3104 - Mapping Single-Cell ecDNA Dynamics in Glioblastoma with CRISPR Lineage Tracing

Purpose/Objective(s):

Extrachromosomal DNA (ecDNA) is a feature of aggressive cancers and is found in over half of glioblastoma (GBM) patients. ecDNA has many pro-oncogenic functions, such as oncogene amplification, rapid copy number changes, and genomic regulation of mobile enhancers and transcription hubs. Interestingly, ecDNA can be “lost” via chromosomal reintegration upon targeted treatment but re-emerge when treatment is stopped. This highly dynamic behavior coupled with its pro-oncogenic roles makes ecDNA an intriguing therapeutic target in cancers like GBM.

While recent efforts have characterized inheritance patterns of ecDNA in bulk cell populations, no tracing of individual ecDNA molecules has been achieved. To reveal single-molecule resolution of ecDNA dynamics through treatment, we utilize a CRISPR lineage tracing (LT) system on native ecDNA molecules. Our approach labels specific ecDNA molecules with a unique, evolving, and heritable identifier. This is the first application of LT to ecDNA and provides novel insight into ecDNA dynamics in GBM in response to therapy.

Materials/Methods:

We utilized GBM39-EC cells with well-characterized ecDNA species containing the EGFRvIII mutation. Homology-directed repair (HDR) was used to integrate the LT construct, which contains a selectable marker (eGFP), a static identifier, and an evolving barcode of 8 Cas9 targets, into native ecDNA. We also introduced a Cas9 adenine base editor to edit the evolving barcode. After HDR, GBM39-EC cells were sorted for eGFP, and correct integration was confirmed via PCR. Clonal lines were derived and LT loci were characterized via DNA sequencing, whole genome sequencing (WGS), and single-cell RNA sequencing (scRNAseq). Engineered GBM39-EC cells were then subject to erlotinib treatment or radiation. During and post-treatment, scRNAseq was employed to track ecDNA inheritance dynamics, and WGS was employed to track ecDNA reintegration and re-excision.

Results:

GBM39-EC cells were successfully engineered with multiple ecDNA LT loci per cell. This allowed for the tracking of ecDNA dynamics in response to targeted (erlotinib) or non-specific treatment (radiation) regimens. This revealed ecDNA-specific patterns of reintegration and re-excision upon targeted treatment. These experiments also uncovered ecDNA responses to radiation that have not been previously reported.

Conclusion:

Our system provides the first instance of high-resolution lineage tracing in ecDNA, tracking single ecDNA molecules through treatment. Further exploration into ecDNA responses to targeted therapy and radiation is needed to characterize how ecDNA contributes to treatment resistance. This is important in cancers like GBM where ecDNA is prevalent, and despite intense therapies, recurrence is common. We hope tools like ours will advance the understanding of ecDNA’s role in GBM, hopefully leading to better patient outcomes.