Mechanisms of action and vulnerabilities associated with the oncogene EZH2 in germinal centre B-cell non-Hodgkin lymphoma

Abstract

Polycomb group (PcG) proteins are highly conserved chromatin regulators that play a critical role as transcriptional repressors in mediating and maintaining cellular identity. PcG proteins assemble into large, distinct, multimeric complexes: Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2). PRC2 comprises core components EZH1/2, EED, SUZ12 and RBBP4/7, with additional substoichiometric components defining mutually exclusive subcomplex assemblies PRC2.1 and PRC2.2. PRC2 core components catalyse post-translational modification of histone H3 by the addition of up to three methyl groups on Lysine at position 27, while substoichiometric components provide varied contributions to PRC2 activity and localisation. EZH2 plays a central role in B-cell lymphopoiesis, though also functions as an oncogene in B-cell lymphoma, whereby recurrent change-of-function hotspot mutations in EZH2 result in increased levels of tri-methylated H3K27. Although enzymatic inhibitors of EZH2 are entering the clinic for the treatment of selected patients with B-cell lymphoma, their utility is limited by inevitable disease progression amongst responders after a short period of time, by as yet undescribed mechanisms. The aim of this PhD thesis was to gain new insight into the mechanism of action of the oncogene EZH2 in lymphoma and to identify novel vulnerabilities in lymphoma amongst other PRC2 components. Firstly, I describe the effects of the change-of-function EZH2 mutation on PRC2 localisation and activity in a lymphoma model and demonstrate that EZH2 asserts its oncogenic role via its methyltransferase activity. Secondly, using a PRC2-directed CRISPR tiling screen, I identify the PRC2.2 component AEBP2 as a specific genetic dependency in germinal centre B-cell lymphoma cell lines and highlight critical domains mediating this dependency. I show that targeting AEBP2 or core PRC2 components can overcome acquired resistance to EZH2 inhibition using a lymphoma cell line model. Finally, through knockdown of AEBP2 in lymphoma cells, I show that AEBP2 inhibits PRC2 methyltransferase activity in vivo, with AEBP2 depletion resulting in activation of PRC2 and further elevation of H3K27me3 in EZH2 mutant lymphoma cells and a disrupted balance of PRC2.1 and PRC2.2 components. Taken together, these data contribute an improved understanding of the role of the oncogene EZH2 in B-cell lymphoma biology and of AEBP2 in Polycomb biology and identify an intriguing novel potential target for treating patients with B-cell non-Hodgkin lymphoma.