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CAN-2409 is a replication-deficient adenovirus encoding herpes simplex virus (HSV) thymidine kinase (tk) currently in clinical trials for treatment of glioblastoma. The expression of tk in transduced cancer cells results in conversion of the pro-drug ganciclovir into a toxic metabolite causing DNA damage, inducing immunogenic cell death and immune activation. We hypothesize that CAN-2409 combined with DNA-damage-response inhibitors could amplify tumor cell death, resulting in an improved response. We investigated the effects of ATR inhibitor AZD6738 in combination with CAN-2409

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.

We previously showed that derivatives of the Chinese traditional medicine indirubin promote survival in murine glioblastoma models (Williams et al. Cancer Research 2011). However, poor drug solubility hampered further development of this approach. Here we introduce PPRX-1701, a 6’-bromoindirubin acetoxime (BiA) containing drug/polymer nanoparticle formulation which can be injected intravenously at relatively high concentrations. Mechanistically, BiA is thought to act as a broadly selective serine-threonine protein kinase inhibitor, with activity against Src family kinases, GSK-3 and JAK2. Our preliminary data show that intravenous administration of PPRX-1701 is well-tolerated and can reach intracranial murine glioblastoma as assessed by luminescent reporter assays. PPRX-1701 administration leads to improved survival in the GL261 glioblastoma mouse model (median survival 30 days (control), 47 days (treated), p < 0.0001). Treatment with PPRX-1701 was associated with alterations in the tumor immune microenvironment, with reduced Tregs and pro-tumor macrophages, and increased CD8+ T cells. Further preliminary mechanistic studies have shown that PPRX-1701/BiA blocks the expression multiple immunosuppressive molecules in GBM downstream of interferon-g (IFNg) including PD-L1 and indoleamine 2,3-dioxygenase 1 (IDO1) - a key enzyme in the tryptophan–kynurenine–aryl hydrocarbon receptor (Trp–Kyn–AhR) immunosuppressive pathway. BiA promoted more effective T-cell mediated tumor cell killing using patient derived glioblastoma ex vivo co-culture models. This data supports further development of PPRX-1701 is a candidate immunotherapeutic agent for glioblastoma treatment. Ongoing pre-clinical studies are investigating combination with other relevant therapies.

Cytomegalovirus (CMV) is widespread in humans and has been implicated in glioblastoma (GBM) and other tumors. However, the role of CMV in GBM remains poorly understood and the mechanisms involved are not well-defined. The goal of this study was to identify candidate pathways relevant to GBM that may be modulated by CMV. Analysis of RNAseq data after CMV infection of patient-derived GBM cells showed significant upregulation of GBM-associated transcripts including the MET oncogene, which is known to play a role in a subset of GBM patients. These findings were validated in vitro in both mouse and human GBM cells. Using immunostaining and RT-PCR in vivo, we confirmed c-MET upregulation in a mouse model of CMV-driven GBM progression and in human GBM. siRNA knockdown showed that MET upregulation was dependent on CMV-induced upregulation of NF-κB signaling. Finally, proneural GBM xenografts overexpressing c-MET grew much faster in vivo than controls, suggesting a mechanism by which CMV infection of tumor cells could induce a more aggressive mesenchymal phenotype. These studies implicate the CMV-induced upregulation of c-MET as a potential mechanism involved in the effects of CMV on GBM growth.

INTRODUCTION Glioblastoma (GBM), an aggressive brain tumor with a poor prognosis, presents an average of 2% of patients surviving beyond 2 years after diagnosis. Therapies to effectively manage glioblastoma are hindered due to the presence of the blood-brain barrier (BBB). Previously, a cell-penetrating peptide, M13, was conjugated to a Pt(IV) cisplatin prodrug, via amide bond formation. The conjugated Pt(IV) releases active cisplatin upon intracellular reduction. Herein, we investigated the BBB-penetrance and biodistribution of M13 conjugated to Pt(IV), as well as its effectiveness against GBM in mouse models. METHODS M13 platinum-conjugate tumor cell killing capacity was assessed by luminescent cell viability assays in vitro. By using Inductively-Coupled Plasma Mass-Spectrometry for platinum detection, BBB penetration and bio-distribution studies were performed in a three-dimensional BBB spheroid in vitro model and in vivo in mouse brain, intracranial tumor, and peripheral organs. Dose-regime studies involved observations of symptomatology and weight variations after bi-weekly injections of platinum compounds at 2mg/kg and 5mg/kg. RESULTS The Pt(IV)-M13 conjugate possesses tumor cell killing effects similar to cisplatin when tested in GBM cell lines in vitro. Platinum increased by using Pt(IV)-M13 when compared to cisplatin in our in vitro BBB-spheroid model (20-fold, p-value=0.0033), in brain tissue (10-fold, p< 0.0001) and GBM tumor-bearing mice models (7.5-fold, p< 0.0001). Bio-distribution of platinum delivered by Pt(IV)-M13 in spleen, heart and blood was significantly different to cisplatin 5hrs. after intravenous injection (p< 0.001). Bi-weekly dose regimes of Pt(IV)-M13 are tolerable in nude mice without toxicity at a similar concentration to reported tolerable cisplatin doses at 5 mg/kg. Finally, Pt(IV)-M13 significantly increased survival in a murine glioblastoma xenograft model compared with controls (median 24 days vs. 29 days, p-value=0.0071). CONCLUSION Overall, our data support the further development of BBB-crossing peptide-drug conjugates for GBM treatment.

Induction of mitotic catastrophe through the disruption of microtubules is an established target in cancer therapy. However, the molecular mechanisms determining the mitotic catastrophe and the following apoptotic or non-apoptotic cell death remain poorly understood. Moreover, many existing drugs targeting tubulin, such as vincristine, have reduced efficacy, resulting from poor solubility in physiological conditions. Here, we introduce a novel small molecule 2-aminoimidazoline derivative&mdash, OAT-449, a synthetic water-soluble tubulin inhibitor. OAT-449 in a concentration range from 6 to 30 nM causes cell death of eight different cancer cell lines in vitro, and significantly inhibits tumor development in such xenograft models as HT-29 (colorectal adenocarcinoma) and SK-N-MC (neuroepithelioma) in vivo. Mechanistic studies showed that OAT-449, like vincristine, inhibited tubulin polymerization and induced profound multi-nucleation and mitotic catastrophe in cancer cells. HeLa and HT-29 cells within 24 h of treatment arrested in G2/M cell cycle phase, presenting mitotic catastrophe features, and 24 h later died by non-apoptotic cell death. In HT-29 cells, both agents altered phosphorylation status of Cdk1 and of spindle assembly checkpoint proteins NuMa and Aurora B, while G2/M arrest and apoptosis blocking was consistent with p53-independent accumulation in the nucleus and largely in the cytoplasm of p21/waf1/cip1, a key determinant of cell fate programs. This is the first common mechanism for the two microtubule-dissociating agents, vincristine and OAT-449, determining the cell death pathway following mitotic catastrophe demonstrated in HT-29 cells.

RATIONALE Herpes virus thymidine kinase (HSV-TK) suicide gene therapy is a well-established approach for in situ tumor cell killing after administration of ganciclovir (GCV), due to the induction of lethal DNA damage in HSV-TK expressing cells. Here we investigated the effects of HSV-TK gene delivery with a non-replicating serotype 5 adenovirus (AdTK) in murine glioblastoma models in combination with the ATR-inhibitor AZD6738 to disrupt the DNA damage response (DDR). METHODS We investigated the effects of disrupted DDR signaling on AdTK therapy in vitro using cytotoxicity, cytokine and flow cytometry assays in glioblastoma cell lines and in vivo with an orthotopic syngeneic murine glioblastoma model. Therapy response was monitored with MRI. Changes in the tumor microenvironment were analyzed with CyTOF. RESULTS The combination of AZD6738 with AdTK was synergistic in cytotoxicity assays, which was complemented by a significant increase of γH2AX foci. Complex modulations of the tumor microenvironment were observed with significantly reduced expression of PD-L1, MICA/B and the pro-tumorigenic cytokines IL1b and IL-4. In vivo, the combination with AZD6738 led to an increase in long-term surviving animals (66.7%) compared to GMCI (50%) and proved to be highly significant in contrast to untreated controls (p=0.0022). However, the combination treatment did not block the growth of tumors upon rechallenge in long-term survivors. CONCLUSION DDR signaling is crucial in the therapeutic efficacy of AdTK/GCV. It significantly enhances cytotoxicity in vitro and in vivo while having complex ramifications at the immunological level, requiring further studies to determine ideal conditions for a maximized therapeutic benefit.

INTRODUCTION Cytomegalovirus (CMV), a member of the betaherpesvirinae subfamily widespread in human populations has been reported to be detectable in glioblastoma (GBM). We have recently shown that CMV latent infection promotes GBM growth in a mouse model. The mechanisms involved are not well-defined and treatment responses have not been evaluated. METHODS To investigate the effects of CMV on GBM cells we performed an RNAseq study. Differentially expressed genes were identified and validated in vitro and in vivo. RESULTS RNAseq analysis showed multiple alterations induced by CMV infection of GBM cells including significant upregulation of c-MET (8.2 log2 fold change) and MGMT (3.9 log2 fold change) transcripts after CMV infection. c-MET is a tyrosine kinase receptor known to promote GBM growth, and MGMT expression leads to resistance to the alkylating agent temozolomide (TMZ). These findings were validated in vitro in both mouse and human GBM cells, and shown to be dependent on CMV-induced upregulation of NF-kB by siRNA knockdown of the essential RelA NF-kB subunit. The c-MET co-receptor CD44 was also upregulated suggesting a role for the c-MET signalling axis in CMV induced tumor progression. CMV-infected tumor cells were resistant to TMZ treatment as expected due to MGMT upregulation. Treatment of our murine GBM model with TMZ led to a significant increase in survival in control mock-infected mice (median survival 38 days control, 48 days TMZ), whereas the CMV infected mice did not respond (median survival 35 days control, 35 days TMZ; p value between TMZ treated groups = 10–4). CONCLUSIONS Our results define novel and relevant mechanisms by which CMV may influence both GBM growth and resistance to treatment. Further therapeutic and mechanistic studies are underway to investigate these pathways in the context of CMV in GBM. These data support further study of CMV targeting in GBM.

Immunotherapeutic approaches to treating glioblastoma (GBM) such as immune checkpoint blockade or dendritic cell vaccines have so far failed in recent clinical trials, despite showing some success in other cancer types. This failure has been attributed partly to the profound immunosuppressive tumor micro-environment (TME), characterized by dysfunctional cytotoxic T cell response and significant infiltration of regulatory lymphocytes and suppressive myeloid cells. We hypothesize that an oncolytic HSV1 (oHSV) which has a direct tumor cytotoxicity and potent in situ immunostimulation may reverse these immune dysfunction state and therefore improve tumor regression. Using several unique reagents, we found that oHSV treatment led to substantial reduction in tumor volume compared to PBS-control mice (oHSV- 9/11 vs PBS- 0/5; p= 0.0022), translating to longer term survival. Interestingly, oHSV led to improved tumor-specific T cells response with an expansion of tumor antigen-specific CD8+ T cells within the TME on both day 3 (oHSV- 4% vs PBS- 2%; p = 0.02) and day 7 (oHSV- 6.51% vs PBS- 1.33%; p = 0.04) following treatment, and a higher expression of IFNγ by CD8+ T cells compared to control (oHSV- 0.21% vs PBS- 5.60%; p = 0.04), suggesting an improved tumor-specific T cell functionality. Importantly, we found that this expansion of tumor-antigen specific CD8+ T cells inversely correlated with the tumor volume (R(2)= 0.41, p= 0.008). oHSV also led to a decrease in microglia and suppressive myeloid cell population which correlate negatively with the number of infiltating tumor-specific CD8+ T cells, suggesting they may play a role in modulating oHSV-mediated expansion of tumor specific T cells. Overall, using these novel tools, we demonstrate for the first time, that oHSV, in addition to its direct cytotoxic effect, play an important immunostimulating role through its modulation of tumor-specific T cells and myeloid cell population within the GBM tumor micro-environment.

Human cytomegalovirus (HCMV) is highly prevalent, and like other herpes viruses, can persist for life in its host in a latent state. However, HCMV can be severely pathogenic in immunocompromised individuals. Interestingly, HCMV proteins and nucleic acids have been identified in up to 90% of patients with the incurable brain tumor glioblastoma (GBM) as well as some other cancers. Accumulating data supports the clinical relevance of HCMV in GBM, with some encouraging responses reported with HCMV-targeted immunotherapies. Although various HCMV proteins increase cell proliferation and invasion, a mechanistic link between HCMV and cancer in vivo has not been established, and the role of HCMV in GBM remains a subject of debate. In the current report we show that perinatal murine CMV (MCMV) infection induces a pro-angiogenic secretome, increasing tumor growth, pericyte accumulation, angiogenesis and tumor blood flow in a murine GBM model. Specifically, we identify platelet-derived growth factor-d (PDGF-D) as a CMV-induced factor essential for tumor growth. In our model, MCMV can be seen in tumor cells and vascular pericytes, a finding that we confirm in human GBM specimens. The anti-viral drug cidofovir improves survival in MCMV-infected mice, inhibiting MCMV activation, PDGF-D expression, pericyte recruitment and tumor angiogenesis. Together these data provide the first mechanistic explanation of how CMV potentiates GBM growth in vivo, identify PDGF-D as a potential therapeutic target, and support the application of anti-viral approaches for GBM therapy.