KS02.5.A THE ONCOMETABOLITE D-2-HYDROXYGLUTARATE PROMOTES DNA HYPERMETHYLATION AT LINEAGE-SPECIFIC ENHANCERS CONTROLLING MICROGLIAL ACTIVATION IN IDH-MUTATED GLIOMAS
Authors: Laurenge A , Pugliese P , Mathon B , Richard Q , Jouannet S , Hayat Y , Scuderi S , Marijon P , Labreche K , Alentorn A , Verreault M , Idbaih A , Birzu C , Huillard E , Pottier N , Desmons A , Fayache I , Kaas G A , Kingsley P J , Marnett L J , Duplus E , El-Habr E , Salas L A , Mokhtari K , Tran S , Suvà M L , Iavarone A , Ceccarelli M , Touat M , Bielle F , Mallat M , Sanson M , Castro-Vega L 
Published: October 2024

Abstract

BACKGROUND
Resident microglia are highly plastic myeloid cells. Their phenotype and immune response are determined by the microenvironment composition. Although microglial cells are highly abundant in IDH-mutated (IDHm) gliomas, how this particular environment modulates the transcriptional programs and cell states of these cells remain largely uncharacterized. Herein we investigated whether and how D-2HG, the oncometabolite produced by the IDH mutation and released into the tumor microenvironment, impinges upon the phenotypic and functional properties of resident microglia.

MATERIAL AND METHODS
We analyzed bulk DNA methylome (Methylation EPIC array) and transcriptome of CD11b+magnetic-purified fractions from IDHm (n= 25) and IDH-wildtype (n=11) gliomas, as well from normal brain tissues (n=6). To determine direct effects of D-2-hydroxyglutarate (D-2HG), the oncometabolite produced by IDHm glioma cells, we optimized human primary microglial cultures obtained from glioma or drug-resistant epilepsy surgery. Microglia exposed to 5mM D-2HG or vehicle for 14 days were analyzed with DNA methylome (LC-MS/MS, EPIC arrays and single-base resolution approaches) and transcriptome (RNA-seq). Using snRNA-seq, we analyzed microglial transcriptome from a paired sample of one IDHm glioma patient before and after treatment with the IDH-inhibitor vorasidenib, which is known to deplete tumor D-2HG levels.

RESULTS
We uncovered that CD11b+ myeloid cells in human IDHm gliomas exhibit DNA hypermethylation predominantly at enhancer elements. Hypermethylation was linked to decreased expression of genes involved in inflammatory responses and glycolytic metabolism, as well as inactivation of transcription factors that regulate microglial responses to environmental stimuli. Prolonged exposure of human primary microglia to D-2HG inhibited TET-mediated 5mC oxidation, resulting in a reduced accumulation of global 5hmC levels. We observed high 5mC/5hmC ratios particularly at lineage-specific enhancers. Consistent with the remodeled enhancer landscape, D-2HG-treated microglia showed reduced proinflammatory capacity and enhanced oxidative phosphorylation. Conversely, depletion of D-2HG levels following vorasidenib treatment in an IDHm glioma patient was associated with overexpression of genes related to microglial activation.

CONCLUSION
Our findings provide a mechanistic rationale for the hyporesponsive state of microglia in IDHm gliomas and support the hypothesis that oncometabolites such as D-2HG may disrupt the function of immune cells residing in the tumor microenvironment.