Specific Histone Deacetylase Inhibitor Research Articles

Abstract 4812: Vorinostat and fenretinide synergize in preclinical models of T-cell lymphoid malignancies via reactive oxygen species

Abstract Introduction: T-cell lymphoid malignancies (TCLMs) are in need of novel and more effective therapies. Pan-histone deacetylase (HDAC) inhibitors vorinostat and belinostat, and class I specific HDAC inhibitor romidepsin have achieved FDA registration as 2nd line therapies for peripheral and/or cutaneous T-cell lymphomas. The cytotoxic retinoid fenretinide achieved durable complete responses against T-cell lymphomas in early-phase clinical trials and T-cell lymphoma patients who failed prior HDAC inhibitor treatment responded to fenretinide (Clinical Cancer Res 23:4550-4555, 2017). Fenretinide is currently being evaluated in a Phase IIa clinical trial for relapsed/refractory PTCL patients (NCT02495415). We have previously shown romidepsin and fenretinide synergize in preclinical models of T-cell lymphoid malignancies (Molecular Cancer Therapeutics 16:649-661, 2017). There exist some key differences in the activity of various classes of HDAC's, which have significantly different chemical structures and metabolic profiles. Therefore we determined if the pan-HDAC inhibitor vorinostat synergizes with fenretinide. Methods and Results: Using the DIMSCAN cytotoxicity assay, we demonstrated cytotoxic synergy between vorinostat and fenretinide in nine TCLM cell lines at clinically-achievable concentrations that lacked cytotoxicity for non-malignant cells (fibroblasts and blood mononuclear cells). In vivo, vorinostat + fenretinide + ketoconazole (enhances fenretinide exposures by inhibiting fenretinide metabolism) showed greater activity in subcutaneous (COG-LL-317m and TX-LY-183x PDX) TCLM xenograft models than single agent vorinostat or fenretinide + ketoconazole. Fenretinide + vorinostat caused a reactive oxygen species (ROS, measured by DCFDA dye)-dependent increase in apoptosis (via TUNEL assay), and histone acetylation (measured by immunoblotting). The synergistic cytotoxicity, apoptosis, and histone acetylation of fenretinide + vorinostat was abrogated by antioxidant vitamin C. Vorinostat + fenretinide activated p38 and JNK via ROS, and shRNA knockdown of p38 and JNK1 significantly decreased the synergistic cytotoxicity and apoptosis. Vorinostat + fenretinide also showed synergistic cytotoxicity for six B-lymphoid malignancy cell lines. Conclusion: Like romidepsin, vorinostat combined with fenretinide achieved synergistic activity in preclinical models of TCLMs, but not in non-malignant cells, via a novel molecular mechanism. As vorinostat is an oral agent and not a PGP substrate it may have advantages in such combination therapy. These data support conducting a clinical trial of vorinostat combined with fenretinide in relapsed and refractory TCLMs. Citation Format: Monish Ram Makena, Thinh H. Nguyen, Balakrishna Koneru, Ashly Hindle, Wan-Hsi Chen, Dattesh U. Verlekar, Min H. Kang, C. Patrick Reynolds. Vorinostat and fenretinide synergize in preclinical models of T-cell lymphoid malignancies via reactive oxygen species [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4812.

Growth Suppression of Glioma Cells Using HDAC6 Inhibitor, Tubacin.

In cancer research, autophagy has been revealed as one of the major ways to maintain the metabolism of cancer cells, including glioma cells, through protein degradation. Meanwhile, autophagy is also regarded as a kind of mechanism to protect glioma cells from a harmful stimulus, such as chemical and radiation treatment. So, the inhibition of autophagy may be very helpful in curing glioma. This study aimed to determine the effect of autophagic inhibition on glioma cells using tubacin, a specific inhibitor of histone deacetylase 6(HDAC6). According to the results, tubacin inhibited the growth of both U251 and LN229 cells, which was accompanied by lower HDAC6 activity and accumulated autophagosome. The inhibition of HDCA6 also led to accumulation of autophagosome and death of glioma cells. Moreover, the combined treatment of tubacin and temozolomide, an alkylating agent used to treat glioblastoma, induced more severe glioma cell death. Thus, it can be concluded that inhibition of HDAC6 suppressed growth and drug resistance of glioma cells in-vitro through autophagic suppression and blocking of fusion of autophagosome and lysosome.

Expression of AhDREB1, an AP2/ERF Transcription Factor Gene from Peanut, Is Affected by Histone Acetylation and Increases Abscisic Acid Sensitivity and Tolerance to Osmotic Stress in Arabidopsis.

Drought stress negatively affects plant growth and development. An increasing number of reports have revealed the involvement of APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors (TFs) in biotic and abiotic stress regulation in plants. However, research on these TFs in the peanut plant (Arachis hypogaea) has been limited. Here, we isolated a full-length coding sequence (CDS) of the AP2/ERF family gene AhDREB1 from the peanut plant and showed that its expression was induced by Polyethylene Glycol (PEG) 6000 and exogenous abscisic acid (ABA) treatment. When overexpressed in Arabidopsis, AhDREB1 increased both ABA levels and ABA sensitivity, affected the ABA signaling pathway and increased the expression of downstream drought stress-related genes RD29A, P5CS1, P5CS2 and NCED1. These results demonstrate that AhDREB1 can improve tolerance to drought via the ABA-dependent pathway in Arabidopsis. In the peanut plant, the specific histone deacetylases (HDACs) inhibitor trichostatin A (TSA) promotes AhDREB1 transcription and the enrichment level of H3ac was increased in regions of the AhDREB1 gene during TSA and PEG treatment. In summary, histone acetylation can affect the expression of AhDREB1 under osmotic stress conditions, thereby improving plant drought resistance.

Silence of HDAC6 suppressed esophageal squamous cell carcinoma proliferation and migration by disrupting chaperone function of HSP90.

Esophageal carcinoma is aggressive in nature and its prognosis is largely dependent on the degree of invasion. Histone deacetylase 6 (HDAC6), as the most unique member of HDACs family, has the positive activity to promote initiation and progression of various cancers via targeting multiple non‐histone proteins in cytoplasm. In this study, we found that HDAC6 was over‐expressed in three esophageal cancer cell lines (KYSE140, KYSE170, KYSE180) when compared to non‐carcinoma esophageal epithelial cell HEEC‐1. Then two HDAC6 specific siRNAs and HDAC6 inhibitor tubastatin A greatly suppressed KYSE140 and KYSE180 cells proliferation and migration, and the inhibition of cell motility was accompanied by elevated acetylation of α‐tubulin, a target of HDAC6. Consistently, the microtubulin skeleton was stabilized after HDAC6 knockdown or inhibition. In addition, acetylation status of HSP90, another HDAC6 target, was also increased towards HDAC6 knockdown or inhibition by co‐immunoprecipitation assay. Besides, co‐treatment of HSP90 inhibitor (PU‐H71) and HDAC6 inhibitor (tubastatin A) induced a stronger cell migration inhibition compared to administration of either drug alone. Furthermore, cell proliferation of KYSE140 and KYSE180 were also compromised in response to combination of HDAC6 and HSP90 inhibitors. Additionally, co‐administration of HSP90 inhibitor and HDAC6 inhibitor strongly inhibited tumor growth in vivo. Taken together, our results indicated that HDAC6 is a promising target by inhibiting HSP90 function in ESCC.

Histone deacetylases in axonal growth and regeneration and their relevance to Parkinson's disease.

Functional decline in Parkinson's disease (PD) is the cumulative result of regulatory alterations affecting large numbers of genes involved in various aspects of neuronal maintenance and function, and it has recently emerged that axonal degeneration plays a key role. Central to the alterations in gene expression that underlie this failure of axon maintenance, is the regulation of gene transcription by enzymes known as histone deacetylases (HDAC) which promote the de‐acetylation of histone proteins to inhibit transcription. alpha‐synuclein, a key protein that accumulates in the brains of patients with the disease, binds histone proteins in vitro & in vivo, and nuclear a‐synuclein reduces acetylated histone 3 (AcH3) levels. However while these molecular insights are important, how they regulate the neuronal structure and axonal growth in neurons affected by Parkinson's, and the relevance of this cell biology approach to the human brain is unknown.Here we used a network approach to identify all genes that had a significant positive (r+) or negative (r−) correlation with the expression of all HDACs using microarray data from the human substantia nigra (SN; GSE 60863). We then conducted a detailed enrichment analysis to identify biological pathways that were over‐represented and may be linked to Parkinson's Disease. These analyses showed that Class‐II but not Class‐I HDACs were significantly correlated with functional pathways linked to Parkinson's Disease (p<0.0001). These included genes linked to familial Parkinson's Disease and oxidative phosphorylation, including those associated with mitochondrial complex I.To link these data with changes in neuronal structure, we next examined the effects of class specific HDAC inhibitors on neuronal structure by examining a range of HDAC inhibitors for their ability to promote axonal growth and branching in dopaminergic and sympathetic neurons which are the two main cell types that undergo progressive axonal degeneration in Parkinson's Disease. We found that a MC1568 (a class II inhibitor) but not MS275 (a class I inhibitor) promoted significant increases in axon growth and branching (p<0.01). Therefore we further examined the therapeutic potential of MC1568, and found it protected against the detrimental effects of MPP+‐ and alpha‐synuclein induced axonal degeneration in a models of Parkinson's disease.This study therefore provides new insights into the regulator mechanisms that control axonal growth in neurons affects by Parkinson's, and highlights the potential of using this approach to identify therapeutic targets for neuroprotection and axonal regeneration.Support or Funding InformationThis work was funded by a Career Development Award from Science Foundation Ireland under the grant number 15/CDA/3498.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Controlled release of an HDAC inhibitor for reduction of inflammation in dry eye disease

Dry eye disease (DED), also known as keratoconjunctivitis sicca, is an ocular surface disease characterized by T-cell-mediated inflammation. Current therapeutics, such as immunosuppressive agents, act to suppress the clinical signs and inflammation. However, long-term usage of these treatments can cause severe side effects. In this study, we present an alternative therapeutic approach that utilizes a histone deacetylase inhibitor (HDACi) to regulate transcription of a variety of immunomodulatory genes. Specifically, HDACi have emerged as a potential anti-inflammatory agent, which can modulate the functions of a subset of suppressive T lymphocytes known as regulatory T cells (Tregs), enhancing FoxP3 acetylation and subsequently guarding the transcription factor from proteasomal degradation. Here, a specific HDACi known as SAHA (suberoylanilide hydroxamic acid) was formulated to controllably release in the lacrimal gland. Intralacrimal gland injection of PLGA-based SAHA microspheres prevented clinical signs of DED in mice with Concanavalin A-induced DED, reduced expression of pro-inflammatory cytokines, and increased expression of FoxP3 in the lacrimal glands. Murine T cell culture experiments also revealed that SAHA decreased effector T cell proliferation and enhanced suppressive function of Tregs in co-cultures of Tregs and effector T cells. Statement of SignificanceIn this study, we demonstrate a therapeutic approach that utilizes a histone deactylase inhibitor (HDACi) to regulate transcription of a variety of immunomodulatory genes. HDACi have emerged as a potential anti-inflammatory agent, which can modulate the functions of a subset of suppressive T lymphocytes known as regulatory T cells (Tregs). Here, HDACi microspheres composed of a biocompatible and biodegradable polymer (poly(lactic-co-glycolic acid) (PLGA)), were able to locally release the HDACi and prevent clinical signs of DED. This work is timely given the recent shift in treatments of DED towards immunological based therapies to reduce ocular inflammation. However, notably, many of these treatments require large amounts of drug, and non-specifically suppress the immune system, leading to several systemic side effects. Instead of merely suppressing or blocking inflammation, the formulation described herein intends to balance the microenvironment promoting immunological homeostasis. This particular drug delivery system may also have broad implications in the field of inflammatory mediated ocular disorders such as uveitis, Sjögren’s syndrome, allergic conjunctivitis.

HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease.

Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.

Histone deacetylase 6 (HDAC6) deacetylates extracellular signal-regulated kinase 1 (ERK1) and thereby stimulates ERK1 activity

Histone deacetylase 6 (HDAC6), a class IIb HDAC, plays an important role in many biological and pathological processes. Previously, we found that ERK1, a downstream kinase in the mitogen-activated protein kinase signaling pathway, phosphorylates HDAC6, thereby increasing HDAC6-mediated deacetylation of α-tubulin. However, whether HDAC6 reciprocally modulates ERK1 activity is unknown. Here, we report that both ERK1 and -2 are acetylated and that HDAC6 promotes ERK1 activity via deacetylation. Briefly, we found that both ERK1 and -2 physically interact with HDAC6. Endogenous ERK1/2 acetylation levels increased upon treatment with a pan-HDAC inhibitor, an HDAC6-specific inhibitor, or depletion of HDAC6, suggesting that HDAC6 deacetylates ERK1/2. We also noted that the acetyltransferases CREB-binding protein and p300 both can acetylate ERK1/2. Acetylated ERK1 exhibits reduced enzymatic activity toward the transcription factor ELK1, a well-known ERK1 substrate. Furthermore, mass spectrometry analysis indicated Lys-72 as an acetylation site in the ERK1 N terminus, adjacent to Lys-71, which binds to ATP, suggesting that acetylation status of Lys-72 may affect ERK1 ATP binding. Interestingly, an acetylation-mimicking ERK1 mutant (K72Q) exhibited less phosphorylation than the WT enzyme and a deacetylation-mimicking mutant (K72R). Of note, the K72Q mutant displayed decreased enzymatic activity in an in vitro kinase assay and in a cellular luciferase assay compared with the WT and K72R mutant. Taken together, our findings suggest that HDAC6 stimulates ERK1 activity. Along with our previous report that ERK1 promotes HDAC6 activity, we propose that HDAC6 and ERK1 may form a positive feed-forward loop, which might play a role in cancer.

Combination of a Histone Deacetylase 6 Inhibitor and a Somatostatin Receptor Agonist Synergistically Reduces Hepatorenal Cystogenesis in an Animal Model of Polycystic Liver Disease

Hepatic cystogenesis in polycystic liver disease (PLD) is associated with abnormalities in multiple cellular processes, including elevated cAMP and overexpression of histone deacetylase 6 (HDAC6). Disease progression in polycystic kidney (PCK) rats (an animal model of PLD) is attenuated by inhibition of either cAMP production or HDAC6. Therefore, we hypothesized that concurrent targeting of HDAC6 and cAMP would synergistically reduce cyst growth. Changes in hepatorenal cystogenesis were examined in PCK rats treated with a pan-HDAC inhibitor, panobinostat; three specific HDAC6 inhibitors, ACY-1215, ACY-738, and ACY-241; and a combination of ACY-1215 and the somatostatin receptor analogue, pasireotide. We also assessed effects of ACY-1215 and pasireotide alone and in combination on cell proliferation, cAMP production, and expression of acetylated α-tubulin invitro in cultured cholangiocytes and the length of primary cilia and the frequency of ciliated cholangiocytes invivo in PCK rats. Panobinostat and all three HDAC6 inhibitors decreased hepatorenal cystogenesis in PCK rats. ACY-1215 was more effective than other HDAC inhibitors and was chosen for combinational treatment. ACY-1215+pasireotide combination synergistically reduced cyst growth and increased length of primary cilia in PCK rats. In cultured cystic cholangiocytes, ACY-1215+pasireotide combination concurrently decreased cell proliferation and inhibited cAMP levels. These data suggest that the combination of drugs that inhibit HDAC6 and cAMP may be an effective therapy for PLD.

Epigenetic mechanisms in Alzheimer's Disease: Therapeutic potential of Class specific HDAC inhibitors in insulin resistance induced cognitive impairments

Epigenetic mechanisms in Alzheimer's Disease: Therapeutic potential of Class specific HDAC inhibitors in insulin resistance induced cognitive impairments

Disruption to schizophrenia-associated gene Fez1 in the hippocampus of HDAC11 knockout mice

Histone Deacetylase 11 (HDAC11) is highly expressed in the central nervous system where it has been reported to have roles in neural differentiation. In contrast with previous studies showing nuclear and cytoplasmic localisation, we observed synaptic enrichment of HDAC11. Knockout mouse models for HDACs 1–9 have been important for guiding the development of isoform specific HDAC inhibitors as effective therapeutics. Given the close relationship between HDAC11 and neural cells in vitro, we examined neural tissue in a previously uncharacterised Hdac11 knockout mouse (Hdac11KO/KO). Loss of HDAC11 had no obvious impact on brain morphology and neural stem/precursor cells isolated from Hdac11KO/KO mice had comparable proliferation and differentiation characteristics. However, in differentiating neural cells we observed decreased expression of schizophrenia-associated gene Fez1 (fasciculation and elongation protein zeta 1), a gene previously reported to be regulated by HDAC11 activity. FEZ1 has been associated with the dendritic growth of neurons and risk of schizophrenia via its interaction with DISC1 (disrupted in schizophrenia 1). Examination of cortical, cerebellar and hippocampal tissue reveal decreased Fez1 expression specifically in the hippocampus of adult mice. The results of this study demonstrate that loss of HDAC11 has age dependent and brain-region specific consequences.

Histone deacetylase inhibition induces odor preference memory extension and maintains enhanced AMPA receptor expression in the rat pup model.

Histone deacetylase (HDAC) plays a role in synaptic plasticity and long-term memory formation. We hypothesized that trichostatin-A (TSA), an HDAC inhibitor, would promote long-term odor preference memory and maintain enhanced GluA1 receptor levels that have been hypothesized to support memory. We used an early odor preference learning model in neonate rat pups that normally produces only 24-h memory to test behavior and examine receptor protein expression. Our behavioral studies showed that intrabulbar infusion of TSA, prior to pairing of the conditioned stimulus (peppermint odor) with the unconditioned stimulus (tactile stimulation), prolonged 24-h odor preference memory for at least 9 d. The prolonged odor preference memory was selective for the paired odor and was also observed using a specific HDAC6 inhibitor, tubacin, supporting a role for histone acetylation in associative memory. Immunoblot analysis showed that GluA1 receptor membrane expression in the olfactory bulbs of the TSA-treated group was significantly increased at 48 h unlike control rats without TSA. Immunohistochemistry revealed significant increase of GluA1 expression in olfactory bulb glomeruli 5 d after training. These results extend previous evidence for a close relationship between enhanced GluA1 receptor membrane expression and memory expression. Together, these findings provide a new single-trial appetitive model for understanding the support and maintenance of memories of varying duration.

Inhibition of HDAC6 increases acetylation of peroxiredoxin1/2 and ameliorates 6-OHDA induced dopaminergic injury.

Histone deacetylase 6 (HDAC6) has been regarded as an unusual HDAC because of its unique properties. It contains two deacetylase catalytic domains and one ubiquitin-binding domain, thus exerting both enzymatic and non-enzymatic actions on cellular function. To date, the ubiquitin-binding activity of HDAC6 has been implicated in several neurodegenerative disorders including Parkinson's disease (PD). However, the deacetylation effect of HDAC6 in PD has not been fully illustrated. Therefore, the aim of the present study was to explore the role of deacetyation activity of HDAC6 in PD. We used an in vivo 6-OHDA induced PD model and a specific HDAC6 inhibitor tubastatin A to investigate the acetylation levels of peroxiredoxin1 (Prx1) and peroxiredoxin2 (Prx2) and to explore the effects of tubastain A on nigrostriatal dopaminergic system. Our results showed that expression of HDAC6 significantly increased in dopaminergic neurons after 6-OHDA injury. Acetylation levels of Prx1 and Prx2 decreased. Pharmacological inhibition of HDAC6 with specific inhibitor tubastatin A increased acetylation of Prx1 and Prx2, reduced ROS production and ameliorated dopaminergic neurotoxicity. Our results for the first time provide evidence that HDAC6 medicated deacetylation of Prx1 and Prx2 contributes to oxidative injury in PD, suggesting that the development of specific HDAC6 inhibitor is required to develop more effective therapeutic strategies to treat PD.

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS.

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography - mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

An inhibitor of histone deacetylase 6 activity, ACY-1215, reduces cAMP and cyst growth in polycystic kidney disease.

Adult-onset autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in either the PKD1 or PKD2 gene, leading to malfunction of their gene products, polycystin 1 or 2. Histone deacetylase 6 (HDAC6) expression and activity are increased in PKD1 mutant renal epithelial cells. Here we studied the effect of ACY-1215, a specific HDAC6 inhibitor, on cyst growth in ADPKD. Treatment with ACY-1215 slowed cyst growth in a mouse model of ADPKD that forms massive cysts within 3 wk after knockout of polycystin 1 function. It also prevented cyst formation in MDCK.2 cells, an in vitro model of cystogenesis, and in an ADPKD cell line derived from the proximal tubules from a pkd1-/-.mouse (PN cells). In PN cells ACY-1215 also reduced the size of already established cysts. We found that ACY-1215 lowered cAMP levels and protein expression of adenylyl cyclase 6. Our results suggest that HDAC6 could potentially serve as a therapeutic target in ADPKD.

Abstract 2326: A high-throughput screen identified cancer selective small molecules that kill independent of mitochondrial apoptosis

Abstract Triple negative breast cancer (TNBC) lacks expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2). Importantly, there have been fewer advances in the treatment of TNBC with the mainstay of treatment being cytotoxic chemotherapy. All too often though tumor responds poorly to chemotherapy, or relapse and resistance follow an initial response. We previously found in a range of hematological and solid tumors that chemoresistant cancers are far from the mitochondrial apoptotic threshold. To identify novel therapeutics for the treatment of chemoresistant breast cancer, we performed a high-throughput screen to identify small molecules that are cancer selective but can kill independent of the mitochondrial pathway of apoptosis. To perform this screen we used TNBC line MDA-MB-231 in which the pro-death proteins BAX/BAK were knocked down. BAX/BAK are essential for mitochondrial apoptosis. To enrich for compounds that were not non-specifically toxic, we simultaneously counter-screened against the non-transformed breast epithelial cell line MCF10a. We screened a total of 30,000 compounds in duplicate across the two cell lines. There was a hit rate of 0.3% in the screen and 85 compounds were retested in the validation cherry pick screen. From the cherry pick screen 18 compounds were further validated with low-throughput assessment for mitochondrial independent killing and selectivity for cancer cells. To identify the mechanism of action of the lead compound we used a genetic approach to generate an RNAi signature for the compound. This signature enables classification of the compound into known or novel class of compounds. The lead compound signature was most like that of histone deacetylase inhibitors (HDAC). Using an in vitro HDAC inhibitor screen, we identified that the compound selectively inhibited HDAC6. Importantly, we validated that the lead compound caused an increase in acetylated tubulin in cells, a target of HDAC6. In conclusion, we have identified a novel HDAC6 specific inhibitor that selectively kills cancer cells independent of mitochondrial apoptosis. Citation Format: Catriona M. Dowling, Micheal Hemann, James Bradner, Anthony Letai, Triona Ni Chonghaile. A high-throughput screen identified cancer selective small molecules that kill independent of mitochondrial apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2326. doi:10.1158/1538-7445.AM2017-2326

Abstract 2386: HDAC6 regulates ERK1’s kinase activity via deacetylation

Abstract Extracellular signal-regulated kinases 1/2 (ERK1/2) are important kinases regulating cell proliferation and cell migration, and have been established as therapeutic targets for cancer treatment. Previously, we found that ERK1 phosphorylates histone deacetylase 6 (HDAC6), a class IIb HDAC, to regulate its enzymatic activity. HDAC6 plays an important role in several types of cancers. HDAC6 governs cancer cells’ migration by cooperating with cytoskeleton proteins and their associated proteins, and HDAC6 is arising as a determinant for cisplatin-resistance in non-small cell lung cancer (NSCLC) and ovarian cancer. Here, we have shown that HDAC6 manipulates ERK1’s kinase activity via deacetylation. We demonstrated that both ERK1 and ERK2 interact with HDAC6 physically via GST pull-down assays. To examine whether ERK1/2 are acetylated, we transfected GST-ERK1 or GST-ERK2 into HEK293T cells, and treated the cells with a pan-HDAC inhibitor, Trichostatin A. We showed that the level of acetylated GST-ERK1/2 increased in a dose- and time-dependent manner. Furthermore, when GST-ERK1 or GST-ERK2-expressed cells were treated with an HDAC6-specific inhibitor, ACY-1215, the level of acetylated GST-ERK1/2 increased. These results suggest that HDAC6 is a deacetylase for ERK1/2. In addition, we determined that acetyltransferases CBP and p300 acetylate ERK1/2. We found a novel acetylation site located in ERK1 N-terminus by mass-spectrometry analysis. We then mutated this lysine (K) site with either Glutamic acid (Q) or Arginine (R) to mimic the acetylation or deacetylation status, and performed kinase assays using ELK1 as a substrate. The acetylation mimicking mutant exhibits a decreased kinase activity toward ELK1, while the deacetylation mimicking mutant exhibits a similar level of kinase activity as the wild-type ERK1 does, suggesting that acetylation/deacetylation alters ERK1 enzymatic activities. Therefore, HDAC6 may regulate ERK1’s kinase activity via deacetylation of this site. Furthermore, we have shown that HDAC6 is overexpressed in lung cancer tissue samples and plays an important role in chemo-sensitivity in lung cancer. We predict that inhibition of the HDAC6-ERK1 pathway may provide clinical benefit for lung cancer patients. Citation Format: Jheng-Yu Wu, Xiaohong Zhang. HDAC6 regulates ERK1’s kinase activity via deacetylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2386. doi:10.1158/1538-7445.AM2017-2386

The Contribution of Necroptosis in Neurodegenerative Diseases.

Over the past decades, cell apoptosis has been significantly reputed as an accidental, redundant and alternative manner of cell demise which partakes in homeostasis in the development of extensive diseases. Nevertheless, necroptosis, another novel manner of cell death through a caspase-independent way, especially in neurodegenerative diseases remains ambiguous. The cognition of this form of cell demise is helpful to understand other forms of morphological resemblance of necrosis. Additionally, the concrete signal mechanism in the regulation of necroptosis is beneficial to the diagnosis and treatment of neurodegenerative diseases. Recent studies have demonstrated that necroptotic inhibitor, 24(S)-Hydroxycholesterol and partial specific histone deacetylase inhibitors could alleviate pathogenetic conditions of neurodegenerative diseases via necroptosis pathway. In this review, we summarize recent researches about mechanisms and modulation of necroptotic signaling pathways and probe into the role of programmed necroptotic cell demise in neurodegenerative diseases such as Parkinson's disease, Multiple sclerosis, Amyotrophic lateral sclerosis.

Epigenetics regulates transcription and pathogenesis in the parasite Trichomonas vaginalis.

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Different T.vaginalis strains vary greatly in their adherence and cytolytic capacities. These phenotypic differences might be attributed to differentially expressed genes as a consequence of extra-genetic variation, such as epigenetic modifications. In this study, we explored the role of histone acetylation in regulating gene transcription and pathogenesis in T.vaginalis. Here, we show that histone 3 lysine acetylation (H3KAc) is enriched in nucleosomes positioned around the transcription start site of active genes (BAP1 and BAP2) in a highly adherent parasite strain; compared with the low acetylation abundance in contrast to that observed in a less-adherent strain that expresses these genes at low levels. Additionally, exposition of less-adherent strain with a specific histone deacetylases inhibitor, trichostatin A, upregulated the transcription of BAP1 and BAP2 genes in concomitance with an increase in H3KAc abundance and chromatin accessibility around their transcription start sites. Moreover, we demonstrated that the binding of initiator binding protein, the transcription factor responsible for the initiation of transcription of ~75% of known T.vaginalis genes, depends on the histone acetylation state around the metazoan-like initiator to which initiator binding protein binds. Finally, we found that trichostatin A treatment increased parasite aggregation and adherence to host cells. Our data demonstrated for the first time that H3KAc is a permissive histone modification that functions to mediate both transcription and pathogenesis of the parasite T.vaginalis.

Declined Expression of Histone Deacetylase 6 Contributes to Periodontal Ligament Stem Cell Aging.

Identification of regulators for aging-associated stem cell (SC) dysfunctions is a critical topic in SC biology and SC-based therapies. Periodontal ligament stem cell (PDLSC), a kind of dental mesenchymal SC with dental regeneration potential, ages with functional deterioration in both in vivo and ex vivo expansion. However, little is known about regulators for PDLSC aging. Expression changes of a potential regulator for PDLSC aging, histone deacetylase 6 (HDAC6), were evaluated within various models. Senescence-associated phenotypic and functional alternations of PDLSC in loss-of-function models for HDAC6 were examined using HDAC6-specific pharmacologic inhibitors or RNA interference-based knockdown. Involvement of p27Kip1 in HDAC6-associated aging was demonstrated by its acetylation and stability changes along with overexpression or functional inhibition of HDAC6. Expression of HDAC6 decreased significantly in replicative senescence and induced SC aging models. Loss-of-function experiments suggested that pharmacologic inhibition of deacetylase activity of HDAC6 accelerated PDLSC senescence and impaired its SC activities, which showed reduced osteogenic differentiation and diminished migration capacities. Examination of markers for proliferative exhaustion of SCs revealed that protein level of p27Kip1 was specifically elevated after HDAC6 inhibition. HDAC6 physically interacted with p27Kip1 and could deacetylate p27Kip1. Importantly, acetylation of p27Kip1 was negatively regulated by HDAC6, which correlated with alteration of p27Kip1 protein levels. Data suggest that HDAC6 plays an important role in PDLSC aging, which is dependent, at least partially, on regulation of p27Kip1 acetylation.