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BackgroundIntratumoral viral oncolytic immunotherapy is a promising new approach for the treatment of a variety of solid cancers. CAN-2409 is a replication-deficient adenovirus that delivers herpes simplex virus thymidine kinase to cancer cells, resulting in local conversion of ganciclovir or valacyclovir into a toxic metabolite. This leads to highly immunogenic cell death, followed by a local immune response against a variety of cancer neoantigens and, next, a systemic immune response against the injected tumor and uninjected distant metastases. CAN-2409 treatment has shown promising results in clinical studies in glioblastoma (GBM). Patients with GBM are usually given the corticosteroid dexamethasone to manage edema. Previous work has suggested that concurrent dexamethasone therapy may have a negative effect in patients treated with immune checkpoint inhibitors in patients with GBM. However, the effects of dexamethasone on the efficacy of CAN-2409 treatment have not been explored.MethodsIn vitro experiments included cell viability and neurosphere T-cell killing assays. Effects of dexamethasone on CAN-2409 in vivo were examined using a syngeneic murine GBM model; survival was assessed according to Kaplan-Meier; analyses of tumor-infiltrating lymphocytes were performed with mass cytometry (CyTOF - cytometry by time-of-flight). Data were analyzed using a general linear model, with one-way analysis of variance followed by Dunnett’s multiple comparison test, Kruskal-Wallis test, Dunn’s multiple comparison test or statistical significance analysis of microarrays.ResultsIn a mouse model of GBM, we found that high doses of dexamethasone combined with CAN-2409 led to significantly reduced median survival (29.0 days) compared with CAN-2409 treatment alone (39.5 days). CyTOF analyses of tumor-infiltrating immune cells demonstrated potent immune stimulation induced by CAN-2409 treatment. These effects were diminished when high-dose dexamethasone was used. Functional immune cell characterization suggested increased immune cell exhaustion and tumor promoting profiles after dexamethasone treatment.ConclusionOur data suggest that concurrent high-dose dexamethasone treatment may impair the efficacy of oncolytic viral immunotherapy of GBM, supporting the notion that dexamethasone use should be balanced between symptom control and impact on the therapeutic outcome.

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.