Investigating the Role of H3.3K27M during Cellular Transformation and Differentiation.

Abstract

Diffuse Intrinsic Pontine Glioma (DIPG) is the most fatal brain tumour in childhood and current therapeutic interventions are failing. There is an urgent need for better understanding of DIPG pathogenesis and novel therapeutic approaches. Discovery of histone 3 variant mutations in DIPG has enabled the development of more faithful disease models and highlighted a role for histone epigenetics in gliomagenesis. The H3.3K27M mutant occurs in 65% of DIPGs and is the focus of this thesis. We have previously demonstrated that H3.3K27M transforms immortalized normal human astrocytes (iNHAs). We expand on that initial finding by testing the ability of these cells to form colonies at high confluency (HC). We find that H3.3K27M iNHAs execute a cell-autonomous program promoting cellular invasiveness, EMT and ECM reorganization through transcriptional dysregulation at HC. Importantly, the transcriptome of H3.3K27M iNHAs significantly overlaps with the astrocytic cell program recently described in DIPG. We identify the cell surface protein FPR3 as a putative H3.3K27M target likely upregulated to support EMT. We speculate that downregulation of cell-cell and cell-matrix adhesion proteins by H3.3K27M is central to colony formation and demonstrate that protocadherin 7 (PCDH7) is reduced in our model as in human glioblastoma. This study lays the necessary groundwork for further exploration of these novel putative H3.3K27M targets. Since our lab and others have determined that H3.3K27M alone is not a potent driver of transformation, we investigated its impact on cell state. We utilized the thoroughly characterized C2C12 myoblast differentiation model. The mechanism underlying H3.3K27M-induced transcriptional activation centres on dysregulation of the PRC2 complex and loss of the repressive H3K27me3 mark. Consistent with this, our investigation of the impact of H3.3K27M in differentiating myoblasts revealed massive H3K27me3 loss throughout differentiation and precocious differentiation through activation of the master muscle regulator factors (MRFs) MyoD and myogenin followed by transient cell cycle exit. Importantly, differentiating H3.3K27M myoblasts did not completely downregulate cell cycle factors cyclin B1 and cyclin D1 likely stalling complete terminal differentiate. This work has important implications for our understanding of how cellular decision-making is impacted by H3.3K27M and hints that mutant expression may disrupt proper cell cycle regulation.Ph.D

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