HDAC Enzymes Research Articles

Hybrid inhibitors of DNA and HDACs remarkably enhance cytotoxicity in leukaemia cells

Chlorambucil is a nitrogen mustard-based DNA alkylating drug, which is widely used as a front-line treatment of chronic lymphocytic leukaemia (CLL). Despite its widespread application and success for the initial treatment of leukaemia, a majority of patients eventually develop acquired resistance to chlorambucil. In this regard, we have designed and synthesised a novel hybrid molecule, chloram-HDi that simultaneously impairs DNA and HDAC enzymes. Chloram-HDi efficiently inhibits the proliferation of HL-60 and U937 leukaemia cells with GI50 values of 1.24 µM and 1.75 µM, whereas chlorambucil exhibits GI50 values of 21.1 µM and 37.7 µM against HL-60 and U937 leukaemia cells, respectively. The mechanism behind its remarkably enhanced cytotoxicity is that chloram-HDi not only causes a significant DNA damage of leukaemia cells but also downregulates DNA repair protein, Rad52, resulting in the escalation of its DNA-damaging effect. Furthermore, chloram-HDi inhibits HDAC enzymes to induce the acetylation of α-tubulin and histone H3.

Design, Synthesis and In Vitro Biological Evaluation Of N-(2-Aminophenyl)-3-Quinolin-4-yl -Prop-2-enamide Derivatives As Novel Colon Anticancer Agents

Acetylation and deacetylation of histone proteins is regulated through two enzymes; acetylation is mediated through histone acetyl transferase (HAT) whereas deacetylation is mediated through histone deacetylase (HDAC). Histone deacetylase removes acetyl group of lysine residue on histone proteinthis makes negatively charged histone able to bind with DNA containing positive centers. In this way DNA remain in compact form with histone proteins. Therefore the transcription or replication enzymes and cofactors not able to bind such compact DNA structure. Histone deacetylase inhibition results into inhibition of various transcription activities which may get accelerated during cancer development. The aim of proposed study is to develop effective isoform selective Histone deacetylase inhibitor. Synthesis of N-(2-Aminophenyl)-3-Quinolin-4-yl-Prop-2-Enamide derivatives from isatin and its derivatives was achieved. Synthesis was carried out in two steps with good yield. 21 Compounds were synthesized and found to be effective with IC50s in between 3.694 - 38.4µmol in human HCT-116, COLO 205 and COLO 320 DM colon cancer cells in vitro. For the cytotoxicity activity study MTT assay method was adopted. Docking studies were performed initially with HDAC8 enzyme using V-life MDS software for synthesized compounds and had selected best fit molecules for synthesis. The synthesized molecules are effective as colon anticancer agents.

Nghiên cứu tương tác của vorinostat với enzyme HDAC8 (1T67) bằng Autodock

Vorinostat is a histone deacetylase inhibitor which was approved by the US FDA in 2006 for the treatment of cutaneous manifestations in patients with cutaneous T-cell lymphoma. Among 18 HDAC enzymes, vorinostat is a potent inhibitor of the activity of HDAC1, HDAC2, HDAC3 and HDAC6. However, there have not been many published papers on the inhibitory capacity against HDAC8 (1T67) of vorinostat. In this study, the interactions of vorinostat with the enzyme HDAC8 (1T67) were performed and described by docking vorinostat into the active zone of the HDAC8 enzyme using Autodock. HDAC8 is a class I histone deacetylase implicated as a therapeutic target in various diseases, including cancer, parasitic infections and Cornelia de Lange syndrome. In invasive breast tumor cells, HDAC8 is among the three HDAC family members that are upregulated and driving invasiveness. The docking analysis shows vorinostat’s interactions with Zn+2 ion, Gly151, Gly304, Asp178, Tyr306, Phe207, Met274 and other less interacting residues. Therefore, the results could act as a momentum for further studies on the design of new isozyme-selective HDAC8 inhibitors.

Discovery of 5-naphthylidene-2,4-thiazolidinedione derivatives as selective HDAC8 inhibitors and evaluation of their cytotoxic effects in leukemic cell lines

Histone deacetylases (HDACs) are being explored as a therapeutic target for interventions in different types of cancer. HDAC8 is a class I HDAC that is implicated as a therapeutic target in various indication areas, including different types of cancer and particularly childhood neuroblastoma. Most previously described HDAC8-selective inhibitors contain a hydroxamate function as zinc binding group (ZBG) to confer potency. However, hydroxamate class HDAC inhibitors have raised increasing concerns about their mutagenic character. Therefore, non-hydroxamate based inhibitors could prove to be safer than hydroxamates. In the present work, a series of novel 5-naphthylidene-2,4-thiazolidinedione was designed and evaluated as potential antiproliferative agents targeting selectively HDAC8 enzyme. Eleven novel derivatives were synthesized, purified and characterized by spectroscopic techniques. Compounds 3k and 3h was found to be most potent selective inhibitors of HDAC8 with IC50 values of 2.7 μM and 6.3 μM respectively. 3a to 3i was found to be most cytotoxic in leukemic cell lines. 3a and 3 h both were found to induce apoptosis and cause cell cycle arrest in G2/M phase.

Design, synthesis and biological evaluation of a series of CNS penetrant HDAC inhibitors structurally derived from amyloid-\u03b2 probes

To develop novel CNS penetrant HDAC inhibitors, a new series of HDAC inhibitors having benzoheterocycle were designed, synthesized, and biologically evaluated. Among the synthesized compounds, benzothiazole derivative 9b exhibited a remarkable anti-proliferative activity (GI50 = 2.01 μM) against SH-SY5Y cancer cell line in a dose and time-dependent manner, better than the reference drug SAHA (GI50 = 2.90 μM). Moreover, compound 9b effectively promoted the accumulation of acetylated Histone H3 and α-tubulin through inhibition of HDAC1 and HDAC6 enzymes, respectively. HDAC enzyme assay also confirmed that compound 9b efficiently inhibited HDAC1 and HDAC6 isoforms with IC50 values of 84.9 nM and 95.9 nM. Furthermore, compound 9b inhibited colony formation capacity of SH-SY5Y cells, which is considered a hallmark of cell carcinogenesis and metastatic potential. The theoretical prediction, in vitro PAMPA-BBB assay, and in vivo brain pharmacokinetic studies confirmed that compound 9b had much higher BBB permeability than SAHA. In silico docking study demonstrated that compound 9b fitted in the substrate binding pocket of HDAC1 and HDAC6. Taken together, compound 9b provided a novel scaffold for developing CNS penetrant HDAC inhibitors and therapeutic potential for CNS-related diseases.

Inhibitors of class I histone deacetylases attenuate thioacetamide-induced liver fibrosis in mice by suppressing hepatic type 2 inflammation.

Chronic liver diseases feature excessive collagen and matrix protein deposition or crosslinking that characterises fibrosis, leads to scar tissue, and disrupts liver functions. There is no effective treatment. This study investigated whether treatment with selective histone deacetylase (HDAC) inhibitors might specifically reduce type 2 inflammation in the injured liver, thereby attenuating fibrogenesis in mice. Thioacetamide (TAA) was used to induce hepatic inflammation, fibrosis, and liver damage in female C57BL/6 mice, similar to the clinical features of chronic human liver disease. We used eight inhibitors of different human HDAC enzymes to probe histological (IHC and TUNEL), biochemical and immunological changes (flow cytometry, qPCR, Legendplex, and ELISA) in pathology, fibrosis, hepatic immune cell flux, and inflammatory cytokine expression. Inhibitors of class I, but not class II, HDAC enzymes potently suppressed chronic hepatic inflammation and fibrosis in mice, attenuating accumulation and activation of IL-33-dependent, but not IL-25-dependent, group 2 innate lymphoid cells (ILC2) and inhibiting type 2 inflammation that drives hepatic stellate cells to secrete excessive collagen and matrix proteins. The results show that potent and selective inhibitors of class I only HDAC enzymes profoundly inhibit hepatocyte death and type 2 inflammation to prevent TAA-induced liver fibrosis in mice. The specific HDAC enzymes identified here may be key promoters of inflammation in chronic liver fibrosis.

Histone deacetylases inhibitor trichostatin A reverses anxiety-like symptoms and memory impairments induced by maternal binge alcohol drinking in mice.

Alcohol exposure during development has detrimental effects, including a wide range of physical, cognitive and neurobehavioural anomalies known as foetal alcohol spectrum disorders. However, alcohol consumption among pregnant woman is an ongoing latent health problem. In the present study, the effects of trichostatin A (TSA) on emotional and cognitive impairments caused by prenatal and lactational alcohol exposure were assessed. TSA is an inhibitor of class I and II histone deacetylases enzymes (HDAC), and for that, HDAC4 activity was determined. We also evaluated mechanisms underlying the behavioural effects observed, including the expression of brain-derived neurotrophic factor (BDNF) in discrete brain regions and newly differentiated neurons in the dentate gyrus (DG). C57BL/6 female pregnant mice were used, with limited access to a 20% v/v alcohol solution as a procedure to model binge alcohol drinking during gestation and lactation. Male offspring were treated with TSA during the postnatal days (PD28-35) and behaviourally evaluated (PD36-55). Early alcohol exposure mice presented increased anxiogenic-like responses and memory deterioration - effects that were partially reversed with TSA. Early alcohol exposure produces a decrease in BDNF levels in the hippocampus (HPC) and prefrontal cortex, a reduction of neurogenesis in the DG and increased activity levels of the HDAC4 in the HPC. Such findings support the participation of HDAC enzymes in cognitive and emotional alterations induced by binge alcohol consumption during gestation and lactation and would indicate potential benefits of HDAC inhibitors for some aspects of foetal alcohol spectrum disorders.

Selective inhibition of histone deacetylase 8 improves vascular hypertrophy, relaxation, and inflammation in angiotensin II hypertensive mice

BackgroundThe dysregulation of histone deacetylase (HDAC) protein expression or its enzyme activity is implicated in a variety of diseases. Cardiac HDAC6 and HDAC8 enzyme activity induced by deoxycorticosterone acetate (DOCA) hypertension was attenuated by sodium valproate, a pan-HDAC inhibitor. However, the HDAC6-selective inhibitor, tubastatin A, did not attenuate angiotensin II-induced hypertension. The purpose of this study was to investigate whether PCI34051, an HDAC8-selective inhibitor, can modulate angiotensin II-induced hypertension and its regulatory mechanism.MethodsAn angiotensin II-regulated mouse model was used in this study. Animals received vehicle or PCI34051 (3 mg·kg − 1·day− 1) via intraperitoneal injection. Systolic blood pressure was measured by the tail-cuff method. Blood vessel thickness was measured following hematoxylin and eosin staining, VCAM-1 immunohistochemistry was performed in the aortas, and mRNA expression of renin-angiotensin system components, inflammation markers, and NADPH oxidase (Nox) was determined by RT-PCR. The effect of PCI34051 on vasorelaxation was studied in rat aortic rings, and its effect on nitric oxide (NO) production was determined using DAF-FM DA, a fluorescent dye, in human umbilical vascular endothelial cells (HUVECs).ResultsPCI34051 administration reduced systolic blood pressure via downregulation of angiotensin II receptor type 1 (AT1) mRNA expression. PCI34051 treatment attenuated vascular hypertrophy by decreasing E2F3 and GATA6 mRNA expression. Vascular relaxation after PCI34051 treatment was more dependent on vascular endothelial cells and it was blocked by an NO synthase (NOS) inhibitor. In addition, NO production increased in HUVECs after PCI34051 treatment; this was decreased by the NOS inhibitor. The expression of inflammatory molecules and adhesion molecules VCAM-1 and ICAM-1 decreased in the aortas of angiotensin II-infused mice after PCI34051 administration. However, PCI34051 did not affect Nox or its regulatory subunits.ConclusionsPCI34051 lowered high blood pressure through modulation of arterial remodeling, vasoconstriction, and inflammation in an angiotensin II-induced hypertension model. We suggest that HDAC8 could be a potential therapeutic target for hypertension.

Selective pharmacological inhibitors of HDAC6 reveal biochemical activity but functional tolerance in cancer models.

Our study investigates the biochemical and functional impact of selective histone deacetylase 6 (HDAC6) inhibitors, a promising class of novel therapeutics, in several cancer models. Selective HDAC6 inhibitors (Tubathian A, Tubastatin A, Tubacin and Ricolinostat) and a non-selective HDAC inhibitor (Vorinostat) were evaluated on cancer cell lines derived from multiple tumour types in both an in vitro and in vivo setting as potential cancer therapeutics. Selective HDAC6 inhibitors resulted in α-tubulin acetylation with no impact on histone acetylation but failed to show any anti-cancer properties. Only the use of high concentrations of selective HDAC6 inhibitors resulted in co-inhibition of other HDAC enzymes and consequently in reduced growth, migratory and/or invasive activity of cancer cells in vitro as well as in vivo. The specificity of HDAC6 inhibition was confirmed using a CRISPR/Cas9 knockout cell line. Our results suggest that selective HDAC6 inhibitors may fall short as potential single agent anti-cancer drugs and prove that many previous data regarding this promising class of compounds need to be interpreted with great care due to their use in high concentrations resulting in low selectivity and potential off-target effects.

Synthesis and Biological Investigation of Phenothiazine-Based Benzhydroxamic Acids as Selective Histone Deacetylase 6 Inhibitors.

The phenothiazine system was identified as a favorable cap group for potent and selective histone deacetylase 6 (HDAC6) inhibitors. Here, we report the preparation and systematic variation of phenothiazines and their analogues containing a benzhydroxamic acid moiety as the zinc-binding group. We evaluated their ability to selectively inhibit HDAC6 by a recombinant HDAC enzyme assay, by determining the protein acetylation levels in cells by western blotting (tubulin vs histone acetylation), and by assessing their effects on various cancer cell lines. Structure-activity relationship studies revealed that incorporation of a nitrogen atom into the phenothiazine framework results in increased potency and selectivity for HDAC6 (more than 500-fold selectivity relative to the inhibition of HDAC1, HDAC4, and HDAC8), as rationalized by molecular modeling and docking studies. The binding mode was confirmed by co-crystallization of the potent azaphenothiazine inhibitor with catalytic domain 2 from Danio rerio HDAC6.

Evaluation of Novel 3-Hydroxyflavone Analogues as HDAC Inhibitors against Colorectal Cancer.

Alteration of epigenetic enzymes is associated with the pathophysiology of colon cancer with an overexpression of histone deacetylase 8 (HDAC8) enzyme in this tissue. Numerous reports suggest that targeting HDAC8 is a viable strategy for developing new anticancer drugs. Flavonols provide a rich source of molecules that are effective against cancer; however, their clinical use is limited. The present study investigated the potential of quercetin and synthetic 3-hydroxyflavone analogues to inhibit HDAC8 enzyme and evaluated their anticancer property. Synthesis of the analogues was carried out, and cytotoxicity was determined using MTT assay. Nonspecific and specific HDAC enzyme inhibition assays were performed followed by the expression studies of target proteins. Induction of apoptosis was studied through annexin V and caspase 3/7 activation assay. Furthermore, the analogues were assessed against in vivo colorectal cancer. Among the synthesized analogues, QMJ-2 and QMJ-5 were cytotoxic against HCT116 cells with an IC50 value of 68 ± 2.3 and 27.4 ± 1.8 µM, respectively. They inhibited HDAC enzyme in HCT116 cells at an IC50 value of 181.7 ± 22.04 and 70.2 ± 4.3 µM, respectively, and inhibited human HDAC8 and 1 enzyme at an IC50 value of <50 µM with QMJ-5 having greater specificity towards HDAC8. A reduction in the expression of HDAC8 and an increase in acetyl H3K9 expression were observed with the synthesized analogues. Both QMJ-2 and QMJ-5 treatment induced apoptosis through the activation of caspase 3/7 evident from 55.70% and 83.55% apoptotic cells, respectively. In vivo studies revealed a significant decrease in colon weight to length ratio in QMJ-2 and QMJ-5 treatment groups compared to DMH control. Furthermore, a reduction in aberrant crypt foci formation was observed in the treatment groups. The present study demonstrated the potential of novel 3-hydroxyflavone analogues as HDAC8 inhibitors with anticancer property against colorectal cancer.

A novel class of anthraquinone-based HDAC6 inhibitors

Histone deacetylase 6 (HDAC6) is an important target for the treatment of diverse diseases including cancer, neurodegenerative diseases, autoimmune disorders, inflammation, drug addiction, and viral infection. Therefore, the discovery of HDAC6-isoform selective inhibitors is of high importance for clinical applications. Here, we present an approach to discover HDAC6-isoform selective inhibitors. To our best knowledge, we for the first time perform a virtual screening campaign in the surface and channel region of HDAC6 enzyme, followed by rational installation of zinc binding group for the development of HDAC6-isoform selective inhibitors. Consequently, this approach establishes the proof of principle for the discovery of HDAC6-isoform selective inhibitors and successfully provides our lead compound 3. In particular, compound 3 inhibits HDAC6 enzyme with an IC50 value of 56 nM and displays an excellent HDAC6 selectivity over other HDAC isoforms in HDAC enzyme assay. Furthermore, the exposure of SH-SY5Y cells with compound 3 significantly promotes the acetylation of α-tubulin at the low concentration of 0.5 μM, but not the acetylation of Histone H3 up to 20 μM. Thus, our lead compound 3 represents a novel HDAC6-isoform selective inhibitor and warrants further studies for therapeutic evaluation.

A Pooled Analysis of Relapsed/Refractory Diffuse Large B-Cell Lymphoma Patients Treated with the Dual PI3K and HDAC Inhibitor Fimepinostat (CUDC-907), Including Patients with MYC-Altered Disease

IntroductionPatients with relapsed or refractory (R/R) MYC-altered DLBCL have poor outcomes, and other than for a subset of patients who may benefit from chimeric antigen receptor T cell therapy, no treatment has shown a significant durable benefit or impact on survival outcomes. Fimepinostat is a first-in-class, well-tolerated, oral small molecule inhibitor of HDAC and PI3K enzymes. There is particular interest in evaluating fimepinostat in patients with MYC-dependent tumors as nonclinical studies demonstrated that fimepinostat inhibits transcription of MYC and a subset of MYC-associated genes. Additionally, MYC protein levels were downregulated by fimepinostat in part through inhibition of PI3K-mediated ubiquitination. Pharmacodynamic inhibition of HDAC and PI3K has also been demonstrated in human studies. Here we report the outcome of R/R DBLCL patients treated with fimepinostat in a Phase 1 and Phase 2 study, with an emphasis on outcomes for patients with MYC-altered disease.Patients and MethodsA total of 105 R/R DLBCL patients were enrolled on the Phase 1 study CUDC-907-101 (n = 37) and the Phase 2 study CUDC-907-201 (n = 68). In CUDC-907-101, 14 patients were found to have MYC-altered disease, defined as presence of MYC rearrangement by either central or local testing by fluorescent in situ hybridization or MYC protein expression ≥40%) by immunohistochemistry (IHC). In CUDC-907-201, 46 patients had confirmed MYC-altered disease by central IHC testing. Across both studies, patients without available tissue or prior test results were deemed as MYC-status unknown (n = 23).ResultsA total of 19 responses (9 CR, 10 partial responses [PR]) were reported across both studies. The objective response rate (ORR) in MYC altered patients was 23.3% (14/60). Responses showed encouraging durability with a median duration of response of 13.6 months (range: 2.8 to Not Calculable [NC]). Five MYC-altered responses were ongoing as of the data-cut. Two MYC-altered patients achieving CR discontinued treatment early to pursue stem cell transplantation.Responses associated with fimepinostat often require multiple cycles of treatment to manifest (median time to first response = 2.5 months for MYC-altered patients), and of patients who were treated for ≥2 months, a large proportion (17/33; 52%) achieved a response. Patients with low disease burden at screening (tumor lesions diameters ≤ 5 cm and lactate dehydrogenase [LDH] < 1.5 x upper limit of normal [ULN]) generally continued treatment longer and were most likely to derive clinical benefit (Table 2).ConclusionsThe biologic rationale, tolerable safety profile, and evidence of durable anti-tumor activity in MYC-altered R/R DLBCL support the continued development of fimepinostat in this poor-prognosis patient population. Patients with low disease burden features may be more likely to have sufficient duration of drug exposure to allow clinical benefit. Future enrollment will focus on patients with screening characteristics most likely to derive the greatest benefit from fimepinostat treatment. [Display omitted] DisclosuresLandsburg:Takeda: Consultancy; Curis: Consultancy, Research Funding. Ramchandren:Seattle Genetics: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Merck: Research Funding; Pharmacyclics LLC an AbbVie Company: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Lugtenburg:Millennium/Takeda: Consultancy, Research Funding; Servier: Consultancy, Research Funding; Roche: Consultancy; BMS: Consultancy; Sandoz: Consultancy; Genmab: Consultancy. Younes:Takeda: Honoraria; Abbvie: Honoraria; BMS: Honoraria, Research Funding; Novartis: Research Funding; Curis: Research Funding; Incyte: Honoraria; Seattle Genetics: Honoraria; Janssen: Honoraria, Research Funding; Bayer: Honoraria; Sanofi: Honoraria; Astra Zeneca: Research Funding; Celgene: Honoraria; J&J: Research Funding; Pharmacyclics: Research Funding; Roche: Honoraria, Research Funding; Merck: Honoraria; Genentech: Research Funding. Tuck:Curis, Inc: Employment, Equity Ownership. Barta:Janssen: Membership on an entity's Board of Directors or advisory committees; Merck, Takeda, Celgene, Seattle Genetics, Bayer: Research Funding.

Berberine attenuate staphylococcal enterotoxin B-mediated acute liver injury via regulating HDAC expression

Staphylococcal enterotoxin B (SEB) has been documented to be implicated in the pathogenesis of liver injury in the experimental models of hepatitis. However, the underlying mechanism of SEB-induced acute liver injury (ALI) remains to be further explored. In our study, we explored the therapeutic effectiveness of berberine (BBR), a natural isoquinoline alkaloid, in the SEB-induced ALI. In our study, we found that injection of SEB into d-galactosamine (d-gal)-sensitized mice induced ALI, as demonstrated by an increase of levels of alanine aminotransferase and aspartate aminotransferase, massive infiltration of immune cells into the liver, and pro-inflammatory cytokine release. However, intragastric administration of BBR attenuated SEB-induced ALI in mice. Meanwhile, we discovered that BBR treatment suppressed activation of splenocytes and pro-inflammatory cytokine release in SEB-stimulated splenocytes. Moreover, mechanistic analyses demonstrated that BBR was effective at inhibiting the expression of class I HDAC, but not class II, in SEB-stimulated splenocytes. Furthermore, trichostatin A, a standard HDAC inhibitor, alleviated activation of splenocytes and pro-inflammatory cytokine release in SEB-stimulated splenocytes. Taken together, we inferred from these results that BBR attenuated SEB-mediated ALI through repressing the class I HDAC enzyme, suggesting that BBR may constitute a novel therapeutic modality to prevent SEB-mediated inflammation and ALI.

Abstract A15: DNA damage response-targeted molecules for breast cancer therapy

Abstract Breast cancer genes 1 and 2 (BRACA1 and BRACA2) are known to be linked to breast cancer risk. BRCA1 is an ATM substrate and plays critical roles in the DNA damage response (DDR) and maintenance of genomic stability. Furthermore, recent studies have shown that the aberrant expression of DNA damage repair transducer, ataxia-telangiectasia mutated (ATM), is associated with breast cancers. To develop target-specific drugs, we rationally designed a dual-function small molecule, SP-1-303, by conferring ATM activation and HDAC inhibition activity. The compound was screened against a panel of Class I and Class II HDAC enzymes in vitro and identified as a pan-HDAC inhibitor at an IC50 of 120 nM. Western analysis revealed that SP-1-303 increased acetylated histone H3/H4, α-tubulin, and ATM activation (p-ATM/S1987). Testing two ER-positive BC cell lines (MCF7 and T-47D), two triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and HCC 1937), and one normal breast epithelial cell line as a control (MCF10A), the effect of SP-1-303 on cell growth was examined by cytotoxicity assays. SP-1-303 demonstrated cytotoxicity (IC50) of ER-positive BC cells in a range of 0.2-0.3 μM, while the values for TNBC cells are in a range of 1-3 μM and 12 μM for normal breast epithelial cells, respectively. Furthermore, proteomic analysis of MCF7 cells following SP-1-303 treatment identified differential effects on expression of various DDR molecules and chromatin regulators, including ATM, BRCA1, TP53BP1, NBN, histones, and chromatin remodelers. Taken together, SP-1-303 shows specificity for ER-positive breast cancers, potentially by modulating the DDR signaling pathway. Citation Format: Alfredo Valenea, Scott Grindrod, Mira Jung. DNA damage response-targeted molecules for breast cancer therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A15.

Squaramides as novel class I and IIB histone deacetylase inhibitors for topical treatment of cutaneous t-cell lymphoma

A series of squaramide-based hydroxamic acids were designed, synthesized and evaluated against human HDAC enzyme. Squaramides were found to be potent in the Hut78 cell line, but initially suffered from low solubility. Leads with improved solubility and metabolic profiles were shown to be class I, IIB and IV selective.

Sex-Dependent Effects of the Histone Deacetylase Inhibitor, Sodium Valproate, on Reversal Learning After Developmental Arsenic Exposure.

Several studies have demonstrated that exposure to arsenic in drinking water adversely affects brain development and cognitive function in adulthood. While the mechanism by which arsenic induces adverse neurological outcomes remains elusive, studies suggest a link between reduced levels of histone acetylation and impaired performance on a variety of behavioral tasks following arsenic exposure. Using our developmental arsenic exposure (DAE) paradigm, we have previously reported reduced histone acetylation and associated histone acetyltransferase enzyme expression in the frontal cortex of C57BL/6J adult male mice, with no changes observed in the female frontal cortex. In the present study, we sought to determine if DAE produced sex-dependent deficits in frontal cortical executive function using the Y-maze acquisition and reversal learning tasks, which are specific for assessing cognitive flexibility. Further, we tested whether the administration of valproic acid, a class I–IIa histone deacetylase inhibitor, was able to mitigate behavioral and biochemical changes resulting from DAE. As anticipated, DAE inhibited acquisition and reversal learning performance in adult male, but not female, mice. Valproate treatment for 2 weeks restored reversal performance in the male arsenic-exposed offspring, while not affecting female performance. Protein levels of HDACs 1, 2, and 5 were elevated following behavioral assessment but only in DAE male mice; restoration of appropriate HDAC levels occurred after valproate treatment and was concurrent with improved behavioral performance, particularly during reversal learning. Female frontal cortical levels of HDAC enzymes were not impacted by DAE or valproate treatment. Finally, mRNA expression levels of brain-derived neurotrophic factor, Bdnf, which has been implicated in the control of frontal cortical flexibility and is regulated by HDAC5, were elevated in DAE male mice and restored to normal levels following HDACi treatment. Levels of mRNA encoding glutamate receptor ionotropic NMDA type subunits, which have been linked to cognitive flexibility, were not related to the reversal learning deficit in the DAE mice and were not altered by HDACi treatments. These findings demonstrate that DAE alters frontal cortical HDAC levels and Bdnf expression in males, but not females, and that these molecular changes are associated with sex-dependent differences in cognitive flexibility in a reversal-learning task.

Abstract 1395: HDAC6 and DNMT inhibition affect immunogenicity of ovarian cancer cells: A rationale for combining epigenetic and immune therapy in ovarian cancer

Abstract Background: Therapies that activate the immune system to fight cancer have shown robust responses in solid tumors. However, most patients, including those with ovarian cancer, do not respond to these therapies alone. Drugs that inhibit epigenetic modifiers increase immune signaling from cancer cells. Epigenetic modifiers DNA methyltransferase inhibitors (DNMTi) and selective histone deacetylase inhibitors (HDACi),in particular selective HDAC6i, modulate immune-related pathways involved in anti-tumor immune responses. HDAC6i downregulate immunosuppressive ligands PD-L1 and PD-L2 via dephosphorylating pSTAT3 and upregulate tumor associated antigens (TAA) and antigen presentation machinery. Similarly, DNMTi activate anti-viral signaling via expression of Endogenous Retroviruses (ERVs) to trigger the type I interferon response, upregulate tumor antigen processing and presentation, and stimulate pro-inflammatory cytokines. The aim of our study is to test if the combination of epigenetic modulators Nexturastat A (Next A), a selective HDAC6i, and 5-azacytidine (AZA), a DNMTi, can be safely used to increase an immune response in ovarian cancer. We hypothesize that these drugs will enhance tumor immunity alone and when combined with immune checkpoint blockades targeting PD-1. Results: HDAC enzymes are differentially expressed in A2780, HEY, OVCAR3, SKOV3, and TYKNu human ovarian cancer cell lines. HDAC1 and HDAC2 proteins were similarly expressed in HEY and SKOV3 whereas HDAC6 was expressed at lower levels in HEY and TYKNu but at higher levels in SKOV3, OVCAR3, and A2780. As previously reported we believe this may be due to the presence of the chromatin modifier ARID1A mutation in the SKOV3 and A2780 cell lines. The upregulation of HDAC6 also correlated with a higher IC50 for NextA treatment in those particular cell lines. Further immunoblots showed that PD-L1, a marker of poor prognosis in ovarian cancer, decreased after treatment with NextA and even more in combination with AZA. Additionally, DNMT1, the known target of AZA, was decreased after treatment with AZA and NextA, both independently and in combination, a finding that has not been previously reported. Conclusions: As shown previously, HDAC6 enzyme levels are higher in cell lines with ARID1A mutations. DNMT1 was decreased after treatment with AZA, as expected, but surprisingly also after treatment with NextA. PD-L1 decreased after treatment with NextA and even more so when combined with AZA. We thus believe that combining these two epigenetic modifiers will lead to an additive effect on immune signaling through stimulation of antiviral signaling (DNMTi), which can upregulate the immunosuppressive ligand PD-L1, which is then reduced by HDAC6 inhibition. We are currently testing the combination of both epigenetic modifiers with anti-PD-1 in an immunocompetent mouse model of ovarian cancer. Citation Format: Aneil P. Srivastava, Sara M. Moufarrij, Melissa Hadley, Sarah Chisholm, Micael Lopez-Acevedo, Alejandro Villagra, Katherine B. Chiappinelli. HDAC6 and DNMT inhibition affect immunogenicity of ovarian cancer cells: A rationale for combining epigenetic and immune therapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1395.

Histone Deacetylase Inhibitor (HDACi) Conjugated Polycaprolactone for Combination Cancer Therapy.

The short chain fatty acid, 4-phenylbutyric acid (PBA), is used for the treatment of urea cycle disorders and sickle cell disease as an endoplasmic reticulum stress inhibitor. PBA is also known as a histone deacetylase inhibitor (HDACi). We report here the effect of combination therapy on HeLa cancer cells using PBA as the HDACi together with the anticancer drug, doxorubicin (DOX). We synthesized γ-4-phenylbutyrate-ε-caprolactone monomer which was polymerized to form poly(γ-4-phenylbutyrate-ε-caprolactone) (PPBCL) homopolymer using NdCl3·3TEP/TIBA (TEP = triethyl phosphate, TIBA = triisobutylaluminum) catalytic system. DOX-loaded nanoparticles were prepared from the PPBCL homopolymer using poly(ethylene glycol) as a surfactant. An encapsulation efficiency as high as 88% was obtained for these nanoparticles. The DOX-loaded nanoparticles showed a cumulative release of >95% of DOX at pH 5 and 37 °C within 12 h, and PBA release was monitored by 1H NMR spectroscopy. The efficiency of the combination therapy can notably be seen in the cytotoxicity study carried out on HeLa cells, where only ∼20% of cell viability was observed after treatment with the DOX-loaded nanoparticles. This drastic cytotoxic effect on HeLa cells is the result of the dual action of DOX and PBA on the DNA strands and the HDAC enzymes, respectively. Overall, this study shows the potential of combination treatment with HDACi and DOX anticancer drug as compared to the treatment with an anticancer drug alone.

Curcumin Derivative Epigenetically Reactivates Nrf2 Antioxidative Stress Signaling in Mouse Prostate Cancer TRAMP C1 Cells.

The carcinogenesis of prostate cancer (PCa) in TRAMP model is highly correlated with hypermethylation in the promoter region of Nrf2 and the accompanying reduced transcription of Nrf2 and its regulated detoxifying genes. We aimed to investigate the effects of (3E,5E)-3,5-bis-(3,4,5-trimethoxybenzylidene)-tetrahydro-thiopyran-4-one (F10) and (3E,5E)-3,5-bis-(3,4,5-trimethoxy-benzylidene)-tetrahydropyran-4-one (E10), two synthetic curcumin derivatives, on restoring Nrf2 activity in TRAMP C1 cells. HepG2-C8 cells transfected with an antioxidant-response element (ARE)-luciferase vector were treated with F10, E10, curcumin, and sulforaphane (SFN) to compare their effects on Nrf2-ARE pathways. We performed real-time quantitative PCR and Western blotting to investigate the effects of F10 and E10 on Nrf2, correlated phase II detoxification genes. We also measured expression and activity of DNMTand HDAC enzymes. Enrichment of H3K27me3 on the promoter region of Nrf2 was explored with a chromatin immunoprecipitation (ChIP) assay. Methylation of the CpG region in Nrf2 promoter was doubly examined by bisulfite genomic sequencing (BGS) and methylation DNA immunoprecipitation (MeDIP). Compared with curcumin and SFN, F10 is more potent in activating Nrf2-ARE pathways. Both F10 and E10 enhanced level of Nrf2 and the correlated phase II detoxifying genes. BGS and MeDIP assays indicated that F10 but not E10 hypomethylated the Nrf2 promoter. F10 also downregulated the protein level of DNMT1, DNMT3a, DNMT3b, HDAC1, HDAC4, and HDAC7 and the activity of DNMTs and HDACs. F10 but not E10 effectively reduced the accumulation of H3k27me3 on the promoter of Nrf2. F10 and E10 can activate the Nrf2-ARE pathway and increase the level of Nrf2 and correlated phase II detoxification genes. The reactivation effect on Nrf2 by F10 in TRAMP C1 may come from demethylation, decrease of HDACs, and inhibition of H3k27me3 accumulation.