STEM-26. COMPREHENSIVE FUNCTIONAL GENOMIC SCREENS REVEAL HUMAN TRANSCRIPTION FACTORS REQUIRED TO MAINTAIN GLIOBLASTOMA OLIGODENDROCYTE PROGENITOR CELL-LIKE IDENTITY

Abstract For glioblastoma (GBM), like other solid tumors, intratumoral heterogeneity is likely a critical factor in promoting tumor growth, homeostasis, and therapeutic response. Recent applications of single-cell genomics techniques have revealed the scope and nature of GBM cell diversity. However, these techniques do not allow for the experimental manipulation of these same tumor subpopulations, raising a critical barrier to understanding tumor biology and developing effective therapies. To overcome this barrier, we developed endogenous gene reporter systems to enable the isolation, characterization, and manipulation of key GBM subpopulations directly in patient tumor isolates. This approach utilizes streamlined techniques for gene editing in GBM stem-like cells (GSCs), which eliminate the need for the cloning of individual cells. As proof of concept, we applied this approach to two genes in hGSC isolates: OLIG1 (GBM-OPC marker) and SOX9 (GSC/NSC stem cell homeostasis). We targeted the 3’ exon of each gene so that EGFP or RFP are in-frame with the upstream exon but separated by a 2A ribosome skip sequence. We then employed these GSC reporter isolates to comprehensively screen human transcription factors using a CRISPR-Cas9 library and LV-ORF library (1836 genes and 3548 isoforms) to identify transcription factors that activate or repress OLIG1 and SOX9. From these screens we identified 15 genes necessary and sufficient for regulating OLIG1 promoter activity, including known and novel regulators. These included SOX9 itself, which we find is a key factor linking OPC identify with self-renewal of GSCs. In addition, we identified dozens of transcription factor isoforms (validated by long read RNA-seq) that impact reporter activity, as well as ectopic factors not normally expressed in our GSCs which, nonetheless, significantly alter OLIG1 and SOX9 expression. At this meeting, we report results of these screens and present a functional genetic map of factors affecting the GBM-OPC cell identity, self-renewal, and plasticity.