P09.02.A DISRUPTION OF DNA METHYLATION UNDERPINS THE NEUROINFLAMMATION INDUCED BY TARGETED CNS RADIOTHERAPY

Abstract BACKGROUND Targeted radiotherapy (RT) is integral to the increasing survival of cancer patients, but has significant side-effects, the underlying cellular and molecular mechanisms of which are ill-defined. It is acknowledged that RT induces epigenetic changes in neoplastic tissue, which impacts tumour evolution, however whether epigenetic deregulation also occurs in surrounding non-neoplastic tissue and contributes to the occurrence of RT side-effects is unknown. MATERIAL AND METHODS We retrospectively identified 14 samples that had undergone stereotactic-RT followed by resection and contained peri-lesional brain tissue, as well as 12 controls. Peri-lesional brain was characterised with DNA methylation profiling, RNAseq and spatial transcriptomics (ST). We explored cerebral organoids (CO) as a model of the response of neurons to irradiation, using DNA methylation profiling, immunofluorescence and single-cell RNAseq. RESULTS We show that DNA methylation changes, which are enriched at promoters, exons and 5’UTRs, impact gene expression in irradiated peri-lesional brain tissue. Using ST allows characterisation of micro-environmental niches in irradiated brain and we find that glial niches show inflammation, vascular and extracellular matrix changes, whilst neurons secrete pro-inflammatory neuropeptides, including tachykinins and opioids. When we examine DNA methylation disruption and expression in an irradiated CO model, we observe changes similar to patient tissue, and there is dysregulation of the methylation machinery, with DNMTs and TETs affected. Additionally, at early timepoints we see that neurons, comparable to those seen in patient tissue, show an inflammatory phenotype in response to DNA damage, which is already established at 24hrs post-irradiation. CONCLUSION We have characterised the DNA methylome in a unique cohort of irradiated peri-lesional brain tissue samples and integrated it with gene expression analysis at the spatial level. We show differences in DNA methylation patterns in irradiated brain tissue and identify specific inflammatory micro-environmental niches and their regulatory neuropeptides after irradiation. Finally, we show in a cerebral organoid model, that the same neuropeptides are upregulated as well as similar DNA methylation alterations and disruption of the DNA methylation machinery, in keeping with the interpretation that epigenetic dysregulation plays a role in neurotoxicity, hence raising the possibility it could represent a novel target for the reduction of radiotherapy side-effects.