Single-cell multiome sequencing reveals distinct transcriptional programs associated with ecDNA amplifications in medulloblastoma

Extrachromosomal DNA (ecDNA) is a key driver of intratumoral heterogeneity and has been linked to therapeutic resistance and poor patient outcomes. However, technical limitations in identifying, isolating, and analyzing ecDNA at the single-cell level have hindered our understanding of its role in tumor development and treatment response. We have previously developed a single-cell multiome(RNA + ATAC) sequencing approach that enables the simultaneous analyses of ecDNA and its gene expression profiles. Initial application of this method in a medulloblastoma tumor of the sonic hedgehog subgroup revealed distinct transcriptional signatures in ecDNA-positive compared to ecDNA-negative and non-malignant cells. Building on these findings, we performed multiome single-cell sequencing in an additional cohort of medulloblastoma specimens harboring ecDNA amplifications. Initial analyses revealed distinct functional characteristics between ecDNA-positive and ecDNA-negative tumor cells. For example, ecDNA-positive tumor cells show up-regulation of DNA replication, recombination, and damage repair pathways, while ecDNA-negative cells demonstrated increased expression of synaptic activity, cell signaling, and morphogenesis pathways. Our ongoing analysis focuses on further defining the transcriptional programs that distinguish ecDNA-positive from ecDNA-negative tumor cells, ultimately informing future functional genetic and pharmacological studies targeting transcriptional dependencies in ecDNA-driven medulloblastoma tumors.