Epigenetic re-establishment of tumor suppression in glioblastoma - PROJECT SUMMARY This project focuses on a new epigenetic vulnerability of human glioblastoma (GBM), the most common and deadly adult primary brain malignancy. We discovered the bromodomain (BD)-containing chromatin regulator BRD8 as essential to GBM lacking p53 mutations (TP53WT), which make up ~71% of cases (Sun et al., Nature 2023) (1). BRD8 maintains malignancy by crippling p53-mediated tumor suppression in a way distinct from previously described mechanisms: it reprograms the p53 network through the EP400 histone acetyltransferase complex and by BD-directed occupancy of the histone variant H2AZ at p53-induced targets, enforcing a repressive chromatin state that prevents p53-mediated transactivation. Central to this application is our finding that targeting BRD8 in TP53WT GBM remodels chromatin by evicting H2AZ and enhancing chromatin accessibility, enabling p53 to bind and transactivate its targets. This chromatin remodeling cascade (referred to as the “BRD8/p53 epigenetic switch” in this proposal) re-establishes p53 activity in TP53WT GBM, normalizing gene expression, evoking cell cycle arrest, inhibiting gliomagenesis, and prolonging survival in xenograft models of TP53WT GBM (1). We advance this work by: (i) revealing that BRD8 opposes p53 function in non-malignant brain cells; (ii) setting up a single-cell gene expression pipeline; (iii) showing that the BRD8 BD is the only BD that selectively reprograms the p53 network; (iv) engineering a GBM-prone mouse model with intact p53 in which we can ablate Brd8; and (v) establishing TP53WT GBM organoids from patients. The above findings support the hypothesis that targeting BRD8 in TP53WT GBM evokes the BRD8/p53 epigenetic switch that re- establishes p53-mediated tumor suppression. Despite these advances, how the BRD8/p53 epigenetic switch works, and whether it will be useful for treating patients with TP53WT GBM, remain obscure. Therefore, the goal of Aim 1 is to define the epigenetic mechanism by which targeting BRD8 re-establishes p53-mediated tumor suppression in TP53WT GBM organoids. This will be accomplished by: (Aim 1.1) defining the set of genes transactivated by the BRD8/p53 epigenetic switch at single-cell resolution and characterizing the genomic and epigenomic features of these loci; (Aim 1.2) identifying pathways induced by the BRD8/p53 epigenetic switch that facilitate p53-mediated cell cycle arrest; and (Aim 1.3) elucidating the dynamics of the BRD8/p53 epigenetic switch and how it is modulated by EP400. The goal of Aim 2 is to demonstrate the preclinical relevance of targeting BRD8 to re-establish p53-mediated tumor suppression in vivo. This will be accomplished by: (Aim 2.1) assessing the effectiveness of targeting Brd8 in engineered GBM-prone mice; (Aim 2.2) establishing the effectiveness of targeting BRD8 in patient-derived xenograft (PdX) models of TP53WT GBM; and (Aim 2.3) evaluating the heterogeneity of p53-induced targets in single cells of GBM from spontaneous and PdX models and defining the cell populations impacted by the BRD8/p53 epigenetic switch.
