Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation

Wei Qi; HoMan Chan; Lin Teng; Ling Li; Shannon Chuai; Ruipeng Zhang; Jue Zeng; Min Li; Hong Fan; Ying Lin; Justin Gu; Ophelia Ardayfio; Ji-Hu Zhang; Xiaoxia Yan; Jialuo Fang; Yuan Mi; Man Zhang; Tao Zhou; Grace Feng; Zijun Chen; Guobin Li; Teddy Yang; Kehao Zhao; Xianghui Liu; Zhengtian Yu; Chris X. Lu; Peter Atadja; En Li

doi:10.1073/pnas.1210371110

Edited* by Rudolf Jaenisch, Whitehead Institute for Biomedical Research, Cambridge, MA, and approved November 12, 2012 (received for review June 18, 2012)

Abstract

Ezh2 (Enhancer of zeste homolog 2) protein is the enzymatic component of the Polycomb repressive complex 2 (PRC2), which represses gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and differentiation during embryonic development. Recently, hot-spot mutations of Ezh2 were identified in diffused large B-cell lymphomas and follicular lymphomas. To investigate if tumor growth is dependent on the enzymatic activity of Ezh2, we developed a potent and selective small molecule inhibitor, EI1, which inhibits the enzymatic activity of Ezh2 through direct binding to the enzyme and competing with the methyl group donor S-Adenosyl methionine. EI1-treated cells exhibit genome-wide loss of H3K27 methylation and activation of PRC2 target genes. Furthermore, inhibition of Ezh2 by EI1 in diffused large B-cell lymphomas cells carrying the Y641 mutations results in decreased proliferation, cell cycle arrest, and apoptosis. These results provide strong validation of Ezh2 as a potential therapeutic target for the treatment of cancer.

Ezh2 is the mammalian homolog of Enhancer of Zeste, the key component of Polycomb repressive complex 2 (PRC2), which represses gene expression in development (1). Ezh2 and the other two proteins, Suz12 and EED, form the core PRC2 complex, which possesses histone methyltransferase (HMT) activity and methylates H3K27 (2, 3). H3K27 methylation has been correlated with transcriptional repression and heterochromatin formation (4). The Drosophila ortholog, E(z), was initially identified as a regulator of homeotic gene expression and body segmentation (5). In mammals, Ezh2 is highly expressed in stem cells and actively proliferating cells, and down-regulated in differentiated cells (6). Ezh2 knockout in mice leads to developmental abnormality and embryonic lethality (7, 8). Ezh2-null ES cells are viable (8) and Ezh2 is involved in maintaining pluripotency and repression of lineage-differentiation genes (8 ⇓–10).

Ezh2 is overexpressed in a broad spectrum of tumors, including prostate cancer, breast cancer, myeloma, hepatocellular carcinoma, gastric cancer, and so forth (1, 6). Overexpression of Ezh2 in mouse mammary gland leads to epithelial hyperplasia (11). Multiple studies using si/shRNA show that reduction of Ezh2 expression in tumor cell lines inhibits cell proliferation (12), migration, and invasion (13) or angiogenesis (14), and leads to apoptosis (15). Ezh2 contains the characteristic SET domain [Suppressor of variegation 3–9 (Suv (3–9), Enhancer of zeste (E(z)) and Trithorax] (16) present in most HMTs. Recurrent somatic mutations in the SET domain of Ezh2 was identified in diffused large B-cell lymphomas (DLBCL) patients (17 ⇓⇓⇓⇓–22). These mutations lead to the change of tyrosine (Y) 641 to phenylalanine (F), serine (S), asparagine (N), histidine (H), cysteine (C), or alanine (A) 677 to glycine (G). The Y641 mutant and A677G proteins show enhanced activity on H3K27me2 peptide (20, 23). Cancer cell lines heterozygous for these Ezh2 mutants show increased H3K27me3 and decreased H3K27me2 level (17, 20, 21, 23). Wild-type and mutant Ezh2 proteins may work cooperatively in cells to maintain a high level of H3K27me3 (23). It is therefore hypothesized that the Ezh2 hot-spot mutations and their enhanced activity toward H3K27me2 promotes DLBCL cell proliferation.

In this study, we developed an S-Adenosyl methionine (SAM) competitive inhibitor of Ezh2, EI1, which inhibits the methyltransferase activity of the Ezh2/PRC2 with high selectivity across an HMT panel. EI1 treated cells exhibited decreased H3K27 methylation without changes of other histone H3 methylation marks. Using this tool inhibitor, we showed that inhibition of Ezh2 in DLBCL cells carrying hot-spot mutations and some Ezh2 overexpressed tumor cell lines results in decreased proliferation, cell cycle arrest, and apoptosis. Thus, inhibition of Ezh2 enzymatic activity may provide a therapeutic option for the treatment of DLBCL and other cancers.

Results

Identification and Biochemical Characterization of EI1 as a Potent and Selective Inhibitor of PRC2.

To identify inhibitors of Ezh2/PRC2, we performed a high-throughput screen using recombinant PRC2 protein complex containing Ezh2, Suz12, EED, RbAP48, and AEBP2 (24). EI1 (Fig. 1A) was designed based on one chemical scaffold identified from the high-throughput screen. This compound demonstrated potent, concentration-dependent inhibition of the enzymatic activity against both Ezh2 wild-type and Y641F mutant enzymes with IC50 of 15 ± 2 nM and 13 ± 3 nM, respectively (Fig. 1B). To understand the mode of inhibition for EI1, SAM competition experiments were carried out under the conditions of saturated substrate peptide. In accordance with the Cheng–Prusoff relationship for a competitive binding mode, the IC50 value of EI1 increased linearly with increasing concentration of SAM. The fitting of the data into the Cheng–Prusoff equation for competitive inhibition using linear regression analysis gave a K_i value of 13 ± 3 nM (Fig. 1C).

Fig. 1.

Biochemical characterization of EI1 inhibition of PRC2 complex. (A) Chemical structure of EI1. (B) Inhibition curves of EI1 for wild-type enzyme with unmethylated H3K27 as the substrate and Y641F enzyme with di-methylated H3K27 as the substrate. Each data point represents the mean of two replicates at each concentration of the compounds. The data are fit to a dose–response equation using PRISM. (C) Plot of IC50 values of EI1 as a function of SAM concentration relative to the Michaelis–Menten constant (K_m) of SAM. Using the Cheng–Prusoff equation for competitive inhibitors [IC50 = K_i (1+[S]/K_m)], the K_i value of EI1 was derived from the linear regression fitting.

Although SAM is the common cofactor for all HMTs, EI1 showed remarkable selectivity against Ezh2 over other HMTs (Table 1). All biochemical reactions were carefully characterized with enzymology studies and the SAM and substrate concentrations were kept at their respective K_m for most of the HMTs. Strikingly, EI1 displayed ∼90-fold selectivity for Ezh2 over Ezh1, and >10,000-fold selectivity over other HMTs (Table 1). In contrast, another well-characterized SAM competitive HMT inhibitor, sinefungin, showed no selectivity toward Ezh2 (Table 1). Taken together, our results indicate that EI1 is a potent and selective inhibitor against Ezh2.

View this table:

Table 1.

HMT Profiling of EI1 with a panel of HMT enzymes

EI1 Inhibits Cellular H3K27 Methylation and Activates Ezh2 Target Gene Expression.

Previous studies using si/shRNA to knock down Ezh2 or other components of PRC2 showed that DLBCL and rhabdoid tumor cells were highly dependent on PRC2 for proliferation (25, 26). Therefore, we tested the effect of EI1 in these cell lines, including the DLBCL cells carrying Ezh2 mutations [WSU-DLCL2 (Ezh2^Y641F), SH-DHL6 (Ezh2^Y641N)] or wild-type Ezh2 [(OCI-LY19 (Ezh2^WT), and GA10 (Ezh2^WT)] and a rhabdoid tumor line G401 (Ezh2^WT). EI1 dramatically inhibited the H3K27me3 and H3K27me2 levels in these cells in a dose-dependent manner, but H3K27me1 was largely unchanged (Fig. 2A and Fig. S1 A and B). The effect was similar in these cell lines, although they have different basal H3K27me3 and H3K27me2 levels. For example, in WSU-DLCL2, SU-DHL6, and Karpas422 with Ezh2 mutations, the H3K27me3 level was much higher than that in DLBCL cells with wild-type Ezh2 (Fig. S1C).

Fig. 2.

EI1 inhibits cellular H3K27 methylation and activates p16 expression. (A) Inhibition of H3K27 methylation by different concentrations of EI1. SU-DHL6 (Ezh2^Y641N) and OCI-LY19 (Ezh2^WT) cells were treated with EI1 for 4 d, and G401 cells were treated with EI1 for 2 d at the indicated concentrations. H3K27me3, H3K27me2, H3K27me1, and H3 were detected by immunoblot. (B) Time course of H3K27 methylation inhibition by EI1. WSU-DLCL2 (Ezh2^Y641F) cells were treated with EI1 at 10 μM for indicated time periods. (C) Immunoblot against histone H3 methylation modifications using WSU-DLCL2 (Ezh2^Y641F) cell lysates treated with indicated concentrations of EI1. (D) qPCR analysis of p16 mRNA level in G401 (Ezh2^WT) cells. p16 mRNA is normalized to GAPDH level and plotted as fold to control base line samples. (Left) The expression of p16 gene in G401 cells treated by different concentrations of EI1 for 2 d. (Right) Time course of p16 expression in G401 cells treated by EI1 (10 μM) for indicated time. (E) Graphic presentation of p16 genomic locus. The regions A, B, and C indicate the primer location for ChIP-PCR. (F) ChIP-PCR using anit-Ezh2 and H3K27me3 antibodies at the indicated regions on p16 promoter before and after 48 h of EI1 treatment. Rabbit and mouse IgG are used as controls. Relative enrichment is calculated as percent of input.

A time-course study was performed in WSU-DLCL2 cells to understand the kinetics of H3K27 methylation inhibition. The H3K27me3 level was decreased 24 h after EI1 treatment, and reached the lowest level after 4–5 d (Fig. 2B). To test the specificity of EI1, we examined other histone H3 lysine methylation marks. As shown in Fig. 2C, the other marks were not changed.

p16 is a well characterized Ezh2 target gene (26 ⇓–28) and its promoter is directly bound by Ezh2 and enriched for H3K27me3 (8, 27). Depletion of Ezh2 in G401 cells leads to up-regulation of p16 expression and senescence (26). We therefore examined the effect of EI1 on p16 expression in G401 and found EI1 could activate p16 expression in a dose-dependent manner (Fig. 2D, Left). Consistently, EI1 also caused growth inhibition of G401 (Fig. S3). In a time-course study, p16 expression was activated 2 d after EI1 treatment and the expression level increased by 20-fold at day 5 (Fig. 2D, Right). The decrease of H3K27me3 was readily observable 24 h after EI treatment (Fig. S1D). These results indicate that the decrease of H3K27me3 precedes the activation of p16 expression.

ChIP experiments were performed to examine the change of H3K27me3 and Ezh2 status on p16 promoter (Fig. 2E). Both Ezh2 and H3K27me3 were enriched at the p16 promoter (Fig. 2F), as previously reported (8, 27). Specific and significant enrichment of H3K27me3 and Ezh2 were observed at region A and B, and the enrichment decreased toward region C (Fig. 2F). Following EI1 treatment, H3K27me3 signals were significantly reduced across the p16 promoter (Fig. 2F). Interestingly, Ezh2 remained bound at the p16 promoter (Fig. 2F). Therefore, EI1 activated p16 expression by suppressing H3K27me3, but not Ezh2 occupancy at the promoter.

EI1 Caused Similar Phenotypes as Ezh2 Knockout in Mouse Embryonic Fibroblasts.

To assess the selectivity of EI1 in cells, we established SV40-T immortalized mouse embryonic fibroblasts (MEFs) carrying the Cre-ERT2 transgene and two floxed Ezh2 loci (29). Treatment with 4-OH-tamoxifen (4-OH-T) led to significant depletion of Ezh2 and H3K27me3 (Fig. 3A). EI1 treatment did not affect Ezh2 protein level but caused similar loss of H3K27me3 (Fig. 3A). The immortalized MEFs showed decreased proliferation (∼40–50%) following Ezh2 depletion by 4-OH-T (Fig. 3B). Consistently, a similar decrease in proliferation was observed in EI1-treated MEF. Importantly, there was no further reduction of proliferation when the MEF was treated with EI1 in combination with 4-OH-T (Fig. 3B). Therefore, EI1 treatment mimics Ezh2 knockout in the MEFs to inhibit H3K27me3 and cell proliferation.

Fig. 3.

EI1 inhibits H3K27me3 similarly as Ezh2 knockout. (A) Inhibition of H3K27me3 by EI1 and Ezh2 knockout induced by 4-OH-T. Immortalized MEF (Ezh2^f/f) cells were treated with 3.3 μM EI1, 4-OH-T, or the combination of EI1 and 4-OH-T. After cell harvesting, indicated proteins were detected by immunoblot. (B) Proliferation of the immortalized MEFs after treatment by 3.3 μM EI1, Ezh2 knockout induced by 4-OH-T, or the combination of EI1 and 4-OH-T. Viable cells were counted at days 3, 6, and 11. The results were plotted on a linear scale.

EI1 Selectively Inhibited the Growth of DLBCL Cells Carrying Ezh2 Mutation.

To test if inhibition of Ezh2 might be efficacious to inhibit cancer cell growth, siRNAs specific to Ezh2 was designed and transfected into OCI-LY19 (Ezh2^WT) and WSU-DLCL2 (Ezh2^Y641F). Two independent siRNAs, named siEzh2-1 and siEzh2-2, knocked down Ezh2 mRNA by ∼80% (Fig. S2). Both siRNAs inhibited the proliferation of WSU-DLCL2 but had little effect on OCI-LY19 (Fig. 4A), suggesting the proliferation of DLBCL cells with Ezh2 mutations is dependent on Ezh2.

Fig. 4.

EI1 selectively inhibits the proliferation and colony formation of DLBCL cell lines carrying Ezh2 Y641 mutations. (A) Proliferation of WSU-DLCL2 (Ezh2^Y641F) and OCI-LY19 (Ezh2^WT) cells. Viable cells were counted every 3 or 4 d in the presence of siRNAs against Ezh2 and results were plotted on a logarithmic scale. (B) Proliferation of WSU-DLCL2 (Ezh2^Y641F), SH-DHL-6 (Ezh2^Y641N), OCI-LY19 (Ezh2^WT), and GA10 (Ezh2^WT) cells in the presence of EI1 at the indicated concentrations. Viable cells were similarly counted as in A. (C) Effect of EI1 on the proliferation of DLBCL cell lines with wild-type Ezh2, or cells carrying Ezh2 Y641 mutations. Cells were similarly cultured and counted as in B and IC50 values were calculated at 14 or 15 d of treatment. The level bars indicate median for each group (see also Fig. S3). (D) Colony formation of WSU-DLCL2 (Ezh2^Y641F) and OCI-LY19 (Ezh2^WT) cells in the presence of EI1 at the indicated concentrations. (Upper) On day14, photomicroscopy of cell colonies was taken at 20× magnification. (Lower) Quantification of colony formation by AlamarBlue. The data are plotted as percentage of DMSO.

To test whether EI1 treatment has a similar effect as Ezh2 knockdown, we measured the proliferation after EI1 treatment and IC50s of EI1 on both wild-type and mutant Ezh2 DLBCL lines. The proliferation of Ezh2 mutant DLBCL cells, including WSU-DLCL2, SU-DHL6, Karpas422, DB, and SU-DHL4, was strongly inhibited by EI1 in a dose-dependent manner (Fig. 4 B and C and Fig. S3). In contrast, Ezh2 wild-type DLBCL cells, including OCI-LY19 and GA10, were not affected by EI1, and Toledo was only weakly affected by EI1 (Fig. 4 B and C and Fig. S3). Thus, specific inhibition of Ezh2 enzymatic activity by EI1 selectively inhibited the growth of DLBCL cells carrying Ezh2 mutations.

Soft agar colony formation evaluates if cancer cells can proliferate in an anchorage-independent manner (30). We tested the effect of EI1 on the colony formation ability of WSU-DLCL2 and OCI-LY19 cells. As shown in Fig. 4D, both the size and the number of WSU-DLCL2 colonies were decreased after EI1 treatment. In contrast, the colony formation of OCI-LY19 was not affected. Taken together, these results indicate that EI1 is not generally toxic to cells, but selectively inhibits the neoplastic properties of DLBCL cells with Ezh2 mutation.

EI1 Causes Cell Cycle Arrest and Apoptosis of DLBCL Cells with Y641 Mutations.

To understand the role of Ezh2 in the regulation of the proliferation of DLBCL lines carrying Ezh2 Y641 mutations, we examined the DNA synthesis and cell cycle progression by BrdU incorporation and FACS analysis. In WSU-DLCL2 cells treated with EI1 for 7 d, significant decrease of BrdU⁺ cells (reduced from 41–8.72%) was detected (Fig. 5A). The percentage of cells in G2/M also decreased, whereas the percentage of cells in G1 was increased (Fig. 5 A and B). Similar results were observed in two other Ezh2 mutant DLBCL cell lines (Fig. 5B). In contrast, the Ezh2 wild-type cells OCI-LY19, GA10, and Toledo did not show significant changes of DNA synthesis or cell cycle distribution (Fig. 5 A and B).

Fig. 5.

EI1 causes cell cycle G0/1 arrest and apoptosis in DLBCL cell lines carrying Ezh2 Y641 mutations. (A) Cell cycle analysis of WSU-DLCL2 (Ezh2^Y641F) and OCI-LY19 (Ezh2^WT) cells after 7-d treatment with DMSO or EI1 (10 μM). The analysis was performed using BrdU incorporation with 7-AAD staining followed by flow cytometry. Annotations indicate distribution of cells to G0/1, S, and G2/M phases of cell cycle. One representative result of multiple experiments is shown. (B) Cell cycle changes analyzed by FACS after treatment as in A. The cell lines are: WSU-DLCL2 (Ezh2^Y641F), SU-DHL6 (Ezh2^Y641N), Karpas422 (Ezh2^Y641N), OCI-LY19 (Ezh2^WT), GA10 (Ezh2^WT), and Toledo (Ezh2^WT). (C) Immunoblot analysis. Cells were similarly cultured and treated as in A (see also Fig. S4). (D) Immunoblot using SU-DHL6 cell lysates treated with 10 μM of EI1 for indicated time periods.

Cell cycle progression is regulated by the activity of cyclin-dependent kinases and levels of various cyclins (31). We therefore surveyed the expression of cyclins in EI1-treated WSU-DLCL2 cells. Cyclin A and B1 protein levels were diminished after 6 d of EI1 treatment (Fig. 5C). Similar effects were observed in other DLBCL cells with Ezh2 mutations (Fig. S4). This finding is consistent with the reduction of the G2/M population from FACS analysis. A mild increase of sub-G1 population was observed in EI1-treated SU-DHL6 and Karpas422, suggesting an increase of apoptosis. To further examine this phenomenon, we carried out the Western blotting of procaspase 3 and cleaved (c)-caspase 3 in EI1-treated SU-DHL6 cells. Indeed, c-caspase 3 level was gradually elevated with increasing treatment time (Fig. 5D). Taking these data together, we show that inhibition of the enzymatic activity of Ezh2 by EI1 can significantly block cell cycle progression and induce apoptosis in Ezh2 mutant DLBCL cells.

Inhibition of Ezh2 Causes Down-Regulation of a Proliferation Gene Signature and Up-Regulation of a Memory B-Cell Signature in DLBCL.

We examined the gene-expression changes in Karpas422 (Ezh2^Y641N) after EI1 treatment in a time-course experiment. EI1-treated cells were compared with the corresponding controls to obtain the significantly differentially expressed gene lists using a two-tailed Student t test at each time point. A cutoff of P value < 0.05 and fold-change > 1.5 was used to select the EI1 regulated genes. The number of genes that showed statistically significant change increased with increasing treatment time (Fig. 6A). More genes were up-regulated than down-regulated between days 1 and 3 after EI1 treatment (Fig. 6A), suggesting the early response to Ezh2 inhibition is primarily gene up-regulation, which is consistent with Ezh2’s role in transcriptional repression. By day 6, the number of down-regulated genes dramatically increased, which could be because of the secondary response (Fig. 6A).

Fig. 6.

Ezh2 inhibition by EI1 causes down-regulation of DLBCL proliferation signature and up-regulation of memory B-cell signature. (A) Gene-expression changes of Karpas422 cell at different time points after EI1 (5 μM) treatment. Changes in gene expression were filtered by fold-change >1.5 or ≤1.5 and by P < 0.05. (B and F). GSEA showing enrichment of “Proliferation signature in DLBCL” (B) (34) and “Nuytten Ezh2 targets down” (F) (37) in genes down-regulated by 6 d of EI1 treatment. (C–E). GSEA showing enrichment of “Splenic marginal zone memory B cell high vs. GC B cell” (C) (36), “genes negative correlated with Ezh2 mRNA in DLBCL” (D) (25), and “Nuytten Ezh2 targets up” (E) (37) in genes up-regulated by 6 d of EI1 treatment. See also Table S1.

To investigate if genes with expression changes at early time points are more likely direct PRC2 targets, two differential expressed gene lists were generated at days 3 and 9 after EI1 treatment, respectively. These lists were compared with the PRC2 and H3K27me3 target genes in human embryonic fibroblasts and centroblasts, respectively (10, 25). The gene list at day 3 showed better enrichment to the known PRC2 target genes than the day 9 gene list (Fig. S5). Almost all these genes are up-regulated, suggesting early response genes are direct Ezh2 targets. We further selected 13 genes that were up-regulated by EI1 from microarray and revalidated their expression by quantitative PCR (qPCR) (Fig. S6A). ChIP experiments were performed to determine if these genes were labeled by H3K27me3 at the promoters. EI1 treatment significantly decreased the H3K27me3 enrichment in these 13 gene promoters, but not the negative control GAPDH promoter (Fig. S6B). These data further strengthen the notion that the depletion of H3K27me3 at target gene promoters correlates to EI1-dependent transcriptional up-regulation.

To further delineate the pathways that are responsive to Ezh2 inhibition, we performed gene set enrichment analysis (GSEA) using the microarray data (P value < 0.05 and fold-change > 1.5) after 6 d of EI1 treatment (32, 33). The analysis revealed that the genes down-regulated by EI1 showed significant enrichment of a proliferation signature in DLBCL (Fig. 6B) (Proliferation_DLBCL signature from Lymphoma/Leukemia Molecular Profiling Project), consistent with our data on cell proliferation and cell cycle (Figs. 4 and 5) (34, 35). Interestingly, genes up-regulated by EI1 showed significant enrichment of a gene set distinguishing splenic marginal zone memory B cell from the germinal center B cell (Splenic_marginal_zone_B cell_gt_GC_B cell signature from Lymphoma/Leukemia Molecular Profiling Project), indicating that Ezh2 inhibition may promote differentiation to memory B-cell (Fig. 6C) (35, 36). We compared our data with a geneset that negatively correlated with Ezh2 mRNA level in DLBCL (25), and found that this gene signature showed significant enrichment in the genes up-regulated by EI1 treatment (Fig. 6D). Intriguingly, our data also displayed significant enrichment of a geneset regulated in prostate cancer cell after siRNA-mediated knockdown of Ezh2 (Fig. 6 E and F and Fig. S7) (37).

Discussion

In this study, we developed a potent small molecule inhibitor, EI1, which inhibits Ezh2 activity through competition with the cofactor SAM. EI1 is highly selective against Ezh2 over its close homolog Ezh1 and other HMTs. The SET domains of Ezh1 and Ezh2 are 90% identical (Fig. S8A). Structural modeling shows the divergent residues I626 and C663 in Ezh2 (T664 and S664 in Ezh1) are around the EI1 binding pocket, even though no direct interaction with EI1 (Fig. S8 B and C), suggesting these residues may contribute to the selectivity. Future structural studies of EI1-Ezh2 cocrystal structure may help to understand how such high selectivity is achieved.

EI1 treated cells show specific reduction of H3K27me2 and H3K27me3 (Fig. 2C). Using Ezh2-floxed MEF cells, we further demonstrated that EI1 had similar effect as Ezh2 knockout on H3K27 methylation and proliferation, indicating that EI1 is a highly specific Ezh2 inhibitor suitable for functional studies. siRNA knockdown of PRC2 complex, such as Ezh2, Eed, and Suz12, could disrupt the complex and block cancer cell proliferation (1, 12 ⇓–14, 37). Here, we demonstrated that inhibition of HMT activity of Ezh2 by EI1 without disrupting the chromatin binding of PRC2 is sufficient to blocks cancer cell proliferation and colony formation, particularly in Ezh2 mutant containing DLBCL cells. Thus, a small molecule inhibitor of Ezh2 can be efficacious in treating cancers.

During the review of our manuscript, Knutson et al. (38) and McCabe et al. (39) published two Ezh2 inhibitors, which also act in a SAM-competitive manner and are highly selective over Ezh1. Although it was not shown whether the gene up-regulation by these compounds correlated with the decrease of H3K27me3 mark, both compounds could inhibit the proliferation of several Ezh2 mutant lymphoma cell lines. This finding is consistent with our findings and independently validates Ezh2 as a potential target for cancer treatment.

All Ezh2 mutations are heterozygous in DLBCL and the mutant cells contain much higher levels of H3K27me3 than wild-type cells (Fig. S1C) (17, 20, 21). Ezh2-Y641 mutants have weak biochemical activity toward unmethylated H3K27 and H3K27me1 peptide substrates, whereas they have much higher catalytic activity converting H3K27me2 to H3K27me3 (20, 23, 40). Wild-type and mutant Ezh2 complexes may collaborate to convert unmethylated H3K27 toward high levels of H3K27me3 (40). Interestingly, although H3K27me2 and H3K27me3 are similarly depleted in DLBCL cells with or without Ezh2 mutation, Ezh2 mutant cells are specifically growth-inhibited by EI1 (Figs. 2A and 4 B and C, and Fig. S3). This finding suggests that the Ezh2 mutation in DLBCL may be an oncogenic driver. Because EI1 is equally active against both the wild-type and Y641 mutant Ezh2, it will be interesting to develop a mutant-selective inhibitor in the future to evaluate if it will be equally efficacious as EI1.

In this study, we conducted a detailed cell cycle analysis of EI1 treated DLBCL cells. Ezh2 mutant cells are growth-arrested by EI1 with G1 arrest (Fig. 5). Expression of cyclin A and B1 is down-regulated (Fig. 5). GSEA analysis showed EI1 can reactivate the expression of PRC2 target genes (Fig. S5) (37). After 6 d of treatment, the proliferation signature was significantly down-regulated (Fig. 6B) (34) and accompanied by the activation of a set of memory B-cell–specific genes (Fig. 6C) (36). These results suggest inhibition of Ezh2 may induce growth arrest and differentiation into memory B cells. We found multiple genes up-regulated by EI1 have H3K27me3 enrichment at their promoters. EI1 effectively reduced the H3K27me3 level on these promoters and activated their expression. At the p16 promoter, EI1 suppressed H3K27me3 level but Ezh2 remained bound to the promoter. To our knowledge, this demonstration that inhibition of histone H3K27me3 can occur without dissociation of the Ezh2 complex from its target gene is unique. Ezh2 can interact with other chromatin modifying enzymes, such as histone deacetylases and DNA methyltransferases (4) to repress transcription. Future studies should investigate if all Ezh2 target genes can be sufficiently reactivated by reversing promoter H3K27me3 level alone. Such knowledge might help to understand how to use Ezh2 inhibitors as single agent or in combination for cancer treatment.

Materials and Methods

Cell Culture.

Cells were maintained in a humidified incubator at 37 °C, 5% (vol/vol) CO₂. OCI-LY19, GA10, Toledo, WSU-DLCL2, DB, SU-DHL4, Karpas422 and SU-DHL6 were cultured in RPMI-1640 (Invitrogen, 11875) with 15% (vol/vol) FBS (Invitrogen, 10099-141). G401 was cultured in DMEM (Invitrogen, 11995) with 10% (vol/vol) FBS and 0.055 mM 2-mercaptoethanol (Sigma, M7522). MEFs were from the Antoine Peters laboratory (41) and cultured in DMEM with 10% (vol/vol) FBS. The immortalized MEF was generated by retroviral transduction of pBABE-SV40 T and selected with hygromycin (Invitrogen). Single clones were selected based on the inducible knockout of Ezh2 in response to 4-OH-T (100 nM).

Biochemical Assay.

PRC2 biochemical reaction was carried out in reaction buffer (20 mM Tris pH 8.0, 0.1% BSA, 0.01% Triton, 0.5 mM DTT). The 20 nM wild-type PRC2 complex, 1 µM SAM, and 1.5 µM H3K27me0 (21–44); or 10 nM Y641F mutant complex, 1 µM SAM, and 3 µM H3K27me2 (21-44) were used, respectively, because they have differential substrate preference (38).

Microarray Analysis.

RNA was purified from Karpas422 after 1, 2, 3, 6, and 9 d after DMSO or EI1 (5 μM) treatment. Triplicate samples were collected and hybridized to U133_Plus 2 Chips (Affymetrix). The gene-expression data were normalized using the Robust Multiarray Averaging method (42). To generate a differentially expressed gene list, EI1-treated samples were compared with the corresponding DMSO controls using a cutoff of fold-change > 1.5 and P value < 0.05 (Student t test). GSEA was performed as previously described (33).

Additional details of biochemical and cellular reagents and protocols are described in SI Materials and Methods. See Tables S2 and S3 for primers used.

Acknowledgments

We thank Youzhen Wang, Jeff Zhang, Jingquan Dai, Wei Yi, Shaolian Zhou, Yaochang Xu, Martin Pfeifer, Daniela Gabriel, Clemens Schuffler, Jan Weiler, Chandra Vargeese, Keith Bowman, Julien Papillon, and Nicholas Keen for support and discussions; and Antoine Peters (Friedrich Miescher Institute) for discussion and reagents.

Footnotes

↵¹W.Q. and H.C. contributed equally to this work.
↵²Present address: Oncology, Novartis Institutes for BioMedical Research, Cambridge, MA 02139.
↵³To whom correspondence should be addressed. E-mail: en.li{at}novartis.com.

Author contributions: W.Q., H.C., C.X.L., P.A., and E.L. designed research; W.Q., L.T., L.L., R.Z., and Y.L. performed research; J.Z., M.L., H.F., J.G., O.A., J.-H.Z., X.Y., J.F., Y.M., M.Z., T.Z., G.F., Z.C., G.L., T.Y., and K.Z. contributed new reagents/analytic tools; W.Q., L.T., S.C., X.L., and Z.Y. analyzed data; and W.Q., H.C., and E.L. wrote the paper.
The authors declare no conflict of interest.
↵*This Direct Submission article had a prearranged editor.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE41315).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1210371110/-/DCSupplemental.

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1. Cao R,
2. et al.
(2002) Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298(5595):1039–1043.
OpenUrl Abstract/FREE Full Text
↵
1. Morey L,
2. Helin K
(2010) Polycomb group protein-mediated repression of transcription. Trends Biochem Sci 35(6):323–332.
OpenUrl CrossRef PubMed
↵
1. Margueron R,
2. Reinberg D
(2011) The Polycomb complex PRC2 and its mark in life. Nature 469(7330):343–349.
OpenUrl CrossRef PubMed
↵
1. Lewis EB
(1978) A gene complex controlling segmentation in Drosophila. Nature 276(5688):565–570.
OpenUrl CrossRef PubMed
↵
1. Sparmann A,
2. van Lohuizen M
(2006) Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer 6(11):846–856.
OpenUrl CrossRef PubMed
↵
1. O’Carroll D,
2. et al.
(2001) The polycomb-group gene Ezh2 is required for early mouse development. Mol Cell Biol 21(13):4330–4336.
OpenUrl Abstract/FREE Full Text
↵
1. Shen X,
2. et al.
(2008) EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell 32(4):491–502.
OpenUrl CrossRef PubMed
↵
1. Boyer LA,
2. et al.
(2006) Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441(7091):349–353.
OpenUrl CrossRef PubMed
↵
1. Lee TI,
2. et al.
(2006) Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125(2):301–313.
OpenUrl CrossRef PubMed
↵
1. Li X,
2. et al.
(2009) Targeted overexpression of EZH2 in the mammary gland disrupts ductal morphogenesis and causes epithelial hyperplasia. Am J Pathol 175(3):1246–1254.
OpenUrl CrossRef PubMed
↵
1. Gonzalez ME,
2. et al.
(2009) Downregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcinoma and requires BRCA1. Oncogene 28(6):843–853.
OpenUrl CrossRef PubMed
↵
1. Au SL,
2. et al.
(2012) Enhancer of zeste homolog 2 epigenetically silences multiple tumor suppressor microRNAs to promote liver cancer metastasis. Hepatology 56(2):622–631.
OpenUrl CrossRef PubMed
↵
1. Lu C,
2. et al.
(2010) Regulation of tumor angiogenesis by EZH2. Cancer Cell 18(2):185–197.
OpenUrl CrossRef PubMed
↵
1. Wu ZL,
2. et al.
(2010) Polycomb protein EZH2 regulates E2F1-dependent apoptosis through epigenetically modulating Bim expression. Cell Death Differ 17(5):801–810.
OpenUrl CrossRef PubMed
↵
1. Schapira M
(2011) Structural chemistry of human SET domain protein methyltransferases. Curr Chem Genomics 5(Suppl 1):85–94.
OpenUrl CrossRef PubMed
↵
1. Bödör C,
2. et al.
(2011) EZH2 Y641 mutations in follicular lymphoma. Leukemia 25(4):726–729.
OpenUrl CrossRef PubMed
↵
1. Capello D,
2. et al.
(2011) Mutations of CD79A, CD79B and EZH2 genes in immunodeficiency-related non-Hodgkin lymphomas. Br J Haematol 152(6):777–780.
OpenUrl CrossRef PubMed
↵
1. Lohr JG,
2. et al.
(2012) Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proc Natl Acad Sci USA 109(10):3879–3884.
OpenUrl Abstract/FREE Full Text
↵
1. McCabe MT,
2. et al.
(2012) Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27) Proc Natl Acad Sci USA 109(8):2989–2994.
OpenUrl Abstract/FREE Full Text
↵
1. Morin RD,
2. et al.
(2010) Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat Genet 42(2):181–185.
OpenUrl CrossRef PubMed
↵
1. Ryan RJ,
2. et al.
(2011) EZH2 codon 641 mutations are common in BCL2-rearranged germinal center B cell lymphomas. PLoS ONE 6(12):e28585.
OpenUrl CrossRef PubMed
↵
1. Yap DB,
2. et al.
(2011) Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation. Blood 117(8):2451–2459.
OpenUrl Abstract/FREE Full Text
↵
1. Cao R,
2. Zhang Y
(2004) SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Mol Cell 15(1):57–67.
OpenUrl CrossRef PubMed
↵
1. Velichutina I,
2. et al.
(2010) EZH2-mediated epigenetic silencing in germinal center B cells contributes to proliferation and lymphomagenesis. Blood 116(24):5247–5255.
OpenUrl Abstract/FREE Full Text
↵
1. Wilson BG,
2. et al.
(2010) Epigenetic antagonism between polycomb and SWI/SNF complexes during oncogenic transformation. Cancer Cell 18(4):316–328.
OpenUrl CrossRef PubMed
↵
1. Agherbi H,
2. et al.
(2009) Polycomb mediated epigenetic silencing and replication timing at the INK4a/ARF locus during senescence. PLoS ONE 4(5):e5622.
OpenUrl CrossRef PubMed
↵
1. Kotake Y,
2. et al.
(2007) pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK4alpha tumor suppressor gene. Genes Dev 21(1):49–54.
OpenUrl Abstract/FREE Full Text
↵
1. Conzen SD,
2. Cole CN
(1995) The three transforming regions of SV40 T antigen are required for immortalization of primary mouse embryo fibroblasts. Oncogene 11(11):2295–2302.
OpenUrl PubMed
↵
1. Shoemaker RH,
2. et al.
(1985) Application of a human tumor colony-forming assay to new drug screening. Cancer Res 45(5):2145–2153.
OpenUrl Abstract/FREE Full Text
↵
1. Malumbres M,
2. Barbacid M
(2009) Cell cycle, CDKs and cancer: A changing paradigm. Nat Rev Cancer 9(3):153–166.
OpenUrl CrossRef PubMed
↵
1. Mootha VK,
2. et al.
(2003) PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34(3):267–273.
OpenUrl CrossRef PubMed
↵
1. Subramanian A,
2. et al.
(2005) Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102(43):15545–15550.
OpenUrl Abstract/FREE Full Text
↵
1. Rosenwald A,
2. et al.,
3. Lymphoma/Leukemia Molecular Profiling Project
(2002) The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med 346(25):1937–1947.
OpenUrl CrossRef PubMed
↵
1. Shaffer AL,
2. et al.
(2006) A library of gene expression signatures to illuminate normal and pathological lymphoid biology. Immunol Rev 210:67–85.
OpenUrl CrossRef PubMed
↵
1. Weller S,
2. et al.
(2004) Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. Blood 104(12):3647–3654.
OpenUrl Abstract/FREE Full Text
↵
1. Nuytten M,
2. et al.
(2008) The transcriptional repressor NIPP1 is an essential player in EZH2-mediated gene silencing. Oncogene 27(10):1449–1460.
OpenUrl CrossRef PubMed
↵
1. Knutson SK,
2. et al.
(2012) A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat Chem Biol 8(11):890–896.
OpenUrl PubMed
↵
1. McCabe MT,
2. et al.
(2012) EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature doi:10.1038/nature11606.
OpenUrl CrossRef
↵
1. Sneeringer CJ,
2. et al.
(2010) Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas. Proc Natl Acad Sci USA 107(49):20980–20985.
OpenUrl Abstract/FREE Full Text
↵
1. Terranova R,
2. et al.
(2008) Polycomb group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted repression in early mouse embryos. Dev Cell 15(5):668–679.
OpenUrl CrossRef PubMed
↵
1. Irizarry RA,
2. et al.
(2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4(2):249–264.
OpenUrl Abstract

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Proceedings of the National Academy of Sciences: 109 (52)

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[1] ↵
Simon JA,
Lange CA
(2008) Roles of the EZH2 histone methyltransferase in cancer epigenetics. Mutat Res 647(1–2):21–29.
OpenUrl PubMed

[2] Simon JA,

[3] Lange CA

[4] ↵
Cao R,
et al.
(2002) Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298(5595):1039–1043.
OpenUrl Abstract/FREE Full Text

[5] Cao R,

[6] et al.

[7] ↵
Morey L,
Helin K
(2010) Polycomb group protein-mediated repression of transcription. Trends Biochem Sci 35(6):323–332.
OpenUrl CrossRef PubMed

[8] Morey L,

[9] Helin K

[10] ↵
Margueron R,
Reinberg D
(2011) The Polycomb complex PRC2 and its mark in life. Nature 469(7330):343–349.
OpenUrl CrossRef PubMed

[11] Margueron R,

[12] Reinberg D

[13] ↵
Lewis EB
(1978) A gene complex controlling segmentation in Drosophila. Nature 276(5688):565–570.
OpenUrl CrossRef PubMed

[14] Lewis EB

[15] ↵
Sparmann A,
van Lohuizen M
(2006) Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer 6(11):846–856.
OpenUrl CrossRef PubMed

[16] Sparmann A,

[17] van Lohuizen M

[18] ↵
O’Carroll D,
et al.
(2001) The polycomb-group gene Ezh2 is required for early mouse development. Mol Cell Biol 21(13):4330–4336.
OpenUrl Abstract/FREE Full Text

[19] O’Carroll D,

[20] et al.

[21] ↵
Shen X,
et al.
(2008) EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell 32(4):491–502.
OpenUrl CrossRef PubMed

[22] Shen X,

[23] et al.

[24] ↵
Boyer LA,
et al.
(2006) Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441(7091):349–353.
OpenUrl CrossRef PubMed

[25] Boyer LA,

[26] et al.

[27] ↵
Lee TI,
et al.
(2006) Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125(2):301–313.
OpenUrl CrossRef PubMed

[28] Lee TI,

[29] et al.

[30] ↵
Li X,
et al.
(2009) Targeted overexpression of EZH2 in the mammary gland disrupts ductal morphogenesis and causes epithelial hyperplasia. Am J Pathol 175(3):1246–1254.
OpenUrl CrossRef PubMed

[31] Li X,

[32] et al.

[33] ↵
Gonzalez ME,
et al.
(2009) Downregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcinoma and requires BRCA1. Oncogene 28(6):843–853.
OpenUrl CrossRef PubMed

[34] Gonzalez ME,

[35] et al.

[36] ↵
Au SL,
et al.
(2012) Enhancer of zeste homolog 2 epigenetically silences multiple tumor suppressor microRNAs to promote liver cancer metastasis. Hepatology 56(2):622–631.
OpenUrl CrossRef PubMed

[37] Au SL,

[38] et al.

[39] ↵
Lu C,
et al.
(2010) Regulation of tumor angiogenesis by EZH2. Cancer Cell 18(2):185–197.
OpenUrl CrossRef PubMed

[40] Lu C,

[41] et al.

[42] ↵
Wu ZL,
et al.
(2010) Polycomb protein EZH2 regulates E2F1-dependent apoptosis through epigenetically modulating Bim expression. Cell Death Differ 17(5):801–810.
OpenUrl CrossRef PubMed

[43] Wu ZL,

[44] et al.

[45] ↵
Schapira M
(2011) Structural chemistry of human SET domain protein methyltransferases. Curr Chem Genomics 5(Suppl 1):85–94.
OpenUrl CrossRef PubMed

[46] Schapira M

[47] ↵
Bödör C,
et al.
(2011) EZH2 Y641 mutations in follicular lymphoma. Leukemia 25(4):726–729.
OpenUrl CrossRef PubMed

[48] Bödör C,

[49] et al.

[50] ↵
Capello D,
et al.
(2011) Mutations of CD79A, CD79B and EZH2 genes in immunodeficiency-related non-Hodgkin lymphomas. Br J Haematol 152(6):777–780.
OpenUrl CrossRef PubMed

[51] Capello D,

[52] et al.

[53] ↵
Lohr JG,
et al.
(2012) Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proc Natl Acad Sci USA 109(10):3879–3884.
OpenUrl Abstract/FREE Full Text

[54] Lohr JG,

[55] et al.

[56] ↵
McCabe MT,
et al.
(2012) Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27) Proc Natl Acad Sci USA 109(8):2989–2994.
OpenUrl Abstract/FREE Full Text

[57] McCabe MT,

[58] et al.

[59] ↵
Morin RD,
et al.
(2010) Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat Genet 42(2):181–185.
OpenUrl CrossRef PubMed

[60] Morin RD,

[61] et al.

[62] ↵
Ryan RJ,
et al.
(2011) EZH2 codon 641 mutations are common in BCL2-rearranged germinal center B cell lymphomas. PLoS ONE 6(12):e28585.
OpenUrl CrossRef PubMed

[63] Ryan RJ,

[64] et al.

[65] ↵
Yap DB,
et al.
(2011) Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation. Blood 117(8):2451–2459.
OpenUrl Abstract/FREE Full Text

[66] Yap DB,

[67] et al.

[68] ↵
Cao R,
Zhang Y
(2004) SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Mol Cell 15(1):57–67.
OpenUrl CrossRef PubMed

[69] Cao R,

[70] Zhang Y

[71] ↵
Velichutina I,
et al.
(2010) EZH2-mediated epigenetic silencing in germinal center B cells contributes to proliferation and lymphomagenesis. Blood 116(24):5247–5255.
OpenUrl Abstract/FREE Full Text

[72] Velichutina I,

[73] et al.

[74] ↵
Wilson BG,
et al.
(2010) Epigenetic antagonism between polycomb and SWI/SNF complexes during oncogenic transformation. Cancer Cell 18(4):316–328.
OpenUrl CrossRef PubMed

[75] Wilson BG,

[76] et al.

[77] ↵
Agherbi H,
et al.
(2009) Polycomb mediated epigenetic silencing and replication timing at the INK4a/ARF locus during senescence. PLoS ONE 4(5):e5622.
OpenUrl CrossRef PubMed

[78] Agherbi H,

[79] et al.

[80] ↵
Kotake Y,
et al.
(2007) pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK4alpha tumor suppressor gene. Genes Dev 21(1):49–54.
OpenUrl Abstract/FREE Full Text

[81] Kotake Y,

[82] et al.

[83] ↵
Conzen SD,
Cole CN
(1995) The three transforming regions of SV40 T antigen are required for immortalization of primary mouse embryo fibroblasts. Oncogene 11(11):2295–2302.
OpenUrl PubMed

[84] Conzen SD,

[85] Cole CN

[86] ↵
Shoemaker RH,
et al.
(1985) Application of a human tumor colony-forming assay to new drug screening. Cancer Res 45(5):2145–2153.
OpenUrl Abstract/FREE Full Text

[87] Shoemaker RH,

[88] et al.

[89] ↵
Malumbres M,
Barbacid M
(2009) Cell cycle, CDKs and cancer: A changing paradigm. Nat Rev Cancer 9(3):153–166.
OpenUrl CrossRef PubMed

[90] Malumbres M,

[91] Barbacid M

[92] ↵
Mootha VK,
et al.
(2003) PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34(3):267–273.
OpenUrl CrossRef PubMed

[93] Mootha VK,

[94] et al.

[95] ↵
Subramanian A,
et al.
(2005) Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102(43):15545–15550.
OpenUrl Abstract/FREE Full Text

[96] Subramanian A,

[97] et al.

[98] ↵
Rosenwald A,
et al.,
Lymphoma/Leukemia Molecular Profiling Project
(2002) The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med 346(25):1937–1947.
OpenUrl CrossRef PubMed

[99] Rosenwald A,

[100] et al.,

[101] Lymphoma/Leukemia Molecular Profiling Project

[102] ↵
Shaffer AL,
et al.
(2006) A library of gene expression signatures to illuminate normal and pathological lymphoid biology. Immunol Rev 210:67–85.
OpenUrl CrossRef PubMed

[103] Shaffer AL,

[104] et al.

[105] ↵
Weller S,
et al.
(2004) Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. Blood 104(12):3647–3654.
OpenUrl Abstract/FREE Full Text

[106] Weller S,

[107] et al.

[108] ↵
Nuytten M,
et al.
(2008) The transcriptional repressor NIPP1 is an essential player in EZH2-mediated gene silencing. Oncogene 27(10):1449–1460.
OpenUrl CrossRef PubMed

[109] Nuytten M,

[110] et al.

[111] ↵
Knutson SK,
et al.
(2012) A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat Chem Biol 8(11):890–896.
OpenUrl PubMed

[112] Knutson SK,

[113] et al.

[114] ↵
McCabe MT,
et al.
(2012) EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature doi:10.1038/nature11606.
OpenUrl CrossRef

[115] McCabe MT,

[116] et al.

[117] ↵
Sneeringer CJ,
et al.
(2010) Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas. Proc Natl Acad Sci USA 107(49):20980–20985.
OpenUrl Abstract/FREE Full Text

[118] Sneeringer CJ,

[119] et al.

[120] ↵
Terranova R,
et al.
(2008) Polycomb group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted repression in early mouse embryos. Dev Cell 15(5):668–679.
OpenUrl CrossRef PubMed

[121] Terranova R,

[122] et al.

[123] ↵
Irizarry RA,
et al.
(2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4(2):249–264.
OpenUrl Abstract

[124] Irizarry RA,

[125] et al.

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