Inhibition Of Histone Deacetylase Activity Research Articles

Common SAR Derived from Multiple QSAR Models on Vorinostat Derivatives Targeting HDACs in Tumor Treatment.

Dysregulation of HDACs has been associated with tumour development and therefore inhibiting HDAC's have surfaced as promising therapeutic strategy in malignancy. Vorinostat analogues with different biological activities were investigated for underlying structure-activity relationship. Out of six activities and their multiple QSAR models, HDAC1 and HDAC8 produced statistically fit, stable and predictive linear (MLR) and non-linear (SVM) QSAR models. In case of HDAC1 activity as end point, linear (R2=0.8089, R2 CV=0.7343) and non-linear (R2=0.9801, R2 CV=0.8952) QSAR models turned reliable to investigate SAR. Similarly, HDAC8 activity based linear (R2=0.9454, R2 CV=0.9049) and non-linear (R2=0.9899, R2 CV=0.9232) QSAR models produced statistically improved and stable models. Molecular descriptors derived from 3-D Morse and Radial Distribution Function indices were found to be selective in all the models. These molecular descriptors which encode common SAR among Vorinostat derivatives were evaluated for their potent HDAC inhibition activity.

Sulforaphane, a natural component of broccoli, inhibits vestibular schwannoma growth in vitro and in vivo.

Vestibular schwannoma (VS) is an intracranial tumor that causes significant morbidity, including hearing loss, tinnitus, dizziness, and possibly even death from brainstem compression. However, FDA-approved pharmacologic treatments for VS do not exist. Sulforaphane (SFN) is a naturally occurring isothiocyanate found in cruciferous vegetables, such as broccoli, with potent chemoprotective effects in several cell types. Our objective was to determine whether SFN is effective against VS in vitro and in vivo. Human primary VS cells, HEI-193 schwannoma cells, and SC4 Nf2−/− Schwann cells were used to investigate the inhibitory effects of SFN in vitro. Cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation, and cell viability and metabolic activity was calculated by MTT assay. Apoptosis was measured by flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and Western blot for cleaved caspases. A mouse model with a murine schwannoma allograft was also used to examine the antitumor activity of SFN. SFN exhibited significant antiproliferative activity in schwannoma cells in vitro, via the inhibition of HDAC activity and the activation of ERK. SFN treatment induced apoptosis and cell cycle arrest at the G2/M phase. SFN also significantly inhibited schwannoma growth in vivo. Our preclinical studies motivate a future prospective clinical study of SFN for the treatment of VS.

Sodium butyrate activates NRF2 to ameliorate diabetic nephropathy possibly via inhibition of HDAC.

Oxidative stress contributes to the pathogenesis of diabetic nephropathy (DN). Nuclear factor erythroid 2-related factor 2 (NRF2) plays a key role in cellular defense against oxidative stress. NRF2 activators have shown promising preventive effects on DN. Sodium butyrate (NaB) is a known activator of NRF2. However, it is unknown whether NRF2 is required for NaB protection against DN. Therefore, streptozotocin-induced diabetic C57BL/6 Nrf2 knockout and their wild-type mice were treated in the presence or absence of NaB for 20 weeks. Diabetic mice, but not NaB-treated diabetic mice, developed significant renal oxidative damage, inflammation, apoptosis, fibrosis, pathological changes and albuminuria. NaB inhibited histone deacetylase (HDAC) activity and elevated the expression of Nrf2 and its downstream targets heme oxygenase 1 and NAD(P)H dehydrogenase quinone 1. Notably, deletion of the Nrf2 gene completely abolished NaB activation of NRF2 signaling and protection against diabetes-induced renal injury. Interestingly, the expression of Kelch-like ECH-associated protein 1, the negative regulator of NRF2, was not altered by NaB under both diabetic and non-diabetic conditions. Moreover, NRF2 nuclear translocation was not promoted by NaB. Therefore, the present study indicates, for the first time, that NRF2 plays a key role in NaB protection against DN. Other findings suggest that NaB may activate Nrf2 at the transcriptional level, possibly by the inhibition of HDAC activity.

A grape seed procyanidin extract inhibits HDAC activity leading to increased Pparα phosphorylation and target-gene expression.

Histone deacetylases (HDACs) have emerged as epigenetic regulators of risk factors associated with the metabolic syndrome (MetS), and certain botanical extracts have proven to be potent HDAC inhibitors. Understanding the role of dietary procyanidins in HDAC inhibition is important in exploring the therapeutic potential of natural products. C57BL/6 mice were gavaged with vehicle (water) or grape seed procyanidin extract (GSPE, 250 mg/kg) and terminated 14h later. Liver and serum were harvested to assess the effect of GSPE on HDAC activity, histone acetylation, Pparα activity and target-gene expression, and serum lipid levels. GSPE increased histone acetylation and decreased Class I HDAC activity in vivo, and dose-dependently inhibited recombinant HDAC2 and 3 activities in vitro. Accordingly, Pparα gene and phosphorylated protein expression were increased, as were target genes involved in fatty acid catabolism, suggesting increased Pparα activity. Serum fibroblast growth factor 21 (Fgf21) was elevated, and triglyceride levels were reduced by 28%. GSPE regulates HDAC and Pparα activities to modulate lipid catabolism and reduce serum triglycerides in vivo.

Design and synthesis of novel HDAC8 inhibitory 2,5-disubstituted-1,3,4-oxadiazoles containing glycine and alanine hybrids with anti cancer activity

Oxadiazole is a heterocyclic compound containing an oxygen atom and two nitrogen atoms in a five-membered ring. Of the four oxadiazoles known, 1,3,4-oxadiazole has become an important structural motif for the development of new drugs and the compounds containing 1,3,4-oxadiazole cores have a broad spectrum of biological activity. Herein, we describe the design, synthesis and biological evaluation of a series of novel 2,5-disubstituted 1,3,4-oxadiazoles (10a–10j) as class I histone deacetylase (HDAC) inhibitors. The compounds were designed and evaluated for HDAC8 selectivity using in silico docking software (Glide) and the top 10 compounds with high dock score and obeying Lipinski’s rule were synthesized organically. Further the biological HDAC inhibitory and selectivity assays and anti-proliferative assays were carried out. In in silico and in vitro studies, all compounds (10a–10j) showed significant HDAC inhibition and exhibited HDAC8 selectivity. Among all tested compounds, 10b showed substantial HDAC8 inhibitory activity and better anticancer activity which is comparable to the positive control, a FDA approved drug, vorinostat (SAHA). Structural activity relation is discussed with various substitutions in the benzene ring connected on 1,3,4-oxadizole and glycine/alanine. The study warranted further investigations to develop HDAC8-selective inhibitory molecule as a drug for neoplastic diseases. Novel 1,3,4-oxadizole substituted with glycine/alanine showed HDAC8 inhibition.

Non-toxic Efficacy of the Combination of Caffeine and Valproic Acid on Human Osteosarcoma Cells In Vitro and in Orthotopic Nude-mouse Models.

We have previously reported that caffeine can enhance chemotherapy efficacy of bone and soft-tissue sarcoma via cell-cycle perturbation. Valproic acid has histone deacetylase (HDAC) inhibitory activity. The present study aimed to investigate the efficacy of the combination of valproic acid and caffeine on human osteosarcoma cells in vitro and in orthotopic nude-mouse models. Human osteosarcoma cell lines (MG63, 143B and SaOS2) were used. Cell survival after a 72 h exposure to valproic acid and caffeine was assessed with a WST-8 assay. Half maximal inhibitory concentration (IC50) values and combination indices (CIs) were calculated. Caspase 3 activity was measured by a fluorochrome inhibitor of caspase (FLICA) assay. 143B cells were also transplanted to the tibia of nude mice and treated with these drugs. Both valproic acid and caffeine caused concentration-dependent cell death of the osteosarcoma cell lines in vitro. Apoptosis induction was observed with the Caspase 3 assay. The combination was synergistic. The combination of valproic acid and caffeine showed effective anti-tumor activity in vivo without the need for conventional anticancer drugs or any observable toxicity Conclusion: Efficacy of combination therapy with caffeine and valproic acid in osteosarcoma was observed in vitro and in vivo without toxicity, suggesting that either or both drugs can be effectively combined with appropriate chemotherapy in the future.

Synthesis of a selective HDAC6 inhibitor active in neuroblasts

In recent years, the role of HDAC6 in neurodegeneration has been partially elucidated, which led some authors to propose HDAC6 inhibitors as a therapeutic strategy to treat neurodegenerative diseases. In an effort to develop a selective HDAC6 inhibitor which can cross the blood brain barrier (BBB), a modified hydroxamate derivative (compound 3) was designed and synthetized. This compound was predicted to have potential for BBB penetration based on in silico and in vitro evaluation of passive permeability. When tested for its HDAC inhibitory activity, the IC50 value of compound 3 towards HDAC6 was in the nM range in both enzymatic and cell-based assays. Compound 3 showed a cell-based selectivity profile close to that of tubastatin A in SH-SY5Y human neuroblastoma cells, and a good BBB permeability profile.

Design, synthesis and in vitro evaluation of amidoximes as histone deacetylase inhibitors for cancer therapy

Amindoximes are geometric isomers of N-hydroxyamidines which are bioisosteres of hydroxamates. Since amindoxime group is capable of chelating transition metal ions including zinc ion, amindoximes should possess histone deacetylases (HDACs) inhibitory activity. In this work, we designed and synthesized a series of amindoximes, examined their inhibitory activities against HDACs, and investigated their cytotoxicity to human cancer cells. Preliminary results demonstrated that amindoximes possessed submicromolar HDACs inhibitory activity, with noteworthy enhancement compared with hydroxamates. Furthermore, the amindoximes arrested HCT116 and A549 cells in G2/M phase and showed good efficacy in inducing cells death. We provided a proof-of-concept that amindoximes could be used as HDACs inhibitors and hold great promise as epigenetic drugs.

Design, synthesis and antiproliferative activity of hydroxyacetamide derivatives against HeLa cervical carcinoma cell and breast cancer cell line

Purpose: To design and develop a new series of histone deacetylase inhibitors (FP1 - FP12) and evaluate their inhibitory activity against hydroxyacetamide (HDAC) enzyme mixture-derived HeLa cervical carcinoma cell and MCF-7.Methods: The designed molecules (FP1 - FP12) were docked using AUTODOCK 1.4.6. FP3 and FP8 showed higher interaction comparable to the prototypical HDACI. The designed series of 2-[[(3- Phenyl/substituted Phenyl-[4-{(4- (substituted phenyl)ethylidine-2-Phenyl-1,3-Imidazol-5-One}](-4H- 1,2,4-triazol-5-yl)sulfanyl]-N-hydroxyacetamide derivatives (FP1-FP12) was synthesized by merging 2- [(4-amino-3-phenyl-4H- 1, 2, 4-triazol-5-yl) sulfanyl]- N-hydroxyacetamide and 2-{[4-amino-3-(2- hydroxyphenyl)-4H-1,2, 4-triazol-5-yl]sulfanyl}-N hydroxyacetamide derivatives with aromatic substitutedoxazolone. The biological activity of the synthesized molecule (FP1-FP12) was evaluated against HDAC enzyme mixture-derived HeLa cervical carcinoma cell and breast cancer cell line (MCF-7).Results: HDAC inhibitory activity of FP10 showed higher IC50 (half-maximal concentration inhibitory activity) of 0.09 μM, whereas standard SAHA molecule showed IC50 of 0.057 μM. On the other hand, FP9 exhibited higher GI50 (50 % of maximal concentration that inhibited cell proliferation) of 22.8 μM against MCF-7 cell line, compared with the standard, adriamycin, with GI50 of (-) 50.2 μM.Conclusion: Synthesis, spectral characterization, and evaluation of HDAC inhibition activity and in vitro anticancer evaluation of novel hydroxyacetamide derivatives against MCF-7 cell line have been achieved. The findings indicate the emergence of potentialanticancer compounds.Keywords: Molecular docking, Hydroxyacetamide derivative, Histone deacetylase inhibition activity, MCF-7 cell line

Microbiota metabolite short chain fatty acids, GPCR, and inflammatory bowel diseases.

Gut microbiota has been well recognized in regulation of intestinal homeostasis and pathogenesis of inflammatory bowel diseases. However, the mechanisms involved are still not completely understood. Further, the components of the microbiota which are critically responsible for such effects are also largely unknown. Accumulating evidence suggests that, in addition to pathogen-associated molecular patterns, nutrition and bacterial metabolites might greatly impact the immune response in the gut and beyond. Short chain fatty acids (SCFA), which are metabolized by gut bacteria from otherwise indigestible fiber-rich diets, have been shown to ameliorate diseases in animal models of inflammatory bowel diseases (IBD) and allergic asthma. Although the exact mechanisms for the action of SCFA are still not completely clear, most notable among the SCFA targets is the mammalian G protein-coupled receptor pair of GPR41 and GPR43. In addition to the well-documented inhibition of histone deacetylases activity mainly by butyrate and propionate, which causes anti-inflammatory activities on IEC, macrophages, and dendritic cells, SCFA has recently been implicated in promoting development of Treg cells and possibly other T cells. In addition to animal models, the beneficial effects have also been reported from the clinical studies that used SCFA therapeutically in controlled trial settings in inflammatory disease, in that application of SCFA improved indices of IBD and therapeutic efficacy was demonstrated in acute radiation proctitis. In this review article, we will summarize recent progresses of SCFA in regulation of intestinal homeostasis as well as in pathogenesis of IBD.

Virtual screening and experimental validation of novel histone deacetylase inhibitors.

BackgroundHistone deacetylases (HDACs) are promising therapeutic targets for the treatment of cancer, diabetes and other human diseases. HDAC inhibitors, as a new class of potential therapeutic agents, have attracted a great deal of interest for both research and clinical applications. Increasing efforts have been focused on the discovery of HDAC inhibitors and some HDAC inhibitors have been approved for use in cancer therapy. However, most HDAC inhibitors, including the clinically approved agents, do not selectively inhibit the deacetylase activity of class I and II HDAC isforms, and many suffer from metabolic instability. This study aims to identify new HDAC inhibitors by using a high-throughput virtual screening approach.MethodsAn integration of in silico virtual screening and in vitro experimental validation was used to identify novel HDAC inhibitors from a chemical database.ResultsA virtual screening workflow for HDAC inhibitors were created by integrating ligand- and receptor- based virtual screening methods. Using the virtual screening workflow, 22 hit compounds were selected and further tested via in vitro assays. Enzyme inhibition assays showed that three of the 22 compounds had HDAC inhibitory properties. Among these three compounds, ZINC12555961 significantly inhibited HDAC activity. Further in vitro experiments indicated that ZINC12555961 can selectively inhibit proliferation and promote apoptosis of cancer cells.ConclusionsIn summary, our study presents three new and potent HDAC inhibitors and one of these HDAC inhibitors shows anti-proliferative and apoptosis-inducing activity against various cancer cell lines. These results suggest that the developed virtual screening workflow can provide a useful source of information for the screening and validation of new HDAC inhibitors. The new-found HDAC inhibitors are worthy to further and more comprehensive investigations.Electronic supplementary materialThe online version of this article (doi:10.1186/s40360-016-0075-8) contains supplementary material, which is available to authorized users.

Abstract 4745: Repurposing of valproic acid and simvastatin combination as anticancer agents in prostate cancer: synergistic interaction with docetaxel and suppression of docetaxel resistance

Abstract Although docetaxel (DTX) remains a standard of care for advanced prostate cancer (PCa), limited long-term responses, side effects and resistant disease suggested the need of novel combination strategies. Increased expression of histone deacetylases (HDAC) and alteration of the mevalonate pathway (MVP) are common aberrations in PCa. In this study, we analyzed the antitumor effect of DTX in combination with valproic acid (VPA), an anticonvulsant with HDAC inhibitory activity, and simvastatin (SIM), a cholesterol-lowering drug inhibiting the rate-limiting enzyme of MVP HMG-CoA reductase, on androgen-dependent 22RV1 and LNCAP and androgen-independent PC3 and DU145 PCa cells, as well as on the highly aggressive SIM-resistant DU145R80 subline developed in our laboratory from DU145 cells (Milone MR et al. Cell Death Dis. 2013; Milone MR et al. Oncotarget 2014). We first showed a potent synergistic anti-proliferative effect of VPA/SIM combination, assessed by calculating combination index (CI) according to the method of Chou and Talalay, on all cell lines, including SIM-resistant cells, whatever schedule of administration (simultaneous vs sequential) we used, confirming our previous data on the combination between the HDAC inhibitor (HDACi) panobinostat and zoledronic acid, the latter also targeting the MVP pathway (Bruzzese F. et al, Cell Death Dis. 2013). Notably, exposure to triple combinations (VPA/SIM/DTX) resulted in a further strong synergistic anti-proliferative effect, with sequential exposure with 24h delay between VPA/SIM and DTX as the best schedule. The synergistic interaction of VPA/SIM and DTX combination involved apoptotic effect, measured by FACS analysis of sub-diploid DNA content and caspase 3/7 cleavage, and DNA damage induction, assessed by γH2AX expression. Synergistic effect of VPA/SIM and DTX combination was confirmed by soft agar clonogenic assay and by 3D culture on self-assembled PCa spheroids. Significantly, VPA/SIM combination was also able to revert DTX-resistance in DTX-resistant PC3 and DU145 sublines developed in our laboratory from the parental cells. All together these findings suggested that the combination of two safe generic drugs such as VPA and SIM can improve DTX efficacy, representing a novel therapeutic approach that warrant clinical investigation in advanced PCa patients. Our study also suggests a new strategy to overcome resistance to standard taxane-based therapy in PCa patients. Citation Format: Federica Iannelli, Rita Lombardi, Biagio Pucci, Maria Rita Milone, Chiara Ciardiello, Alessandra Leone, Elena Di Gennaro, Alfredo Budillon, Francesca Bruzzese. Repurposing of valproic acid and simvastatin combination as anticancer agents in prostate cancer: synergistic interaction with docetaxel and suppression of docetaxel resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4745.

Abstract 4721: Enhancement of doxorubicin cytotoxicity by histone deacetylase inhibition in human sarcoma cells

Abstract Introduction: Soft tissue and bone sarcomas are rare malignancies of mesenchymal origin that account for about 1% of all adult solid tumors. Surgical resection is the first treatment option but is heavily hampered by delayed diagnosis. Due to few therapeutic treatments available, novel and efficacious therapy is urgently needed. Histone deacetylase inhibitors (HDACi) are emerging as a prominent class of therapeutic agents for several cancers; however, little is known about HDACi activity in sarcomas. By using human sarcoma models, we studied the therapeutic activity of ITF2357, a novel histone deacetylase inhibitor, as an anti-cancer agent alone or in combination with doxorubicin, on both wild-type and mutated p53-bearing sarcoma cell lines. Materials and methods: A wide panel of sarcoma cell lines, including both established and patient-derived sarcoma from different histotypes, were used. Western Blot analysis was performed to evaluate the effect of ITF2357 on histone and non-histone protein acetylation and expression of potential downstream targets. Cell viability was tested by MTT and CellTiterGlo assays. Apoptosis induction and cell cycle perturbation were assessed by flow cytometry and Western Blot analysis. Autophagy was assessed by analysis of autophagosome formation in EGFP-LC3B expressing cells and Western Blot analysis. Pharmacological interaction between ITF2357 and Doxorubicin was assessed by conservative isobologram analysis. In vivo efficacy of drug combination was evaluated in nude mice bearing sarcoma xenografts. Results: In sarcoma cell lines, HDAC inhibition induced H3 histone and alpha-tubulin acetylation, cell proliferation inhibition and activation of apoptotic program in a dose-dependent manner. ITF2357 cytotoxicity was independent on p53 status, despite its ability to inhibit the expression of mutated p53 protein. Moreover, ITF2357 induced a canonical-autophagic process, which protects sarcoma cells from apoptotic cell death and enhances cell survival. The combination of doxorubicin and ITF2357 was found to be highly synergistic in terms of cell proliferation inhibition and was related to the engagement of the apoptotic mitochondrial pathway. Noteworthy, treatment with both drugs also showed synergistic inhibition of cell viability in a doxorubicin-resistant osteosarcoma line, as compared with either agent alone. The effect of the drug combination was corroborated in sarcoma stem cells obtained from sarcoma patients. In vivo experiments in mouse xenograft models confirmed that co-treatment increased both inhibition of tumor growth and apoptosis, as compared with either agent alone. Conclusions: Overall, HDAC inhibition in sarcoma may provide an effective therapeutic modality capable of overcoming the doxorubicin resistance, and thus may lead to more durable responses in the clinic. Citation Format: Maria Grazia Tupone, Daniela Trisciuoglio, Marianna Desideri, Marta Di Martile, Simonetta Buglioni, Barbara Antoniani, Rossella Loria, Rita Falcioni, Michele Milella, Virginia Ferraresi, Gemma Di Pompo, Nicola Baldini, Roberto Biagini, Donatella Del Bufalo. Enhancement of doxorubicin cytotoxicity by histone deacetylase inhibition in human sarcoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4721.

Abstract 275: Combining low dose microtubule targeting agents with belinostat potentiates cytotoxic response in HDACi resistant diffuse large b-cell lymphoma

Abstract Patients diagnosed with Diffuse Large B-cell lymphoma have an overall 60% five-year survival rate. New therapeutic approaches are needed to effectively treat aggressive forms of DLBCL that are refractory to the standard treatment or that relapse within two years of treatment. Histone deacetylase inhibitors (HDACi) are novel therapeutics that are well tolerated in humans and are being extensively evaluated in combination with other therapeutics against hematologic malignancies. Rational selection of companion therapeutics has been difficult due to the cell type-specific mechanisms of HDACi action. In order to address this, we have developed a pre-clinical model system of sensitivity and resistance to HDACi-induced cytotoxicity in DLBCL cell lines that share characteristics with aggressive DLBCL tumors. We previously reported that HDACi resistance is associated with reversible arrest in G1 that involves sustained up-regulation of cyclin-dependent kinase inhibitors. In the current study we demonstrate that HDACi-sensitive cell lines undergo mitotic arrest prior to anaphase in response to treatment with the approved HDACi, belinostat, consistent with activation of the spindle assembly checkpoint (SAC). In contrast, HDACi-resistant cell lines are capable of completing mitosis in the presence of belinostat. To force SAC activation in HDACi resistant cell lines, we used low dose microtubule targeting agents (MTA) vincristine and paclitaxel to induce maximal mitotic arrest with minimal cytotoxicity. The combination of these low dose MTAs and belinostat efficiently caused SAC failure, mitotic slippage, and apoptosis in a synergistic manner. A key mechanism associated with resistance to MTAs is their ability to induce aneuploid cell populations that survive and undergo endoreduplication. The addition of belinostat eliminated the accumulation of a vincristine-induced aneuploid population. Pan-caspase inhibition in conjunction with belinostat and vincristine co-treatment resulted in a sustained survival of the aneuploid population. Thus belinostat triggers apoptosis in the aneuploid cells to enhance the cytotoxic effects of vincristine. Our study identifies the use of low dose MTA/HDACi combination as a potential therapeutic strategy for treatment of relapsed or refractory DLBCL. Citation Format: Aaron P. Havas, Anvi Bhakta, Kameron Rodrigues, Catharine L. Smith. Combining low dose microtubule targeting agents with belinostat potentiates cytotoxic response in HDACi resistant diffuse large b-cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 275.

Abstract 388: Pathway-directed high throughput drug screen identifies PI3K inhibitors that synergistically potentiate anti-tumor activity of HDAC inhibitors in mycosis fungoides and Sézary syndrome

Abstract Introduction and Purposes: Mycosis fungoides and Sézary syndrome (MF/SS) represent a group of heterogeneous diseases. Recent studies demonstrated dysregulation of several signaling pathways in MF/SS, including PI3K/AKT, JAK/STAT, RAS and NFκB pathways. We performed a high throughput drug screen to determine the potential of novel agents targeting these pathways for the treatment of MF/SS. Experimental Procedures: We compiled a library of 94 compounds targeting pathways known to be relevant in cancer biology. These included kinases involved in growth factor receptor signaling, HDACs, proteasome, DNA repair and regulators of apoptosis. The compounds were screened for anti-proliferative activity against four MF/SS cell lines in high throughput proliferation assays. Selected hits were further studied in xenograft models of MF/SS and in primary T cell lymphomas. Promising candidates from different classes were also tested in combination therapy assays using a matrix block method across dose gradients of each compound designed to detect synergistic activities. Results: From the high throughput screen, we identified 14 compounds with anti-proliferative activity in MF/SS, including multiple inhibitors of the PI3K pathway. PI3K inhibitors emerged as preliminary hits in this screen and secondary validation assays confirmed the class effect of PI3K inhibitors. From this class, the PI3K inhibitor BKM120 was selected for in vivo studies. In a xenograft model of MF, BKM120 exhibited striking anti-tumor activity measured by a marked suppression of tumor growth and prolonged survival of tumor-bearing mice compared with vehicle control. In a search for even more effective combination therapies, we identified that BKM120 and the HDAC inhibitor class of compounds exhibit synergistic anti-proliferative effects in MF/SS tumor cells. Each of three HDAC inhibitors including LBH, Romidepsin and Vorinostat showed synergistic activity with BKM120, most evident at the GI50 concentrations of each drug, and apparent in both growth inhibition and apoptotic assays. Conclusion: BKM120 is highly active in preclinical models of MF/SS. Furthermore, it synergistically potentiates the effect of HDAC inhibitors against MF/SS tumor cells. These are highly promising approaches for the treatment of MF/SS and warrant clinical investigation. Citation Format: Chen-Yen Yang, Razan Faraj, Taha Rakhshandaroo, Shervin Afghani, Laura Pincus, Sourav Bandyopadhyay, Frank McCormick, Weiyun Ai. Pathway-directed high throughput drug screen identifies PI3K inhibitors that synergistically potentiate anti-tumor activity of HDAC inhibitors in mycosis fungoides and Sézary syndrome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 388.

Abstract 4634: Novel dual HDAC & PI3K inhibitor, CUDC-907, for MYC-driven malignancies

Abstract MYC family genes are among the most frequently deregulated oncogenic drivers in human cancer. Pharmacologic inhibition of HDAC activity and blockade of the PI3K pathway have both been shown to suppress MYC-induced transcription. HDAC activity is critical for MYC gene regulation, as MYC represses transcription of target genes through recruitment of HDACs. HDAC inhibitors have been shown to restore expression of genes suppressed by MYC family members and to induce rapid downregulation of expression of MYC itself. The PI3K pathway plays a central role in regulating MYC at the post-transcriptional level. Activation of PI3K signaling leads to activation of AKT, which phosphorylates and inhibits GSK3β. As GSK3β normally phosphorylates MYC which facilitates the degradation of MYC, activation of PI3K signaling leads to increased stability of MYC, whereas PI3K inhibitors decrease MYC stability. A recent study has demonstrated addiction to MYC signaling and hypersensitivity to PI3K inhibition in PTEN-deficient diffuse large B-cell (DLBCL) cell lines, suggesting that MYC-driven cancers may be particularly sensitive to PI3K inhibition. As HDACs and PI3K regulate MYC protein levels and functions through nonoverlapping mechanisms, simultaneous HDAC and PI3K inhibition may further enhance MYC suppression. CUDC-907 is an orally bioavailable, small-molecule dual HDAC and PI3K inhibitor that primarily inhibits class I and II HDACs and the PI3Kα, β, and δ isoforms. CUDC-907 shows greater anti-tumor activity in vitro than single-target HDAC or PI3K inhibitors, especially in MYC-dependent cell types, such as DLBCL and NUT midline carcinoma (NMC). In preclinical testing, CUDC-907 treatment leads to a dose-dependent decrease in MYC protein levels, and is also more potent in decreasing MYC than the HDAC inhibitor panobinostat and the pan-PI3K inhibitor pictilisib alone or in combination. Significant antitumor effects have been consistently observed in MYC-driven DLBCL xenograft and genetically engineered mouse models exposed to CUDC907. In particular, certain MYC translocation (Daudi), double-hit (concurrent MYC and BLC2 translocation, WSUDLCL2 and DOHH2), double-expresser (expression of MYC and BCL2 proteins, U2932) xenograft models, and the Eμ-Myc transgenic mouse model achieve tumor growth inhibition of 100%, 69%, 56%, 97% and 72%, respectively. These findings raise the possibility that hematologic and solid tumors driven by aberrant overexpression of MYC family genes (e.g., MYC-altered DLBCL and NMC) might be more responsive to simultaneous HDAC and PI3K inhibition with CUDC-907 than they are to single-target therapy. Clinical Phase 1 studies are currently testing CUDC-907 in patients with relapsed/refractory (R/R) DLBCL and advanced MYC-aberrant solid tumors. Preliminary data are encouraging and support the planned Phase 2 study in R/R MYC-altered DLBCL, as well as further testing in other MYC-driven malignancies. Citation Format: Kaiming Sun, Ruzanna Atoyan, Mylissa A. Borek, Steven Dellarocca, Maria E. Samson, Anna W. Ma, Guangxin Xu, Troy Patterson, David P. Tuck, Jaye L. Viner, Ali Fattaey, Jing Wang. Novel dual HDAC & PI3K inhibitor, CUDC-907, for MYC-driven malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4634.

Hybrid Enzalutamide Derivatives with Histone Deacetylase Inhibitor Activity Decrease Heat Shock Protein 90 and Androgen Receptor Levels and Inhibit Viability in Enzalutamide-Resistant C4-2 Prostate Cancer Cells.

Histone deacetylase inhibitors (HDACIs) can disrupt the viability of prostate cancer (PCa) cells through modulation of the cytosolic androgen receptor (AR) chaperone protein heat shock protein 90 (HSP90). However, toxicities associated with their pleiotropic effects could contribute to the ineffectiveness of HDACIs in PCa treatment. We designed hybrid molecules containing partial chemical scaffolds of enzalutamide and suberoylanilide hydroxamic acid (SAHA), with weakened intrinsic pan-HDACI activities, to target HSP90 and AR in enzalutamide-resistant PCa cells. The potency of the new molecules, compounds 2-75 [4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide] and 1005 [(E)-3-(4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluorophenyl)-N-hydroxyacrylamide], as inhibitors of nuclear and cytosolic histone deacetylases was substantially lower than that of SAHA in cell-free and in situ assays. Compounds 2-75 and 1005 antagonized gene activation by androgen without inducing chromatin association of AR. Enzalutamide had no effect on the levels of AR or HSP90, whereas the hybrid compounds induced degradation of both AR and HSP90, similar to (compound 1005) or more potently than (compound 2-75) SAHA. Similar to SAHA, compounds 2-75 and 1005 decreased the level of HSP90 and induced acetylation in a predicted approximately 55 kDa HSP90 fragment. Compared with SAHA, compound 2-75 induced greater hyperacetylation of the HDAC6 substrate α-tubulin. In contrast with SAHA, neither hybrid molecule caused substantial hyperacetylation of histones H3 and H4. Compounds 2-75 and 1005 induced p21 and caused loss of viability in the enzalutamide-resistant C4-2 cells, with efficacies that were comparable to or better than SAHA. The results suggest the potential of the new compounds as prototype antitumor drugs that would downregulate HSP90 and AR in enzalutamide-resistant PCa cells with weakened effects on nuclear HDACI targets.

Cytotoxic effects of peanut phenolics possessing histone deacetylase inhibitory activity in breast and cervical cancer cell lines.

Epigenetic histone modifications are considered as a promising avenue for cancer preventive and therapeutic strategies. The purpose of this study was to evaluate the antiproliferative and histone deacetylase (HDAC) inhibitory activity of selected peanut phenolics, including p-coumaric acid, ferulic acid, sinapinic acid and resveratrol, in MCF-7 and HeLa cells. The cytotoxic and HDAC inhibitory activities were assessed by MTT assays, flow cytometric analyses of cell cycle arrest and apoptosis induction, and western blotting. The results showed that all four phenolics inhibited proliferation of both MCF-7 and HeLa cells in a dose-dependent manner. Among the phenolics tested, resveratrol was the most effective in inhibiting growth of cancer cells. Treatment with all phenolics resulted in histone H3 hyperacetylation in both cell lines, indicating potential for HDAC inhibition. These phenolics induced apoptosis in both MCF-7 and HeLa cells in a concentration-dependent manner. Moreover, all phenolics induced G0/G1-phase arrest of the cell cycle in MCF-7 cells while p-coumaric and ferulic acids caused S-phase arrest in HeLa cells. Exposure to p-coumaric acid increased p53 and p21 expression but decreased CDK4 levels in both cell types, which could result in the observed G0/G1 arrest. Moreover, inhibition of ERK1/2 phosphorylation by ferulic acid and resveratrol contributed to cell growth inhibition. Peanut phenolics appear to influence the extent of histone acetylation in MCF-7 and HeLa cells, and this activity modulates multiple pathways that are implicated in cancer prevention.

PXD101 analogs with L-phenylglycine-containing branched cap as histone deacetylase inhibitors.

Histone deacetylases (HDACs) allow histones to wrap DNA more tightly and finally lead to the repression of some tumor suppressor genes. Histone deacetylase inhibitors (HDACIs) have been proved to have effects on tumorigenesis and tumor progression. In this study, we reported the design, synthesis, and in vitro activity evaluation of novel PXD101 analogs with L-phenylglycine-containing cap as HDACIs. Our results showed that HDACs inhibitory activities of compounds 10k, 10r, and 10s were not only superior to the first approved HDACI SAHA, but also comparable to their parent compound PXD101, a recently approved HDACI in 2014. However, all 6 selected PXD101 analogs exhibited moderate in vitro antiproliferative activities, less potent than PXD101 and SAHA. Representative compound 10s showed similar HDACs isoform selective profile to PXD101, which demonstrated that introduction of L-phenylglycine-containing branched cap group could not change the isoform selectivity of PXD101 dramatically.

HDAC inhibition promotes both initial consolidation and reconsolidation of spatial memory in mice.

Accumulating evidence suggests a critical role for epigenetic regulations in long term memory (LTM) formation. Among them, post-translational modifications of proteins, as histone acetylation, are an important regulator of chromatin remodelling and gene transcription. While the implication of histone acetylation in memory consolidation is widely accepted, less is known about its role in memory reconsolidation i.e. during memory restabilization after its reactivation. In the present study, we investigated the role of histone acetylation during the initial consolidation and the reconsolidation of spatial memory, using a weak massed learning procedure in the Morris water maze paradigm in mice. Usually a weak learning is sufficient for short term memory (STM) formation, but insufficient to upgrade STM to LTM. We found that promoting histone acetylation through intra-hippocampal infusion of a class I selective histone deacetylase (HDAC) inhibitor immediately after a subthreshold spatial learning improved LTM but not STM retention. More importantly, inhibiting HDAC activity after the reactivation of a weak memory promoted specifically LTM reconsolidation without affecting post-reactivation STM. These findings argue in favour of an important role for histone acetylation in memory consolidation, and more particularly during the reconsolidation of spatial memory in mice.