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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Amino Acids. Author manuscript; available in PMC 2014 April 1.
Published in final edited form as:
PMCID: PMC3692581

The re-expression of the epigenetically silenced e-cadherin gene by a polyamine analogue lysine-specific demethylase-1 (LSD1) inhibitor in human acute myeloid leukemia cell lines


Aberrant epigenetic silencing of tumor suppressor genes is a common feature observed during the transformation process of many cancers, including those of hematologic origin. Histone modifications, including acetylation, phosphorylation, and methylation, collaborate with DNA CpG island methylation to regulate gene expression. The dynamic process of histone methylation is the latest of these epigenetic modifications to be described, and the identification and characterization of LSD1 as a demethylase of lysine 4 of histone H3 (H3K4) has confirmed that both the enzyme and the modified histone play important roles as regulators of gene expression. LSD1 activity contributes to the suppression of gene expression by demethylating promoter-region mono- and dimethyl- H3K4 histone marks that are associated with active gene expression. As most posttranslational modifications are reversible, the enzymes involved in the modification of histones have become targets for chemotherapeutic intervention. In this study, we examined the effects of the polyamine analogue LSD1 inhibitor 2d (1,15-bis{N5-[3,3-(diphenyl)propyl]-N1-biguanido}-4,12-diazapentadecane) in human acute myeloid leukemia (AML) cell lines. In each line studied, 2d evoked cytotoxicity and inhibited LSD1 activity, as evidenced by increases in the global levels of mono- and di-methylated H3K4 proteins. Global increases in other chromatin modifications were also observed following exposure to 2d, suggesting a broad response to this compound with respect to chromatin regulation. On a gene-specific level, treatment with 2d resulted in the reexpression of e-cadherin, a tumor suppressor gene frequently silenced by epigenetic modification in AML. Quantitative chromatin immunoprecipitation analysis of the ecadherin promoter further confirmed that this re-expression was concurrent with changes in both active and repressive histone marks that were consistent with LSD1 inhibition. As hematologic malignancies have demonstrated promising clinical responses to agents targeting epigenetic silencing, this polyamine analogue LSD1 inhibitor presents an exciting new avenue for the development of novel therapeutic agents for the treatment of AML.

Keywords: histone, methylation, chromatin, epigenetic, CDH-1, polyamine


Epigenetic regulation of gene expression involves hypermethylation of DNA promoter region CpG islands with subsequent binding of methyl-CpG binding proteins and recruitment of histone-modifying proteins. These histone-modifying enzymes catalyze post-translational modifications of specific residues on the N-terminal tails of histone proteins, including phosphorylation, acetylation, ubiquitination, and methylation, thereby affecting the accessibility of local chromatin structure. The combination of these histone “marks” at a given promoter, along with DNA methylation, ultimately regulates gene transcription (Lachner et al. 2003; Jenuwein and Allis 2001), and tumor cells have been shown to alter these modifications as a means to evade growth, repair, and death control mechanisms. Aberrant epigenetic silencing of tumor suppressor genes is associated with tumorigenesis in most cancer types and is one of the most common molecular changes associated with hematopoietic neoplasia. These observations, along with the fact that epigenetic changes are typically the result of reversible posttranslational modifications, suggest the utility of strategies reversing these modifications in the treatment of cancer. Therefore, several classes of “epi-drugs” have been developed to target specific enzymes that modify chromatin with the goal of restoring the natural growth regulatory pathways of tumor cells through the re-expression of tumor suppressor genes, rather than through overt tumor cell toxicity.

The lysine-specific histone demethylase LSD1 (KDM1, AOF2/BHC110/KIAA0601) is a member of a growing family of proteins that mediate the removal of methyl groups from specific lysine residues on the N-terminal tail of histone protein H3. Recent studies have suggested that LSD1 can act as either a repressor or facilitator of transcription, in a context-dependent manner (Wang et al. 2007). Originally identified as a member of several corepressor complexes (Humphrey et al. 2001; Shi et al. 2003; Hakimi et al. 2002; Tong et al. 1998; You et al. 2001), LSD1 specifically catalyzes the demethylation of mono- and dimethyl lysine 4 of histone H3 (H3K4me1 and H3K4me2) via an FAD-dependent amine oxidase reaction (Shi et al. 2004; Lee et al. 2005; Shi et al. 2005). Because H3K4me2 is a positive chromatin mark associated with the promoters of active genes (Liang et al. 2004; Schneider et al. 2004), LSD1 activity in this capacity is correlated with transcriptional repression, and pharmacologic inhibition of LSD1 in human colon, lung, and breast cancer cell lines has confirmed this correlation (Huang et al. 2007b; Sharma et al. 2010; Zhu et al. 2012). In contrast, evidence also exists linking LSD1 activity to transcriptional activation via nuclear hormone receptors (Garcia-Bassets et al. 2007; Metzger et al. 2005; Wissmann et al. 2007). In this context, LSD1 has been proposed to function with the receptor complex to demethylate lysine 9 of histone H3 (H3K9), resulting in a facilitation of the transcription of nuclear receptormediated target genes. In either regard, it is evident that LSD1 has the potential to be a critical regulator of gene expression via modulation of chromatin structure. Importantly, LSD1 expression and activity have been found to be elevated in multiple cancers, again underscoring the potential of LSD1 as an antitumor target (Lim et al. 2010; Schulte et al. 2009).

The role of LSD1 as a repressor of transcription and the fact that in cancer, one of its substrates, H3K4me2, is depleted in the promoters of genes silenced by aberrant DNA methylation (McGarvey et al. 2006) suggests the potential of inhibiting LSD1 activity as a means of re-expressing these genes for therapeutic intervention. Because the LSD1 protein is significantly homologous to the FAD-dependent polyamine oxidases (Shi et al. 2004; Wang et al. 2001), polyamine analogue-based LSD1 inhibitors have been developed using the structures of known polyamine oxidase inhibitors as starting points (Federico et al. 2001; Bianchi et al. 2006; Sharma et al. 2010). The recently described, biguanide compound 2d (1,15-bis{N5-[3,3-(diphenyl)propyl]-N1-biguanido}-4,12-diazapentadecane) (Fig. 1a) has proven to be an effective inhibitor of LSD1 in human colon cancer cell lines, with treatment resulting in increased activating marks (H3K4me1 and H3K4me2) at the promoters of several epigenetically silenced genes important for cancer development. Importantly, these activating chromatin marks accompanied an ultimate re-expression of several aberrantly silenced genes, including members of the GATA family of transcription factors, as well as the secreted frizzled-related proteins (SFRPs), which are antagonists of the Wnt signaling pathway (Huang et al. 2007b).

Fig. 1
(a) Chemical structure of compound 2d. (b) Effects of 2d on AML cell growth in culture. HL-60, KG1a, HNT-34, and ML-1 cells were seeded and treated in quadruplicate wells of 96-well plates and incubated for 24, 48, 72, or 96 hours. At the specified times, ...

Epigenetic changes are common in hematopoietic malignancies, where the clinical effects of potential epi-drug therapies have been more comprehensively studied than in solid tumors. Acute myeloid leukemia (AML) is a highly malignant hematopoietic neoplasm that is the leading cause of death due to cancer in children and persons younger than 39 years of age. The most common leukemia in adults, it has the lowest survival rate of all leukemia types, with prognosis worsening with age of diagnosis (median survival without treatment of 11–20 weeks) (Deschler and Lubbert 2006). The frequent, aberrant, epigenetic silencing of multiple genes by promoter CpG island hypermethylation, including tumor suppressor genes, genes involved in cell cycle regulation, and adhesion molecule genes, has been well-documented in patients with myeloid leukemia (Toyota et al. 2001), and oncogenic fusion proteins that occur frequently as a cause of acute leukemia have been linked to the aberrant recruitment of DNA methyltransferases (DMNTs) (Di Croce et al. 2002) and histone deacetylases (HDACs) (Wang et al. 1998; Heibert et al. 2001) to target promoters. AML clinical trials with previously identified epi-drugs, including DNMT and HDAC inhibitors, have provided promising results, but with significant limitations. We therefore sought to investigate the therapeutic potential of the cellular inhibition of LSD1 by the polyamine analogue 2d in established AML cell lines.

Materials and Methods

Cell lines, culture conditions, and chemicals

The human AML cell lines ML-1, HNT-34, and KG1a, and the acute promyelocytic leukemia cell line (APL) HL-60 were maintained in RPMI 1640 medium containing 9% fetal bovine serum, penicillin, and streptomycin, at 37°C, 5% CO2. Compound 2d was synthesized as previously described (Bi et al. 2006). Custom primers were synthesized by Invitrogen (Carlsbad, CA).

Cytoproliferation assays

HL-60, HNT-34, and ML-1 cells were seeded in quadruplicate at 5000 cells per well of 96-well plates in 50 µL of medium. KG1a cells were seeded at 10000 cells per well. At the appropriate time points, 50 µL of fresh medium containing the appropriate concentration of 2d was added per well. Following incubation, 20 µL of CellTiter96 aqueous one solution cell proliferation assay reagent (Promega, Madison, WI) was added per well, plates were incubated 1.5 hours at 37°C, and absorbance was measured at 490 nm. Wells containing medium with 2d, but without cells, were used as background controls.

Western blotting

ML-1, HL-60, KG1a, and HNT-34 cells were seeded at 2×105 cells/mL of medium. The LSD1 inhibitor 2d was added at concentrations of 5 and 10 µM and incubated at 37°C for 24 or 48 hours. For Western blot analysis of specific histone modifications, nuclear protein was harvested using NE-PER Nuclear and Cytoplasmic Extraction Reagents according to the manufacturer’s protocol (Pierce Biotechnology, Rockford, IL), and protein was quantified using the BioRad DC assay (Hercules, CA) with absorbance measured at 750 nm. Absorbance was converted to protein concentration using interpolation on a bovine serum albumin standard curve. Nuclear proteins (20 µg per lane) were separated on pre-cast 10% Bis-Tris Novex gels with 1× MES running buffer (Invitrogen) and transferred onto Immun-Blot PVDF membranes (BioRad). Blots were blocked overnight at 4°C in Odyssey blocking buffer (LI-COR, Lincoln, NE), and proteins of interest were visualized using the following antibodies: H3K4me1, H3K4me2, H3K4me3, H3K9me1, H3K9me2, H3K9me3, AcH3K9, and H3K36me3 (all from Millipore, Billerica, MA), with Proliferating Cell Nuclear Antigen (PCNA) (Calbiochem, La Jolla, CA) as a loading control in all experiments except for the 48-h treatment of HL-60 cells with 2d. Following washes, blots were incubated with species-specific, fluorophore-conjugated secondary antibodies to allow the visualization and quantification of immunoreactive proteins using the Odyssey infrared detection system and software (LI-COR).

RNA extraction and gene expression analysis

For gene re-expression studies using RT-PCR, cells were seeded and treated in parallel with those described above for nuclear protein. Following incubation with 2d, total RNA was extracted using TRIzol reagent (Invitrogen) according to the provided protocol. RNA was treated with DNAse I, and cDNA was synthesized using the SuperScript III First Strand Synthesis System (Invitrogen) with oligo-d(T)20 as the primer. PCR primers were as follows: CDH-1, 5’-CAA TCC CAC CAC GTA CAA G-3’ (sense) and 5’-CCT GGG CAG TGT AGG ATG TGA-3’ (antisense); and GapDH, 5’-GAA GAT GGT GAT GGG ATT TC-3’ (sense) and 5’-GAA GGT GAA GGT CGG AGT C-3’ (antisense). A total of 40 cycles of amplification was performed for each of the RT-PCR experiments. GapDH was amplified as an internal control. Amplified products were analyzed on 2% agarose gels with GelStar staining (Lonza, Walkersville, MD).

Quantitative ChIP analysis of gene promoter-specific chromatin marks

HL-60 and KG1a cells were seeded and treated with 10 µM 2d for 24 or 48 hours, respectively. Following incubation, the total cell number of each condition was determined using Trypan blue exclusion. Cells were exposed to formaldehyde (30 minutes at room temperature) to cross-link proteins, rinsed with PBS, pelleted, and frozen at −80°C. For the assay, cell pellets were thawed on ice and resuspended in lysis buffer at a concentration of 1×107 cells/mL. Aliquots of 400 µL were sonicated eight times for 10 seconds each using a duty setting of 2.5 and 40% output. The sonicated lysates were divided into 100 µL aliquots for ChIP assays (1×106 cells per IP) using the reagents and protocol provided in the EZ-ChIP Assay Kit (Millipore). All modified histone antibodies for immunoprecipitation of DNA-protein complexes were used at concentrations of 1 µg per IP and were the same as those described for Western blotting. Chromatin eluted from IPs with IgG was used as a negative control, and chromatin immunoprecipitated with an antibody to pan histone H3 (Abcam, Cambridge, MA) was used as a positive control for normalization. Four previously described primer pairs (Li et al. 2006; Ting et al. 2005) tiling −568 to +155 of the transcriptional start site of the CDH-1 gene were used for SYBR green-mediated qPCR (Quanta Biosciences, Gaithersburg, MD) detection and quantification of eluted DNA on a Bio-Rad MyiQ Single-Color Real-Time PCR Detection System. PCR products were also visualized on 2% agarose gels using GelStar stain and KODAK Digital Science Image Analysis Software (Rochester, NY).

Analysis of the polyamine metabolic pathway in response to 2d

HL-60 and KG1a cells were treated for 24 and 48 hours with 10 µM 2d. Cells were collected and assayed for ornithine decarboxylase (ODC) enzymatic activity as previously described (Seely and Pegg 1983). Samples were also assayed for total protein content using the method of Bradford (Bradford 1976) and intracellular polyamine concentrations were determined by HPLC following pre-column dansylation as described by Kabra et al. (Kabra et al. 1986).


Cytoproliferative responses of AML cells to 2d exposure

The representative AML cell lines HL-60, KG1a, HNT-34, and ML-1 were treated with increasing doses of 2d, and growth response was evaluated every 24 hours over a 96-hour period (Fig. 1b). Each of the four cell lines exhibited significant growth inhibition over the 96-hour exposure, with HNT-34 appearing to be the most sensitive to the antiproliferative effects of 2d. In HL-60 cells, no growth inhibition was detected within 24 hours, and at 48 hours, only the maximum dose (10 µM) of 2d produced any effect (~40% reduction in viable cells). It should be noted, however, that the HL-60 cells consistently demonstrated slightly increased proliferation in response to lower doses of 2d. All cell lines exhibited nearly complete cytotoxicity by the end of the 96-hour treatment.

Global epigenetic chromatin modifications increase in AML cell lines following treatment with 2d

To determine if compound 2d is capable of LSD1 inhibition in cells of AML origin, each of the four cell lines were treated with increasing doses of each compound for 24 hours. In each cell line, this exposure resulted in significant increases in H3K4me2 protein within 24 hours (Fig. 2a). The HNT-34 and ML-1 lysates consistently demonstrated 2 bands corresponding to H3K4me2, possibly due to a degradation product. The HL-60 and KG1a cell lines were therefore chosen for further studies, and lysates were evaluated for additional epigenetically modified histone proteins, as indicated in Figure 2, panels b through e. Surprisingly, in addition to the expected LSD1 substrates H3K4me2 and H3K4me1, treatment of HL-60 (Fig. 2b and c) or KG1a (Fig. 2d and e) cells resulted in substantially increased levels of acetylated, and mono-, di-, and trimethylated H3K9. Levels of H3K4me3 were not detectable in HL-60 cells at 24 hours, but extending the treatment time to 48 hours led to a detectable increase (Fig. 2c). HL-60 cells exhibited dramatic increases in nearly all histone modifications analyzed at 48 hours, however it should be noted that significant decreases in PCNA levels were detected at this time point, suggesting an association with cell death. Levels of β-actin were analyzed as a potential alternative loading control, however, these also decreased with treatment at 48 hours. The levels of the histone modifications in HL-60 cells at 48 hours were therefore not quantified and representative blots are presented as Figure 2c. Levels of PCNA were unchanged in all other experiments. These data are in contrast to those previously reported in HCT116 human colon carcinoma cells treated with 2d, which demonstrated no change in global levels of methylated or acetylated H3K9 over a 48-hour treatment period (Huang et al. 2007b).

Fig. 2
Treatment with 2d alters global chromatin marks in HL-60, KG1a, HNT-34, and ML-1 cells. Cells were treated for 24 or 48 hours with 5 or 10 µM 2d and harvested for nuclear protein. Twenty micrograms of protein were separated per lane, transferred ...

Re-expression of the aberrantly silenced e-cadherin gene by 2d

Because H3K4me2 is frequently depleted in the promoter regions of transcriptionally inactive genes (McGarvey et al. 2006), we sought to determine if the increase in global H3K4 methylation observed following 2d exposure correlated with the re-expression of a candidate epigenetically silenced gene in these AML cell lines. The CDH-1 gene, which encodes e-cadherin, is aberrantly silenced by CpG island methylation in many types of cancers including leukemia (Melki et al. 2000; Corn et al. 2000; Melki et al. 1999), and RT-PCR analysis failed to detect a CDH-1 transcript in untreated KG1a or HL-60 cells. However, treatment of each cell line with 10 µM 2d revealed a substantial re-expression of CDH-1 mRNA within 24 hours for HL-60 cells and within 48 hours for KG1a cells (Fig. 3). This re-expression was diminished in HL-60 cells when exposure time was increased to 48 hours, possibly relating to the growth inhibitory and/or cytotoxic effects suggested by the decreased PCNA levels observed at this time (Fig. 2c).

Fig. 3
Re-expression of the cancer-related CDH-1 gene following exposure to 2d. HL-60 and KG1a cells were treated for 24 or 48 hours with 5 and 10 µM 2d. RNA was extracted and cDNA was produced for RT-PCR analysis of CDH-1 expression. GapDH mRNA expression ...

Altered promoter occupancy by active and repressive chromatin marks following treatment with 2d

To further confirm that the re-expression of CDH-1 by 2d is related to its ability to inhibit LSD1, we performed quantitative ChIP assays to determine the changes in histone H3 modifications at various loci across the CDH-1 promoter of HL-60 cells (Fig. 4a). As shown in Figure 4b, a 24-hour exposure to 2d significantly increased CDH-1 promoter occupancy by the active transcription mark and substrate of LSD1 H3K4me2. Measurable amounts of H3K4me1 were not detectable in untreated HL-60 cells; however, treatment with 2d resulted in enrichment of this activating mark, also a substrate of LSD1, as indicated by the detection of a band at promoter region “D” (Fig. 4c). Furthermore, the transcriptionally repressive chromatin mark H3K9me2 was detected at substantial levels in untreated cells at promoter region “D”. Treatment with 2d diminished occupancy by this mark to a point not accurately quantifiable by qPCR. No significant changes in occupancy were observed with H3K4me3 or AcH3K9. These results provide evidence that treatment with 2d alters the chromatin architecture of the CDH-1 gene promoter to a more transcriptionally permissive state than that of untreated cells, and these chromatin changes are consistent with the inhibition of LSD1.

Fig. 4
Treatment with 2d alters chromatin marks at the CDH-1 promoter in HL-60 cells. (a) CDH-1 promoter-tiling primer locations & sizes. ChIP was performed with antibodies to specific histone modifications and quantitative PCR of the CDH-1 promoter ...

Changes in polyamine metabolism following exposure to 2d

Certain polyamine analogues exert their anti-tumor effects via a dramatic up-regulation of polyamine catabolism that is accompanied by a down-regulation of polyamine biosynthesis and transport, consequently depleting the pools of natural polyamines essential for continued proliferation. In addition to regulating growth, depletion of the longer-chain, cationic polyamines spermidine and spermine has the potential to alter chromatin structure, as these are most commonly found bound to negatively charged molecules such as nucleic acids. We therefore analyzed the effects of treatment with 2d on polyamine metabolism and the resulting changes in intracellular polyamine pools. As shown in Figure 5a, 24-hour 2d exposure resulted in a down-regulation of the basal levels of one of the rate-limiting polyamine biosynthetic enzymes, ODC, in both HL-60 and KG1a cell lines, similar to results previously reported using HCT116 colon adenocarcinoma cells (Huang et al. 2007b). However, this down-regulation of ODC had little effect on polyamine pools (Fig. 5b). Additionally, the spermidine and spermine levels remaining near basal levels throughout the 48-hour treatment indicated that not only were the polyamine pools unaffected by the down-regulation of ODC, but polyamine catabolism was not induced by 2d. These results provide evidence that the cytokinetic effects observed with 2d exposure are not due to depletion of the natural polyamines, nor is it likely that altered polyamine concentrations are contributing to the observed chromatin changes.

Fig. 5
ODC activity is depleted in AML cells treated with 2d. HL60 and KG1a cells were treated with 10µM 2d for 24 or 48 hours, respectively. Cells were quick-frozen and assayed for ODC enzyme activity (a) and intracellular polyamine pool concentrations ...


The aberrant silencing of tumor suppressor and growth regulatory genes is an attractive target for anticancer therapy as it has become apparent that this silencing is reversible. With the discovery that LSD1 is one of the enzymes responsible for removing transcriptional activating marks and thus has the potential to broadly and aberrantly repress gene expression, it has become a logical target for anticancer therapy (Shi et al. 2004; Liang et al. 2004). Additionally, because the active sight of LSD1 is both structurally and catalytically very similar to that of the polyamine oxidases (Wang et al. 2001; Shi et al. 2004), we hypothesized that compounds similar to the polyamine analogues capable of inhibiting the polyamine oxidases would also be capable of inhibiting LSD1; this hypothesis tested true (Huang et al. 2009).

Previous studies with compound 2d have demonstrated that it effectively inhibited the H3K4 demethylase activity of purified LSD1 protein. Using structurally related, but minimally effective inhibitors for comparison, evaluation of colon cancer cell lines treated with 2d confirmed the correlation between cellular inhibition of LSD1 and the consequent increase in global H3K4me1/2 levels and gene re-expression (Huang et al. 2007b). In the current study, the AML cell lines HL-60 and KG1a also responded to treatment with 2d by exhibiting substantial global increases in mono- and dimethylated H3K4 protein. However, in contrast to the colon cancer cell lines previously studied, both AML cell lines also demonstrated increases in dimethyl H3K9 protein, a repressive chromatin mark associated with silenced transcription (Daniel et al. 2005). In the MCF-7 breast cancer cell line, it has been shown that LSD1 can act as a demethylase of H3K9me2 when recruited to the promoters of specific estrogen receptor (ERα) target genes (Garcia-Bassets et al. 2007). Additionally, when in complex with the androgen receptor (AR), LSD1 has been proposed to be part of a complex that demethylates H3K9me2, thereby facilitating transcription of AR target genes (Metzger et al. 2005; Wissmann et al. 2007). Thus, it might be possible that inhibiting LSD1 with 2d results in an increase in the H3K9me2 chromatin mark and transcriptional repression of the target genes. It is therefore possible that LSD1 inhibition by 2d is increasing both H3K4me2 and H3K9me2 abundance when considering the overall changes in total nuclear proteins. These changes are separated however, at the local level, where our ChIP experiments clearly demonstrate that only the H3K4me1/2 substrates are enriched at the CDH-1 promoter following 2d exposure, whereas H3K9me2 is diminished. These changes in promoter-region chromatin architecture are entirely consistent with the observed re-expression of e-cadherin mRNA.

At the global protein level, 2d treatment also increased the abundance of the active chromatin mark AcH3K9. These results were also observed following pharmacologic inhibition or siRNA targeting of LSD1 in breast cancer cells (Huang et al. 2012) and represent the intimate protein interactions between specific members of the HDAC family and LSD1 (Lee et al. 2005; Shi et al. 2005; Lan et al. 2007).

Although well established as a suppressor of metastasis in epithelial cancers (Karayiannakis et al. 1998; Sulzer et al. 1998; Zheng et al. 1999), the precise role of e-cadherin in hematopoiesis and AML has yet to be fully determined. It is clear however, that aberrant hypermethylation of the e-cadherin gene is a common event in AML (Corn et al. 2000; Melki et al. 2000; Melki et al. 1999; Shimamoto et al. 2005; Ekmekci et al. 2004). Shimamoto et al. have demonstrated significantly elevated numbers of leukemic cells in AML patients harboring methylated e-cadherin alleles, suggesting a correlation between the silencing of e-cadherin expression and an enhanced migration of hematopoietic cells (Shimamoto et al. 2005). The e-cadherin protein has been implicated in the interactions between hematopoietic progenitor cells and bone marrow stromal cells, and recent studies have demonstrated that low expression of e-cadherin can decrease adhesion of progenitors to stromal cells, thereby providing a growth advantage, enhancing colony formation, and increasing the likelihood of cell transformation (Rao et al. 2010).

Several reports now exist suggesting a hypermethylator phenotype in AML, in which a subset of tumor-type specific genes including e-cadherin, are concurrently silenced via DNA hypermethylation (Ekmekci et al. 2004; Melki et al. 1999; Toyota et al. 2001). Furthermore, clinical studies have determined that acute myeloid leukemia in pediatric patients, young adults, and older adults are very different diseases. Toyota et al. (Toyota et al. 2001) have described an inverse correlation between the frequency of hypermethylation of a subset of genes in AML and the age of the patient, while Ekmekci et al. (Ekmekci et al. 2004) found a greater number of certain methylated genes in young adults than in children or older patients. Both of these studies suggest the benefit of targeting epigenetic therapies to certain subsets of patients. The fact that treatment with compound 2d results in an increase in the activating chromatin mark H3K4me2 and the re-expression of one of these aberrantly silenced genes validates its potential as a therapeutic agent in the treatment of AML.

In AML clinical trials, DNMT inhibitors, including 5-azacitidine, have shown 30–60% response rates (Issa et al. 2004; Gore et al. 2006; Silverman et al. 2002); however, the promising effects of these compounds are potentially limited by their own cytotoxicity and the possibility of genomic instability due to hypomethylation (Eden et al. 2003; Gaudet et al. 2003). Pre-clinical studies in solid tumor models employing the polyamine analogue-based LSD1 inhibitors have demonstrated increased efficacy with regard to tumor suppressor gene re-expression and tumor growth inhibition when used in combination with other inhibitors of epigenetic modifications, including DNMT and HDAC inhibitors. It is likely that an investigation of these combinations in hematopoietic malignancies may have a similar outcome, thus enabling the use of less toxic doses, potentially stimulating the expression of additional aberrantly silenced tumor suppressor genes, and ultimately suppressing tumor growth. Furthermore, LSD1 inhibition by 2d has recently been shown to reactivate the all-trans-retinoic acid (ATRA) differentiation pathway in non-APL AML, potentially allowing for effective ATRA-treatment for patients not in the APL subtype (Schenk et al. 2012).

In summary, through the inhibition of LSD1 and the associated remodeling of local chromatin, the polyamine analogue 2d induces the re-expression of the aberrantly silenced e-cadherin tumor suppressor gene, thereby validating LSD1 as a potential therapeutic target in the treatment of AML.


This work was funded by NIH Grants CA51085, CA98454, CA149095, and the Samuel Waxman Cancer Research Foundation.


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