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Leukemia (2014) 28,44–49 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu

CONCISE REVIEW EZH2 in normal and malignant hematopoiesis

K Lund1, PD Adams2 and M Copland3

The methyltransferase Enhancer of Zeste Homologue 2 (EZH2), a component of the polycomb group complex, is vital for stem cell development, including hematopoiesis. Its primary function, to deposit the histone mark , promotes transcriptional repression. The activity of EZH2 influences cell fate regulation, namely the balance between self-renewal and differentiation. The contribution of aberrant EZH2 expression to tumorigenesis by directing cells toward a cancer stem cell (CSC) state is increasingly recognized. However, its role in hematological malignancies is complex. Point mutations, resulting in gain-of- function, and inactivating mutations, reported in lymphoma and leukemia, respectively, suggest that EZH2 may serve a dual purpose as an oncogene and tumor-suppressor gene. The reduction of CSC self-renewal via EZH2 inhibition offers a potentially attractive therapeutic approach to counter the aberrant activation found in lymphoma and leukemia. The discovery of small molecules that specifically inhibit EZH2 raises the exciting possibility of exploiting the oncogenic addiction of tumor cells toward this protein. However, interference with the tumor-suppressor role of wild-type EZH2 must be avoided. This review examines the role of EZH2 in normal and malignant hematopoiesis and recent developments in harnessing the therapeutic potential of EZH2 inhibition.

Leukemia (2014) 28, 44–49; doi:10.1038/leu.2013.288 Keywords: EZH2; hematopoiesis; cancer stem cell; lymphoma; EZH2 inhibitors

INTRODUCTION EZH2 AND THE POLYCOMB GROUP COMPLEX Strictly, is defined as a collection of heritable EZH2, a polycomb group (PcG) protein, functions in concert with mechanisms capable of altering genome function other than by other proteins (EED, SUZ12 and RBBP4), which together form the direct alteration of the DNA sequence. Here we use ‘epigenetic’ multi-subunit polycomb repressive complex (PRC)-2. PRC2 acts via more loosely to refer to elements of structure that a number of mechanisms to initiate polycomb-mediated gene control genome function, regardless of whether the control is repression (Figure 1). The mechanisms include direct inhibition of heritable. Epigenetic or chromatin-based regulation contributes to transcriptional machinery via RNA polymerase II and chromatin the control of in normal development and cancer compaction, which inhibits the access and action of states. factors.9 EZH2 has also been suggested to facilitate DNA Histone proteins, which are responsible for packaging and , by acting as a recruitment platform for DNA ordering DNA into the nucleosomal subunits that comprise methyltransferases.10 Importantly, following trimethylation by chromatin fibers, are frequently covalently modified at their the suppressor of variegation 3–9, enhancer of zeste and N-terminal tails. These histone ‘marks’ are defined by the histone, trithorax (SET) domain of EZH2, the histone mark H3K27me3 the modified amino acid, its position in the polypeptide chain and functions as a docking site to recruit a second polycomb complex the nature of the modification, for example, methylation or PRC1, which acts to maintain gene repression via ubiquitination of (for example, trimethylation on 27 of H2AK119.2,11 is abbreviated to H3K27me3). These marks are thought to control DNA transcription and other DNA functions, and have become known as the ‘histone code’ or perhaps, more accurately, ‘histone EZH2 FUNCTION IN STEM CELLS language’.1 EZH2 contributes to the regulation of cell fate decisions, Changes to the structure and/or function result orchestrating gene expression to control the balance between from the activity of histone-modifying enzymes. One of the best self-renewal and differentiation.12 EZH2 has its predominant role described is the histone methyltransferase Enhancer of Zeste in embryonic development and becomes downregulated in some Homologue 2 (EZH2), responsible for trimethylating lysine 27 of adult differentiated tissues.13 However, PcG proteins also have an histone H3 to generate H3K27me3.2 This article will focus on the important role in maintaining the multi-potency of adult stem effect of mutations and other modes of dysregulation of EZH2 in cells in various contexts, including hematopoietic tissues.14 In hematological malignancy. EZH2 is one of several chromatin hematopoietic stem cell (HSC) models, at times of replicative regulatory proteins that have prompted great clinical and stress, or as cells age, the balance shifts toward differentiation with scientific interest as they offer the possibility of new therapeutic potential to ‘exhaust’ the HSC pool.15 However, Ezh2 has been targets in cancer.3–8 shown to stabilize the chromatin structure and maintain

1Department of Epigenetics of Cancer and Aging, Institute of Cancer Sciences, University of Glasgow, Cancer Research UK Beatson Labs, Glasgow, Scotland, UK; 2Department of Epigenetics of Cancer and Aging, Institute of Cancer Sciences, University of Glasgow, Beatson Institute for Cancer Research, Glasgow, Scotland, UK and 3Paul O’Gorman Leukaemia Research Centre, Institute of Cancer Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Gartnavel General Hospital, 1053 Great Western Road, Glasgow, Scotland. Correspondence: Professor M Copland, Paul O’Gorman Leukaemia Research Centre, College of Medical, Veterinary and Life Sciences, University of Glasgow, Gartnavel General Hospital, 1053 Great Western Road, Glasgow, G12 0ZD, Scotland, UK. E-mail: [email protected] Received 7 May 2013; revised 1 September 2013; accepted 20 September 2013; accepted article preview online 7 October 2013; advance online publication, 1 November 2013 EZH2 in normal and malignant hematopoiesis K Lund et al 45

RBP48 EED PRC1 PRC 2 SUZ12 EZH2 SET RING1 PH1

PC1 BMI1 H3K27 H2AK119

Me Ub MeC

Figure 1. The interactions and effects of EZH2 in regulation of transcriptional repression. Polycomb complex 2 (PRC2) exerts methyltransferase activity to H3K27 via the SET domain of the EZH2 subunit. This results in transcriptional repression via various mechanisms, including the recruitment of DNA methyltransferases (DNMT) and PRC1, which ubiquitinates H2AK119.

Figure 2. Regulation of EZH2 in normal and cancer states. HDAC, long-term self-renewal potential of HSCs by switching off ; miR, microRNA; PI3K, phosphatidylinositol 15,16 3-kinase; PTEN, phosphatase and tensin homolog. (a) Homeostatic pro-differentiation genes and promoting a shift toward relationship between c-MYC, EZH2 and the PI3K pathway in proliferation by increasing expression of genes, which facilitate 17 controlled cell growth. (b) In c-MYC driven lymphomagenesis, progression through the cell cycle. c-MYC and EZH2 contribute to each other’s upregulation via the activity of miR-26a and miR-494. EZH2 forms a co- complex with HDAC3 and c-MYC to repress miR-29, which drive cancerous EZH2 AND CANCER STEM CELLS (CSCs) cell growth. The existence of the CSC, first described in the context of acute myeloid leukemia (AML),18 remains controversial, but is recognized as a viable explanation for disease propagation. The CSC hypothesis postulates that tumor tissue comprises a turn suppresses Akt. The inactivation of Akt causes an increase in heterogeneous population of cells and only a small Ezh2-mediated gene repression, including negative autoregulation subpopulation, the CSCs, has the ability to self-renew and give of c-Myc (Figure 2a).28 rise to malignant progeny. In the leukemia stem cell, EZH2 may In certain cancer states, an alteration in the relationship potentially contribute to tumorigenesis by suppressing between EZH2 and c-MYC has been observed.29 In c-MYC driven differentiation genes, thereby directing ‘healthy’ cells toward a lymphomagenesis, c-MYC and EZH2 contribute to each other’s stem cell state.19–22 This makes aberrantly expressed EZH2 an upregulation via the activity of microRNAs. c-MYC represses EZH2 attractive therapeutic target; for example, in breast and pancreatic via miR-26a and EZH2 suppresses the c-MYC-targeting miR-494 to cancer, its inhibition has the potential to deplete the CSC pool.23 maintain high levels of both proteins (Figure 2b). EZH2 forms a PcG target genes identified in embryonic stem cells (ESCs) are co-repressor complex with HDAC3 and c-MYC to repress miR-29, in more likely to be DNA hypermethylated in cancers, as shown in turn facilitating the expression of miR-29 target genes, which drive colon and embryonic carcinoma cell lines.24–26 Perhaps aggressive mantle cell lymphoma growth. Aberrant microRNA accounting for this, is evidence suggesting that EZH2 recruits expression has been shown to regulate EZH2 in other cancer DNA methyltransferases to the promoter region of PcG target contexts; for example, dysregulation of miR-101 and miR-144 is genes resulting in the silencing of PcG targets, including tumor- linked to overexpression of EZH2 in bladder and prostate cancer, suppressor genes.10 This indicates strong cross-talk between respectively.30,31 histone and DNA methylation resulting in a ‘double-lock’ repressive effect. There is further evidence to indicate that this combined epigenetic repressive effect becomes established in response to cancer-promoting DNA damage generated by EZH2 IN HEMATOLOGICAL CANCERS reactive oxygen species.27 By implication, aberrant EZH2 activity, EZH2 ‘gain-of-function’ ESC gene and/or chromatin signatures and reactive oxygen The occurrence of mutations within chromatin regulators in species (or other stresses) conspire to lock adult somatic cells cancer is linked to inappropriate gene expression and genomic into an aberrant CSC state. instability. Overexpression of wild-type (WT) EZH2 has been described in several non-hematological cancers, including prostate and breast cancer32,33 and evidence demonstrate that REGULATORS OF EZH2 IN CANCER this increased expression promotes tumor progression and EZH2 is controlled by a complex array of mechanisms, which may metastasis in these cancers.34 become dysregulated in different cancer types. For example, in In hematological malignancies, overexpression of mutant the healthy state, a co-dependent relationship exists between EZH2 has been recognized in a wide selection of B- and T-cell Ezh2 and c-Myc, which maintains a homeostatic balance, thereby lymphoproliferative disorders.35–38 One landmark study described promoting normal growth; c-Myc indirectly enhances Ezh2 activity a large cohort of diffuse large B-cell lymphoma (DLBCL) and via the stimulation of the PI3K pathway regulator Pten which in follicular lymphoma cases with EZH2 variants. ‘Gain-of-function’

& 2014 Macmillan Publishers Limited Leukemia (2014) 44 – 49 EZH2 in normal and malignant hematopoiesis K Lund et al 46 point mutations resulting in a switch from tyrosine to histidine at model, Ezh2 loss perturbed leukemic progression by reactivating codon 641 (Tyr641) in the catalytically active SET domain of the Ezh2-repressed genes involved in myeloid differentiation, such as EZH2 protein were found in 7.2% and 21.7% of the follicular Egr1, and appeared to convert AML into a MDS/MPN type lymphoma and DLBCL cases, respectively.37 All of the DLBCL cases disease.44 This indicates that the loss of Ezh2 function in this were germinal center (GC) cell phenotype and all appeared to be model converts a high-grade myeloid leukemia to a less heterozygous for the variant. This study demonstrated that, in aggressive MPN, indicating that the presence of Ezh2 confers apparent contrast to the increased EZH2 mRNA levels found in oncogenic activity in the myeloid lineage. Recent studies have breast and prostate cancer, Tyr641 mutations were associated indicated that in Mll-AF9 leukemia, Ezh2 exerts its oncogenic with marked reduction in EZH2 enzymatic activity in vitro. It was effects by inhibiting differentiation. Specifically, Thiel et al. have postulated that these mutations may change EZH2 target gene shown that the trithorax protein menin upregulates Ezh2, resulting specificity and so alter DNA methylation at PcG targets in these in inhibition of C/ebpa-mediated differentiation in Mll-AF9 tumors.37 leukemia.21 However, in a later study, the Tyr641 EZH2 mutation was shown Thus, the contrasting effects of Ezh2 overexpression and to confer an aberrant functional interaction between mutant and deletion, respectively, in these two mouse models ultimately WT gene products. Sneeringer et al. showed that the malignant results in a similar phenotype (that is, a MDS/MPN overlap phenotype arising in Tyr641-mutated B-cell lymphoma occurs due syndrome). In both cases, Ezh2 demonstrates oncogenic activity in to altered cooperation between the mutated and WT EZH2 with the myeloid lineage, but its end point clearly depends on cell and an overall ‘hyper-trimethylating’ effect on H3K27me3, resulting in genetic context. gene repression.39 Hence, the EZH2 Tyr641 mutation appears to be an unusual ‘gain-of-function’ mutation, after all. Massive upregulation of EZH2 expression is evident in GC B cells EZH2 ‘loss-of-function’ when compared with naive B-cell controls and interestingly the Although the evidence from human disease and mouse models EZH2 transcriptional regulatory profile in GC B cells appears to be described above indicates that gain of EZH2 H3K27 trimethylating highly correlated with that seen in human ESCs (ESCs).22 activity promotes lymphoid and myeloid cancers, there is also Chromatin immunoprecipitation (ChIP) of EZH2 from GC B cells, data to suggest that, conversely, loss of H3K27 trimethylation, naive B-cell controls and human ESCs, followed by microarray both directly via EZH2 inactivating mutations and indirectly analysis (ChIP on chip), identified promoters bound by EZH2 and via ASXL1 mutations,45 may also contribute to malignant demonstrated the repression of key cell-cycle-related tumor- hematopoiesis.46,47 This indicates that the EZH2 protein also has suppressor genes (CDKN1a, CDKN1b) in both GC B cells and capacity to work as a tumor suppressor in certain cellular contexts. human ESC as compared with naive B cells. In keeping with this, The landmark studies examining recurrent somatic inactivating small interfering RNA knockdown of EZH2 in DLBCL, a GC B-cell EZH2 mutations in MDS/MPN overlap disorders were performed malignancy, led to significant G1/S cell cycle arrest. This by two groups.46,47 Ernst et al. uncovered 49 mutations experimental evidence is complimentary to other reports (approximately one-third of these were homozygous, indicative indicating that EZH2 upregulation drives proliferative potential of inactivation of a tumor suppressor) in a total of 614 individuals and self-renewal in other B- and T-cell lymphoproliferative with myeloid malignancy–most commonly involving myelofibrosis disorders.35–38 The role of EZH2 in GC formation, and its or MDS/MPN overlap syndromes (chronic myelomonoctyic contribution toward GC-type DLBCL, has been attributed to its leukemia and atypical chronic myeloid leukemia). Nikoloski et al. activity at bivalent domains.40 Through these sites mutant EZH2 sequenced the EZH2 gene in 126 patients with MDS, revealing alleles or overexpression of WT EZH2 resulted in repression of somatic frameshift, nonsense and missense mutations throughout proliferation checkpoint and differentiation genes, thereby the gene. inducing GC hyperplasia and lymphomagenesis. Expression of Specific interest has focused on the link between mutant EZH2 in GC cells was unable to induce DLBCL alone, but 7 abnormalities, common in myeloid cancers, and the location of collaborated with BCL6/BCL2 to cause the disease phenotype. the EZH2 gene, present at position 7q36.1.46 On screening, Ernst A correlation is also described between EZH2 overexpression et al. established that EZH2 mutations in the second allele were and myeloid malignancy.41 Xu et al. examined a heterogeneous not particularly associated with chromosome 7 losses or 7q myelodysplastic syndrome (MDS)/AML population known to deletion. However, they did report finding homozygous EZH2 harbor DNA methylation of tumor-suppressor genes, such as mutations in 75% of individuals with acquired uniparental disomy. p15INK4B. Patients with p15INK4B gene methylation had Univariate analysis in MDS/MPN indicated that EZH2 mutations statistically higher mean relative expression of EZH2 compared were associated with both a poorer overall and progression-free with non-methylated counterparts. The level of EZH2 expression survival when compared with cases without mutations (overall correlated positively with poor disease outcome as judged by the survival: 39 months vs 13 months (P ¼ 0.0006); progression-free International Prognostic Scoring System.42 survival: 30 months vs17 months (P ¼ 0.044)).46 The presence of In order to further investigate the mechanisms by which Ezh2 homozygous mutations conferred a trend toward poorer survival may contribute to myeloid malignancy, recent publications have when compared with heterozygotes, although the difference was used murine models of leukemia to assess the function of the not statistically significant. A similar prognostic picture was protein. A conditional Ezh2 ‘gain-of-function’ mouse model uncovered in a cohort of myelofibrosis patients where loss-of- showed that induction of WT Ezh2 expression increases the function EZH2 mutations were found to independently predict a repopulation potential of HSCs.43 Serial transplantation of these poor overall survival.48 Ezh2-overexpressing HSCs resulted in mice developing a Following assessment of a 469 patients with myeloid malig- myeloproliferative neoplasm (MPN), consistent with the nancies, Khan et al. report that EZH2 mutations were present in 8% hypothesis that MPNs arise from a mutation in the stem cell of cases. Some of these mutations, such as mutations in splicing pool. In this model, a number of HSC maintenance genes were factor genes, U2AF1 and SRSF2, cause dysfunctional processing of regulated by Ezh2, including the transcriptional regulators Evi-1 pre-mRNA and reduced EZH2 expression.20 Khan et al. also and Ntrk3, which are often aberrantly expressed in hematological describe how the stem cell self-renewal HOX gene family is a malignancies.43 In another model, Mll-AF9 transformed AML cells major downstream target of EZH2 and how a similar picture of from WT and Ezh2-deficient mice have been studied in vitro and increased HOXA9 expression occurs as a consequence of both in vivo.44 Deletion of Ezh2 compromised cell cycle progression, inactivating EZH2 mutations or as a consequence of deletion of although cells still maintained some proliferative capacity. In this the whole EZH2 locus in patients harboring -7/del7q abnormality.

Leukemia (2014) 44 – 49 & 2014 Macmillan Publishers Limited EZH2 in normal and malignant hematopoiesis K Lund et al 47 They propose that EZH2 ‘loss-of-function’ mutations contribute to deacetylation inhibitors, which are increasingly used in combina- formation of the CSC, via HOXA9-mediated self-renewal of myeloid tion with conventional therapies,52 a number of other novel small- progenitors.20 molecule inhibitors, including EZH2 inhibitors, are in Evidence of EZH2 ‘loss-of-function’ mutations is not limited to development. aberrant myelopoiesis. Ntziachristos et al. identified EZH2 or SUZ12 mutations in 25% of primary T-acute lymphoblastic leukemia (T-ALL) samples.49 Studies reporting the function of EZH2 in EZH2 INHIBITORS human and mouse T-ALL have used various strategies to Given that evidence points to a role for EZH2 in increasing self- demonstrate that loss-of-function PRC2 mutations contribute to renewal in at least some cancers (including some hematological the pathogenesis of T-ALL, which is predominantly driven by malignancies), therapies that inhibit EZH2 may promote exhaus- oncogenic activation of NOTCH1 signaling.49,50 EZH2 silencing is tion of CSC populations.15 Moreover, if EZH2 has non-stem cell- shown to increase the tumorigenic potential and mortality of specific oncogenic functions, for example, promoting cell T-ALL cells transplanted into NOD-SCID mice.49 proliferation or inhibiting differentiation, its inhibition would also A summary of subtype and frequency of EZH2 mutations in be expected to be of therapeutic benefit. Consequently, myeloid and lymphoid malignancies is provided in Figure 3 and considerable effort has been directed toward development of Table 1. The conflicting findings discussed above highlight the EZH2 inhibitors. complexity of epigenetic regulation, illustrating that polycomb 3-Deazaneplanocin (DZNep), the first drug proposed to inhibit proteins, such as EZH2, can have a dual role as either oncogenes EZH2, acts indirectly via competitive inhibition of S-adenosylho- or tumor-suppressor genes, depending on factors such as gene mocysteine hydrolase. Consequently, there is an accumulation of dosage and cell context. These divergent phenotypes suggest that the enzyme substrate adenosylhomocysteine, which in turn the PcG complexes act on a broad range of target genes resulting inhibits methyltransferases, induces degradation of the PRC2 in potentially opposing downstream effects.51 complex and reduces EZH2 levels.4,5 There is evidence to indicate that the drug has a broad inhibitory effect on protein methyltransferases other than EZH2, and is therefore not a well- 4 EPIGENETIC THERAPIES targeted therapy. However, based on murine studies, the use of DZNep may offer the possibility of therapeutic potential as an A key feature of epigenetic mutations in malignancy is their immuno-modulatory drug in the treatment of graft-versus-host relative plasticity, unlike genetic changes to DNA sequence that disease, a common complication of allogeneic stem cell are essentially permanent. This has important clinical relevance, as transplantation for hematological malignancies.53 Histone this feature can make them more amenable to inhibitor therapies methylation modulates inflammatory T-cell responses and the that reverse the epigenetic alterations. In addition to established use of DZNep in a graft-versus-host disease mouse model agents, such as DNA methyltransferease inhibitors and histone activated pro-apoptotic genes in allo-antigen-activated T cells (thereby suppressing graft-versus-host disease), whereas maintaining the anti-leukemic activity of donor T cells. The discovery of EZH2 mutations in lymphoma (particularly DLBCL) has provided a model system/novel target for the development of EZH2 inhibitors.7,8,54,55 In their work describing microRNA function in regulating EZH2, Zhao et al. coupled DZNep first with histone deacetylation inhibitor Vorinostat, and second with bromodomain and extra-terminal (BET) domain inhibitor JQ1.54 The action of this drug combination resulted in disruption of the c-MYC-miR-EZH2-HDAC3 feedback loop (alluded to in Figure 2b), which in turn increased tumor-suppressor miR-29 Figure 3. EZH2 domain structure and positions of missense/point expression, downregulated miR-29 target genes and reduced mutations identified in hematological cancers. CXC, cysteine-rich lymphoma growth. domain; D1, domain I; D2, domain II; GCB, germinal center B cell; MDS/MPN, myelodyplastic/myeloproliferative neoplasms; SET, sup- Qi et al. developed an Ezh2-selective small-molecule inhibitor pressor of variegation 3–9, enhancer of zeste and trithorax domain; EI1, which competitively binds to the S-adenosylmethionine (SAM) T-ALL;T-cell acute lymphoblastic leukemia. Mutational hot spots pocket of the Ezh2 SET domain in both WT and Tyr641 mutated identified at D2 and SET domains, frameshift and nonsense cells.55 This inhibition of histone H3K27me3 led to G1 growth mutations are more equally distributed across the genome (not arrest, apoptosis and differentiation of Ezh2 mutant cells into shown on this figure). memory B cells.

Table 1. EZH2 pathogenic mutations in hematological cancers

Disease Mutation types identified Effect Frequency (%) Reference

DLBCL-GCB Missense Gain of function 14–21.7 37, 56 Follicular Lymphoma 7.2–22 MDS/MPN Missense frameshift, nonsense Loss of function 8–12 20, 46 MPN 3–13 MDS 2.5 AML 2 T-ALL Missense, frameshift, nonsense Loss of function 18 49 Abbreviations: AML, acute myeloid leukemia; DLBCL-GCB, diffuse large B-cell lymphoma-germinal center type; EZH2, Enhancer of Zeste Homologue 2; MDS/MPN, myelodysplastic/myeloproliferative neoplasm; T-ALL, T-acute lymphoblastic leukemia.

& 2014 Macmillan Publishers Limited Leukemia (2014) 44 – 49 EZH2 in normal and malignant hematopoiesis K Lund et al 48 Two other recent publications have demonstrated further development and clinical trials adopting the new agents that advances in the therapeutic potential of EZH2 inhibition to treat selectively target oncogenic EZH2 have yet to be undertaken. lymphoma.7,8 Two compounds, EPZ005687 and GSK126, However, careful patient selection will be imperative in future independently identified by high-throughput screening, inhibit trials to ensure that the potentially contrary effects of such drugs EZH2 using a similar mechanism to that described for EI1. Both are appropriately exploited, reducing tumor growth rather than compounds are highly selective for EZH2 (500- to 1000-fold, accelerating it. compared with other methyltransferases), reduce H3K27me3 levels and increase transcriptional activation. Both drugs have proven efficacy in inducing apoptotic killing of lymphoma cell CONFLICT OF INTEREST lines harboring Tyr641 mutations with minimal effect on WT cells. The authors declare no conflict of interest. Although the EPZ005687 compound has been limited to in vitro use,7 the GSK126 molecule has also been assessed in mouse xenograft models, where it resulted in complete inhibition of ACKNOWLEDGEMENTS tumor growth and significantly increased survival of the mice.8 In MC is supported by the Scottish Funding Council (SCD/04) and Leukaemia and line with these therapeutic advances, the development of a Lymphoma Research (Grant ref: 11017). PA is supported by Cancer Research UK clinically applicable screening assay to detect Tyr641 mutations to (Grant ref: C10652/A10250). We thank Professor Tessa Holyoake for critical review of guide future ‘genotype targeted therapy’ has been reported.56 the manuscript. Pre-clinical studies clearly show that inhibition of EZH2 warrants further investigation in myeloid malignancy.3,6,44 AML represents AUTHOR CONTRIBUTIONS an important disease in which to investigate epigenetic therapies, KL wrote and revised the manuscript. MC and PA revised the manuscript. All authors and there is also increasing evidence for the use of these agents in checked the final version. myelofibrosis and MDS. However, evidence for inactivating EZH2 mutations in myeloid disease indicates a tumor suppressor function for EZH2 in the myeloid lineage, meaning that EZH2 REFERENCES inhibitors must be applied with caution. It will be necessary to 1 Strahl BD, Allis CD. The language of covalent histone modifications. Nature 2000; identify genetic or other biomarkers to distinguish between 403: 41–45. 2 Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P et al. 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