Specific Histone Deacetylase Inhibitor Research Articles

Single cell proteomics by mass spectrometry reveals deep epigenetic insight into the actions of an orphan histone deacetylase inhibitor.

Epigenetic programming has been shown to play a role in nearly every human system and disease where anyone has thought to look. However, the levels of heterogeneity at which epigenetic or epiproteomic modifications occur at single cell resolution across a population remains elusive. While recent advances in sequencing technology have allowed between 1 and 3 histone post-translational modifications to be analyzed in each single cell, over twenty separate chemical PTMs are known to exist, allowing thousands of possible combinations. Single cell proteomics by mass spectrometry (SCP) is an emerging technology in which hundreds or thousands of proteins can be directly quantified in typical human cells. As the proteins detected and quantified by SCP are heavily biased toward proteins of highest abundance, chromatin proteins are an attractive target for analysis. To this end, I applied SCP to the analysis of cancer cells treated with mocetinostat, a class specific histone deacetylase inhibitor. I find that 16 PTMs can be confidently identified and localized with high site specificity in single cells. In addition, the high abundance of histone proteins allows higher throughput methods to be utilized for SCP than ever described. While quantitative accuracy suffers when analyzing more than 700 cells per day, 9 histone proteins can be measured in single cells analyzed at even 3,500 cells per day, a throughput 10-fold greater than any previous report. In addition, the unbiased global approach utilized herein identifies a previously uncharacterized response to this drug through the S100-A8/S100-A9 protein complex partners. This response is observed in nearly every cell of the over 1,000 analyzed in this study, regardless of the relative throughput of the method utilized. While limitations exist in the methods described herein, current technologies can easily improve upon the results presented here to allow comprehensive analysis of histone PTMs to be performed in any mass spectrometry lab. All raw and processed data described in this study has been made publicly available through the ProteomeXchange/MASSIVE repository system as MSV000093434.

Abstract 1421: The HDAC6-specific inhibitor AVS100 (SS208) blocks M2 polarization of tumor associated macrophages and potentiates immunotherapy in preclinical tumor models

Abstract Inhibition of HDAC6 was associated with an increased proinflammatory tumor microenvironment and an antitumoral response. Here, we show that a highly specific HDAC6 inhibitor AVS100 (SS208), blocks M2 polarization in murine and human macrophages while partially affecting M1 polarization. AVS100 effects were observed as blocked upregulation of M2-related gene signature under M2 polarizing conditions and blocked generation of CD206+ and Arg1+ macrophages. Oral administration of AVS100 had an antitumoral effect in SM1 melanoma and CT26 colon cancer models and increased the efficacy of anti-PD1 treatment, leading to complete remission in melanoma and increased response in colon cancer. Flow cytometry and scRNAseq analysis of tumor-infiltrating immune cells revealed an increase of proinflammatory/anti-inflammatory ratio in tumor-associated macrophages as well as an increase of intratumoral CD8 effector T-cells after AVS100 treatment. Interestingly, cured mice didn’t relapse and became resistant to a subsequent tumor challenge, suggesting acquired antitumoral T-cell immunity. T-cell repertoire analysis of effector/memory T-cells in cured mice revealed a higher number of immunodominant T-cell clones after AVS100 treatment, indicating increased T-cell expansion. Finally, AVS100 has demonstrated no mutagenicity and a strong safety profile in rats and dogs, leading to its recent U.S. FDA clearance of an Investigational New Drug (IND) application and planned initiation of Phase Ia/b clinical trials targeting locally advanced or metastatic solid tumors in the first half of 2024. Altogether, we have performed the preclinical characterization of a novel small molecule inhibitor targeting HDAC6 for solid cancers. AVS100 had an antitumoral effect as single agent and improved the efficacy of immune checkpoint inhibition by blocking the immunoregulatory tumor microenvironment and increasing T-cell immunity. Citation Format: Damian Kovalovsky, Satish Noonepalle, Manasa Suresh, Dileep Kumar, Michael Berrigan, Anelia Horvath, Allen Kim, David Quiceno-Torres, Karthik Musunuri, Alejandro Villagra. The HDAC6-specific inhibitor AVS100 (SS208) blocks M2 polarization of tumor associated macrophages and potentiates immunotherapy in preclinical tumor models [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 1421.

Spinal HDAC6 mediates nociceptive behaviors induced by chronic constriction injury via neuronal activation and neuroinflammation.

Neuropathic pain (NP) is often accompanied by psychiatric comorbidities and currently lacks effective treatment. Prior research has shown that HDAC6 plays a crucial role in pain sensitization, but the specific mechanisms remain unclear. HDAC6 inhibitors have been found to alleviate mechanical allodynia caused by inflammation and peripheral nerve damage. In this study, we investigated the cellular mechanisms of HDAC6 in the development and maintenance of neuropathic pain. Our findings indicate that HDAC6 expression in the spinal cord (SC) is upregulated in a time-dependent manner following chronic constriction injury (CCI). HDAC6 is primarily expressed in neurons and microglia in the spinal cord. CCI-induced HDAC6 production was abolished by intrathecal injection of a microglia inhibitor. ACY-1215, a specific HDAC6 inhibitor, significantly reduced CCI-induced mechanical allodynia, but not thermal hyperalgesia. ACY-1215 also inhibited neuron activation and suppressed CCI-induced pyroptosis and neuroinflammatory responses. In summary, our results suggest that HDAC6 contributes to the development and maintenance of NP through neuronal activation and neuroinflammation. HDAC6 may be a promising target for treating NP.

Epigenetic Modulation Enhances the Therapeutic Potential of All-Trans Retinoic Acid in Acute Myeloid Leukemia

Epigenetic dysregulation has been strongly associated with the development and progression of acute myeloid leukemia (AML). While all-trans retinoic acid (ATRA), a derivative of vitamin A, has demonstrated significant anti-cancer effects in acute promyelocytic leukemia (APL-AML), its clinical efficacy in non-APL AMLs has been limited. This limited response can be attributed, in part, to the epigenetic silencing of genes targeted by or involved in the ATRA pathway, rendering them unresponsive to the drug. Consequently, our research aims to overcome the epigenetic barriers in AML by combining ATRA with small molecules that possess epigenetic modulating properties. To investigate the activation of the retinoic acid pathway in response to ATRA and a library of over 650 small molecules with epigenetic activity, we transduced HEK-293 cells with the p-GreenFire-RARE-Tk-Luc reporter construct. Through the screening process, we have identified 11 compounds that show promise as potential candidates for combination therapy with ATRA in non-APL AML. Notably, when combined with ATRA, two specific compounds, a dual PI3K HDAC inhibitor, and a pan-PKC inhibitor, demonstrated a significant 4-5 fold increase in RARE activity, indicating a synergistic effect. To determine the impact of selected compounds on the functional level, we evaluated the viability, differentiation, and phosphorylation status of ERK1/2, AKT1, MAPK9, p38, and MAPK9 in various cell lines, including HL-60, KG1a, BMNC, and primary MSC and AML cells. The dual PI3K HDAC inhibitor and a pan-PKC inhibitor were noncytotoxic to HL60, BMNC, and primary MSC. Interestingly, dual PI3K HDAC inhibitor with and without ATRA reduced the viability of KG1α by more than 95%. Moreover, dual PI3K HDAC inhibitor and a pan-PKC inhibitor, when combined with ATRA, lead to a 2-3 fold increase in the differentiation of HL60 cells and a 2-4 fold increase in the differentiation of AML cells ex vivo compared to ATRA in combination with the LSD1 inhibitor TCP (treatment control protocol) and untreated cells from AML subtypes M1, M2, and M4. PI3K HDAC inhibitor combined with ATRA significantly reduced p-MAPK and p-p38 levels while increasing the phosphorylation of p53 in HL-60 cells. Furthermore, RNA sequencing and chromatin profiling by Cleavage Under Targets and Tagmentation (CUT&Tag) were performed to gain additional insights into the molecular mechanisms involved. Based on obtained results, the combination of ATRA with specific dual PI3K HDAC and pan-PKC inhibitors holds promise as a potential therapeutic approach for AML. Nevertheless, further investigations, including studies utilizing patient-derived xenograft models, are warranted to validate the efficacy and feasibility of this proposed treatment strategy.

Paxillin/HDAC6 regulates microtubule acetylation to promote directional migration of keratinocytes driven by electric fields

Endogenous electric fields (EFs) have been demonstrated to facilitate wound healing by directing the migration of epidermal cells. Despite the identification of numerous molecules and signaling pathways that are crucial for the directional migration of keratinocytes under EFs, the underlying molecular mechanisms remain undefined. Previous studies have indicated that microtubule (MT) acetylation is linked to cell migration, while Paxillin exerts a significant influence on cell motility. Therefore, we postulated that Paxillin could enhance EF-induced directional migration of keratinocytes by modulating MT acetylation. In the present study, we observed that EFs (200 mV/mm) induced migration of human immortalized epidermal cells (HaCaT) towards the anode, while upregulating Paxillin, downregulating HDAC6, and increasing the level of microtubule acetylation. Our findings suggested that Paxillin plays a pivotal role in inhibiting HDAC6-mediated microtubule acetylation during directional migration under EF regulation. Conversely, downregulation of Paxillin decreased microtubule acetylation and electrotaxis of epidermal cells by promoting HDAC6 expression, and this effect could be reversed by the addition of tubacin, an HDAC6-specific inhibitor. Furthermore, we observed that EFs also mediated the polarization of Paxillin and acetylated α-tubulin, which is critical for directional migration. In conclusion, our study revealed that MT acetylation in EF-guided keratinocyte migration is regulated by the Paxillin/HDAC6 signaling pathway, providing a novel theoretical foundation for the molecular mechanism of EF-guided directional migration of keratinocytes.

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.

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.

Abstract 6235: Discovery of novel HDAC6 inhibitors that enhance proteasomal activity to boost antigen presentation and trigger anti-myeloma T-cell immunity

Abstract Transformative success of cancer immunotherapy has revolutionized cancer treatment and can induce long lasting responses in patients with cancers from a wide range of histologies. However, only a fraction of patients respond to immunotherapy and cancer cells escape immunosurveillance through molecular mechanisms that remain elusive. Proteasomes play a central role in the immune response by generating peptides from intracellular antigens which are presented on the cell surface for recognition by CD8+ cytotoxic T lymphocytes (CTLs). Malignant cells employ strategies to downregulate antigen presentation and impair CTL-mediated tumor recognition and lysis. Recently, we discovered that HDAC6-specific inhibitors increased intracellular proteasome activity and the generation of antigenic class I MHC peptides. However, current HDAC6 inhibitors have pharmacologic liabilities that limit efficacy, e.g., low potency and solubility, poor pharmacokinetic properties and potential genotoxicity. Therefore, we performed a cell-based, high-throughput screen (HTS) of 9,600 HDAC-specific compounds to identify novel compounds that increased proteasomal activity in myeloma cells. We identified 8 hits that increased proteasomal activity at least two-fold. Importantly, two novel molecules identified in the HTS increased proteasome activity more potently and more rapidly than the HDAC6-specific inhibitors Tubastatin-A and ACY-1215 (Ricolinostat) with comparatively low cytotoxicity. Proteasomes degrade chicken ovalbumin (Ova) to generate the immunodominant antigen SIINFEKL presented at the cell surface complexed with the MHC-class I molecule H-2Kb. Treatment of tumor cells with Tubastatin-A and ACY-1215 increased levels of the MHC-class I-SIINFEKL complex by ~3-fold. Tumor cells were pretreated with Tubastatin-A and ACY-1215 and then co-cultured with CTLs that had been genetically-engineered to express a SIINFEKL-restricted T-cell receptor (TCR). Pre-treatment of lymphoma or multiple myeloma cells with novel HDAC6 inhibitors also increased tumor lysis by CTLs that expressed the SIINFEKL-restricted TCR by 2-fold. Taken together, our results support repositioning pharmacologics that inhibit HDAC6 to activate proteasomal generation of antigenic peptides and boost antigen presentation as cancer immunotherapeutics. Citation Format: Priyanka S. Rana, James Ignatz-Hoover, James J. Driscoll. Discovery of novel HDAC6 inhibitors that enhance proteasomal activity to boost antigen presentation and trigger anti-myeloma T-cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6235.

Protective role and mechanism of tubastatin A on renal and intestinal injuries after cardiopulmonary resuscitation in swine

To investigate the protective effect and potential mechanism of tubastatin A (TubA), a specific inhibitor of histone deacetylase 6 (HDAC6), on renal and intestinal injuries after cardiopulmonary resuscitation (CPR) in swine. Twenty-five healthy male white swine were divided into Sham group (n = 6), CPR model group (n = 10) and TubA intervention group (n = 9) using a random number table. The porcine model of CPR was reproduced by 9-minute cardiac arrest induced by electrical stimulation via right ventricle followed by 6-minute CPR. The animals in the Sham group only underwent the regular operation including endotracheal intubation, catheterization, and anesthetic monitoring. At 5 minutes after successful resuscitation, a dose of 4.5 mg/kg of TubA was infused via the femoral vein within 1 hour in the TubA intervention group. The same volume of normal saline was infused in the Sham and CPR model groups. Venous samples were collected before modeling and 1, 2, 4, 24 hours after resuscitation, and the levels of serum creatinine (SCr), blood urea nitrogen (BUN), intestinal fatty acid binding protein (I-FABP) and diamine oxidase (DAO) in serum were determined by enzyme-linked immunoadsordent assay (ELISA). At 24 hours after resuscitation, the upper pole of left kidney and terminal ileum were harvested to detect cell apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL), and the expression levels of receptor-interacting protein 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) were detected by Western blotting. After resuscitation, renal dysfunction and intestinal mucous injury were observed in the CPR model and TubA intervention groups when compared with the Sham group, which was indicated by significantly increased levels of SCr, BUN, I-FABP and DAO in serum. However, the serum levels of SCr and DAO starting 1 hour after resuscitation, the serum levels of BUN starting 2 hours after resuscitation, and the serum levels of I-FABP starting 4 hours after resuscitation were significantly decreased in the TubA intervention group when compared with the CPR model group [1-hour SCr (μmol/L): 87±6 vs. 122±7, 1-hour DAO (kU/L): 8.1±1.2 vs. 10.3±0.8, 2-hour BUN (mmol/L): 12.3±1.2 vs. 14.7±1.3, 4-hour I-FABP (ng/L): 661±39 vs. 751±38, all P < 0.05]. The detection of tissue samples indicated that cell apoptosis and necroptosis in the kidney and intestine at 24 hours after resuscitation were significantly greater in the CPR model and TubA intervention groups when compared with the Sham group, which were indicated by significantly increased apoptotic index and markedly elevated expression levels of RIP3 and MLKL. Nevertheless, compared with the CPR model group, renal and intestinal apoptotic indexes at 24 hours after resuscitation in the TubA intervention group were significantly decreased [renal apoptosis index: (21.4±4.6)% vs. (55.2±9.5)%, intestinal apoptosis index: (21.3±4.5)% vs. (50.9±7.0)%, both P < 0.05], and the expression levels of RIP3 and MLKL were significantly reduced [renal tissue: RIP3 protein (RIP3/GAPDH) was 1.11±0.07 vs. 1.39±0.17, MLKL protein (MLKL/GAPDH) was 1.20±0.14 vs. 1.51±0.26; intestinal tissue: RIP3 protein (RIP3/GAPDH) was 1.24±0.18 vs. 1.69±0.28, MLKL protein (MLKL/GAPDH) was 1.38±0.15 vs. 1.80±0.26, all P < 0.05]. TubA has the protective effect on alleviating post-resuscitation renal dysfunction and intestinal mucous injury, and its mechanism may be related to inhibition of cell apoptosis and necroptosis.

Protective effects of histone deacetylase 6 specific inhibitor tubastatin A on subarachnoid hemorrhage in rats and the underlying mechanisms.

Subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease. Early brain injury (EBI) and cerebral vasospasm are the main reasons for poor prognosis of SAH patients. The specific inhibitor of histone deacetylase 6 (HDAC6), tubastatin A (TubA), has been proved to have a definite neuroprotective effect on a variety of animal models of acute and chronic central nervous system diseases. However, the neuroprotective effect of TubA on SAH remains unclear. This study aims to investigate the expression and localization of HDAC6 in the early stage of SAH, and to evaluate the protective effects of TubA on EBI and cerebral vasospasm after SAH and the underlying mechanisms. Adult male SD rats were treated with modified internal carotid artery puncture to establish SAH model. In the first part of the experiment, rats were randomly divided into 6 groups: a sham group, a SAH-3 h group, a SAH-6 h group, a SAH-12 h group, a SAH-24 h group, and a SAH-48 h group. At 3, 6, 12, and 24 h after SAH modeling, the injured cerebral cortex of rats in each group was taken for Western blotting to detect the expression of HDAC6. In addition, the distribution of HDAC6 in the cerebral cortex of the injured side was measured by immunofluorescence double staining in SAH-24 h group rats. In the second part, rats were randomly divided into 4 groups: a sham group, a SAH group, a SAH+TubAL group (giving 25 mg/kg TubA), and a SAH+TubAH group (giving 40 mg/kg TubA). At 24 h after modeling, the injured cerebral cortex tissue was taken for Western blotting to detect the expression levels of HDAC6, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining to detect apoptosis, and hematoxylin and eosin (HE) staining to detect the diameter of middle cerebral artery. The protein expression of HDAC6 began to increase at 6 h after SAH (P<0.05), peaked at 24 h (P<0.001), and decreased at 48 h, but there was still a difference compared with the sham group (P<0.05). HDAC6 is mainly expressed in the cytoplasm of the neurons. Compared with the sham group, the neurological score was decreased significantly and brain water content was increased significantly in the SAH group (both P<0.01). Compared with the SAH group, the neurological score was increased significantly and brain water content was decreased significantly in the SAH+TubAH group (both P<0.05), while the improvement of the above indexes was not significant in the SAH+TubAL group (both P>0.05). Compared with the sham group, the expression of eNOS was significantly decreased (P<0.01) and the expressions of iNOS and HDAC6 were significantly increased (P<0.05 and P<0.01, respectively) in the SAH group. Compared with the SAH group, the expression of eNOS was significantly increased, and iNOS and HDAC6 were significantly decreased in the SAH+TubA group (all P<0.05). Compared with the SAH group, the number of TUNEL positive cells was significantly decreased and the diameter of middle cerebral artery was significantly increased in the SAH+TubA group (both P<0.05) . HDAC6 is mainly expressed in neurons and is up-regulated in the cerebral cortex at the early stage of SAH. TubA has protective effects on EBI and cerebral vasospasm in SAH rats by reducing brain edema and cell apoptosis in the early stage of SAH. In addition, its effect of reducing cerebral vasospasm may be related to regulating the expression of eNOS and iNOS.

The Class IIA Histone Deacetylase (HDAC) Inhibitor TMP269 Downregulates Ribosomal Proteins and Has Anti-Proliferative and Pro-Apoptotic Effects on AML Cells.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by altered myeloid progenitor cell proliferation and differentiation. As in many other cancers, epigenetic transcriptional repressors such as histone deacetylases (HDACs) are dysregulated in AML. Here, we investigated (1) HDAC gene expression in AML patients and in different AML cell lines and (2) the effect of treating AML cells with the specific class IIA HDAC inhibitor TMP269, by applying proteomic and comparative bioinformatic analyses. We also analyzed cell proliferation, apoptosis, and the cell-killing capacities of TMP269 in combination with venetoclax compared to azacitidine plus venetoclax, by flow cytometry. Our results demonstrate significantly overexpressed class I and class II HDAC genes in AML patients, a phenotype which is conserved in AML cell lines. In AML MOLM-13 cells, TMP269 treatment downregulated a set of ribosomal proteins which are overexpressed in AML patients at the transcriptional level. TMP269 showed anti-proliferative effects and induced additive apoptotic effects in combination with venetoclax. We conclude that TMP269 exerts anti-leukemic activity when combined with venetoclax and has potential as a therapeutic drug in AML.

Histone deacetylase 6 plays an important role in TGF-β-induced murine Treg cell differentiation by regulating cell proliferation

Regulatory T (Treg) cells maintain immune homeostasis by preventing abnormal or excessive immune responses. Histone deacetylase 6 (HDAC6) regulates expression of Foxp3, and thus, Treg cell differentiation; however, its role in Treg cell differentiation is unclear and somewhat controversial. Here, we investigated the role of HDAC6 in TGF-β-induced murine Treg cells. HDAC6 expression was higher in Treg cells than in other T helper cell subsets. Pharmacological inhibitors of HDAC6 selectively inhibited Treg cell differentiation and suppressive function. A specific HDAC6 inhibitor induced changes in global gene expression by Treg cells. Of these changes, genes related to cell division were prominently affected. In summary, HDAC6 plays an important role in TGF-β-induced murine Treg cell differentiation by regulating cell proliferation.

Development of a Cellular Model Mimicking Specific HDAC Inhibitors.

Class I histone deacetylases (HDACs) are important regulators of cellular functions in health and disease. HDAC1, HDAC2, HDAC3, and HDAC8 are promising targets for the treatment of cancer, neurological, and immunological disorders. These enzymes have both catalytic and non-catalytic functions in the regulation of gene expression. We here describe the generation of a genetic toolbox by the CRISPR/Cas9 methodology in nearly haploid human tumor cells. This novel model system allows to discriminate between catalytic and structural functions of class I HDAC enzymes and to mimic the treatment with specific HDAC inhibitors.

Expression and Crystallization of HDAC6 Tandem Catalytic Domains.

Histone deacetylase 6 (HDAC6) is an atypical lysine deacetylase with tandem catalytic domains and an ubiquitin-binding zinc finger domain. HDAC6 is involved in various biological processes, such as cell motility or stress responses, and has been implicated in pathologies ranging from cancer to neurodegeneration. Due to this broad range of functions, there has been considerable interest in developing HDAC6-specific small molecule inhibitors, several of which are already available. The crystal structure of the tandem catalytic domains of zebrafish HDAC6 has revealed an arrangement with twofold symmetry and extensive surface interaction between the catalytic domains. Further dissection of the biochemical properties of HDAC6 and the development of novel inhibitors will benefit from being able to routinely express high-quality protein. We present here our optimized protocol for expression and crystallization of the zebrafish tandem catalytic domains.

Analyzing Lymphoma Development and Progression Using HDACi in Mouse Models.

Besides the physiological role of histone deacetalylases in maintaining normal cellular integrity, the acetylation landscape is changed in cancer cells, which has been implicated as a potential target in cancer therapy. The overexpression of certain HDACs correlates with specific cancer types. Therefore, the development of specific HDAC inhibitors may extend the therapeutic strategy for cancer therapy. Here, we describe how to investigate the therapeutic potential of specific HDACi by treatment in a mouse model for B-cell lymphoma, exemplified by the HDAC6 inhibitor Marbostat-100.

Targeting HDAC6 to Overcome Autophagy-Promoted Anti-Cancer Drug Resistance

Histone deacetylases (HDACs) regulate gene expression through the epigenetic modification of chromatin structure. HDAC6, unlike many other HDACs, is present in the cytoplasm. Its deacetylates non-histone proteins and plays diverse roles in cancer cell initiation, proliferation, autophagy, and anti-cancer drug resistance. The development of HDAC6-specific inhibitors has been relatively successful. Mechanisms of HDAC6-promoted anti-cancer drug resistance, cancer cell proliferation, and autophagy are discussed. The relationship between autophagy and anti-cancer drug resistance is discussed. The effects of combination therapy, which includes HDAC6 inhibitors, on the sensitivity of cancer cells to chemotherapeutics and immune checkpoint blockade are presented. A summary of clinical trials involving HDAC6-specific inhibitors is also presented. This review presents HDAC6 as a valuable target for developing anti-cancer drugs.

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation.

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.

Inhibition of HDAC6 With CAY10603 Ameliorates Diabetic Kidney Disease by Suppressing NLRP3 Inflammasome.

Background: Diabetic nephropathy (DN) is one of the leading causes of chronic kidney disease (CKD) worldwide, tubular injury is the driving force during the pathogenesis and progression of DN. Thus, we aim to utilize the connectivity map (CMap) with renal tubulointerstitial transcriptomic profiles of biopsy-proven DN to identify novel drugs for treating DN. Methods: We interrogated the CMap profile with tubulointerstitial transcriptomic data from renal biopsy-proven early- and late-stage DN patients to screen potential drugs for DN. Therapeutic effects of candidate drug were assessed in Murine model of diabetic kidney disease (STZ-induced CD-1 mice), and HK-2 cells and immortalized bone marrow-derived macrophages (iBMDMs). Results: We identified CAY10603, a specific inhibitor of histone deacetylase 6 (HDAC6), as a potential drug that could significantly reverse the altered genes in the tubulointerstitial component. In DN patients and mice, upregulation of HDAC6 was mainly observed in renal tubular cells and infiltrated macrophages surrounding the diluted tubules. In both early- and late-onset diabetic mice, daily CAY10603 administration effectively alleviated renal dysfunction and reduced macrophage infiltration, tubular injury and tubulointerstitial fibrosis. Mechanistically, CAY10603 suppressed NLRP3 activation in both HK-2 cells and iBMDMs. Conclusion: CAY10603 exhibited therapeutic potential for DN by suppressing NLRP3 inflammasome activation in both tubular cells and macrophages.

Pharmacological inhibition of HDAC6 overcomes cisplatin chemoresistance by targeting cancer stem cells in oral squamous cell carcinoma.

Chemoresistance is associated with recurrence and metastasis in oral squamous cell carcinoma (OSCC). The cancer stem cell (CSC) subpopulation is highly resistant to therapy, and they are regulated by epigenetic mechanisms. HDACs are histone deacetylase enzymes that epigenetically regulate gene expression. HDAC6 acts on several physiological processes, including oxidative stress, autophagy and DNA damage response, and its accumulation is associated with cancer. Here, we investigate the role of HDAC6 in CSC-mediated chemoresistance in oral carcinoma in addition to its application as a therapeutic target to reverse chemoresistance. Wild-type oral carcinoma cell lines (CAL27 WT and SCC9 WT), cisplatin-resistant (CAL27 CisR and SCC9 CisR), and the subpopulations of cancer stem cells (CSC+) and non-stem (CSC-) derived from CisR cells were investigated. HDAC6 accumulation was analyzed by Western blot and immunofluorescence; DNA damage was evaluated by immunofluorescence of phospho-H2A.X; the qPCR for PRDX2, PRDX6, SOD2, and TXN and ROS assay assessed oxidative stress. Apoptosis and CSC accumulation were investigated by flow cytometry. We identified the accumulation of HDAC6 in CisR cell lines and CSC. Cisplatin-resistant cell lines and CSC demonstrated a reduction in DNA damage and ROS and elevated expression of PRDX2. The administration of tubastatin A (a specific HDAC6 inhibitor) increased oxidative stress and DNA damage and decreased PRDX2. Tubastatin A as a monotherapy induced apoptosis in CisR and CSC and reduced the stemness phenotype. High levels of HDAC6 sustain CSC subpopulation and chemoresistance in OSCC, suggesting HDAC6 as a pharmacological target to overcome resistance and perhaps prevent recurrence in OSCC.

HDAC6-specific inhibitor alleviates hashimoto's thyroiditis through inhibition of Th17 cell differentiation

ObjectiveHashimoto's thyroiditis (HT) is one of the commonest autoimmune disorders. This study was performed to investigate the potential effect of histone deacetylase 6-specific inhibitor (HDAC6i) on Th17 cell differentiation in animal model and the underlying mechanism. MethodsExperimental autoimmune thyroiditis (EAT) mouse model was established by subcutaneously immunization of porcine thyroglobulin (pTg) and adjuvant, and the HDACi Tubastatin A (TSA) or HDAC6i (ACY-1215) was intraperitoneally injected into mice in the following. The histological examination and immune analysis in EAT mice were carried out. Next, the CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of EAT mice followed by Th17 cell differentiation assay. The associated factor levels, and the protein interaction between HDAC6 and PKM2 were examined. Subsequently, the effect of STAT3 activation on Th17 cell differentiation was explored. ResultsACY-1215 or TSA treatment reduced lymphocytic infiltration and alleviated thyroid tissue injury in EAT mice. Correspondingly, either ACY-1215 or TSA treatment reduced the levels of anti-thyroglobulin (Tg), anti-thyroid peroxidase (TPO), IL-17A, and IFN-γ in the serum, decreased Th17 cell differentiation, but enhanced TGF-β level and promoted Treg cell differentiation. In vitro, after induction of Th17 cell differentiation from CD4+ T cells, HDAC6 activity and Th17 cell differentiation were significantly decreased when treated with ACY-1215 or TSA. HDAC6 could interact with PKM2, and HDAC6 overexpression promoted the phosphorylation of STAT3 and PKM2 nuclear translocation. Furthermore, the STAT3 activator treatment reversed the effects of ACY-1215 or TSA treatment. ConclusionHDAC6i suppresses Th17 cell differentiation and attenuates HT via PKM2/STAT3 axis.