Broad-spectrum Histone Deacetylase Research Articles

Regulation of Glutamate Transporter Type 1 by TSA and the Antiepileptic Mechanism of TSA

Epilepsy (EP) is a neurological disorder characterized by abnormal, sudden neuronal discharges. Seizures increase extracellular glutamate levels, causing excitotoxic damage. Glutamate transporter type 1 (GLT-1) and its human homologue excitatory amino acid transporter-2 (EAAT2) clear 95% of extracellular glutamate. Studies on neurodegenerative diseases suggest that trichostatin A (TSA), a broad-spectrum histone deacetylase (HDAC) inhibitor, can increase GLT-1/EAAT2 transcription. However, the precise mechanism by which TSA modulates GLT-1/EAAT2 levels remains unclear. This research demonstrated that TSA increases GLT-1/EAAT2 expression through histone acetylation, exerting substantial antiepileptic effects. Our results identify a promising therapeutic strategy for EP involving the modulation of glutamate transporters to mitigate seizures. Future research should explore the specific mechanisms underlying the effects of TSA and its potential clinical applications. Acute and chronic EP models were induced using kainic acid (KA) to assess the effects of TSA on the seizure threshold and frequency. Electrophysiological recordings of the hippocampus were used to evaluate the impact of TSA on neuronal excitability. RNA-Seq was used to analyse changes in glutamate transporter-related gene expression. Western blot analysis and qRT‒PCR were used to assess the influence of TSA on HDAC expression. To validate the role of GLT-1/EAAT2 in the antiepileptic effects of TSA, the impact of the GLT-1/EAAT2 inhibitor dihydrokainic acid (DHK) on the effects of TSA was assessed. Glutamate release was measured, and microdialysis was used to determine the glutamate content in the cerebrospinal fluid. Finally, metabolomics analysis was used to explore changes in amino acid levels in the hippocampus following TSA treatment to further confirm the antiepileptic potential of TSA. TSA effectively inhibited seizures in both acute and chronic models. It reduced the amplitude of excitatory postsynaptic currents (PSCs) and the frequency of spontaneous excitatory PSCs in the hippocampus without affecting inhibitory PSCs. Transcriptome analysis was used to identify glutamate transmission-related targets and revealed significant upregulation of the GLT-1 and EAAT2 genes in the hippocampus, which was confirmed by qRT‒PCR and Western blotting. Acetylation-induced upregulation of GLT-1/EAAT2 was observed, and inhibition of these transporters by DHK reduced the seizure-mitigating effects of TSA, underscoring the role of GLT-1/EAAT2 in clearing glutamate and its contribution to the observed antiepileptic effects of TSA. Our findings highlight the crucial role of GLT-1/EAAT2 in mediating the impact of TSA on glutamatergic transmission and seizure activity. These insights pave the way for the development of novel therapeutic approaches for EP involving the modulation of glutamate transporters.

Development of an UPLC-MS/MS method for quantitative analysis of abexinostat levels in rat plasma and application of pharmacokinetics

Broad-spectrum histone deacetylase inhibitors (HDACi) have excellent anti-tumor effects, such as abexinostat, which was a novel oral HDACi that was widely used in clinical treatment. The purpose of this study was to establish a rapid and reliable method for the detection of abexinostat concentrations in rat plasma using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The mobile phase we used was acetonitrile and 0.1% formic acid, and the internal standard (IS) was givinostat. Selective reaction monitoring (SRM) was used for detection with ion transitions at m/z 397.93 → 200.19 for abexinostat and m/z 422.01 → 186.11 for givinostat, respectively. The intra-day and inter-day precision of abexinostat were less than 11.5% and the intra-day and inter-day accuracy ranged from − 10.7% to 9.7% using this method. During the analysis process, the stability of the test sample was reliable. In addition, the recovery and matrix effects of this method were within acceptable limits. Finally, the method presented in this paper enabled accurate and quick determination of abexinostat levels in rat plasma from the pharmacokinetic study following gavage at a dose of 8.0 mg/kg abexinostat.

Upregulation of HDAC9 in hippocampal neurons mediates depression-like behaviours by inhibiting ANXA2 degradation

Major depressive disorder (MDD) is a pervasive and devastating mental disease. Broad spectrum histone deacetylase (HDAC) inhibitors are considered to have potential for the treatment of depressive phenotype in mice. However, due to its non-specific inhibition, it has extensive side effects and can not be used in clinical treatment of MDD. Therefore, finding specific HDAC subtypes that play a major role in the etiology of MDD is the key to develop corresponding specific inhibitors as antidepressants in the future. Copy number variation in HDAC9 gene is thought to be associated with the etiology of some psychiatric disorders. Herein, we found that HDAC9 was highly expressed in the hippocampus of chronic restraint stress (CRS) mouse model of depression. Upregulation of HDAC9 expression in hippocampal neurons of mice induced depression-like phenotypes, including anhedonia, helplessness, decreased dendritic spine density, and neuronal hypoexcitability. Moreover, knockdown or knockout of HDAC9 in hippocampal neurons alleviated depression-like phenotypes caused by chronic restraint stress (CRS) in WT mice. Importantly, using immunoprecipitation–mass spectrometry (IP–MS), we further found that Annexin A2 (ANXA2) was coupled to and deacetylated by HDAC9. This coupling resulted in the inhibition of ubiquitinated ANXA2 degradation and then mediates depression-like behavior. Overall, we discovered a previously unrecognized role for HDAC9 in hippocampal neurons in the pathogenesis of depression, indicating that inhibition of HDAC9 might be a promising clinical strategy for the treatment of depressive disorders.

Epigenetic regulation of GABAergic differentiation in the developing brain.

In the vertebrate brain, GABAergic cell development and neurotransmission are important for the establishment of neural circuits. Various intrinsic and extrinsic factors have been identified to affect GABAergic neurogenesis. However, little is known about the epigenetic control of GABAergic differentiation in the developing brain. Here, we report that the number of GABAergic neurons dynamically changes during the early tectal development in the Xenopus brain. The percentage of GABAergic neurons is relatively unchanged during the early stages from stage 40 to 46 but significantly decreased from stage 46 to 48 tadpoles. Interestingly, the histone acetylation of H3K9 is developmentally decreased from stage 42 to 48 (about 3.5 days). Chronic application of valproate acid (VPA), a broad-spectrum histone deacetylase (HDAC) inhibitor, at stage 46 for 48 h increases the acetylation of H3K9 and the number of GABAergic cells in the optic tectum. VPA treatment also reduces apoptotic cells. Electrophysiological recordings show that a VPA induces an increase in the frequency of mIPSCs and no changes in the amplitude. Behavioral studies reveal that VPA decreases swimming activity and visually guided avoidance behavior. These findings extend our understanding of histone modification in the GABAergic differentiation and neurotransmission during early brain development.

Using an HDAC Inhibitor to Alter Muscle Growth in Fetal Pigs

A broad spectrum histone deacetylase (HDAC) inhibitor, butyrate, has been shown to promote increased myofiber size by altering satellite cell (SC) activity. The use of dietary supplementation of butyrate to affect muscle development has been hampered by its technical and pharmacokinetic properties. Most of these can be overcome with the use of tributyrin (TB), a butyrate prodrug. Dietary supplementation of TB was able to promote muscle growth by increasing myofiber size in neonatal pigs. We hypothesized that muscle growth during fetal development may be improved by increased fiber number through the dietary supplementation of TB to gestating sows. Nine sows of 53.6 ±1.08 gestational days (gd) were randomly assigned to one of three dietary treatment groups (n=3). These dietary treatments were a basal diet, basal diet + 1% TB, and the basal diet + 2% TB. All diets were made isocaloric with the addition of oil and wheat flour. Fetal piglets were removed at 107.6 ±1.5 gd. Weights and body lengths were recorded for all fetuses. Sections of Longissimus dorsi and Psoas major were collected from the median male, median female, the smallest, and the largest piglets of each litter and frozen for future histological analysis. Two novel methods for objective description of muscle fiber organization were evaluated, including fiber eccentricity (measure of elongation), and proportion of primary myofibers. The most significant difference in cross sectional area (CSA) was found within LD muscle with TB supplementation causing a significant reduction compared to control animals (p value < 0.05). Fibers from 1% TB animals were significantly more elongated than control (p value < 0.05). The proportion of primary to secondary myofibers was significantly higher in muscle from 2% TB treated piglets compared to control (p value < 0.05). The smaller CSA together with a similar body weight across individual piglets (p >0.05) suggest that TB supplementation increased myofiber number. The increased fiber count may be further supported by more elongated myofibers suggesting alterations to muscle fiber organization and an improved development of primary fibers. Further studies to elucidate the impact of TB supplementation during gestation and early postnatal life are warranted.

Combining EGFR-TKI With SAHA Overcomes EGFR-TKI-Acquired Resistance by Reducing the Protective Autophagy in Non-Small Cell Lung Cancer.

Nowadays, lung cancer has the highest mortality worldwide. The emergence of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has greatly improved the survival of patients with non-small cell lung cancer (NSCLC) having EGFR-TKI-sensitive mutations. Unfortunately, acquired resistance happens for most patients. In the present research, we found that EGFR-TKIs (such as gefitinib and osimertinib) can induce autophagy in NSCLC cell lines. Compared with parental sensitive cells, drug-resistant cells have higher autophagy activity. The use of an autophagy inhibitor could enhance the toxicity of gefitinib and osimertinib, which indicates that the enhancement of protective autophagy might be one of the mechanisms of EGFR-TKI resistance in NSCLC. In addition, increased autophagy activity is associated with decreased enhancer of zeste homolog 2 (EZH2) expression. Knockdown of EZH2 or EZH2 inhibitor treatment could lead to increased autophagy in NSCLC cells, indicating that EZH2 is a negative regulator of autophagy. We revealed that the increase in autophagy caused by the reduction of EZH2 was reversed in vitro and in vivo when combining gefitinib or osimertinib with suberoylanilide hydroxamic acid (SAHA), a broad-spectrum histone deacetylase inhibitor (HDACi). In conclusion, our results indicated that the combination of EGFR-TKIs and SAHA may be a new strategy to overcome EGFR-TKIs acquired resistance.

Identifying pathways regulating the oncogenic p53 family member \u0394Np63 provides therapeutic avenues for squamous cell carcinoma

BackgroundΔNp63 overexpression is a common event in squamous cell carcinoma (SCC) that contributes to tumorigenesis, making ΔNp63 a potential target for therapy.MethodsWe created inducible TP63-shRNA cells to study the effects of p63-depletion in SCC cell lines and non-malignant HaCaT keratinocytes. DNA damaging agents, growth factors, signaling pathway inhibitors, histone deacetylase inhibitors, and metabolism-modifying drugs were also investigated for their ability to influence ΔNp63 protein and mRNA levels.ResultsHaCaT keratinocytes, FaDu and SCC-25 cells express high levels of ΔNp63. HaCaT and FaDu inducible TP63-shRNA cells showed reduced proliferation after p63 depletion, with greater effects on FaDu than HaCaT cells, compatible with oncogene addiction in SCC. Genotoxic insults and histone deacetylase inhibitors variably reduced ΔNp63 levels in keratinocytes and SCC cells. Growth factors that regulate proliferation/survival of squamous cells (IGF-1, EGF, amphiregulin, KGF, and HGF) and PI3K, mTOR, MAPK/ERK or EGFR inhibitors showed lesser and inconsistent effects, with dual inhibition of PI3K and mTOR or EGFR inhibition selectively reducing ΔNp63 levels in HaCaT cells. In contrast, the antihyperlipidemic drug lovastatin selectively increased ΔNp63 in HaCaT cells.ConclusionsThese data confirm that ΔNp63-positive SCC cells require p63 for continued growth and provide proof of concept that p63 reduction is a therapeutic option for these tumors. Investigations of ΔNp63 regulation identified agent-specific and cell-specific pathways. In particular, dual inhibition of the PI3K and mTOR pathways reduced ΔNp63 more effectively than single pathway inhibition, and broad-spectrum histone deacetylase inhibitors showed a time-dependent biphasic response, with high level downregulation at the transcriptional level within 24 h. In addition to furthering our understanding of ΔNp63 regulation in squamous cells, these data identify novel drug combinations that may be useful for p63-based therapy of SCC.

Panobinostat reverses HepaCAM gene expression and suppresses proliferation by increasing histone acetylation in prostate cancer.

Increased expression of histone deacetylases (HDACs) affiliated to the epigenetic regulation is common aberration in prostate cancer (PCa). We have confirmed that hepatocyte cell adhesion molecule (hepaCAM), acting as a tumor suppressor gene, is rarely expressed in PCa previously, However, the mechanisms of which is still unknown. The level of histone acetylation reportedly may involve anti-oncogene transcription and expression. In this study, we investigated the effect of panobinostat, the broad-spectrum histone deacetylases inhibitor, on PCa LNCaP and DU145 cell growth, and observed re-expression of hepaCAM when treated with panobinostat. We demonstrated that intranuclear acetylation of lys9 of histone H3 (Ac-H3K9) were increased, while that of both mRNA and protein of HDAC1, HDAC3, and HDAC4 were decreased when the treating concentration of panobinostat increased. We confirmed the relationship between histone acetylation and the expression of hepaCAM and AR in prostate cancer tissues. We also confirmed that panobinostat could overcome the resistance for androgen deprivation therapy (ADT). Further, we combined panobinostat with Ad-hepaCAM, which resulted in significantly increased antitumor activity and significant attenuation of the proliferation-associated genes CCND1 and PCNA compared to each single treatment. In conclusion, panobinostat may enhance the acetylation of lys9 of histone 3 and reverse the hepaCAM expression through its inhibitory effect on HDACs activity in PCa LNCaP and DU145 cells; Ad-hepaCAM combined with panobinostat may synergistically inhibit the growth of LNCaP and DU145 cells, via a potential mechanism associated with the down-regulation of the expression of CCND1 and PCNA. These findings suggest that this therapeutic strategy should be further developed in clinical trials.

High-Throughput Mechanistic Screening of Epigenetic Compounds for the Potential Treatment of Meningiomas.

Background: Meningiomas are the most common primary central nervous system tumors. 20–30% of these tumors are considered high-grade and associated with poor prognosis and high recurrence rates. Despite the high occurrence of meningiomas, there are no FDA-approved compounds for the treatment of these tumors. Methods: In this study, we screened patient-cultured meningiomas with an epigenetic compound library to identify targetable mechanisms for the potential treatment of these tumors. Meningioma cell cultures were generated directly from surgically resected patient tumors and were cultured on a neural matrix. Cells were treated with a library of compounds meant to target epigenetic functions. Results: Although each tumor displayed a unique compound sensitivity profile, Panobinostat, LAQ824, and HC toxin were broadly effective across most tumors. These three compounds are broad-spectrum Histone Deacetylase (HDAC) inhibitors which target class I, IIa, and IIb HDACs. Panobinostat was identified as the most broadly effective compound, capable of significantly decreasing the average cell viability of the sample cohort, regardless of tumor grade, recurrence, radiation, and patient gender. Conclusions: These findings strongly suggest an important role of HDACs in meningioma biology and as a targetable mechanism. Additional validation studies are necessary to confirm these promising findings, as well to identify an ideal HDAC inhibitor candidate to develop for clinical use.

Inhibition of Histone Deacetylases 1, 2, and 3 Enhances Clearance of Cholesterol Accumulation in Niemann-Pick C1 Fibroblasts.

Niemann-Pick disease type C1 (NPC1) is a rare genetic cholesterol storage disorder caused by mutations in the NPC1 gene. Mutations in this transmembrane late endosome protein lead to loss of normal cholesterol efflux from late endosomes and lysosomes. It has been shown that broad spectrum histone deacetylase inhibitors (HDACi's) such as Vorinostat correct the cholesterol accumulation phenotype in the majority of NPC1 mutants tested in cultured cells. In order to determine the optimal specificity for HDACi correction of the mutant NPC1s, we screened 76 HDACi's of varying specificity. We tested the ability of these HDACi's to correct the excess accumulation of cholesterol in patient fibroblast cells that homozygously express NPC1I1061T, the most common mutation. We determined that inhibition of HDACs 1, 2, and 3 is important for correcting the defect, and combined inhibition of all three is needed to achieve the greatest effect, suggesting a need for multiple effects of the HDACi treatments. Identifying the specific HDACs involved in the process of regulating cholesterol trafficking in NPC1 will help to focus the search for more specific druggable targets.

Histone Deacetylase 7 Inhibition in a Murine Model of Gram-Negative Pneumonia-Induced Acute Lung Injury.

Pulmonary infections remain the most common cause of Acute Respiratory Distress Syndrome (ARDS), a pulmonary inflammatory disease with high mortality, for which no targeted therapy currently exists. We have previously demonstrated an ameliorated syndrome with early, broad spectrum Histone Deacetylase (HDAC) inhibition in a murine model of gram-negative pneumonia-induced Acute Lung Injury (ALI), the underlying pulmonary pathologic phenotype leading to ARDS. With the current project we aim to determine if selective inhibition of a specific HDAC leads to a similar pro-survival phenotype, potentially pointing to a future therapeutic target. C57Bl/6 mice underwent endotracheal instillation of 30×10Escherichia coli (strain 19138) versus saline (n = 24). Half the infected mice were administered Trichostatin A (TSA) 30 min later. All animals were sacrificed 6 h later for tissue sampling and HDAC quantification, while another set of animals (n = 24) was followed to determine survival. Experiments were repeated with selective siRNA inhibition of the HDAC demonstrating the greatest inhibition versus scrambled siRNA (n = 24). TSA significantly ameliorated the inflammatory phenotype and improved survival in infected-ALI mice, and HDAC7 was the HDAC with the greatest transcription and protein translation suppression. Similar results were obtained with selective HDAC7 siRNA inhibition compared with scrambled siRNA. HDAC7 appears to play a key role in the inflammatory response that leads to ALI after gram-negative pneumonia in mice.

Reversal of increased mammary tumorigenesis by valproic acid and hydralazine in offspring of dams fed high fat diet during pregnancy

Maternal or paternal high fat (HF) diet can modify the epigenome in germ cells and fetal somatic cells leading to an increased susceptibility among female offspring of multiple generations to develop breast cancer. We determined if combined treatment with broad spectrum DNA methyltransferase (DNMT) inhibitor hydralazine and histone deacetylase (HDAC) inhibitor valproic acid (VPA) will reverse this increased risk. C57BL/6 mouse dams were fed either a corn oil-based HF or control diet during pregnancy. Starting at age 7 weeks, female offspring were administered 3 doses of 7,12-dimethylbenz[a]anthracene (DMBA) to initiate mammary cancer. After last dose, offspring started receiving VPA/hydralazine administered via drinking water: no adverse health effects were detected. VPA/hydralazine reduced mammary tumor multiplicity and lengthened tumor latency in HF offspring when compared with non-treated HF offspring. The drug combination inhibited DNMT3a protein levels and increased expression of the tumor suppressor gene Cdkn2a/p16 in mammary tumors of HF offspring. In control mice not exposed to HF diet in utero, VPA/hydralazine increased mammary tumor incidence and burden, and elevated expression of the unfolded protein response and autophagy genes, including HIF-1α, NFkB, PERK, and SQSTM1/p62. Expression of these genes was already upregulated in HF offspring prior to VPA/hydralazine treatment. These findings suggest that breast cancer prevention strategies with HDAC/DNMT inhibitors need to be individually tailored.

Butyrate stimulates hepatic gluconeogenesis in mouse primary hepatocytes.

Butyrate is a major short-chain fatty acid (SCFA) produced by microbial fermentation of dietary fiber in the gastrointestinal tract. Butyrate is also a well-known broad-spectrum histone deacetylase (HDAC) inhibitor. Butyrate has been reported to improve energy metabolism in rodents, which is associated with its beneficial effects on skeletal muscle, brown fat tissue and pancreatic β-cells. The present study investigated the direct effect of butyrate on hepatic gluconeogenesis in mouse primary hepatocytes and the underlying mechanism. Isolated mouse primary hepatocytes were incubated with sodium butyrate, other HDAC inhibitors and other SCFAs. Hepatic glucose production was measured and gluconeogenic gene expression was detected by polymerase chain reaction analysis. The phosphorylation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) was assessed by western blot analysis. The results revealed that sodium butyrate dose-dependently increased hepatic glucose production and gluconeogenic gene expression in isolated mouse primary hepatocytes. Trichostatin A, a potent broad-spectrum HDAC inhibitor, had the opposite effect. Similar to sodium butyrate, propionate, which is another SCFA, promoted hepatic glucose production and gluconeogenic gene expression in the presence or absence of gluconeogenic substrates, which were further enhanced by cAMP. Furthermore, sodium butyrate also increased the accumulation of intracellular ATP and induced the phosphorylation of CREB in mouse hepatocytes. In conclusion, the present study suggested that butyrate stimulates hepatic gluconeogenesis and induces gluconeogenic gene expression as a substrate and cAMP/CREB signaling activator.

CRISPR Activation Screen for HDAC Inhibitor Resistance

Abstract Panobinostat is an oral broad-spectrum histone deacetylase inhibitor that alters gene expression via epigenetic mechanisms and function of key proteins through changes in their protein acetylation state. Panobinostate was approved a few years ago by the FDA and EMA for use in combination with bortezomib and dexamethasone in patients with multiple myeloma (MM) who have received ≥2 prior regimens, including bortezomib and an immunomodulatory drug. To study the mechanisms which regulate the response vs. resistance of MM cells to panobinostat, we performed genome-scale CRISPR activation screen of MM1S cells. MM1S cells were transduced with dCas9 and pooled lentiviral particles of the Calabrese P65-HSF CRISPR activation library consisting of 2 pooled sgRNA sub-libraries. After selection for viral transduction, cells were continuously cultured with (2 different concentration of 12.5 and 20 nM) or without panobinostat for 5 weeks, before being harvested. At that time point, dose-response curves for panobinostat treatment confirmed that the drug-exposed MM.1S cell populations of our study had become significantly less sensitive to panobinostat than treatment- naive MM.1S cells. Genomic DNA was extracted and next generation sequencing was performed to quantify the abundance of the sgRNA "barcodes" within the tumor cell populations of our study, while rank aggregation algorithms were performed to rank genes according to the magnitude and concordance of enrichment for its different sgRNAs. In MM.1S cells which had developed resistance to the lower dose (12.5 nM) of panobinostat, we observed major sgRNA enrichment (at least 4 of 6 sgRNAs, enrichment p-value <0.05, average log2 fold change of enrichment >1.5) for sgRNAs for a variety of genes, of which the most prominently enriched ones encode for the cell surface ABC transporters ABCB1 (MDR1/p-glycoprotein), to a lesser extent ABCC4 (MRP4) and even less so for ABCG2. In addition, we observed sgRNA enrichment for transcription factors, such as IRF4, POU2AF1, AFF2, IKZF3, AFF3, and RELA, or the transcriptional coregulator MTA1; Bcl2 family members such as BCL2 and BCL2L1; and chromatin remodeling genes such as KAT6A. However, in MM.1S cells which had survived the treatment with higher concentration (25 nM) of panobinostat, the genes with significant and concordant sgRNA enrichment were restricted to ABCB1, ABCC4, and IRF4. These observations indicate that the most efficient mechanism for MM cells to develop resistance to both low and higher concentrations of panobinostat is by increasing its export from the cells, with ABCB1 as the primary, but not sole, transporter which can assume this role. When we transduced MM.1S cells, which already express high levels of IRF4 transcript and protein, with lentiviral construct for IRF4 cDNA, we observed a shift to the right for the panobinostat dose-response curve, further supporting the observation that modulation of IRF4 levels in MM cells can alter the degree of MM cell sensitivity to panobinostat. Proteasome inhibitors suppress the activity of RELA and NFkappaB more broadly, while thalidomide derivatives cause degradation of IKZF3 and can decrease the IRF4 transcript levels: these results may respectively explain, at least in part, the mechanistic basis for the enhanced combined effect of panobinostat with proteasome inhibitors and the favorable clinical results obtained with panobinostat and other broad spectrum HDAC inhibitors in combination with thalidomide derivatives. Our study provides unbiased genome-scale systematic characterization of the mechanisms regulating MM cell response vs. resistance to panobinostat and identify the pronounced and dose-dependent enrichment of these resistance mechanisms for genes contributing to panobinostat export from MM cells, as well as a role for IRF4 and (primarily at lower panobinostat concentrations) for other transcription factors, chromatin remodeling genes and anti-apoptotic BCL-2 family members. These observations also support the need for a more systematic characterization of the regulation of expression of ABC transporters in MM cells; and for development of novel strategies to disrupt more comprehensively IRF4 and other transcription factors for which gain-of-function is associated with decreased responsiveness to panobinostat, with the goal of improving the impact of this agent and potentially other broad spectrum HDAC inhibitors in MM. Disclosures Mitsiades: TEVA: Research Funding; Takeda: Other: employment of a relative; EMD Serono: Research Funding; Abbvie: Research Funding; Janssen/ Johnson & Johnson: Research Funding.

Histone Deacetylase Inhibitor-Induced Autophagy in Tumor Cells: Implications for p53.

Autophagy is an essential process of the eukaryotic cell allowing degradation and recycling of dysfunctional cellular components in response to either physiological or pathological changes. Inhibition of autophagy in combination with chemotherapeutic treatment has emerged as a novel approach in cancer treatment leading to cell cycle arrest, differentiation, and apoptosis. Suberoyl hydroxamic acid (SAHA) is a broad-spectrum histone deacetylase inhibitor (HDACi) suppressing family members in multiple HDAC classes. Increasing evidence indicates that SAHA and other HDACi can, in addition to mitochondria-mediated apoptosis, also promote caspase-independent autophagy. SAHA-induced mTOR inactivation as a major regulator of autophagy activating the remaining autophagic core machinery is by far the most reported pathway in several tumor models. However, the question of which upstream mechanisms regulate SAHA-induced mTOR inactivation that consequently initiate autophagy has been mainly left unexplored. To elucidate this issue, we recently initiated a study clarifying different modes of SAHA-induced cell death in two human uterine sarcoma cell lines which led to the conclusion that the tumor suppressor protein p53 could act as a molecular switch between SAHA-triggered autophagic or apoptotic cell death. In this review, we present current research evidence about HDACi-mediated apoptotic and autophagic pathways, in particular with regard to p53 and its therapeutic implications.

Timing of valproic acid in acute lung injury: prevention is the best therapy?

Acute lung injury and respiratory distress syndrome is characterized by uncontrolled inflammation of the lungs after a severe inflammatory stimulus. We have previously demonstrated an ameliorated syndrome and improved survival in mice with early administration of valproic acid (VPA), a broad-spectrum histone deacetylase inhibitor, while studies in humans have shown no benefit when anti-inflammatories are administered late. The current study tested the hypothesis that early treatment would improve outcomes in our gram-negative pneumonia-induced acute lung injury. Mice (C57BL/6) had 50 × 106Escherichia coli (strain 19,138) instilled endotracheally and VPA (250mg/kg) administered intraperitoneally 3, 4, 6, and 9h (n=12/group) later. Six hours after VPA administration, the animals were sacrificed, and bronchoalveolar lavage (BAL) fluid interleukin-6 (IL-6), tumor necrosis factor, neutrophils and macrophages as well as the E coli colony-forming units were quantified. Plasma IL-6 was also measured. A separate group of mice (n=12/group) were followed prospectively for 7days to assess survival. BAL IL-6 and tumor necrosis factor as well as plasma IL-6 were significantly lower in the animals administered VPA within 3 h (P<0.05) but not when administered later (4,6,9 h). There was no difference in the BAL E coli colony-forming units, macrophage, or neutrophil numbers at any time point. Survival improved only when VPA was administered within 3 h. A narrow therapeutic window exists in this murine model of gram-negative pneumonia-induced acute lung injury and likely explains the lack of response in studies with late administration of anti-inflammatory therapies in clinical studies.

Metabolic inhibitors accentuate the anti-tumoral effect of HDAC5 inhibition

The US FDA approval of broad-spectrum histone deacetylase (HDAC) inhibitors has firmly laid the cancer community to explore HDAC inhibition as a therapeutic approach for cancer treatment. Hitting one HDAC member could yield clinical benefit but this required a complete understanding of the functions of the different HDAC members. Here we explored the consequences of specific HDAC5 inhibition in cancer cells. We demonstrated that HDAC5 inhibition induces an iron-dependent reactive oxygen species (ROS) production, ultimately leading to apoptotic cell death as well as mechanisms of mitochondria quality control (mitophagy and mitobiogenesis). Interestingly, adaptation of HDAC5-depleted cells to oxidative stress passes through reprogramming of metabolic pathways towards glucose and glutamine. Therefore, interference with both glucose and glutamine supply in HDAC5-inhibited cancer cells significantly increases apoptotic cell death and reduces tumour growth in vivo; providing insight into a valuable clinical strategy combining the selective inhibition of HDAC5 with various inhibitors of metabolism as a new therapy to kill cancer cells.

Evaluating the Effect of HDAC8 Inhibition in Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive disease with a dismal prognosis. The disease can occur sporadically or in patients with inherited neurofibromatosis (NF-1). MPNST is typically resistant to therapeutic intervention. Hence, the need for improved therapies is warranted. Several broad spectrum histone deacetylase (HDAC) inhibitors have a high affinity for class I HDAC isoforms. Inhibition of multiple HDAC isoforms often results in undesirable side effects, while inhibiting a single isoform could possibly improve the therapeutic window and limit toxicity. Recently, HDAC8 inhibitors have been developed and in initial preclinical studies, they demonstrate anticancer efficacy. Little is known about the role of HDAC8 in MPNST. We recently revealed an anticancer effect of HDAC8 inhibition in human and murine MPNST models. The goal of our previous study was to determine the potential therapeutic efficacy of HDAC8 inhibition in MPNST. In this chapter, we briefly describe the methods for determining the role of pharmacological HDAC inhibition in MPNST.

Trichostatin A Inhibits Retinal Pigmented Epithelium Activation in an In Vitro Model of Proliferative Vitreoretinopathy

Purpose: Proliferative vitreoretinopathy (PVR) is a blinding disorder that develops after a retinal tear or detachment. Activation of the retinal pigmented epithelium (RPE) is implicated in PVR; however, the mechanisms leading to enhanced RPE proliferation, migration, and contraction remain largely unknown. This study utilized an in vitro model of PVR to investigate the role of acetylation in RPE activation and its contribution to the progression of this disease.Methods: ARPE-19 cells, primary cultures of porcine RPE, and induced pluripotent stem cell-derived RPE (iPS-RPE) were utilized for cellular and molecular analyses. Cells treated with transforming growth factor beta 2 (TGFβ2; 10 ng/mL) alone or in the presence of the broad-spectrum histone deacetylase (HDAC) inhibitor, trichostatin A (TSA; 0.1 μM), were assessed for contraction and migration through collagen contraction and scratch assays, respectively. Western blotting and immunofluorescence analysis were performed to assess α-smooth muscle actin (α-SMA) and β-catenin expression after TGFβ2 treatment alone or in combination with TSA.Results: TGFβ2 significantly increased RPE cell contraction in collagen matrix and this effect was inhibited in the presence of TSA (0.1 μM). In agreement with these data, immunofluorescence analysis of TSA-treated iPS-RPE wounded monolayers revealed decreased α-SMA as compared with control. Scratch assays to assess wound healing revealed TSA inhibited TGFβ2-mediated iPS-RPE cell migration.Conclusions: Our findings indicate a role of acetylation in RPE activation. Specifically, the HDAC inhibitor TSA decreased RPE cell proliferation and TGFβ2-mediated cell contraction and migration. Further investigation of pharmacological compounds that modulate acetylation may hold promise as therapeutic agents for PVR.

Short Communication: The Broad-Spectrum Histone Deacetylase Inhibitors Vorinostat and Panobinostat Activate Latent HIV in CD4(+) T Cells In Part Through Phosphorylation of the T-Loop of the CDK9 Subunit of P-TEFb.

Cessation of highly active antiretroviral therapy (HAART) in HIV-infected individual leads to a rebound of viral replication due to reactivation of a viral reservoir composed largely of latently infected memory CD4(+) T cells. Efforts to deplete this reservoir have focused on reactivation of transcriptionally silent latent proviruses. HIV provirus transcription depends critically on the positive transcription elongation factor b (P-TEFb), whose core components are cyclin-dependent kinase 9 (CDK9) and cyclin T1. In resting CD4(+) cells, the functional levels of P-TEFb are extremely low. Cellular activation upregulates cyclin T1 protein levels and CDK9 T-loop (T186) phosphorylation. The broad-spectrum histone deacetylase inhibitors (HDACis) vorinostat and panobinostat have been shown to reactivate latent virus in vivo in HAART-treated individuals. In this study, we have found that vorinostat and panobinostat activate P-TEFb in resting primary CD4(+) T cells through induction of CDK9 T-loop phosphorylation. In contrast, tacedinaline and romidepsin, HDAC 1 and 2 inhibitors, were unable to activate CDK9 T-loop phosphorylation. We used a CCL19 primary CD4(+) T-cell model HIV latency to assess the correlation between induction of CDK9 T-loop phosphorylation and reactivation of latent HIV virus by HDACis. Vorinostat and panobinostat treatment of cells harboring latent HIV increased CDK9 T-loop phosphorylation and reactivation of latent virus, whereas tacedinaline and romidepsin failed to induce T-loop phosphorylation or reactivate latent virus. We conclude that the ability of vorinostat and panobinostat to induce latent HIV is, in part, likely due to the ability of the broad-spectrum HDACis to upregulate P-TEFb through increased CDK9 T-loop phosphorylation.