Functional Genomics of Chromatin Regulator Dependencies and Resistance Mechanisms in B-cell Lymphoma

Abstract

The polycomb repressive complexes 1 and 2 (PRC1 and 2) in mammals are multi-member protein complexes that catalyse chemical group additions to chromatin, functioning to maintain transcriptional repression and coordinate context-dependent gene programs. Germinal-centre lymphomas, the most common form of lymphoma, arise from B-cells that acquire recurrent mutations in chromatin regulatory genes during B-cell maturation. The catalytic subunit of Polycomb Repressive Complex 2 (PRC2), EZH2, is recurrently mutated in 25% of diffuse large B-cell lymphomas (DLBCL), causing increased H3K27me3 and decreased H3K27me2 levels, leading to hyper-repression of the gene programs required for normal B-cell terminal differentiation and exit from the germinal centre. EZH2 inhibitors provide clinical benefit, but resistance frequently develops, highlighting the need for alternative therapeutic targets. This project identifies multiple targetable chromatin regulatory genes as genetic sensitisers to EZH2 inhibition in B-cell lymphoma, including BRD9, EP300, DOT1L, EHMT2 and MEN1. The PRC2 accessory protein AEBP2 was identified as a specific genetic dependency and EZH2 inhibitor sensitiser in B-cell lymphoma. While AEBP2 acts through PRC2, its essential role is surprisingly independent of canonical H3K27me3-mediated gene silencing. Instead, AEBP2 functions within a PRC2.2 complex lacking JARID2, using its zinc-finger domains to sample intergenic chromatin to sustain H3K27me2. The H3K36 methyltransferases, NSD1 and NSD2, which antagonise Polycomb function, were identified as genetic resistors to EZH2 inhibitors. While NSD2 does not physically interact with PRC2, its loss blunts gene de-repression in response to EZH2 inhibitors, potentially through effects at distal regulatory elements. Notably, loss of AEBP2 or NSD2 caused contrasting changes in intergenic H3K27me2 levels, driving sensitivity or resistance to PRC2 inhibitors, respectively. The work presented here identifies AEBP2-PRC2.2-maintained intergenic H3K27me2 as a therapeutic vulnerability in EZH2-mutant DLBCL and highlight dysregulated H3K27me2 as an underappreciated form of PRC2 dysfunction in cancer, with important therapeutic implications.