Germinal center dysregulation by histone methyltransferase EZH2 promotes lymphomagenesis

Abstract

Protection against deadly pathogens requires the production of high-affinity antibodies by B cells, which are generated in germinal centers (GCs). Alteration of the GC developmental program is common in many B cell malignancies. Identification of regulators of the GC response is crucial to develop targeted therapies for GC B cell dysfunctions, including lymphomas. The histone H3 lysine 27 methyltransferase enhancer of zeste homolog 2 (EZH2) is highly expressed in GC B cells and is often constitutively activated in GC-derived non-Hodgkin lymphomas (NHLs). The function of EZH2 in GC B cells remains largely unknown. Herein, we show that Ezh2 inactivation in mouse GC B cells caused profound impairment of GC responses, memory B cell formation, and humoral immunity. EZH2 protected GC B cells against activation-induced cytidine deaminase (AID) mutagenesis, facilitated cell cycle progression, and silenced plasma cell determinant and tumor suppressor B-lymphocyte-induced maturation protein 1 (BLIMP1). EZH2 inhibition in NHL cells induced BLIMP1, which impaired tumor growth. In conclusion, EZH2 sustains AID function and prevents terminal differentiation of GC B cells, which allows antibody diversification and affinity maturation. Dysregulation of the GC reaction by constitutively active EZH2 facilitates lymphomagenesis and identifies EZH2 as a possible therapeutic target in NHL and other GC-derived B cell diseases.

Figures

Figure 1. EZH2 is upregulated in mouse GC B cells and required for GC formation.

(A) Ezh2 transcript levels in B cell subsets relative to Rplp0. Columns represent mean ± SD of triplicates. Experiments were performed on pools of B cells sorted from 3 mice. (B) FACS analysis of EZH2 protein levels in follicular (FO) and GC B cells. Numbers indicate mean expression. (C) Representative FACS analysis of splenic B cells in NP-CGG–immunized Ezh2 control (Ezh2fl/+:Cγ1-cre) and mutant (Ezh2fl/fl:Cγ1-cre) mice. Numbers indicate percentage of boxed GC B cells. (D) Frequency of splenic GC B cells in NP-CGG–immunized Ezh2 control (Ezh2fl/+:Cγ1-cre [Ezh2+/–]; n = 17) and mutant (Ezh2fl/fl:Cγ1-cre [Ezh2–/–]; n = 22) mice. Symbols represent individual mice; bars refer to mean values. *P = 0.013 (t test). (E) Representative histological analysis of PNA-positive (brown) GCs (arrowheads) in the spleens of NP-CGG–immunized Ezh2 control (Ezh2fl/+:Cγ1-cre; n = 6) and mutant (Ezh2fl/fl:Cγ1-cre; n = 6) mice. Scale bar: 200 μm. (F) Frequency of Ezh2 inactivation in GC B cells of Ezh2 control (Ezh2fl/+:Cγ1-cre; n = 3) and mutant (Ezh2fl/fl:Cγ1-cre; n = 4) animals, quantified by genomic qPCR. (G) Frequency of YFP+ GC B cells in R26-yfp;Ezh2 control (Ezh2fl/+) and mutant (Ezh2fl/fl) mice. (H) Summary of data on frequencies of YFP+ GC B cells in R26-yfp;Cγ1-cre;Ezh2 control (n = 4) and mutant (n = 6) mice. (F and H) Columns indicate mean ± SD. **P = 0.002 (t test). Data represent (C and D) 10, (G) 5, (F, and H) 3, and (B, E, and F) 2 experiments, respectively.

Figure 2. T cell–dependent antibody responses and memory B cells depend on EZH2 catalytic function in GC B cells.

(A) NP-specific IgG1 serum titers in Ezh2 control (Ezh2fl/+:Cγ1-cre [Ezh2+/–]; n = 6) and mutant (Ezh2fl/fl:Cγ1-cre [Ezh2–/–]; n = 7) mice after primary immunization with NP-CGG. Each symbol represents 1 mouse. (B) Frequency of NP-specific IgG1+ memory B cells (NIP+IgG1+PNAloIgκ–CD95–CD38+CD19+) in the spleens of Ezh2 control (Ezh2fl/+:Cγ1-cre; n = 5) and mutant (Ezh2fl/fl:Cγ1-cre; n = 7) mice 60 days after primary NP-CGG immunization measured by FACS. Each symbol represents 1 mouse; bars represent median values. *P = 0.009 (t test). (C) NP-specific high-affinity (NP4) IgG1 serum titers in Ezh2 control (Ezh2fl/+:Cγ1-cre; n = 6; black circles) and mutant (Ezh2fl/fl:Cγ1-cre; n = 6; gray circles) mice at the end of the primary response (day 60) and 1 week after NP-CGG boost (day 67). For the secondary response, 4 controls and 6 Ezh2 mutant mice were used, respectively. (D) FACS analysis of representative Ezh2 control (Ezh2fl/+:Cr2-cre [Ezh2+/–]) and mutant (Ezh2fl/fl:Cr2-cre [Ezh2–/–]) B cell cultures at day 3 of LPS+IL-4 stimulation. Numbers indicate frequency of boxed IgG1+ B cells. Data are representative of (A–C) 2 and (D) 5 experiments.

Figure 3. Ezh2 inactivation increases apoptosis of GC B cells.

(A) Representative FACS analysis of CaspGLOW staining of GC B cells in the spleens of Ezh2 control (Ezh2fl/+:Cγ1-cre [Ezh2+/–]) and mutant (Ezh2fl/fl:Cγ1-cre [Ezh2–/–]) mice after NP-CGG immunization. Numbers indicate percentage of CaspGLOW+ cells. (B) Mean frequency ± SD of CaspGLOW+ GC B cells in Ezh2 control (Ezh2fl/+:Cγ1-cre; n = 6) and mutant (Ezh2fl/fl:Cγ1-cre; n = 4) mice immunized with NP-CGG. **P = 0.002 (t test). (C) qRT-PCR analysis of Cdkn2a transcripts in purified GC B cells of Ezh2 control (Ezh2fl/+:Cγ1-cre; n = 12) and mutant (Ezh2fl/fl:Cγ1-cre; n = 12) mice after NP-CGG immunization. Values are relative to Rplp0 expression. *P = 0.033 (t test). (D) Frequency of splenic GC B cells in Ezh2 control (Ezh2fl/+:Cγ1-cre) and mutant (Ezh2fl/fl:Cγ1-cre) mice in the presence (Cdkn2a+/+; n = 5 or more) or absence (Cdkn2a–/–; n = 3 or more) of Cdkn2a, as measured by flow cytometry after NP-CGG immunization. *P = 0.018; **P = 0.0017 (t test). (E) Proportion of CaspGLOW+ splenic GC B cells revealed by FACS analysis in Cdkn2a-deficient Ezh2 control (Ezh2fl/+:Cγ1-cre; n = 7) and mutant (Ezh2fl/fl:Cγ1-cre; n = 5) mice after NP-CGG immunization. (F) Efficiency of Ezh2 inactivation in purified splenic GC B cells of NP-CGG–immunized Cdkn2a-deficient Ezh2 control (Ezh2fl/+:Cγ1-cre; n = 2) and mutant (Ezh2fl/fl:Cγ1-cre; n = 2) mice. Data are representative of (A, B, and F) 2, (D and E) 4, and (C) 10 experiments, respectively. (C) Columns indicate mean ± SEM. (D and E) Columns indicate mean ± SD.

Figure 4. AID induces apoptosis of Ezh2 mutant GC B cells.

(A) TUNEL+Ezh2 control (Ezh2fl/+:Cr2-cre [Ezh2+/–]; n = 3) and mutant (Ezh2fl/fl:Cr2-cre [Ezh2–/–]; n = 3) B cells 24 hours after IR treatment (mean frequency ± SD). (B) Proportion of Ezh2 control (Ezh2fl/+:Cr2-cre; n = 2) and mutant (Ezh2fl/fl:Cr2-cre; n = 2) B cells with 53BP1+ foci after exposure to IR (average frequency ± SEM). (C) Mean frequency ± SD of CaspGLOW+Ezh2 control (Ezh2fl/+:Cr2-cre; n = 5) and mutant (Ezh2fl/fl:Cr2-cre; n = 7) B cells 3 days after LPS or αRP105 stimulation. (D) Histological analysis of PNA+ GC (blue) within IgD+ B cell follicles (brown) in the spleen of Ezh2 control (Ezh2fl/fl:Cr2-cre) and mutant (Ezh2fl/fl:Cr2-cre) mice. Scale bar: 100 μm. (E) Spleen GC B cell numbers in immunized Ezh2 control (Ezh2fl/+:Cr2-cre) and mutant (Ezh2fl/fl:Cr2-cre) mice before (Ezh2+/–, n = 9; Ezh2–/–, n = 6) and after (Ezh2+/–, n = 8; Ezh2–/–, n = 13) Aicda inactivation. Symbols represent individual mice. (F) Representative FACS analysis of CaspGLOW+ GC B cells in Ezh2 mutant mice (Ezh2fl/fl:Cr2-cre) before and after AID inactivation. (G) Proportion of splenic CaspGLOW+ GC B cells in Ezh2 control (Ezh2fl/+:Cr2-cre) and mutant (Ezh2fl/fl:Cr2-cre) mice before (Ezh2+/–, n = 7; Ezh2–/–, n = 6) and after (Ezh2+/–, n = 11; Ezh2–/–, n = 13) Aicda inactivation (mean ± SD). (H) Efficiency of Ezh2 inactivation in Ezh2 mutant (Ezh2fl/fl:Cr2-cre) follicular and GC B cells before (n = 1) or after (n = 3) Aicda inactivation. Columns indicate mean values. Data represent (E–G) 6, (H) 5, (C) 3, and (A, B, and D) 2 experiments. *P < 0.05; **P < 0.01; ***P < 0.0001.

Figure 5. Genome-wide identification and characterization of H3K27me3 and H3K4me3 target genes in mouse GC B cells.

(A) Venn diagram showing sets of genes marked by H3K27me3 and/or H3K4me3, respectively, in primary GC B cells purified from the spleens of immunized C57BL/6J wild-type mice. (B) Box plot of average mRNA levels of genes marked by H3K27me3 (n = 4,549), H3K4me3 (n = 6,311), or both histone marks (n = 1,747) in GC B cells. ***P < 2.2 × 10–16 (t test). (C) Box plot analysis of transcript levels of candidate genes (relative to Rplp0) quantified in Ezh2 control (Ezh2fl/+:Cγ1-cre [Ezh2+/–]; n = 15) and mutant (Ezh2fl/fl:Cγ1-cre [Ezh2–/–]; n = 17) primary GC B cells. P values are indicated within each plot (Wilcoxon rank-sum test). (B and C) In box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate one and half time the value of the interquartile range added to the 75th percentile, or subtracted to the 25th percentile, respectively. (D) H3K27me3 status in wild-type GC B cells of 67 candidate genes (listed in Supplemental Table 2) tested by qRT-PCR in Ezh2 control and mutant GC B cells described in C. The genes were grouped based on whether expression was upregulated (up) or remained unchanged (no change) in Ezh2 mutant GC B cells. ***P = 0.0009 (Fisher’s exact test).

Figure 6. EZH2 regulates G1-to-S transition in B cells in a p16INK4A-independent fashion.

(A) Representative cell cycle analysis of day 4 Ezh2 control (Ezh2fl/+:Cγ1-cre [Ezh2+/–]; n = 3) and mutant (Ezh2fl/fl:Cγ1-cre [Ezh2–/–]; n = 4) iGB cells stained with propidium iodide (PI) and pulsed with EdU 1 hour prior to analysis. Numbers indicate percentage of cells in G0/G1 (bottom left), S (top), and G2/M (bottom right) phase of the cell cycle, respectively. (B) Summary of data on cell cycle distribution of Ezh2 control (Ezh2fl/+:Cγ1-cre; n = 3) and mutant (Ezh2fl/fl:Cγ1-cre; n = 4) iGB cells at day 4 of stimulation. (C) Transcript levels of cyclin-dependent kinase inhibitors in Ezh2 mutant (Ezh2fl/fl:Cγ1-cre) iGB cells relative to control (Ezh2fl/+:Cγ1-cre) and Rplp0. Expression of p19ARF encoded by Cdkn2a was also assessed. Columns represent mean ± SEM. (D) Summary data on cell cycle distribution of Ckn2a-deficient Ezh2 control (Ezh2fl/+:Cγ1-cre; n = 3) and mutant (Ezh2fl/fl:Cγ1-cre; n = 3) iGB cells at day 4 of stimulation. (B and D) Columns indicate mean ± SD. *P = 0.009; **P = 0.001 (t test).

Figure 7. EZH2 modulates BCL6 repression in GC B cells.

(A) Percentage of a representative set of BCL6 target genes (FDR ≤ 0.0001; Supplemental Table 3) that are significantly (P < 0.05) upregulated in Ezh2 mutant GC B cells, classified on the basis of H3K27me3 status in their wild-type counterparts. **P = 0.004 (Fisher’s exact test). (B) Box plot representation of transcript levels (relative to Rplp0) of a representative set of BCL6 target genes in Ezh2 control (Ezh2fl/+:Cγ1-cre [Ezh2+/–]; n = 11, white) and mutant (Ezh2fl/fl:Cγ1-cre [Ezh2–/–]) GC B cells, respectively. Ezh2 mutant GC B cells were compared before (n = 14, gray) and after (n = 3, blue) complementation with the IμHABCL6 transgene. Significance of the differences in gene expression levels is indicated within each plot (Wilcoxon rank-sum test). Horizontal bars indicate the medians, In box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate one and half time the value of the interquartile range added to the 75th percentile, or subtracted to the 25th percentile, respectively.

Figure 8. H3K27me3 marks Blimp1 and Irf4 in GC B cells and restricts PC differentiation.

(A) Blimp1 and Irf4 H3K27me3 ChIP-qPCR in primary GC B cells. Gapdh was assessed as negative control. Enrichment is relative to input and histone H3 density. Columns represent mean values. (B) Blimp1 and Irf4 H3K27me3 ChIP-qPCR in I.29μ+ B cells before (–) and 48 hours after LPS stimulation and in A20 cells in response to 24 hours of IL-21 stimulation. (C) Immunoblot analysis of H3K27me3 and full-length (Ezh2)/truncated (Ezh2SET) EZH2 levels in representative Ezh2 control (Ezh2fl/+:Cγ1-cre [Ezh2+/–]; n = 3) and mutant (Ezh2fl/fl:Cγ1-cre [Ezh2–/–]; n = 3) iGB cultures 4 days after CD40L/BAFF/IL-4 stimulation. Histone H3 was assessed as loading control. Quantification of H3K27me3 levels relative to controls is shown. (D) Blimp1 and Irf4 H3K27me3 ChIP-qPCR analysis in Ezh2 control (Ezh2fl/+:Cγ1-cre) and mutant ( Ezh2fl/fl:Cγ1-cre) iGB cells stimulated as in C. (E) qRT-PCR analysis of Irf4 and Blimp1 transcripts in Ezh2 control (Ezh2fl/+:Cγ1-cre; n = 3) and mutant (Ezh2–/–; Ezh2fl/fl:Cγ1-cre; n = 4) iGB cells stimulated as in C. Values are relative to Rplp0. Columns indicate mean ± SEM. *P = 0.006 (t test). (F) Representative FACS analysis and (G) frequency of Irf4hiCD138+ PCs in control (Ezh2fl/+:Cγ1-cre; n = 6) and mutant (Ezh2–/–; Ezh2fl/fl:Cγ1-cre; n = 6) iGB cultures 2 days after IL-21 stimulation. ***P = 0.0006 (t test). (H) IgG1 levels in the supernatant of Ezh2 control (Ezh2fl/+:Cγ1-cre; n = 3) and mutant (Ezh2–/–; Ezh2fl/fl:Cγ1-cre; n = 3) iGB cultures after IL-21 stimulation. (G and H) Symbols represent individual samples; bars refer to mean values. (B and D) Fold enrichments are calculated as in A.

Figure 9. Y641 mutant of EZH2 silences BLIMP1 expression through PRC2 to facilitate DLBCL growth.

(A) BLIMP1 transcripts in SU-DHL-4 and SU-DHL-6 DLBCL cells 12 days after treatment with GSK343 or vehicle, as revealed by qRT-PCR analysis. Columns indicate mean ± SD of triplicates. Expression is relative to that of vehicle-treated control cells after normalization for RPLP0 expression. (B) Growth curves of SU-DHL-4 (black line) and SU-DHL-6 (gray line) DLBCL cells treated with either GSK343 (solid line) or vehicle (dotted line). Average cell numbers at the indicated time points ± SEM of 4 replicates is shown. ***P < 0.001 (2-way ANOVA). (C) BLIMP1 transcript levels in SU-DHL-4 DLBCL cells after infection with lentiviruses expressing control (ctrl) or anti-EED (EED) shRNAs, respectively. Results are presented relative to control after normalization for GAPDH expression. Columns indicate mean of 2 independent experiments. (D) Frequency of SU-DHL-4 DLBCL cells expressing shRNA against EED relative to those expressing control shRNA at the indicated days after lentivirus infection. (E) qRT-PCR analysis of Blimp1 transcript levels in WSU-DLCL-2 DLBCL cells transfected with control or BLIMP1 expression vector, respectively. Columns indicate mean value of 2 independent experiments. Values are presented relative to control after normalization for HPRT expression. (F) Comparison of WSU-DLCL-2 cell numbers at the indicated days after transfection with control or Blimp1 expression vector, respectively. Data are representative of 2 experiments.