Our previous results showed that oligodendrocyte development is regulated by both nociceptin and its G-protein coupled receptor, the nociceptin/orphanin FQ receptor (NOR). The present in vitro and in vivo findings show that nociceptin plays a crucial conserved role regulating the levels of the glutamate/aspartate transporter GLAST/EAAT1 in both human and rodent brain astrocytes. This nociceptin-mediated response takes place during a critical developmental window that coincides with the early stages of astrocyte maturation. GLAST/EAAT1 upregulation by nociceptin is mediated by NOR and the downstream participation of a complex signaling cascade that involves the interaction of several kinase systems, including PI-3K/AKT, mTOR, and JAK. Because GLAST is the main glutamate transporter during brain maturation, these novel findings suggest that nociceptin plays a crucial role in regulating the function of early astrocytes and their capacity to support glutamate homeostasis in the developing brain.

Abstract PI3K/AKT/mTOR signaling pathway activation is a common mechanism of tumor progression in diffuse lower grade glioma. Robust and accurate biomarkers are needed to stratify patients for therapies targeting this pathway. To investigate the potential of phosphoprotein quantification to provide a direct and functional pathway readout, we analyzed 90 tumors from 83 patients with IDH-mutant diffuse glioma. The cohort comprised 50 IDH-mutant astrocytomas, 40 IDH-mutant and 1p/19q-codeleted oligodendrogliomas, 7 of whom had paired samples from initial diagnosis and recurrence. We developed and validated a pipeline using multiplex immunofluorescence to quantify tumor cell-specific phospho-protein expression of 3 pathway nodes, ribosomal protein S6 (RPS6), PRAS40, and 4E-BP1. In oligodendroglioma the fraction of tumor cells expressing each of the three phosphorylated proteins increased with tumor grade (p< 0.05). Comparing tumors at initial diagnosis (n=48) and at recurrence (n=42), p-RPS6 and p-PRAS40 increased in tumor cells (p< 0.05) and there was an overall increase in intertumoral heterogeneity of signaling activity at recurrence (p< 0.04). Analysis of paired samples demonstrated increased signaling pathway activity in a subset at recurrence. Robust signaling activity, defined as a phospho-positive tumor cell fraction ≥ median for all three phosphoproteins, was identified in 71.4% of grade 3 IDH-mutant astrocytoma(5/7) and 45.4% of grade 3 IDH-mutant, 1p/19q-codeleted oligodendroglioma(5/11). In a subset of cases analyzed by targeted NGS, robust signaling pathway activity was identified in 38%(11/29) at the protein level while genetic alterations predicted to activate the pathway were present in only 17.2% (5/29). Our results demonstrate robust PI3K/AKT/mTOR signaling activity in a significant fraction of IDH-mutant diffuse glioma, an association with increasing tumor grade in oligodendroglioma, and an increase at recurrence in both oligodendroglioma and astrocytoma. Overall, our data suggest that quantitative evaluation of phosphoproteins may be a sensitive method to detect PI3K/AKT/mTOR pathway activity and may be useful for patient stratification.

Glycosaminoglycans (GAGs) such as heparan sulfate and chondroitin sulfate decorate all mammalian cell surfaces. These mucopolysaccharides act as coreceptors for extracellular ligands, regulating cell signaling, growth, proliferation, and adhesion. In glioblastoma, the most common type of primary malignant brain tumor, dysregulated GAG biosynthesis results in altered chain length, sulfation patterns, and the ratio of contributing monosaccharides. These events contribute to the loss of normal cellular function, initiating and sustaining malignant growth. Disruption of the aberrant cell surface GAGs with small molecule inhibitors of GAG biosynthetic enzymes is a potential therapeutic approach to blocking the rogue signaling and proliferation in glioma, including glioblastoma. Previously, 4-azido-xylose-α-UDP sugar inhibited both xylosyltransferase (XYLT-1) and β-1,4-galactosyltransferase-7 (β-GALT-7)-the first and second enzymes of GAG biosynthesis-when microinjected into a cell. In another study, 4-deoxy-4-fluoro-β-xylosides inhibited β-GALT-7 at 1 mM concentration in vitro. In this work, we seek to solve the enduring problem of drug delivery to human glioma cells at low concentrations. We developed a library of hydrophobic, presumed prodrugs 4-deoxy-4-fluoro-2,3-dibenzoyl-(α- or β-) xylosides and their corresponding hydrophilic inhibitors of XYLT-1 and β-GALT-7 enzymes. The prodrugs were designed to be activatable by carboxylesterase enzymes overexpressed in glioblastoma. Using a colorimetric MTT assay in human glioblastoma cell lines, we identified a prodrug-drug pair (4-nitrophenyl-α-xylosides) as lead drug candidates. The candidates arrest U251 cell growth at an IC₅₀ = 380 nM (prodrug), 122 μM (drug), and U87 cells at IC₅₀ = 10.57 μM (prodrug). Molecular docking studies were consistent with preferred binding of the α- versus β-nitro xyloside conformer to XYLT-1 and β-GALT-7 enzymes.

Background

IDH-mutant diffuse gliomas are heterogeneous, and improved methods for optimal patient therapeutic stratification are needed. PI3K/AKT/mTOR signaling activity can drive disease progression and potential therapeutic inhibitors of the pathway are available. Yet, the prevalence of PI3K/AKT/mTOR signaling pathway activity in IDH-mutant glioma is unclear and few robust strategies to assess activity in clinical samples exist.

Methods

PI3K/AKT/mTOR signaling pathway activity was evaluated in a retrospective cohort of 132 IDH-mutant diffuse glioma (91 astrocytoma and 41 oligodendroglioma, 1p/19q-codeleted) through quantitative multiplex immunoprofiling using phospho-specific antibodies for PI3K/AKT/mTOR pathway members, PRAS40, RPS6, and 4EBP1, and tumor-specific anti-IDH1 R132H. Expression levels were correlated with genomic evaluation of pathway intrinsic genes and univariate and multivariate Cox proportional hazard regression models were used to evaluate the relationship with outcome.

Results

Tumor-specific expression of p-PRAS40, p-RPS6, and p-4EBP1 was common in IDH-mutant diffuse glioma and increased with CNS WHO grade from 2 to 3. Genomic analysis predicted pathway activity in 21.7% (13/60) while protein evaluation identified active PI3K/AKT/mTOR signaling in 56.6% (34/60). Comparison of expression in male versus female patients suggested sexual dimorphism. Of particular interest, when adjusting for clinical prognostic factors, the level of phosphorylation of RPS6 was strongly associated with PFS (P < .005). Phosphorylation levels of both PRAS40 and RPS6 showed an association with PFS in univariate analysis.

Conclusions

Our study emphasizes the value of proteomic assessment of signaling pathway activity in tumors as a means to identify relevant oncogenic pathways and potentially as a biomarker for identifying aggressive disease.

Purpose

The immunosuppressive tumor microenvironment present in the majority of diffuse glioma limits therapeutic response to immunotherapy. As the determinants of the glioma-associated immune response are relatively poorly understood, the study of glioma with more robust tumor-associated immune responses may be particularly useful to identify novel immunomodulatory factors that can promote T-cell effector function in glioma.

Experimental design

We used multiplex immune-profiling, proteomic profiling, and gene expression analysis to define the tumor-associated immune response in two molecular subtypes of glioma and identify factors that may modulate this response. We then used patient-derived glioma cultures and an immunocompetent murine model for malignant glioma to analyze the ability of tumor-intrinsic factors to promote a CD8+ T-cell response.

Results

As compared with isocitrate dehydrogenase (IDH)-mutant astrocytoma, MAPK-activated pleomorphic xanthoastrocytoma (PXA) harbored increased numbers of activated cytotoxic CD8+ T cells and Iba1+ microglia/macrophages, increased MHC class I expression, enrichment of genes associated with antigen presentation and processing, and increased tumor cell secretion of the chemokine CXCL14. CXCL14 promoted activated CD8+ T-cell chemotaxis in vitro, recruited tumor-infiltrating CD8+ T cells in vivo, and prolonged overall survival in a cytotoxic T-cell-dependent manner. The immunomodulatory molecule B7-H3 was also highly expressed in PXA.

Conclusions

We identify the MAPK-activated lower grade astrocytoma PXA as having an immune-rich tumor microenvironment and suggest this tumor may be particularly vulnerable to immunotherapeutic modulation. We also identify CXCL14 as an important determinant of the glioma-associated immune microenvironment, sufficient to promote an antitumor CD8+ T-cell response.

Seizures are a frequent complication of adult-type diffuse gliomas, and are often difficult to control with medications. Gliomas with mutations in isocitrate dehydrogenase 1 or 2 (IDHmut) are more likely than IDH-wild type (IDHwt) gliomas to cause seizures as part of their initial clinical presentation. However, whether IDHmut is also associated with seizures during the remaining disease course, and whether IDHmut inhibitors can reduce seizure risk, are unclear. Clinical multivariable analyses showed that preoperative seizures, glioma location, extent of resection, and glioma molecular subtype (including IDHmut status) all contributed to postoperative seizure risk in adult-type diffuse glioma patients, and that postoperative seizures were often associated with tumor recurrence. Experimentally, the metabolic product of IDHmut, d-2-hydroxyglutarate, rapidly synchronized neuronal spike firing in a seizure-like manner, but only when non-neoplastic glial cells were present. In vitro and in vivo models recapitulated IDHmut glioma-associated seizures, and IDHmut inhibitors currently being evaluated in glioma clinical trials inhibited seizures in those models, independent of their effects on glioma growth. These data show that postoperative seizure risk in adult-type diffuse gliomas varies in large part by molecular subtype, and that IDHmut inhibitors could play a key role in mitigating such risk in IDHmut glioma patients.