EPCO-23. LONGITUDINAL TRACKING AND LONG-READ CHROMATIN FIBER SEQUENCING OF HUMAN ASTROCYTES WITH TELOMERE DYSFUNCTION REVEALED ALTERED CHROMATIN ACCESSIBILITY AND INCREASED STRUCTURAL VARIATION

Abstract Glioma is an aggressive cancer of the central nervous system that is genetically characterized by aneuploidy and frequent structural variation (SV). We sought to investigate the role of telomere dysfunction (TD) in shaping the SV landscape in glioma. Normal human astrocytes (NHA) were engineered to express viral E6E7 oncoproteins which are known to spontaneously trigger TD by suppressing p53/pRb. We hypothesized that NHA-E6E7 cells gradually accumulate SVs and aneuploidy over time because of progressive TD. We therefore longitudinally tracked bulk and single-cell clones of NHA-E6E7 cells for a period of over 6 months since viral infection. Overall, NHA-E6E7 cells demonstrate a growth benefit over primary NHA cells. We obtained Illumina short-read sequencing for NHA-E6E7 cells at multiple time points. Three time points were additionally selected for both Oxford Nanopore and PacBio HiFi long-read sequencing using the single-molecule chromatin fiber sequencing (fiber-seq) protocol. Copy number variation (CNV) analysis of short-read data showed deletion events in chromosome 13 as early as population doubling level (PDL) 48. K-mer based CNV analysis using the long-read data confirmed this finding and also detected deletion events in chromosome 7 at PDL 50. From the long-read data, we found an elevated number of SVs in NHA-E6E7 cells compared to the primary NHA cells. In particular, we detected telomere junctions and found that telomere-associated SVs were more prevalent in NHA-E6E7 cells compared to primary cells. Next, chromatin accessibility and regulatory elements were predicted from fiber-seq data with exogenous N6-methyladenosine (m6A) stencils. We observed large-scale chromatin actuation changes in various regulatory elements across the genome that intensified in later passages of NHA-E6E7 cells. For example, we found a decrease in the accessibility in FBXO7 which is a gene that maintains chromosome stability. Our preliminary results indicated that TD is an important mediator of structural variation in glioma.