Abstract 3884: Uneven inheritance and functional heterogeneity derived from extrachromosomal DNA enable rapid adaptation of cancer cells

Abstract The amplification of oncogenes on extrachromosomal DNA (ecDNA) is common in cancer and associated with poor outcomes. Uneven inheritance of ecDNA during cell division leads to intratumoral ecDNA copy number heterogeneity. However, the relationship between shifts in ecDNA copy number and the rapid environmental adaptation of cancer cells remains unclear. In this study, we utilized imaging and single-cell multi-omics analyses to demonstrate that the transcript and protein expression of ecDNA oncogenes, along with functional heterogeneity, correlate with ecDNA copy number heterogeneity. Using fluorescence-activated cell sorting (FACS) and live-cell imaging, we found that cells with “middle” ecDNA copy numbers display the highest growth rates compared to cells with the lowest or highest copy number statuses, indicating greatest fitness is achieved by the balance of promoting cell proliferation while minimizing heightened replication stress arising from increasing copy numbers of ecDNA oncogenes. Through simulations based on random segregation and optimal copy number models, we found that ecDNA-bearing cells can rapidly adjust their copy numbers to achieve optimal growth in new environments. Notably, compared to chromosomally amplified cells, those with ecDNA exhibited superior growth following xenograft injection and treatment with single drugs targeting replication stress or oncogenes, aligning with our simulation findings. Our results reveal that functional heterogeneity resulting from uneven ecDNA inheritance enhances cancer cells' adaptability to tumor microenvironments and rapid acquisition of drug resistance, warranting that the development of combinatorial therapies that synergistically target a wide range of copy numbers is critical for effectively treating ecDNA-containing cancers. Citation Format: Shu Zhang, Aditi Gnanasekar, Ellis J. Curtis, Jun Tang, Vishnu Shankar, Ivy Tsz-Lo Wong, Xiaowei Yan, Rui Li, Lu Yang, Howard Y. Chang, Paul S. Mischel. Uneven inheritance and functional heterogeneity derived from extrachromosomal DNA enable rapid adaptation of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3884.