EZH2抑制剂治疗急性髓细胞白血病进展

Acute myeloid leukemia (AML), a malignant disease of the bone marrow, is characterized by the clonal expansion of myeloid progenitor cells and a block in differentiation. The high heterogeneity of AML significantly impedes the development of effective treatment strategies. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the polycomb repressive complex 2 (PRC2), regulates the expression of downstream target genes through the trimethylation of lysine 27 on histone 3 (H3K27me3). Increasing evidence suggests that the dysregulation of EZH2 expression in various cancers is closely associated with tumorigenesis. In the review, we examine the role of EZH2 in AML, highlighting its crucial involvement in regulating stemness, proliferation, differentiation, immune response, drug resistance and recurrence. Furthermore, we summarize the application of EZH2 inhibitors in AML treatment and discuss their potential in combination with other therapeutic modalities. Therefore, targeting EZH2 may represent a novel and promising strategy for the treatment of AML.

The methylation of lysine 27 on histone H3 (H3K27me3) is a chromatin mark associated with nucleosome condensation and gene expression silencing. EZH2 is a lysine methyltransferase that catalyzes H3K27me3. In this issue of Cancer Research, Porazzi and colleagues report that pretreatment with EZH2 inhibitors opened up the H3K27me3-marked chromatin of acute myeloid leukemia (AML) cells, which enhanced DNA damage and apoptosis induced by chemotherapeutic agents, in particular the topoisomerase II inhibitors, doxorubicin and etoposide. The EZH2 inhibitor/doxorubicin combination also enabled the expression of proapoptotic genes, potentially contributing to the death of AML cells. This study has significant implications for improving the efficacy of DNA-damaging cytotoxic agents in AML, thereby enabling lower chemotherapy doses and reducing treatment-related side effects. See related article by Porazzi et al., p. 458

Dear Editor, Despite recent therapeutic advances, overall survival of acute myeloid leukemia (AML) patients remains poor. Cytarabine (Ara-C), in combination with anthracyclines, induces complete remissions in 60 – 80% of AML patients, with only a fraction of them achieving long-term survival, and 60 – 90% of refractory AML patients succumb to the disease. Treatment failure is mainly attributed to the insuf fi cient eradication of cell-kinetically quiescent leukemia stem/progenitor cells (LSPCs) that reside in the bone marrow (BM) niche. Therefore, overcoming the dormancy of LSPCs should improve survival of AML patients [1]. Quiescent LSPCs are dependent on enhancer of zeste homolog (EZH)2 and EZH1, the key catalytic subunits of the polycomb repressive complex 2 (PRC2). Genetic ablation of EZH2/1 in an AML model depleted quiescent LSPCs [2]. In this study, we observed that inhibition of EZH2/1 by valemetostat tosylate (DS-3201b or valemetostat) monotherapy in a phase 1 clinical trial induced proliferation and mobilization of immature blasts into circulation in AML patients. Preclinical experiments in vitro and in vivo recapitulated the fi ndings and indicated the therapeutic potential of valemetostat in combination with Ara-C. We assessed blast counts in AML patients enrolled in the U-102 study before and after valemetostat treatment (patient characteristics are described in Supplementary Table S1). Circulating blasts increased 0.14-to 474.1-fold with a mean increase of 65.42 ± 51.23 (SEM) fold (Fig. 1A, Supplementary Fig. S1A) after valemetostat administration. To study cell kinetics

All-trans-retinoic acid (ATRA)-based differentiation therapy of acute promyelocytic leukemia (APL) represents one of the most clinically effective examples of precision medicine and the first example of targeted oncoprotein degradation. The success of ATRA in APL, however, remains to be translated to non-APL acute myeloid leukemia (AML). We previously showed that aberrant histone modifications, including histone H3 lysine 4 (H3K4) and lysine 27 (H3K27) methylation, were associated with this lack of response and that epigenetic therapy with small molecule inhibitors of the H3K4 demethylase LSD1/KDM1A could reprogram AML cells to respond to ATRA. Serving as the enzymatic component of Polycomb Repressive Complex 2, EZH2/KMT6A methyltransferase plays a critical role in normal hematopoiesis by affecting the balance between self-renewal and differentiation. The canonical function of EZH2 is methylation of H3K27, although important non-canonical roles have recently been described. EZH2 mutation or deregulated expression has been conclusively demonstrated in the pathogenesis of AML and response to treatment, thus making it an attractive therapeutic target. In this study, we therefore investigated whether inhibition of EZH2 might also improve the response of non-APL AML cells to ATRA-based therapy. We focused on GSK-343, a pyridone-containing S-adenosyl-L-methionine cofactor-competitive EZH2 inhibitor that is representative of its class, and HKMTI-1-005, a substrate-competitive dual inhibitor targeting EZH2 and the closely related G9A/GLP H3K9 methyltransferases. We found that treatment with HKMTI-1-005 phenocopied EZH2 knockdown and was more effective in inducing differentiation than GSK-343, despite the efficacy of GSK-343 in terms of abolishing H3K27 trimethylation. Furthermore, transcriptomic analysis revealed that in contrast to treatment with GSK-343, HKMTI-1-005 upregulated the expression of differentiation pathway genes with and without ATRA, while downregulating genes associated with a hematopoietic stem cell phenotype. These results pointed to a non-canonical role for EZH2, which was supported by the finding that EZH2 associates with the master regulator of myeloid differentiation, RARα, in an ATRA-dependent manner that was enhanced by HKMTI-1-005, possibly playing a role in co-regulator complex exchange during transcriptional activation. In summary, our results strongly suggest that addition of HKMTI-1-005 to ATRA is a new therapeutic approach against AML that warrants further investigation.

… EZH2 is a promising treatment strategy for AML. Here we examined the effects of genetic and pharmacologic inhibition of Ezh1 and Ezh2 … By contrast, GSK126, a selective EZH2 inhibitor…

Internal tandem duplication mutations in fms-like tyrosine kinase 3 ( FLT3-ITD ) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3 - ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Epigenetic dysregulation plays important roles in leukemogenesis and the progression of acute myeloid leukemia (AML). Histone acetyltransferases (HATs) and histone deacetylases (HDACs) reciprocally regulate the acetylation and deacetylation of nuclear histones. Aberrant activation of HDACs results in uncontrolled proliferation and blockade of differentiation, and HDAC inhibition has been investigated as epigenetic therapeutic strategy against AML. Cell growth was assessed with CCK-8 assay, and apoptosis was evaluated by flow cytometry in AML cell lines and CD45 + and CD34 + CD38- cells from patient samples after staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). EZH2 was silenced with short hairpin RNA (shRNA) or overexpressed by lentiviral transfection. Changes in signaling pathways were detected by western blotting. The effect of chidamide or EZH2-specific shRNA (shEZH2) in combination with adriamycin was studied in vivo in leukemia-bearing nude mouse models. In this study, we investigated the antileukemia effects of HDAC inhibitor chidamide and its combinatorial activity with cytotoxic agent adriamycin in AML cells. We demonstrated that chidamide suppressed the levels of EZH2, H3K27me3 and DNMT3A, exerted potential antileukemia activity and increased the sensitivity to adriamycin through disruption of Smo/Gli-1 pathway and downstream signaling target p-AKT in AML cells and stem/progenitor cells. In addition to decreasing the levels of H3K27me3 and DNMT3A, inhibition of EZH2 either pharmacologically by chidamide or genetically by shEZH2 suppressed the activity of Smo/Gli-1 pathway and increased the antileukemia activity of adriamycin against AML in vitro and in vivo. Inhibition of EZH2 by chidamide has antileukemia activity and increases the chemosensitivity to adriamycin through Smo/Gli-1 pathway in AML cells (Fig. 5). These findings support the rational combination of HDAC inhibitors and chemotherapy for the treatment of AML.

Acute myeloid leukemia (AML) is one of the most common hematological malignancies with high heterogeneity, characterized by a differentiating block at the early progenitor stage. The selective BCL-2 inhibitor, Venetoclax (Ven), has shown exciting clinical results in a certain group of AML patients. However, Ven alone is insufficient to reach an enduringly complete response, which leads to the concern of Ven resistance. Alternative combined therapies with Ven are demanded in AML. Here, we reported the synergistic effect and molecular mechanism of the enhancer of zeste homolog 2 (EZH2) inhibitor DZNeP with Ven in AML cells. Results showed that the combination of DZNeP with Ven significantly induces cell proliferation arrest compared to single-drug control in AML cells and primary samples, and CalcuSyn analysis showed their significant synergy. The combination also significantly promotes apoptosis and increases the expression of pro-apoptotic proteins. The whole transcriptome analysis showed that phosphoinositide-3-kinase-interacting protein1 (PIK3IP1), the PI3K/AKT/mTOR signaling suppressor, is upregulated upon DZNeP treatment. Moreover, EZH2 is upregulated but PIK3IP1 is downregulated in 88 newly diagnosed AML cohorts compared to 70 healthy controls, and a higher expression of EZH2 is associated with poor outcomes in AML patients. Particularly, the combination of DZNeP with Ven dramatically eliminated CD117 (c-KIT) (+) AML blasts, suggesting the effect of the combination on tumor stem cells. In summary, our data indicated that DZNeP increases the sensitivity of Ven in AML by affecting PI3K and c-KIT signaling in AML. Our results also suggested that the therapeutic targeting of both EZH2 and BCL-2 provides a novel potential combined strategy against AML.

… lymphoma and leukemia, respectively, suggest that EZH2 may … The reduction of CSC self-renewal via EZH2 inhibition offers … described in the context of acute myeloid leukemia …

Our understanding of the significance of epigenetic dysregulation in the pathogenesis of myeloid malignancies has greatly advanced in the past decade. Enhancer of Zeste Homolog 2 (EZH2) is the catalytic core component of the Polycomb Repressive Complex 2 (PRC2), which is responsible for gene silencing through trimethylation of H3K27. EZH2 dysregulation is highly tumorigenic and has been observed in various cancers, with EZH2 acting as an oncogene or a tumor-suppressor depending on cellular context. While loss-of-function mutations of EZH2 frequently affect patients with myelodysplastic/myeloproliferative neoplasms, myelodysplastic syndrome and myelofibrosis, cases of chronic myeloid leukemia (CML) seem to be largely characterized by EZH2 overexpression. A variety of other factors frequently aberrant in myeloid leukemia can affect PRC2 function and disease pathogenesis, including Additional Sex Combs Like 1 (ASXL1) and splicing gene mutations. As the genetic background of myeloid malignancies is largely heterogeneous, it is not surprising that EZH2 mutations act in conjunction with other aberrations. Since EZH2 mutations are considered to be early events in disease pathogenesis, they are of therapeutic interest to researchers, though targeting of EZH2 loss-of-function does present unique challenges. Preliminary research indicates that combined tyrosine kinase inhibitor (TKI) and EZH2 inhibitor therapy may provide a strategy to eliminate the residual disease burden in CML to allow patients to remain in treatment-free remission.

… zeste homolog 2 (EZH2) inhibitor tazemetostat (… acute myeloid leukemia (AML). Although this finding may be interpreted as an idiosyncratic liability of a single agent, the biology of EZH2 …

Chemotherapy resistance and disease relapse are major determinants of treatment failure in acute myeloid leukemia (AML). Therapy-induced senescence (TIS) is one outcome of chemotherapy, but its immunological consequences in AML remain unclear. Here we show that ex vivo chemotherapy induces senescence in a subset of therapy-naïve AML samples. TIS is marked by elevated interferon signaling, upregulation of human leukocyte antigen (HLA) class I and II molecules, and increased presentation of leukemia- and senescence-associated peptides, conferring AML cells antigen-presenting cell-like features. These changes enhance autologous CD4+ and CD8+ T cell responses against AML, both ex vivo and in patient-derived xenograft models. TIS also restores AML sensitivity to immune checkpoint blockade therapy. Mechanistically, we identify reduced Polycomb Repressive Complex 2 (PRC2) activity as central to TIS induction and its immunogenicity. PRC2 inhibition reactivates senescence-related genes and HLA expression in non-senescent AML cells, enabling T cell activation. These findings uncover a senescence-driven immune mechanism with potential to improve therapy outcomes in AML. Acute myeloid leukemia responses to chemotherapy vary, and the effects of therapy induced senescence on anti-tumor immunity remain unclear. Here, the authors show that chemotherapy-induced senescence increases antigen presentation and T-cell recognition of leukemia cells and enhances responses to immune checkpoint blockade, while EZH2 inhibition can restore these immune features in less responsive leukemias.

Epigenetic regulators, such as EZH2, are frequently mutated in cancer, and loss-of-function EZH2 mutations are common in myeloid malignancies. We have examined the importance of cellular context for Ezh2 loss during the evolution of acute myeloid leukemia (AML), where we observed stage-specific and diametrically opposite functions for Ezh2 at the early and late stages of disease. During disease maintenance, WT Ezh2 exerts an oncogenic function that may be therapeutically targeted. In contrast, Ezh2 acts as a tumor suppressor during AML induction. Transcriptional analysis explains this apparent paradox, demonstrating that loss of Ezh2 derepresses different expression programs during disease induction and maintenance. During disease induction, Ezh2 loss derepresses a subset of bivalent promoters that resolve toward gene activation, inducing a feto-oncogenic program that includes genes such as Plag1, whose overexpression phenocopies Ezh2 loss to accelerate AML induction in mouse models. Our data highlight the importance of cellular context and disease phase for the function of Ezh2 and its potential therapeutic implications.

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 functions as a catalytic subunit of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Tumorigenic functions of EZH2 and its synthetic lethality with some subunits of SWItch/Sucrose Non‐Fermentable (SWI/SNF) chromatin remodeling complexes have been observed. However, little is known about the function of EZH1 in tumorigenesis. Herein, we developed novel, orally bioavailable EZH1/2 dual inhibitors that strongly and selectively inhibited methyltransferase activity of both EZH2 and EZH1. EZH1/2 dual inhibitors suppressed trimethylation of histone H3 lysine 27 in cells more than EZH2 selective inhibitors. They also showed greater antitumor efficacy than EZH2 selective inhibitor in vitro and in vivo against diffuse large B‐cell lymphoma cells harboring gain‐of‐function mutation in EZH2. A hematological cancer panel assay indicated that EZH1/2 dual inhibitor has efficacy against some lymphomas, multiple myeloma, and leukemia with fusion genes such as MLL‐AF9, MLL‐AF4, and AML1‐ETO. A solid cancer panel assay demonstrated that some cancer cell lines are sensitive to EZH1/2 dual inhibitor in vitro and in vivo. No clear correlation was detected between sensitivity to EZH1/2 dual inhibitor and SWI/SNF mutations, with a few exceptions. Severe toxicity was not seen in rats treated with EZH1/2 dual inhibitor for 14 days at drug levels higher than those used in the antitumor study. Our results indicate the possibility of EZH1/2 dual inhibitors for clinical applications.

TET2 and EZH2 play important roles in the epigenetic regulation in many cancers. However, their specific roles in acute myeloid leukemia (AML) pathogenesis remain unknown. Here, the expression, methylation or mutation of EZH2 and TET2 was determined and further correlated with the levels of the chromosome instability (CIN) genes MAD2 and CDC20. We down-regulated EZH2 and TET2 in AML cell lines and assessed the effect on CIN using fluorescence in situ hybridization (FISH). Our results showed that TET2, EZH2, MAD2 and CDC20 were aberrantly expressed in AML patients. The expression level of MAD2 or CDC20 was positively correlated with that of TET2 or EZH2. Hypermethylation of the TET2 gene down-regulated its transcription. Down-regulation of EZH2 or TET2 expression inhibited apoptosis, affected MAD2 and CDC20 expression, and promoted CIN in AML cells. Decitabine treatment restored TET2 methylation and EZH2 transcription and ameliorated CIN in AML. Therefore, TET2 and EZH2 play a tumor-inhibiting role in AML that affects CIN via MAD2 and CDC20.

Key Points Boolean implications are a useful computational algorithm to mine mutation-specific methylation relationships in large cancer data sets. Mutant WT1 is associated with DNA hypermethylation of PRC2 targets in AML, and inhibition of EZH2 induces myeloid differentiation.

Next-generation genomic sequencing has identified multiple novel molecular alterations in cancer. Since the identification of DNA methylation and histone modification, it has become evident that genes encoding epigenetic modifiers that locally and globally regulate gene expression play a crucial role in normal development and cancer progression. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) is the enzymatic catalytic subunit of the polycomb-repressive complex 2 (PRC2) that can alter gene expression by trimethylating lysine 27 on histone 3 (H3K27). EZH2 is involved in global transcriptional repression, mainly targeting tumor-suppressor genes. EZH2 is commonly overexpressed in cancer and shows activating mutations in subtypes of lymphoma. Extensive studies have uncovered an important role for EZH2 in cancer progression and have suggested that it may be a useful therapeutic target. In addition, tumors harboring mutations in other epigenetic genes such as ARID1A, KDM6, and BAP1 are highly sensitive to EZH2 inhibition, thus increasing its potential as a therapeutic target. Recent studies also suggest that inhibition of EZH2 enhances the response to tumor immunotherapy. Many small-molecule inhibitors have been developed to target EZH2 or the PRC2 complex, with some of these inhibitors now in early clinical trials reporting clinical responses with acceptable tolerability. In this review, we highlight the recent advances in targeting EZH2, its successes, and potential limitations, and we discuss the future directions of this therapeutic subclass.

Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the Polycomb repressive complex 2 (PRC2), catalyzes the methylation of lysine 27 of histone H3 (H3K27) up to its trimethylated form (H3K27me), inducing by this way block of transcription and gene silencing. High levels of H3K27me3 have been found in both hematological malignancies and solid cancers, due to EZH2 overexpression and/or EZH2 mutation. From 2012, a number of highly potent and selective catalytic inhibitors of EZH2 have been reported, almost all bearing a 2-pyridone group in their structure. Typically, 2-pyridone inhibitors are selective for EZH2 over other methyltransferases, and some of them are specific for EZH2 over EZH1, others behave as dual EZH2/EZH1 inhibitors. The 2-pyridone moiety was crucial for the enzyme inhibition, as revealed later by crystallographic studies because it occupies partially the site for the co-substrate SAM (or the by-product, SAH) in the binding pocket of the enzyme, accounting for the SAM-competitive mechanism of action displayed by all the 2-pyridone inhibitors. The 2-pyridone warhead is linked to a support substructure, that can be either a bicyclic heteroaromatic ring (such as indazole, see for instance EPZ005687 and UNC1999, or indole, see for instance GSK126, EI1, and the more recent CPI-1205) or a simple monocyclic (hetero) aromatic ring (tazemetostat, MC3629, (R)-OR-S1/2), eventually annulated with the amide chain carrying the 2-pyridone group (3,4-dihydroisoquinoline-1(2H)-ones). Different substitutions at the support moiety influence the pharmacokinetics and pharmacodynamics of the compounds as well as their water solubility. In cancer diseases, the first reported 2-pyridone inhibitors displayed high antiproliferative effects in vitro and in vivo in lymphomas characterized by mutant EZH2 (such as Y641N), but the most recent compounds exert their anticancer activity against tumors with wild-type EZH2 as well. The dual EZH2/1 inhibitors have been recently reported to be more effective than EZH2 selective inhibitors in specific leukemias including leukemias cancer stem cells.

Valemetostat tosilate (valemetostat; EZHARMIA®), a selective dual inhibitor of histone-lysine N-methyltransferases enhancer of zeste homolog 1 and 2 (EZH1/2), is being developed by Daiichi Sankyo Company, Ltd for the treatment of various haematological malignancies and solid tumours, including types of non-Hodgkin lymphomas (NHL). Valemetostat was approved in Japan in September 2022 for the treatment of patients with relapsed or refractory adult T-cell leukaemia/lymphoma (R/R ATL), a subtype of NHL. This article summarizes the milestones in the development of valemetostat leading to this first approval for R/R ATL.

Epigenetic modifications in cancer stem cells largely result in phenotypic and functional heterogeneity in many solid tumors. Increasing evidence indicates that enhancer of zeste homolog 2 (EZH2), the catalytic subunit of Polycomb repressor complex 2, is highly expressed in cancer stem cells of numerous malignant tumors and has a critical function in cancer stem cell expansion and maintenance. Here, we review up-to-date information regarding EZH2 expression patterns, functions, and molecular mechanisms in cancer stem cells in various malignant tumors and discuss the therapeutic potential of targeting EZH2 in tumors.

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that is of great interest in human cancer. It has been shown to have a dual nature, as it can act as a gene repressor or activator. Studies have highlighted the various roles of EZH2 in the pathophysiology of multiple myeloma (MM). It was also shown to have a role in the development of drug resistance in MM. There are several ongoing clinical trials of EZH2 inhibitors in haematological malignancies. Pre-clinical studies have provided a rationale for the therapeutic relevance of EZH2 inhibitors in MM. This paper reviews the evidence supporting the role of EZH2 in MM pathophysiology and drug resistance, with an emphasis on interactions between EZH2 and microRNAs, as well as the prognostic significance of EZH2 expression in MM. Furthermore, results from the pre-clinical studies of EZH2 inhibition in MM and currently available interim results from clinical trials of EZH2 inhibitors in haematological malignancies are presented. Preliminary data exploring anticipated mechanisms of resistance to EZH2 inhibitors are also reviewed. There is therefore strong evidence to support the relevance of targeting EZH2 for the treatment of MM.

Epigenetic alterations are major drivers of follicular lymphomagenesis, and these alterations are frequently caused by mutations in or upregulation of EZH2, a histone methyltransferase responsible for PRC2-mediated gene repression. EZH2 hyperactivation increases proliferation of B cells and prevents them from exiting the germinal center, favoring lymphomagenesis. The first FDA-approved EZH2 inhibitor is tazemetostat, which is orally available and targets both mutant and wild-type forms of the protein to induce cell cycle arrest and apoptosis of lymphoma cells in preclinical models. Phase II trials have shown objective response rates of 69% for patients with lymphoma-carrying EZH2 mutations and 35% for those with wild-type EZH2 without major toxicity, leading to tazemetostat approval for this cancer by the US FDA in June 2020.

Abundant evidence has illustrated that long non-coding RNA (lncRNA) plays a vital role in the regulation of tumor development and progression. Most lncRNAs have been proven to have biological and clinical significance in acute myeloid leukemia (AML), but further investigation remains necessary. In this study, we investigated lncRNA NR-104098 in AML and its specific mechanism. The microarray analysis was performed on NB4 cells. Based on the related analysis results, we identified that lncRNA NR-104098 is a suppressor gene that is significantly upregulated in AML cells. LncRNA NR-104098 could inhibit proliferation and induce differentiation in AML cells in vitro and also play main role in the mouse xenografts. Mechanically, it was confirmed that lncRNA NR-104098 may effectively inhibit EZH2 transcription by directly binding to E2F1 and recruiting E2F1 to the EZH2 promoter. In addition, ATPR can significantly increase the expression of lncRNA NR-104098, whereas knocking down NR104098 can inhibit the inhibitory effect of ATPR on the proliferation and induction differentiation of AML cells. Taken together, these results lead to deeper insight into the mechanism of ATPR-induced AML differentiation and prevent proliferation by inhibiting EZH2 on the transcriptional level.

Abstract 57 The polycomb group proteins function in gene silencing through histone modifications. They have been characterized as a general regulator of stem cells, but also play a critical role in cancer. EZH2 is a catalytic component of the polycomb repressive complex 2 (PRC2) and tri-methylates histone H3 at lysine 27 to transcriptionally repress the target genes. Although EZH2 is over-expressed in various cancers including hematological malignancies, it remains unknown how EZH2 contributes to the initiation and/or progression of acute myeloid leukemia (AML). To understand the role of EZH2 in AML, we transformed granulocyte macrophage progenitors (GMPs) from Cre-ERT;Ezh2+/+ and Cre-ERT;Ezh2flox/flox mice with the MLL-AF9 fusion gene. Then, Ezh2 was deleted by inducing nuclear translocation of Cre by adding tamoxifen to culture. We found that proliferation of Ezh2δ/δ transformed cells was severely compromised upon deletion of Ezh2 (Ezh2δ/δ) in liquid culture. They gave rise to a significantly reduced number of colonies in replating assays. Of note, while Ezh2+/+ cells formed compact colonies composed of immature myeloblasts, Ezh2δ/δ cells formed dispersed colonies composed of differentiated myeloid cells. We next transplanted Cre-ERT;Ezh2+/+ and Cre-ERT;Ezh2flox/flox GMPs transformed by MLL-AF9 into recipient mice. All the recipient mice developed AML by 3 weeks after transplantation. At 3 weeks after transplantation, we depleted Ezh2 by intraperitoneal injection of tamoxifen. Deletion of Ezh2 significantly prolonged the survival of the recipient mice (60 days vs. 76 days, p<0.0001), although all the mice eventually died of leukemia. Nonetheless, as was detected in vitro, Ezh2δ/δ AML cells in BM were apparently differentiated in morphology compared with the control. Ezh2δ/δ AML cells in BM gave rise to 10-fold fewer colonies in methylcellulose medium compared with Ezh2+/+ AML cells, and again showed an obvious tendency of differentiation. These observations imply that Ezh2 is critical for the progression of MLL-AF9 AML and maintains the immature state of AML cells. To elucidate the mechanism how Ezh2 promotes the progression of MLL-AF9-induced AML, we examined the genome-wide distribution of tri-methylation of histone H3 at lysine 27 (H3K27me3) by ChIP-sequencing and microarray-based expression analysis. ChIP-sequencing using Ezh2+/+ and Ezh2δ/δ BM AML cells identified 3525 and 89 genes exhibiting a ≧ 10-fold enrichment in H3K27me3 levels in Ezh2+/+ and Ezh2δ/δ AML cells, respectively, confirming a drastic reduction in the levels of global H3K27me3 in the absence of Ezh2. Microarray analysis using lineage marker (except for Mac1)−Sca-1−c-Kit+FcγRII/IIIhi BM AML cells revealed 252 upregulated and 154 downregulated genes (≧ 2-fold) in Ezh2δ/δ AML cells compared with Ezh2+/+ AML cells. Of interest, the absence of Ezh2 did not affect the transcriptional activation of the major target genes of MLL-AF9, including HoxA9 and Meis1. Because Ezh2 functions as transcriptional repressor, de-repressed genes could be direct targets of Ezh2. Based on these data, we are now engaged in further comprehensive analysis to narrow down the direct target genes of Ezh2 responsible for the progression of AML. Collectively, our findings suggest that Ezh2 is the major enzyme for H3K27me3 in AML and contributes to the progression of AML by regulating transcription a cohort of genes that are supposedly relevant to the self-renewal capacity and perturbed differentiation of AML stem cells. Disclosures: No relevant conflicts of interest to declare.

Enhancer of zeste homolog 2 (EZH2) is a part of the polycomb repressive complex and catalyzes the trimethylation of lysine 27 on histone H3 (H3K27me3). EZH2 inhibition has a complex role in the pathogenesis of acute myeloid leukemia (AML), in that it has been shown to be either a tumor suppressor or an oncogene depending on the stage of AML development and the genes that EZH2 is regulating during each stage. Unlike follicular and diffuse large B-cell lymphoma where EZH2 mutations result in gain of function, EZH2 mutations are typically loss of function in myeloid diseases. However, we hypothesized that in AML patients without EZH2 mutations, loss of EZH2 function may produce a phenotype that would allow for therapeutic targeting without influencing normal hematopoiesis. We used EPZ011989 (EPZ), an EZH2 inhibitor tool compound, to inhibit H3K27me3 in our studies. We started by treating the MOLM-13 AML cell line with EPZ and confirmed a decrease in H3K27me3. This reduction in H3K27me3 resulted in a slight decrease in metabolic activity via MTS assays as well as decreased colony formation in methocult. These studies were followed up with EPZ inhibition in primary AML samples in vitro. We found that EZH2 inhibition resulted in decreased self-renewal of primary AML samples but not of CD34+ bone marrow cells from normal donors. Furthermore, we found that after 7-day treatment with EPZ, primary AML samples undergo moderate differentiation as suggested by an increase in CD11b surface expression via flow cytometry. These results are further supported by the morphological changes seen after 14-days of EPZ treatment in vitro. Based on these results, we hypothesize that EZH2 inhibition in primary AML samples promotes the differentiation of AML blasts. Furthermore, our preliminary data suggests that daily treatment with 150 mg/kg of EPZ results in a survival advantage and reduced disease burden in the MOLM-13-luciferase murine xenograft model. Despite loss of function EZH2 mutations portending poor outcomes in myeloid malignancies, we demonstrate that pharmacologic EZH2 inhibition reduces AML blast stemness and promotes differentiation into mature myeloid cells. In contrast, no change in normal CD34+ stem cells occurs with EZH2 inhibition, offering the opportunity to selectively target myeloid leukemia. Citation Format: Sydney Fobare, Ola A. Elgamal, Emily H. Stahl, Abeera Mehmood, Jean Truxall, Mariah L. Johnson, Amina Abdul-Aziz, John C. Byrd, Erin Hertlein. EZH2 inhibition induces blast differentiation in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1824.

Myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML) link to unfavourable prognoses. We explored the mechanism of enhancer of zeste homologue 2/histone H3 of lysine 27 (EZH2/H3K27me3) downregulating C‐X‐C motif chemokine 10 (CXCL10) to affect CD8+ T‐cell exhaustion, participating in MDS‐to‐AML transformation. NHD13 mice were treated with GSK126 (EZH2 inhibitor) and CXCL10 neutralizing antibody, with transformation time, blood cell counts and CD8+ T cell determined. SKM‐1 cells treated with short hairpin‐EZH2, overexpressing‐EZH2, GSK126 and CXCL10 were co‐cultured with CD8+ T cells. EZH2, CXCL10, H3K27me3 and EZH2 levels and EZH2 enzyme activity were assessed. CD8+ T‐cell cytotoxicity, exhaustion, apoptosis and SKM‐1 cell malignant behaviours were evaluated. In vivo, EZH2 inhibition upregulated CXCL10, decelerating MDS to AML transformation and delaying CD8+ T‐cell exhaustion. EZH2 inhibition elevated peripheral blood cells, alleviated splenomegaly, reduced CD8+ T cells, elevated CD8+ T cytotoxicity and abated CD8+ T‐cell exhaustion in NHD13 mice. CXCL10 neutralizing antibody accelerated AML transformation by inhibiting CD8+ T‐cell exhaustion via EZH2. In vitro, EZH2 overexpression facilitated CD8+ T‐cell exhaustion and SKM‐1 cell malignant behaviours. EZH2‐mediated H3K27me3 curbed CXCL10 transcription and secretion. Collectively, EZH2/H3K27me3 downregulates CXCL10 to facilitate CD8+ T‐cell exhaustion, accelerating transformation from MDS to AML.

… that occur during haematopoietic differentiation, researchers … an increased risk of transformation to AML; these data suggest a … of reported EZH2 mutations in de novo AML. Similarly, …

… of either myeloid (acute myeloid leukemia [AML]) or lymphoid (acute lymphoblastic leukemia [… to a marked expansion of ETPs and subsequent differentiation block. This, together with a …

Chemotherapy resistance is the main impediment in the treatment of acute myeloid leukaemia (AML). Despite rapid advances, the various mechanisms inducing resistance development remain to be defined in detail. Here we report that loss-of-function mutations (LOF) in the histone methyltransferase EZH2 have the potential to confer resistance against the chemotherapeutic agent cytarabine. We identify seven distinct EZH2 mutations leading to loss of H3K27 trimethylation via multiple mechanisms. Analysis of matched diagnosis and relapse samples reveal a heterogenous regulation of EZH2 and a loss of EZH2 in 50% of patients. We confirm that loss of EZH2 induces resistance against cytarabine in the cell lines HEK293T and K562 as well as in a patient-derived xenograft model. Proteomics and transcriptomics analysis reveal that resistance is conferred by upregulation of multiple direct and indirect EZH2 target genes that are involved in apoptosis evasion, augmentation of proliferation and alteration of transmembrane transporter function. Our data indicate that loss of EZH2 results in upregulation of its target genes, providing the cell with a selective growth advantage, which mediates chemotherapy resistance.

In this paper, we have focused on the investigation of the expression level of the enhancer of zeste homolog 2 (EZH2) gene in bone marrow mononuclear cells of acute myeloid leukemia (AML) patients and analyze the relationship between EZH2 gene expression and EMI. The expression of EZH2mRNA in bone marrow mononuclear cells of 26 patients with incipient AML was detected by qRT-PCR, and the relationship between EZH2mRNA expression and clinical characteristics was analyzed. EZH2 mRNA expression was increased in 26 AML patients. EZH2 gene expression in male patients was significantly higher than that in female patients. The expression of EZH2 in the group with extramedullary infiltration (EMI) was significantly higher than that in the group without EMI. The patients were divided into different groups according to the chromosomal karyotype and prognosis. Statistical analysis showed that the expression level of the medium-risk group was significantly higher than that of the low-risk group, while there was no statistical difference in other groups (P > 0.05). The expression of EZH2 gene in AML patients was closely related to EMI, and the expression of EZH2 in AML cells was closely related to cell migration ability. EZH2 is expected to be one of the indicators of disease recurrence.

Key Points We characterize active vs inactive analog compounds suitable for inhibition of both PRC2-EZH2 and PRC2-EZH1 ex vivo and in vivo. This study is the first to show oral delivery of an EZH2 and EZH1 dual inhibitor as promising therapeutics for MLL-rearranged leukemia.

… in MDS and AML, while in cases with a diploid EZH2 locus, … or haploinsufficient EZH2 expression, we investigated EZH2 … hypotonic conditions allow differentiation of chromatin states …

Most acute promyelocytic leukemia (APL) patients express PML-RARA fusion; in rare cases, RARA is rearranged with partner genes other than PML. To date, only 2 patients presenting features similar to APL showing the RARG gene rearrangement have been described. We report an acute myeloid leukemia patient with morphology resembling APL without involvement of the RARA gene. Molecular and fluorescent in situ hybridization analyses excluded PML-RARA fusion and variant rearrangements involving RARA and RARG loci. Targeted next-generation sequencing showed EZH2- D185H mutation. As this mutation involved the region of interaction with DNA methyltransferases, we speculate an epigenetic alteration of genes involved in the APL-like phenotype. Expression analysis by droplet digital polymerase chain reaction revealed downregulation of the RARA and RARG genes. We hypothesize a novel mechanism of EZH2 function alteration, which may be responsible for an acute myeloid leukemia with APL-like phenotype featuring dysregulation of the RARA and RARG genes.

Purpose We recently found that the tetraspanin family member, CD82, which is aberrantly expressed in chemotherapy-resistant CD34+/CD38− acute myelogenous leukemia (AML) cells, negatively regulates matrix metalloproteinase 9, and plays an important role in enabling CD34+/CD38− AML cells to adhere to the bone marrow microenvironment. This study explored novel functions of CD82 that contribute to AML progression. Materials and Methods We employed microarray analysis comparing the gene expression profiles between CD34+/CD38− AML cells transduced with CD82 shRNA and CD34+/CD38− AML cells transduced with control shRNA. Real-time RT-PCR and western blot analysis were performed to examine the effect of CD82 knockdown on the expression of the polycomb group member, enhancer of zeste homolog 2 (EZH2), in leukemia cells. A chromatin immunoprecipitation assay was performed to examine the effect of CD82 expression on the amount of EZH2 bound to the promoter regions of tumor suppressor genes in leukemia cells. We also utilized methylation-specific PCR to examine whether CD82 expression influences the methylation status of the tumor suppressor gene promoter regions in leukemia cells. Results Microarray analysis revealed that levels of EZH2 decreased after shRNA-mediated depletion of CD82 in CD34+/CD38− AML cells. Moreover, the antibody-mediated blockade of CD82 in leukemia cells lowered EZH2 expression via activation of p38 MAPK signaling, decreased the amount of EZH2 bound to the promoter regions of the tumor suppressor genes, and inhibited histone H3 lysine 27 trimethylation in these promoter regions, resulting in upregulation of the tumor suppressors at both the mRNA and protein levels.

Key Points • Single cell multiomics in PLZF-RARA leukemic cells resolves the retinoic acid resistance network.• Targeting pan-EZH2 activities (canonical/noncanonical) eradicates leukemia relapse-initiating cells.

In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction of histone H3K27 trimethylation as a novel pathway of acquired resistance to tyrosine kinase inhibitors (TKIs) and cytotoxic drugs in AML. Low EZH2 protein levels correlated with poor prognosis in AML patients. Suppression of EZH2 protein expression induced chemoresistance of AML cell lines and primary cells in vitro and in vivo. Low EZH2 levels resulted in derepression of HOX genes, and knockdown of HOXB7 and HOXA9 in the resistant cells was sufficient to improve sensitivity to TKIs and cytotoxic drugs. The endogenous loss of EZH2 expression in resistant cells and primary blasts from a subset of relapsed AML patients resulted from enhanced CDK1-dependent phosphorylation of EZH2 at Thr487. This interaction was stabilized by heat shock protein 90 (HSP90) and followed by proteasomal degradation of EZH2 in drug-resistant cells. Accordingly, inhibitors of HSP90, CDK1 and the proteasome prevented EZH2 degradation, decreased HOX gene expression and restored drug sensitivity. Finally, patients with reduced EZH2 levels at progression to standard therapy responded to the combination of bortezomib and cytarabine, concomitant with the re-establishment of EZH2 expression and blast clearance. These data suggest restoration of EZH2 protein as a viable approach to overcome treatment resistance in this AML patient population.

Enhancer of zeste homolog 2 (EZH2) is enzymatic catalytic subunit of polycomb repressive complex 2 (PRC2) that can alter downstream target genes expression by trimethylation of Lys-27 in histone 3 (H3K27me3). EZH2 could also regulate gene expression in ways besides H3K27me3. Functions of EZH2 in cells proliferation, apoptosis, and senescence have been identified. Its important roles in the pathophysiology of cancer are now widely concerned. Therefore, targeting EZH2 for cancer therapy is a hot research topic now and different types of EZH2 inhibitors have been developed. In this review, we summarize the structure and action modes of EZH2, focusing on up-to-date findings regarding the role of EZH2 in cancer initiation, progression, metastasis, metabolism, drug resistance, and immunity regulation. Furtherly, we highlight the advance of targeting EZH2 therapies in experiments and clinical studies.

Summary: In this issue, Maganti and colleagues described an epigenetic link between reduced abundance of Polycomb-related protein MTF2 and chemotherapy resistance in refractory acute myeloid leukemia. MTF2 deficiency impaired expression of the PRC2 complex and deposition of H3K27me3 at many genes, including the key target gene MDM2, leading to increased MDM2 expression that in turn depleted p53 and thereby conferred chemoresistance.

… are resistant to the enzymatic inhibition of EZH2 because of … from an MDS/AML patient with EZH2-Y641C mutation [45, 46]… EZH2 and a potential therapeutic target for mutant EZH2-…

Mutations modifying RAS (RAS) occur frequently in myeloid neoplasms (MN) and play a key role in myeloid leukemogenesis [1, 2]. The most commonly observed RAS in MN comprise aberrations in NRAS and KRAS, as well as in three genes that modulate the levels of active RAS-GTP (NF1, PTPN11, and CBL) [3]. Mechanistically, RAS activate a multitude of downstream signaling cascades, with the MAPK/ERK module being considered one of the major RAS-effector pathways [4]. Consequently, pharmacologic MAPK/ERK inhibition—i.e., by MEK inhibitors—is an appealing therapeutic approach. Indeed, the development of MN in Ras mice can be effectively attenuated by treatment with these substances [2]. Unfortunately, these promising results could not be translated into human MN, with disappointing results in clinical trials [5]. One potential reason is the fact that RAS do not exist as solitary events within these tumors [1, 6]. The existence of co-occurring mutational and non-mutational aberrations has the potential to further influence the activating effects of RAS, which ultimately aggravates or inhibits RAS-driven leukemogenesis and thereby changes the dependency on activated RAS-signaling [6, 7]. Consequently, these co-occurring events might also change the sensitivity to MEK inhibitors, as recently shown for the coexistence of mutations in NRAS and TET2 [7]. Enhancer of zeste homolog 2 (EZH2) is the core component of the Polycomb Repressive Complex 2 (PRC2). It regulates the expression of a broad range of genes and thereby controls a variety of basic cellular functions [8]. In more detail, EZH2 serves as histone methyltransferase that catalyzes trimethylation of lysine 27 of histone H3 (H3K27me3), which in turn causes the transcriptional repression of its target genes. Inactivation of EZH2 (EZH2)-either by mutation, deletion or a decrease in EZH2 expression-can be These authors contributed equally: Veronica Caraffini, Armin Zebisch

The chromatin repressor PRC2 induces HLA class II silencing in acute myeloid leukemia leading to immune escape and posttransplantation relapse, while its chemical inhibition rescues HLA expression and triggers T cell–mediated leukemia eradication.

OBJECTIVE This review aims to summarize current progress in targeted therapy for acute myeloid leukemia (AML) with KMT2A rearrangement (KMT2Ar). This subtype of AML often shows resistance to chemotherapy and has a poor prognosis. The purpose is to emphasize potential therapeutic strategies and explore drugs currently under clinical development. Methods: We reviewed studies on the molecular characteristics of KMT2Ar AML and examined targeted drugs that can block key genetic and epigenetic mechanisms. Information on drug mechanisms, preclinical findings, and clinical trials was collected and analyzed. RESULTS Several new agents targeting KMT2A-related pathways are being explored. Menin inhibitors show encouraging clinical activity, while other inhibitors, such as those targeting DOT1L, BET, and EZH2, have produced promising preclinical results. Early data suggest that combination therapy may be more effective in overcoming drug resistance than monotherapy. DISCUSSION Providing a new therapeutic direction for the abnormal molecular networks in KMT2Ar AML offers a promising approach. However, most therapies are still in the early stages and clinical translation is limited. Further research is needed to improve the safety and long-term efficacy of the treatment. CONCLUSION There is an urgent need for effective targeted drugs for KMT2Ar AML. Continuous research and clinical trials will be key to improving patient prognosis and advancing precise treatment for this challenging leukemia subtype.

Myelodysplastic syndrome (MDS) can progress to acute myeloid leukemia (AML), and conventional chemotherapy (decitabine) does not effectively inhibit tumor cells. Enhancer of zeste homologue 2 (EZH2) and Heme oxygenase-1 (HO-1) are two key factors in patients resistance and deterioration. In total, 58 MDS patients were divided into four groups. We analyzed the difference in HO-1 and EZH2 expression among the groups by real-time PCR. After treatment with Hemin or Znpp IX, flow cytometry was used to detect apoptosis and assess the cell cycle distribution of tumor cells. Following injection of mice with very high-risk MDS cells, spleen and bone marrow samples were studied by immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining. MDS cells overexpressing EZH2 and HO-1 were analyzed by high-throughput sequencing. The effect of HO-1 on the pRB-E2F pathway was analyzed by Western blotting. The effects of decitabine on P15INK4B and TP53 in MDS cells after inhibiting HO-1 were detected by Western blotting. Real-time PCR results showed that EZH2 and HO-1 expression levels were higher in MDS patients than in normal donors. The levels of HO-1 and EZH2 were simultaneously increased in the high-risk and very high-risk groups. Linear correlation analysis and laser scanning confocal microscopy results indicated that EZH2 was related to HO-1. MDS cells that highly expressed EZH2 and HO-1 infiltrated the tissues of experimental mice. IHC results indicated that these phenomena were related to the pRB-E2F pathway. High-throughput sequencing indicated that the progression of MDS to AML was related to EZH2. Using the E2F inhibitor HLM006474 and the EZH2 inhibitor JQEZ5, we showed that HO-1 could regulate EZH2 expression. HO-1 could stimulate the transcription and activation of EZH2 through the pRB-E2F pathway in MDS patients during chemotherapy, which reduced TP53 and P15INK4B expression. EZH2 was associated with HO-1 in high-risk and very high-risk MDS patients. HO-1 could influence MDS resistance and progression to AML.

Epigenetic modifications work in concert with genetic mechanisms to regulate transcriptional activity in normal tissues and are often dysregulated in disease. Although they are somatically heritable, modifications of DNA and histones are also reversible, making them good targets for therapeutic intervention. Epigenetic changes often precede disease pathology, making them valuable diagnostic indicators for disease risk or prognostic indicators for disease progression. Several inhibitors of histone deacetylation or DNA methylation are approved for hematological malignancies by the US Food and Drug Administration and have been in clinical use for several years. More recently, histone methylation and microRNA expression have gained attention as potential therapeutic targets. The presence of multiple epigenetic aberrations within malignant tissue and the abilities of cells to develop resistance suggest that epigenetic therapies are most beneficial when combined with other anticancer strategies, such as signal transduction inhibitors or cytotoxic treatments. A key challenge for future epigenetic therapies will be to develop inhibitors with specificity to particular regions of chromosomes, thereby potentially reducing side effects.

… of EZH2 inhibition, there has been interest in the inhibition of … inhibition has been shown to reduce H3K79 methylation and demonstrate activity in patients with relapsed/refractory AML …

Standard induction chemotherapy, consisting of an anthracycline and cytarabine, has been the first-line therapy for many years to treat acute myeloid leukemia (AML). Although this treatment induces complete remissions in the majority of patients, many face a relapse (adaptive resistance) or have refractory disease (primary resistance). Moreover, older patients are often unfit for cytotoxic-based treatment. AML relapse is due to the survival of therapy-resistant leukemia cells (minimal residual disease, MRD). Leukemia cells with stem cell features, named leukemic stem cells (LSCs), residing within MRD are thought to be at the origin of relapse initiation. It is increasingly recognized that leukemia “persisters” are caused by intra-leukemic heterogeneity and non-genetic factors leading to plasticity in therapy response. The BCL2 inhibitor venetoclax, combined with hypomethylating agents or low dose cytarabine, represents an important new therapy especially for older AML patients. However, often there is also a small population of AML cells refractory to venetoclax treatment. As AML MRD reflects the sum of therapy resistance mechanisms, the different faces of treatment “persisters” and LSCs might be exploited to reach an optimal therapy response and prevent the initiation of relapse. Here, we describe the different epigenetic, transcriptional, and metabolic states of therapy sensitive and resistant AML (stem) cell populations and LSCs, how these cell states are influenced by the microenvironment and affect treatment outcome of AML. Moreover, we discuss potential strategies to target dynamic treatment resistance and LSCs.

EZH2, a catalytic component of the polycomb repressive complex 2, trimethylates histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Although EZH2 is overexpressed in various cancers, including some hematologic malignancies, the role of EZH2 in acute myeloid leukemia (AML) has yet to be examined in vivo. In the present study, we transformed granulocyte macrophage progenitors from Cre-ERT;Ezh2flox/flox mice with the MLL-AF9 leukemic fusion gene to analyze the function of Ezh2 in AML. Deletion of Ezh2 in transformed granulocyte macrophage progenitors compromised growth severely in vitro and attenuated the progression of AML significantly in vivo. Ezh2-deficient leukemic cells developed into a chronic myelomonocytic leukemia–like disease with a lower frequency of leukemia-initiating cells compared with the control. Chromatin immunoprecipitation followed by sequencing revealed a significant reduction in the levels of trimethylation at H3K27 in Ezh2-deficient leukemic cells, not only at Cdkn2a, a known major target of Ezh2, but also at a cohort of genes relevant to the developmental and differentiation processes. Overexpression of Egr1, one of the derepressed genes in Ezh2-deficient leukemic cells, promoted the differentiation of AML cells profoundly. Our findings suggest that Ezh2 inhibits differentiation programs in leukemic stem cells, thereby augmenting their leukemogenic activity.

Acute myeloid leukemia (AML) is a clonal malignant disorder originating from a small number of leukemic stem cells (LSCs). AML relapse after conventional chemotherapy is caused by a remaining population of drug-resistant LSCs. Selective targeting of LSCs is a promising strategy for the prevention and treatment of AML relapse. Polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are important epigenetic regulators that maintain the stemeness of ES cells and hematopoietic stem cells. Enhancer of zeste homolog 1 and 2 (EZH1/2) is a catalytic component of PRC2 that trimethylates histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Mutations and overexpression of EZH1/2 are associated with cancers including hematopoietic malignancies. Here, we used genetic deletion of EZH1/2 or a novel dual inhibitor of EZH1/2 activity to show that loss or inhibition of EZH1/2 eradicates dormant AML stem cells. To examine the effects of genetic deletion of EZH1/2 on AML cells, Ezh1-null, Ezh2-conditional and double knock-out mice were generated. Hematopoietic stem/progenitor cells prepared from single knock-out mice or double knock-out mice were transduced with various types of AML fusion-genes, such as MOZ-TIF2, MLL fusions, AML1-ETO, and others by retroviral infection, and cultured in vitro or transplanted into irradiated recipient mice to induce AML in vivo. When the cells were cultured in vitro, double deletion of Ezh1/2 induced cell differentiation and apoptosis more severely than single deletions in all subtypes of AML tested, resulting in complete loss of cells. In AML mice, deletion of Ezh1/2 induced AML cell differentiation and complete remission of AML, which was not achieved by single deletion of Ezh1 or Ezh2. Genetic deletion of both Ezh1 and Ezh2 on the LSC fraction dramatically reduced the number of LSCs (Lin-c-Kit+ CD16/32+ CD34+, L-GMP), especially quiescent LSCs, whereas deletion of either Ezh1 or Ezh2 did not have such a strong effect. The transcriptional profiles of LSCs deficient in both Ezh1 and Ezh2 were characterized by the upregulation of cell cycle-related genes such as Cyclin D1/D2, which is the main regulator of G0/G1 transition, along with differentiation-related genes. These results suggested that deletion of both Ezh1 and Ezh2 is required for eradication of LSCs. To investigate whether pharmacologic inhibition of EZH1/2 could serve as a therapeutic strategy in AML, we developed a novel EZH1/2 dual inhibitor with potent inhibitory activity against both EZH1 and EZH2. The drug induced cell differentiation and apoptosis in most subtypes of AML tested in vitro and its effects were similar to those of genetic depletion of EZH1/2. A selective EZH2 inhibitor did not affect the growth and survival of AML cells to the same extent as the dual inhibitor. Oral administration of the EZH1/2 dual inhibitor reduced the number of LSCs effectively in AML mice in a manner similar to the effect of genetic deletion of EZH1/2. Taken together, these results strongly suggest that dual inhibition of EZH1 and EZH2 is a promising therapeutic strategy to eradicate LSCs in a wide range of AMLs, which could lead to important advances in the treatment of AML. Disclosures Honma: Daiichi Sankyo Co., Ltd: Employment. Adachi:Daiichi Sankyo Co., Ltd: Employment. Araki:Daiichi Sankyo Co., Ltd.: Employment. Kitabayashi:Daiichi Sankyo Co., Ltd.: Research Funding.

Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the Polycomb repressive complex 2, inhibits gene expression through methylation on lysine 27 of histone H3. EZH2 regulates normal hematopoietic stem cell self-renewal and differentiation. EZH2 also controls normal B cell differentiation. EZH2 deregulation has been described in many cancer types including hematological malignancies. Specific small molecules have been recently developed to exploit the oncogenic addiction of tumor cells to EZH2. Their therapeutic potential is currently under evaluation. This review summarizes the roles of EZH2 in normal and pathologic hematological processes and recent advances in the development of EZH2 inhibitors for the personalized treatment of patients with hematological malignancies.

Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the polycomb repressive complex 2 (PRC2), is a highly conserved histone methyltransferase that targets lysine-27 of histone H3. EZH2 is one of the key determinants of cellular sensitivity to DNA damage, and therefore an abnormal accumulation of EZH2 represses tumor suppressors, including genes related to cell cycle inhibition, apoptosis, senescence, and differentiation. Similar to EZH2, EZH1 is part of the PRC2 complex and shares target genes. Acute myeloid leukemia (AML) is a heterogeneous disease associated with high mortality, dependent on molecular subtype and therapy. Bcl-2 family proteins have been established as key mediators of apoptosis in AML with venetoclax, a small molecule BH3 mimetic, selectively inhibiting Bcl-2. Combinatorial therapy of venetoclax and hypomethylating agents (Ven/HMA) has revolutionized the treatment of elderly patients with AML, yielding response rates over 70%, but overall survival remains short. Since PRC2 is required for AML cell survival (Basheer et al., 2019; Neff et al., 2012), targeting both EZH1 and EZH2 (EZH1/2) concomitantly could be efficient in disrupting oncogenic signals. A recent study using a murine AML model demonstrated that genetic deletion of EZH1/2 depleted quiescent leukemic stem cell (LSC) and prolonged survival of leukemia bearing mice (Fujita at al., 2018). AML LSC are located in BM niches and are mainly non-cycling. Thus, overcoming LSC dormancy as well as LSC mobilization from the BM may be critical steps that improve long-term survival of patients with AML. Here, we hypothesized that EZH1/2 is required to regulate LSC dormancy and increase efficacy of Ven/HMA therapy in AML. To test this hypothesis, we utilized pharmacological inhibition of EZH1/2 by a potent and selective EZH1/2 dual inhibitor, DS-3201 (valemetostat; Daiichi Sankyo, Inc.). Granulocyte colony-stimulating factor (G-SCF) has been shown to not only recruit leukemia cells into cell cycle (Andreeff et al, 1990), but also disrupt AML-stromal interactions for stem cell mobilization. Clinically, G-CSF is a critical component of the FLAG-Ida protocol (Petti et al, 1997). Therefore, we hypothesized that G-CSF will also recruit quiescent LSC into cycle, and the alternative mechanism may act synergistically with EZH1/2 inhibition. To assess if inhibition of EZH1/2 results in recruitment of LSC into the cell cycle and subsequently improves efficacy of Ven/HMA in AML in vitro, we first confirmed the nuclear expression of EZH1/2 in human AML cell lines. All cell lines expressed EZH1/2, although at different levels. These cell lines with various genetic backgrounds were treated with DS-3201 or G-CSF 24 hours prior to their exposure of Ven/HMA. A dual inhibitor of EZH1/2, or G-CSF, in combination with Ven/HMA significantly amplified AML cell death compared to Ven/HMA alone treated counterparts. In a clinical phase 1 trial of DS-3201 in AML, EZH1/2 inhibition promoted cell cycle progression. The proliferative fraction of leukemic cells was assessed by measuring Ki67/DNA in sequential samples obtained from DS-3201 treated patients. Baseline Ki67 positivity of LSC was 11.20%, and the maximized average increased to 59.3% and to over 90% in one patient (Fig 1), validating the concept that EZH1/2 inhibition can indeed recruit leukemia stem cells effectively into cell cycle. Further, a primary sample from a R/R AML patient (resistant to DNR/Ara-C 7+3, Ven/HMA, and Ipilimumab/nivolumab) was ex vivo primed for 5 days with combination of DS-3201 and G-CSF, then exposed to AraC/DNR based treatment. Concomitant treatment of G-CSF and EZH1/2 inhibition increased LSC proliferation and subsequently enhanced AraC/DNR induced apoptosis. Conclusion: we here demonstrate that pharmacological inhibition of EZH1/2 in a clinical trial with DS-3201 resulted in massive recruitment of quiescent AML LSC into the cell cycle. In vitro, EZH1/2 inhibition and G-CSF both enhanced Ven/HMA-induced leukemia cell apoptosis. Experiments are ongoing to test the hypothesis that EZH1/2 inhibition combined with G-CSF receptor activation may sensitize resistant primary AML LSC to apoptosis induction by chemotherapy and/or Bcl-2 inhibition. If successful, this concept could further enhance the activity of FLAG-Ida chemotherapy, which was developed a generation ago along the same concept, and of BH-3 mimetic, apoptosis targeting therapies. Dos Santos: Daiichi Sankyo, Inc.: Current Employment. Slosberg:Daiichi Sankyo, Inc.: Current Employment. Daver:Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Servier: Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novimmune: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trovagene: Research Funding; Fate Therapeutics: Research Funding; ImmunoGen: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trillium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Andreeff:Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding.

Human enhancer of zeste 2 (EZH2) protein belongs to the multiprotein polycomb repressive complex 2, which also includes suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). The polycomb repressive complex 2 complex possesses histone methyltransferase activity mediated by the Su(var)3-9, enhancer of zeste, and trithorax domain of EZH2, which methylates histone H3 on lysine (K)-27 (H3K27). In the present studies, we determined that treatment with the hydroxamate histone deacetylase inhibitor LBH589 or LAQ824 depleted the protein levels of EZH2, SUZ12, and EED in the cultured (K562, U937, and HL-60) and primary human acute leukemia cells. This was associated with decreased levels of trimethylated and dimethylated H3K27, with concomitant depletion of the homeobox domain containing HOXA9 and of MEIS1 transcription factors. Knockdown of EZH2 by EZH2 small interfering RNA also depleted SUZ12 and EED, inhibited histone methyltransferase activity, and reduced trimethylated and dimethylated H3K27 levels, with a concomitant loss of clonogenic survival of the cultured acute myelogenous leukemia (AML) cells. EZH2 small interfering RNA sensitized the AML cells to LBH589-mediated depletion of EZH2, SUZ12, and EED; loss of clonogenic survival; and LBH589-induced differentiation of the AML cells. These findings support the rationale to test anti-EZH2 treatment combined with hydroxamate histone deacetylase inhibitors as an antileukemia epigenetic therapy, especially against AML with coexpression of EZH2, HOXA9, and MEIS1 genes. [Mol Cancer Ther 2006;5(12):3096–104]

Acute Myeloid Leukemia (AML) with rearranged PICALM::MLLT10 is a rare and poorly characterized entity. Here, we describe a patient with this rearrangement, and compare this case to the literature. We observed a trend towards young age, male sex, extramedullary involvement (particularly mediastinal myelosarcoma), trisomy 4, trisomy 19 and aberrant CD7-expression. It was suggested that upregulation of DOT1l or BMI1 is a key effector for subsequent leukemogenesis. However, molecular data are not available for most published cases. Interestingly, two different EZH2-mutations were detected in our case, while generally being rare in AML, which is concordant with recent reports on the occurrence of EZH2mut in this AML subtype. As a synergistic effect of BMI1 and EZH2 has already been demonstrated in other neoplasms, we hypothesize that acquiring an EZH2 mutation might be a crucial proliferation advantage in PICALM::MLLT10 positive cells. This may explain the high percentage of EZH2 mutated cases in this entity, but also supports the hypothesis of BMI1-mediated leukemogenesis.

Acute myeloid leukemia (AML) is the most common hematological malignancy in the world. Long noncoding RNAs (lncRNAs) play an important role in the development of physiology and pathology. Many reports have shown that lncRNA HOXA cluster antisense RNA 2 (HOXA-AS2) is a carcinogen and plays an important role in many tumors, but little is known about its role in AML. The aim of this study was to explore the potential mechanism and role of HOXA-AS2 in AML. HOXA-AS2 was upregulated in AML cell lines and tissues, and the overexpression of HOXA-AS2 is negatively correlated with the survival of patients. Silencing HOXA-AS2 can inhibit the proliferation and induce differentiation of AML cells in vitro and in vivo. Overexpressing HOXA-AS2 showed the opposite result. Moreover, more in-depth mechanism studies showed that carcinogenicity of HOXA-AS2 exerted mainly through binding with the epigenetic inhibitor Enhancer of zeste homolog 2 (EZH2) and then inhibiting the expression of Large Tumor Suppressor 2 (LATS2). Taken together, our findings highlight the important role of HOXA-AS2 in AML, suggesting that HOXA-AS2 may be an effective therapeutic target for patients with AML.

BACKGROUND: Although the biological insight of acute myeloid leukemia (AML) has increased in the past few years, the discovery of novel discriminative biomarkers remains of utmost value for improving outcome predictions. Systematical studies concerning the clinical implications and genetic correlations of HOXA9 aberrations in patients with AML are relatively promising. MATERIALS AND METHODS: Here, we investigated mutational status and the mRNA levels of the HOXA9 gene in 258 patients with AML. Furthermore, hematological characteristics, chromosome abnormalities, and genetic mutations associated with AML were analyzed, followed by the assessment of clinical survival. Besides, the expression level and mutational status of MEIS1, a cofactor of HOXA9, were also detected in patients with AML with the aim of a deeper understanding about the homeodomain-containing transcription factors associated with hematological characteristics. RESULTS: HOXA9 and MEIS1 mutations were detected in 4.26% and 3.49% AML cases, respectively. No correlations were detected between mutation status and clinical characteristics, cytogenetic and genetic aberrations, and clinical survival. Higher HOXA9 expression levels were correlated with white blood cell count and closely associated with unfavorable karyotype as well as MLL-PTD and EZH2 mutations, whereas, there was an inverse correlation with the French-American-British M3 subtype. Compared with patients with lower HOXA9 expression levels, those with higher HOXA9 expression levels had a lower complete remission rate and inferior survivals in both AML and cytogenetically normal AML. CONCLUSION: HOXA9 expression may serve as a promising biomarker to ameliorate a prognostic model for predicting clinical outcome and consummating individualized treatment in patients with AML.

… LSD1 and EZH2 were abnormally over-expressed in the AML patient … inhibit LSD1 and EZH2 as a potential new therapeutic … Neither single drug nor the combination treatment caused …

We report a comprehensive drug synergy study in acute myeloid leukemia (AML). In this work, we investigate a panel of cell lines spanning both MLL-rearranged and non-rearranged subtypes. The work comprises a resource for the community, with many synergistic drug combinations that could not have been predicted a priori, and open source code for automation and analyses. We base our definitions of drug synergy on the Chou-Talalay method, which is useful for visualizations of synergy experiments in isobolograms, and median-effects plots, among other representations. Our key findings include drug synergies affecting the chromatin state, specifically in the context of regulation of the modification state of histone H3 lysine-27. We report open source high throughput methodology such that multidimensional drug screening can be accomplished with equipment that is accessible to most laboratories. This study will enable preclinical investigation of new drug combinations in a lethal blood cancer, with data analysis and automation workflows freely available to the community.

Background GL-V9 is a flavonoid derivative from the natural product wogonin with elegant anti-tumor activity, while the effect of GL-V9 in acute leukemia has no report. Enhancer of zeste homolog 2 (EZH2) catalyzes trimethylation on the K27 residue of histone H3 (H3K27Me3), which facilitates non-homologous DNA end joining (NHEJ). EZH2 inhibitor could induce chromatin decondensation and promote the sensitivity of tumor cells towards DNA damage drugs. In the present study, we evaluated the anti-leukemia activity of GL-V9 and its synergy with EZH2 inhibitor DZNeP in acute myeloid leukemia (AML). Methods Cell proliferation and cytotoxicity were measured by Cell Counting Kit-8(CCK-8) in U937, THP-1, and MV4-11 AML cells. RNA-seq was performed in U937 cells treated with 2μM DZNeP, MV4-11 cells treated with 4μM GL-V9, and vehicle for 48 hours. The clone formation rate was measured by a plate clone formation assay. Cell cycle and apoptosis were measured by flow cytometry analysis. Morphology examination was conducted with Fluorescence (IF) microscopy. Expression of target genes was detected by western blot and RT-qPCR. Results GL-V9 had a dose-/time-dependent effect on cell viability with IC50 at 3-4μM in U937 and THP-1 cells (Fig.1A); it also significantly induced the G2/M arrest and apoptosis of the cells (Fig.1B, C). IF staining showed GL-V9 triggers micro-, multi-nucleation, and cytoskeleton abnormalities, the feature of mitotic catastrophe in U937 cells 4hrs after the treatment (Fig1.D). These data indicated that GL-V9 exerts the anti-leukemia effect by inducing mitotic catastrophe. We further analyzed the whole genome transcriptome upon GL-V9 treatment in U937 cells, and GSEA analysis of the differentially expressed genes (DEGs) showed that GL-V9 treatment negatively correlated with genes involved in sister chromatid separation, microtubule cytoskeleton organization, DNA double-strand break repair, mismatch repair, and cell cycle checkpoint signaling (Fig1.E). These data revealed that GL-V9 may induce DNA damage-mediated mitotic catastrophe in the AML cells. H3K27me3 and EZH2 play an important role in the early DNA damage response. We thus carried out the combination therapy of EZH2 inhibitor DZNeP with GL-V9. Results showed that co-treatment of GL-V9 with DZNeP exerted synergistic effects on reduced cell viability and clone formation versus single-drug in U937 and MV4-11 cells (Fig2.AB). DZNeP induced G0/G1 arrest; and GL-V9 induced G2/M arrest; whereas the combination significantly decreased the number of cells in the S-phase (Fig2.C). Also, the percentage of the apoptotic cells significantly increased upon the GL-V9+DZNeP treatment versus the controls in the cells (Fig2.D) as well as in ASXL1 mutant AML-M5 and AML-M2 primary cells (Fig2.E). Consistently, the combination significantly induced the expression changes of cell cycle regulator CCNE2 and p21, and dramatically up-regulated apoptotic effectors PARP, Caspase-3, and DNA damage marker γH2AX versus single-drug (Fig2.F). Furthermore, the overlapped DEGs were identified with 242 genes regulated in homo-direction but 25 regulated in the opposite treated with GL-V9 or DZNeP, these genes mainly influenced DNA replication, chromatin structures, and DNA damage response (Fig2.G). GL-V9 and/or DZNeP down-regulated the EZH2 expression, induced the decrease of p-AKT and c-Myc, and the increase of p53 in U937 cells (Fig. 2H). These data indicate that GL-V9+DZNeP may exert the anti-leukemia effect by inducing DNA damage via targeting EZH2/ PI3K/AKT signaling and the p53-c-Myc axis in the cells. EZH2 inhibition modifies the chromatin structure and decondensed the H3K27me3-marked chromatin, making AML cells more accessible to the nuclear-attacked agent GL-V9. The mechanism model is depicted in Fig. 2I. Conclusions GL-V9 is an effective novel nature compound against AML and exhibited strong synergistic effects with the epigenomic drug EZH2 inhibitor DZNeP potentially by inducing DNA damage-mediated mitotic catastrophe through targeting EZH2/ PI3K/AKT signaling and p53-cMyc axis. Our results provide pre-clinical evidence for the new combination as a promising clinical option for AML patients.

Enhancer of Zeste 2 (EZH2) is the enzymatic subunit of Polycomb Repressive Complex 2 (PRC2) that is highly upregulated and related to poor progress in AML patients. We observed the synergistic effects of EZH2 inhibitor DZNeP with newly synthesized flavonoid compound GL-V9 on apoptosis and cell proliferation arrest in AML cell lines and AML primary cells in vitro. In this study, we further explore the anti-tumor effect of DZNeP with GL-V9 on AML in vivo. The 5-6-week NSG mice (GemPharmatech) were inoculated via the tail vein with U937 (1×10^6 cells) for the human leukemia xenograft (CDX) mouse model, and the two AML patients' samples (3x10^7) for the PDX mouse model, respectively. Once leukemia engraftment was confirmed by flow cytometry of the mice's peripheral blood, the animals were randomly divided into 4 groups (16 mice/group) for treatment: vehicle (N.S.), DZNeP alone (8 mg/kg every other day, i.p.), GL-V9 alone (300mg/kg daily, oral gavage), and combo. Treatments were performed for 8 weeks in two PDXs and 2 weeks in U937 CDX. The 8 mice per group were euthanized by CO2 inhalation once the vehicle group reached the early removal criteria according to IACUC (Institutional Animal Care and Use Committee) protocols. Bone marrow (BM) and spleen were harvested. For tumor burden analysis, cells from BM and spleen were stained with anti-human CD45 and CD33 and analyzed with flow cytometry (Attune NxT Cytometer), and immunohistochemistry (IHC) was also applied to detect human CD45-positive cells in spleen tissue. The remaining 8 mice per group were used for survival analysis, and the dead mice were recorded daily and statistically analyzed using the Kaplan-Meier curve. In the U937 CDX model, the combination of DZNeP with GL-V9 significantly extended the survival, and reduced the spleen size, spleen weight, and % hCD45+hCD33+ cells in spleen and BM compared to vehicle and either single control. IHC data showed the combo group significantly reduced the % hCD45+ cells in the spleen compared to the control. For the PDX mouse models, the first patient was diagnosed as MDS-derived AML with complex karyotype and mutations in the genes TET2 (82.48%), TP53 (48.21%), U2AF1 (42.77%), WT1 (5.26%), and RUNX1 (49.16%). The second patient is a newly diagnosed AML-M5a, with MLL-AF6 fusion gene, KRAS missense mutations: p.G13D (27.19%) and p.G12D (7.62%). We successfully established PDX models via tail vein injection of the two AML patient samples, as evidenced by the flow analysis of the peripheral blood of the mice. Results showed that the combination of DZNeP with GL-V9 significantly extended the survival, reduced the spleen size, spleen weight, and % hCD45+hCD33+ cells in spleen and BM, and the % hCD45+ cells in the spleen slides (IHC) compared to vehicle and either single control. Overall, our findings suggest the combination of DZNeP with GL- V9 exhibits potential antileukemic activity in PDX mouse models with diverse genetic backgrounds. Our results demonstrated the in vivo anti-leukemia efficacy of DZNeP in combination with GL-V9 in one CDX and two PDX mouse models. Our findings strongly indicate that the combined therapy holds potential as a clinical therapeutic strategy for AML patients.

Mutations in the EZH2 gene are recurrently found in patients with myeloid neoplasms and are associated with a poor prognosis. We aimed to characterize genetic and epigenetic alterations of EZH2 in 58 patients (51 with acute myeloid leukemia and 7 with myelodysplastic or myeloproliferative neoplasms) by integrating data on EZH2 mutational status, co-occurring mutations, and EZH2 copy number status with EZH2 protein expression, histone H3K27 trimethylation, and EZH2 promoter methylation. EZH2 was mutated in 6/51 acute myeloid leukemia patients (12%) and 7/7 patients with other myeloid neoplasms. EZH2 mutations were not overrepresented in patients with chromosome 7q deletions or losses. In acute myeloid leukemia patients, EZH2 mutations frequently co-occurred with CEBPA (67%), ASXL1 (50%), TET2 and RAD21 mutations (33% each). In EZH2-mutated patients with myelodysplastic or myeloproliferative neoplasms, the most common co-mutations were in ASXL1 (100%), NRAS, RUNX1, and STAG2 (29% each). EZH2 mutations were associated with a significant decrease in EZH2 expression (p = 0.0002), which was similar in patients with chromosome 7 aberrations and patients with intact chromosome 7. An association between EZH2 protein expression and H3K27 trimethylation was observed in EZH2-unmutated patients (R2 = 0.2, p = 0.01). The monoallelic state of EZH2 was not associated with EZH2 promoter hypermethylation. In multivariable analyses, EZH2 mutations were associated with a trend towards an increased risk of death (hazard ratio 2.51 [95% confidence interval 0.87–7.25], p = 0.09); similarly, low EZH2 expression was associated with elevated risk (hazard ratio 2.54 [95% confidence interval 1.07–6.04], p = 0.04). Perturbations of EZH2 activity in AML/MDS occur on different, genetic and non-genetic levels. Both low EZH2 protein expression and, by trend, EZH2 gene mutations predicted inferior overall survival of AML patients receiving standard chemotherapy.

Accumulating studies have proved EZH2 dysregulation mediated by mutation and expression in diverse human cancers including AML. However, the expression pattern of EZH2 remains controversial in acute myeloid leukaemia (AML). EZH1/2 expression and mutation were analysed in 200 patients with AML. EZH2 expression was significantly decreased in AML patients compared with normal controls but not for EZH1 expression. EZH2 mutation was identified three of the 200 AML patients (1.5%, 3/200), whereas none of the patients harboured EZH1 mutation (0%, 0/200). EZH2 expression and mutation were significantly associated with −7/del(7) karyotypes. Moreover, lower EZH2 expression was associated with older age, higher white blood cells, NPM1 mutation, CEBPA wild‐type and WT1 wild‐type. Patients with EZH2 mutation showed shorter overall survival (OS) and leukaemia‐free survival (LFS) than patients without EHZ2 mutation after receiving autologous or allogeneic haematopoietic stem cell transplantation (HSCT). However, EZH2 expression has no effect on OS and LFS of AML patients. Notably, in EZH2 low group, patients undergone HSCT had significantly better OS and LFS compared with patients only received chemotherapy, whereas no significant difference was found in OS and LFS between chemotherapy and HSCT patients in EZH2 high group. Collectively, EZH2 dysregulation caused by mutation and under‐expression identifies specific subtypes of AML EZH2 dysregulation may be acted as potential biomarkers predicting prognosis and guiding the treatment choice between transplantation and chemotherapy.

Intermediate-risk acute myeloid leukemia (IR-AML) is a clinically heterogeneous disease, for which optimal post-remission therapy is debated. The utility of next-generation sequencing information in decision making for IR-AML has yet to be elucidated. We retrospectively studied 100 IR-AML patients, defined by European Leukemia Net classification, who had mutational information at diagnosis, received intensive chemotherapy and achieved complete remission (CR) at Cleveland Clinic (CC). The Cancer Genome Atlas (TCGA) data were used for validation. In the CC cohort, median age was 58.5 years, 64% had normal cytogenetics, and 31% required >1 induction cycles to achieve CR1. In univariable analysis, patients carrying mutations in DNMT3A, U2AF1, and EZH2 had worse overall and relapse-free survival. After adjusting for other variables, the presence of these mutations maintained an independent effect on survival in both CC and TCGA cohorts. Patients who did not have the mutations and underwent hematopoietic cell transplant (HCT) had the best outcomes. HCT improved outcomes for patients who had these mutations. RUNX1 or ASXL1 mutations did not predict survival, and performance of HCT did not confer a significant survival benefit. Our results provide evidence of clinical utility in considering mutation screening to stratify IR-AML patients after CR1 to guide therapeutic decisions.

Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase and a catalytic component of PRC2, catalyzes tri-methylation of histone H3 at Lys 27 (H3K27me3) to regulate gene expression through epigenetic machinery. EZH2 also functions both as a transcriptional suppressor and a transcriptional co-activator, depending on H3K27me3 or not and on the different cellular contexts. Unsurprisingly, numerous studies have highlighted the role of EZH2 in cancer development and progression. Through modulating critical gene expression, EZH2 promotes cell survival, proliferation, epithelial to mesenchymal, invasion, and drug resistance of cancer cells. The tumor suppressive effects of EZH2 are also identified. What is more, EZH2 has decisive roles in immune cells (for example, T cells, NK cells, dendritic cells and macrophages), which are essential components in tumor microenvironment. In this review, we aim to discuss the molecular functions of EZH2, highlight recent findings regarding the physiological functions and related regulation of EZH2 in cancer pathogenesis. Furthermore, we summarized and updated the emerging roles of EZH2 in tumor immunity, and current pre-clinical and clinical trials of EZH2 inhibitors in cancer therapy.

The discussion in this review centers around the significant relationships between EZH2 and the initiation, progression, metastasis, metabolism, drug resistance, and immune regulation of cancer. Polycomb group (PcG) proteins, which encompass two primary Polycomb repressor complexes (PRC1 and PRC2), have been categorized. PRC2 consists mainly of four subunits, namely EZH2, EED, SUZ12, and RbAp46/48. As the crucial catalytic component within the PRC2 complex, EZH2 plays a pivotal role in controlling a wide range of biological processes. Overexpression/mutations of EZH2 have been detected in a wide variety of tumors. Several mechanisms of EZH regulation have been identified, including regulation EZH2 mRNA by miRNAs, LncRNAs, accessibility to DNA via DNA‐binding proteins, post‐translational modifications, and transcriptional regulation. EZH2 signaling triggers cancer progression and may intervene with anti‐tumor immunity; therefore it has charmed attention as an effective therapeutic target in cancer therapy. Numerous nucleic acid‐based therapies have been used in the modification of EZH2. In addition to gene therapy approaches, pharmaceutical compounds can be used to target the EZH2 signaling pathway in the treatment of cancer. EZH2‐associated tumor cells and immune cells enhance the effects of the immune response in a variety of human malignancies. The combination of epigenetic modifying agents, such as anti‐EZH2 compounds with immunotherapy, could potentially be efficacious even in the context of immunosuppressive tumors. Summary, understanding the mechanisms underlying resistance to EZH2 inhibitors may facilitate the development of novel drugs to prevent or treat relapse in treated patients.