ID #819 Targeting reactive oxygen species to modulate the epigenome of diffuse midline glioma

Abstract

Diffuse midline gliomas (DMGs) are aggressive paediatric brain tumours with a survival of less than one year, with palliative radiotherapy the mainstay of treatment. DMGs are characterised by H3K27-alterations driving global chromatin hypomethylation, with approximately 36% of cases harbor receptor tyrosine kinase (RTK) alterations, including PDGFRA, which correlate with worse outcomes. Non-genomic influences also contribute to DMG pathogenesis with metabolic dysregulation a hallmark that contributes to elevated levels of reactive oxygen species (ROS) driving extreme levels of oxidative stress. While radiotherapy induces DNA damage by increasing ROS, emerging evidence indicates that excessive ROS can paradoxically sustain RTK activity, impair tumour suppressor function, and reinforce oncogenic signaling, forming a feedforward loop that drives tumour progression. In prior studies of RTK-driven cancers, we demonstrated that NADPH oxidase 2 (NOX2)-derived ROS oxidises DNA methyltransferase DNMT1, impairing its activity, reducing DNA methylation at oncogenes such as PI3K/Akt, and promoting therapy resistance. Pharmacologic inhibition of NOX2 restored DNMT1 function, rescued methylation, and enhanced responses to tyrosine kinase inhibitors, establishing a mechanistic link between ROS, epigenetic dysregulation, and oncogenic signalling. Building on these findings, our preliminary DMG data shows high NOX2 expression suggesting elevated ROS levels in DMG cells, accompanied by global DNA hypomethylation. We hypothesise that NOX2-derived ROS oxidise epigenetic regulators such as DNMT1 in DMG, driving hypomethylation of chromatin and oncogene activation at the promoter region. To test this, we have generated constitutive and inducible NOX2 knockout/knockdown (CRISPR/CRISPRi) models in PDGFRA-mutant and wild-type DMGs. We will quantify ROS, assess DNA damage, and perform redox proteomics to identify reversibly oxidised proteins and phosphoproteins. Global DNA methylation profiling using Illumina EPIC arrays will identify differentially methylated regions linking oxidative stress, DNMT1 oxidation, and epigenetic dysregulation. This study will define a novel redox-epigenetic mechanism and help identify therapeutic strategies to restore epigenetic silencing, enhance radiotherapy, and improve targeted therapy responses.