Histone Deacetylase 6 Activity Research Articles

Identification of Potential Inhibitors of Histone Deacetylase 6 Through Virtual Screening and Molecular Dynamics Simulation Approach: Implications in Neurodegenerative Diseases

Background: Histone deacetylase 6 (HDAC6) plays a crucial role in neurological, inflammatory, and other diseases; thus, it has emerged as an important target for therapeutic intervention. To date, there are no FDA-approved HDAC6-targeting drugs, and most pipeline candidates suffer from poor target engagement, inadequate brain penetration, and low tolerability. There are a few HDAC6 clinical candidates for the treatment of mostly non-CNS cancers as their pharmacokinetic liabilities exclude them from targeting HDAC6-implicated neurological diseases, urging development to address these challenges. They also demonstrate off-target toxicity due to limited selectivity, leading to adverse effects in patients. Selective inhibitors have thus been the focus of development over the past decade, though no selective and potent HDAC6 inhibitor has yet been approved. Methods: This study involved an integrated virtual screening against HDAC6 using the DrugBank database to identify repurposed drugs capable of inhibiting HDAC6 activity. The primary assessment involved the determination of the ability of molecules to bind with HDAC6. Subsequently, interaction analyses and 500 ns molecular dynamics (MD) simulations followed by essential dynamics were carried out to study the conformational flexibility and stability of HDAC6 in the presence of the screened molecules, i.e., penfluridol and pimozide. Results: The virtual screening results pinpointed penfluridol and pimozide as potential repurposed drugs against HDAC6 based on their binding efficiency and appropriate drug profiles. The docking results indicate that penfluridol and pimozide share the same binding site as the reference inhibitor with HDAC6. The MD simulation results showed that stable protein–ligand complexes of penfluridol and pimozide with HDAC6 were formed. Additionally, MMPBSA analysis revealed favorable binding free energies for all HDAC6–ligand complexes, confirming the stability of their interactions. Conclusions: The study implies that both penfluridol and pimozide have strong and favorable binding with HDAC6, which supports the idea of repositioning these drugs for the management of neurodegenerative disorders. However, further in-depth studies are needed to explore their efficacy and safety in biological systems.

Inhibition of HDAC6 mitigates high-fat diet-induced kidney inflammation and hypertension via reduced infiltration of macrophages

Obesity-mediated hypertension is a worldwide problem. Recent research has indicated that chronic inflammation is associated with the pathogenesis of obese hypertension. Activated immune cells infiltrate target organs, such as arteries, kidneys, and brain, causing end-organ damage and hypertension. Histone deacetylase 6 (HDAC6) regulates the inflammatory cell activity mediating the production of inflammatory cytokines and may play a role in the crosstalk between inflammation and hypertension. In this study, we investigated the roles of HDAC6 in high-fat diet (HFD)-induced kidney inflammation and hypertension. Nine-week-old male C57BL/6 mice were fed either a normal diet (ND) or HFD for 15 weeks. HFD-induced hypertension with increased HDAC6 activities in the kidney and bone marrow-derived macrophages (BMDM). When HFD group reached the hypertensive phase, each group of mice was intraperitoneally injected with vehicle or selective HDAC6 inhibitor Tubacin (1 mg/kg/day) for 14 days. Tubacin treatment lowered blood pressure (BP) of HFD-fed mice to the normal level with successful inhibition of HDAC6 activity. Immunohistochemical staining of F4/80, which is known as a macrophage marker, revealed that HFD promoted macrophage infiltration into the kidney. Consequently, pro-inflammatory factors TNFα and IL-6 gene expressions in the kidney were increased by HFD. Tubacin canceled HFD-induced macrophage infiltration and inflammation in the kidney. HDAC6 gene silencing and Tubacin treatment in Raw 264.7 cells also blocked the chemoattractant-stimulated cell migration in vitro. The results reveal the novel role of HDAC6 in BMDM migration, kidney inflammation, and high BP induced by HFD providing HDAC6 inhibitors as a therapeutic option for obesity-mediated hypertension.

Genetic deletion or pharmacologic inhibition of histone deacetylase 6 protects the heart against ischaemia/reperfusion injury by limiting tumour necrosis factor alpha-induced mitochondrial injury in experimental diabetes.

The histone deacetylase 6 (HDAC6) inhibitor, tubastatin A (TubA), reduces myocardial ischaemia/reperfusion injury (MIRI) in type 1 diabetic rats. It remains unclear whether HDAC6 regulates MIRI in type 2 diabetic animals. Diabetes augments the activity of HDAC6 and the generation of tumour necrosis factor alpha (TNF-α) and impairs mitochondrial complex I (mCI). Here, we examined how HDAC6 regulates TNF-α production, mCI activity, mitochondria, and cardiac function in type 1 and type 2 diabetic mice undergoing MIRI. HDAC6 knockout, streptozotocin-induced type 1 diabetic, and obese type 2 diabetic db/db mice underwent MIRI in vivo or ex vivo in a Langendorff-perfused system. We found that MIRI and diabetes additively augmented myocardial HDAC6 activity and generation of TNF-α, along with cardiac mitochondrial fission, low bioactivity of mCI, and low production of adenosine triphosphate. Importantly, genetic disruption of HDAC6 or TubA decreased TNF-α levels, mitochondrial fission, and myocardial mitochondrial nicotinamide adenine dinucleotide levels in ischaemic/reperfused diabetic mice, concomitant with augmented mCI activity, decreased infarct size, and improved cardiac function. Moreover, HDAC6 knockout or TubA treatment decreased left ventricular dilation and improved cardiac systolic function 28 days after MIRI. H9c2 cardiomyocytes with and without HDAC6 knockdown were subjected to hypoxia/reoxygenation injury in the presence of high glucose. Hypoxia/reoxygenation augmented HDAC6 activity and TNF-α levels and decreased mCI activity. These negative effects were blocked by HDAC6 knockdown. HDAC6 is an essential negative regulator of MIRI in diabetes. Genetic deletion or pharmacologic inhibition of HDAC6 protects the heart from MIRI by limiting TNF-α-induced mitochondrial injury in experimental diabetes.

Central inhibition of HDAC6 re-sensitizes leptin signaling during obesity to induce profound weight loss

Leptin resistance during excess weight gain significantly contributes to the recidivism of obesity to leptin-based pharmacological therapies. The mechanisms underlying the inhibition of leptin receptor (LepR) signaling during obesity are still elusive. Here, we report that histone deacetylase 6 (HDAC6) interacts with LepR, reducing the latter's activity, and that pharmacological inhibition of HDAC6 activity disrupts this interaction and augments leptin signaling. Treatment of diet-induced obese mice with blood-brain barrier (BBB)-permeable HDAC6 inhibitors profoundly reduces food intake and leads to potent weight loss without affecting the muscle mass. Genetic depletion of Hdac6 in Agouti-related protein (AgRP)-expressing neurons or administration with BBB-impermeable HDAC6 inhibitors results in a lack of such anti-obesity effect. Together, these findings represent the first report describing a mechanistically validated and pharmaceutically tractable therapeutic approach to directly increase LepR activity as well as identifying centrally but not peripherally acting HDAC6 inhibitors as potent leptin sensitizers and anti-obesity agents.

Abstract 2883: HDAC6 mediates the radioresistance of NSCLC through LSD1, independent of its own deacetylation activity

Abstract Resistance to radiation therapy significantly affects the prognosis of solid tumors. Histone deacetylase 6 (HDAC6) is a stress-responsive lysine deacetylase that has emerged as a promising target for cancer therapy, with numerous clinical trials investigating interventions to modulate its activity. While HDAC6 appears to be linked to responses to DNA-damaging therapeutics, the reported responses and underlying mechanisms remain intriguing and varied. In this study, we elucidate a novel mechanism of radioresistance mediated by HDAC6 in non-small cell lung cancer (NSCLC). We classified nine NSCLC cell lines into three groups based on their radioresistance using survival assays and correlated this resistance with the induced expression of six deacetylases. We conducted gain-of-function experiments (GOF) using plasmid transfection and loss-of-function experiments (LOF) employing inhibitors or RNAi. Furthermore, we quantified the repair of damaged dsDNA through FACS-based GFP assays and assessed the mechanism of homologous recombination repair (HRR) end-resection via single-strand quantitative PCR. Co-immunoprecipitation assays, cancer stem cell enrichment, DNA damage-induced senescence assays, reverse-phase protein assays (RPPA), and RNA sequencing were also performed to investigate gene expression changes resulting from the loss of HDAC6. Our findings revealed that HDAC6 induction by radiation is strongly correlated with NSCLC resistance to radiation leading to CSC survival and escape from radiation led senescence LOF of HDAC6 through RNAi or inhibitors suppressed resistance, while GOF achieved through wild-type or deacetylase activity-deficient mutant transfection, induced resistance. Importantly, this resistance mechanism was independent of deacetylation activity. RNAi-mediated loss of HDAC6 reduced both HRR and non-homologous end-joining, whereas pharmacological inhibition of HDAC6 activity did not. Notably, DNA end-resection, a critical step in HRR, was decreased by RNA interference but not affected by the inhibitors. RPPA assays demonstrated that Histone demethylase 1A (LSD1/KDM1A) levels decreased significantly upon HDAC6 RNA interference but remained unaffected by pharmacological intervention. Loss of LSD1 alone resulted in decreased HRR and end-resection, likely mediated through HDAC6-induced ubiquitination. Moreover, overexpression of LSD1 rescued the HDAC6 loss-induced sensitization of NSCLCs. In summary, our study unveils a novel mechanism by which HDAC6 mediates HRR after radiation through the stabilization of LSD1. This mechanism operates independently of deacetylase activity. Therefore, our findings suggest that interventions targeting both deacetylation and non-deacetylation roles of HDAC6 should be considered to overcome HDAC6-derived radioresistance in NSCLCs and enhances the radiation therapy efficacy. Citation Format: Sojung Ha, Hyejin Kim, Hani Lee, Seokgyeong Choi, Ho-young Lee, Woo-Young Kim. HDAC6 mediates the radioresistance of NSCLC through LSD1, independent of its own deacetylation activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2883.

Unveiling the modulation of Pseudomonas aeruginosa virulence and biofilm formation by selective histone deacetylase 6 inhibitors.

Bacterial infections represent a key public health issue due to the occurrence of multidrug-resistant bacteria. Recently, the amount of data supporting the dynamic control of epigenetic pathways by environmental cues has triggered research efforts toward the clarification of their role in microbial infections. Among protein post-translational modifications, reversible acetylation is the most implicated in the feedback to environmental stimuli and in cellular homeostasis. Accordingly, the latest studies identified the histone deacetylase 6 (HDAC6) enzyme as a crucial player in the complex molecular machinery underlying bacterial clearance or killing. A very important milestone for the elucidation of the consequence of HDAC6 activity in bacterial infections is herein described, unveiling for the first time the role of a potent HDAC6 inhibitor in interfering with biofilm formation and modulating virulence factors of P. aeruginosa. We demonstrated that compound F2F-2020202 affected the production of some important virulence factors in P. aeruginosa, namely pyocyanin and rhamnolipids, clearly impairing its ability to form biofilm. Furthermore, evidence of possible QS involvement is supported by differential regulation of specific genes, namely RhlI, phAz1, and qsrO. The data herein obtained also complement and in part explain our previous results with selective HDAC6 inhibitors able to reduce inflammation and bacterial load in chronic infection models recapitulating the cystic fibrosis (CF) phenotype. This study fosters future in-depth investigation to allow the complete elucidation of the molecular mechanisms underlying HDAC6's role in bacterial infections.

Piezo1 Mediates the Regulation of Substrate Stiffness on Primary Cilia in Chondrocytes

To investigate how substrate stiffness regulates the morphology of primary cilia in chondrocytes and to illustrate how Piezo1 mediates the morphology regulation of primary cilia by substrate stiffness. Polydimethylsiloxane (PDMS) curing agent and the main agent (Dow Corning, Beijing, China) were mixed at the ratio of 1∶10 (stiff), 1∶50 (medium stiffness), and 1∶70 (soft), respectively, to prepare substrate films with the thickness of 1 mm at different levels of stiffness, including stiff substrate of (2.21±0.12) MPa, medium-stiffness substrate of (54.47±6.06) kPa, and soft substrate of (2.13±0.10) kPa. Chondrocytes were cultured with the substrates of three different levels of stiffness. Then, the cells were treated with Tubastatin A (Tub A) to inhibit histone deacetylase 6 (HDAC6), Piezo1 activator Yoda1, and inhibitor GsMTx4, respectively. The effects of HDAC6, Yoda1, and GsMTx4 on chondrocyte morphology and the length of primary cilia were analyzed through immunofluorescence staining. The stiff substrate increased the spread area of the chondrocytes. Immunofluorescence assays showed that the cytoskeleton and the nuclear area of the cells on the stiff substrate were significantly increased (P<0.05) and the primary cilia were significantly extended (P<0.05) compared with those on the medium-stiffness and soft substrates. However, the presence rate of primary cilia was not affected. The HDAC6 activity of chondrocytes increased with the decrease in substrate stiffness. When the activity of HDAC6 was inhibited, the cytoskeletal area, the nuclei area, and the primary cilium length were increased more significantly on the stiff substrate (P<0.05). Further testing showed that Piezo1 activator and inhibitor could regulate the activity of HDAC6 in chondrocytes, and that the length of primary cilia was significantly increased after treatment with the activator Yoda1 (P<0.05). On the other hand, the length of primary cilia was significantly shortened on the stiff substrate after treatment with the inhibitor GsMTx4 (P<0.05). Both substrate stiffness and Piezo1 may affect the morphology of chondrocyte primary cilia by regulating HDAC6 activity.

Sulforaphane, an NRF2 agonist, alleviates ferroptosis in acute liver failure by regulating HDAC6 activity

ObjectiveAcute liver failure (ALF) is characterized by severe liver dysfunction, rapid progression and high mortality and is difficult to treat. Studies have found that sulforaphane (SFN), a nuclear factor E2-related factor 2 (NRF2) agonist, has anti-inflammatory, antioxidant and anticancer effects, and has certain protective effects on neurodegenerative diseases, cancer and liver fibrosis. This paper aimed to explore the protective effect of SFN in ALF and it possible mechanisms of action. MethodsLipopolysaccharide and D-galactosamine were used to induce liver injury in vitro and in vivo. NRF2 agonist SFN and histone deacetylase 6 (HDAC6) inhibitor ACY1215 were used to observe the protective effect and possible mechanisms of SFN in ALF, respectively. Cell viability, lactate dehydrogenase (LDH), Fe2+, glutathione (GSH) and malondialdehyde (MDA) were detected. The expression of HDAC6, NRF2, glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) were detected by Western blotting and immunofluorescence. ResultsOur results show that NRF2 was activated by SFN. LDH, Fe2+, MDA and ACSL4 were downregulated, while GSH, GPX4 and SLC7A11 were upregulated by SFN in vitro and in vivo, indicating the inhibitory effect of SFN on ferroptosis. Additionally, HDAC6 expression was decreased in the SFN group, indicating that SFN could downregulate the expression of HDAC6 in ALF. After using the HDAC6 inhibitor, ACY1215, SFN further reduced HDAC6 expression and inhibited ferroptosis, indicating that SFN may inhibit ferroptosis by regulating HDAC6 activity. ConclusionSFN has a protective effect on ALF, and the mechanism may include reduction of ferroptosis through the regulation of HDAC6.Please cite this article as: Zhang YQ, Shi CX, Zhang DM, Zhang LY, Wang LW, Gong ZJ. Sulforaphane, an NRF2 agonist, alleviates ferroptosis in acute liver failure by regulating HDAC6 activity. J Integr Med. 2023; 21(5): 464–473.

HDAC6 inhibition: a significant potential regulator and therapeutic option to translate into clinical practice in renal transplantation.

Histone deacetylase 6 (HDAC6), an almost exclusively cytoplasmic enzyme, plays an essential role in many biological processes and exerts its deacetylation-dependent/independent effects on a variety of target molecules, which has contributed to the flourishing growth of relatively isoform-specific enzyme inhibitors. Renal transplantation (RT) is one of the alternatively preferred treatments and the most cost-effective treatment approaches for the great majority of patients with end-stage renal disease (ESRD). HDAC6 expression and activity have recently been shown to be increased in kidney disease in a number of studies. To date, a substantial amount of validated studies has identified HDAC6 as a pivotal modulator of innate and adaptive immunity, and HDAC6 inhibitors (HDAC6i) are being developed and investigated for use in arrays of immune-related diseases, making HDAC6i a promising therapeutic candidate for the management of a variety of renal diseases. Based on accumulating evidence, HDAC6i markedly open up new avenues for therapeutic intervention to protect against oxidative stress-induced damage, tip the balance in favor of the generation of tolerance-related immune cells, and attenuate fibrosis by inhibiting multiple activations of cell profibrotic signaling pathways. Taken together, we have a point of view that targeting HDAC6 may be a novel approach for the therapeutic strategy of RT-related complications, including consequences of ischemia-reperfusion injury, induction of immune tolerance in transplantation, equilibrium of rejection, and improvement of chronic renal graft interstitial fibrosis after transplantation in patients. Herein, we will elaborate on the unique function of HDAC6, which focuses on therapeutical mechanism of action related to immunological events with a general account of the tantalizing potential to the clinic.

Pharmacological inhibition of HDAC6 suppresses NLRP3 inflammasome-mediated IL-1β release

The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome is an important regulator of inflammation and immune responses. Histone deacetylase 6 (HDAC6) has been implicated in the assembly and activation of the NLRP3 inflammasome in mouse cells, however, the role in human immune cells remains poorly understood. Here, we investigated the effect of HDAC6 deficiency on NLRP3-mediated interleukin (IL)-1β release using proteolysis targeting chimeras (PROTAC) technology. We designed an HDAC6 PROTAC (A6) composed of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the E3 ligase ligand thalidomide and a control PROTAC (non-degrading control, nc-A6) that binds to HDAC6 but lacks the ability to induce HDAC6 degradation. A6 but not nc-A6 reduced HDAC6 levels in THP-1 macrophages without affecting cell viability. PROTAC A6 and nc-A6 significantly reduced the release of IL-1β in a concentration-dependent manner, suggesting that HDAC6 deficiency is not necessary for inhibition of NLRP3 inflammasome-mediated IL-1β release. We found that inhibition of the catalytic domain with HDAC inhibitor SAHA or the specific HDAC6 inhibitor tubastatin A is sufficient to reduce IL-1β release indicating that the enzymatic activity of HDAC6 is critical for NLRP3 inflammasome function. Mechanistically, the observed effects of HDAC6 inhibition on NLRP3-mediated inflammatory responses could be attributed to its interaction with Toll-like receptor (TLR) signaling. Tubastatin A did not affect IL-1β levels when added after TLR-mediated priming. Collectively, our findings indicate that HDAC6 inhibitors show potent anti-inflammatory activity and suppress IL-1β release by human macrophages, independent of NLRP3 assembly and activation.

Shortening of primary cilia length is associated with urine concentration in the kidneys.

The primary cilium, a microtubule-based cellular organelle present in certain kidney cells, functions as a mechano-sensor to monitor fluid flow in addition to various other biological functions. In kidneys, the primary cilia protrude into the tubular lumen and are directly exposed to pro-urine flow and components. However, their effects on urine concentration remain to be defined. Here, we investigated the association between primary cilia and urine concentration. Mice either had free access to water (normal water intake, NWI) or were not allowed access to water (water deprivation, WD). Some mice received tubastatin, an inhibitor of histone deacetylase 6 (HDAC6), which regulates the acetylation of α-tubulin, a core protein of microtubules. WD decreased urine output and increased urine osmolality, concomitant with apical plasma membrane localization of aquaporin 2 (AQP2) in the kidney. After WD, compared with after NWI, the lengths of primary cilia in renal tubular epithelial cells were shortened and HDAC6 activity increased. WD induced deacetylation of α-tubulin without altering α-tubulin levels in the kidney. Tubastatin prevented the shortening of cilia through increasing HDAC6 activity and consequently increasing acetylated α-tubulin expression. Furthermore, tubastatin prevented the WD-induced reduction of urine output, urine osmolality increase, and apical plasma membrane localization of AQP2. WD shortens primary cilia length through HDAC6 activation and α-tubulin deacetylation, while HDAC6 inhibition blocks the WD-induced changes in cilia length and urine output. This suggests that cilia length alterations are involved, at least in part, in the regulation of body water balance and urine concentration.

HDAC6 Degrades nsp8 of Porcine Deltacoronavirus through Deacetylation and Ubiquitination to Inhibit Viral Replication.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that has the potential to infect humans. Histone deacetylase 6 (HDAC6) is a unique type IIb cytoplasmic deacetylase with both deacetylase activity and ubiquitin E3 ligase activity, which mediates a variety of cellular processes by deacetylating histone and nonhistone substrates. In this study, we found that ectopic expression of HDAC6 significantly inhibited PDCoV replication, while the reverse effects could be observed after treatment with an HDAC6-specific inhibitor (tubacin) or knockdown of HDAC6 expression by specific small interfering RNA. Furthermore, we demonstrated that HDAC6 interacted with viral nonstructural protein 8 (nsp8) in the context of PDCoV infection, resulting in its proteasomal degradation, which was dependent on the deacetylation activity of HDAC6. We further identified the key amino acid residues lysine 46 (K46) and K58 of nsp8 as acetylation and ubiquitination sites, respectively, which were required for HDAC6-mediated degradation. Through a PDCoV reverse genetics system, we confirmed that recombinant PDCoV with a mutation at either K46 or K58 exhibited resistance to the antiviral activity of HDAC6, thereby exhibiting higher replication compared with wild-type PDCoV. Collectively, these findings contribute to a better understanding of the function of HDAC6 in regulating PDCoV infection and provide new strategies for the development of anti-PDCoV drugs. IMPORTANCE As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has sparked tremendous attention. Histone deacetylase 6 (HDAC6) is a critical deacetylase with both deacetylase activity and ubiquitin E3 ligase activity and is extensively involved in many important physiological processes. However, little is known about the role of HDAC6 in the infection and pathogenesis of coronaviruses. Our present study demonstrates that HDAC6 targets PDCoV-encoded nonstructural protein 8 (nsp8) for proteasomal degradation through the deacetylation at the lysine 46 (K46) and the ubiquitination at K58, suppressing viral replication. Recombinant PDCoV with a mutation at K46 and/or K58 of nsp8 displayed resistance to the antiviral activity of HDAC6. Our work provides significant insights into the role of HDAC6 in regulating PDCoV infection, opening avenues for the development of novel anti-PDCoV drugs.

Repurposing bempedoic acid as a histone deacetylase 6 inhibitor

The dysregulation of the activity of histone deacetylase 6 (HDAC6) is observed in a number of human diseases. Targeting HDAC6 has been identified as an ideal treatment strategy in epigenetic‑based therapies. Bempedoic acid is an FDA‑approved cholesterol‑lowering medication. In the present study, bempedoic acid was repurposed as an HDAC6 inhibitor for the first time, at least to the best of our knowledge. Bempedoic acid inhibited the activity of HDAC6 <em>in vitro</em> with an IC₅₀ value of ~0.8 mM. <em>In silico</em> analysis predicted the formation of hydrogen bonds and hydrophobic interactions between HDAC6 residues and bempedoic acid, which may be attributed to its HDAC6 inhibitory potential. The results of the present study provide new opportunities to bring bempedoic acid into epigenetic‑based drug discovery platforms.

Advances in the Mechanistic Study of the Control of Oxidative Stress Injury by Modulating HDAC6 Activity

Oxidative stress is defined as an injury resulting from a disturbance in the dynamic equilibrium of the redox environment due to the overproduction of active/radical oxygen exceeding the antioxidative ability of the body. This is a key step in the development of various diseases. Oxidative stress is modulated by different factors and events, including the modification of histones, which are the cores of nucleosomes. Histone modification includes acetylation and deacetylation of certain amino acid residues; this process is catalyzed by different enzymes. Histone deacetylase 6 (HDAC6) is a unique deacetylating protease that also catalyzes the deacetylation of different nonhistone substrates to regulate various physiologic processes. The intimate relationship between HDAC6 and oxidative stress has been demonstrated by different studies. The present paper aims to summarize the data obtained from a mechanistic study of HDAC6 and oxidative stress to guide further investigations on mechanistic characterization and drug development.

HDAC6, une désacétylase très spécifique porteuse d’espoir thérapeutique

The cytoplasmic histone deacetylase 6 (HDAC6) is defined today as a new key player in the treatment of many diseases. Overexpression of HDAC6 was observed in a variety of diseases. Over the past ten years, plenty of new selective inhibitors of HDAC6 activity have been synthesized and characterized. Many studies have shown the high efficiency and beneficial effects of HDAC6 inhibitors in many diseases such as cancers, neurodegenerative, inflammatory, or neuromuscular diseases. The mechanisms of HDAC6 action that explain the benefit of its inhibition in various pathologies are still unknown. We have recently shown that HDAC6, via the regulation of the microtubule network, plays a role at the level of neuromuscular junctions by controlling acetylcholine receptor delivery.

Telomere Shortening in Hypertensive Heart Disease Depends on Oxidative DNA Damage and Predicts Impaired Recovery of Cardiac Function in Heart Failure.

Heart failure (HF) coincides with cardiomyocyte telomere shortening. Arterial hypertension is the most prominent risk factor for HF. Both HF and arterial hypertension are associated with dysregulation of the neurohormonal axis. How neurohormonal activation is linked to telomere shortening in the pathogenesis of HF is incompletely understood. Cardiomyocyte telomere length was assessed in a mouse model of hypertensive HF induced by excess neurohormonal activation (AngII [angiotensin II] infusion, high salt diet, and uninephrectomy), in AngII-stimulated cardiomyocytes and in endomyocardial biopsies from patients with HF. Superoxide production, expression of NOX2 (NADPH oxidase 2) and PRDX1 (peroxiredoxin 1) and HDAC6 (histone deacetylase 6) activity were assessed. Telomere shortening occurred in vitro and in vivo, correlating with both left ventricular (LV) dilatation and LV systolic function impairment. Telomere shortening coincided with increased superoxide production, increased NOX2 expression, increased HDAC6 activity, loss of the telomere-specific antioxidant PRDX1, and increased oxidative DNA-damage. NOX2 knockout prevented PRDX1 depletion, DNA-damage and telomere shortening confirming this enzyme as a critical source of reactive oxygen species. Cotreatment with the NOX inhibitor apocynin ameliorated hypertensive HF and telomere shortening. Similarly, treatment with the HDAC6 inhibitor tubastatin A, which increases PRDX1 bioavailability, prevented telomere shortening in adult cardiomyocytes. To explore the clinical relevance of our findings, we examined endomyocardial biopsies from an all-comer population of patients with HF with reduced ejection fraction. Here, cardiomyocyte telomere length predicted the recovery of cardiac function. Cardiomyocyte telomere shortening and oxidative damage in heart failure with reduced ejection fraction induced by excess neurohormonal activation depends on NOX2-derived superoxide and may help to stratify HF therapy.

Modification of cardiac disease by transgenically altered histone deacetylase 6

Histone deacetylase 6 (HDAC6) is known to deacetylate amino acid lysine in alpha-tubulin. However, the functional role of HDAC6 in the progression of cardiac disease remains uncertain. The functional role of HDAC6 in the hearts was examined using transgenic (TG) mice expressing either human wild-type HDAC6, deacetylase inactive HDAC6 (HDAC6H216A, H611A), and human HDAC6 replaced all serine or threonine residues with aspartic acid at N-terminal 1- 43 amino acids (HDAC6NT-allD) specifically in the hearts. Overexpression of wild-type HDAC6 significantly reduced acetylated tubulin levels, and overexpression of HDAC6H216A, H611A significantly increased it in the mouse hearts. Detectable acetylated tubulin disappeared in HDAC6NT-allD TG mouse hearts. Neither histological alteration nor alteration of cardiac function was observed in the HDAC6 TG mouse hearts. To analyze the role of HDAC6 and acetylated tubulin in disease conditions, we examined HDAC6 in isoprenaline-induced hypertrophy or pressure-overload hypertrophy (TAC). No obvious alteration in the heart weight/body weight ratio or gene expressions of hypertrophic markers between NTG and HDAC6NT-allD mice was observed following treatment with isoprenaline. In contrast, a marked reduction in the shortening fraction and dilated chamber dilatation was detected in the HDAC6NT-allD TG mouse hearts 2 weeks after TAC. A sustained low level of acetylated tubulin and acetylated cortactin was observed in the TAC HDAC6NT-allD TG mouse hearts. Cardiac HDAC6 activity that can regulate acetylated levels of tubulin and cortactin may be critical factors involved in cardiac disease such as pressure-overload hypertrophy.

Autophagic LC3+ calcified extracellular vesicles initiate cartilage calcification in osteoarthritis.

Pathological cartilage calcification plays an important role in osteoarthritis progression but in which the origin of calcified extracellular vesicles (EVs) and their effects remain unknown. Here, we demonstrate that pathological cartilage calcification occurs in the early stage of the osteoarthritis in which the calcified EVs are closely involved. Autophagosomes carrying the minerals are released in EVs, and calcification is induced by those autophagy-regulated calcified EVs. Autophagy-derived microtubule-associated proteins 1A/1B light chain 3B (LC3)–positive EVs are the major population of calcified EVs that initiate pathological calcification. Release of LC3-positive calcified EVs is caused by blockage of the autophagy flux resulted from histone deacetylase 6 (HDAC6)–mediated microtubule destabilization. Inhibition of HDAC6 activity blocks the release of the LC3-positive calcified EVs by chondrocytes and effectively reverses the pathological calcification and degradation of cartilage. The present work discovers that calcified EVs derived from autophagosomes initiate pathological cartilage calcification in osteoarthritis, with potential therapeutic targeting implication.

Microtubule depolymerization limits porcine betacoronavirus PHEV replication

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a typical neurotropic betacoronavirus causing digestive disease and/or neurological dysfunction in neonatal pigs. Actin filaments have been identified to implicate in PHEV invasion, but the effects of viral infection on microtubules (MTs) cytoskeleton are unknown. Here, we observed that PHEV infection induced MT depolymerization and was accompanied by the disappearance of microtubule organizing centers. Depolymerization of MTs induced by nocodazole significantly inhibited viral RNA replication, but over-polymerization of MTs induced by paclitaxel did not substantially affect PHEV infection. The expression of histone deacetylase 6 (HDAC6), an important regulator of MT acetylation, progressively increased during PHEV infection. Tramstatin A could alter HDAC6 deacetylase activity to enhance the acetylation of the substrate α-tubulin and MT polymerization, but does not increase PHEV proliferation. These findings suggest that PHEV could subvert host MT cytoskeleton to facilitate infection, and that MT depolymerization negatively affects viral replication independently of HDAC6 activity.

Ricolinostat enhances adavosertib‑induced mitotic catastrophe in TP53‑mutated head and neck squamous cell carcinoma cells.

TP53 mutation is one of the most frequent gene mutations in head and neck squamous cell carcinoma (HNSCC) and could be a potential therapeutic target. Recently, the WEE1 G2 checkpoint kinase (WEE1) inhibitor adavosertib (Adv) has attracted attention because of its selective cytotoxicity against TP53-mutated cells and has shown promising activity in early phase clinical trials. In the present study, it was demonstrated that combined treatment with Adv and a selective histone deacetylase 6 (HDAC6) inhibitor, ricolinostat (RCS), synergistically enhanced cell death induction in four out of five HNSCC cell lines with TP53 mutation (CAL27, SAS, HSC-3, and OSC-19), one HNSCC cell line with impaired TP53 function by HPV-infection (UPCI-SCC154), and TP53-knockout human lung cancer cell line (A549 TP53-KO), but not in TP53 wild-type A549 cells. Time-lapse imaging showed that RCS enhanced the Adv-induced mitotic catastrophe. Consistent with this, RCS treatment suppressed checkpoint kinase 1 (Chk1) (Ser345) phosphorylation and co-administration of RCS with Adv suppressed cyclin-dependent kinase 1 (Tyr15) phosphorylation along with increased expression of γ-H2A.X, a marker of DNA double-strand breaks in CAL27 cells. These data showed that RCS enhanced Adv-induced premature mitotic entry and cell death induction in the mitotic phase. However, although HDAC6 knockdown enhanced Adv-induced cell death with γ-H2A.X elevation, HDAC6 knockdown did not repress Chk1 phosphorylation in CAL27 cells. Our data demonstrated that the co-administration of RCS with Adv in HNSCC cells resulted in the suppression of Chk1 activity, leading to synergistically enhanced apoptosis via mitotic catastrophe in a p53-dependent manner. This enhanced cell death appeared to be partially mediated by the inhibition of HDAC6 activity by RCS.