Inhibition Of Histone Deacetylase Activity Research Articles

The addition of Valproic acid to concurrent radiation therapy and temozolomide improves patient outcome: a Correlative analysis of RTOG 0525, SEER and a Phase II NCI trial.

Valproic Acid (VPA) is an antiepileptic agent with HDACi (histone deacetylase inhibitor) activity shown to radiosensitize glioblastoma (GBM) cells. We evaluated the addition of VPA to standard radiation therapy (RT) and temozolomide (TMZ) in an open-label, phase II study (NCI-06-C-0112). The intent of the current study was to compare our patient outcomes with modern era standard of care data (RTOG 0525) and general population data (SEER 2006-2013). 37 patients with newly diagnosed GBM were treated in a phase II NCI trial with daily VPA (25 mg/kg) in addition to concurrent RT and TMZ (2006 - 2013) and 411 patients with newly diagnosed GBM were treated in the standard TMZ dose arm of RTOG 0525 (2006 - 2008). Using the SEER database, adult patients (age > 15) with diagnostic codes 9440-9443 (third edition (IDC-O-3) diagnosed between 2006 - 2013 were identified and 6083 were included in the analysis. Kaplan-Meier method was used to estimate OS and PFS. The effect of patient characteristics and clinical factors on OS and PFS was analyzed using univariate analysis and a Cox regression model. A landmark analysis was performed to correlate recurrence to OS and conditional probabilities of surviving an additional 12 months at diagnosis, 6, 12, 18, 24 and 30 months were calculated for both the trial data and the SEER data. Updated median OS in the NCI cohort was 30.9m (22.2- 65.6m), compared to RTOG 0525 18.9m (16.8-20.3m) (p= 0.007) and the SEER cohort of 11m. Median PFS in the NCI cohort was 11.1m (6.6 - 49.6m) compared to RTOG 0525 with a median PFS of 7.5m (6.9-8.2m) (p = 0.004). Younger age, class V RPA and MGMT status were significant for PFS in both the NCI cohort and the RTOG 0525 cohort, in addition KPS was also significant for OS. In comparison to RTOG 0525, the population in the NCI cohort had a more favorable KPS and RPA, and a higher proportion of patients receiving bevacizumab after protocol therapy however with the exception of RPA (V) (8% vs 18%) (0.026), the effects of these factors on PFS and OS were not significantly different between the two cohorts. Previously reported improvements in PFS and OS with the addition of VPA to concurrent RT and TMZ in the NCI phase II study were confirmed by comparison to both a trial population receiving standard of care (RTOG 0525) and a contemporary SEER cohort. These results provide further justification of a phase III trial of VPA/RT/TMZ.

Emodin and emodin-rich rhubarb inhibits histone deacetylase (HDAC) activity and cardiac myocyte hypertrophy

Pathological cardiac hypertrophy is a classical hallmark of heart failure. At the molecular level, inhibition of histone deacetylase (HDAC) enzymes attenuate pathological cardiac hypertrophy in vitro and in vivo. Emodin is an anthraquinone that has been implicated in cardiac protection. However, it is not known if the cardio-protective actions for emodin are mediated through HDAC-dependent regulation of gene expression. Therefore, we hypothesized that emodin would attenuate pathological cardiac hypertrophy via inhibition of HDACs, and that these actions would be reflected in an emodin-rich food like rhubarb. In this study, we demonstrate that emodin and Turkish rhubarb containing emodin inhibit HDAC activity in vitro, with fast-on, slow-off kinetics. Moreover, we show that emodin increased histone acetylation in cardiomyocytes concomitant to global changes in gene expression; gene expression changes were similar to the well-established pan-HDAC inhibitor trichostatin A (TSA). We additionally present evidence that emodin inhibited phenylephrine (PE) and phorbol myristate acetate (PMA)-induced hypertrophy in neonatal rat ventricular myocytes (NRVMs). Lastly, we demonstrate that the cardioprotective actions of emodin are translated to an angiotensin II (Ang) mouse model of cardiac hypertrophy and fibrosis and are linked to HDAC inhibition. These data suggest that emodin blocked pathological cardiac hypertrophy, in part, by inhibiting HDAC-dependent gene expression changes.

Histone Deacetylase Inhibitory and Cytotoxic Activities of theConstituents from the Roots of Sophora Pachycarpa.

Four prenylated flavonoids, including isosophoranone, sophoraflavanone G, alopecurone J, alopecurone P, and a resveratrol derivative HPD (2-(4-hydroxyphenyl)-2,3-dihydrobenzo[b] furan-3,4,6-triol), were isolated from the roots of Sophora pachycarpa. The cytotoxic activity of obtained compounds was evaluated against A2780, A549, HeLa, and HCT116 human cancer cell lines. We also evaluated their histone deacetylase (HDAC) inhibitory activities. Of all compounds tested, alopecurone J was the most active with IC50 values in the range of 9.97−30.91 μM against four cancer cell lines with potent pan-HDAC inhibitory activity (IC50 = 0.08−3.85 μM). Molecular docking experiments of these compounds with HDAC8 displayed potential selective HDAC inhibitory. Molecular docking data showed consistent results in the in-vitro experiments with high selectivity towards HDAC8. The Resveratrol group plays an essential role in HDAC inhibition.

An In Vitro Study Determining the anti-inflammatory activities of sinapinic acid-containing extracts generated from Irish rapeseed meal

Sinapinic acid (SA) has been shown to possess various bioactive properties including anti-diabetic, anti-inflammatory and histone deacetylase inhibitory activities. However, the amount obtained from our diet is insufficient to produce beneficial effects on health. Therefore, isolating this bioactive phenolic from a natural source, such as rapeseed meal, could generate extracts containing concentrated amounts of SA which could be consumed as a functional food ingredient to prevent health-related disease, particularly inflammation. Inflammation is a multi-faceted pathology, which plays a role in numerous diseases including cardiovascular disease, diabetes, arthritis and cancer. Current therapies such as non-steroidal anti-inflammatories (NSAIDs) are associated with various adverse side effects, with the result being an increase in research aiming to identify natural compounds which possess anti-inflammatory activity. In this work, an in vitro study assessed the anti-inflammatory activities of two sinapinic acid-containing extracts on human-derived peripheral blood mononuclear cells (PBMCs). Both extracts were found to significantly reduce the levels of key pro-inflammatory cytokines including TNF-alpha, IL-12 and IL-6. Importantly, these extracts were found to be more potent than commercial SA in terms of their anti-inflammatory activities. Results demonstrate the potential of these extracts as anti-inflammatory agents.

Randomized phase II study of valproic acid in combination with bevacizumab and oxaliplatin/fluoropyrimidine regimens in patients with RAS-mutated metastatic colorectal cancer: the REVOLUTION study protocol.

Background:Despite effective treatments, metastatic colorectal cancer (mCRC) prognosis is still poor, mostly in RAS-mutated tumors, thus suggesting the need for novel combinatorial therapies. Epigenetic alterations play an important role in initiation and progression of cancers, including CRC. Histone-deacetylase inhibitors (HDACi) have shown activity in combination with chemotherapy in the treatment of solid tumors. Owing to its HDACi activity and its safe use for epileptic disorders, valproic acid (VPA) is a good candidate for anticancer therapy that we have largely explored preclinically translating our findings in currently ongoing clinical studies. We have shown in CRC models that HDACi, including VPA, induces synergistic antitumor effects in combination with fluoropyrimidines. Furthermore, unpublished results from our group demonstrated that VPA induces differentiation and sensitization of CRC stem cells to oxaliplatin. Moreover, preclinical and clinical data suggest that HDACi may prevent/reverse anti-angiogenic resistance.Methods/Design:A randomized, open-label, two-arm, multicenter phase-II study will be performed to explore whether the addition of VPA to first line bevacizumab/oxaliplatin/fluoropyrimidine regimens (mFOLFOX-6/mOXXEL) might improve progression-free survival (PFS) in RAS-mutated mCRC patients. A sample size of 200 patients was calculated under the hypothesis that the addition of VPA to chemotherapy/bevacizumab can improve PFS from 9 to 12 months, with one-sided alpha of 0.20 and a power of 0.80. Secondary endpoints are overall survival, objective response rate, metastases resection rate, toxicity, and quality of life. Moreover, the study will explore several prognostic and predictive biomarkers on blood samples, primary tumors, and on resected metastases.Discussion:The “Revolution” study aims to improve the treatment efficacy of RAS-mutated mCRC through an attractive strategy evaluating the combination of VPA with standard cancer treatment. Correlative studies could identify novel biomarkers and could add new insight in the mechanism of interaction between VPA, fluoropyrimidine, oxaliplatin, and bevacizumab.Trial Registration:EudraCT: 2018-001414-15; ClinicalTrials.gov identifier: NCT04310176

QSAR and molecular docking study on the biological activity of levofloxacin and thiodiazole histone deacetylase inhibitors

Using quantitative structure-activity relationship (QSAR) and molecular docking to study the interaction between levofloxacin and thiadiazole histone deacetylase inhibitors. The molecular electrical distance vector (MEDV) and multiple linear regression (MLR) were used to study the relationship between the structure and activity of compounds. The three equations obtained by multiple linear regression, their R2 = 0.976, 0.985 and 0.976, and their Rcv2 = 0.949, 0.977 and 0.932. The structures that affect HDAC1 and HDAC6 activity are -O- and -S-, and the structures that affect HDAC2 are -C- and -N-. Finally, molecular docking is used to study the binding of receptors and drug molecules to provide guidance for future drug design.

Withdrawal: Histone deacetylase inhibitors suppress the induction of c-Jun and its target genes including COX-2.

Cyclooxygenase-2 (COX-2) is considered to be a target for anticancer therapy. Histone deacetylase (HDAC) inhibitors exhibit antitumor activity, but the mechanisms of action are incompletely understood. We investigated whether HDAC inhibitors blocked AP-1-mediated activation of COX-2 transcription. Trichostatin A and suberoylanilide hydroxamic acid, two structurally related inhibitors of HDAC activity, blocked AP-1-mediated induction of COX-2 expression and prostaglandin E2 biosynthesis. Chromatin immunoprecipitation assays indicated that HDAC inhibitors suppressed c-Jun binding to the COX-2 promoter and thereby blocked transcription. The observed reduction in binding reflected reduced levels of c-Jun. HDAC inhibitors suppressed the induction of c-jun transcription by blocking the recruitment of the preinitiation complex (RNA polymerase II and TFIIB) to the c-jun promoter. HDAC3 but not HDAC1 or HDAC2 was required for AP-1-mediated stimulation of c-jun expression. Because HDAC inhibitors suppressed the induction of c-jun gene expression, resulting in reduced COX-2 transcription, it was important to determine whether other known AP-1 target genes were also modulated. Cyclin D1 and collagenase-1 are AP-1-dependent genes that have been implicated in carcinogenesis. HDAC inhibitors suppressed the induction of both cyclin D1 and collagenase-1 transcription by inhibiting the binding of c-Jun to the respective promoters. Taken together, these results suggest that HDAC inhibitors block the induction of c-jun transcription by inhibiting the recruitment of the preinitiation complex to the c-jun promoter. This led, in turn, to reduced expression of several activator protein-1-dependent genes (COX-2, cyclin D1, collagenase-1). These findings provide new insights into the mechanisms underlying the antitumor activity of HDAC inhibitors.

Peanut testa extracts enhance anticancer effect of cisplatin against human cholangiocarcinoma cells via modulation of histone deacetylase inhibitory activity

Objective: To investigate the effect of combination treatments of cisplatin and KK4 and ICG15042 peanut testa extracts against cholangiocarcinoma cells in vitro. Methods: The growth inhibition, cell cycle arrest and apoptosis of cholangiocarcinoma cells were determined by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry analysis, respectively. The levels of proteins involved in apoptosis were assessed using Western blotting assays. The caspase activity was assessed using a colorimetric caspase activity assay. Results: Cisplatin and peanut (KK4 and ICG15042) testa extracts inhibited the growth of cholangiocarcinoma cell lines (KKU- M214 and KKU-100 cells) in a dose- and time-dependent manner. The combination treatments reduced cell viability and induced apoptosis of cholangiocarcinoma cells more efficiently than singledrug treatments. Cancer cell death synergistically mediated by cisplatin and peanut testa extracts was observed in KKU-M214 cells (combination index 1.0). The combination treatments also increased the sub- G1 population and caused KKU-M214 cell cycle arrest at S and G2/ M phases, which were the combined effects of cisplatin (S phase arrest) and peanut testa extracts (G2/M phase arrest). In addition, pERK1/2, Ac-H3, Bcl-2 and proteins related to apoptosis, including Bax and caspases 3, 8, 9, exhibited enhanced expression in KKU- M214 cells. The combination treatments caused down-regulation of p53, whereas the expression of p21 was fairly constant when compared with cisplatin single drug treatment. Conclusions: Peanut testa extracts in combination with cisplatin synergistically reduce cell viability and induce apoptosis through stimulation of caspases 3, 8 and 9 in KKU-M214 cells.

Discovery of histone deacetylase 3 (HDAC3)-specific PROTACs.

Histone deacetylases (HDACs) are validated drug targets for cancer treatment. Increased HDAC isozyme selectivity and novel strategies to inhibit HDAC activity could lead to safer and more effective drug candidates. Nonetheless, it is quite challenging to develop isozyme-specific HDACi due to the highly conserved catalytic domain. We discovered XZ9002, a first-in-class HDAC3-specific PROTAC that potently degraded HDAC3. Importantly, XZ9002 is more effective to inhibit cancer cell proliferation than its proteolysis-inactive counterpart, suggesting HDAC3 degradation is a novel and promising anticancer approach.

TLR2/EGFR Are Two Sensors for pBD3 and pEP2C Induction by Sodium Butyrate Independent of HDAC Inhibition.

Host defense peptides (HDPs) are vital mucosal defense effectors of the innate immune response. The expression of HDPs is inducible in epithelial cells by potent endogenous inducers. Herein, our results demonstrate that sodium butyrate (NaB) induces the expression of porcine β-defensin-3 (pBD3) and porcine epididymis protein 2 splicing variant C (pEP2C) in a dose- and time-dependent manner, without modifying the production of proinflammatory cytokines, in porcine intestinal epithelial cells (IPEC J2). Moreover, NaB promotes toll-like receptor 2 (TLR2) expression. TLR2 silencing inhibits the pBD3 and pEP2C expression induced by NaB but does not abolish the histone deacetylase (HDAC) inhibitory activity of NaB. We found that NaB activated the nuclear factor-κB (NF-κB) pathway. Importantly, the degree of cell confluence governs the regulatory responses but does not affect the HDAC activity of NaB. Furthermore, epidermal growth factor receptor (EGFR), but not the mitogen-activated protein kinase (MAPK) pathway, is vital during the NaB-induced pBD3 and pEP2C regulation process. We also demonstrated that pBD3 overexpression increases interleukin-18 levels. This study showed that NaB simultaneously induces pBD3 and pEP2C via TLR2 and EGFR in IPEC J2 cells without increasing the risk of a harmful inflammatory response.

Histone deacetylase activity and vitamin D-dependent gene expressions in relation to sulforaphane in human breast cancer cells.

This study employed a combinatorial chemopreventive strategy to investigate the impact of dietary histone deacetylase (HDAC) inhibitor, that is, sulforaphane on chromatin remodeling in BC. To understand the epigenetics-mediated changes in gene expression, MCF-7 cells were exposed for 24hr to 1,25(OH)2D3 (100nM) either alone or in combination with L-sulforaphane and TSA (20μM and 1μM, respectively) at 70% confluency. Changes in VDR, CYP24A1, CYP27B1, and TRPV6 gene expressions were quantified using real-time PCR-based assays. HDAC inhibitor activity was assessed using HDAC I/II assay that measured global changes in acetylation status. Cell viability was measured using ATP and MT assays. Clonogenic and migration assays were performed to analyze the ability of single cells to grow into colonies and % closure (migration ability) upon treatments, respectively. Results were expressed as ΔCT±standard error of means (SEM) from One-way ANOVA analyses for mRNA expressions and mean±SEM for all other assays. In MCF-7 cells, treatment with 1,25(OH)2D3 tended to decrease VDR (13±0.4) and CYP27B1 (12±0.96), while significantly increased TRPV6 (p=.02, 14±0.1) and CYP24A1 (p<.0001, 0.38±0.12) expression. D alone and D+TSA group had the opposite effects on HDAC inhibition from SFN alone, D+SFN, and TSA alone. The clonogenic assay showed a significant decrease in colony formation with no colonies for D+TSA (p<.03) and TSA alone groups (p<.03). Cell viability tended to decrease with D alone and in combination with TSA. These data suggest that the effects of 1,25(OH)2D3 and sulforaphane are selective and gene-specific in MCF-7 cells. Breast cancer (BC) affects a large number of the U.S. population each year. Like most cancers, nutrition does play a role in the prevention of BC. However, dietary advice that includes reducing alcohol intake, red meat, and saturated fat consumption, while increasing the intake of heart-healthy fats, dietary fiber, and lean protein, etc., is difficult to apply to all cancers from a preventative standpoint. Vitamin D has been implicated in BC, mostly as a protective factor, with mixed findings. This research focuses on the role of vitamin D as a protective intervention in human BC, along with a dietary bioactive compound-sulforaphane. The idea is to combine the known benefits of a micronutrient with potential benefits of the bioactive compound to establish a stronger intervention against BC progression, irrespective of the subtype.

4-phenylbutyric acid promotes migration of gastric cancer cells by histone deacetylase inhibition-mediated IL-8 upregulation

ABSTRACT Histone acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). It is associated with gene transcription and expression. 4-Phenylbutyric acid (4-PBA), an HDAC inhibitor (HDACi), can inhibit cancer cell proliferation by increasing the level of histone acetylation. However, 4-PBA did not show any efficacy in clinical trials. In this study, we found that 4-PBA induced epithelial–mesenchymal transition (EMT) in gastric cancer cell lines MGC-803 and BGC-823 with ectopic E-cadherin expression. Based on the expression profile microarray, IL-8 was the most significantly up-regulated gene by 4-PBA, and was selected for further investigation. Knockdown of IL-8 partially prevented 4-PBA-induced-EMT by blocking the activation of the downstream Gab2-ERK pathway. Furthermore, CHIP assay confirmed that acetyl-H3 directly combined with the promoter region of IL-8 to promote its transcription. Therefore, the results of this study demonstrated that 4-PBA-mediated inhibition of HDAC activity could induce EMT in gastric cancer cells via acetyl-histone-mediated IL-8 upregulation, and the downstream Gab2/ERK activation. These data indicated the possible reason for the failure of 4-PBA in clinical trials.

Current status of histone deacetylase inhibitors in cutaneous T-cell lymphoma.

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin's lymphoma with a heterogenous presentation and highly variable disease course. The most common subtypes of CTCL are mycosis fungoides (MF) and Sézary Syndrome (SS). Treatment varies based on the stage of the disease with skin directed therapies typically utilized for early stage disease, and systemic therapies employed for more advanced disease. There are few highly effective treatments available, and systemic therapies have limited response rates. Histone deacetylase inhibitors have emerged as mainstream treatments for MF/SS over the past several years. Here, we discuss the mechanism of action of histone deacetylase inhibitors in relation to the pathogenesis of MF/SS, evaluate the clinical trials that led to Food and Drug Administration approval of two of the histone deacetylase inhibitors for MF/SS and describe the results for those still under investigation. Additionally, we discuss the potential for combination therapies in order to optimize outcomes of treatment with histone deacetylase inhibitors.

Histone Deacetylase Inhibitors and Antioxidants From the Root of Gluta usitata

A new glycoside, glutacoside (1), as well as 6 known compounds was isolated and identified from the root of Gluta usitata. Their structures were determined by Infrared spectroscopy, Mass spectroscopy, and 1-Dimensional and 2-dimensional nuclear magnetic resonance spectroscopy data. The histone deacetylase (HDAC) inhibitory and antioxidant activities of the obtained compounds were evaluated. Molecular docking experiments of the selected compound with representatives of class I (HDAC2 and HDAC8) and class II (HDAC4 and HDAC7) HDAC isoforms displayed potential isoform-selective HDAC inhibitors. Molecular docking data showed consistent results to the in vitro experiments with the highest potency against HDAC8. The docking studies suggested that the phenolic and carbonyl group can be favorably accommodated at the gorge region of the binding site. Furthermore, the phenolic groups also acted as major roles for antioxidant activities.

Arsenic Sulfide Nanoformulation Induces Megakaryocytic Differentiation through Histone Deacetylase Inhibition

AbstractArsenic sulfide (As4S4) is a mineral drug that can be administrated orally and has been applied in several Chinese medicine formulations for the treatment on some kinds of myeloid leukemia. However, mechanisms of As4S4 are not demonstrated adequately due to the poor water solubility of As4S4 in its original crystal form. The authors previously fabricated water dissolvable arsenic sulfide nanoformulation (ee‐As4S4), which downregulated intracellular reactive oxygen species (ROS). As ROS is closely associated with the histone deacetylase (HDAC), in this study the effect of ee‐As4S4 on the HDAC activity in chronic myeloid leukemia (CML) cells and whether the effect can induce subsequent megakaryocytic differentiation and underlying mechanisms are investigated. Results show that ee‐As4S4 inhibits HDAC activity in CML cells, resulting in the suppression of cellular respiration. The inhibition of HDAC activity also leads to the transcriptional activation of RUNX1, which induces megakaryocytic differentiation in CML cell lines, evidenced by the significantly multiple nuclei and giant nuclear structure, upregulation of CD41a and the increased phosphorylation of ERK1/2. In conclusion, ee‐As4S4 is able to inhibit HDAC activity, and therefore induce significant megakaryocytic differentiation in CML cells through RUNX1‐dependent pathways, which suggests its clinical potential as a complementary drug option for CML patients.

Restore the Secretion and Activity of Factor VIII Mutants By Proteostasis Regulator

Quantitative or qualitative deficiency in factor VIII (FVIII) result in hemophilia A. FVIII deficiency is caused by broad spectrum mutations, and missense mutation is the most common type of mutation, which lead to various defects of FVIII from decreased secretion to impaired function. Cellular protein homeostasis is disturbed by FVIII mutants, and thus excessive misfolding and degradation of FVIII mutant result in FVIII deficiency. We hypothesized that folding and/or trafficking of FVIII can be enhanced by proteostasis regulators, leading to increased coagulation factor levels sufficient to restore hemostasis. We performed a cell-based screening using a FDA-approved drug library and a natural compound library looking for compounds that enhance secretion of FVIII with a B domain deletion. We identified SAHA (Suberanilohydroxamic acid) as a compound that increased both wild-type (WT) and a missense mutant FVIII. SAHA is a histone deacetylase inhibitor that was also reported to enhance proteostaisis of mutant CFTR, a-1 antitrypsin, lysosomal glucocerebrosidase and the GABA receptor. Here we show that SAHA can increase the secretion and activity of FVIII mutants by enhancing their folding and trafficking as well transcription. To confirm the ability of SAHA to enhance FVIII mutant secretion, we first used the WT FVIII A2 domain and the M614T mutant A2 domain to detect the proper working time and concentration of SAHA in HEK 293T cells. M614T is a missense mutation that was shown to cause secretion defect of FVIII in our previous study. Western blot showed a dose-dependent increase in levels of both A2-WT and A2-M614T in cell culture media between 0 and 2.5ug/ml of SAHA after 24-hour treatment. Levels of both WT and mutant A2 domain dropped when SAHA concentration exceeded 5 ug/ml due to cellular toxicity. SAHA enhanced the secretion of A2-M614T much more than that of A2-WT. Of note, protein levels of both A2-WT and A2-M614T in cell lysate were also slightly elevated by SAHA. Time course study showed that the maximum effect on secretion was achieved after 24 hours of SAHA treatment. We next studied the effect of SAHA on activity and secretion of several full-length FVIII mutants (A469S, R527W, R531G, I566T, N582D, S584T, R593C, M614T, R1997G, R2150C), which were shown to decrease FVIII secretion due to various defects in intracellular trafficking in our previous studies. Except for N582D, activity levels of all FVIII mutants in cell culture media were elevated 3-14 folds after a 24-hour SAHA treatment (P&lt;0.05), whereas the activity level of FVIII WT only increased by ~2 folds (P&lt;0.05). Antigen levels of FVIII mutants in both cell lysates and cell culture media increased by ~3 folds following SAHA treatment (P&lt;0.05). The increase in activities of several FVIII mutants (I566T, S584T, R593C, R1997G, R2150C) is higher than the increase in antigen levels of the same mutants in cell culture media, which indicates that, in addition to enhancing secretion, SAHA treatment also increased the proportions of functional proteins in secreted FVIII mutants in cell culture media. To investigate the mechanism, we studied the interactions of WT FVIII and several responsive mutant FVIII (I566T, S584T, R593C and R1997C) with endoplasmic reticulum (ER) chaperones BiP and calreticulin by co-Immunoprecipitation. The protein levels of BiP and calreticulin in HEK 293T cells were not affected by SAHA. However, the interactions of BiP with both WT FVIII and FVIII mutants were remarkably enhanced by SAHA, while no significant differences in the interactions of calreticulin with FVIII WT or FVIII mutants were observed. The effect of SAHA on FVIII secretion can be blocked by simultaneously treating cells with a BiP inhibitor SubAB. We next measured mRNA levels of BiP as well as FVIII in cells expressing FVIII WT and FVIII mutants with or without SAHA treatment via real time PCR. Similar to protein levels, BiP mRNA levels were not changed by SAHA. However, FVIII mRNA levels were elevated by 1.2 to 3.1 folds (P&lt;0.05) by SAHA, in keeping with the histone deacetylase inhibitor activity of SAHA. In summary, we have shown that SAHA increases FVIII antigen and activity levels in cell culture media by increasing FVIII mRNA levels and by enhancing its folding and trafficking via specifically promoting its interaction with BiP. SAHA is a potential proteostaisis regulator that can increase the secretion and restore the function of certain FVIII missense mutants. Disclosures No relevant conflicts of interest to declare.

This Is a Title in Title Case: A Combination of Low-Dose Decitabine and Chidamide Resulted in Synergistic Effects on the Proliferation and Apoptosis of Human Myeloid Leukemia Cell Lines

Introduction: Myeloid leukemia is a malignant disease caused by both genetic and epigenetic changes. Aberrant DNA methylation and histone modifications are prevalent in cancers including leukemia and the process of epigenetic modifications is reversible, which makes them potential treatment targets using specific inhibitors. In this study, we sought to determine the antileukemic effects of low-dose decitabine (one of the most widely used DNA methyltransferase inhibitor) combined with chidamide (a novel orally active histone deacetylase inhibitor) on myeloid leukemia cells by detecting cell proliferation, cell cycle distribution and cell apoptosis to provide a promising regimen for clinical application. Methods: We conducted CCK-8 assay to determine the effect of low-dose decitabine combinated with chidamide on cell viability, cell cycle distribution analysis by use of flow cytometry accompanied with RT-qPCR and western blotting analysis to reveal its inhibitory mechanism of cell growth, and cell apoptotic analysis also by use of flow cytometry accompanied with RT-qPCR, western blotting analysis, intracellular ROS production and mitochondrial transmembrane potential assay to determine the apoptotic mechanism of combination of low-dose decitabine and chidamide in K562 and THP-1 cells. Result: Low-dose decitabine and chidamide alone played an inhibitory role in the proliferation of K562 and THP-1 cells in a dose- and time-dependent manner, and the combination of the two enhanced this effect, accompanied by induction of p21 expression and cell cycle arrest at G0/G1 phase. Chidamide instead of decitabine had pronounced effects on the histone H3 acetylation levels of leukemia cells. Meanwhile, combination of the two significantly induced cell apoptosis via mitochondrial transmembrane potential loss, resulting in upregulation of the cell apoptosis-related gene Bax and the protein cleaved PARP-1 and downregulation of antiapoptosis gene Bcl-2, and proteins, including pro-caspase 9, pro-caspase 3 and PARP-1. Conclusion: Our results provided evidence for the antileukemia effects of low-dose DNA methyltransferase inhibitor decitabine combinated with histone deacetylase inhibitor chidamide in preclinical myeloid leukemia models and suggested that combination of the two showed therapeutic potential for myeloid leukemia treatment. Disclosures No relevant conflicts of interest to declare.

Design, synthesis and evaluation of belinostat analogs as histone deacetylase inhibitors.

Aim: Histone deacetylase (HDAC) is an attractive target for antitumor therapy. Therefore, the development of novel HDAC inhibitors is warranted. Materials & methods: A series of HDAC inhibitors based on N-hydroxycinnamamide fragment was designed as the clinically used belinostat analog using amide as the connecting unit. All target compounds were evaluated for their in vitro HDAC inhibitory activities and some selected compounds were tested for their antiproliferative activities. Conclusion: Among them, compound 7e showed an IC50 value of 11.5nM in inhibiting the HDAC in a pan-HDAC assay, being the most active compound of the series.

N-alkyl-hydroxybenzoyl anilide hydroxamates as dual inhibitors of HDAC and HSP90, downregulating IFN-γ induced PD-L1 expression

Novel dual inhibitors of histone deacetylase (HDAC) and heat-shock protein 90 (HSP90) are synthesized and evaluated. These compounds are endowed with potent HDAC and HSP90 inhibitory activities with IC50 values in nanomolar range with Compound 20 (HDAC IC50 = 194 nM; HSP90α IC50 = 153 nM) and compound 26 (HDAC IC50 = 360 nM; HSP90α IC50 = 77 nM) displaying most potent HDAC and HSP90α inhibitory activities. Both of these compounds induce HSP70 expression and down regulate HSP90 client proteins which play important roles in the regulation of survival and invasiveness in cancer cells. In addition, compounds 20 and 26 induce acetylation of α-tubulin and histone H3. Significantly, compounds 20 and 26 could effectively reduce programmed death-ligand 1 (PD-L1) expression in IFN-γ treated lung H1975 cells in a dose dependent manner. These findings suggest that dual inhibition of HDAC and HSP90 that can modulate immunosuppressive ability of tumor area may provide a better therapeutic strategy for cancer treatment in the future.

Abstract 034: Differential Regulation of Renal Injury in Npr1 Gene-Targeted Male and Female Mice: Role of Class I-Specific HDAC Inhibitor

The objective of the present study was to gain insight into the effect of class I-specific histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103; MGCD) on Npr1 (coding for natriuretic peptide receptor-A; NPRA) gene expression and regulation of renal pathology. Adult male and female Npr1 haplotype (1-copy; Npr1 +/- ), wild-type (2-copy; Npr1 +/+ ), and gene-duplicated (3-copy; Npr1 ++/+ ) mice were injected intraperitoneally with MGCD (2 mg/kg) at alternate days for 2-weeks. The renal NPRA protein levels were significantly increased in the treated male (1-copy, 2.1-fold; 2-copy, 2.3-fold; and 3-copy, 1.5-fold; p &lt; 0.05) and female mice (1-copy, 3.6-fold; 2-copy, 2.1-fold; and 3-copy, 1.7-fold; p &lt; 0.05) compared with untreated animals. After MGCD treatment, the renal guanylyl cyclase-A (GC-A) activity significantly increased in male mice (1-copy, 77.4 ± 0.5 vs. control 30.7 ± 0.5; 2-copy 126.1 ± 5.0 vs. control 76.3 ± 6.3; and 3-copy, 182.7 ± 2.8 vs. control 141.2 ± 6.4; p &lt; 0.05; cGMP/mg protein/ 30 min) and female mice (1-copy, 108.2 ± 6.8 vs. control 51.9 ± 3.0; p &lt; 0.05). Male mice exhibited significantly higher systolic blood pressure (SBP) than females and treatment with MGCD decreased SBP in both 1-copy male (106 ± 0.6 vs. control, 129 ± 1.9; mmHg) and female mice (97 ± 2.2 vs. control, 110 ± 2.1, p &lt; 0.001). Higher urinary albumin to creatinine ratio was detected in male mice compared with female mice, which was attenuated by MGCD treatment in male mice (1-copy, 0.39 ± 0.04 vs. untreated 0.71 ± 0.05; 2-copy 0.31 ± 0.01 vs. untreated 0.46 ± 0.05; and 3-copy, 0.2 ± 0.02 vs. untreated 0.21 ± 0.02; p &lt; 0.05). HDAC activity was significantly higher in male mice (1-copy, 24.4 ± 2.8; 2-copy, 15.9 ± 1.2; and 3-copy, 6.4 ± 0.7; ng/min/mg protein) than female mice (1-copy, 13.7 ± 1.4; 2-copy, 8.5 ± 0.9; and 3-copy, 4.7 ± 0.9, p &lt; 0.05); however, MGCD treatment significantly attenuated HDAC activity in both male and female animals (p &lt; 0.05). The present results indicate MGCD upregulates Npr1 expression in 1-copy haplotype mice via inhibition of HDAC activity, lowers SBP and repairs renal injury differentially in male vs. female mice. These findings will have important implications for treatment of hypertension and renal injury in humans in a sex-related manner.