Katerina Kraft, Sedona E. Murphy, Matthew G. Jones, Quanming Shi, Aarohi Bhargava-Shah, Christy Luong, King L. Hung, Britney Jiayu He, Rui Li, Seung Kuk Park, Michael T. Montgomery, Natasha E. Weiser, Yanbo Wang, Jens Luebeck, Vineet Bafna, Jef D. Boeke, Paul S. Mischel, Alistair N. Boettiger, Howard Y. Chang
Extrachromosomal DNA (ecDNA) drives oncogene amplification and intratumoural heterogeneity in aggressive cancers. While transposable element reactivation is common in cancer, its role on ecDNA remains unexplored. Here we map the 3D architecture of MYC-amplified ecDNA in colorectal cancer cells and identify 68 ecDNA-interacting elements-genomic loci enriched for transposable elements that are frequently integrated onto ecDNA. We focus on an L1M4a1#LINE/L1 fragment co-amplified with MYC, which functions only in the ecDNA-amplified context. Using CRISPR-CATCH, CRISPR interference and reporter assays, we confirm its presence on ecDNA, enhancer activity and essentiality for cancer cell fitness. These findings reveal that repetitive elements can be reactivated and co-opted as functional rather than inactive sequences on ecDNA, potentially driving oncogene expression and tumour evolution. Our study uncovers a mechanism by which ecDNA harnesses repetitive elements to shape cancer phenotypes, with implications for diagnosis and therapy.