Targeting of cancer stem cells by inhibitors of DNA and histone methylation.

Review

Expert Opin. Investig. Drugs Downloaded from informahealthcare.com by CDL-UC San Diego on 06/11/15 For personal use only.

Targeting of cancer stem cells by inhibitors of DNA and histone methylation 1.

Introduction

2.

Cancer stem cells

3.

DNA methylation and cancer

4.

Inhibition of DNA

Richard L Momparler† & Sylvie Coˆte Universit e de Montr e al, Centre de recherche, D e partement de Pharmacologie, Qu e bec, Canada

methylation by 5-Aza-2’-deoxycytidine for use in cancer therapy 5.

Polycomb repressive complex and cancer

6.

The use of EZH2 inhibitors for cancer therapy

7.

Cross-talk between DNA and histone methylation: a rationale for targeting CSCs

8.

Preclinical data that support the targeting of DNA and histone methylation

9. 10.

An innovative design for clinical trials Expert opinion

Introduction: Curative chemotherapy should target cancer stem cells (CSCs). The key characteristics of CSCs are a block in differentiation and an epigenetic signature similar to embryonic stem cells (ESCs). Differentiation by ESCs and CSCs is suppressed by gene silencing through the polycomb repressive complex 2 (PRC2) and/or DNA methylation. PRC2 contains the EZH2 subunit, which catalyzes the trimethylation of histone 3 lysine 27, a gene silencing marker. It is possible to reverse this ‘double lock’ mechanism using a combination of inhibitors of EZH2 and DNA methylation (5-aza-2’-deoxycytidine), which exhibits remarkable synergistic antineoplastic activity in preclinical studies. Areas covered: The authors discuss several specific EZH2 inhibitors that have been synthesized with antineoplastic activity. One such inhibitor, EPZ-6438 (E7438), has been shown to be effective against lymphoma in a Phase I study. The indirect EZH2 inhibitor, 3-deazaneplanocin-A (DZNep), also exhibits remarkable anticancer activity due to its inhibition of methionine metabolism. Expert opinion: Agents that target EZH2 warrant Phase I trials. Due to its positive pharmacodynamics, DZNep merits a high priority for clinical investigation. Agents that show positive results in Phase I studies should be advanced to clinical trials for use in combination with 5-aza-2’-deoxycytidine due to the interesting potential of this epigenetic therapy to target CSCs. Keywords: 3-deazaneplanocin-A, 5-aza-2’-deoxycytidine, cancer stem cells, decitabine, embryonic stem cells, epigenetic therapy, EPZ-6438 (E7438), EZH2 inhibitors, GSK-126, histone methyltransferase, polycomb repressive complex 2 Expert Opin. Investig. Drugs [Early Online]

1.

Introduction

Research on how embryonic stem cells (ESCs) program terminal differentiation can provide insight into the mechanisms of neoplastic transformation and present possible targets for chemotherapy. In pluripotent ESCs, the developmental genes that are poised for activation contain the bivalent marks: H3K4me3 for gene activation and H3K27me3 for gene suppression [1,2]. The methylation of H3K4 is catalyzed by the histone methyltransferases of the Trithorax complex. The trimethylation of H3K27 is catalyzed by EZH2, which is the methyltransferase of the PRC2 complex [3]. To differentiate, ESCs remove the H3K27me3 marker and retain the H3K4me3 marker of the target genes to be activated for a specific differentiation program [3-5]. A model of these events is illustrated in Figure 1. Most undesirable differentiation programs are silenced permanently by retaining the H3K27me3 gene silencing marker and recruiting histone deacetylase (HDAC) to convert chromatin to a compact configuration. In addition, some developmental genes can also undergo DNA methylation, which confers a more permanent form of gene silencing. These epigenetic alterations, which occur during malignancy, are the main focus of this review because they represent interesting targets for experimental chemotherapy. 10.1517/13543784.2015.1051220 © 2015 Informa UK, Ltd. ISSN 1354-3784, e-ISSN 1744-7658 All rights reserved: reproduction in whole or in part not permitted

1

R. L. Momparler & S. Coˆte

Article highlights. . .

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A blockage in cancer stem cell (CSC) differentiation is a hallmark of cancer. Similar to embryonic stem cells, CSCs silence the genes that program differentiation by the EZH2-mediated trimethylation of histone H3 lysine 27 and/or DNA methylation. Because these gene silencing mechanisms are reversible, they are attractive targets for chemotherapeutic intervention. Several EZH2 inhibitors with antineoplastic activity have been synthesized. The EZH2 inhibitor E7438 exhibits clinical activity against lymphoma ‘in a Phase I trial’. Positive pharmacodynamic properties of the EZH2 inhibitor, 3-deazaneplanocin-A, suggest that it warrants clinical investigation. Combination of inhibitors of EZH2 and DNA methylation (5-aza-2’-deoxycytidine) exhibits synergistic antineoplastic activity against CSCs. This form of epigenetic therapy merits clinical investigation.

This box summarizes key points contained in the article.

2.

Cancer stem cells

It is widely accepted that the generation and progression of malignancy is driven by a subpopulation of cancer stem cells (CSCs) [6]. According to this hypothesis, only a small fraction of leukemia or tumor cells are able to form cancer in vivo. CSCs were first identified in acute myeloid leukemia (AML) [7]. The origin of CSCs is unclear, although their formation depends on both genetic and epigenetic alterations [1,2]. CSCs possess hallmarks of ESCs, namely, the capacity for self-renewal and an undifferentiated phenotype [6]. An understanding of the molecular mechanisms by which ESCs program differentiation can provide insight into the events that might occur in CSCs during neoplastic transformation. CSCs can block differentiation by suppressing gene expression through H3K27me3 modification and/or DNA methylation, in a manner that is similar to that seen in ESCs [8]. Because both these epigenetic modifications are reversible, they represent attractive targets for chemotherapeutic intervention. In this review, we discuss the chemotherapeutic potential of EZH2 inhibitors, both when used as single agents and when used in combination with the DNA methylation inhibitor, 5-aza-2’-deoxycytidine (decitabine, 5-AZA-CdR), to specifically target CSCs by reversing their blocking of terminal differentiation. 3.

DNA methylation and cancer

DNA methylation is an epigenetic mechanism that is used for the long-term silencing of gene expression [9] and can maintain differential gene expression patterns in a tissue-specific and developmental-stage-specific manner. 2

Methylation patterns are established early during development and are normally maintained throughout the life of an individual [10]. DNA methylation is catalyzed by DNA methyltransferases (DNMT) at the 5-position of the cytosine ring using S-adenosylmethionine as the methyl donor, primarily at the CpG positions in the promoter regions of target genes [11]. Several forms of DNMTs exist: DNMT3A and DNMT3B function as de novo methyltransferases of unmethylated DNA and play an important role during embryonic development [9]. The primary function of DNMT1 is to maintain the DNA methylation signature after DNA replication using hemimethylated DNA as the template [9]. DNA methylation near the transcription start site inhibits gene expression. This inhibition is mediated by the recruitment of methyl-binding proteins (MBDs) to 5-methylcytosines in the promoter regions of target genes. This protein complex directly inhibits transcription by blocking the binding of transcriptional factors and the recruitment of HDACs, which convert chromatin to a compact configuration [12]. Many genes that suppress malignancy are silenced by aberrant DNA methylation in both leukemia [13] and solid tumors [14]. DNA methylation in the gene body can also effect the action of 5-AZA-CdR on gene expression [15]. Demethylation of the promoter region of target genes by 5-AZA-CdR can lead to gene reactivation, but demethylation of the gene body can decrease overexpression of genes. Restoration of the high levels of gene expression requires remethylation by DNMT3B. These epigenetic alterations can possibly influence the anticancer action of 5-AZA-CdR. The potential reversibility of DNA methylation patterns suggests that these might represent useful targets for cancer therapy. One goal of epigenetic therapy is to restore normal DNA methylation patterns and to reactivate silent tumor suppressor genes. Treatment of cancer cells with a specific DNA methylation inhibitor can be used to reverse the silencing of genes that suppress malignancy.

Inhibition of DNA methylation by 5-Aza-2’deoxycytidine for use in cancer therapy

4.

The importance of aberrant DNA methylation in cancer is illustrated by the interesting antineoplastic action of 5-AZA-CdR, a specific and potent DNA methylation inhibitor. 5-AZA-CdR is a prodrug that is activated by phosphorylation by deoxycytidine kinase [11]. The antineoplastic action of this deoxycytidine analogue is due to its incorporation into methylated CpG sites during DNA replication and its irreversible inactivation of DNMT1 during its attempt to methylate cytosine. 5-AZA-CdR has been shown to suppress malignancy by eliminating the proliferative potential of cells and by inducing differentiation, apoptosis and/or senescence [11]. 5-AZA-CdR has been approved for the treatment of myeloid leukemia [16]. However, most patients who obtain remission after treatment will eventually relapse [17-22]. DNA methylation inhibitors, including 5-AZA-CdR, have also exhibited interesting responses in

Expert Opin. Investig. Drugs (2015) 24(8)

Targeting of cancer stem cells by inhibitors of DNA and histone methylation

Differentiated cell Type A ON

OFF

Self-renewal genes

Stem cell


ON

Self-renewal genes

OFF

Lineage A genes

Lineage A genes

OFF

OFF

Lineage C genes

Lineage B genes

Differentiated cell Type B OFF

Lineage B genes

OFF

Lineage C genes

OFF

Self-renewal genes

OFF

Lineage A genes

ON

Lineage B genes

OFF

Lineage C genes

Differentiated cell Type C OFF

Self-renewal genes

OFF

Lineage A genes

OFF

Lineage B genes

ON

Lineage C genes

Figure 1. Programing differentiation by embryonic stem cells (ESCs). In pluripotent ESCs the genes that program differentiation are silenced by the polycomb repressive complex 2 (PRC2), which trimethylates H3K27me3 by EZH2 methyltransferase. Removal of the PRC2 gene silencing mark from selective genes programs terminal differentiation to a specific phenotype. Gene expression of undesired programs of differentiation remain silent due to the retention of the PRC2 marker and, in some cases, by the acquisition of DNA methylation (not shown). Cancer stem cells use similar epigenetic mechanisms to block differentiation in malignant cells.

patients with advanced lung cancer [23,24]. The full role of 5-AZA-CdR in cancer therapy remains to be determined. However, this nucleoside analogue does not fully reactivate all methylated genes in malignant cells, thus limiting its chemotherapeutic effectiveness [25,26], especially if the target genes contain histone markers that silence gene expression [27-29]. The full potential of 5-AZA-CdR in cancer therapy might only be realized when used in combination with other epigenetic agents that complement its action, thus maximizing gene reactivation [29-31]. 5.

Polycomb repressive complex and cancer

Polycomb group (PcG) proteins are involved in the epigenetic regulation of gene expression and play an important role in the maintenance and differentiation of ESCs [32,33]. Two major PcG protein complexes exist: PRC1 and PRC2. PRC2 comprises EZH2, SUZ12, EED plus additional accessory proteins that are thought to modulate gene targeting and the histone methyltransferase EZH2, which catalyzes the trimethylation of lysine 27 of histone H3 (H3K27me3), a repressive marker for gene expression [34]. The trimethylation

of H3K27 plays a critical role in regulating the expression of genes that determine the balance between cell differentiation and proliferation. Alterations in the levels of this histone modification occur frequently in cancer. EZH2 overexpression has been observed in many cancer types and correlates with poor prognosis [35-40]. In addition, EZH2 can attenuate the DNA damage response in cells due to the transcriptional repression of FBXO32, a pro-apoptotic protein [41]. A gain-of-function mutation of EZH2 (Tyr641) was identified in B-cell lymphoma, supporting the oncogenic action of this protein [42]. However, loss-of-function mutations of EZH2 have also been reported in hematologic malignancies [40-43]. These results suggest that EZH2 might act as either a tumor suppressor or as an oncogene. Most data support the oncogenic function of EZH2 when it is aberrantly overexpressed. The putative tumor suppressor activity of loss-of-function mutations in EZH2 requires further clarification because other epigenetic alterations might play an important role in this process, including the possible replacement of EZH2 function by EZH1. The second complex, PRC1, comprises four core components: RING1B, BMI1, HPH and CBX [3,33,34] and


3


DZN

5AZA

*

Loss clonogenicity (%)

75

*

50

25

ZN D + ZA 5A

10 ZN D

ZA

10

00

0

nM

nM

0

5A


100

Figure 2. Clonogenic assay of HL-60 leukemic cells after sequential treatment with 5-AZA-CdR (5AZA) and/or DZNep (DZN). The cells were treated with 100 nM 5-AZA-CdR and/or at 24 h 1000 nM DZNep was added to the medium. At 48 h cells were placed in soft agar medium for colony assay. The results are expressed as mean ± s.e.m., n = 3. Data analysis: 5AZA* vs 5AZA + DZN*, p < 0.05 (one-way ANOVA). Reproduced from [31] with permission of Elsevier.

exhibits E3 ubiquitin ligase activity, which catalyzes the mono-ubiquitination of histone H2A at lysine 119 (H2AK119ub1). This complex recognizes and reinforces the suppression of gene expression that is initiated by PRC2. The interaction between these two complexes is crucial for the maintenance of gene silencing. Whereas PRC2 and PRC1 are associated with gene silencing, the Trithorax complex is implicated in gene activation [3]. Trithorax complex catalyzes the trimethylation of lysine 4 of histone H3 (H3K4me3), a marker associated with gene transcription. This Trithorax action is antagonistic to the gene silencing functions of PRC2 and PRC1. Other histone modifications can work jointly with the gene repression action of PRC2. For example, a reduction in the activity of UTX, the histone demethylase for H3K27me3, can enhance oncogenesis by retaining this gene suppression marker [44]. The full understanding of the complex interactions of the enzymes involved in histone chemical modifications and their role in neoplasia requires further study.

The use of EZH2 inhibitors for cancer therapy 6.

cellular accumulation of S-adenosylhomocysteine, which acts as a competitive inhibitor of S-adenosyl-L-methionine, the methyl donor for methyltransferases [45]. DZNep is a global inhibitor of methyltransferases [46]. The action of DZNep on gene activation, reduction in H3K27me3 and growth inhibition of different types of cancer cells suggests that its inhibition of EZH2 is more important than its inhibition of other histone methyltransferases (Figure 2) [29]. The targeting of EZH2 by DZNep might be related to the Km of S-adenosyl-L-methionine and the Ki of S-adenosylhomocysteine for this enzyme. There are many reports that DZNep exhibits antineoplastic action in leukemic (Table 1) and tumor cells (Table 2), supporting the notion that EZH2 is a key target of DZNep. This analogue has been shown to inhibit the growth of several cancer types in mouse models (Table 3). These results indicate that H3K27me3 plays an important role in the malignant phenotype of these cancers and that its reduction by DZNep suppresses malignancy. Notably, DZNep was also demonstrated to induce leukemic cell differentiation [47]. In addition, DZNep treatment of neoplastic cells induces a remarkable upregulation of many gene classes, as shown by microarray analysis [29,31]. Exposure of MCF-7 breast carcinoma cells to DZNep led to the upregulation of 751 genes [48]. Gene ontology analysis revealed an enrichment of genes that act in growth inhibition or apoptosis. Other investigators have reported that DZNep upregulates 30 genes in this cell line [46]. A study in glioblastoma cells revealed that DZNep upregulated 823 genes and downregulated many other genes, including the c-myc oncogene [35]. In MCF-7 breast carcinoma cells, DZNep treatment upregulated 372 genes, including FBXO32, a proapoptotic gene [49]. Treatment of AML-3 leukemic cells with DZNep also upregulated FBXO32, p16, p21 and p27 tumor suppressor genes [47]. DZNep treatment of HL-60 myeloid leukemic cells upregulated 426 genes, including several genes that suppress malignancy [29,31]. The action of DZNep on other histone methyltransferases might modulate some of its antineoplastic activity. In breast carcinoma cells, DZNep reduces the level of H4K20me3, a repressive marker for gene expression [48,49]. This effect on tumor growth is not fully understood. The inhibition of DOT1L (the histone methyltransferase for H3K79me3, a gene activation marker) by DZNep suppresses the growth of breast carcinoma cells [50]. Aberrant methylation of H3K79me3 by DOT1L in MLL-rearranged leukemia can result in the activation of HoxA genes, thus promoting leukemic cell proliferation [51]. The MLL is frequently translocated in leukemia [52]. Many MLL fusion partners result in leukemic transformation by the involvement of transcriptional regulation through chromatin remodeling. EPZ-6438 (E7438) EPZ-6438 is a potent and selective EZH2 inhibitor. The activity of EPZ-6438 has been characterized in preclinical models of non-Hodgkin lymphoma (NHL) [53]. EPZ-6438 6.2

6.1

3-Deazaneplanocin-A

3-Deazaneplanocin-A (DZNep) is an adenosine analogue that inhibits S-adenosylhomocysteine hydrolase, resulting in the 4




Table 1. In vitro antineoplastic action of 3-deazaneplanocin-A (DZNep) on leukemic cells. Type of cancer

Cell lines

Antineoplastic action by DZNep

Ref.

Leukemia

HL-60

[29]

Leukemia

AML-3

Leukemia

HL-60

Leukemia

AML-3

Leukemia

MOLM-14 MV4-11 Kazumi-1 TF-1 Mono-Mac-1 K562

1000 nM 24 h 35% reduction colony formation 1000 nM 24 h 30% inhibition of growth 1000 nM 24 h 15% induction of apoptosis 500 nM 24 h 20% reduction colony formation 500 nM 24 h 15% inhibition of growth 500 nM 24 h 10% induction of apoptosis 500 nM 72 h 30% induction of apoptosis 1000 nM 72 h 35% induction of apoptosis 200 nM 48 h 65% reduction colony formation 500 nM 48 h 90% reduction colony formation 200 nM 72 h 8% induction of apoptosis 500 nM 72 h 35% induction of apoptosis 200 nM 48 h 60% reduction colony formation 500 nM 48 h 80% reduction colony formation 4.2 µM 48 h 50% induction of apoptosis 6.3 µM 48 h 50% induction of apoptosis 4.8 µM 48 h 50% induction of apoptosis 12.5 µM 48 h 50% induction of apoptosis 15.0 µM 48 h 50% induction of apoptosis 1.0 µM 72 h 60% inhibition of growth 1.0 µM 72 h 16% induction of erythroid differentiation 2.0 µM 48 h 32% induction of apoptosis 2.0 uM 48 h 35% induction of apoptosis

Leukemia (Erythro-) Lymphoma Mantle cell

MO2058 JeKo-1

selectively inhibits H3K27 methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation leads to selective cell death in human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after treatment in two EZH2-mutant xenograft models. EPZ-6438, also designated as E7438, is currently under study in a Phase I trial in advanced solid tumors and in relapsed and refractory B-cell lymphomas that have failed all standard therapies [54]. Preliminary results obtained from this clinical trial were presented at the 26th EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics in Barcelona, November 2014 [54]. Of 10 E7438-treated patients with lymphoma, 1 exhibited a complete response (i.e., all signs of cancer disappeared) and 3 exhibited a partial response (i.e., tumor shrinkage). Antitumor effects were seen at different doses. In addition, 1 patient with a malignant rhabdoid tumor in a group of 11 patients with advanced solid tumors exhibited a partial response. Skin samples showed a decrease in cellular H3K27me3 expression. The researchers reported that E7438 was well tolerated by most patients at all doses up to 1600 mg twice a day (b.i.d.). The most common mild side effects were weakness, decreased appetite and nausea. The only severe treatment-related side effect observed was low platelet levels in one patient.

[29]

[47]

[47]

[71]

[79] [80]

CPI-169 CPI-169 is a potent EZH2 inhibitor that acts synergistically with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) in vivo and achieves complete regression in B-cell lymphoma (DLBCL) xenograft models [55]. CPI-169 inhibits the catalytic activity of PRC2 with an IC50 of < 1 nM and decreases cellular levels of H3K27me3 with an EC50 of 70 nM. This inhibitor triggers cell cycle arrest and apoptosis in a variety of cell lines. Administered subcutaneously at 200 mg/kg b.i.d., CPI-169 was well tolerated in mice with no observed toxic effect or body weight loss. CPI-169 treatment led to tumor growth inhibition in an EZH2 mutant KARPAS-422 DLBCL xenograft. The highest dose used, 200 mg/kg b.i.d., led to complete tumor regression. Because CHOP is the standard treatment for advanced DLBCL, a suboptimal dose of CPI-169 (100 mg/kg b.i.d.) was combined with a single dose of CHOP in the KARPAS-422 model. After a week of combined treatment, the tumors rapidly regressed and became unpalpable. 6.3

GSK-126 GSK-126 is a selective, S-adenosyl-methionine-competitive small molecule inhibitor of EZH2 methyltransferase activity that exhibits higher affinity for EZH2 than EZH1. The Ki of GSK-126 for EZH2 is 0.57 nM as compared to its Ki for EZH1 of 9.9 nM [42]. At concentrations of 7 -- 252 nM, GSK-126 inhibits global H3K27 trimethylation levels and reactivates silenced PRC2 target genes in lymphoma cells [42]. 6.4


5


Table 2. In vitro antineoplastic action of 3-deazaneplanocin-A (DZNep) on tumor cells. Type of cancer

Cell lines

Antineoplastic action by DZNep

Ref.

Breast cancer

MCF-7 MB-468 MDA-MB-231 MCF-7 KB1P (BRCA1+) KP (BRCA1-) HCT-116 HT-29 (Sox2 Hi SC) IM95, YCC6, AZ521,AGS IM-95 BT-1, BT-2, BT-3 (primary) H1299, H460, A549 LU130 H209 H1299 H1975 A549 SB-MES-1 H2452 RPMI-8226

5 µM 72 h 48% induction of apoptosis 5 µM 96 h 50% induction of apoptosis 235 nM 6 days 50% inhibition of growth 194 nM 6 days 50% inhibition of growth 5 µM 4 days 90% reduction of growth 5 µM 4 days 45% reduction of growth 5 µM 72 h 60% induction of apoptosis 5 µM 24 h 60% induction of apoptosis 0.3 -- 2.0 µM 72 h 50% inhibition of growth 5 uM 48 h 40% induction of apoptosis 5 µM 5 days 80% reduction in colony formation 10 uM 48 h 50% inhibition of growth 5 µM 6 days 40% inhibition of growth 5 µM 6 days 40% inhibition of growth 0.21 µM 72 h 50% inhibition of growth 0.08 µM 72 h 50% inhibition of growth 0.24 µM 72 h 50% inhibition of growth 5 µM 72 h 50% inhibition of growth 5 µM 72 h 45% inhibition of growth 10 µM 72 h 60% inhibition of growth 10 µM 72 h 25% induction of apoptosis 10 µM 72 h 40% inhibition of growth 10 µM 72 h 18 % induction of apoptosis 1 µM 72 h 50% inhibition of growth 1 µM 72 h 50% inhibition of growth 0.5 µM 72 h 15% induction of apoptosis 0.5 µM 72 h 23% induction of apoptosis 5 µM 24 h 11% induction of apoptosis 5 µM 24 h 18% induction of apoptosis 1.0 µM 72 h 50% inhibition of growth 0.1 µM 72 h 50% inhibition of growth 5 µM 72 h 16.5% induction of apoptosis 5 µM 72 h 17.5% induction apoptosis 2.5 µM 72 h 20% induction of apoptosis 2.5 µM 72 h 10% induction of apoptosis

[48]

Breast cancer Breast cancer


Colon cancer Colon cancer Gastric cancer Glioblastoma Lung cancer Lung cancer (SCLC) Lung cancer (NSCLC) Mesothelioma Multiple myeloma

U266-1984 Multiple myeloma

Osteosarcoma pancreatic cancer

Prostate Cancer

MM1S NCI-H929 MM1S NCI-H929 U2OS (p53+) SAOS-2 (p53-) MIA-PaCa-2 LPC006 MIA-PaCa-2 LPC006 DU-145 LNCaP

[81] [82] [48] [73] [83] [35] [84] [39] [85]

[86] [87]

[88]

[89] [90]

[91]

SC: Stem cells; SCLC: Small cell lung cancer.

Furthermore, GSK-126 can inhibit the proliferation of EZH2 mutant DLBCL cell lines (IC50 = 28 -- 61 nM) and the growth of EZH2 mutant DLBCL xenografts in mice receiving a daily dose of 50 mg/kg. GSK-343 GSK-343 is a highly potent, selective, SAM-competitive and cell-active small molecule EZH2 inhibitor. Among the breast and prostate cell lines that were evaluated by Verma et al. [56], the prostate cancer cell line LNCaP was the most sensitive to EZH2 inhibition by GSK-343 (growth IC50 value, 2.9 µM). This growth IC50 value is ~ 20-fold higher than that for H3K27me3 reduction, suggesting that nearly complete erasure of the H3K27me3 mark is required for growth inhibition. A ~ 10-fold range of growth inhibition potency was observed among the remaining cell lines, suggesting that the dependence on EZH2 activity varies. In rat pharmacokinetic studies, GSK-343 exhibited high clearance rates. The rapid clearance of GSK-343 suggests that this inhibitor is not 6.5

6

suitable for in vivo studies [56]. Treatment of epithelial ovarian cancer (EOC) cells with GSK-343 decreases the level of H3K27me3. However, GSK-343 exhibited limited effects on the growth of EOC cells in conventional two-dimensional (2D) culture. In contrast, GSK-343 significantly suppressed the growth of EOC cells when cultured in 3D Matrigel extracellular matrix, which more closely mimics the tumor microenvironment in vivo. Notably, GSK-343 induced the apoptosis of EOC cells in 3D but not in 2D culture. In addition, GSK343 significantly inhibited the invasion of EOC cells [57].

EI1 EI1 is another potent and selective small molecule inhibitor of EZH2 enzymatic activity. EI1-treated cells exhibit a genomewide loss of the H3K27-mediated methylation and activation of PRC2 target genes. EZH2 inhibition by EI1 in diffused large B-cell lymphomas cells carrying the Y641 mutations 6.6




Table 3. In vivo antineoplastic action of 3-deazaneplanocin-A (DZNep). Type of cancer

Cell line

Mouse model

Dose schedule

Antineoplastic activity

Ref.

Leukemia Leukemia

HL-60 MOLM-14

NOD/SCID NOD/SCID

1 mg/kg 2 days/wk 2 wk 2 mg/kg daily

Jeko-1 Huh 7

NOD/CSID NOD/SCID

Colon cancer (SC) Mesothelioma

HT-29/SOX2Hi MES-1

NOD/SCID Nude mice

Glioblastoma

U87

Nude mice

50% decrease tumor wt.

[93]

Glioblastoma

BT-1 CSC BT-2 CSC BT-3 CSC DU-145

Mice xenografts

1 mg/kg 2 wk 1 mg/kg 2x/wk 5 mg/kg 2x/wk 5 uM (ex vivo) 2.5 mg/kg b.d. qdx3 q7d 0.07 mg/kg days 3, 5, 7 followed by 1 q wk 5 uM 5 days ex vivo

increase survival decrease tumor vol. increase survival decrease tumor vol. decrease tumor vol. decrease tumor vol. decrease tumor vol. decrease tumor wt.

[47] [71]

Mantle cell lymphoma Hepatic cancer

19% 79% 27% 50% 67% 91% 50% 50%

50% increase survival

[35]

33% decrease tumor vol.

[91]

Prostate Cancer

Mice xenografts

1 mg/kg every other day over 38 days

[80] [92] [73] [86]

CSC: Cancer stem cells; SC: Stem cells; vol: Volume; wk: Week; wt: Weight.

EED

DMNT EZH2

SUZ12 me H3K27

me

OFF

CG

Figure 3. Epigenetic silencing of genes that block differentiation by the ‘double lock’ mechanism of DNA methylation and H3K27me3. Since both these epigenetic marks are reversible, they are interesting targets for chemotherapeutic intervention. The compaction of chromatin due to the action of HDAC can also contribute to gene silencing (not shown).

results in decreased proliferation, cell cycle arrest and apoptosis [58].

Cross-talk between DNA and histone methylation: a rationale for targeting CSCs

7.

PRC2 target genes in stem cells are 12-fold more likely to exhibit cancer cell-specific promoter DNA hypermethylation than non-target genes [59]. The cited study identified 195 genes exhibiting promoter DNA methylation in colon cancer cells; 177 (90%) of these genes were methylated by PRC2; 77 genes (43%) displayed evidence of cancerassociated DNA methylation. These observations support a stem cell origin for cancer in which reversible gene repression (i.e., by H3K27me3) is replaced by permanent silencing (DNA methylation), thereby locking the cell into a perpetual

state of self-renewal. The role of EZH2 in promoting selfrenewal and impeding ESC differentiation suggests that similar events can occur in CSCs. DNA hypermethylation might involve the recruitment of DNMTs to PRC2 target genes [60,61]. In CSCs, the genes that program terminal differentiation can be silenced by two different epigenetic mechanisms: aberrant DNA methylation and H3K27 methylation by EZH2 (Figure 3). The interaction between these epigenetic events provides a rationale to use inhibitors of DNA and histone methylation to target CSCs.

Preclinical data that support the targeting of DNA and histone methylation

8.

Treatment of various malignant cell types with either 5-AZACdR or DZNep alone inhibits their growth (Tables 1 and 2) [62]. These observations suggest that the molecular mechanism of action of each of these analogues is similar for several cancer types: the reversal of epigenetic gene silencing. However, the potential of these compounds for use as single agents to cure cancer is very limited. The full gene reactivation provided by 5-AZA-CdR or DZNep alone is insufficient to completely eradicate all the CSCs. The existence of cross-talk between DNA and histone methylation suggests that the combination of 5-AZA-CdR and DZNep might exhibit enhanced anticancer activity. Our experimental results support this hypothesis. We reported that 5-AZA-CdR treatment in combination with DZNep produced a synergistic reduction in colony formation by human myeloid leukemic cells (Figure 2) [29,31]. In addition, the combination exhibited a positive interaction in mice with leukemia [31]. Treatment with 5-AZA-CdR in combination with DZNep resulted in a synergistic reactivation of many genes that suppress malignancy [31]. Microarray analysis


7



revealed that 5-AZA-CdR increased the expression of 571 transcripts and DZNep increased 426 transcripts; however, the 5-AZA-CdR/DZNep combination increased the expression of 1574 transcripts [31]. The combination of these drugs produced a synergistic reactivation of CDKN1A (p21), CDKN1B (p27) and FBXO32 as determined using quantitative RT-PCR [31]. Some investigators have criticized the possible use of DZNep in cancer therapy because it is not a specific inhibitor of EZH2 [40,43,46,63]. However, this analysis does not consider the kinetics of enzyme inhibition, which depends on the Km and Ki values of the substrate and inhibitor, respectively, as discussed above. The notion that EZH2 is the major target of DZNep is also supported by the finding of the synergistic antineoplastic activity of the 5-AZA-CdR/DZNep combination (Figure 2) [29,31]. In addition, when used alone, DZNep can reactivate many silent genes, which implicates the removal of the H3K27me3 repressive marker [29,31,48,49]. 9.

An innovative design for clinical trials

To improve the effectiveness of chemotherapy in cancer treatment, it is important to find novel targets in CSCs that can be exploited using new agents. The block in the differentiation of CSCs is one of the hallmarks of cancer. In this regard, the preclinical studies cited above suggest that silencing of the genes that program CSC differentiation by DNA methylation and/ or histone methylation (H3K27me3) is a potential target for chemotherapy. The combination of 5-AZA-CdR and an EZH2 inhibitor merits clinical investigation in patients with advanced cancer. Because the epigenetic mechanisms that silence developmental genes are similar for both hematologic malignancies and tumors, all types of cancer should be responsive to the combinations of inhibitors of DNA and histone methylation. In addition, the use of such combinations reduces the probability of developing of drug resistance. Preclinical data suggest that EZH2 inhibitors lack the potency required for curative cancer therapy when used alone. The full clinical value of these inhibitors might only be realized when used in combination with other agents, such as DNA methylation inhibitors. The key challenge is to determine which EZH2 inhibitor has the greatest potential to interact with 5-AZA-CdR to completely eradicate CSCs. Preclinical studies can guide the selection of an appropriate EZH2 inhibitor for use with 5-AZA-CdR. First, the in vitro antineoplastic activity of various EZH2 inhibitors can be compared using a colony assay. This assay can provide valuable information with regard to the concentration and duration of exposure that are required to eliminate the self-renewal potential of CSCs. Second, an important aspect of the selection of EZH2 inhibitors is their appropriate anatomical distribution in body fluids to target all CSCs present. Dose-response curves of the various inhibitors in mouse models of different cancer types can provide insights into this question. Third, the toxicity of 8

5-AZA-CdR when combined with EZH2 inhibitors should be evaluated in animal models. The major side effect of 5-AZA-CdR in patients with cancer is granulocytopenia [17,23]. Therefore, it is important to determine whether EZH2 inhibitors increase the level and/or duration of 5-AZA-CdRinduced granulocytopenia. For a Phase I study, a sequential schedule of drug administration is recommended (first 5-AZA-CdR, then the EZH2 inhibitor). The blockage of cell cycle progression at S phase entry by EZH2 inhibitors has the potential to interfere with the antineoplastic action of 5-AZA-CdR, an S phasespecific agent. A dose-schedule that would be interesting to investigate is 5-AZA-CdR administered as a 24 h intravenous (i.v.) infusion, followed by the EZH2 inhibitor administered by oral or i.v. infusion over 24 h. The selected dose should result in a plasma level of ~ 1 µM for each agent. Preclinical studies have demonstrated that a continuous i.v. infusion is an excellent way to administer 5-AZA-CdR [64]. Interesting responses have been observed in patients with acute leukemia and lung cancer who received i.v. infusions of 5-AZA-CdR [17,23]. A key aim of future studies will be to determine whether the addition of an EZH2 inhibitor increases the level and duration of the response produced by 5-AZA-CdR. It is important to answer the following question: Will prolonged infusions of 5-AZA-CdR produce unacceptable hematopoietic toxicity? Because 5-AZA-CdR is an S phasespecific agent, it targets only the proliferating normal hematopoietic stem cells (HSCs) in the S phase. The HSCs that are not in S phase during treatment and resting non-proliferating HSCs will survive, thus allowing hematopoietic recovery. Currently, the EZH2 inhibitor E7438 (EPZ-6438) is available for clinical investigation. In a recent Phase I study, E7438 exhibited good antitumor activity against lymphoma with minimal toxicity [54]. This report facilitates the initiation of a pilot clinical trial using E7438 in combination with 5-AZA-CdR in patients with advanced cancer. Other active EZH2 inhibitors can be introduced into clinical trials as soon as they become available. 10.

Expert opinion

Both genetic and epigenetic alterations play important roles in cancer development [2,65]. Changes in the genome and epigenome can target EZH2. Genomic loss of miR-101 in cancer can lead to EZH2 overexpression [66]. Loss-of-function mutations in the KDM6A histone demethylase, which targets H3K27me3, occurs in some medulloblastomas [67]. EZH2 is overexpressed in various cancer types [35-40]. Both gain-offunction and loss-of-function mutations take place in EZH2 [40-43]. These observations indicate that EZH2 is an interesting target for chemotherapeutic intervention. In preclinical studies, EZH2 inhibitors were shown to inhibit the growth of various cancer types (Tables 1 and 2) [42,53,55-58]. EPZ-6438 (E7438) is the first EZH2 inhibitor to have been introduced recently into clinical trials and produced




responses in patients with lymphoma with minimal signs of toxicity [54]. Additional clinical studies on patients with cancer should be performed using EPZ-6438 (E7438). The preclinical data suggest that other EZH2 inhibitors also merit investigation. DZNep is an interesting EZH2 inhibitor, but has been criticized as not ideal for clinical study in cancer because it is not a specific EZH2 inhibitor [40,43,46,63]. The inhibition of methionine metabolism by DZNep leads to an accumulation of S-adenosyl-homocysteine, which competes with S-adenosyl-methionine, the methyl donor for methyltransferases [45]. The sensitivity of histone methyltransferases to DZNep depends on the Km of S-adenosyl-methionine and the Ki of S-adenosyl-homocysteine. It is of interest to compare the antineoplastic activity of DZNep with specific inhibitors of EZH2, such as GSK-126 and EPZ-6438 (E7438). Because GSK-126 and EPZ-6438 (E7438) specifically targets EZH2, they may be expected to be more potent than DZNep. GSK-126 is a pentacyclic molecule with a molecular weight of 526.67 daltons, a weight that is approximately double that of DZNep (262.26 daltons). The larger size of GSK-126 might limit its penetration into cells, and its complex molecular structure might undergo rapid enzymatic degradation before it reaches its target, EZH2. In contrast, DZNep uses the nucleoside transport system for rapid entry into cells [68,69]. This is a major advantage because drug penetration into cells is an important factor that often limits the efficacy of anticancer drugs [70]. Because EPZ-6438 (E7438) is also a pentacyclic molecule, its penetration into cells might also be slow. This may explain why high doses of EPZ-6438 (E7438) (i.e., up to 200 mg b.i.d. in continuous use) were needed to obtain a clinical response. In vitro studies demonstrate that EPZ-6438 (E7438) also requires a longer exposure time than DZNep to produce significant antineoplastic activity. A 11-day exposure by EPZ-6438 (E7438) was used to analyze growth inhibition of lymphoma cells [53], whereas a 2-day exposure of DZNep inhibited the growth of LSCs [71]. Smaller specific EZH2 inhibitors should be synthesized and their antineoplastic activity evaluated. The difference in the antineoplastic activity of various EZH2 inhibitors should be investigated in animal models of cancer. The most potent inhibitors can be selected for clinical investigation. Eradication of CSCs is essential for curative chemotherapy. In this regard, DZNep exhibits the capacity to target both LSCs [71,72] and tumor stem cells [35,73,74]. In addition, DZNep has the potential to inhibit angiogenesis [75] and tumor metastasis [76]. All types of cancer cells are sensitive to the antineoplastic action of DZNep (Tables 1 and 2). In

animal models with cancer, DZNep treatments showed minimal toxicity (Table 3). These observations suggest that DZNep should be given priority for clinical investigation in patients with advanced cancer. Treatment of cancer with a single epigenetic agent might be limited by its capacity to reactivate a sufficient number of genes to abolish the self-renewal function of all of the CSCs that are present. In the case of 5-AZA-CdR, < 2% of the promoters demethylated by this analogue assume an open chromatin configuration suitable for gene expression [77]. The induction of complete remission in patients with AML by 5-AZA-CdR [78] indicates that this inhibitor of DNA methylation can target LSCs. The combination of 5-AZA-CdR with DZNep reactivates more genes than either agent alone [31], suggesting that the combination has greater potential to target CSCs than single drug treatments. In addition, DZNep exhibits a remarkable synergy in combination with 5-AZACdR against leukemic cells (Figure 2). An explanation for this synergy is that it reverses the ‘double lock’ mechanism that silences developmental genes by DNA and histone methylation (i.e., via H3K27me3), and enables CSC differentiation. It is interesting to note that > 50% of genes that are silenced by DNA methylation in CSCs contain the H3K27me3 marker in ESCs. A large number of these genes are key regulators of differentiation and this may contribute to the stem-like state of cancer [8]. The importance of this combination is that it can elicit a maximal de-repression of the cancer epigenome. This novel therapy, which has the potential to target CSCs, merits clinical investigation. This approach is very promising and will hopefully lead to treatments that are more effective for all types of cancer.

Acknowledgments We thank V Marquez for valuable discussion on DZNep and G Chabot for the inspiration to write this review. The authors are also grateful to NPG language editing for their help in improving the accuracy and clarity of the language.

Declaration of interest The authors are supported by the Canadian Cancer Society Research Institute grant number. 700795. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.


9


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Affiliation

Richard L Momparler† & Sylvie Coˆte † Author for correspondence Universite de Montreal, Centre de recherche, Departement de Pharmacologie, CHU-SaintJustine, Montreal, Quebec, Canada E-mail: [email protected]


13

Epigenetic therapy of cancer stem and progenitor cells by targeting DNA methylation machineries.

Reversible Regulation of Promoter and Enhancer Histone Landscape by DNA Methylation in Mouse Embryonic Stem Cells.

Histone modifications: Targeting head and neck cancer stem cells.

Targeting histone methylation for colorectal cancer.

Targeting histone methylation for cancer therapy: enzymes, inhibitors, biological activity and perspectives.

Therapeutic potential of mTOR inhibitors for targeting cancer stem cells.

In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse.

Mechanism of DNA methylation-directed histone methylation by KRYPTONITE.

Compendium of aberrant DNA methylation and histone modifications in cancer.

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Targeting leukemia stem cells with HDAC inhibitors and modulators of the DNA damage response.

DNA methylation and histone modifications regulate SOX11 expression in lymphoid and solid cancer cells.

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DNA promoter and histone H3 methylation downregulate NGX6 in gastric cancer cells.

Understanding the relationship between DNA methylation and histone lysine methylation.

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Targeting cancer stem cells by curcumin and clinical applications.

Chemotherapy targeting cancer stem cells.

SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation.

Synergetic effects of DNA methylation and histone modification during mouse induced pluripotent stem cell generation.

Targeting tumor-initiating cells: eliminating anabolic cancer stem cells with inhibitors of protein synthesis or by mimicking caloric restriction.

Histone Methylation by Temozolomide; A Classic DNA Methylating Anticancer Drug.

Anti-Cancer Phytometabolites Targeting Cancer Stem Cells.

Neuronal developmental gene and miRNA signatures induced by histone deacetylase inhibitors in human embryonic stem cells.