UC San Diego

ecDNA is an important driver of aggressive adult cancers, but its role in pediatric tumors is not yet well-understood. To survey the genomic landscape of ecDNA amplifications across pediatric cancers, we applied computational methods to detect ecDNA in the genomes of a cohort of 1670 pediatric tumors from 1481 patients. ecDNA was detected in at least 10% of pediatric ETMR, osteosarcoma, rhabdomyosarcoma, high-grade glioma, retinoblastoma, and medulloblastoma (MB) tumors. To assess the clinical importance of ecDNA in MB, the most common malignant pediatric brain tumor, we applied the same methods to a cohort of 468 MB patients. ecDNA was detected in 18% of MB tumors and carried a threefold greater risk of mortality within 5 years. Affected genomic loci harbor up to hundredfold amplification of oncogenes including MYC, MYCN, TERT, and other novel putative oncogenes. Between sequential patient biopsies at initial diagnosis and subsequent relapse, we observed structural variation at ecDNA loci and generation of new ecDNA sequences. To elucidate the functional regulatory landscapes of ecDNAs in MB, we generated transcriptional (RNA-seq), accessible chromatin (ATAC-seq), and chromatin interaction (Hi-C) profiles of 6 MB tumor samples. In each case, we identified regulatory interactions that cross fusion breakpoints on the ecDNA, representing potential “enhancer rewiring” events which may contribute to transcriptional activation of co-amplified oncogenes. To test this hypothesis, we conducted an in vitro CRISPRi screen targeting regulatory regions on the ecDNA of a MB cell line and identified distal enhancers which promote proliferation. Using single-cell sequencing, we have also developed methods to explore intratumoral heterogeneity of ecDNA in a p53-mutant SHH MB patient tumor and its corresponding PDX model. In summary, ourstudy analyzes the frequency, diversity, and functional relevance of ecDNA across MB subgroups and provides strong justification for continued mechanistic studies of ecDNA in MB with the potential to uncover new therapeutic approaches.