HDAC-mediated control of ERK- and PI3K-dependent TGF-β-induced extracellular matrix-regulating genes

Histone deacetylases (HDACs) regulate the acetylation of histones in the control of gene expression. Many non-histone proteins are also targeted for acetylation, including TGF-ß signalling pathway components such as Smad2, Smad3 and Smad7. Our studies in mouse C3H10T1/2 fibroblasts suggested that a number of TGF-ß-induced genes that regulate matrix turnover are selectively regulated by HDACs. Blockade of HDAC activity with trichostatin A (TSA) abrogated the induction of a disintegrin and metalloproteinase 12 (Adam12) and tissue inhibitor of metalloproteinases-1 (Timp-1) genes by TGF-ß, whereas plasminogen activator inhibitor-1 (Pai-1) expression was unaffected. Analysis of the activation of cell signalling pathways demonstrated that TGF-ß induced robust ERK and PI3K activation with delayed kinetics compared to the phosphorylation of Smads. The TGF-ß induction of Adam12 and Timp-1 was dependent on such non-Smad signalling pathways and, importantly, HDAC inhibitors completely blocked their activation without affecting Smad signalling. Analysis of TGF-ß-induced Adam12 and Timp-1 expression and ERK/PI3K signalling in the presence of semi-selective HDAC inhibitors valproic acid, MS-275 and apicidin implicated a role for class I HDACs. Furthermore, depletion of HDAC3 by RNA interference significantly down-regulated TGF-ß-induced Adam12 and Timp-1 expression without modulating Pai-1 expression. Correlating with the effect of HDAC inhibitors, depletion of HDAC3 also blocked the activation of ERK and PI3K by TGF-ß. Collectively, these data confirm that HDACs, and in particular HDAC3, are required for activation of the ERK and PI3K signalling pathways by TGF-ß and for the subsequent gene induction dependent on these signalling pathways

Combinations of isoform-targeted histone deacetylase inhibitors and bryostatin analogues display remarkable potency to activate latent HIV without global T-cell activation

AbstractCurrent antiretroviral therapy (ART) for HIV/AIDS slows disease progression by reducing viral loads and increasing CD4 counts. Yet ART is not curative due to the persistence of CD4+ T-cell proviral reservoirs that chronically resupply active virus. Elimination of these reservoirs through the administration of synergistic combinations of latency reversing agents (LRAs), such as histone deacetylase (HDAC) inhibitors and protein kinase C (PKC) modulators, provides a promising strategy to reduce if not eradicate the viral reservoir. Here, we demonstrate that largazole and its analogues are isoform-targeted histone deacetylase inhibitors and potent LRAs. Significantly, these isoform-targeted HDAC inhibitors synergize with PKC modulators, namely bryostatin-1 analogues (bryologs). Implementation of this unprecedented LRA combination induces HIV-1 reactivation to unparalleled levels and avoids global T-cell activation within resting CD4+ T-cells.</jats:p

Mutation of the co-chaperone Tsc1 in bladder cancer diminishes Hsp90 acetylation and reduces drug sensitivity and selectivity

The molecular chaperone Heat shock protein 90 (Hsp90) is essential for the folding, stability, and activity of several drivers of oncogenesis. Hsp90 inhibitors are currently under clinical evaluation for cancer treatment, however their efficacy is limited by lack of biomarkers to optimize patient selection. We have recently identified the tumor suppressor tuberous sclerosis complex 1 (Tsc1) as a new co-chaperone of Hsp90 that affects Hsp90 binding to its inhibitors. Highly variable mutations of TSC1 have been previously identified in bladder cancer and correlate with sensitivity to the Hsp90 inhibitors. Here we showed loss of TSC1 leads to hypoacetylation of Hsp90-K407/K419 and subsequent decreased binding to the Hsp90 inhibitor ganetespib. Pharmacologic inhibition of histone deacetylases (HDACs) restores acetylation of Hsp90 and sensitizes Tsc1-mutant bladder cancer cells to ganetespib, resulting in apoptosis. Our findings suggest that TSC1 status may predict response to Hsp90 inhibitors in patients with bladder cancer, and co-targeting HDACs can sensitize tumors with Tsc1 mutations to Hsp90 inhibitors

Identification and Characterization of AES-135, a Hydroxamic Acid-Based HDAC Inhibitor That Prolongs Survival in an Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, incurable cancer with a 20% 1 year survival rate. While standard-of-care therapy can prolong life in a small fraction of cases, PDAC is inherently resistant to current treatments, and novel therapies are urgently required. Histone deacetylase (HDAC) inhibitors are effective in killing pancreatic cancer cells in in vitro PDAC studies, and although there are a few clinical studies investigating combination therapy including HDAC inhibitors, no HDAC drug or combination therapy with an HDAC drug has been approved for the treatment of PDAC. We developed an inhibitor of HDACs, AES-135, that exhibits nanomolar inhibitory activity against HDAC3, HDAC6, and HDAC11 in biochemical assays. In a three-dimensional coculture model, AES-135 kills low-passage patient-derived tumor spheroids selectively over surrounding cancer-associated fibroblasts and has excellent pharmacokinetic properties in vivo. In an orthotopic murine model of pancreatic cancer, AES-135 prolongs survival significantly, therefore representing a candidate for further preclinical testing

HDAC1 inhibition by MS-275 in mesothelial cells limits cellular invasion and promotes MMT reversal

Peritoneal fibrosis is a pathological alteration of the peritoneal membrane occurring in a variety of conditions including peritoneal dialysis (PD), post-surgery adhesions and peritoneal metastases. The acquisition of invasive and pro-fibrotic abilities by mesothelial cells (MCs) through induction of MMT, a cell-specific form of EMT, plays a main role in this process. Aim of this study was to evaluate possible effects of histone deacetylase (HDAC) inhibitors, key components of the epigenetic machinery, in counteracting MMT observed in MCs isolated from effluent of PD patients. HDAC inhibitors with different class/isoform selectivity have been used for pharmacological inhibition. While the effect of other inhibitors was limited to a partial E-cadherin re-expression, MS-275, a HDAC1-3 inhibitor, promoted: (i) downregulation of mesenchymal markers (MMP2, Col1A1, PAI-1, TGFβ1, TGFβRI) (ii) upregulation of epithelial markers (E-cadherin, Occludin), (iii) reacquisition of an epithelial-like morphology and (iv) marked reduction of cellular invasiveness. Results were confirmed by HDAC1 genetic silencing. Mechanistically, MS-275 causes: (i) increase of nuclear histone H3 acetylation (ii) rescue of the acetylation profile on E-cadherin promoter, (iii) Snail functional impairment. Overall, our study, pinpointing a role for HDAC1, revealed a new player in the regulation of peritoneal fibrosis, providing the rationale for future therapeutic opportunities

Potent and selective inhibitors of histone deacetylase-3 containing chiral oxazoline capping groups and a N-(2-Aminophenyl)-benzamide binding unit

A novel series of potent chiral inhibitors of histone deacetylase (HDAC) is described that contains an oxazoline capping group and a N-(2-aminophenyl)-benzamide unit. Among several new inhibitors of this type exhibiting Class I selectivity and potent inhibition of HDAC3-NCoR2, in vitro assays for the inhibition of HDAC1, HDAC2, and HDAC3-NCoR2 by N-(2-aminophenyl)-benzamide 15k gave respective IC50 values of 80, 110, and 6 nM. Weak inhibition of all other HDAC isoforms (HDAC4, 5, 6, 7, and 9: IC50 > 100 000 nM; HDAC8: IC50 = 25 000 nM; HDAC10: IC50 > 4000 nM; HDAC11: IC50 > 2000 nM) confirmed the Class I selectivity of 15k. 2-Aminoimidazolinyl, 2-thioimidazolinyl, and 2-aminooxazolinyl units were shown to be effective replacements for the pyrimidine ring present in many other 2-(aminophenyl)-benzamides previously reported, but the 2-aminooxazolinyl unit was the most potent in inhibiting HDAC3-NCoR2. Many of the new HDAC inhibitors showed higher solubilities and lower binding to human serum albumin than that of Mocetinostat. Increases in histone H3K9 acetylation in the human cell lines U937 and PC-3 was observed for all three oxazolinyl inhibitors evaluated; those HDAC inhibitors also lowered cyclin E expression in U937 cells but not in PC-3 cells, indicating underlying differences in the mechanisms of action of the inhibitors on those two cell lines

Epigenomic regulation of heart failure: integrating histone marks, long noncoding RNAs, and chromatin architecture.

Epigenetic processes are known to have powerful roles in organ development across biology. It has recently been found that some of the chromatin modulatory machinery essential for proper development plays a previously unappreciated role in the pathogenesis of cardiac disease in adults. Investigations using genetic and pharmacologic gain- and loss-of-function approaches have interrogated the function of distinct epigenetic regulators, while the increased deployment of the suite of next-generation sequencing technologies have fundamentally altered our understanding of the genomic targets of these chromatin modifiers. Here, we review recent developments in basic and translational research that have provided tantalizing clues that may be used to unlock the therapeutic potential of the epigenome in heart failure. Additionally, we provide a hypothesis to explain how signal-induced crosstalk between histone tail modifications and long non-coding RNAs triggers chromatin architectural remodeling and culminates in cardiac hypertrophy and fibrosis

Epigenetic Regulation of Matrix Metalloproteinase-1 and -3 Expression in Mycobacterium tuberculosis Infection.

In pulmonary tuberculosis (TB), the inflammatory immune response against Mycobacterium tuberculosis (Mtb) is associated with tissue destruction and cavitation, which drives disease transmission, chronic lung disease, and mortality. Matrix metalloproteinase (MMP)-1 is a host enzyme critical for the development of cavitation. MMP expression has been shown to be epigenetically regulated in other inflammatory diseases, but the importance of such mechanisms in Mtb-associated induction of MMP-1 is unknown. We investigated the role of changes in histone acetylation in Mtb-induced MMP expression using inhibitors of histone deacetylases (HDACs) and histone acetyltransferases (HAT), HDAC siRNA, promoter-reporter constructs, and chromatin immunoprecipitation assays. Mtb infection decreased Class I HDAC gene expression by over 50% in primary human monocyte-derived macrophages but not in normal human bronchial epithelial cells (NHBEs). Non-selective inhibition of HDAC activity decreased MMP-1/-3 expression by Mtb-stimulated macrophages and NHBEs, while class I HDAC inhibition increased MMP-1 secretion by Mtb-stimulated NHBEs. MMP-3 expression, but not MMP-1, was downregulated by siRNA silencing of HDAC1. Inhibition of HAT activity also significantly decreased MMP-1/-3 secretion by Mtb-infected macrophages. The MMP-1 promoter region between -2,001 and -2,942 base pairs from the transcriptional start site was key in control of Mtb-driven MMP-1 gene expression. Histone H3 and H4 acetylation and RNA Pol II binding in the MMP-1 promoter region were increased in stimulated NHBEs. In summary, epigenetic modification of histone acetylation via HDAC and HAT activity has a key regulatory role in Mtb-dependent gene expression and secretion of MMP-1 and -3, enzymes which drive human immunopathology. Manipulation of epigenetic regulatory mechanisms may have potential as a host-directed therapy to improve outcomes in the era of rising TB drug resistance

AKT activation controls cell survival in response to HDAC6 inhibition.

HDAC6 is emerging as an important therapeutic target for cancer. We investigated mechanisms responsible for survival of tumor cells treated with a HDAC6 inhibitor. Expression of the 20 000 genes examined did not change following HDAC6 treatment in vivo. We found that HDAC6 inhibition led to an increase of AKT activation (P-AKT) in vitro, and genetic knockdown of HDAC6 phenocopied drug-induced AKT activation. The activation of AKT was not observed in PTEN null cells; otherwise, PTEN/PIK3CA expression per se did not predict HDAC6 inhibitor sensitivity. Interestingly, HDAC6 inhibitor treatment led to inactivating phosphorylation of PTEN (P-PTEN Ser380), which likely led to the increased P-AKT in cells that express PTEN. Synergy was observed with phosphatidylinositol 3-kinases (PI3K) inhibitor treatment in vitro, accompanied by increased caspase 3/7 activity. Furthermore, combination of HDAC6 inhibitor with a PI3K inhibitor caused substantial tumor growth inhibition in vivo compared with either treatment alone, also detectable by Ki-67 immunostaining and (18)F-FLT positron emission tomography (PET). In aggregate AKT activation appears to be a key survival mechanism for HDAC6 inhibitor treatment. Our findings indicate that dual inhibition of HDAC6 and P-AKT may be necessary to substantially inhibit growth of solid tumors