3-Deazaneplanocin A (DZNep), an Inhibitor of S-Adenosylmethionine-dependent Methyltransferase, Promotes Erythroid Differentiation*

Background: S-adenosylmethionine-dependent methyltransferase inhibitor, DZNep, targets the degradation of histone methyltransferase EZH2 that catalyzes H3K27 trimethylation. Results: DZNep induced erythroid-related genes, which may not be directly related to EZH2 inhibition but may be partly associated with reduced protein level of hematopoietic corepressor ETO2. Conclusion: DZNep has the capacity to induce erythroid differentiation. Significance: Our data may be exploited for therapeutic applications for hematological diseases, including anemia.


EZH2, a core component of polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through histone H3 Lys-27 trimethylation and is involved in various biological processes, including hematopoiesis. It is well known that 3-deazaneplanocin A (DZNep), an inhibitor of S-adenosylme-
thionine-dependent methyltransferase that targets the degradation of EZH2, preferentially induces apoptosis in various hematological malignancies, suggesting that EZH2 may be a new target for epigenetic treatment. Because PRC2 participates in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation, inhibition of EZH2 may influence erythropoiesis. To explore this possibility, we evaluated the impact of DZNep on erythropoiesis. DZNep treatment significantly induced erythroid differentiation of K562 cells, as assessed by benzidine staining and quantitative RT-PCR analysis for representative erythroid-related genes, including globins. When we evaluated the effects of DZNep in human primary erythroblasts derived from cord blood CD34-positive cells, the treatment significantly induced erythroid-related genes, as observed in K562 cells, suggesting that DZNep induces erythroid differentiation. Unexpectedly, siRNA-mediated EZH2 knockdown had no significant effect on the expression of erythroid-related genes. Transcriptional profiling of DZNep-treated K562 cells revealed marked up-regulation of SLC4A1 and EPB42, previously reported as representative targets of the transcriptional corepressor ETO2. In addition, DZNep treatment reduced the protein level of ETO2. These data suggest that erythroid differentiation by DZNep may not be directly related to EZH2 inhibition but may be partly associated with reduced protein level of hematopoietic corepressor ETO2. These data provide a better under-standing of the mechanism of action of DZNep, which may be exploited for therapeutic applications for hematological diseases, including anemia.
The differentiation of hematopoietic stem cells into specific progenitor cells and ultimately into diverse blood cell types is controlled by various transcription factors, represented by the GATA factors (1). In the context of erythropoiesis, GATA-1 activates or represses the expression of various genes by recognizing the DNA binding consensus sequence (A/T)GATA(A/G) through dual zinc finger motifs, which are characteristic of the GATA family (2). GATA-1 forms a complex with another master regulator of hematopoiesis, the basic-helix-loop-helix transcription factor SCL/TAL1 (1,3,4). SCL/TAL1 is a master regulator of hematopoiesis that binds E-boxes and the non-DNA-binding components of LMO2 and LDB1 (1,(3)(4)(5)(6)(7)(8)(9)(10), which contributes to the activation of GATA-1 target genes.
Although it has been known that a subset of genes could be directly repressed by GATA-1, the partners for GATA-1 in gene repression are less clear (11). Hematopoietic corepressor ETO2 associates with the GATA-1⅐SCL/TAL1 complex and confers transcriptional repression mediated through interaction with histone deacetylases (HDACs) 2 (12)(13)(14)(15)(16). GATA-1 is also thought to facilitate gene repression via association with the NuRD (nucleosome remodeling and deacetylation) complex, which may be mediated by FOG-1 (friend of GATA-1) (17,18), as well as via the transcriptional corepressor Gfi-1 (19). GATA-1 can recruit polycomb repressive complex 2 (PRC2), a multiunit complex comprising the EZH2 histone methyltransferase and SUZ12 and EED structural components, leading to transcriptional repression through trimethylation of histone H3 at lysine 27 (H3K27me3) (20). 3-Deazaneplanocin A (DZNep) is the cyclopentanyl analog of 3-deazaadenosine that inhibits the activity of S-adenosylhomocysteine hydrolase, the enzyme responsible for reversible hydrolysis of S-adenosylhomocysteine to adenosine and homocysteine (21,22), leading to the indirect inhibition of various S-adenosylmethionine-dependent methylation reactions (23). Thus, DZNep can be considered as an inhibitor of S-adenosylmethionine-dependent methytransferase. DZNep has recently been reported to decrease protein levels of PRC2 (24). Because DZNep can induce efficient apoptotic cell death in cancer cells but not in normal cells (24), it has been widely examined as a possible epigenetic therapeutic agent for the treatment of various cancers, including lung cancer (25), gastric cancer (26), myeloma (27), acute myeloid leukemia (28), and lymphoma (29). However, regardless of the evidence for the potential association of PRC2 with GATA-1 as described above, the impact of DZNep on erythropoiesis has remained unknown. In the present study, we examined the effects of DZNep in the K562 erythroid cell line and further extended the analysis to human primary erythroblasts.

EXPERIMENTAL PROCEDURES
Reagents-DZNep was purchased from Cayman Chemical Co. (Ann Arbor, MI) and was reconstituted with ethanol or DMSO (Sigma).
The use of cord blood samples for the study was approved by the ethics committee of Tohoku University. Ethical considerations according to the Declaration of Helsinki were followed.
Assay of Erythroid Differentiation with Benzidine Staining-Erythroid differentiation of K562 cells as well as primary erythroblasts was scored by benzidine staining as described previously (32). Benzidine (o-dianisidine) was obtained from Sigma. Benzidine staining-positive cells were quantified by light microscopy (n ϭ 600). Viable cells were counted with trypan blue stain solution (Invitrogen).
Gene Transfer and Vectors-Overexpression of ETO2 was conducted with pBABE-puro vector as described previously (16). Briefly, the expression vector (10 g) was transfected into K562 cells by Amaxa Cell Line Nucleofector II with the program T-016 (Amaxa Biosystems, Köln, Germany), according to the manufacturer's instructions.
Silencing of Gene Expression by siRNA-For siRNA-mediated transient human ETO2 and EZH2 knockdown in K562 cells, siGENOME siRNA SMART pool (Thermo Scientific Dharmacon, Lafayette, CO) was used. The antisense sequences of the siRNA for human CBFA2T3 (which encodes ETO2 protein) were UGAAUGAGGUGAAGCGGCA, CAGCGGAGGUGA-AGACGCA, CCAAAGAGAACGGGUCAGA, and CAUUGA-CGAUCGAGGAGUU, and for human EZH2, they were CAA-AGAAUCUAGCAUCAUA, GAGGACGGCUUCCCAAUAA, GCUGAAGCCUCAAUGUUUA, and GAAUGGAAACAGC-GAAGGA. As a negative control, siGENOME non-targeting siRNA pool 1 (Thermo Scientific Dharmacon) was used. siRNA was transfected into K562 cells using an Amaxa Cell Line Nucleofector II (Amaxa Biosystems) with the T-016 program as described previously (16). siRNA was transfected twice at 0 and 24 h, and the cells were harvested at 48 h.
Real-time Quantitative RT-PCR-Total RNA was purified with TRIzol (Invitrogen), and aliquots of 1 g of purified total RNA were used to synthesize cDNA with ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Tokyo, Japan). Reaction mixtures (20 l) for real-time quantitative RT-PCR consisted of 2 l of cDNA, 10 l of Quantitect SYBR Green PCR kit (Qiagen, Hilden, Germany), and appropriate primers. Primer sequences are available upon request. Product accumulation was monitored by measuring SYBR Green fluorescence and normalized relative to GAPDH or 28 S mRNA (10,16,30).
Microarray Analysis-For expression profiling to identify the DZNep-regulated and ETO2-regulated gene ensemble, SurePrint G3 Human GE 8ϫ60K Microarrays (G4851B) were . Statistical analyses were conducted by comparing with control signal (defined as 1). *, p Ͻ 0.05. mRNA expression was normalized relative to that of GAPDH (n ϭ 4, mean Ϯ S.E.). C, Western blotting to assess H3K27me3 level in DZNep-treated K562 cells. K3K27me3 level was quantified and normalized to that of H3 (n ϭ 3, mean Ϯ S.E.). Statistical analyses were conducted by comparing with control signal (defined as 1). D, changes in total cell number after DZNep treatment. Statistical significance was calculated by comparison with the control (0 M) condition at each time point. *, p Ͻ 0.05; **, p Ͻ 0.01. E, Western blotting to detect p27 protein in DZNep-treated K562 cells. p27 protein signal was quantified and normalized to that of ␣-tubulin (n ϭ 3, mean Ϯ S.E.). Statistical analyses were conducted by comparing with control signal (defined as 1). *, p Ͻ 0.05. F, effect of DZNep (1 and 5 M, respectively, for 24 h) on cell cycle in K562 cells. The percentage of cells in each cell cycle was measured using a flow cytometer. Representative data of three independent experiments are shown. In all analyses, we observed increased G 1 as well as decreased G 2 phase cells.

mRNA Expression (Relative Units)
DZNep used according to the manufacturer's instructions (Agilent, Palo Alto, CA) (12,16). Gene ontology analysis was conducted using the DAVID bioinformatics program. For expression profiling to identify the EZH2-regulated gene ensemble, a Human Oligo Chip 25k (Toray, Tokyo, Japan) was used essentially as described previously (30). For global normalization, the background value was subtracted and subsequently adjusted to the average signal value of 25.
Quantitative Chromatin Immunoprecipitation (ChIP) Analysis-Real-time PCR-based quantitative ChIP analysis was performed essentially as described (10,16). Cells were cross-linked with 1% formaldehyde for 10 min at room temperature. The nuclear lysate was sonicated to reduce DNA fragment length using a Sonifier cell disruptor (Branson, Danbury, CT). The protein-DNA complexes were immunoprecipitated by specific antibody and Protein A-or Protein G-Sepharose (Sigma). Immunoprecipitated DNA fragments were quantified by realtime PCR to amplify regions of 75-150 bp overlapping with the appropriate motif. The product was measured by SYBR Green fluorescence in 20-l reactions, and the amount of product was determined relative to a standard curve generated from titration of input chromatin. Analysis of postamplification dissociation curves indicated that primer pairs generated single products. Primer sequences are available upon request.
Flow Cytometry-The cells collected from cultures were washed twice with PBS (Sigma). The cells were then incubated with relevant antibodies, washed twice with PBS, and analyzed using FACSCalibur or LSRFortessa TM (BD Biosciences). The collected data were processed with FlowJo software.
Cell Cycle Analysis-The cells were washed twice with PBS and fixed in 70% ethanol at Ϫ20°C. Fixed cells were subjected to centrifugation and then resuspended in 0.1% Triton X-100 (Sigma), 200 g/ml RNase A (Invitrogen), and 20 g/ml propidium iodide (Sigma) to label the DNA. DNA content was measured using LSRFortessa TM (BD Biosciences).
Statistics-Statistical significance was assessed by two-sided Student's t test. To rank the gene ontology terms, corrected p values were calculated based on the Benjamini-Yakutieli method as described previously (33). In all analyses, p Ͻ 0.05 was taken to indicate statistical significance.

DZNep Treatment Depletes EZH2 Protein and Inhibits Cell
Growth in K562 Cells-We first examined the effects of DZNep treatment based on K562 erythroleukemia cells. Cells were treated with DZNep at a concentration of 0.2 or 1 M for 72 h, employing the dosages reported previously (25,28). As shown in Fig. 1A, we confirmed the significant reduction of EZH2 protein levels, whereas EZH2 mRNA expression was not significantly affected (Fig. 1B), suggesting that EZH2 level was regulated at the posttranscriptional level. Although DZNep treatment did not alter H3K27me3 levels (Fig. 1C), the treatment inhibited cell growth (Fig. 1D), accompanied by significant accumulation of the cell cycle regulator p27 (Fig. 1E) as well as G 1 cell cycle arrest (Fig. 1F). These observations were similar to those of the previous study based on a lung cancer cell line (25). Intriguingly, we did not observe a significant increase in the frequency of apoptotic cells under identical conditions  (data not shown). These results suggested that DZNep-mediated inhibition of cell growth is independent of its effect on H3K27me3.
DZNep Treatment Induced Erythroid Differentiation of K562 Cells-Interestingly, we found that DZNep treatment induced hemoglobinization of K562 cells, as determined from the percentage of cells showing positive benzidine staining ( Fig. 2A). We next conducted transcriptional profiling with untreated and DZNep-treated K562 cells (1 M), demonstrating that 788 and 698 genes were up-regulated and down-regulated (Ͼ2fold), respectively (supplemental Table 1). The DZNep-in-duced gene ensemble included prototypical erythroid genes, such as SLC4A1, EPB42, and ALAS2 (Fig. 2B) (10), which were confirmed by quantitative RT-PCR analysis (Fig. 2C). Gene ontology analysis revealed significant enrichment of genes related to "oxygen transport" (p ϭ 0.0062) in the DZNep-induced gene ensemble (Table 1). Quantitative RT-PCR analysis confirmed that DZNep treatment significantly increased globin gene expression (Fig. 2D). Taken together, these data suggested that DZNep induced the expression of representative erythroid genes and contributed to the erythroid differentiation of K562 cells. . Statistical analyses were conducted by comparing with control signal (defined as 1). *, p Ͻ 0.05. mRNA expression was normalized relative to that of GAPDH (n ϭ 3, mean Ϯ S.E.). *, p Ͻ 0.05. C, quantitative RT-PCR analysis for erythroid genes (analyzed in Fig. 2, C and D). mRNA levels were normalized relative to GAPDH (n ϭ 3, mean Ϯ S.E.). D, microarray profiling based on the Human Oligo Chip 25K (Toray). There was a high degree of similarity in the expression profile between control and EZH2 siRNA-treated K562 cells (r ϭ 0.977).

EZH2 Inhibition Has No Significant Effect on Erythroid Gene Expression in K562
Cells-To examine whether the observed results of DZNep treatment (Fig. 2) were due to the direct inhibition of EZH2 or to some hitherto unrecognized effects, we conducted siRNA-mediated knockdown of EZH2 in K562 cells (Fig. 3, A and B). Quantitative RT-PCR analysis demonstrated that EZH2 knockdown had no significant effect on the expres-sion of erythroid lineage-related genes (Fig. 3C). Concomitantly, the knockdown did not exhibit significant changes in the percentages of benzidine staining-positive cells (data not shown). Furthermore, transcription profiles of the genes in the quantitative range of the array were similar between control and EZH2 siRNA-treated K562 cells (r ϭ 0.977) (Fig. 3D), except for obvious down-regulation of EZH2 (2.96-fold). Taken

DZNepregulated
together with our observation that DZNep treatment did not affect global H3K27me3 level in K562 cells (Fig. 1C), we consider that DZNep-mediated erythroid differentiation may not be directly related to EZH2 inhibition. DZNep Reduced Protein Level of the Hematopoietic Corepressor, ETO2, in K562 Cells-The molecular mechanisms by which DZNep induced erythroid differentiation remain unclear. However, the preferential up-regulation of SLC4A1 and EPB42 among prototypical erythroid-related genes (Fig. 2,  B and C) suggests that transcriptional corepressor ETO2 may play an important role, because we demonstrated previously that Slc4a1 and Epb4.2 were highly sensitive to ETO2 knockdown in murine erythroid cells (G1E-ER-GATA-1 cells (34)) (12). Therefore, we examined ETO2 expression in DZNeptreated K562 cells. The results indicated a significant reduction of ETO2 protein level, whereas ETO2 mRNA expression was not significantly affected (Fig. 4, A and B), implying the involvement of posttranscriptional regulation mediated by DZNep. We also confirmed that the expression of class I HDACs, which interacts with ETO2 (13), was not significantly compromised by DZNep treatment (Fig. 4C).
Quantitative ChIP analysis demonstrated that EZH2 occupancy at promoters of DZNep-induced erythroid-related genes was unchanged by DZNep treatment (Fig. 4D). On the other hand, the treatment significantly increased acetylation of histone H3 at lysine 9 (H3K9ac) at the SLC4A1 and EPB42 loci without influencing this marker at globin as well as control promoters, including inactive and active NECDIN and RPII215 promoters, respectively (Fig. 4D). Interestingly, H3K27me3 levels were also significantly decreased at the SLC4A1 and EPB42 loci (Fig. 4D).
To determine the contribution of the decrease in ETO2 due to DZNep treatment to the total DZNep-regulated gene ensemble, we conducted expression profiling to identify ETO2regulated genes, which were subsequently compared with the DZNep-regulated gene ensemble (Fig. 2). ETO2 was transiently knocked down in K562 cells with anti-ETO2 siRNA. As shown in Fig. 5A, we confirmed significant decreases in ETO2 mRNA and protein levels. In addition, the percentage of benzidine staining-positive cells and expressions of globin genes were significantly increased after ETO2 knockdown (Figs. 5B and 6). Transcriptional profiling of K562 cells with control and anti-ETO2 siRNA demonstrated that 94 and 73 genes were up-regulated and down-regulated (Ͼ2-fold), respectively (supplemental Table 2). We confirmed that SLC4A1 and EPB42 were strongly up-regulated after ETO2 knockdown (Fig. 5, C and D), which were repressed again by overexpression of ETO2 (Fig. 6), suggesting the specificity of ETO2 knockdown. Interestingly, 69.5 and 37.5% of ETO2 knockdown-up-regulated and -downregulated genes, respectively, were also regulated by DZNep treatment (Fig. 5E and supplemental Table 2). To further address if DZNep-mediated ETO2 decrease is truly responsible for erythroid differentiation of K562 cells, we rescued ETO2 expression after DZNep treatment (Fig. 7, A and B). As shown in Fig. 7C, we confirmed that the expressions of erythroid-related genes were induced by DZNep treatment, which was inhibited by ETO2 overexpression. Therefore, we consider that ETO2 may be at least one of the important factors contributing to the processes.
DZNep Induces Erythroid Gene Expression in CD34-positive Cell-derived Primary Erythroblasts-We analyzed the effects of DZNep in the K562 erythroleukemia cell line, but the phenotypic and karyotypic differences form normal erythroblasts are difficult to interpret. Therefore, we moved to an ex vivo model of human erythroid differentiation, starting from cord bloodderived CD34-positive cells. In the present study, isolated CD34-positive cells were first expanded in serum-free medium containing stem cell factor, IL-3, and GM-CSF for 5 days and subsequently underwent erythroid differentiation based on serum-free medium as described previously (16,31). Erythroid differentiation was evaluated by measuring the proportion of cell surface CD71-and glycophorin A-positive cells by flow cytometry analysis (Fig. 8A). We performed quantitative RT-PCR analysis to analyze the expression pattern of genes involved in the erythroid pathway. As shown in Fig. 7B, erythroid-related genes, such as the ␤-globin gene (HBB), were strongly induced along with differentiation. It has been demonstrated that, prior to the increase in GATA-1 expression during erythroid differentiation, GATA-2 is expressed in hematopoietic stem cells (4), and GATA-1 directly represses GATA-2 transcription at the GATA-2 locus (termed the "GATA switch") (1). Likewise, we confirmed the down-regulation of GATA-2 and subsequent increase of GATA-1 in our analysis (Fig. 8B).
Based on the primary erythroid differentiation system, cells were treated with DZNep at 0.01 and 0.1 M for 96 h. Similar to the analysis based on K562 cells (Fig. 2), the cell pellet appeared reddish in color and showed a significantly higher percentage of benzidine-positive cells (Fig. 8C). Western blotting analysis confirmed the significant decrease of EZH2 as well as ETO2 protein level at 0.1 M (Fig. 8D). Furthermore, quantitative RT-PCR analysis confirmed up-regulation of erythroid genes, including SLC4A1, EPB42, ALAS2, HBG, HBB, HBA, HBZ, and HBE, whereas GATA-1 and ETO2 were unaffected (Fig. 8E). Taken together, these data suggested that DZNep has the capacity to induce differentiation of primary erythroblasts, and the decrease of ETO2 may be partly involved in this process.

DISCUSSION
Epigenetic drugs have been tested in preclinical and clinical cancer models. DNA methyltransferase inhibitors and histone deacetylase inhibitors have been approved for the treatment of some hematological malignancies (35). On the other hand, an inhibitor of S-adenosylmethionine-dependent methytransferase, DZNep, showed promising results against lung cancer (25), gastric cancer (26), myeloma (27), acute myeloid leukemia (28), and lymphoma (29). Furthermore, DZNep has been reported to modulate allogeneic T cell responses and may represent a novel therapeutic approach for treatment of graft ver- sus host disease (36). Despite these promising results, there has been little focus on the effects of DZNep on normal hematopoiesis, especially erythropoiesis. Although we confirmed that DZNep treatment reduced EZH2 protein level in K562 cells, the treatment did not affect H3K27me3 level (Fig. 1, A-C). Similar unexpected results were observed in myeloma as well as lung cancer cell lines (25,27). These observations suggest that proteins other than EZH2 may be responsible for changes in H3K27me3. Alternatively, DZNep may also affect other histone methyltransferases that are not responsible for H3K27me3, such as those responsible for histone H3 Lys-9 trimethylation, histone H3 Lys-4 trim- ethylation, and histone H3 Lys-20 trimethylation (37), which may have an influence on K562 cells. One of the most important findings of the present study was that DZNep treatment promotes erythroid differentiation (Figs. 2 and 8). Unexpectedly, siRNA-mediated direct inhibition of EZH2 had little influence on erythroid differentiation of K562 cells (Fig. 3), suggesting the dispensable role of EZH2. Consistent with this suggestion, a previous study in Ezh2 conditional knock-out mice demonstrated that depletion of Ezh2 in adult bone marrow did not significantly affect erythropoiesis (38). On the other hand, another report suggested that EZH2 is required for the transient repression of Hes1, a downstream target of the Notch signaling pathway, in murine erythroid cells (39), which supported a positive effect of EZH2 on erythroid differentiation. However, we did not observe the activation of HES1 after EZH2 knockdown (Fig. 3D), suggesting that HES1 may not be solely regulated by EZH2 in K562 cells. Taken together, these observations suggest that DZNep promotes erythroid differentiation of K562 cells, presumably through a mechanism that is not directly related to EZH2 inhibition.
Although the molecular mechanisms by which DZNep treatment promotes erythroid differentiation have remained elusive, we speculated that the decrease in hematopoietic corepressor ETO2 level might promote erythroid differentiation in K562 cells as well as primary erythroblasts (Figs. 4B and 8D). Our hypothesis is supported by the obvious overlap between the DZNep-activated and ETO2-repressed gene ensemble (Fig.  5, C-E), including preferential up-regulation of SLC4A1 and EPB42, reported as ETO2-sensitive genes (10,12), by DZNep treatment (Figs. 2B and 5D). Interestingly, DZNep treatment significantly increased H3K9ac but decreased H3K27me3 at the SLC4A1 and EPB42 loci (Fig. 4D), although DZNep treatment did not affect global H3K27me3 level (Fig. 1C). Unresponsiveness of these histone marks at globin gene promoters (Fig. 4C) might possibly be explained by the relatively weak induction of mRNA levels in our experimental conditions. Nevertheless, the findings were quite similar to those of previous reports based on ETO2 knockdown in G1E-ER-GATA-1 cells, indicating that H3K27me3 could be affected at ETO2-sensitive gene loci (12). Given its binding properties with HDACs (13), the increase in H3K9ac is consistent with this mechanism. On the other hand, the molecular mechanism by which ETO2 confers changes in H3K27me3 levels at the target gene loci remains unknown. Nevertheless, the present data based on K562 cells support the possibility that it may be a universal or physiological phenomenon that ETO2 regulates H3K27me3 levels at these loci, and further investigations are warranted to determine the underlying molecular mechanisms.
To our knowledge, there is no direct evidence supporting our hypothesis that DZNep decreases ETO2 protein level in erythroid cells. The decrease of ETO2 protein, with no effect on its mRNA level, suggests the possible involvement of posttranscriptional regulation by DZNep (Fig. 4, A and B). Because DZNep has been reported to induce ubiquitin ligases, which contribute to inhibition of EZH2 protein in a proteasome-dependent manner (24,40), these ubiquitin ligases may have an impact on ETO2 protein level. Alternatively, given the RNA binding properties of ETO2 (41), DZNep-regulated RNA components may affect protein stability of ETO2.
An important implication of this study is that DZNep may be a new differentiation-inducing compound (42,43), because the treatment significantly induced various globin genes, including fetal hemoglobin, in K562 cells (Fig. 2) as well as primary erythroblasts (Fig. 8). Known inducers of erythroid differentiation of K562 cells, such as hydroxyurea and 5-azacytidine, are also capable of inducing fetal globin induction when administered to normal erythroid cells alone or in combination (43), and these inducers have already been applied in clinical settings. Alternatively, DZNep may be applicable in "differentiation therapy" to treat various hematological and perhaps non-hematological malignancies, as represented by all-trans-retinoic acid for acute promyelocytic leukemia (44). It has already been demonstrated that DZNep treatment induces the differentiation of acute myeloid leukemia cell lines (HL-60 and OCI-AML3) (21,28). Further experiments, including in vivo studies in experimental animals, are still necessary to conclusively determine the efficacy of DZNep. Furthermore, because DZNep may alter the expression of many genes that are essential for normal hematopoiesis, determining the precise mechanism underlying the promotion of erythroid differentiation by DZNep may lead to the development of more specific and effective drugs.
In conclusion, we have demonstrated that DZNep promotes erythroid differentiation. Our findings highlight the therapeutic potential of DZNep as a differentiation-inducing drug.