Histone Deacetylase Enzymatic Activity Research Articles

A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV-infected cells

A long-lived latent reservoir of HIV-1-infected CD4 Tcells persists with antiretroviral therapy and prevents cure. We report that the emergence of latently infected primary CD4 Tcells requires the activity of histone deacetylase enzymes HDAC1/2 and HDAC3. Data from targeted HDAC molecules, an HDAC3-directed PROTAC, and CRISPR-Cas9 knockout experiments converge on a model where either HDAC1/2 or HDAC3 targeting can prevent latency, whereas all three enzymes must be targeted to achieve latency reversal. Furthermore, HDACi treatment targets features of memory Tcells that are linked to proviral latency and persistence. Latency prevention is associated with increased H3K9ac at the proviral LTR promoter region and decreased H3K9me3, suggesting that this epigenetic switch is a key proviral silencing mechanism that depends on HDAC activity. These findings support further mechanistic work on latency initiation and eventual clinical studies of HDAC inhibitors to interfere with latency initiation.

Enhancing Therapeutic Efficacy: Harnessing the Synergy of Histone Deacetylase and BCL6 Inhibition in DLBCL

Background Diffuse large B-cell lymphoma (DLBCL) is characterized by its aggressive nature and resistance to standard chemotherapy, necessitating the development of new therapeutic approaches. Histone acetylation, a crucial epigenetic modification, is globally decreased in DLBCL. Chidamide, a histone deacetylase (HDACs) inhibitor, has shown promise in certain lymphomas but its effectiveness in DLBCL remains under investigation. Methods RNA-seq and gene enrichment analysis were performed to identify chidamide's impact on the biological processes in DLBCL. The key factor influencing chidamide sensitivity was determined through correlation analysis, utilizing gene expression levels from the Cancer Cell Line Encyclopedia and IC50 values of four HDAC inhibitors across multiple cancer cell lines sourced from the Genomics of Drug Sensitivity database. Histone acetylation levels, HAT and HDAC enzyme activity analysis, and co-immunoprecipitation were performed to investigate the underlying mechanism through which BCL6 influences chidamide sensitivity. A search among small molecule drugs in clinical investigation aimed to find drugs inhibiting BCL6 to enhance DLBCL sensitivity to chidamide. Synergy between HDAC and BCL6 inhibition was explored through synergistic effects, ubiquitination, and proteasomal degradation analysis. DLBCL mouse models were utilized to evaluate the therapeutic potential of combining chidamide and lenalidomide in vivo. Additionally, chidamide's effects on macrophage polarization were investigated through RNA-seq analysis of M1, M2, and chidamide-treated M2 macrophages. Results Chidamide exhibited potent suppression of DLBCL both in vitro and in vivo (Figure 1A), exerting a significant impact on diverse biological processes in DLBCL. BCL6 inhibited chidamide-induced histone acetylation by recruiting HDACs, leading to drug resistance in DLBCL cells. Lenalidomide could target BCL6 degradation through the ubiquitination pathway and restored the sensitivity of drug-resistant DLBCL to chidamide (Figure 1B). Additionally, chidamide had an impact on the tumor microenvironment. Chidamide inhibited the activity of M2-like tumor-associated macrophages and redirected the polarization of macrophages toward the M1 phenotype. Moreover, we conducted a single-arm multicenter clinical trial to evaluate the safety and efficacy of chidamide in combination with lenalidomide in DLBCL. Conclusions These findings highlighted HDACs as a viable therapeutic strategy for DLBCL, providing valuable insights into chidamide's global impact on the disease. Furthermore, BCL6 was a pivotal determinant of chidamide sensitivity and a reliable biomarker for treatment response. Integrating HDAC and BCL6 inhibition held potential for personalized and effective DLBCL treatments.

The Histone Deacetylase Inhibitory Potential of Chicken Egg Yolk Fat and Their Fatty Acid Composition.

Histone deacetylation is a key biochemical event associated with transcriptional regulation. Histone deacetylases (HDACs) mediate the deacetylation of histones. Fatty acids have been reported to function as histone deacetylase inhibitors (HDACi). The present instigation reports the HDAC inhibitory activity of egg yolks and egg yolk-derived fat of country and farm chicken for the first time. Egg yolks and fatty acids derived from both country (CCEF) and farm chicken (FCEF) demonstrated significant HDAC enzyme activity inhibition. Furthermore, egg yolks, CCEF, and FCEF exhibited DPPH free radical scavenging effects. The analysis of fatty acid profiles revealed varying degrees of saturated, mono-, and polyunsaturated fatty acids in the egg yolks. Palmitic acid (C16 : 0) was found to be the most abundant saturated fatty acid in both CCEF and FCEF. Among the monounsaturated fatty acids, oleic acid (C18 : 1) was the most abundant in both CCEF and FCEF. In terms of polyunsaturated fatty acids, a significant difference was observed in the content of linoleic acid (C18 : 2), an omega-6 fatty acid, and docosahexaenoic acid (C22 : 6), an omega-3 fatty acid, between CCEF and FCEF. These findings present exciting prospects for the development of histone deacetylase inhibitors based on egg yolk fat.

Anticancer effects of the HDAC inhibitor, 3β,6β‑dihydroxyurs‑12‑en‑27‑oic acid, in MCF‑7 breast cancer cells via the inhibition of Akt/mTOR pathways.

Astilbechinensis (A.chinensis) is a perennial herb that is used to treat chronic bronchitis and pain. The anticancer activity of 3β,6β‑dihydroxyurs‑12‑en‑27‑oic acid (ACT‑3), a major component isolated from A.chinensis, has not yet been investigated in detail. The purpose of the present study was to investigate the histone deacetylase (HDAC) inhibitory and anticancer activities of ACT‑3 compared with suberoylanilide hydroxamic acid (SAHA) in MCF‑7 human breast cancer cells. The purity of ACT‑3 was determined using high‑performance liquid chromatography. In the present study, the effects of ACT‑3 on anticancer effects of MCF‑7 cells were determined by measuring the level of apoptotic cell death and cell cycle regulator using flow cytometry analysis and western blot analysis, respectively. The effects of ACT‑3 on HDAC enzyme activity were measured using assay kits. ACT‑3 and SAHA increased the levels of acetylated histone H3 and reduced the levels of HDAC1 and HDAC3 in MCF‑7 cells. ACT‑3 significantly decreased the cell viability in a concentration‑dependent manner and induced different morphological changes at high concentrations. ACT‑3 and SAHA significantly inhibited the colony formation in MCF‑7 cells. ACT‑3 inhibited total HDAC activity in a dose‑dependent manner. ACT‑3 significantly reduced the expression levels of cyclin D1 and cyclin‑dependent kinase 4, and upregulated the expression levels of p21WAF1 and p53. A significant increase in the G1 phase cell population was observed in MCF‑7 cells and ACT‑3 induced apoptosis by reducing the ratio of B‑cell lymphoma‑2 (Bcl‑2)/Bcl‑2‑associated X (Bax) and releasing cleaved caspase 9. Additionally, ACT‑3 significantly increased autophagic cell death by inhibiting the serine‑threonine kinase/mammalian target of the rapamycin pathway. Autophagy induction was confirmed via acridine orange staining. ACT‑3 significantly increased the pERK1/2 and p21 in MCF‑7 cells. Thus, the activated ERK pathway played an important role in cell cycle arrest and apoptosis via ERK‑dependent induction of p21 in MCF‑7 cells. These data indicated that ACT‑3 can be used as a promising anticancer agent to overcome the limitations and reduce the side effects of conventional anticancer drugs.

Early Transcriptional Activity of Vtr-297 - Results from an Ongoing Phase I Clinical Trial

VTR-297 (Trichostatin A) is a pan-inhibitor of histone deacetylases types 1, 2, 4, 6, and 10. Histone deacetylase inhibitors (HDACi) are well-known small molecules that increase the acetylation of lysine residues by inhibiting the activity of histone deacetylase enzymes. These anticancer agents affect epigenetic and non-epigenetic transcriptional regulation and are already part of effective clinical practice and hematology regimens. As HDACi are known for their relatively wide transcriptional modulation, we have conducted a high throughput screen to elucidate early transcriptional effects related to exposure to Trichostatin A (TSA) on patient-derived, clinical time-series samples. NCT03838926 is an ongoing, open-label, dose-escalation, phase I clinical trial for subjects with relapsed or refractory hematologic malignancies. Enrolled patients were assigned to receive one currently tested dose (1.25 / 2.50 / 5.00 mg; IV infusion) in 28-day cycles, 3-times a week for three weeks with one week off (IV administration days 1,3,5,8,10,12,15,17 and 19) until disease progression, unacceptable toxicity, or withdrawal of consent. The primary endpoint was safety/tolerability and maximum tolerated dose (MTD) of VTR-297 (TSA). Currently, twelve patients enrolled in the study, including Multiple Myeloma (MM, n=5), Non-Hodgkin's Lymphoma (NHL, n=5), and Hodgkin's Lymphoma (HL, n=2) across the first three dose-escalation cohorts that completed (1.25 / 2.50 / 5.00mg). No significant drug-related toxicities were reported, and MTD was not reached (recruitment is currently ongoing in the fourth dose cohort of 10mg). As a part of the exploratory analysis of whole blood (PBMC), patient samples were collected on cycle1 day1 (C1D1; pre-infusion, 15min-post, 30min-post, 1h-post, 2h-post, and 6h-post infusion) to capture any transcriptional changes (RNAseq) related to VTR-297 treatment. In the current analysis, samples were collected and analyzed in respective batches (15min / 30min / 1h / 2h / 6h) and compared with combined baseline samples (pre-infusion). Using Deseq2 to ascertain transcriptionally affected genes (pval<0.05, up/down relative fold-change 1.5x), we detected 136 differentially expressed genes across at least one of the time point analyzed. Among the detected genes, we found novel and previously described genes related to the core pathophysiology of myeloid and lymphoid disease (e.g., iron metabolism, vascular metastasis, and tyrosine kinase activity). In conclusion, these preliminary signatures offer early biomarker information related to a specific transcriptional activity elicited by intravenously delivered VTR-297 in the clinical setting. In an ongoing clinical study, we will further monitor, validate, and expand on these findings. Further observations at higher, safe, and clinically meaningful doses will complement our current, early picture of VTR-297 activity and how it can be used to optimize TSA-based treatments.

Transcriptome Analysis of Beet Webworm Shows That Histone Deacetylase May Affect Diapause by Regulating Juvenile Hormone.

Simple SummaryDiapause is a seasonal adaptation to stress in insects, including in Lepidoptera. Juvenile hormone (JH) is the key factor affecting larval diapause. The diapause of beet webworm, an important agricultural pest, is induced by photoperiod, but the mechanism is unknown. Transcriptome sequencing was performed in five different stages of beet webworm. The results showed that 393 genes in the red module were strongly related to JH, and the hub gene of the red module was histone deacetylase (HDAC). After injecting HDAC chemical inhibitors into beet webworms, we found that HDAC enzyme had an effect on JH content in beet webworms under photoperiod that induced diapause. Therefore, HDAC may be involved in diapause in beet webworms by altering JH levels.The beet webworm (Loxostege sticticalis L.) is an important agricultural pest and can tolerate harsh environmental conditions by entering diapause. The diapause mechanism of beet webworm is unknown. Therefore, we conducted a transcriptomic study of the process from diapause induction to diapause release in beet webworms. The results revealed 393 gene modules closely related to the diapause of beet webworm. The hub gene of the red module was the HDACI gene, which acts through histone deacetylase (HDAC) enzymes. HDAC enzyme activity was regulated by the light duration and influenced the JH content under induced beet webworm diapause conditions (12 h light:12 h dark). In addition, transcriptomic data suggested that circadian genes may not be the key genes responsible for beet webworm diapause. However, we showed that the photoperiod affects HDAC enzyme activity, and HDAC can regulate the involvement of JH in beet webworm diapause. This study provided a new module for studying insect diapause and links histone acetylation and diapause at the transcriptome level.

Enriched environment causes epigenetic changes in hippocampus and improves long-term cognitive function in sepsis

Sepsis is defined as a life-threatening organ dysfunction caused by an inappropriate host response to infection. The presence of oxidative stress and inflammatory mediators in sepsis leads to dysregulated gene expression, leading to a hyperinflammatory response. Environmental conditions play an important role in various pathologies depending on the stimulus it presents. A standard environment condition (SE) may offer reduced sensory and cognitive stimulation, but an enriched environment improves spatial learning, prevents cognitive deficits induced by disease stress, and is an important modulator of epigenetic enzymes. The study evaluated the epigenetic alterations and the effects of the environmental enrichment (EE) protocol in the brain of animals submitted to sepsis by cecal ligation and perforation (CLP). Male Wistar rats were divided into sham and CLP at 24 h, 72 h, 10 days and 30 days after sepsis. Other male Wistar rats were distributed in a SE or in EE for forty-five days. Behavioral tests, analysis of epigenetic enzymes:histone acetylase (HAT), histone deacetylase (HDAC) and DNA methyltransferase (DNMT), biochemical and synaptic plasticity analyzes were performed. An increase in HDAC and DNMT activities was observed at 72 h, 10 days and 30 days. There was a positive correlation between epigenetic enzymes DNMT and HDAC 24 h, 10 days and 30 days. After EE, HDAC and DNMT enzyme activity decreased, cognitive impairment was reversed, IL1-β levels decreased and there was an increase in PSD-95 levels in the hippocampus. Interventions in environmental conditions can modulate the outcomes of long-term cognitive consequences associated with sepsis, supporting the idea of the potential benefits of EE.

The Hydroxamic Acids as Potential Anticancer and Neuroprotective Agents.

Hydroxamic acids are a promising class of chemical compounds with proven antitumor potential, primarily due to their ability to inhibit the activity of histone deacetylase enzymes. The analysis of modern experimental data shows a wide range of biological activities of hydroxamic acids, which make them equally worthy candidates for the fight against neuropathologies. A characteristic feature of hydroxamic acids is their ability to act simultaneously on several promising molecular targets for the correction of both neuropathologies and oncological diseases, thereby exhibiting multifunctionality. This review discusses the effect of hydroxamic acids on key parts of cancer and neurodegenerative disorders' pathogenesis. Pathological changes in the processes associated with oxidative stress, the functioning of mitochondria, and the activity of metal enzymes of the class of histone deacetylases, as the main links in the epigenetic regulation of pathological conditions, are such molecular targets.

The effects of increasing fruit and vegetable intake in children with asthma: A randomized controlled trial.

A high fruit and vegetable (F&V) diet reduces asthma exacerbations in adults; this has not been examined in children to date. To investigate the effect of a 6-month, high F&V diet on the time to first asthma exacerbation in children with asthma, in a parallel-group, randomized, controlled trial. Children (aged 3-11years) with asthma, history of exacerbations and usual low F&V intake (≤3 serves/day) were randomized to the intervention (high F&V diet) or control group (usual diet) for 6months. The primary outcome was time to first exacerbation requiring medical intervention. Secondary outcomes included exacerbation rate, lung function, plasma TNF-α, CRP, and IL-6, faecal microbiota and peripheral blood mononuclear cell (PBMC) histone deacetylase (HDAC) activity and G-protein coupled receptor (GPR) 41/43 and HDAC (1-11) expression. 67 children were randomized between September 2015 and July 2018. F&V intake (difference in change (∆): 3.5serves/day, 95% CI: [2.6, 4.4] p<0.001) and plasma total carotenoids (∆: 0.44µg/ml [0.19, 0.70] p=0.001) increased after 6months (intervention vs control). Time to first exacerbation (HR: 0.81, 95% CI: [0.38, 1.69], p=0.569; control vs. intervention) and exacerbation rate (IRR: 0.84, [0.47, 1.49], p=0.553; control vs. intervention) were similar between groups. In per-protocol analysis, airway reactance z-scores increased in the intervention versus control group (X5 ∆: 0.76 [0.04, 1.48] p=0.038, X20 ∆: 0.93 [0.23, 1.64] p=0.009) and changes in faecal microbiota were observed though there was no difference between groups in systemic inflammation or molecular mechanisms. In the control group, CRP and HDAC enzyme activity increased, while GPR41 expression decreased. No adverse events attributable to the interventions were observed. A high F&V diet did not affect asthma exacerbations over the 6-month intervention, though warrants further investigation as a strategy for improving lung function and protecting against systemic inflammation in children with asthma.

Histone Deacetylase 5 Is an Early Epigenetic Regulator of Intermittent Hypoxia Induced Sympathetic Nerve Activation and Blood Pressure.

Intermittent hypoxia (IH) is a hallmark manifestation of obstructive sleep apnea (OSA). Long term IH (LT-IH) triggers epigenetic reprogramming of the redox state involving DNA hypermethylation in the carotid body chemo reflex pathway resulting in persistent sympathetic activation and hypertension. Present study examined whether IH also activates epigenetic mechanism(s) other than DNA methylation. Histone modification by lysine acetylation is another major epigenetic mechanism associated with gene regulation. Equilibrium between the activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) determine the level of lysine acetylation. Here we report that exposure of rat pheochromocytoma (PC)-12 cells to IH in vitro exhibited reduced HDAC enzyme activity due to proteasomal degradation of HDAC3 and HDAC5 proteins. Mechanistic investigations showed that IH-evoked decrease in HDAC activity increases lysine acetylation of α subunit of hypoxia inducible factor (HIF)-1α as well as Histone (H3) protein resulting in increased HIF-1 transcriptional activity. Trichostatin A (TSA), an inhibitor of HDACs, mimicked the effects of IH. Studies on rats treated with 10 days of IH or TSA showed reduced HDAC activity, HDAC5 protein, and increased HIF-1 dependent NADPH oxidase (NOX)-4 transcription in adrenal medullae (AM) resulting in elevated plasma catecholamines and blood pressure. Likewise, heme oxygenase (HO)-2 null mice, which exhibit IH because of high incidence of spontaneous apneas (apnea index 72 ± 1.2 apnea/h), also showed decreased HDAC activity and HDAC5 protein in the AM along with elevated circulating norepinephrine levels. These findings demonstrate that lysine acetylation of histone and non-histone proteins is an early epigenetic mechanism associated with sympathetic nerve activation and hypertension in rodent models of IH.

Histone Deacetylase 1 Reduces Lipogenesis by Suppressing SREBP1 Transcription in Human Sebocyte Cell Line SZ95.

Proper regulation of sebum production is important for maintaining skin homeostasis in humans. However, little is known about the role of epigenetic regulation in sebocyte lipogenesis. We investigated histone acetylation changes and their role in key lipogenic gene regulation during sebocyte lipogenesis using the human sebaceous gland cell line SZ95. Sebocyte lipogenesis is associated with a significant increase in histone acetylation. Treatment with anacardic acid (AA), a p300 histone acetyltransferase inhibitor, significantly decreased the lipid droplet number and the expression of key lipogenic genes, including sterol regulatory-binding protein 1 (SREBP1), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). In contrast, treatment with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, increased the expression of these genes. Global HDAC enzyme activity was decreased, and HDAC1 and HDAC2 expression was downregulated during sebaceous lipogenesis. Interestingly, HDAC1 knockdown increased lipogenesis through SREBP1 induction, whereas HDAC1 overexpression decreased lipogenesis and significantly suppressed SREBP1 promoter activity. HDAC1 and SREBP1 levels were inversely correlated in human skin sebaceous glands as demonstrated in immunofluorescence images. In conclusion, HDAC1 plays a critical role in reducing SREBP1 transcription, leading to decreased sebaceous lipogenesis. Therefore, HDAC1 activation could be an effective therapeutic strategy for skin diseases related to excessive sebum production.

A novel HDAC1 inhibitor, CBUD‑1001, exerts anticancer effects by modulating the apoptosis and EMT of colorectal cancer cells.

Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies and is a leading cause of cancer‑related mortality worldwide. Histone deacetylases (HDACs) are a class of enzymes responsible for the epigenetic regulation of gene expression. Some HDAC inhibitors have been shown to be efficient agents for cancer treatment. The aim of the present study was to discover a novel, potent HDAC inhibitor and demonstrate its anticancer effect and molecular mechanisms in CRC cells. A novel fluorinated aminophenyl‑benzamide‑based compound, CBUD‑1001, was designed to specifically target HDAC1, and it was then synthesized and evaluated. CBUD‑1001 exerted a potent inhibitory effect on HDAC enzyme activity and exhibited anticancer potency against CRC cell lines. Molecular docking analysis rationalized the high potency of CBUD‑1001 by validating its conformation in the HDAC active site. Further investigation using CRC cells demonstrated that CBUD‑1001 inhibited HDAC activity by hyper‑acetylating histones H3 and H4, and it exerted an apoptotic effect by activating a mitochondrial‑dependent pathway. Of note, it was found that CBUD‑1001 attenuates the cell motility of CRC cells by downregulating the EMT signaling pathway. Thus, CBUD‑1001 may prove to be a promising novel drug candidate for CRC therapy.

Repurposing natural products as novel HDAC inhibitors by comparative analysis of gene expression profiles

BackgroundExtracts derived from natural products have been used to produce health supplements or therapeutic agents in oriental medicine. Although these extracts contain various bioactive compounds, their applications are generally limited to a few previously known diseases. To effectively expand their use for the treatment of other conditions, systematic analysis should be conducted for repurposing. PurposeThe purpose of the present study was to investigate the new therapeutic efficacies of the Platycodon grandiflorum and ginseng extract using the CMAP-based gene expression analysis. MethodsIn the present study, we analyzed the expression patterns of differentially expressed genes (DEGs) from extracts as the basis for drug repurposing. Cells were treated with extracts or single compounds derived from nine natural products. DEG analysis indicated that the gene expression patterns of cells treated with P. grandiflorum and ginseng extracts were highly similar to those of cells treated with different types of Histone deacetylase (HDAC) inhibitors. To identify the new mechanism of these extracts, we carried out cell viability assay, TUNEL assay, HDAC enzyme activity assay and immunoblot analysis. ResultsIn vitro experiments at the dose of 50 µg/ml of each extract did not affect cell death rate but significantly inhibited HDAC activity. Each extract was found to inhibit HDAC enzymatic activity and induce the expression of the p21. Furthermore, our results revealed that each extract stimulated cell death and inhibited cell proliferation. ConclusionThese findings demonstrate the HDAC-inhibiting activity of P. grandiflorum and ginseng extracts and further validate the effectiveness of DEG similarity-based repurposing of natural products.

Abstract 2038: Reactive oxygen species-mediated synergism of fenretinide and romidepsin in preclinical models of T-cell lymphoid malignancies

Abstract Introduction: T-cell lymphoid malignancies (TCLMs) are in need of novel and more effective therapies. Romidepsin is a histone deacetylase (HDAC) inhibitor that achieved FDA registration as 2nd line therapy for peripheral and cutaneous T-cell lymphomas. The cytotoxic retinoid fenretinide achieved durable complete responses against T-cell lymphomas in early-phase clinical trials and T-cell lymphoma patients who failed prior romidepsin treatment responded to fenretinide. Fenretinide is currently being evaluated in a Phase IIa clinical trial for relapsed/refractory PTCL patients (NCT02495415). We investigated the potential for using these two agents in combination in TCLMs. Methods and Results: Using the DIMSCAN assay, we demonstrated cytotoxic synergy between romidepsin and fenretinide in fifteen TCLM cell lines at clinically-achievable concentrations that lacked cytotoxicity for non-malignant cells (fibroblasts and blood mononuclear cells). In vivo, romidepsin + fenretinide + ketoconazole (enhances fenretinide exposures by inhibiting fenretinide metabolism) showed greater activity in subcutaneous (COG-LL-317m and TX-LY-183x PDX) and disseminated (COG-LL-317m Luc expressing the luciferase gene) TCLM xenograft models than single agent romidepsin or fenretinide + ketoconazole. Fenretinide + romidepsin caused a reactive oxygen species (ROS, measured by DCFDA dye)-dependent increase in pro-apoptotic proteins (Bim, tBid, Bax and Bad), apoptosis (via TUNEL assay), and inhibition of HDAC enzymatic activity, which achieved a synergistic increase in histone acetylation. The synergistic cytotoxicity, apoptosis, and histone acetylation of fenretinide + romidepsin was abrogated by antioxidants (vitamins C or E). Romidepsin + fenretinide activated p38 and JNK via ROS, and shRNA knockdown of p38 and JNK1 significantly decreased the synergistic cytotoxicity and apoptosis. Romidepsin + fenretinide also showed synergistic cytotoxicity for B-lymphoid malignancy cell lines, but did not increase ROS, acetylation of histones, activation of p38 + JNK, or cytotoxicity in fibroblasts and blood mononuclear cells. Conclusion: Romidepsin + fenretinide achieved synergistic activity in preclinical models of TCLMs, but not in non-malignant cells, via a novel molecular mechanism. These data support conducting clinical trials of romidepsin + fenretinide in relapsed and refractory TCLMs. Citation Format: Monish Ram Makena, Balakrishna Koneru, Min H. Kang, C. Patrick Reynolds. Reactive oxygen species-mediated synergism of fenretinide and romidepsin in preclinical models of T-cell lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2038. doi:10.1158/1538-7445.AM2017-2038

Reactive Oxygen Species-Mediated Synergism of Fenretinide and Romidepsin in Preclinical Models of T-cell Lymphoid Malignancies.

T-cell lymphoid malignancies (TCLM) are in need of novel and more effective therapies. The histone deacetylase (HDAC) inhibitor romidepsin and the synthetic cytotoxic retinoid fenretinide both have achieved durable clinical responses in T-cell lymphomas as single agents. We investigated the potential for using these two agents in combination in TCLMs. We demonstrated cytotoxic synergy between romidepsin and fenretinide in 15 TCLM cell lines at clinically achievable concentrations that lacked cytotoxicity for nonmalignant cells (fibroblasts and blood mononuclear cells). In vivo, romidepsin + fenretinide + ketoconazole (enhances fenretinide exposures by inhibiting fenretinide metabolism) showed greater activity in subcutaneous and disseminated TCLM xenograft models than single-agent romidepsin or fenretinide + ketoconazole. Fenretinide + romidepsin caused a reactive oxygen species (ROS)-dependent increase in proapoptotic proteins (Bim, tBid, Bax, and Bak), apoptosis, and inhibition of HDAC enzymatic activity, which achieved a synergistic increase in histone acetylation. The synergistic cytotoxicity, apoptosis, and histone acetylation of fenretinide + romidepsin were abrogated by antioxidants (vitamins C or E). Romidepsin + fenretinide activated p38 and JNK via ROS, and knockdown of p38 and JNK1 significantly decreased the synergistic cytotoxicity. Romidepsin + fenretinide also showed synergistic cytotoxicity for B-lymphoid malignancy cell lines, but did not increase ROS, acetylation of histones, activation of p38 + JNK, or cytotoxicity in nonmalignant cells. Romidepsin + fenretinide achieved synergistic activity in preclinical models of TCLMs, but not in nonmalignant cells, via a novel molecular mechanism. These data support conducting clinical trials of romidepsin + fenretinide in relapsed and refractory TCLMs. Mol Cancer Ther; 16(4); 649-61. ©2017 AACR.

Marine-derived chromopeptide A, a novel class I HDAC inhibitor, suppresses human prostate cancer cell proliferation and migration.

Histone deacetylases (HDACs), especially HDAC1, 2, 3 and 4, are abundantly expressed and over-activated in prostate cancer that is correlated with the poor prognosis. Thus, inhibition of HDAC activity has emerged as a potential alternative option for prostate cancer therapy. Chromopeptide A is a depsipeptide isolated from the marine sediment-derived bacterium Chromobacterium sp. HS-13-94; it has a chemical structure highly similar to FK228, a class I HDAC inhibitor that is approved by FDA for treating T-cell lymphoma. In this study, we determined whether chromopeptide A, like FK228, acted as a class I HDAC inhibitor, and whether chromopeptide A could inhibit the growth and migration of human prostate cancer in vitro and in vivo. HDAC enzyme selectivity and kinetic analysis revealed that chromopeptide A selectively inhibited the enzymatic activities of HDAC1, 2, 3 and 8 in a substrate non-competitive manner with comparable IC50 values for each HDAC member as FK228 in vitro. Importantly, chromopeptide A dose-dependently suppressed the proliferation of human prostate cancer cell lines PC3, DU145 and LNCaP with IC50 values of 2.43±0.02, 2.08±0.16, and 1.75±0.06 nmol/L, respectively, accompanied by dose-dependent inhibition of HDAC enzymatic activity in PC3 and DU145 cells. Chromopeptide A (0.2-50 nmol/L) caused G2/M phase arrest and induced apoptosis in the prostate cancer cell lines. Moreover, chromopeptide A dose-dependently inhibited the migration of PC3 cells. In mice bearing PC3 prostate cancer xenografts, intravenous injection of chromopeptide A (1.6, 3.2 mg/kg, once a week for 18 d) significantly suppressed the tumor growth, which was associated with increased expression levels of Ac-H3 and p21 in tumor tissues. Our results identify chromopeptide A as a novel class I HDAC inhibitor and provide therapeutic strategies that may be implemented in prostate cancer.

Chemical and structural biology of protein lysine deacetylases.

Histone acetylation is a reversible posttranslational modification that plays a fundamental role in regulating eukaryotic gene expression and chromatin structure/function. Key enzymes for removing acetyl groups from histones are metal (zinc)-dependent and NAD+-dependent histone deacetylases (HDACs). The molecular function of HDACs have been extensively characterized by various approaches including chemical, molecular, and structural biology, which demonstrated that HDACs regulate cell proliferation, differentiation, and metabolic homeostasis, and that their alterations are deeply involved in various human disorders including cancer. Notably, drug discovery efforts have achieved success in developing HDAC-targeting therapeutics for treatment of several cancers. However, recent advancements in proteomics technology have revealed much broader aspects of HDACs beyond gene expression control. Not only histones but also a large number of cellular proteins are subject to acetylation by histone acetyltransferases (HATs) and deacetylation by HDACs. Furthermore, some of their structures can flexibly accept and hydrolyze other acyl groups on protein lysine residues. This review mainly focuses on structural aspects of HDAC enzymatic activity regulated by interaction with substrates, co-factors, small molecule inhibitors, and activators.

Context and Auditory Fear are Differentially Regulated by HDAC3 Activity in the Lateral and Basal Subnuclei of the Amygdala.

Histone acetylation is a fundamental epigenetic mechanism that is dynamically regulated during memory formation. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) compete to modulate histone acetylation, allowing for rapid changes in acetylation in response to a learning event. HDACs are known to be powerful negative regulators of memory formation, but it is not clear whether this function depends on HDAC enzymatic activity per se. Here, we tested whether the enzymatic activity of an individual Class I HDAC, HDAC3, has a role in fear memory formation in subregions of the hippocampus and amygdala. We found that fear conditioning drove expression of the immediate early genes cFos and Nr4a2 in the hippocampus, which coincided with reduced HDAC3 occupancy at these promoters. Using a dominant-negative, deacetylase-dead point mutant virus (AAV-HDAC3(Y298H)-v5), we found that selectively blocking HDAC3 deacetylase activity in either the dorsal hippocampus or basal nucleus of the amygdala enhanced context fear without affecting tone fear. Blocking HDAC3 activity in the lateral nucleus of the amygdala, on the other hand, enhanced tone, but not context fear memory. These results show for the first time that the enzymatic activity of HDAC3 functions to negatively regulate fear memory formation. Further, HDAC3 activity regulates different aspects of fear memory in the basal and lateral subregions of the amygdala. Thus, the deacetylase activity of HDAC3 is a powerful negative regulator of fear memory formation in multiple subregions of the fear circuit.

Changes in histone acetylation as potential mediators of pupal diapause in the flesh fly, Sarcophaga bullata

The growing appreciation that epigenetic processes are integral to the responses of many organisms to changes in the environment suggests a possible role for epigenetics in coordination of insect diapause. The results we present suggest that histone modification may be one type of epigenetic process that contributes to regulation of pupal diapause in the flesh fly, Sarcophaga bullata. Reduction in total histone H3 acetylation in diapausing pupae, shifts in mRNA expression profiles of genes encoding histone acetyltransferase (HAT) and histone deacetylase (HDAC) in pre-diapause, diapause and post-diapause flies compared to their nondiapause counterparts, and alterations in HDAC enzyme activity during and post-diapause lend support to the hypothesis that this specific type of histone modification is involved in regulating diapause programming, maintenance, and termination. Transcription of genes encoding HDAC1, HDAC3, HDAC6, and Sirtuin2 were all upregulated in photosensitive first instar larvae programmed to enter pupal diapause, suggesting that histone deacetylation may be linked to the early decision to enter diapause. A 50% reduction in transcription of hdac3 and a corresponding 30% reduction in HDAC activity during diapause suggest that removal of acetyl groups from histones primarily occurs prior to diapause entry and that further histone deacetylation is not necessary to maintain diapause. Transcription of the HDAC genes was quickly elevated when diapause was terminated, followed by an increase in enzyme activity after a short delay. A maternal effect operating in these flies prevents pupal diapause in progeny whose mothers experienced pupal diapause, even if the progeny are reared in strong diapause-inducing short-day conditions. Such nondiapausing pupae had HDAC transcription profiles nearly identical to the profiles seen in nondiapausing pupae generated under a long-day photoperiod. Together, these results provide consistent evidence for histone acetylation and deacetylation as regulators of this insect's developmental trajectory.

A Novel Combinatorial Epigenetic Therapy Using Resveratrol and Pterostilbene for Restoring Estrogen Receptor-α (ERα) Expression in ERα-Negative Breast Cancer Cells.

Breast cancer is the second most common cancer and a leading cause of cancer death in women. Specifically, estrogen receptor-α (ERα)-negative breast cancers are clinically more aggressive and normally do not respond to conventional hormone-directed therapies such as tamoxifen. Although epigenetic-based therapies such as 5-aza-2’-deoxycytidine and/or trichostatin A as DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, respectively, can regulate the expression of ERα, this can often lead to a number of side effects. Plant-based dietary compounds such as resveratrol and pterostilbene in novel combinatorial therapy provides new avenues to target these side effects and provide similar results with a higher level of safety. Here, we report that combinatorial resveratrol and pterostilbene leads to the reactivation of ERα expression in ERα-negative breast cancer cells in a time-dependent manner. Chromatin immunoprecipitation analysis of the ERα promoter in each cell type revealed an increase in enrichment of acetyl-H3, acetyl-H3lysine9 (H3K9) and acetyl-H4 active chromatin markers in the ERα promoter region after combinatorial treatment. This treatment also resulted in a significant change in HDAC and histone acetyl transferase (HAT) enzyme activity in these cells after 3 days of treatments. The combination resulted in a significant decrease in DNMT enzyme activity and 5-methylcytosine levels in MDA-MB-157 breast cancer cells. Moreover, reactivation of ERα expression by resveratrol combined with pterostilbene was found to sensitize ERα-dependent response to 17β-estradiol (E2)-mediated cellular proliferation and antagonist 4-hydroxytamoxifen (4-OHT)-mediated inhibition of cellular proliferation in ERα-negative breast cancer cells. E2 and 4-OHT further affected the ERα-responsive downstream progesterone receptor (PGR) gene in ERα reactivated MDA-MB-157 cells. Collectively, our findings provide a new and safer way of restoring ERα expression by regulating epigenetic mechanisms with the use of phytochemicals in combinatorial therapy. This combination can further provide effective treatment options for hormonal refractory breast cancer with available anti-hormonal therapy.