Synthetic cell model reveals the coupling of ecDNA maintenance and genomic instability

Tumor cells commonly harbor highly amplified extrachromosomal DNA (ecDNA), which encompasses oncogenes along with large noncoding regulatory regions. However, it remains ambiguous whether ecDNA maintenance in cancer cells relies on specific intrinsic sequence elements and associated pathways. Based on the analysis of 6021 glioma cases from the Cancer Genome Atlas (TCGA), we found recurrent focal amplification at 7p11.2 centered on epidermal growth factor receptor ( EGFR ). Therefore, we developed a synthetic ecDNA (syn-ecDNA) model that contained the defined EGFR DNA sequences in ecDNA-negative (ecDNA–) cells. An approximately 15 kb fragment derived from the high-frequency EGFR amplicon was cloned into a circular synthetic vector. Notably, syn-ecDNA was stably maintained for at least 30 days and retained high syn-ecDNA copy number. RNA-seq analysis further revealed the activation of the FOS/activator protein 1 (AP-1) transcription factor, suggesting its involvement in the stable maintenance of syn-ecDNA. Consistent with the DNA damage response (DDR)-inducing properties of endogenous ecDNA, our syn-ecDNA models also induced phosphorylated H2AX (γH2AX) enrichment, engaged the ATM-CHK2 checkpoint, and elevated genomic instability. These findings are consistent with DNA synthesis and topoisomerase-linked lesions. In summary, we demonstrate that syn-ecDNA mimics key features of endogenous ecDNA and enables direct testing of how defined sequence elements influence episome maintenance and DNA damage signaling. • syn-ecDNA model recapitulates key endogenous ecDNA features in ecDNA– cells. • EGFR -based syn-ecDNA shows long-term maintenance and sustains high syn-ecDNA copy number. • Transcription factor FOS may play a crucial role in ecDNA maintenance. • syn-ecDNA activates ATM-CHK2 signaling and genomic instability linked to DNA lesions.