SUMMARY Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. While the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obsure. Here, we report the identification of novel N(6)-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer, glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase, ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator, ALKBH1, in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification, N6-mA.

Despite aggressive multi-modality treatment with surgery, radiation and chemotherapies, malignant glioma inevitably recurs and has dismal survival rates. Recent progress in immunotherapy has led to a resurgence of interest, and immunotherapies are being investigated for treatment of glioma. However, the unique brain anatomy and a highly immunosuppressive glioma microenvironment pose significant challenges to achieving efficacy. Thus, there is a critical need for assessment of next-generation immunotherapies for glioma. In this study, we have investigated the efficacy of the nanoparticle platform technology based on plant-derived Cowpea mosaic virus like particles (empty CPMV or eCPMV) to instigate a potent immune response against intracranial glioma. CPMV immunotherapy has been shown to efficiently reverse the immunosuppressive tumor microenvironments in pre-clinical murine models of dermal melanoma and metastatic melanoma, metastatic breast cancer, intraperitoneal ovarian cancer and in canine patients with oral melanoma. In the present study, we demonstrate that in situ administration of CPMV immunotherapy in the setting of glioma can effectively recruit unique subset of effector innate and adaptive immune cells to the brain parenchyma while reducing immune suppressive cellular population, leading to regression of intracranial glioma. The in situ CPMV nanoparticle vaccine offers a potent yet safe and localized immunotherapy for intracranial glioma.