A conserved enhancer locus in ecDNA and HSRs activates MYC transcription in group 3 medulloblastoma

MYC is amplified on extrachromosomal DNA (ecDNA) or homogenously staining regions (HSRs) in Group 3 medulloblastoma (G3-MB), conferring a poor prognosis, but the underlying mechanisms controlling MYC expression within ecDNA and HSRs are poorly understood. Using a structure-function approach, we identified and characterized a novel enhancer ( ecMYC E1 ) that drives MYC activation specifically in G3-MB with MYC -amplified ecDNA or HSRs. The ecMYC E1 locus exhibits enhancer hallmarks exclusively in MYC -amplified G3-MB but not in other MYC -dependent cancer cell lines, including those with MYC amplification. Silencing of the ecMYC E1 enhancer significantly reduced MYC transcription, which was compensated by increases in ecDNA copy number, but not in HSR-driven G3-MB tumor. NeuroD1 and BRD4 interact with each other and bind to ecMYC E1 , looping to the enhancer to the MYC promoter, and defining a novel mechanism that regulates amplified MYC gene expression within ecDNA or HSRs specifically in G3-MB.