EPCO-54. DEFINING THE MULTI-SCALE ORGANIZATION AND REGULATION OF EGFR EXTRACHROMOSOMAL DNA IN GLIOBLASTOMA WITH MULTI-MODAL MERFISH

Abstract The standard of care for glioblastoma (GBM) has not changed since 2005, which is due, in part, to its hallmark heterogeneity. Oncogene amplifications are key mediators of GBM cellular states. Extrachromosomal DNA (ecDNA), large circular DNA elements harboring oncogenes, are a class of amplifications with oncogenic properties due to their structure. Without centromeres, ecDNA segregate randomly into daughter cells, promoting genetic heterogeneity, exceptionally high copy number, and aberrantly high transcription. ecDNA are present in over 50% of GBM tumors, more than any other cancer type, and the most prevalent genes found on ecDNA are well-characterized cellular state drivers like EGFR. ecDNA studies have traditionally relied on bulk sequencing or low-throughput, low-resolution imaging. However, given the intratumoral and possible intermolecular heterogeneity of ecDNA, mechanistic studies of ecDNA would ideally visualize its higher order chromatin structure, multi-scale organization, and RNA expression at a single-molecule resolution in a high-throughput manner. Several multi-modal Multiplex Error-Robust Fluorescence in-situ Hybridization (mm-MERFISH) strategies were leveraged to investigate the multi-scale organization of ecEGFR: from their 3D conformation to their subcellular localization to their distribution across the higher order tumor architecture. In an orthotopic mouse model, there were distinct differences in ecDNA copy number, expression, and localization. Several cell populations had decreased ecEGFR copy number and expression and were localized to the tumor rim. One population diffusively infiltrated cortical neurons, expressed a Proneural transcriptional program, contained low ecEGFR copy number, and expressed the lowest levels of EGFR in the entire tumor. This spatial organization was also observed in multi-region primary GBM samples. Testing whether the microenvironment can impact ecDNA phenotype, GBM39 cells grown with neurons had dramatic morphological changes and decreased ecDNA copy number. These results suggest that ecEGFR dosage and expression are spatially organized and that the unique properties of ecDNA contribute to transcriptional heterogeneity in GBM.