EZH2 and BCL6 Cooperate to Assemble CBX8-BCOR Complex to Repress Bivalent Promoters, Mediate Germinal Center Formation and Lymphomagenesis

Abstract

The EZH2 histone methyltransferase mediates the humoral immune response and drives lymphomagenesis through formation of bivalent chromatin domains at critical germinal center (GC) B cell promoters. Herein we show that the actions of EZH2 in driving GC formation and lymphoma precursor lesions require site-specific binding by the BCL6 transcriptional repressor and the presence of a non-canonical PRC1-BCOR-CBX8 complex. The chromodomain protein CBX8 is induced in GC B cells, binds to H3K27me3 at bivalent promoters, and is required for stable association of the complex and the resulting histone modifications. Moreover, oncogenic BCL6 and EZH2 cooperate to accelerate diffuse large B cell lymphoma (DLBCL) development and combinatorial targeting of these repressors results in enhanced anti-lymphoma activity in DLBCLs.

Figure 1. EZH2 is Required for BCL6 to Drive GC Hyperplasia

(A–E) Ezh2^fl/fl, Ezh2^fl/fl;Cγ1-cre, IµBcl6, and Ezh2^fl/fl;Cγ1-cre;IµBcl6 mice (n = 5 per group) were immunized with SRBC to induce germinal center (GC) formation and sacrificed 10 days later. (A) Flow cytometry plot of one representative mouse spleen per group. The gated area shows the percentage of GC B cells (GL7+FAS+) within live B cells (B220+DAPI−). (B) Average of GC B populations of each group of mice quantified by flow cytometry. Each dot represents the percentage of GC B cells within splenic live B cells of one mouse. (C) Splenic tissue was stained for peanut agglutinin (PNA), Ki67, EZH2, and B220. (D and E) Quantification of PNA staining from (C). The #GC/spleen section is the count of all GCs per spleen section (D). The GC area/total spleen area is the quantified area of each individual GC divided by the total area of the spleen section (E). (F–H) IµBcl6 mice were immunized with SRBC, treated daily with GSK503(150 mg/kg/day, n = 7) or vehicle (n = 7) and sacrificed 10 days after immunization. (F) Representative flow cytometry plot of splenic GC B cells (left) and quantification (right) as in (A) and (B). (G) Splenic tissue was stained for PNA, Ki67, EZH2, and B220. (H) Quantification of PNA staining from (G) as in (D) and (E). Values in (B), (D), (E), (F), and (H) are shown as means ± SEM. t test, *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S1.

Figure 2. BCL6 Is Required for Mutant EZH2^Y641 to Drive GC Hyperplasia

(A) Generation of Ezh2(Y641F)^fl conditional mice. A mini-gene comprising exons 16–20 flanked by loxP sites and the pGK-Neo cassette flanked by FRT sites were inserted into intron 15–16 of the Ezh2 allele. The Neo cassette was removed by crossing to FLP deleter strain. Prior to Cre expression, the WT product is generated from the mini-gene. CRE-mediated deletion of the mini-gene results in expression of the mutant form (represented with a red asterisk). (B–F) Ezh2(Y64lF)^fl/WT crossed with Cγ1-cre positive (n = 5) or negative control mice (n = 5) were immunized with SRBC and sacrificed 10 days later. (B and C) Percentages of splenic GC B cells were measured by flow cytometry (B) and quantified (C) as in Figures 1A and 1B. (D) Splenic tissue was stained for PNA. (E) Quantification of PNA staining from (D). (F) Immunoblotting with anti-EZH2 and H3K27me3 antibodies was performed in sorted GC B splenocytes, using βactin and histone 3 as loading controls. (G–K) Bcl6^fl/fl, Bcl6^fl/fl;Cγ1-cre, Ezh2(Y641F)^fl/WT;Cγ1-cre and Bcl6^fl/fl;Ezh2(Y641F)^fl/WT;Cγ1-cre mice (n = 3 to 5 per group) were immunized with SRBC and sacrificed 10 days later. (G and H) Representative flow cytometry plot of splenic GC cells (G) and quantification (H) as shown in Figures 1A and 1B. (I) Splenic tissue was stained for PNA, Ki67, EZH2, and B220. (J and K) Quantification of GC number (J) and area (K) based on PNA staining in (I). Values in (C), (E), (H), (J) and (K) are shown as means ± SEM. t test, *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S2.

Figure 3. EZH2 and BCL6/BCOR Complexes Are Both Required to Repress Key De Novo GC B Cell Bivalent Promoters

(A and B) Percentage and number of de novo bivalent promoters (H3K27me3+H3K4me3, n = 1,011) (A) and H3K27me3 monovalent promoters (n = 5,798) (B) overlapping ChIP-seq peaks of BCL6 and BCOR in GC B cells. (C) Heatmap of over-represented gene categories among genes with BCL6+BCOR-occupied de novo bivalent promoters compared with non-bivalent genes and de novo bivalent genes without BCL6 or BCOR. Enrichment was measured using hypergeometric p values. (D and E) CDKN1B (D) and PRDM1 (E) gene loci showing H3K4me3, H3K27me3, BCL6, and BCOR ChIP-seq read density in naive B cells (NB) and germinal center B cells (GCB). Green, red, blue, and black bars: H3K4me3, H3K27me3, BCL6, and BCOR peaks, respectively. (F) Heatmap of the gene expression level and GSEA of de novo bivalent genes with BCL6+BCOR in 4 NB and 4 GCB samples. NES, normalized enrichment score; FDR, false discovery rate. (G) Heatmap of over-represented de novo bivalent genes with and without BCL6- and BCOR-occupied promoters for genes induced by EZH2 or BCL6 inhibitors (2 µM GSK343 for 5 days and 25 µM FX1 for 12 hr) or shRNAs for EZH2 (7 days) or siRNAs for BCL6 (2 days) in OCI-Ly1, OCI-Ly7, SUDHL5, SUDHL6, Farage, WSU-DLCL2, and Pfeiffer DLBCL cell lines. Enrichment measured using hypergeometric p values. (H and I) Heatmap of the gene expression level (H) and RT-qPCR (I) of canonical and non-canonical PRC1 components in 4 NB and 4 GCB samples. Values in (I) are shown as means ± SEM. t test, **p < 0.01. (J) Immunoblotting of whole-cell lysates from NB and GCB samples. See also Figure S3 and Table S1.

Figure 4. Mutant EZH2 Fails to Induce GC Hyperplasia in the Absence of BCOR in a BCL6-Dependent Manner

(A–E) Bcor^fl/Y, Bcor^fl/Y;Cγ1-cre, Ezh2(Y641F)^fl/WT;Cγ1-cre, and Bcor^fl/Y;Ezh2(Y641F)^fl/WT;Cγ1-cre mice (n = 4 to 6 per group) were immunized with SRBC and sacrificed 10 days later. Note that Bcor is on the X chromosome; hence male mice have only one floxed Bcor allele and Y indicates the Y chromosome. (A) Representative flow cytometry plot of splenic GC B cells as in Figure 1A. (B) Quantification of GC B cells by flow cytometry as in Figure 1B. (C) Splenic tissue was stained for PNA, Ki67, EZH2, and B220. (D and E) Quantification of GC number (D) and area (E) based on PNA staining in (C). (F–J) Ezh2(Y641F)^fl/WT, Ezh2(Y641F)^fl/WT;Cγ1-cre, Bcl6^BTBmut;Cγ1-cre and Bcl6^BTBmut;Ezh2(Y641F)^fl/WT;Cγ1-cre mice (n = 4 per group) were immunized with SRBC and sacrificed 10 days later. (F) Representative flow cytometry plot of splenic GC B cells as in Figure 1A. (G) Quantification of GC B cells by flow cytometry as in Figure 1B. (H) Splenic tissue was stained for PNA, Ki67, EZH2, and B220. (I and J) Quantification of GC number (I) and area (J) based on PNA staining in (H). Values in (B), (D), (E), (G), (I), and (J) are shown as means ± SEM. t test, *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S4.

Figure 5. PRC1-BCOR Complex Requires Both PRC2 and BCL6 for Stable Association and Repression of Bivalent Promoters

(A) ChIP re-ChIP in the bivalent promoters of CDKN1B and PRDM1 in OCI-Ly7 cells using the indicated antibodies for the first ChIP (1° ab) and the sequential ChIP (2° ab). As negative control, qPCR was performed using primers for a region in chromosome 6 where no BCOR, EZH2, or BCL6 enrichment was found by ChIP-seq read density in GC B cells. (B and C) qChIP in CDKN1B promoter of SUDHL6 cells treated with (B) 2 µM EZH2 inhibitor GSK343 or control compound GSK669 for 72 hr, and (C) 25 µM BCL6 inhibitor FX1 or vehicle for 6 hr. NC-PRC1, non-canonical PRC1. Values are shown as means of triplicates or quadruplicates ± SD. t test, *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.

Figure 6. CBX8 Recruits BCOR Complex to H3K27me3 Marked Bivalent Genes and Is Required for the Biological Actions of EZH2

(A) Expression level in FPKM (fragments per kilobase of transcript per million mapped reads) of CBX genes in four naive B cells (NB) and four germinal center B cells (GCB) samples. Values are means ± SEM. (B) RT-qPCR of CBX8 in four NB and four GCB samples. Values are means ± SEM. (C) Immunoblotting of whole-cell lysates from NB and GCB cell samples. (D) Myc immunoblotting of Flag IPs using whole-cell extracts from Sf9 insect cells transfected with expression vectors for the indicated proteins, m, amino terminal myc epitope tag; f, flag tag; mf, both tags. (E) Immunoblotting of immunoprecipitation (IP) using whole-cell lysates from the indicated cell lines. (F) Immunoblotting of IP using whole-cell lysates from primary GCB from human tonsils. (G) CBX8 qChIP in OCI-Ly7 cells treated with 2 µM GSK343 or GSK669 for 72 hr. qPCR was performed using primers targeting the promoters of the indicated genes. (H) Immunoblotting of whole-cell lysates from OCI-Ly7 cells expressing two independent CBX8 shRNAs or control. (I) BCOR and H2AK119ub qChIP was done in cells from (H). Values in (G) and (I) are shown as means of triplicates or quadruplicates ±SD. t test, *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S6, Tables S2, and S3.

Figure 7. CBX8 Phenocopies the EZH2 Loss-of-Function Phenotype In Vitro and In Vivo

(A) RT-qPCR of the indicated mRNAs from OCI-Ly7 cells expressing two independent CBX8 shRNAs or control used in Figures 6H and 6I. p Values shown are compared with the shControl. (B) Viability of OCI-Ly7 cells from (A) was evaluated 7 days after infection using cell titer blue. (C) WSU-DLCL2 cells were infected with two independent CBX8 shRNAs or control for 5 days, and CD20, CD138, and immunoglobulin (Ig) expression levels were examined by flow cytometry. (D) Quantification of mean fluorescence intensity (MFI) from (C) (n = 3). (E) Representative images of WSU-DLCL2 cells infected as in (C). (F–J) Cbx8^fl/fl (n = 8) and Cbx8^fl/fl;Cγ1-cre mice (n = 4) were immunized with SRBC and sacrificed 10 days later. (F) Representative flow cytometry plot of splenic GC B cells as in Figure 1A. (G) Quantification of GC B cells by flow cytometry as in Figure 1B. (H) Splenic tissue was stained for PNA, Ki67, EZH2, and B220. (I and J) Quantification of GC number (I) and area (J) based on PNA staining in (H). Values in (A) and (B) are shown as means of triplicates or quadruplicates ± SD. Values in (D), (G), (I), and (J) are means ± SEM. t test, *p < 0.05, **p< 0.01, ***p < 0.001. See also Figure S7.

Figure 8. Mutant EZH2 and Constitutive BCL6 Cooperate to Induce Lymphomagenesis, and Combinatorial Targeting of EZH2 and BCL6 Yields Enhanced Anti-Lymphoma Effect

(A) Bone marrow transplantation was performed using IµBcl6, Ezh2(Y641F)^fl/WT;Cγ1-cre, Ezh2(Y641F)^fl/WT;Cγ1-cre;IµBcl6, and control negative littermate donor mice. BM, bone marrow. (B) Survival curve of transplanted mice. (C) Representative pictures of spleens from mice sacrificed 223 days after transplantation and quantification of the spleen weight (control, n=4; Ezh2(Y641F)^fl/WT;Cγ1-cre, n = 4; IµBcl6, n = 5; Ezh2(Y641F)^fl/WT;Cγ1-cre;IµBcl6, n = 12). (D) Representative pictures of submandibular lymph nodes from mice sacrificed in (C). (E) Percentage and number of mice from cohort used in (C) that did or did not develop lymphoma. (F) Dose reduction plot for GSK343 at 90% growth inhibition after exposure of cells to increasing concentrations of GSK343 for 6 days and FX1 for 2 days. Data represent means of triplicate experiments. (G) RT-qPCR of the indicated mRNAs from OCI-Ly7 treated with 2 µM GSK343 for 72 hr, 25 µM FX1 for 12 hr, or the combination. Values are means of triplicates ±SD. (H and I) Tumor growth curves and area under the curve (AUC) for SUDHL6 (H) and WSU-DLCL2 (I) xenografted mice treated with vehicle (SUDHL6, n = 9; WSU-DLCL2, n = 11), GSK126 (80 mg/kg/day, SUDHL6, n = 5; WSU-DLCL2, n = 10), FX1 (12 mg/kg/day, SUDHL6, n = 4; WSU-DLCL2, n = 10), or the combination of GSK126 and FX1 (SUDHL6, n = 6; WSU-DLCL2, n = 12). Values in (C), (H), and (I) are means ± SEM. t test, *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S8.