Abstract 2564: Tracing extrachromosomal DNA inheritance patterns in glioblastoma using CRISPR

Abstract

Abstract Glioblastoma (GBM) is the most lethal brain tumor; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of 10%. Recurrence after therapy for GBM is unavoidable. There are substantial differences among the cells of GBM tumors in the abundance and types of genetic material. This heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. A recent study has suggested that the majority of GBM activate oncogenes through amplification extrachromosomal DNA (ecDNA). Despite the speculation that ecDNA is a key factor of tumor heterogeneity, how ecDNA is propagated and distributed among—and how it behaves within—cancer cells is completely unknown. To address this gap in knowledge, this study focused on developing a novel cytogenetic tool that enables visualization and tracking ecDNA behavior in live GBM cells. We found breakpoint sequences resulting from genome rearrangements during ecDNA formation by performing computational analysis whole genome sequencing data from a pair of primary/recurrent GBM neurospheres. Each breakpoint represents a unique target sequence for ecDNA-specific labeling. Breakpoint were validated by breakpoint-PCR, focusing on breakpoints shared by the primary and recurrent neurosphere. We determined location and quantity of all ecDNA breakpoints through breakpoint-FISH (BP-FISH) analysis, which showed that all signal was found outside of the conventional chromosomes in the metaphase spread, validating the extrachromosomal nature of the ecDNA breakpoints. The breakpoint quantities, reflecting the number of ecDNA elements per cell, varied between primary and recurrent neurosphere suggesting that ecDNAs quantitatively changed during GBM evolution. These results emphasize the necessity of tracking ecDNA dynamics to answer the question of how ecDNA is distributed into the daughter cells. Our findings will be strong evidence to make ecDNA-specific CRISPR system in further research. Tracing ecDNA dynamics will provide new insight into the impact of ecDNA on cancer evolution. Citation Format: Eunhee Yi, Roel Verhaak. Tracing extrachromosomal DNA inheritance patterns in glioblastoma using CRISPR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2564.