Induce and Image extrachromosomal DNA in human glioblastoma avatar

SUMMARY/ ABSTRACT Glioblastoma (GBM) is an isocitrate dehydrogenase wildtype cancer that accounts for 80% of all primary malignant brain tumors in adults with no effective treatment. One major factor contributing to therapeutic resistance is cell state heterogeneity. However, the molecular mechanisms behind this phenomenon are not well understood, posing a significant challenge to addressing therapeutic failure. One of the most common genetic lesions in GBM patients is the epidermal growth factor receptor (EGFR) gene amplification, typically in the form of extrachromosomal DNA (ecDNA). The circular, megabase-sized ecDNA contains oncogenes like EGFR and regulatory elements such as enhancers, leading to significant oncogene amplification and expression. ecDNA does not have centromeres and is believed to randomly segregate into daughter cells thereby conferring cancer cell heterogeneity, promoting cancer genome evolution, and mediating therapy resistance. Moreover, ecDNA can also facilitate genetic recombination, generating new regulatory circuitry and expressing variants, including clinically common variant EGFRvIII. For these reasons, ecDNA has been found inversely correlated with poor survival of GBM patients. Despite the exciting yet presumptive connection of ecDNA to GBM, fundamental questions of ecDNA in GBM pathobiology remain surprisingly unanswered. First, does EGFR ecDNA causally fuel GBM tumorigenesis? Second, how does centromereless ecDNA propagate to daughter cells during mitotic inheritance? Third, what are the molecular mechanisms by which ecDNA promotes GBM heterogeneity? These knowledge gaps are due to i) lack of a clinically relevant, genetically modifiable model system to recapitulate GBM pathobiology, ii) shortage of effective methods to de novo engineer ecDNA synthesis in non-transformed cells, and iii) paucity of unbiased ecDNA labeling and optimized imaging modality to longitudinally trace the dynamics of ecDNA during mitosis in living cells. This R21 exploratory application utilizes the principal investigator’s considerable expertise in CRISPR engineering, ecDNA imaging, stem cell biology, and single-cell genomics. We propose to ab initio engineer the induction of ecDNA from genetically normal human induced pluripotent stem cells (hiPSCs) and to orthotopically engraft hiPSC-derived neural progenitor cells to directly assess the role of ecDNA in GBM tumorigenesis. We will combine live-cell super-resolution imaging and single- cell sequencing to evaluate the role of EGFR ecDNA to GBM heterogeneity. Unlike cell lines or common mouse models that typically lack of tumor heterogeneity, our engineered hiPSC system provides a novel platform to examine the causality and mechanism of EGFR ecDNA in GBM transformation and heterogeneity. The live-cell super-resolution imaging and quantitative analysis framework will provide comprehensive tracing of ecDNA dynamics during mitotic inheritance and extraction of potentially new regulatory principles for genetic propagation. This genetically defined 'avatar' system offers a new way to model GBM etiology in the lab and is set to find new therapeutic targets to tackle GBM's heterogeneity for better treatments.