Uncovering transcriptomic landscape alterations of CAN-2409 in in vitro and in vivo glioma models.

Rationale: CAN-2409 is a locally delivered oncolytic therapy, which results in vaccination against the injected tumor. CAN-2409 consists of a non-replicating adenovirus armed with the Herpes virus thymidine kinase, which metabolizes ganciclovir into a phosphorylated nucleotide that is incorporated into the tumor cell's genome, thereby inflicting immunogenic cancer cell death. While CAN-2409's immunological impact has been well characterized, its effects on the tumor cells transcriptome remains unknown. We compared the transcriptomic landscape after treatment of glioblastoma models with CAN-2409 in vitro and in vivo to assess how the interplay with the tumor microenvironment influences CAN-2409-mediated transcriptome alterations. Methods: We performed RNA-Seq with CAN-2409 treated patient-derived glioma stem-like cells and tumors of C57/BL6 mice and compared KEGG pathway usage and differential gene expression focusing on immune cell and cytokine profiles. T-cell -killing assays were performed to assess candidate effectors. Results: PCA analysis showed distinct clustering of control and CAN-2409 samples under both conditions. KEGG pathway analysis revealed significant enrichment for p53 signaling and cell cycle pathway, with similar dynamics for key regulators of both pathways in vitro and in vivo, including MYC, CCNB1, PLK1 and CDC20. Selected alterations (PLK1 and CCNB1) were validated at the protein level. Cytokine expression analysis revealed upregulation of pro-inflammatory IL12a under both conditions; immune cell gene profiling showed reduction of myeloid associated genes. T-cell-killing assays showed increased killing in the presence of IL-12. Conclusion: CAN-2409 significantly alters the transcriptome both in vitro and in vivo. Comparison of pathway enrichment revealed mutual and differential utilization of pathways under both conditions, suggesting a modulating influence on the cell cycle in tumor cells, and of the tumor microenvironment on the transcriptome in vivo. IL-12 synthesis likely depends on interactions with the tumor microenvironment, and it facilitates CAN-2409 cell killing. This dataset provides potential to understand resistance mechanisms and identify potential biomarkers for future studies.

PPRX-1701, a nanoparticle formulation of 6'-bromoindirubin acetoxime, improves delivery and shows efficacy in preclinical GBM models.

Derivatives of the Chinese traditional medicine indirubin have shown potential for the treatment of cancer through a range of mechanisms. This study investigates the impact of 6'-bromoindirubin-3'-acetoxime (BiA) on immunosuppressive mechanisms in glioblastoma (GBM) and evaluates the efficacy of a BiA nanoparticle formulation, PPRX-1701, in immunocompetent mouse GBM models. Transcriptomic studies reveal that BiA downregulates immune-related genes, including indoleamine 2,3-dioxygenase 1 (IDO1), a critical enzyme in the tryptophan-kynurenine-aryl hydrocarbon receptor (Trp-Kyn-AhR) immunosuppressive pathway in tumor cells. BiA blocks interferon-γ (IFNγ)-induced IDO1 protein expression in vitro and enhances T cell-mediated tumor cell killing in GBM stem-like cell co-culture models. PPRX-1701 reaches intracranial murine GBM and significantly improves survival in immunocompetent GBM models in vivo. Our results indicate that BiA improves survival in murine GBM models via effects on important immunotherapeutic targets in GBM and that it can be delivered efficiently via PPRX-1701, a nanoparticle injectable formulation of BiA.

Perturbing DDR signaling enhances cytotoxic effects of local oncolytic virotherapy and modulates the immune environment in glioma.

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 in vitro using cytotoxicity, cytokine, and fluorescence-activated cell sorting (FACS) assays in glioma cell lines and in vivo with an orthotopic syngeneic murine glioma model. Tumor immune infiltrates were analyzed by cytometry by time of flight (CyTOF). In vitro, we observed a significant increase in the DNA-damage marker γH2AX and decreased expression of PD-L1, pro-tumorigenic cytokines (interleukin-1ß [IL-1ß], IL-4), and ligand NKG2D after combination treatment compared with monotherapy or control. In vivo, long-term survival was increased after combination treatment (66.7%) compared with CAN-2409 (50%) and control. In a tumor re-challenge, long-term immunity after combination treatment was not improved. Our results suggest that ATR inhibition could amplify CAN-2409's efficacy in glioblastoma through increased DNA damage while having complex immunological ramifications, warranting further studies to determine the ideal conditions for maximized therapeutic benefit.

Systemic high-dose dexamethasone treatment may modulate the efficacy of intratumoral viral oncolytic immunotherapy in glioblastoma models.

BACKGROUND: Intratumoral 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. METHODS: In 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. RESULTS: In 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. CONCLUSION: Our 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.

HSV-1 Oncolytic Viruses from Bench to Bedside: An Overview of Current Clinical Trials.

Herpes simplex virus 1 (HSV-1) provides a genetic chassis for several oncolytic viruses (OVs) currently in clinical trials. Oncolytic HSV1 (oHSV) have been engineered to reduce neurovirulence and enhance anti-tumor lytic activity and immunogenicity to make them attractive candidates in a range of oncology indications. Successful clinical data resulted in the FDA-approval of the oHSV talimogene laherparepvec (T-Vec) in 2015, and several other variants are currently undergoing clinical assessment and may expand the landscape of future oncologic therapy options. This review offers a detailed overview of the latest results from clinical trials as well as an outlook on newly developed HSV-1 oncolytic variants with improved tumor selectivity, replication, and immunostimulatory capacity and related clinical studies.