Epigenetic maintenance of the Injury Response state in Glioblastoma Stem Cells

Abstract Background Glioblastoma (GBM) is a common and highly lethal type of primary brain tumor in adults. Therapeutic failure is partly attributed to a fraction of Glioblastoma Stem Cells (GSCs) that show high levels of heterogeneity and plasticity. GSCs exist in a transcriptional gradient between two states: Developmental (Dev) and Injury Response (IR) in which IR-GSCs exhibit more invasive behaviors. While previous studies have identified fitness genes in GSCs, the genes required to establish and maintain the Dev and IR states remain poorly defined. Methods To identify the regulators of the IR GSC state, we performed a phenotypic genome-wide CRISPR-Cas9 knockout screen in patient-derived GSCs based on cell surface expression of the IR marker CD44. Validation of EP300 in regulation of IR state was performed using CRISPR gene editing and A-458 inhibitor treatment. RNA-seq, CUT&RUN, invasion assays, sphere forming assays and a mouse GBM model were used to characterize the phenotypic consequences of EP300 perturbation. Results We found that perturbation of the histone acetyltransferase EP300 led to decreased CD44 cell surface expression, and loss IR transcriptional state identity through dysregulation of the epigenome. Functional studies demonstrated that this loss of state identity coincides with decreased self-renewal and invasion in GSCs and delayed tumor initiation and progression in a mouse GBM model. Conclusion Collectively, our results establish EP300 as a key regulator of IR state identity in GSCs and provide a mechanistic basis for therapeutic targeting of aggressive cellular states in GBM.