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 post-translational 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{N ⁵-[3,3-(diphenyl)propyl]-N ¹-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 re-expression of e-cadherin, a tumor suppressor gene frequently silenced by epigenetic modification in AML. Quantitative chromatin immunoprecipitation analysis of the e-cadherin 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.     

组蛋白去甲基化酶LSD1及其生物学功能

赖氨酸特异性组蛋白去甲基化酶1(Lysine specific demethylase 1, LSD1) 的发现, 表明组蛋白的甲基化修饰是一个动态可调节的过程。结构分析显示, LSD1 是一个黄素腺嘌呤二核苷酸(Flavin adenine dinulcleotide, FAD) 依赖性胺氧化酶, 它能够特异性脱去单甲基化和二甲基化组蛋白H3第4位赖氨酸(H3K4) 和H3K9 位点上的甲基基团。功能研究显示, LSD1 定位于细胞核内, 调控着基因转录的激活和抑制, 被誉为细胞深处的基因“开关”, 在胚胎发育和肿瘤发生过程中起着重要的作用。文章主要综述了LSD1 的结构、作用机制及其调控作用研究的新进展。     

Polyamine analogs modulate gene expression by inhibiting lysine-specific demethylase 1 (LSD1) and altering chromatin structure in human breast cancer cells

Aberrant epigenetic repression of gene expression has been implicated in most cancers, including breast cancer. The nuclear amine oxidase, lysine-specific demethylase 1 (LSD1) has the ability to broadly repress gene expression by removing the activating mono- and di-methylation marks at the lysine 4 residue of histone 3 (H3K4me1 and me2). Additionally, LSD1 is highly expressed in estrogen receptor α negative (ER-) breast cancer cells. Since epigenetic marks are reversible, they make attractive therapeutic targets. Here we examine the effects of polyamine analog inhibitors of LSD1 on gene expression, with the goal of targeting LSD1 as a therapeutic modality in the treatment of breast cancer. Exposure of the ER-negative human breast cancer cells, MDA-MB-231 to the LSD1 inhibitors, 2d or PG11144, significantly increases global H3K4me1 and H3K4me2, and alters gene expression. Array analysis indicated that 98 (75 up and 23 down) and 477 (237 up and 240 down) genes changed expression by at least 1.5-fold or greater after treatment with 2d and PG11144, respectively. The expression of 12 up-regulated genes by 2d and 14 up-regulated genes by PG11144 was validated by quantitative RT-PCR. Quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated that up-regulated gene expression by polyamine analogs is associated with increase of the active histone marks H3K4me1, H3K4me2 and H3K9act, and decrease of the repressive histone marks H3K9me2 and H3K27me3, in the promoter regions of the relevant target genes. These data indicate that the pharmacologic inhibition of LSD1 can effectively alter gene expression and that this therapeutic strategy has potential.     

LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells

We identify LSD1 (lysine-specific demethylase 1; also known as KDM1A and AOF2) as a key histone modifier that participates in the maintenance of pluripotency through the regulation of bivalent domains, a chromatin environment present at the regulatory regions of developmental genes that contains both H3K4 di/trimethylation and H3K27 trimethylation marks. LSD1 occupies the promoters of a subset of developmental genes that contain bivalent domains and are co-occupied by OCT4 and NANOG in human embryonic stem cells, where it controls the levels of H3K4 methylation through its demethylase activity. Thus, LSD1 has a role in maintaining the silencing of several developmental genes in human embryonic stem cells by regulating the critical balance between H3K4 and H3K27 methylation at their regulatory regions.     

Discovery of tranylcypromine analogs with an acylhydrazone substituent as LSD1 inactivators: Design,synthesis and their biological evaluation

Lysine specific demethylase 1 (LSD1), the first identified histone demethylase, plays an important role in epigenetic regulation of gene activation and repression, has been reported to be up-regulated and involved in numbers of solid malignant tumors. In this study, we identified a series of phenylalanyl hydrazones based LSD1 inhibitors, and the most potent one, compound 4q, can inactivate LSD1 with IC₅₀?=?91.83?nM. In cellular level, compound 4q can induce the accumulation of CD86 as well as H3K4me2, and inhibit gastric cancer cell proliferation by inactivating LSD1. Our findings indicated that compound 4q may serve as a potential leading compound to target LSD1 overexpressed gastric cancer.     

LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

LSD1 is essential for the maintenance of pluripotency of embryonic stem (ES) or embryonic carcinoma/teratocarcinoma (EC) cells. We have previously developed novel LSD1 inhibitors that selectively inhibit ES/EC cells. However, the critical targets of LSD1 remain unclear. Here, we found that LSD1 interacts with histone deacetylase 1 (HDAC1) to regulate the proliferation of ES/EC cells through acetylation of histone H4 at lysine 16 (H4K16), which we show is a critical substrate of HDAC1. The LSD1 demethylase and HDAC1 deacetylase activities were both inactivated if one of them in the complex was chemically inhibited in ES/EC cells or in reconstituted protein complexes. Loss of HDAC1 phenocopied the selective growth-inhibitory effects and increased the levels of H3K4 methylation and H4K16 acetylation of LSD1 inactivation on ES/EC cells. Reduction of acetylated H4K16 by ablation of the acetyltransferase males absent on the first (MOF) is sufficient to rescue the growth inhibition induced by LSD1 inactivation. While LSD1 or HDAC1 inactivation caused the downregulation of Sox2 and Oct4 and induction of differentiation genes, such as FOXA2 or BMP2, depletion of MOF restored the levels of Sox2, Oct4, and FoxA2 in LSD1-deficient cells. Our studies reveal a novel mechanism by which LSD1 acts through the HDAC1- and MOF-mediated regulation of H4K16 acetylation to maintain the pluripotency of ES/EC cells.