Specific Histone Deacetylase Inhibitor Research Articles

Histone deacetylase 6 promotes growth of glioblastoma through inhibition of SMAD2 signaling.

Histone deacetylases (HDACs) play a role in the tumorigenesis of glioblastoma multiforme (GBM), whereas the underlying mechanism has not been elucidated. Here, we reported significantly higher HDAC6 levels in GBM from the patients. GBM cell growth was significantly inhibited by ACY-1215, a specific HDAC6 inhibitor. Further analyses show that HDAC6 may promote growth of GBM cells through inhibition of SMAD2 phosphorylation to downregulate p21. Thus, our data demonstrate a previously unrecognized regulation pathway in that HDAC6 increases GBM growth through attenuating transforming growth factor β (TGFβ) receptor signaling.

Inhibiting histone deacetylase 6 partly protects cultured rat cortical neurons from oxygen‑glucose deprivation‑induced necroptosis.

Necroptosis has an important role in ischemia-reperfusion damage. The expression of histone deacetylase 6 (HDAC6) is upregulated in neurons following ischemia-reperfusion, however, whether HDAC6 is closely involved in the necroptosis, which occurs during ischemia-reperfusion damage remains to be elucidated. In the present study, the roles of HDAC6 in the necroptosis of cultured rat cortical neurons were investigated in a oxygen-glucose deprivation (OGD) model. The results demonstrated that OGD induced marked necroptosis of cultured rat cortical neurons and upregulated the expression of HDAC6 in the cultured neurons, compared with the control (P<0.05). The necroptosis inhibitor, necrostatin-1 (Nec-1), decreased The expression of HDAC6 in the OGD-treated cultured neurons, accompanied by the inhibition of necroptosis. Further investigation revealed that, compared with OGD treatment alone, inhibiting the activity of HDAC6 with tubacin, a specific HDAC6 inhibitor, reduced the OGD-induced necroptosis of the cultured rat cortical neurons (P<0.05), which was similar to the change following treatment with Nec-1 (P>0.05). In addition, inhibiting the activity of HDAC6 reversed the OGD-induced increase of reactive oxygen species (ROS) and the OGD-induced decrease of acetylated tubulin in the cultured rat cortical neurons (P<0.05), compared with the neurons treated with OGD alone). The levels of acetylated tubulin in the cultured neurons following treatment with OGD and tubacin were significantly higher than those in the control (P<0.05). These results suggested that HDAC6 was involved in the necroptosis of neurons during ischemia-reperfusion by modulating the levels of ROS and acetylated tubulin.

The Class I HDAC inhibitor RGFP963 enhances consolidation of cued fear extinction.

Evidence indicates that broad, nonspecific histone deacetylase (HDAC) inhibition enhances learning and memory, however, the contribution of the various HDACs to specific forms of learning is incompletely understood. Here, we show that the Class I HDAC inhibitor, RGFP963, enhances consolidation of cued fear extinction. However, RGFP966, a strong inhibitor of HDAC3, does not significantly enhance consolidation of cued fear extinction. These data extend previous evidence that demonstrate the Class I HDACs play a role in the consolidation of long-term memory, suggesting that HDAC1 and/or HDAC2, but less likely HDAC3, may function as negative regulators of extinction retention. The development of specific HDAC inhibitors, such as RGFP963, will further illuminate the role of specific HDACs in various types of learning and memory. Moreover, HDAC inhibitors that enhance cued fear extinction may show translational promise for the treatment of fear-related disorders, including post-traumatic stress disorder (PTSD).

Suppression of monosodium urate crystal-induced cytokine production by butyrate is mediated by the inhibition of class I histone deacetylases

ObjectivesAcute gouty arthritis is caused by endogenously formed monosodium urate (MSU) crystals, which are potent activators of the NLRP3 inflammasome. However, to induce the release of active interleukin (IL)-1β, an...

Sulforaphane Inhibits Metastatic Events in Breast Cancer Cells through Genetic and Epigenetic Regulation

Metastasis is a deadly event in carcinogenesis, which is regulated both genetically as well as epigenetically. Regulation of key molecules involved in this phenomenon could be a promising strategy in cancer control. The epigenetic enzyme HDAC6 along with telomerase and HSP90, two genetic markers of carcinogenesis are implicated in the metastatic pathway. Therefore, modulation of these markers may aid in prevention of spread of cancer to distant parts. Plant derived molecules are apparently nontoxic and stud with anticancer activities. Present study aims to investigate the effect of sulforaphane (Sfn), an organosulfur compound on these markers, which might be helpful in inhibition of metastasis. It was observed that sulforaphane significantly inhibited HDAC6 expression, both at protein and genetic level in metastatic breast cancer cell MDA-MB-231. Inhibition of HDAC6 was associated with increased acetylation of HSP90 and diminished expression of transcription factor c-myc. These results were further confirmed by using tubacin, a specific HDAC6 inhibitor. Activity and expression of human telomerase reverse transcriptase, a main determinant of catalytic activity of the enzyme was found to be inhibited by Sfn. Repression of c-myc led to transcriptional down-regulation of hTERT mRNA and de-repression of p21. Modulation of these proteins led to down-regulation of VEGF and MMPs (2 and 9), two key players of the metastatic event. Regulation of these proteins by Sfn decelerates migration and invasion of the metastatic breast cancer cells, thereby showing anti-metastatic potential. Sulforaphane, by virtue of its modulatory role on HDAC6 and other associated proteins may lead to inhibition of metastasis in breast cancer cells.

Drug Combinations with HDAC Inhibitors in Antitumor Therapy.

The treatment of tumor cells with HDAC inhibitors (HDACi) induces a range of effects including apoptosis, cell cycle arrest, differentiation and senescence, modulation of immune response, and altered angiogenesis. The single-agent activities of several HDACi have been tested in preclinical and clinical studies and are currently the subject of ongoing clinical trials. Although HDACi have been shown to be effective as a single agent against a defined subset of hematological tumors, less convincing results have been found in the treatment of solid tumors. Since current clinical trials of single-agent HDACi showed limited efficacy, in this review we focus on drug combinations including HDACi with conventional chemotherapeutic agents and novel targeted agents. Particular emphasis has been devoted to combinations effective in solid tumors and to combinations between HDACi and immunotherapies. An outline of novel combination strategies, including a new generation of more potent and specific HDACi (e.g., compounds with adamantine and noradamantane as scaffolds) as well as chemical hybrid molecules, has been provided. The paradoxical role of HDACs as tumor suppressors in developing tumors and as therapeutic targets in established neoplasms has also been considered.

Modulation of immune responses by histone deacetylase inhibitors.

Recent studies have demonstrated that histone deacetylase (HDAC) inhibitors (HDACi) have potential immunomodulatory activity since they affect the immune surveillance by regulating the production of cytokines, alter the activity and function of macrophages and dendritic cells (DC), regulate the transcription of a variety of immune-stimulating genes, and can modulate the activity of immune effector cells of both the innate and adaptive immune system. Besides their immunostimulatory activity, HDACi can induce growth arrest and cell death, and modulate a subset of cellular functions such as cell motility or differentiation. This makes HDACi interesting therapeutic candidates for the treatment of a variety of human diseases like cancer, autoimmune, and graft versus host diseases. Besides these, HDACs have been shown to be involved in virus replication and pathogenesis, and it was recently shown that HDACi provide therapeutic effects in the treatment of oncogenic virus infections and associated malignancies. This review will further give information about the different families of HDACs and their opponents, the histone acetylases (HATs), about the classes and function of specific HDACi, and their use in the treatment of human diseases.

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Objectives Human cytomegalovirus (HCMV) infection of the placenta is associated with significant pathological consequences, including pregnancy loss, intrauterine growth restriction, preeclampsia and preterm labor. It is thought that some of these pathological anomalies are attributed to the inability of extravillous cytotrophoblasts (EVT) to adequately invade the maternal uterine wall during first trimester pregnancy resulting in impaired remodeling of maternal spiral arteries. Our earlier studies show that HCMV inhibits cytotrophoblast invasion, although the mechanism(s) involved are not fully understood. We hypothesize that HCMV inhibits activity of histone deacetylase 6 (HDAC6) that is involved in the balanced control of deacetylation/acetylation of alpha-tubulin and cortactin that is essential for invadosome formation and function. The purpose of this study is to determine whether HCMV inhibits invasion of EVT through dysregulation of HDAC6-dependent invadosome formation and function. Methods An organotypic, 3-dimensional (3-D) model system using primary first trimester trophoblast progenitor cells (TBPCs) or the SGHPL-4 cell line was used to induce differentiation of cytotrophoblasts into an invasive phenotype. Invasive cytotrophoblasts were plated on fluorescein-labeled gelatin and analyzed for invadosome formation and function by confocal microscopy. Cells were infected with HCMV or treated with HDAC6-specific inhibitors to determine the ability of both to abrogate EVT invasion and to increase levels of acetylated alpha-tubulin, a cytoplasmic substrate of HDAC6, the latter of which has been shown, in cancer cells, to be required for invadosome formation and function. Results Cytotrophoblasts cultured in 3-D form actin-rich invasive protrusions that degrade extracellular matrix. HDAC6-specific inhibition and HCMV infection inhibit cytotrophoblast invasion and increase levels of acetylated alpha-tubulin. Conclusions These data suggest that HCMV impairs trophoblast invadosome formation and function through dysregulation of HDAC6. Elucidating the mechanism(s) by which HCMV impairs placentation may be key to understanding and inhibiting fetal and maternal pathologies, such as preeclampsia. Disclosures K.F. Swan: None. M. Ferris: None. G. Pridjian: None. D.E. Sullivan: None. C.A. Morris: None.

Histone Deacetylase 6 (HDAC6) Regulates Gene Transcription and Enucleation during Erythroid Differentiation

The deacetylation of histone and non-histone proteins by histone deacetylase (HDACs) plays a critical role in gene transcription and many other cellular processes in eukaryotic cells. Class II deacetylase HDAC6 is mainly localized in the cytoplasm and deacetylates tubulin and other cytoplasmic proteins. Earlier studies from our laboratory showed that HDAC6 can be transported to the nucleus, and can interact and deacetylate nuclear proteins, such as histones. In this study, we investigate the function of HDAC6 during erythroid differentiation. In proerythroblast cells, HDAC6 is detected in the nucleus and gradually migrates to the cytoplasm upon the induction of differentiation, indicating different HDAC6 functions during erythropoiesis. Inhibition of HDAC6 in mouse fetal liver erythroblasts impairs differentiation and enucleation. β-globin gene transcription is reduced by HDAC6 specific inhibitor, Tubastatin A, in cultured fetal erythroblasts. HDAC6 knockout mice also showed impaired globin gene transcription. HDAC6 specifically interacts with serine 2 phosphorylated Pol II and is recruited mainly at the transcribed region of β-globin, correlating with RNA Pol II recruitment. These results suggest that HDAC6 promotes erythroblast differentiation through the regulation of transcription elongation. HDAC6 also plays a role in regulating the enucleation process during the late stage of erythropoiesis. The formation of the contractile actin ring is disrupted and the enucleation process is blocked in cultured mouse fetal erythroblasts treated with the HDAC6 inhibitor. We further investigated the molecular mechanism of HDAC6 regulated enucleation. We found that HDAC6 interacts with and deacetylates mDia2, an effector of Rho GTPases that is required for erythroblast enucleation. Deacetylation of mDia2 is required for the formation of the contractile actin ring and subsequent enucleation. Altogether, our results identify the important role of HDAC6 in regulating transcription and enucleation during the different stages of mammalian erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

HDAC Inhibitors As Potential New Agents Improving the Efficacy of Monoclonal Antibodies

HDAC (histone deacetylase) inhibitors belong to a novel class of antitumor agents widely explored in hematology. So far two of them - romidepsin and vorinostat have been registered in cutaneous T-cell lymphoma, while panobinostat awaits FDA decision concerning its registration in multiple myeloma. Several other agents are currently being tested in clinical trials in lymphoma and myeloma patients and preliminary results suggest that they may be most effective when used in combinations with other anti-cancer drugs. Reports from others (Sugimoto et al. Biochem Biophys Res Commun 2009 Shimizu et al. Leukemia 2010) and the results from our group show that HDAC inhibitors augment the efficacy of anti-CD20 monoclonal antibodies (mAbs) in complement-dependent cytotoxicity (CDC) by up-regulating CD20 level. In our previous reports we have shown that HDAC6 isoform is involved in the regulation of CD20 degradation and trafficking. Since the efficacy of mAbs depends not only on the level of target molecule but is also regulated by the expression of complement regulatory proteins (mCRPs) - CD46, CD55 and CD59, we sought to investigate the influence of HDAC inhibitors on the expression of those molecules. Moreover, given the fact that HDAC inhibitors have been reported to regulate NK cells activity, we investigated their influence on antibody-dependent cell-mediated cytotoxicity (ADCC) which is one of the mechanisms of action of mAbs in vivo.The purpose of this study was to thoroughly investigate the influence of HDAC inhibition on the level of surface molecules – CD20 and CD38 – targets already used and tested in clinical trials in immunotherapy of B-cell malignancies as well as the expression of mCRPs. Using non-selective pan-inhibitors as well as selective inhibitors of particular HDAC classes and isoforms we sought to show the inhibition of which HDAC enzyme is the most effective in potentiating the outcome of mAbs based anti-tumor therapies. Since the results of clinical trials show that the use of HDAC inhibitors has several non-specific adverse effects, we hypothesize that selective inhibition of particular isoforms may considerably diminish these toxicities.Using flow cytometry we performed experiments assessing the level of surface CD20 CD38 and mCRPs – CD46, CD55 and CD59. We used established lymphoma, chronic lymphocytic leukemia (CLL) and myeloma cell lines as well as primary cells isolated from the patients suffering from CLL. We observed that the use of HDAC pan-inhibitors – trichostatin A (TsA) and vorinostat (Fig.1A) and selective HDAC6 inhibitors – tubacin and ACY1215 (ricolinostat) (Fig.1B) considerably improves the outcome of anti-CD20 mAbs – rituximab and ofatumumab in CDC in several B-cell lymphoma cell lines and in CLL primary samples (Fig. 1C). In our experiments we show that selective inhibition of HDAC6 in Raji cells does not influence the expression of mCRPs (Fig. 1D). However, we observed that pre-incubation with tubacin considerably increases the expression of CD38 (Fig. 1 D). Therefore, in our further studies we aim at expanding our observations to primary samples from multiple myeloma patients, especially since novel anti-CD38 mAbs are currently intensively explored in the therapy of this malignancy.Moreover, using flow cytometry we determined the influence of HDAC inhibition on degranulation of NK cells isolated from healthy donors in ADCC in vitro assay with rituximab and ofatumumab. Contrary to the reports of others, we observed no negative regulation of NK cells activity and their ability to kill target tumor cells. Therefore, since HDAC inhibitors seem to influence mainly CDC, we investigated the influence of specific HDAC inhibitors on the level of mCRPs. Those results suggest that specific inhibition of HDAC3 (Fig. 1E) and HDAC1/2 (Fig. 1F) are effective therapeutic strategies as they considerably decreased the expression of complement inhibitors and concomitantly increased the level of CD20 antigen. However, further studies including silencing of particular HDAC isoforms with shRNA are needed to confirm these findings. Altogether, our results strongly suggest that HDAC inhibitors potentiate the outcome of mAbs used in hematological malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Short-Chain Fatty Acids Enhance Adipocyte Differentiation in the Stromal Vascular Fraction of Porcine Adipose Tissue

Background: Short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are the main products of microbial fermentation in the gut and might mediate some of the effects of gut microbiota and nutrition on development, metabolism, and pathogenesis of obesity and other diseases.Objective: The objective of this study was to determine the effects of SCFAs on adipocyte differentiation and the underlying mechanism.Methods: The stromal vascular fraction (SVF) of the porcine subcutaneous fat was used as the preadipocyte model. Adipocyte differentiation was assessed by Oil Red O staining and gene expression analysis of adipocyte markers. Chromatin immunoprecipitation was used to assess the histone acetylation amounts at the peroxisome proliferator-activated receptor γ (PPARG) and CCAAT/enhancer binding protein α (CEBPA) promoters.Results: Compared with control, propionate and butyrate enhanced the formation of adipocytes by 10–20% and mRNA expression of adipocyte markers by 20–200% in porcine SVF undergoing adipocyte differentiation. Compared with control, short-term treatment of propionate and butyrate enhanced PPARG and CEBPA mRNA expression in porcine SVF by 50–100%. Neither free fatty acid receptor (FFAR) 2 nor FFAR3 mRNA was detectable in porcine SVF before or during differentiation. Neither a cAMP analogue nor an activator of AMP-activated protein kinase (AMPK) affected propionate- or butyrate-enhanced expression of PPARG or CEBPA mRNA. Trichostatin A, a specific inhibitor of histone deacetylases (HDACs), enhanced the formation of adipocytes in porcine SVF by nearly 100% and the expression of PPARG and CEBPA mRNAs by 150% and 50%, respectively. Butyrate increased whereas propionate had no significant effect on histone H3 acetylation at the CEBPA promoter in porcine SVF.Conclusions: Propionate and butyrate enhance adipocyte differentiation in porcine SVF. These effects are unlikely mediated through FFAR2, FFAR3, cAMP, or AMPK. The effect of butyrate may be partially mediated by its HDAC inhibitory activity, whereas that of propionate is independent of its HDAC inhibitory activity.

Abstract 20085: Inhibition of Hdac Induces Protective Effects Against Diabetic Cardiomyopathy

Background: It is known that inhibition of histone deacetylases (HDACs) protects the heart against ischemic injury. However, it remains unknown whether HDAC inhibition produces the cardioprotective effect in the diabetic heart. Objective: We sought to determine whether HDAC inhibition preserves cardiac performance and suppresses cardiac remodeling in diabetic cardiomyopathy. Methods: Adult ICR male mice received an intraperitoneal injection (i.p.) of streptozotocin (STZ, 200mg/kg) to establish the diabetic model. Once hyperglycemia was confirmed, diabetic mice received sodium butyrate, a specific HDAC inhibitor (1%) in drinking water on a daily basis. Myocardial function was assessed by echocardiography. Cardiac hypertrophy, fibrosis, and myocyte death were evaluated by histological analysis. Western blot was employed to determine the proteins of signaling pathways. Results: Left ventricular function was depressed in diabetic mice as compared with control group. HDAC inhibition resulted in an improvement in cardiac function in STZ-injected mice (Table1). Likewise, HDAC inhibition attenuates cardiac hypertrophy, as evidenced by a reduced heart/tibia ratio and areas of cardiomyocytes. Additionally, sodium butyrate reduced interstitial fibrosis and apoptosis, but increased angiogenesis in diabetic myocardium. Notably, glucose transporter type 1 and 4 (GLUT1, GLUT4) and superoxide dismutase (SOD 1) were elevated following HDAC inhibition in the diabetic myocardium. GLUT1 acetylation was increased in diabetic myocardium following HDAC inhibition. Conclusion: HDAC inhibition plays a critical role to reduce cardiac damage in diabetic mice.

Histone deacetylase inhibitor sodium butyrate attenuates gentamicin-induced hearing loss in vivo

ObjectiveAlthough histone deacetylase (HDAC) inhibition has been shown to protect against gentamicin (GM)-induced hearing loss in vitro, its protective effect has not been proven in vivo. In the present study, the aim was to investigate the protective effect of sodium butyrate (NaB), a specific HDAC inhibitor, on GM-induced ototoxicity in vivo. MethodsForty 8-week-old albino guinea pigs were divided into two experimental groups. Group 1 (n=10) underwent bilateral ear surgery to place sponges (0.3mm3) permeated with NaB (10μl, 100mg/ml) and physiological saline (10μl; control) in the right and left round window niches, respectively. The sponges were left in place for 15days to evaluate the effects of NaB at the applied concentration. Group 2 (n=30) underwent the same bilateral ear surgery described for Group 1, except three days after surgery, the animals received intramuscular GM injections (200mg/kg/day) for 5 consecutive days. Seven days after the final GM injection, the protective effects of NaB were examined. ResultsAfter 15days of NaB treatment (10μl, 100mg/ml), an increase in histone acetylation was detected in Corti organ samples. Auditory brainstem response (ABR) threshold shifts and hair cell loss were also reduced in NaB-treated ears after GM administration. Furthermore, GM treatment increased HDAC1 expression in outer hair cells (OHCs) in vivo, and NaB blocked this action. ConclusionGM increases HDAC1 expression in OHCs, and NaB is able to block this action. Thus, it appears that the HDAC inhibitor, NaB, attenuates GM-induced hearing loss in guinea pigs.

Histone deacetylase 6 inhibition impairs effector CD8 T-cell functions during skin inflammation

Broad-spectrum histone deacetylase (HDAC) inhibitors are useful in the treatment of allergic and autoimmune diseases and malignancy. However, use of more specific HDAC inhibitors might limit the toxicities caused by HDAC inhibition. HDAC6, a member of the HDAC family, is highly expressed on CD8 T cells and has been shown to regulate immune responses through interactions between T cells and antigen-presenting cells. However, the mechanism by which HDAC6 inhibition affects the activation and functions of CD8 T cells is unclear. We investigated the role or roles of HDAC6 in CD8 T-cell activation and functions during skin inflammation in vitro and in vivo and examined the mechanism by which HDAC6 inhibition modifies T-cell receptor signaling in vitro. We assessed the clinical and biological effects of ACY-1215, an HDAC6-specific inhibitor, by using murine CD8 T cell-related skin disease models, including contact hypersensitivity (CHS) and experimental graft-versus-host disease (GVHD)-like disease. ACY-1215, an HDAC6 inhibitor, prevented the development of CHS and GVHD-like disease in vivo by modulating CD8 T-cell activation and functions; abrogated the induction of effector T cells from naive CD8 T cells by means of anti-CD3/CD28 antibody- or antigen-specific stimulation in vitro; and enhanced the binding of acetylated heat shock protein 90 to lymphocyte-specific protein tyrosine kinase in vitro, disrupting lymphocyte-specific protein tyrosine kinase phosphorylation and leading to impairment of the mitogen-activated protein kinase pathway. HDAC6, a key modifier of T-cell receptor signaling, might represent a novel target for the treatment of CD8 T cell-related skin diseases, including CHS and GVHD.

ET-27 * REPLICATION AND SPREAD OF ONCOLYTIC HERPES VIRUS IN GLIOMA STEM CELLS CAN BE ENHANCED BY SPECIFIC INHIBITION OF HISTONE DEACETYLASE 6

High-grade gliomas remain clinically challenging and under current therapies the prognosis is generally dismal. Intratumoral heterogeneity, therapy resistance, and tumor recurrence likely caused by glioma stem cells (GSCs) still represent major challenges, which need to be addresses by novel, multimodal therapies. We attempted to combine rQnestin34.5, an oncolytic herpes simplex virus expressing its ICP34.5 virulence gene under the glioma-specific nestin promoter, with specific inhibition of histone deacetylase 6 (HDAC6). We have previously reported that pan-HDAC inhibition using valproic acid enhances rQnestin34.5's antitumor activity and our data obtained in established glioma cell lines indicate that sole inhibition of HDAC6 can at least in part be accounted for this effect. We now analyzed the influence of the specific HDAC6 inhibitor Tubacin on rQnestin34.5 replication in GSC lines originating from proneural, classic, and mesenchymal glioma subtypes. Time-lapse microscopy revealed that plaque formation and spread were accelerated within the first 48 hours after infection when cells were pre-treated with Tubacin, translating into significantly increased cytotoxicity in the respective time frame. Viral DNA replication was enhanced after combination treatment as increased viral copy numbers per cell were detected by qPCR. Furthermore, HDAC6 inhibition increased mRNA expression levels of immediate early and late genes. Importantly, the positive effect of Tubacin on rQnestin34.5 replication and spread in GSCs was critically dependent on drug pre-treatment regimens and very low infectious titers. We also noted that the benefit of rQnestin34.5 oncolysis from HDAC6 inhibition varied quantitatively between GSCs. While responsiveness was not determined by glioma subtype or HDAC6 expression levels, we are currently investigating whether the choice of the exact viral entry mechanism can be used as a predictive marker. Future studies will also aim at establishing a therapeutic regimen demonstrating the potential benefit of HDAC6 inhibition in addition to herpes simplex oncolysis in a GSC-derived orthotopic glioma model.

Abstract 4086: Histone deacetylase 6 (HDAC6) as a new target modulating the proliferation and immune-related pathways in melanoma

Abstract Histone deacetylases (HDACs), originally described as histone modifiers, have more recently been demonstrated to modify a variety of other proteins involved in diverse cellular processes unrelated to the chromatin environment. This includes the deacetylation of multiple non-histone targets, such as proteins involved in cell cycle/apoptosis and immune regulation. Specifically, HDACs have garnered significant interest due to the availability of drugs that selectively inhibits HDACs. We recently identified that the pharmacological or genetic abrogation of a single HDAC, HDAC6, modifies the immunogenicity and proliferation of melanoma in both in vitro and in vivo models. Using specific HDAC6 inhibitors (HDAC6i) we observed decreased proliferation and G1 cell cycle arrest in all melanoma cell lines measured by MTS assay and flow cytometry. These results were also observed in stable HDAC6 knockdown melanoma cell lines (HDAC6KD) generated by specific lentiviral shRNA for HDAC6. In addition to the effects observed in proliferation and apoptosis after inhibiting HDAC6, we found important changes in the expression of immune-related pathways, including increased expression of MHC, co-stimulatory molecules, and specific melanoma tumor associated antigens such as gp100, MART-1, Tyrp1 and Tyrp2. Our in vitro results were further supported by in vivo tumor growth studies. We observed a delayed tumor growth of inoculated B16 melanoma cells in C57BL/6 mice treated with selective HDAC6i. A similar outcome was identified after inoculation of HDAC6KD B16 melanoma cells in C57BL/6 mice. Such an effect was reverted partially in CD4+ and CD8+ depleted C57BL/6 mice challenged with HDAC6KD cells, suggesting that the disruption of HDAC6 enhances immune system recognition of melanoma cells. This delay in tumor growth could be a reflection of their diminished proliferation and an increase in their immunogenicity leading to improved immune recognition and clearance. These studies provide critical insights into the molecular pathways that are involved in the regulatory role of HDAC6 in cell proliferation, survival, and cytokine signaling of human melanoma cells. Collectively, our data has identified HDAC6 as an attractive therapeutic target in melanoma. Citation Format: Patricio Perez-Villarroel, Maritza Lienlaf, Calvin Lee, Fengdong Cheng, David Woods, Kelly Barrios, Karrune Woan, Jorge Canales, Tessa Knox, Danay Marante, Hongwei Wang, Pedro Horna, Keiran Smalley, Esteban Celis, Ed Seto, Jeffrey S. Weber, Eduardo M. Sotomayor, Alejandro Villagra. Histone deacetylase 6 (HDAC6) as a new target modulating the proliferation and immune-related pathways in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4086. doi:10.1158/1538-7445.AM2014-4086

Abstract 5541: Proteasome inhibitor synergizes with histone deacetylase inhibitor to trigger ROS- and ER stress-induced apoptosis of nasopharyngeal carcinoma independent of aggresome disruption

Abstract Proteasome inhibitor and histone deacetylase inhibitor (HDACi) can synergistically induce apoptosis of cancer cells through aggresome disruption or induction of endoplasmic reticulum (ER) stress. We previously reported that suberoylanilide hydroxamic acid (SAHA), a pan-HDACi, and bortezomib, a proteasome inhibitor can synergistically induce apoptosis of nasopharyngeal carcinoma (NPC) cells through reactive oxygen species (ROS)-driven caspase-dependent mechanism. Here, we wish to investigate the role of aggresome disruption or ER stress in the process. We first tested the effect of bortezomib combining with a more specific HDACi, including MS-275, apicidin or romidepsin, on proliferation of NPC cells. Bortezomib could synergize with each of the specific HDACis to induce killing of the NPC cells. Strong apoptosis was demonstrated by the proteolytic cleavage of PARP and caspase-3, -8 and -9 as well as the high percentage of annexin V/propidium iodide (AV/PI)-positive NPC cells upon treatment with combination of bortezomib at 7.5 to 15 nM and romidepsin at 2.5 to 5 nM. Consistently, the apoptosis was ROS- and caspase-dependent. Interestingly, α-tubulin, a key substrate of histone deacetylase 6, was not acetylated, arguing against the involvement of aggresome disruption. In contrast, ER stress induction might be involved because ATF-4 and CHOP, which are markers of ER stress, were up-regulated by combined bortezomib/romidepsin and inhibition of caspase-4 could suppress the apoptosis. Furthermore, addition of ROS scavenger, N-acetyl-cysteine, suppressed the expression of ATF-4 and CHOP. We conclude that proteasome inhibitor can synergize with HDACi to trigger ROS- and ER stress-induced apoptosis of NPC cells, independent of aggresome disruption. This project is funded by NPC Area of Excellence (AoE/M 06/08 Center for Nasopharyngeal Carcinoma Research), CRCG (#10401264) and Epstein-Barr virus research (# 20004525) grants of A.K.S. Chiang. Citation Format: Alan K. Chiang, Kwai Fung Hui. Proteasome inhibitor synergizes with histone deacetylase inhibitor to trigger ROS- and ER stress-induced apoptosis of nasopharyngeal carcinoma independent of aggresome disruption. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5541. doi:10.1158/1538-7445.AM2014-5541

Abstract 4090: Inhibition of class I histone deacetylases promotes robust and durable enhancement of PDL1 expression in melanoma: Rationale for combination therapy

Abstract Histone deacetylase inhibitors (HDACi) have shown remarkable anti-tumor activity, leading to FDA approval of two HDACi for the treatment of CTCL and several others currently at various stages of clinical development for the treatment of both solid and hematological malignancies. Previous work from our lab has shown that treatment with HDACi results in increased expression of pro-inflammatory promoting surface markers on melanoma cells, promoting enhanced T-cell activation. Recent clinical trial data has shown that blockade of the PD1/PDL1 interaction is effective in the treatment of melanoma, renal cell and non-small cell lung cancer. Importantly, responses to PD1 blocking antibodies were preferentially seen in patients with tumors expressing PDL1. Here we report that HDACi targeting class I HDACs, but not class II, augments expression of PDL1 in melanoma cells. Two murine and five human melanoma cell lines were treated for up to 72 hours with DMSO, LBH589 (pan-HDACi), MS275 (class I inhibitor), MGCD0103 (class I inhibitor), an HDAC6 specific inhibitor, or a class IIa inhibitor. Using flow cytometry, dose dependent, increases in PDL1 expression were found in the LBH589, MS275 and MGCD0103 treated groups, but not in those receiving HDAC6i or class IIa inhibitor, relative to DMSO. Increased expression was noted as early as 24 hours after treatment and peaked at 72 to 96 hours post-treatment. As IFN-γ is known to upregulate the expression of PDL1 in both normal and transformed cells, we evaluated whether these results were associated with induction of IFN-γ expression by the melanoma cells. However, no detectable levels of IFN-γ were seen in either non-treated, class I HDACi, or class II HDACi-treated cells. Melanoma cells treated with HDACi in addition to IFN-γ have enhanced expression of PDL1 relative to either treatment alone. To further gain insight into the specific HDAC regulating the expression of PDL1, preliminary experiments utilizing knockdowns (KD) of individual class I HDACs were performed. In all KD melanoma cells no increase in PDL1 expression was seen, suggesting that the increased expression of PDL1 is dependent on inhibition of multiple class I HDACs. Supporting this conclusion, treatment of class I HDAC-KDs with HDACi recapitulates the increased PDL1 expression seen with WT melanoma. Finally, in preliminary in vivo experiments combining treatment of melanoma bearing mice with anti-PDL1 antibodies, mice receiving the combination treatment had a survival advantage over those receiving PDL1 blocking antibodies or HDACi alone. These results provide a strong rationale for the evaluation of combination therapies utilizing PDL1 or PD1 blocking antibodies in combination with HDACi. Furthermore, these results support the need for further development and investigation of iso-specific HDACi in order to obtain more directed therapeutic efficacy. Citation Format: David M. Woods, Andressa L. Sodre, Eva Sahakian, John Powers, Maritza Lienlaf-Moreno, Patricio Perez-Villarroel, Alejandro Villagra, Javier Pinilla-Ibarz, Eduardo Sotomayor. Inhibition of class I histone deacetylases promotes robust and durable enhancement of PDL1 expression in melanoma: Rationale for combination therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4090. doi:10.1158/1538-7445.AM2014-4090

Class I and IIa histone deacetylases have opposite effects on sclerostin gene regulation.

Adult bone mass is controlled by the bone formation repressor sclerostin (SOST). Previously, we have shown that intermittent parathyroid hormone (PTH) bone anabolic therapy involves SOST expression reduction by inhibiting myocyte enhancer factor 2 (MEF2), which activates a distant bone enhancer. Here, we extended our SOST gene regulation studies by analyzing a role of class I and IIa histone deacetylases (HDACs), which are known regulators of MEF2s. Expression analysis using quantitative PCR (qPCR) showed high expression of HDACs 1 and 2, lower amounts of HDACs 3, 5, and 7, low amounts of HDAC4, and no expression of HDACs 8 and 9 in constitutively SOST-expressing UMR106 osteocytic cells. PTH-induced Sost suppression was associated with specific rapid nuclear accumulation of HDAC5 and co-localization with MEF2s in nuclear speckles requiring serine residues 259 and 498, whose phosphorylations control nucleocytoplasmic shuttling. Increasing nuclear levels of HDAC5 in UMR106 by blocking nuclear export with leptomycin B (LepB) or overexpression in transient transfection assays inhibited endogenous Sost transcription and reporter gene expression, respectively. This repressor effect of HDAC5 did not require catalytic activity using specific HDAC inhibitors. In contrast, inhibition of class I HDAC activities and expression using RNA interference suppressed constitutive Sost expression in UMR106 cells. An unbiased comprehensive search for involved HDAC targets using an acetylome analysis revealed several non-histone proteins as candidates. These findings suggest that PTH-mediated Sost repression involves nuclear accumulation of HDAC inhibiting the MEF2-dependent Sost bone enhancer, and class I HDACs are required for constitutive Sost expression in osteocytes.

Involvement of transcription factor XBP1s in the resistance of HDAC6 inhibitor Tubastatin A to superoxidation via acetylation-mediated proteasomal degradation

HDAC6 is a major cytoplasmic deacetylase. XBP1s is a basic-region leucine zipper (bZIP) transcriptional factor. Despite their mutual involvement in the anti-oxidative process, there are no reports about their inter-protein interactions so far. Here we identified a direct link between HDAC6 inhibition and XBP1s transcription activity in anti-oxidative damage. We showed that the specific HDAC6 inhibitor Tubastatin A could up-regulate XBP1s transcriptional activity, thereby increasing anti-oxidative genes expression. Moreover, knock down of XBP1s could significantly abolish the cell growth protection afforded by Tubastatin A. We hypothesize that Tubastatin A acts to increase XBP1s protein levels that are dependent on its HDAC6 deacetylase inhibition via a mechanism involving acetylation-mediated proteasomal degradation, providing novel mechanistic insight into the anti-oxidative effects of HDAC6 inhibition.