Class IIa HDACs Research Articles

Discovery of novel tubulin/HDAC dual-targeting inhibitors with strong antitumor and antiangiogenic potency

A novel series of cis-diphenylethene and benzophenone derivatives as tubulin/HDAC dual-targeting inhibitors were designed and synthesized. Among them, compound 28g exhibited the most potent antiproliferative activities against six different human cancer cell lines, 28g could not only inhibited tubulin polymerization, disrupted cellular microtubule networks but also selectively inhibited class IIa HDACs, especially HDAC7 activity. Further molecular docking demonstrated 28g could occupy the binding pockets of tubulin and HDAC7 meanwhile. Cellular mechanism studies revealed that 28g could induce G2/M phase arrest by down-regulated expression of p-cdc2 and cell apoptosis by regulating mitochondrial membrane potential, reactive oxygen species (ROS) levels and apoptosis-related proteins (PARP, Caspase families) in a dose-dependent manner. Importantly, 28g significantly inhibited HUVEC tube formation, proliferation, migration and invasion. The inhibitory effect against angiogenesis invivo was confirmed by zebrafish xenograft. Furthermore, 28g could effectively suppress the proliferation and metastasis of MGC-803cells invitro and in zebrafish xenograft. All above results indicated that 28g can act as a promising antitumor and antiangiogenic agent via targeting tubulin and class IIa HDACs.

Histone deacetylase 4 deletion broadly affects cardiac epigenetic repression and regulates transcriptional susceptibility via H3K9 methylation

Histone deacetylase 4 (HDAC4) is a member of class IIa histone deacetylases (class IIa HDACs) and is believed to possess a low intrinsic deacetylase activity. However, HDAC4 sufficiently represses distinct transcription factors (TFs) such as the myocyte enhancer factor 2 (MEF2). Transcriptional repression by HDAC4 has been suggested to be mediated by the recruitment of other chromatin-modifying enzymes, such as methyltransferases or class I histone deacetylases. However, this concept has not been investigated by an unbiased approach.Therefore, we studied the histone modifications H3K4me3, H3K9ac, H3K27ac, H3K9me2 and H3K27me3 in a genome-wide approach using HDAC4-deficient cardiomyocytes. We identified a general epigenetic shift from a ‘repressive’ to an ‘active’ status, characterized by an increase of H3K4me3, H3K9ac and H3K27ac and a decrease of H3K9me2 and H3K27me3.In HDAC4-deficient cardiomyocytes, MEF2 binding sites were considerably overrepresented in upregulated promoter regions of H3K9ac and H3K4me3. For example, we identified the promoter of Adprhl1 as a new genomic target of HDAC4 and MEF2. Overexpression of HDAC4 in cardiomyocytes was able to repress the transcription of the Adprhl1 promoter in the presence of the methyltransferase SUV39H1. On a genome-wide level, the decrease of H3K9 methylation did not change baseline expression but was associated with exercise-induced gene expression.We conclude that HDAC4, on the one hand, associates with activating histone modifications, such as H3K4me3 and H3K9ac. A functional consequence, on the other hand, requires an indirect regulation of H3K9me2. H3K9 hypomethylation in HDAC4 target genes (‘first hit’) plus a ‘second hit’ (e.g., exercise) determines the transcriptional response.

Abstract 3096: A novel ZIP4-NOTCH3-HDAC4 axis in ovarian cancer stem cells

Abstract High grade serous ovarian cancer (HGSOC) is one of the most deadly and heterogenic cancers. We have recently shown that ZIP4 (gene name SLC39A4), a zinc transporter, is a novel cancer stem cell (CSC) marker in HGSOC. 100-200 ZIP4+, but not ZIP4-, cells from both PE04 and PEA2 cells formed larger tumors than those from 100-200 ALDH+ cells in mice. Mechanistically, we found that ZIP4 was an upstream regulator of another CSC-marker, NOTCH3, in HGSOC cells. NOTCH3 was functionally involved in spheroid formation in vitro and tumorigenesis in vivo in HGSOC. Drug-resistance is one of the main characteristics of CSCs, While ZIP4 converts drug-resistance to cisplatin (CDDP) and doxorubicin (DOX) as we reported previously, we unexpectedly found that ZIP4 induced a sensitization of HGSOC cells to histone deacetylase inhibitors (HDACis). In particular, only those HDACis against the Class IIa HDACs showed ZIP4-dependent sensitization. ZIP4 selectively up-regulated HDAC IIa HDACs, including HDAC4 and 5, with little or no effects to HDACs in other classes. ZIP4 knockout (KO) and HDAC4 knockdown (KD) increased cell resistance to LMK-235, a selective HDAC4/5 inhibitor. LMK-235 and HDAC4 knockdown (KD) inhibited spheroid formation in vitro and tumor development in vivo. Collectively, we revealed a novel ZIP4-NOTCH3-HDAC4 axis, which is functionally involved and important in CSC-related activities in vitro and tumorigenesis in vivo, and provide an innovative targeting strategy to CSC. Citation Format: Yan Xu, Qipeng Fan, Robert Emerson. A novel ZIP4-NOTCH3-HDAC4 axis in ovarian cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3096.

Phase 1 study of OKI-179, an oral class 1-selective depsipeptide HDAC inhibitor, in patients with advanced solid tumors: Final results.

3075 Background: OKI-179 is a novel, oral pro-drug analog of largazole, a compound in the romidepsin-depsipeptide class of natural products. OKI-006, the active metabolite of OKI-179, inhibits HDAC 1,2,3 (IC50 = 1.2, 2.4, 2.0 nM, respectively), with no significant inhibition of Class IIa HDACs and has shown promising activity in preclinical models of solid tumors. We conducted a first-in-human dose escalation study of OKI-179 in patients with advanced solid tumors. Methods: Patients with advanced solid tumors, ECOG ≤1, normal QTc, and disease refractory to or with no available standard therapy options were treated with OKI-179 with either intermittent dosing (once daily for 4 days on 3 days off) or continuous dosing (once daily). Dose escalation was conducted using a standard 3+3 design. Pharmacokinetic (PK) and pharmacodynamic (PD) testing was performed at various time points after dosing. Results: As of Feb 4, 2021, 26 patients (19 female, 7 male) were enrolled with mean age of 63 (range 41-83). Patients received a median of 5 (range 1-11) prior lines of therapy and most common tumor types included pancreatic (N = 5), breast (N = 4), lung (N = 4), and ovarian cancer (N = 4). Twenty patients were treated in intermittent dosing cohorts from 30-450 mg. One DLT (Grade 2 [G2] thrombocytopenia) occurred in the 450 mg cohort which was expanded to 6 patients without subsequent DLTs. Six patients were treated in 2 continuous dosing cohorts of 200 mg and 300 mg. Two of 3 patients in the 300 mg cohort had DLTs of G3-4 thrombocytopenia and no DLTs were observed in 3 patients treated at 200 mg PO daily. The most common adverse events (AEs) were nausea (62%), fatigue (42%), anemia (39%), anorexia (27%), and vomiting (23%). These AE’s were G1-2 except for G3 anemia (12%), G3 fatigue (12%), and G3 anorexia (4%). No other G4-5 treatment-related AEs occurred. Median time on study was 79 days and best response was stable disease (SD) in 10 of 24 patients evaluable for efficacy (42%). Prolonged SD was observed in patients with platinum-resistant serous ovarian cancer (446 days) and adenoid cystic nasopharyngeal carcinoma (256 days). OKI-006 achieved consistent exposure with Cmax > 2,000 ng/ml and AUC > 8,000 hr*ng/ml, well above the targeted exposure for efficacy based on pre-clinical studies in murine models. Tmax was 2 hours and T1/2 was 6-8 hours. OKI-179 treatment resulted in > 3X increased T cell histone H3K9 and H3K27 acetylation within circulating PBMCs at doses of 180 - 450 mg. Conclusions: OKI-179 has a manageable safety profile, with thrombocytopenia being the on-target DLT. It has a favorable PK profile and demonstrated on-target PD effects at tolerable doses. The MTD and RP2D for OKI-179 was 450 mg daily for intermittent dosing and 200 mg daily for continuous dosing. Phase 2 studies are being designed, with a focus on combination with endocrine therapy in ER+ breast cancer and in NRAS-mutant melanoma. Clinical trial information: NCT03931681.

Protein Kinase D-Dependent Downregulation of Immediate Early Genes through Class IIA Histone Deacetylases in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death in children and adolescents, and cure rates for relapsed/refractory ALL remain dismal, highlighting the need for novel targeted therapies. To identify genome-wide metabolic-stress regulated genes, we used RNA-sequencing in ALL cells treated with AICAR, an AMPK activator. RNA-sequencing identified the immediate early genes (IEGs) as a subset of genes downregulated by AICAR. We show that AICAR-induced IEGs downregulation was blocked by an adenosine uptake inhibitor indicating AICAR was responsible for IEGs reprogramming. Using pharmacologic and genetic models we established this mechanism was AMPK-independent. Further investigations using kinase assays, PKD/PKC inhibitors and rescue experiments, demonstrated that AICAR directly inhibited PKD kinase activity and identified PKD as responsible for IEGs downregulation. Mechanistically, PKD inhibition suppressed phosphorylation and nuclear export of class IIa HDACs, which lowered histone H3 acetylation and decreased NFκB(p65) recruitment to IEGs promoters. Finally, PKD inhibition induced apoptosis via DUSP1/DUSP6 downregulation eliciting a DNA damage response. More importantly, ALL patient cells exhibited the same PKD-HDACs-IEGs-mediated mechanism. As proof of principle of the therapeutic potential of targeting PKD, we established the in vivo relevance of our findings using an NSG ALL mouse model. In conclusion, we identified a previously unreported PKD-dependent survival mechanism in response to AICAR-induced cellular stress in ALL through regulation of DUSPs and IEGs' expression. IMPLICATIONS: PKD mediates early transcriptional responses in ALL cells as an adaptive survival mechanism to overcome cellular stress.

HDAC4 degradation during senescence unleashes an epigenetic program driven by AP-1/p300 at selected enhancers and super-enhancers

BackgroundCellular senescence is a permanent state of replicative arrest defined by a specific pattern of gene expression. The epigenome in senescent cells is sculptured in order to sustain the new transcriptional requirements, particularly at enhancers and super-enhancers. How these distal regulatory elements are dynamically modulated is not completely defined.ResultsEnhancer regions are defined by the presence of H3K27 acetylation marks, which can be modulated by class IIa HDACs, as part of multi-protein complexes. Here, we explore the regulation of class IIa HDACs in different models of senescence. We find that HDAC4 is polyubiquitylated and degraded during all types of senescence and it selectively binds and monitors H3K27ac levels at specific enhancers and super-enhancers that supervise the senescent transcriptome. Frequently, these HDAC4-modulated elements are also monitored by AP-1/p300. The deletion of HDAC4 in transformed cells which have bypassed oncogene-induced senescence is coupled to the re-appearance of senescence and the execution of the AP-1/p300 epigenetic program.ConclusionsOverall, our manuscript highlights a role of HDAC4 as an epigenetic reader and controller of enhancers and super-enhancers that supervise the senescence program. More generally, we unveil an epigenetic checkpoint that has important consequences in aging and cancer.

A Novel Salt Inducible Kinase 2 Inhibitor, ARN-3261, Sensitizes Ovarian Cancer Cell Lines and Xenografts to Carboplatin.

Simple SummaryCarboplatin and paclitaxel constitute first-line treatment for ovarian cancer, producing tumor shrinkage in 70% of patients, but curing less than 20% with advanced stage disease. Previous studies have shown that treatment with ARN-3261, a small molecule inhibitor of the enzyme salt-induced kinase 2, can improve the response to paclitaxel in human ovarian cancer cells grown in culture and in immunocompromised mice. Here we have found that ARN-3261 also increases carboplatin’s ability to kill ovarian cancer cells grown in culture and in immunocompromised mice, causing additional DNA damage and decreasing levels of survivin, a protein that protects cancer cells from programmed cell death. Our studies encourage clinical evaluation of ARN-3261 and a Phase I clinical trial has been initiated to test the drug in ovarian cancer patients.Salt-induced kinase 2 (SIK2) is a serine-threonine kinase that regulates centrosome splitting, activation of PI3 kinase and phosphorylation of class IIa HDACs, affecting gene expression. Previously, we found that inhibition of SIK2 enhanced sensitivity of ovarian cancer cells to paclitaxel. Carboplatin and paclitaxel constitute first-line therapy for most patients with ovarian carcinoma, producing a 70% clinical response rate, but curing <20% of patients with advanced disease. We have asked whether inhibition of SIK2 with ARN-3261 enhances sensitivity to carboplatin in ovarian cancer cell lines and xenograft models. ARN-3261-induced DNA damage and apoptosis were measured with γ-H2AX accumulation, comet assays, and annexin V. ARN-3261 inhibited growth of eight ovarian cancer cell lines at an IC50 of 0.8 to 3.5 µM. ARN-3261 significantly enhanced sensitivity to carboplatin in seven of eight ovarian cancer cell lines and a carboplatin-resistant cell line tested. Furthermore, ARN-3261 in combination with carboplatin produced greater inhibition of tumor growth than carboplatin alone in SKOv3 and OVCAR8 ovarian cancer xenograft models. ARN-3261 enhanced DNA damage and apoptosis by downregulating expression of survivin. Thus, a SIK2 kinase inhibitor enhanced carboplatin-induced therapy in preclinical models of ovarian cancer and deserves further evaluation in clinical trials.

Activation of Tripartite Motif Containing 63 Expression by Transcription Factor EB and Transcription Factor Binding to Immunoglobulin Heavy Chain Enhancer 3 Is Regulated by Protein Kinase D and Class IIa Histone Deacetylases.

RationaleThe ubiquitin–proteasome system (UPS) is responsible for skeletal muscle atrophy. We showed earlier that the transcription factor EB (TFEB) plays a role by increasing E3 ubiquitin ligase muscle really interesting new gene-finger 1(MuRF1)/tripartite motif-containing 63 (TRIM63) expression. MuRF 1 ubiquitinates structural proteins and mediates their UPS-dependent degradation. We now investigated how TFEB-mediated TRIM63 expression is regulated.ObjectiveBecause protein kinase D1 (PKD1), histone deacetylase 5 (HDAC5), and TFEB belong to respective families with close structural, regulatory, and functional properties, we hypothesized that these families comprise a network regulating TRIM63 expression.Methods and ResultsWe found that TFEB and transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3) activate TRIM63 expression. The class IIa HDACs HDAC4, HDAC5, and HDAC7 inhibited this activity. Furthermore, we could map the HDAC5 and TFE3 physical interaction. PKD1, PKD2, and PKD3 reversed the inhibitory effect of all tested class IIa HDACs toward TFEB and TFE3. PKD1 mediated nuclear export of all HDACs and lifted TFEB and TFE3 repression. We also mapped the PKD2 and HDAC5 interaction. We found that the inhibitory effect of PKD1 and PKD2 toward HDAC4, HDAC5, and HDAC7 was mediated by their phosphorylation and 14-3-3 mediated nuclear export.ConclusionTFEB and TFE3 activate TRIM63 expression. Both transcription factors are controlled by HDAC4, HDAC5, HDAC7, and all PKD-family members. We propose that the multilevel PKD/HDAC/TFEB/TFE3 network tightly controls TRIM63 expression.

JUP/plakoglobin is regulated by salt-inducible kinase 2, and is required for insulin-induced signalling and glucose uptake in adipocytes

BackgroundSalt-inducible kinase 2 (SIK2) is abundant in adipocytes, but downregulated in adipose tissue from individuals with obesity and insulin resistance. Moreover, SIK isoforms are required for normal insulin signalling and glucose uptake in adipocytes, but the underlying molecular mechanisms are currently not known. The adherens junction protein JUP, also termed plakoglobin or γ-catenin, has recently been reported to promote insulin signalling in muscle cells. ObjectiveThe objective of this study was to analyse if JUP is required for insulin signalling in adipocytes and the underlying molecular mechanisms of this regulation. MethodsCo-expression of SIK2 and JUP mRNA levels in adipose tissue from a human cohort was analysed. siRNA silencing and/or pharmacological inhibition of SIK2, JUP, class IIa HDACs and CRTC2 was employed in 3T3-L1- and primary rat adipocytes. JUP protein expression was analysed by western blot and mRNA levels by qPCR. Insulin signalling was evaluated by western blot as levels of phosphorylated PKB/Akt and AS160, and by monitoring the uptake of 3H-2-deoxyglucose. ResultsmRNA expression of SIK2 correlated with that of JUP in human adipose tissue. SIK2 inhibition or silencing resulted in downregulation of JUP mRNA and protein expression in 3T3-L1- and in primary rat adipocytes. Moreover, JUP silencing reduced the expression of PKB and the downstream substrate AS160, and consequently attenuated activity in the insulin signalling pathway, including insulin-induced glucose uptake. The known SIK2 substrates CRTC2 and class IIa HDACs were found to play a role in the SIK-mediated regulation of JUP expression. ConclusionsThese findings identify JUP as a novel player in the regulation of insulin sensitivity in adipocytes, and suggest that changes in JUP expression could contribute to the effect of SIK2 on insulin signalling in these cells.

A small molecular compound CC1007 induces cross-lineage differentiation by inhibiting HDAC7 expression and HDAC7/MEF2C interaction in BCR-ABL1\u2212 pre-B-ALL

Histone deacetylase 7 (HDAC7), a member of class IIa HDACs, has been described to be an important regulator for B cell development and has a potential role in B cell acute lymphoblastic leukemia (B-ALL). CC1007, a BML-210 analog, is designed to indirectly inhibit class IIa HDACs by binding to myocyte enhancer factor-2 (MEF2) and blocking the recruitment of class IIa HDACs to MEF2-targeted genes to enhance the expression of these targets. In this study, we investigated the anticancer effects of CC1007 in breakpoint cluster region-Abelson 1 fusion gene-negative (BCR-ABL1−) pre-B-ALL cell lines and primary patient-derived BCR-ABL1− pre-B-ALL cells. CC1007 had obvious antileukemic activity toward pre-B-ALL cells in vitro and in vivo; it also significantly prolonged median survival time of pre-B-ALL-bearing mice. Interestingly, low dose of CC1007 could inhibit proliferation of BCR-ABL1− pre-B-ALL cells in a time-dependent manner not accompanied by significant cell apoptosis, but along with cross-lineage differentiation toward monocytic lineage. From a mechanistic angle, we showed that HDAC7 was overexpressed in BCR-ABL1− pre-B-ALL cells compared to normal bone marrow samples, and CC1007 could reduce the binding of HDAC7 at the promoters of monocyte–macrophage-specific genes via inhibition of HDAC7 expression and HDAC7:MEF2C interaction. These data indicated that CC1007 may be a promising agent for the treatment of BCR-ABL1− pre-B-ALL.

Abstract 5210: Novel SIK2 inhibitors sensitize ovarian and breast cancer to PARP inhibitors

Abstract Genomic instability is a recognized hallmark of cancer. Germline mutations in critical DNA-repair and DNA-damage response genes predispose to cancer development, but also create vulnerabilities that can be exploited for cancer therapy. Poly (ADP-ribose) polymerase (PARP) inhibitors are selectively active in cells with homologous recombination deficiency (HRD) caused by mutations in BRCA1, BRCA2, and other DNA repair genes. PARP inhibitors elicit significant responses in ovarian and breast cancers from BRCA1 or BRCA2 mutation carriers. However, many cancers that initially respond to PARP inhibitors eventually develop drug resistance. Thus, it is important to develop new strategies to enhance PARP inhibitor sensitivity and to increase the duration of response. We have identified Salt Induced Kinase 2 (SIK2) inhibitors (ARN3236 and ARN3261) that induce double strand breaks (DSBs) in DNA of HR-competent cells and produce synthetic lethality with multiple PARP inhibitors. SIK2 is an AMP-activated protein kinase (AMPK)-related protein kinase that is required for ovarian cancer cell proliferation and metastasis. SIK2 is overexpressed and correlates with poor prognosis in patients with high-grade serous ovarian carcinoma and triple negative breast cancer. SIK2 inhibition enhances paclitaxel sensitivity in both cancer types. We have demonstrated that olaparib-induced-growth inhibition was significantly enhanced by concurrent treatment with either ARN3236 or ARN3261 in each of 12 ovarian and breast cancer cell lines tested, but not in 3 non-tumorigenic cell lines. Co-administration of olaparib with SIK2 inhibitors suppressed tumor growth and increased the survival of mice with human ovarian (OC316) and breast (MDA-MB-231) cancer xenografts without affecting animal weight. ARN3261 produced little toxicity in preclinical toxicology studies. SIK2 inhibitors decrease the phosphorylation of class-IIa HDAC4/5/7 and abolish class-IIa HDAC 4/5/7-associated transcriptional activity of Myocyte Enhancer Factor 2D (MEF2D). Genome-wide chromatin immunoprecipitation (CHIP) sequencing revealed that SIK2 inhibitors reduce MEF2D binding to regulatory regions with high-chromatin accessibility in DNA repair genes, including FANCD2, EXO1 and XRCC4, resulting in repression of critical genes in DNA DSB repair pathway and induction of apoptosis. In addition, SIK2 inhibitors significantly decreased olaparib-mediated PARP activity and enhanced olaparib-induced cytotoxicity. Together, our data argue that the combination of a SIK2 inhibitor and a PARP inhibitor has the potential to increase the magnitude and duration of PARP inhibitor activity with tolerable toxicity. Use of a SIK2 inhibitor in combination with a PARP inhibitor provides a novel therapeutic strategy for ovarian and triple negative breast cancers with or without BRCA gene mutation. Citation Format: Zhen Lu, Weiqun Mso, Janice M. Santiago-O'Farrill, Hailing Yang, Lan Pang, Ahmed A. Ahmed, Hariprasad Vankayalapati, Robert C. Bast Jr. Novel SIK2 inhibitors sensitize ovarian and breast cancer to PARP inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5210.

HDAC7 is an actionable driver of therapeutic antibody resistance by macrophages from CLL patients.

Resistance, to therapeutic antibodies used to treat chronic lymphocytic leukemia (CLL) patients is common. Monocyte-derived macrophages (MDMs) are a major effector of antitumour responses to therapeutic antibodies and we have previously reported that resistance to therapeutic antibodies, by MDMs, increases as CLL disease progresses. In this study, we examine the effect of a Class IIa-selective HDAC inhibitor (TMP195) on the phagocytic response to opsonised tumor cells or non-opsonised targets by MDMs derived from CLL patients. We report that TMP195 enhances phagocytic responses to antibody-opsonised CLL cells and E. coli within 30 min of treatment. The enhanced response is phenocopied by knockdown of the Class IIa HDAC, HDAC7, or by low concentrations of the pan-HDAC inhibitor, vorinostat. HDAC7 knockdown and inhibition induces hyperacetylation and hyperphosphorylation of Bruton's tyrosine kinase (BTK). Moreover, BTK inhibitors abrogated the enhanced response to HDAC7 inhibition. Our data show that HDAC7 is an actionable driver of resistance to therapeutic antibodies by MDMs derived from CLL patients.

A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression.

Salt-inducible kinase 1 maintains HDAC7 stability to promote pathologic cardiac remodeling.

Salt-inducible kinases (SIKs) are key regulators of cellular metabolism and growth, but their role in cardiomyocyte plasticity and heart failure pathogenesis remains unknown. Here, we showed that loss of SIK1 kinase activity protected against adverse cardiac remodeling and heart failure pathogenesis in rodent models and cardiomyocytes derived from human induced pluripotent stem cells. We found that SIK1 phosphorylated and stabilized histone deacetylase 7 (HDAC7) protein during cardiac stress, an event that is required for pathologic cardiomyocyte remodeling. Gain- and loss-of-function studies of HDAC7 in cultured cardiomyocytes implicated HDAC7 as a prohypertrophic signaling effector that can induce c-Myc expression, indicating a functional departure from the canonical MEF2 corepressor function of class IIa HDACs. Taken together, our findings reveal what we believe to be a previously unrecognized role for a SIK1/HDAC7 axis in regulating cardiac stress responses and implicate this pathway as a potential target in human heart failure.

Synthesis and biological evaluation of acridine-based histone deacetylase inhibitors as multitarget agents against Alzheimer’s disease

Multitarget agents simultaneously trigger molecules in functionally complementary pathways, and are therefore considered to have potential in effectively treating Alzheimer’s disease (AD), which has a complex pathogenetic mechanism. In this study, the HDAC inhibitor core is incorporated into the acetylcholine esterase (ACE) inhibitor acridine–derived moiety and resulted in compounds that exhibited higher class IIa HDAC (4, 5, 7, and 9)- and class IIb HDAC6-inhibiting activity when compared to the pan-HDAC inhibitor SAHA in clinical practice. One of these compounds, 11b, displayed greater selectivity toward HDAC6 than other isoform enzymes. In contrast, the activity of compound 6a was selective toward class IIa HDAC and HDAC6. These two compounds exhibited strong activity against Aβ-aggregation as well as significantly disrupted Aβ-oligomer. Additionally, 11b and 6a strongly inhibited AChE. These experimental findings demonstrate that compounds 11b and 6a are HDAC-Aβ-aggregation-AChE inhibitors. Notably, they can enhance neurite outgrowth, but with no significant neurotoxicity. Further biological evaluation revealed the various cellular effects of multitarget compounds 11b and 6a, which have the potential to treat AD.

P048 Mucosal macrophages express elevated levels of HDAC9 in inflamed and uninflamed mucosa of Crohn’s disease, but not ulcerative colitis

Abstract Background Histone deacetylases (HDACs) are a group of enzymes that control histone and non-histone deacetylation and influence inflammatory gene transcription. Certain members of the HDAC family control the function of macrophages and play an important role in immune response. In this study, we aimed to study the expression of HDACs in mucosal macrophages isolated from inflammatory bowel diseases (IBD) patients. Methods Both macroscopically inflamed and non-inflamed colon resection tissue were collected from 15 Crohn’s disease (CD) and nine ulcerative colitis (UC) patients operated on for therapy refractory disease. Of the CD patients, 53% had ileal and 47% ileocolonic disease. Of the UC patients, 44% had left-sided colitis and 56% pancolitis. Lamina propria was separated from the muscularis externa, and a targeted array for epigenetic enzymes was performed. To assess the relevance of HDAC9 gene expression in terms of protein level, immunofluorescence staining of HDAC9 protein was undertaken in tissue sections from inflamed and non-inflamed mucosa. CD68 was used as a pan-macrophage marker. Results From our array, expression of HDAC9 was significantly higher in the inflamed mucosa of CD patients compared with UC patients (p = 0.005). Gene expression of HDAC9 in non-inflamed mucosa from CD was elevated compared with non-inflamed mucosa from UC. In addition, in CD, HDAC9 mRNA level was increased in inflamed tissue in comparison to non-inflamed tissue (p = 0.046). In conjunction with the expression data, HDAC9 protein was found highly expressed in inflamed tissue. HDAC9 was predominantly localised in the cytoplasmic compartment of macrophages in non-inflamed tissue whilst HDAC9 localised to the nucleus of macrophages in inflamed tissue. Conclusion HDAC9 is member of class IIA HDAC superfamily that exerts pro-inflammatory properties. The inhibition of HDAC9 in experimental murine colitis clearly enhances regulatory T-cell function, suggesting a critical role for HDAC9 in breaching immune homeostasis (de Zoeten EF et al, 2009). We suggest here that HDAC9 can serve as an additional marker to distinguish CD from UC in tissue biopsies. Furthermore, we show for the first time that HDAC9 protein is expressed in mucosal macrophages of CD patients, indicating its potential in mediating macrophage inflammatory function in IBD. Further studies are currently being undertaken to elucidate the role of HDAC9 in CD pathogenesis.

The Role of Class IIa HDACs in the Expression of E3 Ligases ATROGIN-1/MAFbx and MuRF1 under Muscle Unloading

The role of histone deacetylases 4/5 (HDAC4/5) in the activation of myogenin and E3 ligases (MuRF1 and MAFbx) under functional unloading of m. soleus was studied. For this purpose, trichostatin A, a histone deacetylase inhibitor, was used in suspended rats. Wistar rats (24 three-month-old males) were divided into three groups (eight in each). One group served as a control (C), the second (HS) and third groups were suspended for 3 days (the rats of group 3 were intraperitoneally administered trichostatin A at a dose of 0.6 mg/kg of body mass per day (HST)). The rats were killed with an overdose of nembutal (75 mg/kg of mass), and m. soleus was immediately frozen in liquid nitrogen. In the HST group, a significant decrease in the HDAC4 protein content in the nuclear fraction (in contrast to the HS group) and its increase in the cytoplasmic fraction (relative to the control (p < 0.05)) were found. The administration of trichostatin A prevented changes in the HDAC4 content in the nucleus (relative to the C group), whereas the HDAC5 content was significantly reduced (p < 0.05) in the HS group. Thus, the drug prevented the effect of functional unloading on the nuclear localization of HDAC 4/5. The level of myogenin transcription factor in the HST group of rats did not differ from that in the control, while it was significantly higher in the HS group (p < 0.05). The expression of mRNA of E3 ligase atrogin-1/MAFbx in the HST rats was the same as in the control group and was significantly higher in the HS group (p < 0.05). The expression of mRNA of E3 ligase MuRF1in both groups was increased compared to the controls (regardless of the drug administration). Conclusion: HDAC4/5 regulates the expression of myogenin and E3 ligase atrogin-1/MAFbx. Inhibition of HDAC4/5 does not affect the regulation of the expression of E3 ligase MuRF1.

Different class IIa HDACs repressive complexes regulate specific epigenetic responses related to cell survival in leiomyosarcoma cells.

Transcriptional networks supervising class IIa HDAC expression are poorly defined. Here we demonstrate that MEF2D is the key factor controlling HDAC9 transcription. This control, which is part of a negative feed-back loop during muscle differentiation, is hijacked in cancer. In leiomyosarcomas the MEF2D/HDAC9 vicious circuit sustains proliferation and cell survival, through the repression of the death receptor FAS. Comprehensive genome-wide studies demonstrate that HDAC4 and HDAC9 control different genetic programs and show both specific and common genomic binding sites. Although the number of MEF2-target genes commonly regulated is similar, only HDAC4 represses many additional genes that are not MEF2D targets. As expected, HDAC4−/− and HDAC9−/− cells increase H3K27ac levels around the TSS of the respective repressed genes. However, these genes rarely show binding of the HDACs at their promoters. Frequently HDAC4 and HDAC9 bind intergenic regions. We demonstrate that these regions, recognized by MEF2D/HDAC4/HDAC9 repressive complexes, show the features of active enhancers. In these regions HDAC4 and HDAC9 can differentially influence H3K27 acetylation. Our studies describe new layers of class IIa HDACs regulation, including a dominant positional effect, and can contribute to explain the pleiotropic actions of MEF2 TFs.

HDAC4 Controls Muscle Homeostasis through Deacetylation of Myosin Heavy Chain, PGC-1α, and Hsc70

HDAC4, a class IIa histone deacetylase, is upregulated in skeletal muscle in response to denervation-induced atrophy. When HDAC4 is deleted postnatally, mice are partially protected from denervation. Despite the name "histone" deacetylase, HDAC4 demonstrably deacetylates cytosolic and non-histone nuclear proteins. We developed potent and selective class IIa HDAC inhibitors. Using these tools and genetic knockdown, we identified three previously unidentified substrates of HDAC4: myosin heavy chain, peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1α), and heat shock cognate 71kDa protein (Hsc70). HDAC4 inhibition almost completely prevented denervation-induced loss of myosin heavy chain isoforms and blocked the action of their E3 ligase, MuRF1. PGC-1α directly interacts with class IIa HDACs; selective inhibitors increased PGC-1α protein in muscles. Hsc70 deacetylation by HDAC4 affects its chaperone activity. Through these endogenous HDAC4 substrates, we identified several muscle metabolic pathways that are regulated by class IIa HDACs, opening up new therapeutic options to treat skeletal muscle disorders and potentially other disease where these specific pathways are affected.

Psychostimulants are not identical pharmacological agents: Distinct effects of psychostimulant drugs on the regulation of class IIa HDACs in the mouse mesocorticolimbic and striatal systems

Psychostimulants are not identical pharmacological agents: Distinct effects of psychostimulant drugs on the regulation of class IIa HDACs in the mouse mesocorticolimbic and striatal systems