EZH2 Gain-of-Function Mutations Generate a Lymphoma-Permissive Immune Niche

Abstract

The polycomb histone methyltransferase protein EZH2 is highly expressed and mutated in approximately 25% of the two most common subtypes of non-Hodgkin's lymphoma: follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL). Both of these tumor types originate from germinal center (GC) B-cells, which are highly proliferative cells that form transiently during the T-cell dependent humoral immune response. Wild type EZH2 plays a critical role in the formation of GC B-cells and mutant EZH2 drives malignant transformation of these cells in vivo. These lymphoma-associated mutations of EZH2 are gain-of-function and enhance EZH2 methyltransferase activity, which cause abnormally efficient H3K27me3, enhancing repression of transcription. Of note, EZH2 mutant lymphomas manifest aberrant suppression of antigen presentation pathways, such as loss or reduced expression of MHC-I and MHC-II, and reduced T-cell infiltrates. We show that mice engineered to conditionally express mutant Ezh2Y641F specifically in GC B-cells exhibited massive GC hyperplasia with expansion of the "light zone" -the GC niche where B-cells are selected based on the affinity of their antibodies- and extended follicular dendritic cells network. With chimeric bone marrow transplantation experiments we found that Ezh2Y641F provides a competitive advantage to activated B-cells in seeding and expanding the T-cell dependent GC reaction. Liquid chromatography separation and mass spectrometry of histone tryptic peptides revealed a massive increase in H3K27me3 in Ezh2Y641F GC B-cells, at the expense of a reduction in H3K27me2. Remarkably, we observed increased unmodified and decreased H3K27me1, indicating a loss-of-function mono-methylase activity of Ezh2Y641F. We confirmed an extensive global gain of H3K27me3 by ChIP-seq, with massive spreading of the mark both upstream and downstream TSS. RNA-seq analysis showed differential expression of genes involved in antigen processing and presentation and immune response, suggesting that B-cell selection process is altered in Ezh2Y641F GC light zone. Therefore, we hypothesized that, through an epigenetic mechanism, Ezh2 mutation makes B-cells less dependent of T-cells by aberrantly suppressing genes involved in the immune synapse. Indeed, inhibition of the interaction between TFH and GC B-cell in vivo using anti CD40L antibody, abrogated GC reaction in WT but not in Ezh2Y641F mice. Of note, EZH2 mutant GCs manifest a massive expansion of the follicular dendritic cell network in spite of lack of T-cell dependence, a finding that reflects low grade FL histology. Hence, EZH2 mutant lymphomas might progress due to acquired immune escape, which is a critical consideration when designing precision, tailored immune-therapy regimens. Disclosures Melnick: Constellation: Consultancy; Janssen: Research Funding; Epizyme: Consultancy.