In order to prioritize available immune therapeutics, immune profiling across glioma grades was conducted, followed by preclinical determinations of therapeutic effect in immune-competent mice harboring gliomas. T cells and myeloid cells were isolated from the blood of healthy donors and the blood and tumors from patients with glioma and profiled for the expression of immunomodulatory targets with an available therapeutic. Murine glioma models were used to assess therapeutic efficacy of agents targeting the most frequently expressed immune targets. In patients with glioma, the A2aR/CD73/CD39 pathway was most frequently expressed, followed by the PD-1 pathway. CD73 expression was upregulated on immune cells by 2-hydroxyglutarate in IDH1 mutant glioma patients. In murine glioma models, adenosine receptor inhibitors demonstrated a modest therapeutic response; however, the addition of other inhibitors of the adenosine pathway did not further enhance this therapeutic effect. Although adenosine receptor inhibitors could recover immunological effector functions in T cells, immune recovery was impaired in the presence of gliomas, indicating that irreversible immune exhaustion limits the effectiveness of adenosine pathway inhibitors in patients with glioma. This study illustrates vetting steps that should be considered before clinical trial implementation for immunotherapy-resistant cancers, including testing an agent’s ability to restore immunological function in the context of intended use.

MiRNAs can silence a wide range of genes, which may be an advantage for targeting heterogenous tumors like glioblastoma. Osteopontin (OPN) plays both an oncogenic role in a variety of cancers and can immune modulate macrophages. We conducted a genome wide profiling and bioinformatic analysis to identify miR-181a/b/c/d as potential miRNAs that target OPN. Luciferase assays confirmed the binding potential of miRNAs to OPN. Expression levels of miR-181a/b/c/d and OPN were evaluated by using quantitative real-time PCR and enzyme-linked immunosorbent assay in mouse and human glioblastomas and macrophages that showed these miRNAs were downregulated in Glioblastoma associated CD11b+ cells compared to their matched blood CD14b+ cells. miRNA mimicking and overexpression using lentiviruses showed that MiR-181a overexpression in glioblastoma cells led to decreased OPN production and proliferation and increased apoptosis in vitro. MiR-181a treatment of immune competent mice bearing intracranial glioblastoma demonstrated a 22% increase in median survival duration relative to that of control mice.

Glioblastoma (GBM) is one of the most common, aggressive, and invasive human brain tumors. There are few reliable mechanism-based therapeutic approaches for GBM patients. The transcriptional repressor RE1 silencing transcriptional factor (REST) regulates the oncogenic properties of a class of GBM stem-like cells (high-REST [HR]-GSCs) in humans. However, it has been unclear whether REST represses specific targets to regulate specific oncogenic functions or represses all targets with overlapping functions in GSCs. We used genome-wide, biochemical, and mouse intracranial tumorigenic assays to identify and determine functions of microRNA (miR) targets of REST in 2 independent HR-GSC lines. Here we show that REST represses 2 major miR gene targets in HR-GSCs: miR-203, a new target, and miR-124, a known target. Gain of function of miR-124 or miR-203 in HR-GSCs increased survival in tumor-bearing mice. Importantly, the increased survival of tumor-bearing mice caused by knockdown of REST in HR-GSCs was reversed by double knockdown of REST and either miR-203 or miR-124, indicating that these 2 miRs are critical tumor suppressors that are repressed in REST-mediated tumorigenesis. We further show that while miR-124 and the REST–miR-124 pathways regulate self-renewal, apoptosis and invasion, miR-203 and the REST–miR-203 pathways regulate only invasion. We further identify and validate potential mRNA targets of miR-203 and miR-124 in REST-mediated HR-GSC tumor invasion. These findings indicate that REST regulates its miR gene targets with overlapping functions and suggest how REST maintains oncogenic competence in GSCs. These mechanisms could potentially be utilized to block REST-mediated GBM tumorigenesis.