Targeting aberrant DNA methylation across subtypes of pediatric high-grade glioma inhibits tumor growth and RUNX3 represents a targetable methylated tumor suppressor.

Aberrant DNA methylation is one of the most prevalent epigenetic alterations in cancer, and reversing DNA methylation holds promise as a therapeutic strategy. However, the function and underlying mechanisms of DNA methylation in pediatric high-grade glioma (pHGG) remain poorly understood. Here, we demonstrate that aberrant DNA methylation patterns are present in pHGG. Additionally, the overall level of DNA methylation is higher in H3/IDH wild-type pHGGs than in the H3K27-altered or H3G34R-mutant subtypes. Importantly, 5-Azacytidine, an inhibitor of DNA methyltransferases (DNMTs), significantly reduced the self-renewal capacity of pHGG cells in vitro and suppressed tumor growth in vivo by reversing DNA methylation. Furthermore, RUNX3, a putative tumor suppressor that is silenced in H3/IDH wild-type and H3K27-altered subtypes, was markedly reactivated by pharmacological or genetic induction of promoter demethylation, leading to reduced tumor growth of pHGG cells in vitro and in vivo. Notably, the antitumoral effects of combination of RUNX3 and temozolomide (TMZ) is superior to TMZ treatment alone in pHGG-derived xenografts. Collectively, our findings suggest that targeting aberrant DNA methylation may attenuate cancer stemness and inhibit tumor growth, which is associated with RUNX3 re-expression. These findings provide mechanistic insights that may inform future therapeutic development.