Double sword role of EZH2 in leukemia

Biomedicine & Pharmacotherapy 98 (2018) 626–635

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Double sword role of EZH2 in leukemia a,b

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T c,d

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Sahar Safaei , Behzad Baradaran , Majid Farshdousti Hagh , Mohammad Reza Alivand , ⁎ Mehdi Talebic, Tohid Gharibia,f, Saeed Solalig,a,b, a

Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Department of Immunology, Division of Hematology and Transfusion Medicine, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran d Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran e Department of Medical Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran f Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran g Molecular Medicine Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b c

A R T I C L E I N F O

A B S T R A C T

Keywords: EZH2 Leukemias Gene expression

Enhancer of zeste homolog 2 (EZH2), the core component of the polycomb group complex, plays a major role in normal hematopoiesis. The molecular function of EZH2 is to establish H3K27me3 mark on specific genes by which promotes transcriptional repression of target genes. The activity of EZH2 affects the balance between selfrenewal and differentiation of hematopoietic stem cells. In addition, EZH2 contributes to the cell cycle regulation in mature lymphocytes. A large number of studies have been performed to identify the implication of EZH2 in tumor development of leukemia. Aberrant expression of EZH2 is increasingly recognized in leukemic malignancies. To clarify its therapeutic potential in hematopoietic malignancies it should be determined whether EZH2 is involved in the pathology of these neoplasms. This paper reviews the current knowledge of the role of EZH2 in the pathogenesis of myeloid and lymphoid leukemia. We will discuss the mechanisms in which microRNAs regulate the expression of EZH2 in different types of leukemias that may provide a means to alter cancer epigenetics associated to tumorogenesis to achieve therapeutic benefits.

1. Introduction Transcriptional regulation of gene expression is controlled primarily by the expression of DNA-binding transcription factors and chromatin remodeling [1]. Cellular critical processes such as gene expression, DNA synthesis and cell cycle progression are related to chromatin structure. Epigenetic regulations based on chromatin modifications contributes to gene expression programs in normal and cancerous cells [1,2]. Epigenetic dysregulations are now found as critical events in cancer initiation, promotion and progression [3]. Hypermethylation of DNA within CpG islands is the most well-defined alteration that is

catalyzed by DNA methyltransferase (DNMT)s [3]. Promoter hypermethylation of tumor suppressor genes often occurs in the most human cancers and contributes to loss of tumor suppressor activity in these cancers [4]. In addition, there is growing evidence that histone associated chromatin modifications and their relevant enzymes are involved in tumor development [5]. A large number of histone modifications have been recognized to play a role in cancer. Polycomb group (PcG) proteins are a group of proteins which influence chromatin structure by specific histone alterations. PcG genes were initially discovered in Drosophila but then human homologs were also found with functions like Drosophila homologs. Polycomb-repressive complex

Abbreviations: EZH1,2, enhancer of Zeste 1 and 2; SUZ12, suppressor of zeste 12 homolog; EED, embryonic ectoderm development; AEBP2, adipocyte enhancer-binding protein 2; RBAP46/48, retinoblastoma binding proteins 46 and 48; PRC, polycomb-repressive complex; PcG, Polycomb group; HDAC, histone deacetylase; HSC, hematopoietic stem cells; MPNs, myeloproliferative neoplasms; MDS, myelodysplastic syndrome; AML, acute myeloid leukaemia; ASXL1, additional sex combs like transcriptional regulator 1; TET2, ten-eleven translocation 2; MECOM/EVI1, MDS1 and EVI1 complex locus protein EVI1/ ecotropic virus integration site 1 protein homolog; U2AF1, U2 small nuclear ribonucleoprotein auxiliary factor; SF3B1, splicing factor 3B subunit 1; SRSF2, splicing factor arginine/serine-rich 2; CMML, chronic myelomonocytic leukemia; TKI, tyrosine-kinase inhibitor; STAT, signal transducer and activator of transcription; PI3K, phosphatidylinositol-45-bisphosphate 3-kinase; MAPK, mitogen-activated protein kinase; ERKs, extracellular signal–regulated kinases; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; MLL, mixed lineage leukaemia; C/EBP, CCAAT-enhancer-binding protein; DOT1L, disruptor of telomeric silencing 1-like; ESCs, embryonic stem cells; B-ALL, B-cell acute lymphoblastic leukemia; PTEN, phosphatase and tensin homolog; RYBP, RING1 and YY1-binding protein; SETD2, SET domain containing 2; EP300, E1A-associated protein p300; CDKN2A, cyclin-dependent kinase Inhibitor 2A; IL-7R, interleukin-7 receptor; 3′-UTR, three prime untranslated region; TGF-β, transforming growth factor beta; LSC, leukemia stem cell; siRNA, small interfering RNA; shRNA, small hairpin RNA ⁎ Corresponding author at: Department of Immunology, Division of Hematology and Transfusion Medicine, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. E-mail address: [email protected] (S. Solali). https://doi.org/10.1016/j.biopha.2017.12.059 Received 17 October 2017; Received in revised form 11 December 2017; Accepted 14 December 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.

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3. The correlation of EZH2 with myeloid leukemias

(PRC) 1 and 2 are two well-characterized PcG proteins present in mammalian cells. Both PRCs have essential repressive roles on many genes which encode for transcription factors important for development of embryonic stem cells [6]. The member of PcG proteins mostly implicated in human cancer pathogenesis is EZH2, the functional catalytic subunit of PRC2 complex [7]. EZH2 is believed to regulate gene expression at transcriptional level by altering chromatin conformation, nucleosome modification and via interactions with transcription factors [8]. The EZH2 protein serves as a histone methyltransferase that catalyzes H3 methylation on lysine 27 when assembled in PRC2 complex. This process requires two other components including suppressor of zeste 12 homolog (SUZ12) and embryonic ectoderm development (EED) to form the complete multimeric structure [9]. Aberrant activity of PRC2 as a result of over expressed EZH2, has been frequently seen in a wide range of human cancers including breast and prostate tumors [10]. However, leukemia-associated epigenetic aberrations are not fully understood [7]. Herein, we will overview the current knowledge of epigenetic alterations associated with PRC2, in particular EZH2 in leukemic malignancies. We also discuss on therapeutic agents targeting EZH2 and mechanisms in which microRNAs (miRNAs) control the expression of EZH2. Elucidation of these mechanisms may propose additional treatment approaches to overcome EZH2 aberration in leukemias.

Our understanding of the genetic basis of myeloid malignancies has been profoundly improved in recent years. Studies have revealed new recurrent somatic mutations in myeloid malignancies, including myeloproliferative neoplasms (MPNs), myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Mutations in several tumor suppressors and oncogenes were identified in patients with MDS. Epigenetic regulatory genes involved in modifications of histones (EZH2 and ASXL1) have been identified to bear mutations in people with MDS [19]. Conversely, a genetic study investigating mutations in genes involved in epigenetic modifications in 153 patients of Chinese descent with MDS reported that mutations in TET2 (involved in DNA modification) and ASXL1 (a histone modifier) were common whereas EZH2 mutations were rare [20]. However, previously, inactivating mutations of EZH2 have been identified in myelodysplastic syndromes supporting the notion that EZH2 functions as a tumor suppressor in certain cellular contexts [21,22]. Wang et al. found that EZH2 mutations were associated with poor leukemia-free survival but were not an independent predictor of survival in multivariate analyses [20]. In several other studies the clinical effects of EZH2 mutations have also been reported to be associated with decreased survival of patients with myeloid malignancies [21,23–25]. Myelodysplastic syndromes represent many common features with acute myeloid leukemias [26]. At the molecular genetics level, chromosome 7 loss (-7) or deletion on 7q (7q-) is common in both AML and myelodysplastic syndromes and it is an adverse prognostic factor [27]. EZH2, located at Cr.7q36.1 has been recognized as associated gene on chromosome 7 in MDS [22] although AML patients with del7 or 7q deletions were initially found with no mutation in EZH2 [21]. Whereas a recent study has found EZH2 to be downregulated in individuals with chemoresistant AML with -7/7qwho are associated with poor prognosis [28], on the other hand it was reported that loss of EZH2 prevents transformation to AML in EZH2 mutant MDS mouse models [29]. Given that ectopic expression of Hoxa9 has potential to induce transformation to AML, resistance to transformation in EZH2-null MDS cells is believed to be partly related to repression of Hoxa9 and MECOM/EVI1 expression [2930]. Concerning the associated molecular events, it is clear that EZH2 absence can lead to activation of its target genes while EZH2 target genes in EZH2_null MDS cells are at transcriptionally active state, suggesting that the complementary function of Ezh1 may account for this gene repression [29]. In contrast to the studies correlating EZH2 loss with MDS, it has also been shown that EZH2 overexpression leads to myeloproliferative neoplasms [31]. Similarly, Xu et al. reported that patients with MDS commonly show overexpression of EZH2, which is linked with unfavorable prognosis and pathogenesis of myelodysplastic syndromes [32]. Nevertheless, most studies suggest that EZH2 loss rather than overexpression is more associated with MDS. EZH2 mutations in myeloid neoplasms, have been described in 10–13% of poor-prognosis myelodysplasia-myeloproliferative neoplasms (MDS/MPN), 13% of myelofibrosis (MF), and 6% of MDS [22]. However, the prevalence and prognostic value of EZH2 mutations in individuals with AML is still uncertain. Apart from MDS-derived AML, it is reported that the frequency of EZH2 mutations was 1.8% in an analysis of 714 patients with de novo AML [33] which implies that loss of function mutations of EZH2 are rare in de novo AML. Among patients analyzed in this study, nonsense, frame shift, and missense mutations were detected in 7.1%, 28.6% and 64.3% of all of EZH2 mutations, respectively, displaying a heterozygous pattern [33]. To our knowledge, although mutations of EZH2 appears to be a rare event, there is a clinical evidence describing that specific cases of childhood AML might harbor EZH2 mutations [34]. So larger studies are required to assess EZH2 mutations in childhood AML patients particularly in children carrying t(8;21) translocation [34]. Overall, it is not yet fully clear whether EZH2 is affected and contributes to the disease in MDS and AML patients. Various types of gene mutations and chromosome aberrations in

2. PRC2: the structure, molecular and physiological functions PRC2 is a class of multimeric PcG complexes that is linked to posttranslational histone marks specially H3K27 trimethylation whereby maintains the transcription repression of its target genes [9]. EZH2 is the catalytic component and EED, SUZ12, RBAP46/48 and AEBP2 are the other members of PRC2 complex. Although their exact function has not been well characterized, it is clear that cooperative activity is needed to exert PRC2 function. EED and SUZ12 are required to retain catalytic activity of EZH2 in vitro while the other partners AEBP2 and RBAP46/48 stimulate EZH2 enzymatic activity [9]. H3K27 trimethylation of histone tails by EZH2 allows for the other major class of PcG protein PRC1 to bind to the chromatin and then catalyze monoubiquitinylation of K119 of H2A (H2AK119ub1). Thus it has been proposed that PRC1 acts downstream of PRC2 conferring further suppression of target gene expression [1]. As well as ubiquitination of H2AK119, EZH1, the catalytic subunit of PRC1, harbors the activity of methyl group addition to H3K27 and maintains much more repression of PRC2 target genes [11]. In addition, PRC2-mediated gene repression involves histone deacetylation which leads to a decrease in the expression of particular genes [12]. EZH2 can physically interact with histone deacetylases (HDAC) 1 and 2 proteins and as a result, deacetylation of histones on specific residues can occur [10]. EZH2 is a regulator of the balance between self-renewal and differentiation thereby contributes to the destination of cell division [13]. EZH2 has important roles in embryonic development [14]. It has also been shown to be implicated in maintenance of long-term self-renewal capacity in hematopoietic stem cells (HSC) by turning off the expression of differentiation genes through stabilization of chromatin structure [15] and promoting cell proliferation by increasing gene expression programs related to the cell cycle progression [16]. In addition to involvement in early development, EZH2 has predominant role in various physiological processes including X chromosome inactivation, myogenesis and B cell development [11]. In lymphoid compartment, EZH2 is upregulated in proliferating cells such as germinal center B cells, circulating lymphocytes and plasmablasts, indicating its key role in controlling the cell cycle and lymphocyte division [17]. On the other hand, EZH2 is down-regulated during B-cell differentiation and maturation. During lymphopoiesis, EZH2 acts as a key component of early checkpoint mechanism in B cell development whose expression controls transition from pro-B to pre-B cell [18]. 627

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to MLL⁄AF9- transformed leukemia in murine models [53,56,57] and a recent work has shown that inhibition of EZH2 in MLL-induced leukemia has treatment advantages by decreasing leukemia initiating cells (LIC) subpopulations via upregulation of p16 [58]. These results reveal that p16 is a real target of EZH2 and suppression of p16 is required to maintain MLL-derived leukemia. Ueda et al. demonstrated the in vivo efficacy of EZH2 inhibition for the treatment of mice with MLL-derived leukemia in which the survival of mice were prolonged [58]. More recently, concurrent inhibition of both EZH2 and EZH1 using an oral selective inhibitor conferred increased survival in MLL-induced leukemia of murine models, further confirming that targeting EZH2 (and EZH1) is a prospective therapeutic approach in MLL-induced leukemias [59]. Targeting the other polycomb group protein, Bmi1, a component of PRC1, that is required for initiating MLL⁄AF9 leukemia, appeared to have no therapeutic advantages [60], suggesting that EZH2 acts as an important therapeutic target among PcG proteins [58]. Moreover, in the previous studies, 3-deazaneplanocin A (DZNep), an inhibitor of EZH2, was reported to have killing effects on leukemic cells [61,62]. However, pharmacological inhibition of Dot1L, the enzyme implicated in di-methylation of histone H3 on K79, the histone mark that bear dysregulated methylation in MLL fusion leukemia [61,62] was shown to be less effective in mouse models of MLL-AF9 leukemia [63]. On the other hand, the results from serial transplantation experiments on murine models highlighted the importance of EZH2 in cancer progression but not essential requirement for MLL-AF9 AML [56]. It was found that genetic loss of EZH2 restricts, but does not terminate, leukemia growth. MLL-AF9 leukemic cells with loss of EZH2, maintain H3K27me mark on many genes, proposing EZH1 as an alternative factor, as previously [11] was shown in ESCs [56]. In contrast, complete loss of PRC2 function by inactivation of EED fully abrogates self-renewal of leukemia [56]. Overall, the authors declared a role for EZH1 in MLL-AF9 leukemia that needs detailed studies in the future [56]. Murine models of AML were used to identify differentially expressed proteins following treatment with the EZH2 inhibitor, DZnep. Sandow and coworkers demonstrated that inhibition of EZH2 induces leukemia cell cycle arrest through regulation of cyclin-dependent kinases and increased expression of p53, a crucial tumor suppressor that regulates proliferation and survival [64]. Results of an in vitro study in 2015 has shown that administration of arsenic trioxide (ATO), a chemotherapeutic agent, is able to induce EZH2 expression, which inhibits apoptosis and mediates chemotherapy resistance in AML cell lines [65]. Knockdown of EZH2 enhanced the ATO-induced apoptosis of leukemic cell lines, through downregulation of Wnt/βcatenin activation. The Wnt signaling is an important pathway in cancer biology which is implicated in AML development as well as self-renewal and survival of leukemic cells [65]. EZH2 was found to regulate Wnt/βcatenin signaling by promoting GSK-3β phosphorylation. These findings suggest that EZH2 may serve as a potential therapeutic target for myeloid malignancies, in particular AML. In contrast, a recently published literature suggested that restoring EZH2 protein is a therapeutic approach to overcome therapy resistance in AML [66]. Chemoresistance of AML cell lines and primary cells in a mouse model as well as in vitro was induced when EZH2 protein was suppressed. It was detected that proteasomal degradation of EZH2 which involves interaction with CDK1 and subsequent phosphorylation of EZH2 followed by ubiquitination is implicated in decreased expression of EZH2 and chemoresistance [66]. Treatment with inhibitors of CDK1 or proteasome effectively restored the protein levels of EZH2 and redirected the cells to chemosensitivity [66]. The consequences resulted from these studies is controversial and further research is needed to elucidate the role of EZH2 in chemoresistance of AML cells. Since Myelodysplastic syndromes and myeloproliferative neoplasms can progress to AML [67], and as described above, EZH2 mutations are linked with poor survival in MDS but not with increased transformation to AML; opposing role for PRC2 function in chronic and acute myeloid malignancies is indicated, which is supported by the absence of EZH2

EZH2 gene have been described in myeloid neoplasms affecting epigenetic regulation of target genes on H3K27. In addition to homozygous nonsense or frameshift mutations, haploinsufficiency of EZH2 in cases with monosomy 7 is another event in deficient gene expression in myeloid disorders [35]. Another mechanism may be splicing failure of EZH2 pre-mRNA in patients carrying mutations in spliceosome genes. U2AF1, SF3B1 and SRSF2 are components of spliceosome machinery which are frequently mutated in MDS, primary and secondary AML and MDS/MPN leading to EZH2 loss of function similar to former mechanisms [36,37]. Inactivation of the other components of PRC2 analogous to EZH2 such as SUZ12 and EED [22,38,39] involved in gene silencing through trimethylation of H3K27 were also identified in patients with myeloid neoplasms [40,41]. However, SUZ12 and EED deletions or mutations have scarcely been detected in AML, MDS and MPN [40,42]. Loss of EZH2 function as a consequence of nonsense or frameshift mutations has also been detected in a cohort study conducted by Jankowska et al. in chronic myelomonocytic leukemia (CMML) patients, as an early event in cancer development [43]. Although, a recent study in Japan described increased levels of EZH2 in bone marrow mononuclear cells (BMMNCs) isolated from patients with CML compared with normal individuals and Philadelphia chromosome-negative myeloproliferative neoplasms [44]. Moreover, a research investigating gene expression profile of human CML cells revealed that CML cells and their respective leukemia-initiating cells (LICs) are dependent on EZH2 [45]. Notably, expression of EZH2 is sustained by BCR–ABL1 signaling in CML cells as TKIs (imatinib or dasatinib) diminish EZH2 protein levels [45]. A crucial molecular feature of CML is BCR/ABL fusion, the product of a chromosomal translocation that activates cell signaling pathway components and transcription factors such as STAT5, (PI3K)/ AKT, RAS/MAPK/ERK and NF-κB in these malignant cells [44]. The members of STAT family transcription factors include STAT1, 2, 3, 4, 5A, 5B and 6 [46]. It was shown that STAT5A is associated with promoter of EZH2 gene and correlates with an aberrant increase in transcription of EZH2 in Ph-positive leukemia cells, suggesting STAT5A as a positive regulator of EZH2 in leukemia cells [44]. To confirm the requirement of LICs for EZH2, the consequences of EZH2 loss in LICs were assessed in the engineered mouse model of CML [45]. The results showed that inactivation of EZH2 eliminated the functional LICs, impaired initiation and development of leukemia and led to markedly improved survival of mouse [45]. Trithorax (Trx) group proteins have counteracting roles with PcG in setting epigenetic regulation of genes involved in establishment of stem cell state and development [47]. The Trx group member, MLL, maintain HOX gene activity in hematopoietic precursors, partly by H3K4 trimethylation [48]. A high level of HOX gene expression is present in hematopoietic precursors and differentiation of these cells is associated with reduced expression of HOX genes [49]. The most frequent mutation of MLL, is a chromosomal rearrangement which results in a fusion transcript through binding to one of a large spectrum of partner genes, often to AF9 in AML [50]. MLL-fusion AML is characterized with an abnormal differentiation block in myeloid lineage and inappropriate self-renewal capacity in c-Kit/CD117 positive leukemia stem cell subpopulations [51,52]. Studies performed in a murine model have demonstrated that the complex of PRC2, along with MLL-AF9 are responsible for blockade of myeloid differentiation in AML leukemia [53]. The suppression of differentiation is at least partly influenced by HOX genes expression mediated by MLL-AF9, and PcG protein EZH2 regulates C/EBPα and contributes to differentiation block in MLL-fusion leukemia in collaboration with MLL [54]. C/EBPα is a critical factor that controls myeloid differentiation [55]. EZH2 interaction with promoter region of C/EBPα target genes renders suppression of C/EBPα target genes in MA9 cells [54]. Fig. 1 depicts the genetic events resulting to MLL-derived AML transformation. Consistent with this finding, several works reported that genetic depletion of EZH2 promotes primary leukemia cell differentiation and reduces susceptibility 628

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Fig. 1. The role of epigenetic regulating enzymes, EZH2 and MLL in transformation to AML in a mouse model. MLL and EZH2 cooperate to promote leukemia. MLL-AF9 fusion protein can cause a block in myeloid lineage at granulocyte-macrophage progenitor (GMP) stage. The protein Menin recruits wild-type MLL and MLL-AF9 to the promoter of EZH2 and upregulates EZH2 expression. C/EBPα is a transcription factor that drives myeloid differentiation. EZH2 interacts with C/EBPα and suppresses the C/EBPα target genes leading to blockade of MA9 cell differentiation. All together these events hinder myeloid differentiation and enhance self-renewal capacity of progenitors leading to AML transformation. EZH2 inhibition, Menin loss and/or induction of C/EBPα promote primary leukemia cell differentiation.

cell proliferation and prolonged survival. Microarray analysis of gene expression profiling in B-ALL Nalm-6 cells and normal B cells, revealed a higher level of EZH2 expression in Nalm-6 cells than normal B cells and that the protein upregulation negatively affects the expression of some tumor suppressor genes such as p53, p21 and PTEN in these cells. Overexpression of EZH2 in B-ALL cells was further confirmed by quantitative real time PCR and western blot analysis [70]. Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy with accumulation of mature B cells that show apoptosis inhibition [71]. CLL disease is divided into two subgroups based on the observation that the rearranged Ig variable gene in CLL cells can either represent with presence or absence of mutation [72]. Ig-unmutated ((U-CLL)) and Igmutated ((M-CLL)) CLL patients exhibit different clinical courses that further supports the “two diseases” model of CLL disease [73]. The observation that the absence of mutations in IgVH gene is associated with increased ZAP-70 expression and lower levels of ZAP-70 is correlated to mutated rearranged IgVH gene suggests that the two subtypes of CLL can be distinguished with distinct expression of genes such as ZAP-70 [74,75]. It has been shown that overexpression of EZH2 is strongly correlated to high ZAP-70 expression in CLL patients with worse prognosis [76]. In addition, in that study, CLL patients with elevated expression of EZH2 showed high white blood cell count, an indicator of disease progression [76]. Moreover, chromosomal abnormalities which are related to worse prognosis in CLL patients, correlate with higher expression of EZH2 [76]. EZH2 expression at mRNA and protein levels has been found to be significantly higher in (U-CLL) cases with adverse prognosis compared to patients with (M-CLL) and

mutations in de novo AML [7]. Heterogeneity of AML associated with different cytogenetics may be linked with EZH2 gene function based on evidences indicating that MLL-rearranged (t (9;22)) leukemias harbor EZH2 hyperactivity; conversely translocation 8;21 may be accompanied by EZH2 loss of function mutations in specific cases [34,56], suggesting new considerations in therapeutic approaches in myeloid malignancies. More research is needed to fully elucidate the in vivo effects of PRC2 overexpression or inactivation in normal and malignant conditions to improve our knowledge of the role of PRC2 proteins in particular EZH2 in myeloid leukaemogenesis. 4. The correlation of EZH2 in lymphoid leukemia 4.1. EZH2 in B-cell malignancies In the case of lymphoid malignancies, many studies have revealed mutations in several genes involved in epigenetic modulations. Overexpression of mutant EZH2 has been identified in B cell lymphomas; including diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma [39]. Early studies found that eukaryotic histone methyltransferase (Eu-HMTaseI) was upregulated in B-cell leukemia along with disease progression [68]. B-cell acute lymphoblastic leukemia (BALL), the most common malignancy in children, is responsible for 30% of all cancers and 80% of all leukemias [69]. Numerous genetic abnormalities have been identified to drive B-ALL development, including aneuploidies, chromosomal translocations and alterations at specific genes that cause differentiation block in B cell precursor, deregulated 629

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gene is Cdkn2a. Repression of CDKN2A tumor suppressor gene by enzymatic activity of EZH2 has been described to induce murine models of MLL derived acute myeloid leukemia (AML) [56]. While in EZH2 null ETP-ALL leukemias genetic inactivation of Cdkn2a by mechanisms other than the function of EZH2 allows for the establishment of ETPALL in human [95]. Inactivation of cdkn2a by genetic deletion has been scarcely reported ruling out the event of cdkn2a gene deletion in majority cases of ETP-ALL [94,96]. It remains unknown that how this gene is controlled in the absent of EZH2 function. ETP-ALL malignant cells present the phenotype of myeloid and stem cells including CD33, CD117, CD13, and CD34 and are both CD4 and CD8 negative, with gene expression program similar to HSC and myeloid progenitors [97]. EZH2 inactivation in early T cell precursors results in increased expression of stem cell-associated genes such as late HoxA-cluster genes that impedes transition from early thymic precursor to double positive (DP) thymocyte and causes a differentiation block [95,98]. Stem cell associated gene expression program in human leukemia is in causal link with inferior outcomes [99]. ETP-ALL cells have been described with stem cell gene expression profile compared to non-ETP T-ALL cells [94]. Inactivation of EZH2 has revealed to associate with deregulation of signaling pathways in hematopoietic malignancies. There is growing evidences that JAK/STAT signaling pathway is implicated in the pathogenesis of hematopoietic malignancies. A recent work has found a novel mechanism controlling the function of EZH2 by phosphorylation of tyrosine 244 residue by a tyrosine kinase activated downstream of cytokine receptors, JAK3, resulting to a switch in EZH2 activity and increase in proliferation and survival of NK/T-cell lymphoma cells [100]. Inhibition of JAK3-EZH2 interaction in NK/T-cell lymphoma cells via suppression or knocking down of JAK3 lead to increased trimethylation of H3K27 indicating that JAK3 mediated phosphorylation of EZH2 may reduce EZH2 methylation activity [100]. Phosphorylation of EZH2 can cause decrease in its methyltransferase activity, while increase the cancer cell growth in NK/T-cell lymphoma. Moreover, mutations of JAK genes and IL-7R have been described in T cell lineage ALL [94,101]. Notably, in human ETP-ALL frequently generated signaling abnormalities (SH2B3 alterations and FLT3-ITD) have been linked to overactive STAT3 signaling [102,103]. A recent study indicated that increased phosphorylation of STAT3 is linked to inactivation of EZH2 which leads to higher expression of IL-6Ra and hyperactivation of STAT3 in response to IL-6 [95]. Importantly, STAT3 hyperactivation together with stem cell like transcription pattern are two major factors associated with poor prognosis independently [95]. Taken together, these data indicate that aberrant expression of EZH2 contributes to leukemogenesis in B cell and T cell lineages in different manners. Table 1 summarizes the results from studies investigating the EZH2 expression in patient samples of different leukemias.

indolent disease course, indicating an association of increased EZH2 expression and poor prognosis in CLL. Since a subset of (M-CLL) with aggressive features has a low level of EZH2 similar to the less aggressive subsets of (M-CLL), it is suggested that EZH2 expression is not the sole mechanism of aggressiveness in CLL [77]. EZH2 expression has also been shown to associate with CLL cell proliferation and resistance to apoptosis in (U-CLL) clones [77]. To our knowledge acute and chronic B cell malignancies are associated with high expression of EZH2; in particular, EZH2 overexpression is related to aggressive features and inferior outcome in these cases. 4.2. EZH2 in T-cell malignancies Adult T-cell leukemia/lymphoma (ATLL) is a rare peripheral T cell malignancy with aggressive features that associates with the human Tcell lymphotropic virus 1 (HTLV-1) infection which can represent as a leukemia or lymphoma [78]. ATL is subclassified into four subtypes: smoldering type and chronic type as indolent subtypes, and acute and lymphoma types as aggressive subtypes [79]. ATL clones significantly express elevated levels of EZH2 and RYBP (RING1 and YY1 binding protein) transcripts than normal CD4-positive T cells from control groups [80]. ATL cells from acute subtype of disease show higher levels of EZH2 transcripts than chronic type ATL cells, which implies that aberrant expression of PcG proteins plays essential role in disease progression in addition to tumor initiation of ATL [80]. During normal development of T cells, hematopoietic progenitor cells in a precisely controlled process migrate from the bone marrow to the thymus and then early thymocytes mature toward functional T cells. This normal process can transform into neoplastic growth through oncogenic cooperation between oncogenes and tumor suppressor genes which can drive uncontrolled expansion of early T cell progenitors and lead to T-cell acute lymphoblastic leukemia [81]. ALL comprise the main cause of mortality from disease in children and young adults [69,82]. Approximately 10–15% of children with ALL tend to present with T cell phenotype [83]. Studies have revealed several genetic alterations in cell signaling pathways and cell cycle components in 75 percent of pediatric ALL patients [84–87]. Epigenetic regulating gene aberrations in pediatric ALL cases have drawn attention in recent years. Previous studies have described that inactivation of EZH2 could drive T-cell leukemia [88] and MDS/MPN-like conditions in mice [89]. However molecular investigation of PRC2 genes, EZH2, SUZ12 and EED in 152 childhoods ALL patients did not identified high incidence of EZH2 mutations in pediatric ALL and early T-cell precursor (ETP)-ALL cases and also found no mutation in SUZ12 and EED genes [90]. This finding was in contrast with data from adult T-ALL which reported SUZ12 mutations more frequently (in 4.4%) in these patients [91]. Although mutations in PRC2 genes appeared to be a rare event in childhood ALL, clinical investigation of specific EZH2 mutant cases revealed association of EZH2 mutation and poor prognosis [90]. For the first time Schäfer et al. reported the promoter hypermethylation as a mechanism of EZH2 loss of function and low level of H3K27 trimethylation in ALL which needs larger studies for further evaluation [90]. Therefore, in malignant cells, loss of EZH2 function may result from genetic and epigenetic events. However, a recent study by Angelo et al. showed contrasting results with previous studies indicating an overexpression of EZH2 and EED in samples of pediatric patients with T cells ALL and a lower probability of disease free survival (DFS) in these cases [92]. Genomic characterization of early T-cell precursor (ETP) ALL as a high risk subtype of T-ALL associated with poor prognosis [93] detected activating mutations in RAS signaling and cytokine receptor with impaired hematopoietic development and mutations inactivating histone modifying genes such as EZH2, EED, SUZ12, SETD2 and EP300 [94]. In particular, genetic EZH2 alterations are in relation to inferior outcomes in this neoplasm. There is a paucity of information on the identification of PRC2 and the associated genes in human T-ALL. A known PRC2 and PRC1 target

5. Targeting EZH2 in leukemia The most common therapeutic strategy to inhibit EZH2 is DZNep, which can affect the cellular EZH2 protein amount. It is a pharmacological approach to decrease EZH2 protein level in cancer cells [104]. DZNep provides depletion of EZH2 through SAH-hydrolase inhibition. Elevation in intracellular concentration of SAH is a negative feedback mechanism leading to PRC2 complex degradation. Therefore, DZNep is not a specific inhibitor of EZH2, since EED and SUZ12 components are also affected by DZNep [105]. Due to the appropriate selective effect of DZNep on cancer cells, it is a suitable therapeutic agent. DZNep is able to arise de-repression of PRC2 target genes and induce apoptosis in brain, breast, colorectal, liver, lung, and prostate cancer cells with no toxicity as normal CD4 + T cells were not sensitive to DZNep [106,107]. Studies have shown that ATL cell lines treated with DZNep also display attenuated proliferation. Treatment of T-ALL cell lines with DZNep showed similar sensitivities, indicating the notion that DZNep toxicity toward lymphoma and leukemia cells is not strictly related to 630

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Table 1 EZH2 in patients with different leukemias. Sample size

Malignancy

EZH2 gene/protein status

Reference

153 patients of Chinese descent with MDS 614 individuals with myeloid disorders 102 subjects individuals with chemoresistant AML with -7/7q54 MDS patients 714 patients with de novo AML childhood AML (a case report) 72 well-characterized patients with CMML 12 individuals in the chronic phase of CML 15 patients acute type 22 cases, chronic type 19 cases 152 childhood ALL patients 20 pediatric patients with T cell ALL 12 ETP ALL cases & 94 T-cell ALL cases

MDS MDS MDS AML MDS AML AML CMML CML B-CLL ATL pediatric ALL & ETP-ALL T-ALL ETP-ALL

Inactivating mutations Inactivating mutations Inactivating mutations Downregulation Overexpression Loss of function mutation Somatic mutation Loss of EZH2 function Overexpression Overexpression Overexpression Inactivating mutations Overexpression Inactivating mutations

Wang et al [18]. Ernst et al [19]. Nikoloski et al [20]. Göllner et al. [26] Xu et al [30]. Wang et al [31]. Ernst et al [32]. Jankowska et al [41]. Nishioka et al [42]. Harikrishnan et al [65]. Sasaki et al [77]. Schäfer et al [87]. Angelo et al [89]. Zhang et al [91].

regulating EZH2. miRNAs are a large class of conserved regulatory RNAs that participate in translational repression of messenger RNA controlling target gene expression [121]. Growing evidences have indicated defects in miRNA in different tumor types in human that are linked to patient’s survival and disease outcome [122,123]. miRNAs have emerged to exert either oncogenic or tumor suppressor function and appear to have important roles in cancer development [124]. It has been reported that miR-101 functions as a tumor suppressor in nonsmall cell lung cancer (NSCLC) and suppresses the expression of EZH2 via binding to 3´-UTR end of EZH2 in NSCLC cells [125]. Similarly, downregulation of miR-101 has been reported to associate with upregulation of EZH2 in bladder and prostate cancers [126]. ATL cells show decreased levels of miR101 expression compared to cells from HTLV-1 carriers, the phenomenon that is not caused by genomic loss of miR-101 gene, while, in contrast such a decrease is caused by genomic loss of the miR-101 gene in prostate cancer [80]. Decreased expression of miR101 promotes overexpression of EZH2, offering miR-101 as a negative regulator of EZH2 in ATL cells. In that study miR-128a showed clearly the same pattern like miR-101, as underexpression of miR-128a also correlated with increased expression of EZH2 in ATL [80]. Sequencing of 3′-UTR of EZH2 mRNA revealed two predicted binding site for miR101 and one for miR-26a [127]. By same analyzes it has been found that there is also one target site for miR-128a near the miR-101 target sites [80]. Nevertheless, miR-26a was shown with no decrease in ATL cells and no correlation was observed between miR-26a and EZH2 expression [80]. However, in normal physiological conditions miR-26a was associated with EZH2 in cell differentiation [80]. More recently Correia et al. reported that miR-101 is decreased in T-ALL patient and T-ALL cell lines and may be implicated in pathology of acute T-lymphoblastic leukemia [128] that was further supported by Qian et al. [129]. Hyperactivation of Notch1 pathway can give rise to transformation into T-ALL in murine models. In more than 50% of acute T-cell lymphoblastic leukemias oncogenic activating mutations of Notch1 have been detected and inactivation of Notch1 signaling impairs leukemia cell proliferation and promotes apoptosis in the T-ALL cells [129]. Qian et al. identified that Notch1 is directly regulated by miR-101 [129]. These data indicate that a critical mechanism underlying miR-101 effects on T-ALL is at least in part through suppression of Notch1. Moreover, in vitro experiments demonstrated that miR-101 is related to chemoresistancy of Jurkat cells to Adriamycin, a chemotherapy agent [129]. Thus, miR-101 can be considered as a potent target for therapy in T-ALL. It is reported that miR26A1 is altered in several cancer types for example Burkitt’s lymphoma and thyroid anaplastic carcinoma operating as a tumor suppressor [130,131]. It was also reported that several signaling pathways, such as p53 and TGF-β signaling, and some tumor-associated genes including SMAD1 [132] and EZH2 [130] are regulated by miR26A1. Apparent effects of high EZH2 expression on adverse prognosis and aggressiveness of B cell leukemias bring forward

histone modification .[81]. In acute myeloid leukemia, DZNep mediates apoptosis by inhibiting thioredoxin activity, reactivating TXNIP and increasing reactive oxygen species [108]. The multiple action of DZNep, limits its use as a targeted probe to study EZH2 in PRC2 complex [109]. GSK343 is an efficient blocker of EZH2 demonstrated in cellular and enzyme assays [110] which exhibits a high potency and more selectivity against EZH2 much more than its selectivity against other methyltransferases [109]. The other inhibitor of EZH2, GSK126 has been found to be the most potent inhibitor so far. This small-molecule component displays [111] activity against EZH2 by a mechanism other than PRC2 protein degradation [112]. GSK126 demonstrate good inhibitory activity on DLBCL cell lines bearing EZH2 mutation through proliferation arrest and show intensive effects in EZH2-mutant DLBCL bearing mice of xenograft models [111]. EZH2 interaction with HADC1 and HDAC2 through the EED subunit is known to recruit histone deacetylases [113,114]. Given that PRC2mediated repression of transcription may be partly dependent on the function of HDACs in specific cell contexts [115], the leukemia cell treatment with hydroxamic acid analogue pan-HDAC inhibitors (HAHDI), results in decreased PRC2 components and downregulated HMTase activity of EZH2 leading to lower level of H3K27di and trimethylation [116]. PRC2-mediated recruitment of HDACs to H3K27 [117], can also promote acetylation of H3K27 following depletion of the PRC2 components by HA-HDI and/or siRNA [116]. Because of multi subunit construction of the PRC2 complex, new strategies for targeting EZH2 would comprise novel designs including inhibition of critical subunit interactions and/or EZH2 active site. In recent works, intervention with the interaction between EZH2 and the other PRC2 proteins has emerged to be a more potent specific therapeutic approach in various tumors. Stabilized alpha-helix of EZH2 (SAH-EZH2) peptides are able to disrupt molecular interactions of the PRC2 complex through which decrease the EZH2 protein thereby leading to decreased activity of histone methylation which induce growth inhibition and differentiation in MLL-AF9 leukemic cells [118]. Similarly, Astemizole is a small inhibitor that prevents EZH2 binding to EED in a competitive manner and can contribute to cell cycle arrest in lymphoma cells [119]. Given that DZNep is a non-specific inhibitor of EZH2, which limits its clinical use, and that GSK343 a highly selective inhibitor of EZH2 also display selectivity against EZH1, still effort is under way to identify new molecules able to target EZH2 and no FDA approved drug have been developed in hematopoietic malignancies [120]. Therefore, development of new strategies through modulation of miRNAs implicated in suppression or enhancement of EZH2 may open new opportunities in therapeutic modalities for cancer. 6. MiRNA-mediated regulation of EZH2 in leukemia Recently miRNAs (miR) have been known to play key roles in 631

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Table 2 miRNAs regulating EZH2 expression in leukemias. . miRNA

Malignancy

miR expression

Effects

miR-101 miR-128a miR26A1 miR-101 miR-217

ATL ATL B-CLL CLL K562DR cells

Downregulated Downregulated Downregulated Downregulated Downregulated

Overexpression Overexpression Overexpression Overexpression Overexpression

References of of of of of

EZH2 EZH2 EZH2 EZH2 EZH2

Sasaki et al. [77]. Sasaki et al [77]. Kopparapu et al. [133]. Papakonstantinou et al. [134]. Nishioka et al. [42].

independent from STAT5 signaling. Notably, miR-217 expression level in K562DR cells were less than those in parental K562 cells. As recent studies report that EZH2 is a direct target of miR-217 in gastric cancer cells [144], this miRNA may exert modulating impacts on expression of EZH2 in K562DR cells [44]. Taken together, data show that BCR/ABL work as an important factor which positively regulates the expression of EZH2 in an STAT5-dependant manner and supports the leukemic cell survival. Thus EZH2 may be a useful target for targeted eradication of CML cells. Table 2 gives a summary of the miRNAs which regulate the expression of EZH2 in leukemia.

identifying the miRNAs that potentially mediate regulation of EZH2 and provide novel therapeutic approaches for treatment of B cell leukemia. The expression level of miR26A1 in patients with CLL is lower than that in normal B cells [133,134]. Indeed, it was shown that two subtypes of CLL, mutated and unmutated subgroups, have different methylation state of miR26A1 promoter. Severe forms of disease with IgV-unmutated gene and poor prognosis represent with significantly higher methylation level as shown in a number of high-throughput studies [133,135–137]. These findings suggest that DNA hypermethylation of miR26A1 leading to gene silencing, appears to be an epigenetic regulating mechanism associated with EZH2 overexpression in pathobiology of CLL disease [137]. Indeed, miR26A1 overexpressing cell lines, display decreased level of EZH2 which result in induction of apoptotic cell death confirming the important role of miR26A1 in tumor suppression in this malignancy. Interestingly, loss of miR-101 is implicated in EZH2 overexpression in aggressive CLLs [138]. Despite the CLL cells, miR-101 is upregulated in chronic phases of individuals with chronic myeloid leukemia [139]. However, in the case of acute myeloid malignancies downregulation of miR-101a was shown in MLL-rearranged AML stem cells and ectopic expression of this miRNA disrupted LSC functions through altering epigenetic profile and suppressing survival signaling which leads to leukemia stem cell apoptosis. Thereby induction of miR-101a overexpression may develop a new strategy to eradicate LSCs with drug-resistance properties [140]. Whereas miR-26a function as a tumor suppressor miRNA in AML, experimental data showed that EZH2 is not a direct target of miR-26a in acute myeloid leukemia cells [141]. In myeloid leukemia cells, EZH2 is transcriptionally regulated by c-Myc, causing cancer by enforcing E2F1 activity and participating in the increased EZH2 level in leukemia cells [141]. Chemotherapy resistance and relapse are probably the most problematic issue in the treatment process of patients with acute myeloid leukemia [28]. More recently, Göllner et al. reported that loss of EZH2 and reduction of histone methyltransferase activity with subsequently decreased level of H3K27 trimethylation contribute to chemoresistancy in AML patients with del(7)/del(7q) [28]. Noticeably, reducing the protein levels of EZH2 via treatment with H3K27 methyltransferase inhibitors or lentiviral knockdown, revealed chemoresistant properties in Normal Karyotype (NK)-AML blasts and cell lines in vitro and in a xenograft mouse model [28]. These results suggest that EZH2 protein deposition for example via proteasome inhibitors may provide a means for restoration of EZH2 function and thereby may be hopeful to overcome drug-resistance in AML. Moreover, long term treatment of leukemia cells with imatinib activates AKT, ERK and STAT5 signaling pathway and induce upregulation of EZH2 protein in patients with CML, Ph + ALL and human eosinophilic leukemia cell line (EOL-1 cells) [142]. So, resistance to tyrosine kinase inhibitor (TKI) imatinib in leukemia cells is mediated by the expression of EZH2 [142] suggesting that in particular conditions anti-epigenetic agents may be a promising approach to improve therapy response in patients receiving tyrosine kinase inhibitors. This group have previously established K562 cells resistant to dasatinib (K562DR) in which both BCR/ABL and STAT5 and not ERK and AKT, were inactivated in contrast to parent K562 cells [143]. K562DR cells were also resistant to growth inhibition by imatinib and increased EZH2 was

7. Conclusion Cell fate decision is regulated by genetic and epigenetic regulation of gene expression. In particular dynamic chromatin remodeling mediated by polycomb repressive complex controls cellular transcription profile through modification of specific histone residues. PRC2 activity promotes the cellular processes by altering the expression of functional genes that participate in lineage commitment, differentiation and cell cycle progression. Accumulated evidence demonstrates that dysregulation of EZH2, the catalytic subunit of the complex, as a master regulator of chromatin is frequently observed in a majority of human cancers particularly hematopoietic malignancies. Aberrant expression of EZH2 differentially contributes to the tumor initiation in different types of leukemia. EZH2 acts as a double facet factor either as a tumor suppressor or oncogene in acute and chronic leukemias of myeloid and/ or lymphoid origin which suggest the complexity of EZH2-mediated regulation of mammalian gene expression. Several molecules are implicated in the regulation of EZH2. MiRNA-dependent regulation of EZH2 is involved in multiple cancers. Therefore, specific strategies for modulation of EZH2 by manipulating miRNAs regulating the expression and function of EZH2 protein may lead to production of more specific and effective therapeutic agents to achieve a precise therapy. Pointing to the multi-faceted role of EZH2 in different types of leukemia, development of context specific strategy for example by using siRNAs or shRNAs [145] could be considered in future personalized targeted medicine for leukemia. However, to establish the appropriate therapy we should elucidate the true EZH2 dependency of tumor cells by a deeper understanding based on large scale genetic and epigenetic studies. References [1] K. Lund, P. Adams, M. Copland, EZH2 in normal and malignant hematopoiesis, Leukemia. 28 (1) (2014) 44–49. [2] M.R. Alivand, Z.S. Soheili, M. Pornour, S. Solali, F. Sabouni, Novel epigenetic controlling of hypoxia pathway related to overexpression and promoter hypomethylation of TET1 and TET2 in RPE cells, J. Cell. Biochem. 9999 (2017) 1–12. [3] M. Rahmani, M. Talebi, M.F. Hagh, A.A.H. Feizi, S. Solali, Aberrant DNA methylation of key genes and acute lymphoblastic leukemia, Biomed. Pharmacother. 97 (2018) 1493–1500. [4] M. Esteller, Cancer epigenomics: DNA methylomes and histone-modification maps, Nat. Rev. Genet. 8 (4) (2007) 286–298. [5] P.A. Marks, R.A. Rifkind, V.M. Richon, R. Breslow, T. Miller, W.K. Kelly, Histone deacetylases and cancer: causes and therapies, Nat. Rev. Cancer 1 (3) (2001) 194–202. [6] L.A. Boyer, K. Plath, J. Zeitlinger, T. Brambrink, L.A. Medeiros, T.I. Lee, et al., Polycomb complexes repress developmental regulators in murine embryonic stem cells, Nature. 441 (7091) (2006) 349–353.

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