Extrachromosomal DNA Couples with ATM-mediated DNA Damage Response for Genome Instability in Tumors

Abstract

Extrachromosomal DNA (ecDNA) drives the evolution of cancer cells by conferring survival advantages over normal cells. However, the functional importance of ecDNA replication and maintenance is yet to be evaluated, and the molecular components that contributed to these processes remain largely unknown. Here, using CRISPR-C (circularization of genes and chromosomes by CRISPR) technology and synthetic circular DNA mimicking ecDNA identified in tumors, we generated ecDNA-positive (ecDNA+) cell models. Using these models, we identified the proteins involved in ecDNA replication and maintenance and found DNA damage response (DDR)-related pathways were significantly enriched. We further demonstrated that ecDNA replication led to increased DDR and genome instability and DDR was also essential for ecDNA maintenance, consistent with observations that ecDNA+ tumors exhibited higher DDR scores than ecDNA-negative (ecDNA–) tumors in patients. ATM, MDC1, and CHK2, but not ATR, were activated in ecDNA+ cells compared to ecDNA– cells. Moreover, ecDNA+ cells were more sensitive to ATM and CHK2 inhibition. We further found that type II topoisomerase TOP2B and DNA ligase III (LIG3) were critical regulators for ecDNA replication and maintenance and related DDR. In summary, our findings of the reciprocal interactions between ecDNA and DDR provide new insights into detection and therapy for ecDNA+ tumors.