Recent advances in class IIa histone deacetylases research

Epigenetic control plays an important role in gene regulation through chemical modifications of DNA and post-translational modifications of histones. An essential post-translational modification is the histone acetylation/deacetylation-process which is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The mammalian zinc dependent HDAC family is subdivided into three classes: class I (HDACs 1-3, 8), class II (IIa: HDACs 4, 5, 7, 9; IIb: HDACs 6, 10) and class IV (HDAC 11). In this review, recent studies on the biological role and regulation of class IIa HDACs as well as their contribution in neurodegenerative diseases, immune disorders and cancer will be presented. Furthermore, the development, synthesis, and future perspectives of selective class IIa inhibitors will be highlighted.     

Complementary roles for histone deacetylases 1, 2, and 3 in differentiation of pluripotent stem cells

Abstract In eukaryotic cells, covalent modifications to core histones contribute to the establishment and maintenance of cellular phenotype via regulation of gene expression. Histone acetyltransferases (HATs) cooperate with histone deacetylases (HDACs) to establish and maintain specific patterns of histone acetylation. HDAC inhibitors can cause pluripotent stem cells to cease proliferating and enter terminal differentiation pathways in culture. To better define the roles of individual HDACs in stem cell differentiation, we have constructed "dominant-negative" stem cell lines expressing mutant, Flag-tagged HDACs with reduced enzymatic activity. Replacement of a single residue (His→Ala) in the catalytic center reduced the activity of HDACs 1 and 2 by 80%, and abolished HDAC3 activity; the mutant HDACs were expressed at similar levels and in the same multiprotein complexes as wild-type HDACs. Hexamethylene bisacetamide-induced MEL cell differentiation was potentiated by the individual mutant HDACs, but only to 2%, versus 60% for an HDAC inhibitor, sodium butyrate, suggesting that inhibition of multiple HDACs is required for full potentiation. Cultured E14.5 cortical stem cells differentiate to neurons, astrocytes, and oligodendrocytes upon withdrawal of basic fibroblast growth factor. Transduction of stem cells with mutant HDACs 1, 2, or 3 shifted cell fate choice toward oligodendrocytes. Mutant HDAC2 also increased differentiation to astrocytes, while mutant HDAC1 reduced differentiation to neurons by 50%. These results indicate that HDAC activity inhibits differentiation to oligodendrocytes, and that HDAC2 activity specifically inhibits differentiation to astrocytes, while HDAC1 activity is required for differentiation to neurons.     

The Development Process: from SAHA to Hydroxamate HDAC Inhibitors with Branched CAP Region and Linear Linker

Histone deacetylases (HDACs) belong to a group of epigenetic regulatory enzymes that participate in modulating the acetylation level of histone lysine residues as well as non‐histone proteins, and they play a key role in the regulation of gene expression. HDACs are potential anticancer drug targets highly expressed in various kinds of cancer cells. So far, five small molecules targeting HDACs have been approved for the therapy of cancer, and over 20 inhibitors of HDACs are under different phases of clinical trials. Among them, hydroxamate‐based HDAC inhibitors (HDACis) represent a well‐investigated series of chemical entities. The current review covers the recent progress in the discovery process, form SAHA to hydroxamate HDAC inhibitors with branched CAP region and linear linker. At the same time, the pharmacological and structure‐activity relationship (SAR) studies of the specific derivatives from SAHA and the HDACis with branched CAP region and linear linker are also introduced.     

Coordinated expression of p300 and HDAC3 upregulates histone acetylation during dentinogenesis

Cellular differentiation is caused by highly controlled modifications in the gene expression but rarely involves a change in the DNA sequence itself. Histone acetylation is a major epigenetic factor that adds an acetyl group to histone proteins, thus altering their interaction with DNA and nuclear proteins. Illumination of the histone acetylation during dentinogenesis is important for odontoblast differentiation and dentinogenesis. In the current study, we aimed to discover the roles and regulation of acetylation at histone 3 lysine 9 (H3K9ac) and H3K27ac during dentinogenesis. We first found that both of these modifications were enhanced during odontoblast differentiation and dentinogenesis. These modifications are dynamically catalyzed by histone acetyltransferases (HATs) and deacetylases (HDACs), among which HDAC3 was decreased while p300 increased during odontoblast differentiation. Moreover, overexpression of HDAC3 or knockdown p300 inhibited odontoblast differentiation in vitro, and inhibition of HDAC3 and p300 with trichostatin A or C646 regulated odontoblast differentiation. Taken together, the results of our present study suggest that histone acetylation is involved in dentinogenesis and coordinated expression of p300- and HDAC3-regulated odontoblast differentiation through upregulating histone acetylation.     

Role of class I and class II histone deacetylases in carcinoma cells using siRNA

The role of the individual histone deacetylases (HDACs) in the regulation of cancer cell proliferation was investigated using siRNA-mediated protein knockdown. The siRNA for HDAC3 and HDAC1 demonstrated significant morphological changes in HeLa S3 consistent with those observed with HDAC inhibitors. SiRNA for HDAC 4 or 7 produced no morphological changes in HeLa S3 cells. HDAC1 and 3 siRNA produced a concentration-dependent inhibition of HeLa cell proliferation; whereas, HDAC4 and 7 siRNA showed no effect. HDAC3 siRNA caused histone hyperacetylation and increased the percent of apoptotic cells. These results demonstrate that the Class I HDACs such as HDACs 1 and 3 are important in the regulation of proliferation and survival in cancer cells. These results and the positive preclinical results with non-specific inhibitors of the HDAC enzymes provide further support for the development of Class I selective HDAC inhibitors as cancer therapeutics.     

Array-Based Approaches for the Identification of Epigenetic Silenced Tumor Suppressor Genes

Carcinogenesis involves the inactivation or inhibition of genes that function as tumor suppressors. Deletions, mutations, or epigenetic silencing of tumor suppressor genes can lead to altered growth, differentiation, and apoptosis. DNA methylation and histone modifications are important epigenetic mechanisms of gene regulation and play essential roles both independently and cooperatively in tumor initiation and progression. Realization that many tumor suppressor genes are silenced by epigenetic mechanisms has stimulated discovery of novel tumor suppressor genes. One of the most useful of these approaches is an epigenetic reactivation screening strategy that combines treatment of cancer cells in vitro with DNA methyltransferase and/or histone deacetylase (HDAC) inhibitors, followed by global gene expression analysis using microarrays, to identify upregulated genes. This approach is most effective when complemented by microarray analyses to identify genes repressed in primary tumors. Recently, using cancer cell lines treated with a DNA methylation inhibitor and/or a HDAC inhibitor in conjunction with cDNA microarray analysis, candidate tumor suppressor genes, which are subject to epigenetic silencing, have been identified in endometrial, colorectal, esophageal, and pancreatic cancers. An increasing number of studies have utilized epigenetic reactivation screening to discover novel tumor suppressor genes in cancer. The results of some of the most recent studies are highlighted in this review.     

HDACs, histone deacetylation and gene transcription： from molecular biology to cancer therapeutics

Histone deacetylases （HDACs） and histone acetyl transferases （HATs） are two counteracting enzyme families whose enzymatic activity controls the acetylation state of protein lysine residues, notably those contained in the N-terminal extensions of the core histones. Acetylation of histones affects gene expression through its influence on chromatin conformation. In addition, several non-histone proteins are regulated in their stability or biological function by the acetylation state of specific lysine residues. HDACs intervene in a multitude of biological processes and are part of a multiprotein family in which each member has its specialized functions. In addition, HDAC activity is tightly controlled through targeted recruitment, protein-protein interactions and post-translational modifications. Control of cell cycle progression, cell survival and differentiation are among the most important roles of these enzymes. Since these processes are affected by malignant transformation, HDAC inhibitors were developed as antineoplastic drugs and are showing encouraging efficacy in cancer patients.     

Dual targeting of epigenetic therapy in cancer

Aberrant epigenetic silencing of tumor suppressor genes by promoter DNA hypermethylation and histone deacetylation plays an important role in the pathogenesis of cancer. The potential reversibility of epigenetic abnormalities encouraged the development of pharmacologic inhibitors of DNA methylation and histone deacetylation as anti-cancer therapeutics. (Pre)clinical studies of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors have yielded encouraging results, especially against hematologic malignancies. Recently, several studies demonstrated that DNMT and HDAC inhibitors are also potent angiostatic agents, inhibiting (tumor) endothelial cells and angiogenesis in vitro and in vivo. By reactivation of epigenetically silenced tumor suppressor genes with angiogenesis inhibiting properties, DNMT and HDAC inhibitors might indirectly - via their effects on tumor cells - decrease tumor angiogenesis in vivo. However, this does not explain the direct angiostatic effects of these agents, which can be unraveled by gene expression studies and examination of epigenetic promoter modifications in endothelial cells treated with DNMT and HDAC inhibitors. Clearly, the dual targeting of epigenetic therapy on both tumor cells and tumor vasculature makes them attractive combinatorial anti-tumor therapeutics. Here we review the therapeutic potential of DNMT and HDAC inhibitors as anti-cancer drugs, as evaluated in clinical trials, and their angiostatic activities, apart from their inhibitory effects on tumor cells.     

Reversible acetylation of chromatin: Implication in regulation of gene expression,disease and therapeutics

The eukaryotic genome is a highly dynamic nucleoprotein complex that is comprised of DNA, histones, nonhistone proteins and RNA, and is termed as chromatin. The dynamicity of the chromatin is responsible for the regulation of all the DNA-templated phenomena in the cell. Several factors, including the nonhistone chromatin components, ATP-dependent remodeling factors and the chromatin-modifying enzymes, mediate the combinatorial post-translational modifications that control the chromatin fluidity and, thereby, the cellular functions. Among these modifications, reversible acetylation plays a central role in the highly orchestrated network. The enzymes responsible for the reversible acetylation, the histone acetyltransferases (HATs) and histone deacetylases (HDACs), not only act on histone substrates but also on nonhistone proteins. Dysfunction of the HATs/HDACs is associated with various diseases like cancer, diabetes, asthma, cardiac hypertrophy, retroviral pathogenesis and neurodegenerative disorders. Therefore, modulation of these enzymes is being considered as an important therapeutic strategy. Although substantial progress has been made in the area of HDAC inhibitors, we have focused this review on the HATs and their small-molecule modulators in the context of disease and therapeutics. Recent discoveries from different groups have established the involvement of HAT function in various diseases. Furthermore, several new classes of HAT modulators have been identified and their biological activities have also been reported. The scaffold of these small molecules can be used for the design and synthesis of better and efficient modulators with superior therapeutic efficacy.     

Structure and property based design, synthesis and biological evaluation of γ-lactam based HDAC inhibitors

Histone deacetylases (HDACs) are involved in post-translational modification and gene expression. Cancer cells recruited amounts of HDACs for their survival by epi-genetic down regulation of tumor suppressor genes. HDACs have been the promising targets for treatment of cancer, and many HDAC inhibitors have been investigated nowadays. In previous study, we synthesized δ-lactam core HDAC inhibitors which showed potent HDAC inhibitory activities as well as cancer cell growth inhibitory activities. Through QSAR study of the δ-lactam based inhibitors, the smaller core is suggested as more active than larger one because it fits better in narrow hydrophobic tunnel of the active pocket of HDAC enzyme. The smaller γ-lactam core HDAC inhibitors were designed and synthesized for biological and property optimization. Phenyl, naphthyl and thiophenyl groups were introduced as the cap groups. Hydrophobic and bulky cap groups increase potency of HDAC inhibition because of hydrophobic interaction between HDAC and inhibitors. In overall, γ-lactam based HDAC inhibitors showed more potent than δ-lactam analogues.     

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts. This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones. This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types. This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.     

The use of diversity profiling to characterize chemical modulators of the histone deacetylases

Target specificity and off-target liabilities are routinely monitored during the early phases of drug discovery for most kinase projects. Typically these criteria are evaluated using a profiling panel comprised of a diverse collection of in vitro kinase assays and relates compound structure to potency and selectivity. The success of these efforts has led to the design of similar panels for phosphatase, protease, and epigenetic targets. Here the implementation of an epigenetic profiling panel, comprised of eleven histone deacetylases (HDACs) and one histone acetyltransferase (HAT), was used to evaluate chemical modulators of these enzymes. HDAC inhibitors (HDACi) such as sodium butyrate and trichostatin A demonstrate diverse biological effects which have led to broad speculation about their therapeutic potential in multiple disease states. Some HDACi have demonstrated tumor suppression in vivo and recently Zolinza was the first HDACi approved by the FDA for the treatment of cutaneous T-cell lymphoma. While HDACi have demonstrated therapeutic utility, many of the first generation compounds are pan-inhibitors. Thus, use of an HDAC profiling panel will be essential in achieving isoform specificity of the next generation of inhibitors. To this end, twenty-one compounds, twelve of which are known to have activities against the HDACs, were tested to evaluate the utility of the epigenetic panel. Additionally, these compounds were tested against a larger 72 member enzyme panel comprised of kinase, phosphatase and protease activities. This effort represents the first time these compounds have been profiled with such a broad range of biochemical activities.