EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation

Abstract

The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B cells and targeted by somatic mutations in B cell lymphomas. Here, we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GC B cell (GCB)-type diffuse large B cell lymphomas (DLBCLs) are mostly addicted to EZH2 but not the more differentiated activated B cell (ABC)-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

Figure 1. EZH2 Is Required for Germinal Center Formation

(A-C) EZH2^fl/fl, EZH2^−/− and WT C57BL6 control mice (n=5 per group) were immunized with SRBC to induce GC formation. (D-F) C57BL6 mice were immunized with SRBC and treated with GSK503 (150 mg/kg/day) or vehicle (n=5 per group). (A and D) Representative flow cytometric plot of splenic GC cells and quantification. (B and E) Splenic tissue was stained with PNA, EZH2, Ki67 and B220. (C and F) Quantitative imaging of PNA staining from (B) and (E) respectively. Values in A, C, D and F are shown as mean±SEM. t test ***p<0.001, **p<0.01, *p<0.05. See also Figure S1 and Tables S1-S4.

Figure 2. Mutant EZH2 Induces Germinal Center Hyperplasia

EZH2^fl/fl and EZH2^Y641N mice were immunized with NP-KLH and treated with GSK503 (150 mg/kg/day) or vehicle (n=6 per group). (A) Representative flow cytometric plot of splenic GC cells and quantification. (B) Splenic tissue was stained with PNA, EZH2, Ki67 and B220. (C) Quantitative imaging of PNA from (B). (D) Immunoblotting from whole cell lysates from sorted splenic GC B cells (GL7⁺/FAS⁺/B220⁺) Values in A, C and D are shown as mean±SEM. t test ***p<0.001, **p<0.01. See also Figure S2.

Figure 3. Mutant EZH2 Enhances Proliferation Effects Through Repression of CDKN1A

(A) Immunoblotting from nuclear extracts from GFP+ BCL1 cells expressing GFP-FLAG-tagged EZH2^Y641F, EZH2^Y641N, or WT EZH2. (B) Mass spectrometry measurement of H3K27me3 mark. (C) Viability using trypan blue exclusion. (D and F) Colony counts and representative pictures. (E) Immunoblotting was performed as in (A) with cells treated as indicated for 3 days. (G) Immunoblotting from whole cell lysates from YFP+ DLBCL cells expressing 2 independent EZH2 shRNA or control (H) Viability of DLBCL YFP+ by flow cytometry using annexinV and DAPI exclusion. Green, WT EZH2; blue, mutant EZH2 cell lines. (I) RT-qPCR of CDKN1A in DLBCL cell lines treated with 2μM GSK for 7 days. Green and blue code as in (H). (J) Pfeiffer cells were co-transduced with the indicated shRNAs and viable YFP+ cells counted as in (H). (K) Pfeiffer cells expressing 2 independent shCDKN1A or control were treated with 2μM GSK for 6 days and viability was measured as in (H). Values in B, C, D, F, H, I, J and K are mean of duplicate or triplicate ±SD. t test ***p<0.001, **p<0.01, *p<0.05. EV, empty vector; WT, WT EZH2. See also Figure S3.

Figure 4. EZH2 Mediates Differentiation Blockade

(A) GSEA in WT vs. mutant EZH2 BCL1 transduced cells. (B) Cells were treated with 20ng/mL IL2 and IL5 for 5 days and viability measured using trypan blue exclusion. (C) RT-qPCR of Prdm1. (D) Cells were treated with 2μM GSK343 or control GSK669 for 7 days and Ig expression levels were examined by flow cytometry. (E) Representative pictures of cells treated as in (D). (F) RT-qPCR of indicated mRNAs in cells treated with 2μM GSK for 7 days. Green, WT EZH2; blue, mutant EZH2 cell lines. (G) Heat map of over-represented gene categories among genes up-regulated by EZH2 shRNA or 2μM GSK343 for 7 days in Ly7, Ly1, SUDHL5, Farage, WSU-DLCL2 and Pfeiffer cells. Enrichment measured using hypergeometric p-values. (H) GSEA in GCB-DLBCL patient samples with WT vs. mutant EZH2. Values in B, C, D and F are mean of duplicate or triplicate ±SD. t test ***p<0.001, **p<0.01, *p<0.05. See also Figure S4 and Tables S5-S6.

Figure 5. Mutant EZH2 Aberrantly Represses Genes by Increasing H3K27me3 at Promoters and EZH2 Generates GC B-cell Specific Bivalent Genes Involved in Differentiation

(A) ChIP-seq density plot. (B) Heat map of change in expression of genes with increased H3K27me3 within promoters in BCL1 cells treated with 0.5μM GSK for 3 days. (C and G) GSEA in WT vs. mutant EZH2 BCL1 cells. (D) State of GCB cell bivalent promoters within naïve B cells (NBC). (E) Density strip representations of normalized EZH2 ChIP-seq reads within DLBCL cell line promoters. (F) Gene expression level of bivalent, H3K4me3 monovalent, H3K27me3 monovalent, and promoters not exhibiting either of these marks in GCB cells. (H) GSEA in GCB-DLBCL patient samples with WT vs. mutant EZH2. (I) Heat map of relative expression of GCB cell bivalent genes repressed in WT and mutant EZH2 GCB-DLBCL patient samples. See also Figure S5.

Figure 6. EZH2 Cooperates With BCL2 To Generate Germinal Center Derived Lymphomas

(A) Bone marrow transplantation was performed using VavP-Bcl2 transgenic donor mice. (B) Representative pictures of spleens from mice sacrificed 111 days after transplantation and quantification of the spleen weight (n=10 per group). (C) Immunoblotting from whole cell lysates from splenocytes of transplanted mice. (D) Quantification of the liver weight of transplanted mice (n=10 per group). (E) Splenic tissue from transplanted mice was stained with H&E, B220 and EZH2. (F) Tumor clonality analysis by RT-PCR performed in B220+ sorted splenocytes. Sample lanes separated by thin white lines were run on the same gel but were noncontiguous. (G) Dose reduction plot for Obatoclax and ABT737 at GI₉₀ after exposure of cells to increasing concentrations of GSK343 for 6 days. Data represent mean of triplicate experiments. (H) Area under the curve (AUC) of the tumor growth curves for 20 days in SUDHL4 and SUDHL6 xenografted mice treated with vehicle (n=7), Obatoclax (2 mg/kg/d, n=8 in SUDHL4 and n=7 in SUDHL6), GSK503 (150 mg/kg/d, n=8 in SUDHL4 and n=7 in SUDHL6), or the combination of Obatoclax and GSK503 (n=8 in SUDHL4 and n=7 in SUDHL6). P values were calculated by t test. Values in B, C, D and H are mean±SEM. t test ***p<0.001, **p<0.01, *p<0.05. BM, bone marrow. See also Figure S6.

Figure 7. EZH2 Targeted Therapy Preferentially Affects GCB But Not ABC DLBCL Cells

(A) Heat map of relative expression of GCB-specific H3K27me3 target genes repressed in mutant EZH2 GCB-DLBCL patient samples. (B-C) GSEA in ABC-DLBCL vs. WT EZH2 GCB-DLBCL (B) and mutant EZH2 GCB-DLBCL (C). (D-E) 4 ABC-DLBCL (HBL-1, Ly3, U2932, TMD8) and 9 GCB-DLBCL cell lines (5 WT EZH2: Ly7, Ly19, Farage, Ly18, SUDHL5, and 4 mutant EZH2: Pfeiffer, WSU-DLCL2, SUDHL4, SUDHL6) were exposed to increasing concentrations of GSK343 and GSK669 for 6 days. (D) Concentration of GSK343 required to inhibit 50% of growth (GI₅₀) relative to GSK669. (E) Cell viability at 10μM GSK. Data are mean with 95% confidence interval for duplicate. (F) When naïve B-cells are activated EZH2 expression is highly induced. EZH2 is required for germinal center (GC) formation and Ig affinity maturation. EZH2 levels decrease as B cells exit the GC reaction, enabling expression of genes that mediate terminal differentiation (e.g. IRF4, PRDM1, NFkB). B-cells in this “exiting” phase such as plasmablasts are believed to give rise to ABC-DLBCLs. However, the occurrence of EZH2 somatic mutations aberrantly sustains repression of proliferation checkpoint and differentiation genes, resulting in GC hyperplasia, and the presence of other oncogenic hits, such as BCL2, enables transformation to GCB-type DLBCL or follicular lymphoma (FL). A possible alternative route leading to GCB-DLBCL could involve overexpression or aberrant maintenance of WT EZH2 expression. GCB-DLBCLs and FLs but not ABC-DLBCLs require EZH2 to maintain their proliferation and survival. Thus, EZH2 methyltransferase inhibitors suppress GCB-DLBCLs but not ABC-DLBCLs. See also Figure S7.