DC with autologous T cells also enhanced T cell cytokine secretion (IL-2 and IFN-γ) and induced non-Ag restricted tumor cell killing. These data demonstrate for the first time that reovirus directly activates human DC and that reovirus-activated DC stimulate innate killing by not only NK cells, but also T cells, suggesting a novel potential role for T cells in oncolytic virus-induced local tumor cell death. Hence reovirus recognition by DC may trigger innate effector mechanisms to complement the virus’s direct cytotoxicity, potentially enhancing the efficacy of reovirus as a therapeutic agent.

Much of today's higher education landscape, particularly for vocational training providers, is market driven and highly reflexive to consumer needs. Industry and employers who require specific professional credentials have a strong influence on programme design and curriculum development. In this chapter, we will explore New Zealand's first and only qualification for offshore and onshore professionals working with future immigrants. This qualification draws on features of open learning courses, and illustrates a pathway for education delivery that moves beyond traditional models into a 21st Century modality. The student demographic comprises a large number of mature learners, who have enrolled to gain formal credentials in their field, are moving to a new career, or may be seeking additional expertise to complement a suite of skills to offer their organisation, or as self-employed contractors/consultants. This population is a good example of lifelong learning applied to personal and professional lifestyle choices.

Membrane-presented CD40 agonists can induce apoptosis in carcinoma, but not normal homologous epithelial cells, whereas soluble agonists are growth inhibitory but not proapoptotic unless protein synthesis is blocked. Here we demonstrate that membrane-presented CD40 ligand (CD154) (mCD40L), but not soluble agonists, triggers cell death in malignant human urothelial cells via a direct mechanism involving rapid upregulation of TNFR-associated factor (TRAF)3 protein, without concomitant upregulation of TRAF3 mRNA, followed by activation of the c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway and induction of the caspase-9/caspase-3-associated intrinsic apoptotic machinery. TRAF3 knockdown abrogated JNK/AP-1 activation and prevented CD40-mediated apoptosis, whereas restoration of CD40 expression in CD40-negative carcinoma cells restored apoptotic susceptibility via the TRAF3/AP-1-dependent mechanism. In normal human urothelial cells, mCD40L did not trigger apoptosis, but induced rapid downregulation of TRAF2 and 3, thereby paralleling the situation in B-lymphocytes. Thus, TRAF3 stabilization, JNK activation and caspase-9 induction define a novel pathway of CD40-mediated apoptosis in carcinoma cells.

Comparing normal human urothelial (NHU) cells to a panel of six representative urothelial cell carcinoma (UCC)-derived cell lines, we showed that while TRAIL receptor expression patterns were similar, susceptibility to soluble recombinant crosslinked TRAIL fell into three categories. 4/6 carcinoma lines were sensitive, undergoing rapid and extensive death; NHU and 253J cells were partially resistant and HT1376 cells, like normal fibroblasts, were refractory. Both normal and malignant urothelial cells underwent apoptosis via the same caspase-8/9-mediated mechanism. Rapid receptor downregulation was a mechanism for evasion by some UCC cells. TRAIL resistance in malignant urothelial cells was partially dependent on FLIP(L) and was differentially mediated by p38(MAPK), whereas in normal cells, resistance was mediated by NF-kappaB. Importantly, extensive killing of UCC cells could be induced using noncrosslinked TRAIL after prolonged exposure, with no damage to their homologous, normal urothelial cell counterparts.

BACKGROUND: Small molecule inhibitors of the mitotic kinases, including the Aurora kinase inhibitor MLN8237, are being investigated as novel therapeutic agents for the treatment of solid cancers including malignant brain tumours. However, resistance has been observed and anti-apoptotic proteins of the BCL-2 family have been shown to promote cell survival following treatment. In this study, we investigated the effect of disrupting anti-apoptotic protein function in medulloblastoma and pediatric glioblastoma (pGBM) models using BH3-mimetics in combination with MLN8237. METHODS: The effects of single agent and combination treatment on cell viability were determined by MTT/WST-1 assays and flow cytometry. Effects on cell phenotype, cell cycle progression and cellular ploidy were determined by live cell imaging and DNA content analysis. Induction of apoptosis was monitored in real-time, by time-lapse microscopy, using a fluorescent, polarity-sensitive phosphatidylserine probe, and confirmed by measuring caspase-3/7 activity. Knockdown of BAX/BAK was used to confirm these effects. Results: ABT-263 in combination with MLN8237 reduced mitotic slippage and polyploidy, significantly reduced cell viability and promoted the elimination of mitotically defective cells. This occurred through a BAX/BAK-dependent, caspase-mediated apoptotic pathway. The BCL-xL antagonist, WEHI-539, which targets BCL-2, significantly augmented tumour cell killing when used in combination with MLN8237. It also sensitized resistant brain tumour cells to a novel BAX activator, SMBA1. In addition, siRNA mediated knockdown of BCL-xL sensitized pGBM and medulloblastoma cells to MLN8237, and mimicked the effect of combination drug treatment. CONCLUSION: Combination treatment using a pro-apoptotic agent enhanced the cytotoxicity of MLN8237 in paediatric tumours and may overcome an important resistance mechanism.

Pediatric high grade glioma is refractory to conventional multimodal treatment, highlighting a need to develop novel efficacious therapies. We investigated tumor metabolism as a potential therapeutic target in a panel of diverse pediatric glioma cell lines (SF188, KNS42, UW479 and RES186) using metformin and 2-deoxyglucose. As a single agent, metformin had little effect on cell viability overall. SF188 cells were highly sensitive to 2-deoxyglucose however, combination of metformin with 2-deoxyglucose significantly reduced cell proliferation compared to either drug alone in all cell lines tested. In addition, the combination of the two agents was associated with a rapid decrease in cellular ATP and subsequent AMPK activation. However, increased cell death was only observed in select cell lines after prolonged exposure to the drug combination and was caspase independent. Anti-apoptotic BCL-2 family proteins have been indicated as mediators of resistance against metabolic stress. Therefore we sought to determine whether pharmacological inhibition of BCL-2/BCL-xL with ABT-263 could potentiate apoptosis in response to these agents. We found that ABT-263 increased sensitivity to 2-deoxyglucose and promoted rapid and extensive cell death in response to the combination of 2-deoxyglucose and metformin. Furthermore, cell death was inhibited by the pan-caspase inhibitor, z-VAD-FMK suggesting that ABT-263 potentiated caspase-dependent cell death in response to 2-deoxyglucose or its combination with metformin. Overall, these data provide support for the concept that targeting metabolic and anti-apoptotic pathways may be an effective therapeutic strategy in pediatric glioma.

Oncolytic viruses (OV) are promising treatments for cancer, with several currently undergoing testing in randomised clinical trials. Measles virus (MV) has not yet been tested in models of human melanoma. This study demonstrates the efficacy of MV against human melanoma. It is increasingly recognised that an essential component of therapy with OV is the recruitment of host anti-tumour immune responses, both innate and adaptive. MV-mediated melanoma cell death is an inflammatory process, causing the release of inflammatory cytokines including type-1 interferons and the potent danger signal HMGB1. Here, using human in vitro models, we demonstrate that MV enhances innate antitumour activity, and that MV-mediated melanoma cell death is capable of stimulating a melanoma-specific adaptive immune response.

Background As well as inducing direct oncolysis, reovirus treatment of melanoma is associated with activation of innate and adaptive anti-tumour immune responses. Results Here we characterise the effects of conditioned media from reovirus-infected, dying human melanoma cells (reoTCM), in the absence of live virus, to address the immune bystander potential of reovirus therapy. In addition to RANTES, IL-8, MIP-1α and MIP-1β, reovirus-infected melanoma cells secreted eotaxin, IP-10 and the type 1 interferon IFN-β. To address the mechanisms responsible for the inflammatory composition of reoTCM, we show that IL-8 and IFN-β secretion by reovirus-infected melanoma cells was associated with activation of NF-κB and decreased by pre-treatment with small molecule inhibitors of NF-κB and PKR; specific siRNA-mediated knockdown further confirmed a role for PKR. This pro-inflammatory milieu induced a chemotactic response in isolated natural killer (NK) cells, dendritic cells (DC) and anti-melanoma cytotoxic T cells (CTL). Following culture in reoTCM, NK cells upregulated CD69 expression and acquired greater lytic potential against tumour targets. Furthermore, melanoma cell-loaded DC cultured in reoTCM were more effective at priming adaptive anti-tumour immunity. Conclusions These data demonstrate that the PKR- and NF-κB-dependent induction of pro-inflammatory molecules that accompanies reovirus-mediated killing can recruit and activate innate and adaptive effector cells, thus potentially altering the tumour microenvironment to support bystander immune-mediated therapy as well as direct viral oncolysis.

Oncolytic virotherapy may mediate antitumor effects via direct oncolysis or immune-mediated tumor regression. Although the ability of oncolytic viruses to generate adaptive antitumor immunity has been characterized, their interactions with the innate immune system are relatively unclear. Using a human in vitro system, this study investigates the innate immunological consequences of reovirus therapy and its potential to activate NK cell-mediated antitumor activity. Dendritic cells (DC) loaded with reovirus-infected human melanoma Mel888 cells (DC-MelReo), but not reovirus-infected tumor cells alone, induced IFN-γ production within the NK cell population upon coculture with PBMC, in a cell-to-cell contact-dependent manner. DC-MelReo secreted the chemokines CCL2, 3, 4, 5, 7, 8, 11, and CXCL10; these culture supernatants induced NK cell chemotaxis. Coculture of DC-MelReo with purified NK cells induced reciprocal contact-dependent phenotypic DC maturation, while DC-MelReo elicited up-regulation of the activation marker CD69 on NK cells, in a partially contact and partially IL-12 dependent manner. Significantly, DC-MelReo induced NK cell cytotoxicity toward tumor cells by a type I IFN dependent mechanism. These data demonstrate that tumor infection by reovirus can act via DC to induce NK cell recruitment, activation, and cytotoxicity, along with reciprocal DC maturation. These findings suggest that reciprocal DC-NK cell interactions, following reovirus therapy, may play an important role in altering the immune milieu of the tumor microenvironment and mediating tumor regression.

Purpose: Reovirus is a naturally occurring oncolytic virus in clinical trials. Although tumor infection by reovirus can generate adaptive antitumor immunity, its therapeutic importance versus direct viral oncolysis is undefined. This study addresses the requirement for viral oncolysis and replication, and the relative importance of antitumor immunity and direct oncolysis in therapy. Experimental Design: Nonantigen specific T cells loaded with reovirus were delivered i.v. to C57BL/6 and severe combined immunodeficient mice bearing lymph node and splenic metastases from the murine melanoma, B16ova, with assessment of viral replication, metastatic clearance by tumor colony outgrowth, and immune priming. Human cytotoxic lymphocyte priming assays were done with dendritic cells loaded with Mel888 cells before the addition of reovirus. Results: B16ova was resistant to direct oncolysis in vitro, and failed to support reovirus replication in vitro or in vivo. Nevertheless, reovirus purged lymph node and splenic metastases in C57BL/6 mice and generated antitumor immunity. In contrast, reovirus failed to reduce tumor burden in severe combined immunodeficient mice bearing either B16ova or reovirus-sensitive B16tk metastases. In the human system, reovirus acted solely as an adjuvant when added to dendritic cells already loaded with Mel888, supporting priming of specific antitumor cytotoxic lymphocyte, in the absence of significant direct tumor oncolysis; UV-treated nonreplicating reovirus was similarly immunogenic. Conclusion: The immune response is critical in mediating the efficacy of reovirus, and does not depend upon direct viral oncolysis or replication. The findings are of direct relevance to fulfilling the potential of this novel anticancer agent.

INTRODUCTION: There is a pressing need for new therapeutic approaches for the treatment of glioblastoma (GBM). MicroRNAs are single-stranded 22-24nt non-coding RNAs which function by reducing translation or causing degradation of target mRNAs. MiR-1300 was identified in a high-throughput miRNA mimic library screen as a promising therapeutic candidate, inducing cell death. METHOD: To validate the cytotoxic effect of miR-1300, a panel of four established cell lines (U251,LN229,U373,KNS42)and two patient-derived stem-like cell lines were utilised for RTqPCR, imaging and flow-cytometry to assess endogenous expression levels, cellular morphology, apoptosis, and cell cycle delay respectively. Bioformatic approaches identified potential miR-1300 targets as mediators of this effect and validation of this involvement in the cytotoxic phenotype using siRNA knock-down is being investigated. Validation of the direct targeting by miR-1300 is being performed using 3' UTR Luciferase assays. RESULTS: Expression, of miR-1300, by transfection of oligonucleotide mimics, in our panel of GBM cells consistently led to G2M arrest followed by apoptosis within 72h. The associated phenotype is binucleation, indicative of cytokinesis failure.Preliminary data suggest this may be mediated through ECT2, a predicted target of miR-1300 known to be involved in formation of the cleavage furrow. CONCLUSION: We have identified a highly potent pro-apoptotic microRNA that may be exploitable for GBM therapy. Importantly, there is no detectable endogenous miR-1300 expression in any GBM samples or cell lines. On-going work will confirm the targets of miR-1300 that drive this effect as well as exploring potential approaches to deliver this as a biotherapeutic agent.