Investigating Extrachromosomal DNA (ecDNA) Segregation and Repair in Cancer

Extrachromosomal DNA (ecDNA) plays a critical role in cancer biology, contributing to tumor progression and resistance to treatment by enabling rapid genetic adaptation through oncogene amplification. Unlike chromosomal DNA, ecDNA exists as circular molecules that replicate independently and segregate randomly during cell division, leading to significant genetic diversity within tumors. This unique non-Mendelian inheritance pattern allows cancer cells to quickly adapt to environmental pressures, including therapeutic interventions, by altering oncogene copy numbers. Recent studies have identified ecDNA across various cancer types, where it is associated with aggressive tumor growth, increased genetic diversity, and shorter patient survival. This collaborative project investigates the mechanisms underlying ecDNA segregation and repair, focusing on how their circular topology affects ecDNA function and maintenance. We propose that proper ecDNA segregation is facilitated by RNA-dependent physical tethering to mitotic chromosomes, thus protecting against cytosolic mis-segregation and chromosomal integration (Aim 1). Using innovative genetic tools and advanced imaging techniques, we will explore how ecDNA-encoded RNA contributes to its tethering and segregation during mitosis. We will also explore the pathways that promote ecDNA re-integration into the chromosome following cytosolic mis-segregation. In Aim 2, we uncover that ecDNA uniquely depends on the mutagenic pathway of microhomology-mediated end-joining (MMEJ) for its repair, distinguishing ecDNA from chromosomal DNA repair. We hypothesize that rampant ecDNA transcription, which leads to transcription- replication conflicts, generates DNA lesions that create a dependency on MMEJ activity for ecDNA maintenance. We will test the hypothesis that blocking MMEJ activity will prevent ecDNA accumulation and block drug resistance. Last, we will design ecDNA to incorporate a palindromic sequence, facilitating its linearization while safeguarding it from degradation (Aim 3). This will enable us to investigate the functional importance of ecDNA circular topology, which has been proposed to allow increased transcription of embedded oncogenes and test its impact on ecDNA segregation and repair processes. By revealing the mechanisms behind ecDNA segregation, maintenance, and integration, we aim to discover novel therapeutic strategies to combat drug resistance resulting from ecDNA amplification. Project Number: 1R01CA304441-01 | Fiscal Year: 2025 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Agnel Sfeir (+1 co-PI) | Institution: SLOAN-KETTERING INST CAN RESEARCH, NEW YORK, NY | Award Amount: $3,201,530 | Activity Code: R01 | Study Section: Cancer Genetics Study Section[CG] View on NIH RePORTER: https://reporter.nih.gov/project-details/11205915