P16.09.A TGFB1 INDUCED TET3 DEPENDENT REGULATION OF OTX2 SUPER ENHANCER HYPOMETHYLATION PROMOTES GROUP3 MEDULLOBLASTOMA PROGRESSION

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Abstract BACKGROUND DNA methylation patterns have been extensively utilized for medulloblastoma (MB) classification. However, the functional implications of enhancer methylation in MB remain poorly understood. METHODS We conducted bisulfite-based genome-wide sequencing on 189 human MB cases. Upon molecular classification, 80 cases was compared against 8 normal cerebellums, revealing differentially methylated regions (DMRs). Integration with gene expression data facilitated the identification of DMR target genes, subsequently validated in the 109 cases. Further, leveraging sn-ATACseq, we evaluated the association between DMRs in chromatin accessibility regions and tumorigenesis. Finally, we investigatedd the impact of upstream epigenetic events involving aberrant methylation on tumor development. RESULTS We observed a negative correlation between the methylation levels of DMR overlapping super enhancers (seDMRs) and the expression of targeted genes, particularly in Group_3 MB (G3-MB), the most aggressive subgroup. Genes targeted by hypomethylated seDMRs were significantly enriched in early-stage cerebellum development hallmarks, specifically the rhombic lip subventricular zone (RLSVZ), but not in the late stage. Hypomethylated seDMRs of OTX2 were associated with elevated chromatin accessibility and oncogenic activation of progenitor-like MB tumor cells, leading to worse prognosis. Integrative epigenetic analysis revealed highly active OTX2 binding sites within its own hypomethylated seDMRs, indicating feedback auto-regulation. Deletion of motif at this enhancer region reduced OTX2 expression and attenuated tumor progression. Further, we found that ten-eleven translocation 3 (TET3), a DNA methylation eraser, was recruited by OTX2 to catalyze active DNA demethylation at binding motifs, thereby organizing chromatin and stimulating gene expression. TET3 was upregulated by TGF-β1/Smad2 signaling. Based on these findings, nanoparticle-coated small interfering RNAs (siRNA) targeting TET3 were designed, effectively suppressing xenograft progression. CONCLUSION Our study highlights the increased transcriptional activity of OTX2 through cell-type-specific super enhancer demethylation, which is dependent on TGF-β1/Smad-induced TET3. This identifies a potential therapeutic strategy for G3-MB.

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