Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. In this study, we couple a CRISPR-based method for ecDNA induction with extensive characterization of newly formed ecDNAs to examine their biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with the formation of 800 kb, 1 Mb, and 1.8 Mb ecDNAs reaching or exceeding 15%. We show nonhomologous end joining and microhomology-mediated end joining both contribute to ecDNA formation, whereas inhibition of DNA-PK catalytic subunit and ATM have opposing impacts on ecDNA formation. ecDNA and the corresponding chromosomal excision scar can form at significantly different rates and respond differently to DNA-PK catalytic subunit and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double-strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.

Significance:

Our study harnesses a CRISPR-based method to examine ecDNA biogenesis, uncovering efficient circularization between double-strand breaks. ecDNAs and their corresponding chromosomal scars can form via nonhomologous end joining or microhomology-mediated end joining, but the ecDNA and scar formation processes are distinct. Based on our findings, we establish a mechanistic model of excisional ecDNA formation.

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©2024 The Authors; Published by the American Association for Cancer Research
2024
American Association for Cancer Research
This open access article is distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License .
Attribution-NonCommercial-NoDerivatives

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Supplementary data

Supplementary Data Set 1

sgRNA, primer, and probe sequences.

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Supplementary Figures 1-11

Supplementary Figure S1. PCR detects circle junctions for all MYC ecDNA guide pairs tested. Supplementary Figure S2. Hi-C contact frequency, sgRNA indel efficiency, and ecDNA circularization rates in Hap1 and K562 cells. Supplementary Figure S3. ecDNA and scar formation frequencies for initial MYC CRISPR-C guide screen. Supplementary Figure S4. Junction type and microhomology frequency in Hap1, K562, and FTE5 organoids. Supplementary Figure S5. Impact of different MH thresholds on MH frequency across loci and cell types and insertion length distribution at TCGA ecDNA junctions. Supplementary Figure S6. ATR inhibition has no impact on DHFR ecDNA or scar formation efficiency. Supplementary Figure S7. Impact of DNA-PKi and ATMi on ecDNA and scar formation efficiency and MH frequency at various loci. Supplementary Figure S8. Impact of DNA-PKi and ATMi on ecDNA and scar formation efficiency and MH frequency in K562 cells. Supplementary Figure S9. Impact of pharmacologic or genetic manipulation of DDR pathways on the most common circle and scar junction sequences for MYC ecDNA. Supplementary Figure S10. Impact of pharmacologic or genetic manipulation of DDR pathways on the most common circle and scar junction sequences for ecDNA generated from various loci. Supplementary Figure S11. A, Schematic of how the rate of DSB repair can promote or disfavor ecDNA formation. B, Schematic of two models for excisional ecDNA and scar formation.

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