Manfred Jung

Histone deacetylase (HDAC) inhibitors in recent clinical trials for cancer therapy

Heritable changes in gene expression that are not based upon alterations in the DNA sequence are defined as epigenetics. The most common mechanisms of epigenetic regulation are the methylation of CpG islands within the DNA and the modification of amino acids in the N-terminal histone tails. In the last years, it became evident that the onset of cancer and its progression may not occur only due to genetic mutations but also because of changes in the patterns of epigenetic modifications. In contrast to genetic mutations, which are almost impossible to reverse, epigenetic changes are potentially reversible. This implies that they are amenable to pharmacological interventions. Therefore, a lot of work in recent years has focussed on the development of small molecule enzyme inhibitors like DNA-methyltransferase inhibitors or inhibitors of histone-modifying enzymes. These may reverse misregulated epigenetic states and be implemented in the treatment of cancer or other diseases, e.g., neurological disorders. Today, several epigenetic drugs are already approved by the FDA and the EMEA for cancer treatment and around ten histone deacetylase (HDAC) inhibitors are in clinical development. This review will give an update on recent clinical trials of the HDAC inhibitors used systemically that were reported in 2009 and 2010 and will present an overview of different biomarkers to monitor the biological effects.

SIRT1 regulates HIV transcription via Tat deacetylation.

The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation.

Inhibitors of histone deacetylase as new anticancer agents.

Inhibitors of histone deacetylase (HDAC) are an emerging class of anticancer agents. They induce hyperacetylation in chromatin usually resulting in activation of certain genes. They induce terminal cell differentiation and/or apoptosis in cancer cells. Histone deacetylase activity is recruited by co-repressor proteins to certain regions of the chromatin and aberrant histone acetylation caused by that recruitment is responsible for the pathogenesis of certain cancers on a molecular level. Inhibitors of HDAC have been identified in natural sources and also synthetic inhibitors are available. The best studied inhibitor is trichostatin A, a hydroxamic acid that exerts its activity by complexation of a zinc ion that is supposed to mediate the acetamide cleavage at the catalytic site. There are several synthetic hydroxamic acids that bear resemblance to trichostatin. Another class of potent inhibitors are naturally occurring and synthetic cyclotetrapeptides that all contain an unusual amino acid with an epoxyketone, ketone or hydroxamic acid function in the side chain. Phenylacetate, phenylbutyrate, butyrate and similar short chain fatty acids are also weak inhibitors. Further inhibitors from natural sources are the epoxide depudecin and depsipeptide FR 901228. The benzamide MS-275 belongs to a new class of synthetic HDAC inhibitors and displays oral activity in animal models. First clinical studies have shown that histone hyperacetylation can be achieved safely in humans and that treatment of cancer is possible. Thus, inhibitors of HDAC are one of the most promising class of new anticancer agents. New screening assays are useful tools that will facilitate identification of further inhibitors.

The emerging therapeutic potential of histone methyltransferase and demethylase inhibitors.

Epigenetics is defined as heritable changes to the transcriptome that are independent of changes in the genome. The biochemical modifications that govern epigenetics are DNA methylation and posttranslational histone modifications. Among the histone modifications, acetylation and deacetylation are well characterized, whereas the fields of histone methylation and especially demethylation are still in their infancy. This is particularly true with regard to drug discovery. There is strong evidence that these modifications play an important role in the maintenance of transcription as well as in the development of certain diseases. This article gives an overview of the mechanisms of action of histone methyltransferases and demethylases, their role in the formation of certain diseases, and available inhibitors. Special emphasis is placed on the strategies that led to the first inhibitors which are currently available and the screening approaches that were used in that process.

Structure-activity studies on suramin analogues as inhibitors of NAD+-dependent histone deacetylases (sirtuins).

Suramin is a symmetric polyanionic naphthylurea originally used for the treatment of trypanosomiasis and onchocerciasis. Suramin and diverse analogues exhibit a broad range of biological actions in vitro and in vivo, including, among others, antiproliferative and antiviral activity. Suramin derivatives usually target purinergic binding sites. Class III histone deacetylases (sirtuins) are amidohydrolases that require nicotinamide adenine dinucleotide (NAD+) as a cofactor for their catalytic mechanism. Deacetylation of the target proteins leads to a change in conformation and alters the activity of the proteins in question. Suramin was reported to inhibit human sirtuin 1 (SIRT1). We tested a diverse set of suramin analogues to elucidate the inhibition of the NAD+‐dependent histone deacetylases SIRT1 and SIRT2 and discovered selective inhibitors of human sirtuins with potency in the two‐digit nanomolar range. In addition, the structural requirements for the binding of suramin derivatives to sirtuins were investigated by molecular docking. The recently published X‐ray crystal structure of human SIRT5 in complex with suramin and the human SIRT2 structure were used to analyze the interaction mode of the novel suramin derivatives.

Enhancement of radiation sensitivity of human squamous carcinoma cells by histone deacetylase inhibitors.

Abstract Zhang, Y., Jung, M., Dritschilo, A. and Jung, M. Enhancement of Radiation Sensitivity of Human Squamous Carcinoma Cells by Histone Deacetylase Inhibitor. Radiat. Res. 161, 667–674 (2004). Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents with potential for disruption of critical cellular processes in cancer cells. Transcriptional regulation, differentiation, cell cycle arrest, radiation sensitization, and apoptosis have been observed in response to exposure to HDAC inhibitors. In the present study, we observed that several potent HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, M344 (an analogue of hydroxamic acid), and the cyclic tetrapeptide, depsipeptide (FR90228), modulate cellular responses to ionizing radiation in cells of two human squamous carcinoma lines (SQ-20B and SCC-35), previously characterized as intrinsically resistant to radiation. Also exposure to IC50 concentrations of these inhibitors, radiation sensitivities were enhanced in both cell lines. Depsipeptide exhibited the greatest effect on SQ-20B cells, decreasing D0 values from 2.62 Gy to 1.64 Gy. M344 was the most active drug in sensitizing SCC-35 cells, decreasing D0 values from 1.91 Gy to 1.21 Gy. The mechanisms underlying HDAC inhibitor-induced radiosensitization were further investigated by extending trichostatin A studies to assess cell cycle distributions and levels of apoptosis. Treatment of SQ-20B cells with radiosensitizing concentrations of trichostatin A resulted in cell cycle arrest in G1 phase (>70%) and inhibition of DNA synthesis. Contrary to previous reports, induction of apoptosis was very low and caspase 3 and 9 were not activated. Taken together, these results implicate G1 arrest and inhibition of DNA synthesis in the mechanisms underlying radiation sensitization by trichostatin A and support the use of HDAC inhibitors for targeting radioresistant cancers.

Structure–Activity Studies on Splitomicin Derivatives as Sirtuin Inhibitors and Computational Prediction of Binding Mode

NAD (+)-dependent histone deacetylases (sirtuins) are enzymes that cleave acetyl groups from lysines in histones and other proteins. Potent selective sirtuin inhibitors are interesting tools for the investigation of the biological functions of those enzymes and may be future drugs for the treatment of cancer. Splitomicin was among the first two inhibitors that were discovered for yeast sirtuins but showed rather weak inhibition on human enzymes. We present detailed structure-activity relationships on splitomicin derivatives and their inhibition of recombinant Sirt2. To rationalize our experimental results, ligand docking followed by molecular mechanics Poisson-Boltzmann/surface area (MM-PBSA) calculations were carried out. These analyses suggested a molecular basis for the interaction of the beta-phenylsplitomicins with human Sirt2. Protein-based virtual screening resulted in the identification of a novel Sirt2 inhibitor chemotype. Selected inhibitors showed antiproliferative properties and tubulin hyperacetylation in MCF7 breast cancer cells and are promising candidates for further optimization as potential anticancer drugs.

Targeting histone methyltransferases and demethylases in clinical trials for cancer therapy

The term epigenetics is defined as heritable changes in gene expression that are not due to alterations of the DNA sequence. In the last years, it has become more and more evident that dysregulated epigenetic regulatory processes have a central role in cancer onset and progression. In contrast to DNA mutations, epigenetic modifications are reversible and, hence, suitable for pharmacological interventions. Reversible histone methylation is an important process within epigenetic regulation, and the investigation of its role in cancer has led to the identification of lysine methyltransferases and demethylases as promising targets for new anticancer drugs. In this review, we describe those enzymes and their inhibitors that have already reached the first stages of clinical trials in cancer therapy, namely the histone methyltransferases DOT1L and EZH2 as well as the demethylase LSD1.

Inhibitors of histone deacetylases as potential therapeutic tools for high-risk embryonal tumors of the nervous system of childhood.

The origin of malignant embryonal tumors is incompletely understood and certain risk groups remain difficult to treat. The epigenetic structure of DNA and its lesions play a role in the origin of these neoplasms. Manipulation of the epigenome may offer novel treatment options. The authors evaluated the cytotoxicity of histone deacetylase inhibitors (HDI) [MS‐275, SAHA, TSA, M344, M360, D85, SW55, SW187 and valproic acid (VPA)] on 13 embryonal tumor cell lines [4 medulloblastomas, 5 neuroblastomas, 2 atypical teratoid/rhabdoid tumors (AT/RT), and 2 malignant rhabdoid tumors of the kidney (RTK)] in MTT assay. In addition, HDI effects on hyperacetylation, reexpression of growth regulatory genes and apoptosis were characterized by Western analysis, RT‐PCR and annexin‐V staining. All HDI inhibited cell proliferation in a time‐ and dose‐dependent manner. VPA was least cytotoxic with GI50 values after 72 hr ranging from 53.6 to 332.9 μM, while TSA was most efficient with GI50 values after 72 hr ranging from 0.01 to 8.8 μM. M344 and M360 were also highly effective. Western blot revealed hyperacetylation of histone H4 after HDI treatment. Reactivation of several genes including the proapoptotic CASP8 was identified by RT‐PCR. Annexin‐V staining demonstrated a dose and time dependent induction of apoptosis. HDI inhibited the growth of medulloblastoma, neuroblastoma and rhabdoid tumors in vitro. Treatment with HDI induced the reactivation of growth regulatory genes and consequently apoptosis. Our results warrant further studies and may help in the design of new protocols geared at the treatment of high risk embryonal tumors.

Characterization of novel inhibitors of histone acetyltransferases

Modification of proteins by histone acetyltransferases (HAT) or histone deacetylases plays an important role in the control of gene expression, and its dysregulation has been linked to malignant transformation and other diseases. Although histone deacetylase inhibitors have been extensively studied and several are currently in clinical trials, there is little information available on inhibitors of HATs (HATi). Starting from the natural product lead HATi anacardic acid, a series of 28 analogues was synthesized and investigated for HAT-inhibitory properties and effects on cancer cell growth. The compounds inhibited up to 95% HAT activity in vitro, and there was a clear correlation between their inhibitory potency and cytotoxicity toward a broad panel of cancer cells. Interestingly, all tested compounds were relatively nontoxic to nonmalignant human cell lines. Western blot analysis of MCF7 breast carcinoma cells treated with HATi showed significant reduction in acetylation levels of histone H4. To directly show effect of the new compounds on HAT activity in vivo, MCF7 cells were cotransfected with the p21 promoter fused to firefly luciferase and a full-length p300 acetyltransferase, and luciferase activity was determined following treatment with HATi. Significant inhibition of p300 activity was detected after treatment with all tested compounds except one. Effects of the new HATi on protein acetylation and HAT activity in vivo make them a suitable tool for discovery of molecular targets of HATs and, potentially, for development of new anticancer therapeutics. [Mol Cancer Ther 2007;6(9):2391–8]