Review

Acta Pharmacologica Sinica (2014) 35: 161–174; doi: 10.1038/aps.2013.161; published online 23 Dec 2013

EZH2: biology, disease, and structure-based drug discovery
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Jin-zhi Tan1, Yan Yan1, Xiao-xi Wang1, Yi Jiang1 and H Eric Xu1,2

  1. 1VARI-SIMM Center, Center for Structure and Function of Drug Targets, Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
  2. 2Laboratory of Structural Sciences, Center for Structural Biology and Drug Discovery, Van Andel Research Institute, 333 Bostwick Ave, NE, Grand Rapids, MI 49503, USA

Correspondence: Jin-zhi Tan, E-mail tanjinzhi@gmail.com; H Eric Xu, Eric.Xu@vai.org

Received 14 August 2013; Accepted 28 September 2013
Advance online publication 23 December 2013

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Abstract

EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2), which is a highly conserved histone methyltransferase that methylates lysine 27 of histone 3. Overexpression of EZH2 has been found in a wide range of cancers, including those of the prostate and breast. In this review, we address the current understanding of the oncogenic role of EZH2, including its PRC2-dependent transcriptional repression and PRC2-independent gene activation. We also discuss the connections between EZH2 and other silencing enzymes, such as DNA methyltransferase and histone deacetylase. We comprehensively address the architecture of the PRC2 complex and the crucial roles of each subunit. Finally, we summarize new progress in developing EZH2 inhibitors, which could be a new epigenetic therapy for cancers.

Keywords:

EZH2; PRC2; transcriptional repression; gene activation; anticancer drug; crystal structure; SET domain; methyltransferase inhibitor; epigenetic therapy

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Introduction

In eukaryotes, the posttranslational modifications of histones play crucial roles in regulating chromatin structure and gene expression. This epigenetic alteration is different from the usual genetic alterations because once the DNA sequence is altered by mutations, it is very difficult to restore the former sequence. However, epigenetic changes can be reversed by specific inhibitors that target enzymes such as DNA methyltransferase, histone methyltransferase, and histone deacetylase1. Because epigenetic abnormalities are common in human cancer and play a key role in tumor progression, an understanding of epigenetic alterations can benefit the process of drug design and discovery for cancer treatment2.

EZH2, as a catalytic subunit of the polycomb repressive complex 2 (PRC2), represses gene expression by methylating lysine 27 of histone 3 (H3K27). EZH2-mediated methylation is a potential independent mechanism for epigenetic silencing of tumor suppressor genes in cancer. Recent studies have found that EZH2 is overexpressed in a wide range of cancers such as those of the prostate, breast, and bladder, which makes EZH2 an attractive anti-cancer drug target. In this review, we focus on the PRC2 machinery, including new advances in understanding how the five PRC2 subunits interact with each other. We also review new findings that suggest that EZH2 acts as transcriptional gene activator rather than a gene silencer. New progress on inhibitor design has been made by targeting the conserved SET domain of EZH2, which may contribute to the development of novel treatment strategies against different cancers.

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Biological function of EZH2

PRC2 is an essential chromatin modifier that is conserved across organisms from plants to flies and humans3. This complex represses the transcription of target genes through trimethylation of lysine 27 on histone 3 (H3K27me3), which is currently viewed as its predominant function in vivo4. The human PRC2 complex includes five subunits: EZH2, EED, SUZ12, RbAp46/48, and AEBP2 (Figure 1). A common biological function of PRC2 is the transcriptional silencing of genes involved in differentiation.

Figure 1.
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Possible architecture of the PRC2 complex. (A) Models of the fly and human PRC2 complexes. The subunits and interactions between them are shown. (B) Domain organizations of each subunit in the human PRC2 complex. Domain “1”, binding region for PHF1 in human cells and PCL in flies; domain “2”, binding region for SUZ12; CXC, cysteine-rich domain; SANT, domain that allows chromatin remodeling protein to interact with histones; SET, catalytic domain of EZH2; VEFS, VRN2-EMF2-FIS2-SUZ12 domain; WD, WD-40 domain; WDB, WD-40 binding domain; Zn, Zn-finger region.

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PRC2 catalyzes three sequential methylation reactions at H3K27, resulting in mono-, di-, and trimethylated H3K27 (H3K27me1, H3K27me2, and H3K27me3). EZH2 is the catalytic subunit of the PRC2 complex, and its C-terminal SET domain exhibits methyltransferase activity. EZH2 lacks enzymatic function on its own; however, it gains robust histone lysine methyltransferase activity when it complexes with two other noncatalytic subunits of the PRC2 complex, namely the zinc-finger-containing SUZ12 and the WD40-repeat protein EED5. These two subunits are required to maintain the integrity of PRC2, and mutations in either gene may destabilize EZH26,7,8.

Regarding the other subunits, it has been reported that the PRC2 subcomplex without the RbAp48 subunit can maintain substantial enzymatic activity, suggesting that the RbAp48 subunit is not required for the histone methyltransferase activity of EZH23. The AEBP2 subunit acts as a cofactor that interacts with the other four subunits by binding to the center of the PRC2 complex, which helps to stabilize the overall architecture of PRC2. The AEBP2 subunit also facilitates the PRC2 complex targeting to specific DNA sites and enhances its methyltransferase activity.

EZH2 as an epigenetic silencer

Currently, EZH2 is believed to function predominately as a transcriptional repressor that silences an array of target genes, including more than 200 tumor suppressors5. This view is supported by the finding that many PRC2-repressed genes are linked to poor outcomes in prostate cancer patients9. It is known that the PRC1 complex, which comprises B lymphoma Mo-MLV insertion region 1 and the ring finger proteins RING1 and RING2, functions cooperatively with PRC2 during epigenetic silencing by ubiquitinating lysine 119 of histone H2A10. The PRC2-mediated trimethylation of H3K27 is believed to recruit PRC1 to target gene loci, leading to a more condensed chromatin configuration. In support of this coordinated recruitment and function, the expression of PRC1 and PRC2 proteins can be integrated through a network of regulatory microRNAs11, such as miR-200, which can be transcriptionally repressed by EZH2. Because these miRNAs are involved in the regulation of PRC1, their repression by EZH2 can upregulate PRC1. In summary, it is suggested that there is a molecular link between these two protein complexes, which play important roles in regulating the state of chromatin and therefore affect the transcription of target genes, through the miRNA network.

Cooperation with other epigenetic silencing enzymes

The EZH2 histone methyltransferase usually cooperates with other epigenetic silencing enzymes. Recent studies have shown that there are physical and functional links between EZH2, DNA methyltransferases (DNMTs)12, and histone deacetylases (HDACs)13,14, suggesting the potential interplay between these different classes of epigenetic modulation enzymes in the control of gene expression.

Figure 2 illustrates a model for the cooperation of epigenetic silencing enzymes. Initially, the PRC2 complex methylates lysine 27 on histone 3 (H3K27me) and silences target genes. However, if K27 is acetylated, a histone deacetylase may be required. HDACs can help to modify the local histone code for silencing by deacetylating H3K27 and other lysines including H3K29, H3K14, and H4K8, thereby making the ε-amino group of lysine side chains available for methylation by PRC2. PRC2 can also cooperate with DNMTs to convert the target genes into a more deeply and permanently silenced chromatin state. During cellular transformation, certain genes that acquire methylation marks due to the action of the PRC2 complex will later become CpG-hypermethylated. Thus, the PRC2 complex can cooperate with HDACs to alter histone marks from acetylation to methylation, and it can also recruit DNMTs to produce a denser chromatin state. Functional links between EZH2, HDACs, and DNMTs have been found in colon, prostate, liver, lung, ovarian, and breast tumors, and all three types of epigenetic silencing machinery may contribute to the control of abnormal gene expression in cancer cells5.

Figure 2.
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A model for the collaboration of epigenetic silencing enzymes, including core of the PRC2 complex, DNA methyltransferase (DNMT), and histone deacetylase (HDAC). In this model, if K27 is pre-acetylated, HDAC may first deacetylates it, and then the target genes are silenced through the methylation of K27 by PRC2. DNMTs may also be recruited by PRC2, and after methylating CpG DNA of target genes, making the chromatin state more deeply silenced. Ac, acetylation; and Me, methylation.

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Mutations in EZH2

EZH2 has been reported to harbor various heterozygous mutations at tyrosine 641 (Y641) in the C-terminal SET domain; such mutations are found in 7% of follicular lymphomas and 22% of germinal center B-cell and diffuse large B-cell lymphomas (DLBCLs)15. Although initially reported to be a loss-of-function mutation, subsequent studies have shown that the Y641 mutation actually results in a gain of function16,17. In contrast to the wild type, which has a substrate preference for unmethylated H3K27 and monomethylated H3K27me1, the Y641 mutants (including Y641F, Y641N, Y641S, Y641H, and Y641C) have enhanced catalytic efficiency for dimethylated H3K27me216,17. Thus, both the wild type and the Y641 mutants can work together to increase the levels of H3K27me3.

Because the crystal structure of EZH2 is not yet available, a homology model was constructed to predict the substrate specificities of the wild type and the Y641 mutant18. The model shows that there is little room in the wild-type binding pocket for dimethylated lysine to rotate into the position to accept a third methyl group. Thus, the Y641 residue may play two roles: first, it may participate in the orientation of unmethylated and monomethylated lysine, and second, it may sterically restrict activity with a dimethylated substrate.

Another mutant, A677G, is also found in lymphoma cell lines, albeit at a low frequency (below 2%–3%). Characterization of this mutant protein has shown that the replacement of alanine 677 with glycine leads to increased activity with H3K27me2 substrates. This result is similar to that obtained with the Y641 mutant; however, mechanistic differences are evident. Relative to the Y641 mutant, which loses its activity with nonmethylated H3K27 substrate, the A677G mutant retains the crucial interactions with H3K27 substrate that are present in the wild-type EZH2, leading to efficient use of all three methylation substrates (H3K27, H3K27me1, and H3K27me2).

EZH2 functions independently as a gene activator

In addition to its role as a transcriptional repressor, several studies have shown that EZH2 may also function in target gene activation19,20,21. Recently, Xu et al reported that EZH2 plays an important role in castration-resistant prostate cancer, and its oncogenic function does not depend on silencing but rather on transcriptional induction of its target genes21. These authors found that a subset of EZH2-bound genes did not bind the PRC2 subunit SUZ12 or display H3K27me3. Many of these genes were downregulated upon EZH2 knockdown, suggesting that the role of EZH2 as an activator was independent of the PRC2 complex. Xu et al also showed that the methyltransferase activity of EZH2 was required for both EZH2-dependent gene activation and androgen-independent growth, which differs from the findings of early reports indicating that EZH2 functions as a gene activator19,20. The latter findings were observed in breast cancer cells, where EZH2 activates NF-κB target genes through the formation of a ternary complex with the NF-κB components RelA and RelB that does not require other PRC2 subunits19. EZH2 overexpression can also lead to its interaction with Wnt signaling components and subsequent activation of the c-myc and cyclin D1 genes; again, this function is independent of its methyltransferase activity20. It has been suggested that EZH2 may act as a multifaceted molecule; ie, it may function as either a transcriptional repressor or activator in promoting breast tumorigenesis22.

In castration-resistant prostate cancer, EZH2-mediated transcriptional activation may occur via methylation of the androgen receptor (AR) or other associated proteins, which would indicate a new role for EZH2 in the methylation of nonhistone proteins. Xu et al21 showed that phosphorylation of EZH2 at Ser21, mediated directly or indirectly by the PI3K-Akt pathway, can alter its function from a polycomb repressor to a transcriptional co-activator of AR and (potentially) other proteins (Figure 3). This finding indicates the potential for the development of inhibitors that can specifically target the activator function of EZH2 while sparing its PRC2 repressive function.

Figure 3.
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A model of the EZH2 functional switch from a polycomb repressor to a transcriptional activator in castration-resistant prostate cancer. PI3K/AKT pathway activation can lead to phosphorylation of EZH2 at Ser21. This phosphorylation event shifts EZH2 from a transcriptional repressor associated with PRC2 to a transcriptional co-activator cooperating with AR, using an intact SET methyltransferase domain.

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EZH2 and cancer

Hyperactivation of EZH2, either by overexpression or mutations, is found in a variety of malignancies including prostate, breast, uterine, gastric, and renal cell cancers in addition to melanoma5. EZH2 expression is correlated with aggressiveness, metastasis, and poor prognosis in most of these cancers. A recent study also found that EZH2 was overexpressed in non-small cell lung cancers and lymphoma23. The functional consequence of increased EZH2 in cancer tissues includes the silencing of genes that promote differentiation and restrain proliferation. Table 1 summarizes the extensive reports describing EZH2 hyperactivation in various cancers.


EZH2 in prostate cancer

A gene profiling study showed that EZH2 upregulated oncogenes in metastatic prostate cancer and that loss of EZH2 inhibited the growth of prostate cancer cells25. According to this study, EZH2 overexpression could be a valuable prognostic indicator of patient outcome. Another recent report focused on the relationship between EZH2 and androgen signaling pathways24, showing that EZH2 expression is repressed by androgens. This repression requires a functional androgen receptor and is mediated through retinoblastoma (RB)/p130-dependent pathways. Further, EZH2 may activate AR-repressed genes in an androgen-deprived environment because EZH2 is frequently overexpressed in metastatic prostate cancers35.

EZH2 in breast cancer

Breast cancer is the most common malignancy and the second leading cause of cancer-related death among women. Abnormally elevated EZH2 levels have been found to be highly correlated with invasiveness and increased proliferation rates of breast carcinomas26,30. These studies also proposed that EZH2 is a promising biomarker for aggressive breast cancers with poor prognosis and that it can be an independent indicator of clinical outcome. Overexpression of EZH2 is correlated with many signaling pathways in breast cancer, such as the pRB-E2F, PI3K/Akt, and estrogen receptor pathways27,28,29. For example, Gonzales et al showed that EZH2 overexpression in breast cancer cells can activate the PI3K/Akt pathway, especially through activation of the Akt isoform28. Based on experimental evidence, Deb et al proposed that EZH2 may function as a co-activator when it is overexpressed during malignancy and that it can be recruited to the estrogen signaling pathway to enhance estrogen signaling and promote proliferation22.

EZH2 in B-cell lymphomas

Lymphogenesis represents a special case wherein EZH2 is repressed in resting naive B cells but is highly upregulated in primary lymphoid follicles during B cell activation and germinal center (GC) formation23. EZH2 is overexpressed in GC-derived lymphomas, such as DLBCL32. Moreover, mutations in the SET domain of EZH2 that favor the formation of trimethylated H3K27 — such as Y641F — have been frequently identified in both DLBCL and follicular lymphoma15,31. In addition, DLBCLs are dependent on the oncogenic function of EZH2 independent of its mutational state because impairments in PRC2 enzyme activity can abolish tumorigenesis by both mutant and wild-type cancer cells. Thus, EZH2 is a promising drug target that can be specifically inhibited by small molecules (see below).

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Progress in drug discovery

DZNep as an indirect inhibitor

Over the past few years, several potent inhibitors of EZH2 have been discovered. Among these inhibitors, 3-deazaneplanocin A (DZNep), a S-adenosylhomocysteine hydrolase inhibitor (Figure 7), depletes EZH2 and the associated H3K27me3 and can induce apoptosis in breast and colon cancer cells63. DZNep interferes with S-adenosylmethionine and SAH metabolism and can indirectly inhibit the methylation reaction. DZNep-induced apoptosis is partially related to its ability to inhibit the PRC2 pathway, although the exact mechanism has not yet been elucidated. In addition, it has been reported that DZNep is synergistic with histone deacetylase inhibitors and DNA methyltransferase inhibitors in the activation of silenced genes64. DZNep has minimal toxicity in vivo as an antiviral compound65, and together with its importance in cancer epigenetic pathways, it may be a promising drug candidate for anti-cancer treatment.

Figure 7.
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PRC2 subunit composition and modes of inhibition. Three types of inhibitors are indicated: SAM competitive inhibitors, SAH, and DZNep as an SAH hydrolase inhibitor.

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SAM-competitive inhibitor

SAH, a universal product of SAM hydrolysis, has a Ki value of 75 μmol/L against EZH2 and an IC50 value of 0.1–20 μmol/L among PMTs. SAH-dependent methylation is involved in many cellular processes66; consequently, SAH has low selectivity against other methyltransferases63. Sinefungin is a nonspecific SAM analog that has similar potency67. Because neither of these compounds shows specificity against EZH2, significant efforts have been made over the past few years to obtain compounds that are potent and highly selective for PRC2 (Table 2)68,69,70,71.


To identify inhibitors of EZH2 methyltransferase activity, high-throughput biochemical screening experiments have been performed with the PRC2 complex. Several potent inhibitors were identified with Ki values in the low nanomolar range. Although the structure of the EZH2 active site has not yet been determined, the conserved SET domain architecture predicts two essential binding pockets: one for the SAM methyl donor and another for the Lys27 substrate. Because more than 50 SET domain proteins have been identified in humans thus far, the selectivity of the inhibitors is crucial for minimizing off-target effects2.

At the end of 2012, several SAM-competitive inhibitors were announced. The compound EPZ005687 has a Ki value of 24 nmol/L and is over 500-fold more selective for EZH2 versus 15 other PMTs and 50-fold more selective for EZH2 versus the closely related enzyme EZH168. EPZ005687 can also inhibit H3K27 methylation by the EZH2 mutants Y641 and A677, and it has been shown to selectively kill lymphoma cells that are heterozygous for one of these EZH2 mutations68.

EI1, another inhibitor of EZH2, was developed by Novartis71 and shows very good selectivity with a low Ki value (approximately 13 nmol/L). Loss of the H3K27 methylation function and activation of PRC2 target genes have been observed in EI1-treated cells. EI1 is equally active against both wild type and the Y641 mutant form of EZH2, and the inhibition of the EZH2 Y641 mutant in B-cell lymphomas leads to decreased proliferation, cell cycle arrest, and apoptosis71.

The most potent inhibitor of EZH2 thus far is GSK126, which has a Ki of 0.5–3 nmol/L70. The selectivity of GSK126 for EZH2 is more than 1000-fold higher than its selectivity for 20 other human methyltransferases containing SET or non-SET domains, and it is over 150-fold more selective for EZH2 than for EZH1. GSK126 effectively inhibits the proliferation of EZH2 mutants in DLBCL cell lines. More importantly, at the animal level, GSK126 markedly inhibits the growth of EZH2-mutant DLBCL xenografts in mice, which was the first animal model for studying the antitumor effects of EZH2. Thus, GSK126 provides a valuable means for evaluating whether EZH2 activity is required for the survival of tumors in which EZH2 overexpression has been linked to poor prognosis. Taken together, these data suggest that pharmacological inhibition of EZH2 activity may be a feasible strategy for treating DLBCLs and non-indolent follicular lymphomas that harbor activating mutations in EZH2.

UNC1999, an analogue of GSK126, is the first orally bioavailable inhibitor that has high in vitro potency against wild type and mutant EZH2 over a broad range of epigenetic and non-epigenetic targets. However, UNC1999 shows less selectivity for EZH1 than the inhibitors mentioned above. Because no crystal structure is available, it is not clear how structural changes contribute to the high selectivity of GSK126 (over 150-fold) versus UNC1999 (approximately 10-fold) for EZH2/EZH1. UNC1999 potently reduced H3K27me3 levels in cells (IC50<50 nmol/L) and selectively killed DLBCL cell lines harboring the Y641N mutation69. Because it is orally bioavailable in mice, UNC1999 could function as a chemical probe for investigating the role of EZH2 in chronic animal studies.

Many competitive inhibitors bind to the SAM pocket; however, a large number of inhibitors can also bind to the protein-substrate binding site, engaging recognition elements within the amino acid channel. BIX-01294 is one such inhibitor (Table 2 and Figure 6) and was found to be a potent inhibitor of G9a with an IC50 of approximately 1.0 μmol/L72.

The SAM competitive inhibitors were also tested against vSET, a viral lysine methyltransferase that is the smallest protein unit capable of catalyzing H3K27 methylation. The sequence identity between the SET domains of EZH2 and vSET is only 23%, and there are significant differences in the active sites of these enzymes73. In addition, vSET folds into a tertiary structure as a homodimer that can methylate H3K2774. Because EZH2 requires at least other two subunits (EED and SUZ12) for its activity, several key differences exist between these two enzymes, including the size of the protein, overall structure, and substrate recognition. Thus, it is not surprising that highly potent inhibitors, such as EPZ005687 and GSK126, are not able to inhibit vSET73. The data clearly reflect significant structural differences between the two enzymes with respect to their SAM binding pockets, which precludes the use of vSET as a meaningful tool for structure-based EZH2 inhibitor design.

In summary, the function of PRC2 as transcriptional repressor or gene activator has been explored as the basis for drug discovery. Although no compounds are currently approved for treatment or clinical trials, much effort has been made to develop EZH2 methyltransferase inhibitors. Because EZH2 is an attractive anti-tumor target, additional research aimed at the discovery and design of EZH2 inhibitors is warranted.

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Future directions

Over the past few years, significant progress has been made in the study of the anti-tumor mechanism of PRC2 and the design of inhibitors against this target. Mechanistic studies have shown that PRC2 catalyzes H3K27 methylation and contains a recognition site for binding to this modification. Additionally, PRC2 also harbors a control module that triggers inhibition of this activity to prevent H3K27 trimethylation in transcriptionally active genes. PRC2 can thus integrate information provided by preexisting histone modifications to accurately tune its enzymatic activity within a particular chromatin context. Nevertheless, important questions still remain.

To fully reveal the functions of each subunit, the structure of the PRC2 complex must be determined; however, this is still a major challenge. At the very least, the core structure of PRC2 (ie, EZH2-EED-SUZ12) should be determined to better understand the interactions between the core subunits . However, the mechanisms of allosteric inhibition need to be addressed by protein dynamics studies (for example, molecular dynamics simulations) to determine how conformational changes could deliver functional effects between the PRC2 subunits. Furthermore, although it is still early in the development of methyltransferase inhibitors for clinical application, drug resistance and low selectivity are common issues of concern for all targeted cancer drugs. The ability to effectively combine the current anti-tumor agents, such as DNA methyltransferase inhibitors and histone deacetylase inhibitors, with EZH2 inhibitors will be a major clinical challenge in the future.

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Acknowledgements

We thank David NADZIEJKA for scientific editing and critical comments. This work was supported in part by the Jay and Betty Van Andel Foundation, Amway (USA), the National Natural Science Foundation of China (NSFC 81123004), and Ministry of Science and Technology (China) grants 2012CB910403 and 2013CB910601.

This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/.