Release of hypoacetylated and trimethylated histone H4 is an epigenetic marker of early apoptosis

Nuclear events such as chromatin condensation, DNA cleavage at internucleosomal sites, and histone release from chromatin are recognized as hallmarks of apoptosis. However, there is no complete understanding of the molecular events underlying these changes. It is likely that epigenetic changes such as DNA methylation and histone modifications that are involved in chromatin dynamics and structure are also involved in the nuclear events described. In this report we have shown that apoptosis is associated with global DNA hypomethylation and histone deacetylation events in leukemia cells. Most importantly, we have observed a particular epigenetic signature for early apoptosis defined by a release of hypoacetylated and trimethylated histone H4 and internucleosomal fragmented DNA that is hypermethylated and originates from perinuclear heterochromatin. These findings provide one of the first links between apoptotic nuclear events and epigenetic markers

Expression of NES-hTERT in Cancer Cells Delays Cell Cycle Progression and Increases Sensitivity to Genotoxic Stress

Telomerase is a reverse transcriptase associated with cellular immortality through telomere maintenance. This enzyme is activated in 90% of human cancers, and inhibitors of telomerase are currently in clinical trials to counteract tumor growth. Many aspects of telomerase biology have been investigated for therapy, particularly inhibition of the enzyme, but little was done regarding its subcellular shuttling. We have recently shown that mutations in the nuclear export signal of hTERT, the catalytic component of telomerase, led to a mutant (NES-hTERT) that failed to immortalize cells despite nuclear localization and catalytic activity. Expression of NES-hTERT in primary fibroblast resulted in telomere-based premature senescence and mitochondrial dysfunction. Here we show that expression of NES-hTERT in LNCaP, SQ20B and HeLa cells rapidly and significantly decreases their proliferation rate and ability to form colonies in soft agar while not interfering with endogenous telomerase activity. The cancer cells showed increased DNA damage at telomeric and extra-telomeric sites, and became sensitive to ionizing radiation and hydrogen peroxide exposures. Our data show that expression of NES-hTERT efficiently counteracts cancer cell growth in vitro in at least two different ways, and suggest manipulation with the NES of hTERT or its subcellular shuttling as a new strategy for cancer treatment

Abstract 1433: Mice Overexpressing Histone Deacetylase 9 Display Abnormal B Cell Development and Proliferation.

Histone acetylation plays a key role in regulating chromatin structure and gene expression. The activities of histone deacetylases (HDAC), enzymes that remove acetyl groups from lysines located at the amino-terminal tails of core histones, have been implicated in the pathogenesis of hematological malignancies. Consequently, inhibitors of these enzymes (HDACi) are considered an important new class of drugs for use in anti-cancer therapy and are now in various stages of development and clinical trials. For example, depsipeptide has now been granted a fast track designation by the FDA for development as a treatment for CTCL. To date, eleven HDACs have been identified and grouped on the basis of sequence homology to class I (HDACs 1–3 and 8), IIa (4, 5, 7 and 9) and IIb (6, 10 and 11). Depsipeptide and other currently used HDACi lack class and enzyme specificities. Additionally, the contribution of each HDAC to the pathogenesis of a given malignant disease is poorly understood. Improvements in HDACi specificities, together with advances in the understanding of the roles that individual HDACs play in normal hematopoiesis and distinct hematological neoplasms, are required for an effective use of these agents in anti-cancer therapies. We have previously identified and characterized HDAC9 as a class IIa enzyme that within the hematopoietic system is preferentially expressed in the lymphoid lineage. HDAC9 appears to be overexpressed in B lymphoid malignancies and associates at physiological levels with the BCL6 oncoprotein ( Petrie et al, JBC 278:16059–72, 2003), a transcriptional repressor implicated in the pathogenesis of non-Hodgkin’s lymphoma, suggesting a role for this chromatin modifying enzyme in B-cell transformation. In order to examine the function of HDAC9 in lymphoid development and cancer, we have generated transgenic mice that express the protein throughout the B lymphoid compartment under the control of the immunoglobulin heavy chain enhancer (Eμ). Initial data show that at an average of seven months these mice display splenomegaly and alterations to the splenic and bone marrow lymphoid populations. The presence of immature lymphocytes was also detected in peripheral blood. Consistent with these results and the above hypothesis, knockdown of HDAC9 results in strong inhibition of cell growth. These data indicate that aberrant expression of HDAC9 confers a proliferative advantage and leads to a phenotype resembling a pre-malignant condition

Interference with Sin3 function induces epigenetic reprogramming and differentiation in breast cancer cells

Sin3A/B is a master transcriptional scaffold and corepressor that plays an essential role in the regulation of gene transcription and maintenance of chromatin structure, and its inappropriate recruitment has been associated with aberrant gene silencing in cancer. Sin3A/B are highly related, large, multidomian proteins that interact with a wide variety of transcription factors and corepressor components, and we examined whether disruption of the function of a specific domain could lead to epigenetic reprogramming and derepression of specific subsets of genes. To this end, we selected the Sin3A/B-paired amphipathic α-helices (PAH2) domain based on its established role in mediating the effects of a relatively small number of transcription factors containing a PAH2-binding motif known as the Sin3 interaction domain (SID). Here, we show that in both human and mouse breast cancer cells, the targeted disruption of Sin3 function by introduction of a SID decoy that interferes with PAH2 binding to SID-containing partner proteins reverted the silencing of genes involved in cell growth and differentiation. In particular, the SID decoy led to epigenetic reprogramming and reexpression of the important breast cancer-associated silenced genes encoding E-cadherin, estrogen receptor α, and retinoic acid receptor β and impaired tumor growth in vivo. Interestingly, the SID decoy was effective in the triple-negative M.D. Anderson-Metastatic Breast-231 (MDA-MB-231) breast cancer cell line, restoring sensitivity to 17β-estradiol, tamoxifen, and retinoids. Therefore, the development of small molecules that can block interactions between PAH2 and SID-containing proteins offers a targeted epigenetic approach for treating this type of breast cancer that may also have wider therapeutic implications

Identification of (1H)-pyrroles as histone deacetylase inhibitors with antitumoral activity

Histone deacetylases (HDACs) play a key role in the regulation of gene expression and chromatin structure, and drugs targeting these enzymes might have an important impact in the treatment of human cancer. Herein, we report the characterization of (1H)-pyrroles as a new subfamily of HDAC inhibitors obtained by computational modeling of class-I human HDACs. From a functional standpoint, (1H)-pyrroles are powerful inductors of acetylation of histones H3 and H4, and restore the expression of growth-inhibitory genes. From a cellular view, these compounds cause a marked decrease in the viability of cancer cells in vitro and in vivo, associated with a cell-cycle arrest at G2/M and an inhibition of angiogenesis. Thus, (1H)-pyrroles emerge as a novel group of HDAC inhibitors with promising antitumoral features. © 2009 Macmillan Publishers Limited All rights reserved.This work was supported by Grants SAF2007-00027-65134, Consolider CSD2006-49, the Spanish Association Against Cancer (AECC), FP6 Grant SMARTER, Ikerchem Ltd., The Universidad del País Vasco-Euskal Herriko Unibertsitatea (Grant UE07/16), the Gobierno Vasco-Eusko Jaurlaritza (Grant 9/UPV00170.215-13548) and the Spanish Ministerio de Educación y Ciencia (CTQ2007-67528/BQU). SR is a ‘Ramón y Cajal’ Researcher. ME is an ICREA Research Professor.Peer Reviewe

Abstract 1046: Modulation of Histone H3K4 and H3K27 Methylation Levels Via Pharmacological Inhibition of LSD1 and Degradation of the EZH2-Containing Polycomb Repressive Complex 2 Stimulates ATRA-Mediated Differentiation of AML Cells.

During hematopoiesis, all-trans-retinoic acid (ATRA), a natural derivative of vitamin A, has been shown to induce both myelomonocytic progenitor/stem cell differentiation and self-renewal. Although these opposing effects are likely to be partly due to developmental differences, it has been shown that pro- and anti-differentiation effects of ATRA are mediated by distinct retinoic acid receptor isotypes (RARαa and RARγ, respectively). With the exception of acute promyelocytic leukemia (APL) ATRA treatment as a single agent has not been successful in other types of acute myeloid leukemia (AML). We have hypothesized that one of the underlying reasons for poor response of non-APL AML to ATRA (pan-RAR agonist) is aberrant expression and/or activities of RAR isotypes favoring RARγ and cell growth versus differentiation. Consistently, we have reported that expression of RARαa isoforms, particularly ATRA-inducible RARαa2, are down-regulated in AML (Blood 2008; 111:2374). Epigenetic analysis of patient samples revealed that relative to normal CD33+ cells, the loss of RARαa2 in AML is associated with a diminution in histone H3K4me2 and an increase H3K27me3 on the RARA2 promoter (modifications associated with transcriptional activation and silencing, respectively). Interestingly, H3K4 demethylase LSD1 (AOF2) and the polycomb represive complex 2 (PCR2)-associated H3K27 methyltransferase EZH2 are highly expressed in AML (www.proteinatlas.org). Small molecules that target these enzymes are in development and, given the above results, we predict that the use of such agents in combination with ATRA will enhance the effects of ATRA-mediated induction of gene expression and differentiation of AML cells. To test this hypothesis, we used ATRA-responsive HL-60 AML cells and the TEX cell line. TEX cells are derived from primitive human cord blood cells immortalized by expression of the TLS-ERG oncogene. These cells, the ATRA-responsiveness of which is not known, mimic features of primary human AML and leukemia initiating cells (Leukemia. 2005; 19:1794). LSD1 activity was inhibited using monoaminoxidase inhibitor (MAOI) trans-2-phenylcyclopropylamine (Parnate, 1μM) in combination with pharmacological (1μM) and sub-optimal (0.1μM) concentrations of ATRA. Co-treatment with Parnate potentiated the HL-60 response to sub-optimal ATRA concentration. While ATRA appeared to be a less potent inducer of TEX cell differentiation, Parnate nevertheless enhanced their maturation at pharmacological ATRA concentrations and sensitized these cells to differentiation induction under sub-optimal ATRA levels. Additionally, we investigated the biguanide polyamine analogue 1,15-bis[N5-[3,3-(diphenyl) propyl]-N1-biguanido]-4,12-diazapentadecane (2d), which is structurally unrelated to Parnate, obtaining similar results. Biguanide polyamine analogue inhibitors of LSD1 may have several benefits over MAOIs, including DNA targeting due their cationic nature. We also tested 3-deazaneplanocin A (DZNep), which diminishes levels of H3K27 trimethylation via depletion of the EZH2 catalytic subunit of the PCR2. Consistent with our hypothesis and the above data, co-treatment of HL-60 and TEX cells with DZNep (0.05μM) and ATRA (0.1μM and/or 1μM) led to more robust differentiation response than when ATRA was used as a single agent. The use of ATRA in combination with DZNep and LSD1 inhibitors at the same time led to a better differentiation response, as measured by CD11b/CD11c expression, morphology and superoxide production (NBT assay), than when either drug alone was used with ATRA. The effects of these drug combinations on AML cell maturation were paralleled by synergistic induction of endogenous ATRA target genes and expected changes in the levels of H3K4/K27 methylation. At the concentrations used with ATRA neither Parnate, 2d nor DZNep induced differentiation when used as single agents, however, when used at higher concentrations both singly and in combination with ATRA, these drugs exerted cytotoxic effects. Importantly, the above described combination treatments were specific for AML blasts as they had no cytotoxic effects on normal CD33+/CD34+ cell populations. These data demonstrate existence of therapeutically relevant crosstalks between the ATRA-induced differentiation pathway and histone H3K4 and K27 methylation and that targeting LSD1 and/or EZH2 in combination with ATRA may represent a promising treatment for AML

Release of hypoacetylated and trimethylated histone H4 is an epigenetic marker of early apoptosis

11 p.-5 fig.-1 fig. supl.Nuclear events such as chromatin condensation, DNA cleavage at internucleosomal sites, and histone release from chromatin are recognized as hallmarks of apoptosis. However, there is no complete understanding of the molecular events underlying these changes. It is likely that epigenetic changes such as DNA methylation and histone modifications that are involved in chromatin dynamics and structure are also involved in the nuclear events described. In this report we have shown that apoptosis is associated with global DNA hypomethylation and histone deacetylation events in leukemia cells. Most importantly, we have observed a particular epigenetic signature for early apoptosis defined by a release of hypoacetylated and trimethylated histone H4 and internucleosomal fragmented DNA that is hypermethylated and originates from perinuclear heterochromatin. These findings provide one of the first links between apoptotic nuclear events and epigenetic markers.This work was supported by Grants SAF 2001-0059, BFU2004-02073/BMC and Ramon y Cajal Programme (MCYT), and GR/SAL/0224/2004 (Government of Madrid).Peer reviewe