Enhancer activation from transposable elements in extrachromosomal DNA.
Extrachromosomal DNA (ecDNA) drives oncogene amplification and intratumoral heterogeneity in aggressive cancers. While transposable element (TE) 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 (EIEs)-genomic loci enriched for TEs 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 tumor evolution. Our study uncovers a mechanism by which ecDNA harnesses repetitive elements to shape cancer phenotypes, with implications for diagnosis and therapy.