2026 - Differential Redox Capacity In Pediatric and Adult High-Grade Gliomas Reveals Therapeutic Vulnerabilities

Purpose/Objective(s): Diffuse intrinsic pontine glioma (DIPG) is a rare pediatric brain tumor arising in the pons, precluding surgical resection. As a result, radiotherapy (RT) ± ONC201 remains the standard treatment, though it is not curative. Despite transient responses to RT, its mechanisms of radioresistance are poorly understood compared with adult glioblastoma multiforme (GBM). RT efficacy can be suppressed by high intracellular antioxidant activity (e.g. catalase, glutathione, thioredoxin) by scavenging RT-induced reactive oxygen species (ROS), blunting oxidative cellular damage and promoting tumor cell survival. We hypothesize that DIPG cells exhibit a distinct antioxidant profile (compared to GBM) contributing to differential treatment response, and that pharmacological inhibition of key antioxidant pathways will reveal DIPG-specific vulnerabilities exploitable for radiosensitization.

Materials/Methods: DIPG and GBM cell lines were assessed for baseline levels of ROS by H₂DCFDA oxidation using flow cytometry; hydrogen peroxide (H₂O₂) removal rate using a H₂O₂ fluorometric assay; and catalase (H₂O₂ scavenger), glutathione (GSH, hydroperoxide removal) and thioredoxin reductase (TR, hydroperoxide removal) activity using spectrophotometric assays. Clonogenic survival was assessed after bolus doses of H₂O₂, buthionine sulfoximine (BSO, GSH inhibitor), and auranofin (AUR, TR inhibitor). Normal human astrocytes (NHAs) were used as non-tumor controls.

Results: DIPG cells exhibited higher rates of H₂O₂ removal compared to GBM cells. Increasing bolus doses of H₂O₂ led to significantly more dose-dependent clonogenic cell death in DIPG cells compared to GBM and NHA cells, likely due to DIPG’s higher baseline ROS. Baseline catalase activity did not differ significantly between DIPG and GBM cells suggesting a non-catalase mechanism in the enhanced H₂O₂ removal in DIPG cells. BSO depleted GSH levels in DIPG cells. However, DIPG cells were less sensitive to BSO-induced clonogenic cell killing compared to GBM cells suggesting that GSH may not be the major antioxidant responsible for intracellular H₂O₂ removal in DIPG. AUR suppressed intracellular TR activity in DIPG cells, which were significantly more sensitive to AUR-induced clonogenic cell killing compared to GBM cells, suggesting that TR may play a prominent antioxidant role specifically in DIPG cells.

Conclusion: Given DIPG's distinct biology relative to GBM, antioxidant metabolism dysregulation warrants exploration. Altogether, the increased sensitivity of DIPG cells to AUR (versus GBM) identifies TR as a key therapeutic target with direct implications for radiosensitivity. These findings support further investigation of AUR and other inhibitors of the thioredoxin antioxidant system as a strategy to overwhelm the redox buffering capacity of DIPG cells and improve long-term tumor response to RT.