Isoform-selective Histone Deacetylase Research Articles

The histone deacetylase inhibitor entinostat (SNDX-275) induces apoptosis in Hodgkin lymphoma cells and synergizes with Bcl-2 family inhibitors

Based on promising invitro and invivo activity of several histone deacetylase inhibitors in Hodgkin lymphoma (HL), we investigated SNDX-275, an oral class 1 isoform-selective histone deacetylase inhibitors in HL-derived cell lines. Proliferation and cell death were examined by MTS assay, Annexin V/propidium iodide, and fluorescence-activated cell sorting analysis. Gene and protein expression were measured by reverse transcriptase polymerase chain reaction, Western blotting, and immunohistochemical analysis. A multiplex assay was used to determine cytokines and chemokines. SNDX-275 induced cell death in a dose- and time-dependent manner with an IC(50) at the sub- and lower micromolar range at 72 hours. At the molecular level, SNDX-275 increased histone H3 acetylation, upregulated p21 expression, and activated the intrinsic apoptosis pathway by downregulating the X-linked inhibitor of apoptosis protein. SNDX-275 downregulated expression of antiapoptotic Bcl-2 and Bcl-xL proteins without altering Mcl-1 or Bax levels. Combination studies demonstrated that two Bcl-2 inhibitors (ABT-737 and obatoclax) significantly enhanced the effect of SNDX-275. SNDX-275 modulated the level of several cytokines and chemokines, including interleukin-12 p40-70, interferon-inducible protein-10, RANTES (regulated on activation, normal T expressed and secreted), interleukin-13, interleukin-4, and thymus and activation-regulated chemokine and variably induced the cancer/testis antigen expression of MAGE-A4 and survivin in HL cell lines. SNDX-275 has antiproliferative activity in HL cell lines, involving several mechanisms: induction of apoptosis, regulation of cytokines and chemokines, and alteration of cancer/testis antigens. Clinical investigation of SNDX-275 alone or in combination with Bcl-2 inhibitors is warranted in patients with HL. Phase 2 studies with SNDX-275 in HL are ongoing, and future clinical studies should investigate combinations with SNDX-275.

Zinc chelation with hydroxamate in histone deacetylases modulated by water access to the linker binding channel.

It is of significant biological interest and medical importance to develop class- and isoform-selective histone deacetylase (HDAC) modulators. The impact of the linker component on HDAC inhibition specificity has been revealed but is not understood. Using Born-Oppenheimer ab initio QM/MM MD simulations, a state-of-the-art approach to simulating metallo-enzymes, we have found that the hydroxamic acid remains to be protonated upon its binding to HDAC8, and thus disproved the mechanistic hypothesis that the distinct zinc-hydroxamate chelation modes between two HDAC subclasses come from different protonation states of the hydroxamic acid. Instead, our simulations suggest a novel mechanism in which the chelation mode of hydroxamate with the zinc ion in HDACs is modulated by water access to the linker binding channel. This new insight into the interplay between the linker binding and the zinc chelation emphasizes its importance and gives guidance regarding linker design for the development of new class-IIa-specific HDAC inhibitors.

Short-Chain HDAC Inhibitors Differentially Affect Vertebrate Development and Neuronal Chromatin

Carboxylic acids with known central nervous system and histone deacetylase (HDAC) inhibitory activities were converted to hydroxamic acids and tested using a suite of in vitro biochemical assays with recombinant HDAC isoforms, cell based assays in human cervical carcinoma Hela cells and primary cultures from mouse forebrain, and a whole animal (Xenopus laevis) developmental assay. Relative to the parent carboxylic acids, two of these analogs exhibited enhanced potency, and one analog showed altered HDAC isoform selectivity and in vivo activity in the Xenopus assay. We discuss potential uses of these novel hydroxamic acids in studies aimed at determining the utility of HDAC inhibitors as memory enhancers and mood stabilizers.

Non-Peptide Macrocyclic Histone Deacetylase Inhibitors Derived from Tricyclic Ketolide Skeleton

Inhibition of histone deacetylase (HDAC) function is a validated therapeutic strategy for cancer treatment. Of the several structurally distinct small molecule histone deacetylase inhibitors (HDACi) reported, macrocyclic depsipeptides possess the most complex cap groups and have demonstrated excellent HDAC inhibition potency and isoform selectivity. Unfortunately, the development of macrocyclic depsipeptides has been hampered in part because of development problems characteristic of large peptides and the complex reaction schemes required for their synthesis. Herein we report that tricyclic ketolide TE-802 is an excellent mimetic for the peptide backbone of macrocyclic HDACi. Compounds derived from this template are particularly selective against HDACs 1 and 2 with nanomolar inhibitory activity. Interrogation of the association between a subset of these compounds and key HDAC isoforms, using AutoDock, enables a molecular description of the interaction between the HDAC enzyme's outer rim and the inhibitors' macrocyclic cap group that are responsible for compound affinity and presumably isoform selectivity.

ENGAGE-501: Phase II study investigating the role of epigenetic therapy with entinostat (SNDX-275) in relapsed and refractory Hodgkin's lymphoma (HL).

TPS298 Background: Entinostat is an oral, class 1 isoform selective histone deacetylase inhibitor (HDACi) with a half-life of 80- 100 hours in humans and has been well tolerated as a single agent and in combination with other agents tested to date. In preclinical studies entinostat demonstrated a dual effect on apoptotic and immunomodulatory pathways that resulted in significant antitumor activity in HL cell lines and primary patient samples. A phase II study was initiated in HL patients to investigate the hypothesis that entinostat will exhibit antitumor activity through the induction of p21 and TRAIL death receptors, downregulation of STAT-6 expression, modulation of cytokines and chemokines, and upregulation of expression of B cell and other antigens. Methods: ENGAGE-501 is an open-label, multicenter two-stage design. Entry criteria mandates that patients must have failed a primary therapy and if eligible, stem cell transplantation. A dose of entinostat of 10 mg on day 1 and day 15 in 28-day cycles was selected. If 1/9 pts responded in the first stage, an additional 15 patients were to be enrolled for a total of 24 evaluable. The primary endpoint is objective response rate over 6 cycles. Duration of response, progression free survival and overall survival will also be measured. Biomarker evaluations on peripheral blood samples (pre-dose: cycle 1 d1, day 15; d8 at anytime; predose Cycle 3) will include: HDAC activity measured by protein lysine acetylation, RNA expression of genes related to mechanism (e.g., p21, TRAIL death receptors, STAT), cytokines/chemokine array analysis (e.g., TARC, IL-12, IL-5), and flow cytometric analysis of antigens on circulating lymphocyte subsets. Pharmacokinetic evaluation will measure entinostat levels in cycle 1 at pre-dose, 15, 30, 60 and 120 minutes, day 8 and day 15 post entinostat. The study is currently enrolling for the second stage. Based on the initial tolerability, the protocol was amended to allow a dose escalation to 15 mg on day 1 and day 15. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Syndax Abbott Laboratories, Amgen, Biogen Idec, Genentech, MethylGene, Novartis, Pharmion, Sandoz, sanofi-aventis, Seattle Genetics, Trubion, Xencor Syndax Abbott Laboratories, Amgen, Biogen Idec, Genentech, MethylGene, Novartis, Pharmion, Sandoz, sanofi-aventis, Seattle Genetics, Trubion, Xencor

A double-blind, randomized, placebo-controlled phase II study of the steroidal aromatase inhibitor exemestane with and without the isoform selective histone deacetylase inhibitor (HDACi) entinostat in metastatic breast cancer (MBC).

TPS128 Background: Anti-estrogentherapies, including aromatase inhibitors, are effective in the treatment of MBC patients who are estrogen receptor (ER) positive, but acquired resistance to these agents eventually develops in virtually all patients, leading to disease progression. Decreased ER expression levels and increase in growth factor signaling both contribute to resistance to aromatase inhibitors (Brodie et al 2009 J Steroid Biochem Mol Biol). Preclinically, HDACi reverse ER expression silencing (reviewed in Stearns et al 2007 Cancer Invest) and inhibit the growth factor signaling pathways that drive breast cancer growth (Huang et al 2009 Canc Res). Entinostat is an oral class 1 HDAC selective inhibitor that has been well tolerated to date and may be combined with aromatase inhibitors. The purpose of this ongoing clinical trial is to assess whether the addition of entinostat to the steroidal aromatase inhibitor exemestane can overcome and/or delay the acquired resistance to aromatase inhibitors in MBC. Methods: Post-menopausal females with ER+ MBC progressing on a non-steroidal aromatase inhibitor are eligible for this study. Target patient population consists of 80% of patients having measurable disease with 20% of patients having nonmeasurable disease only. 114 patients (pts) are planned to be randomized in a double-blind, placebo-controlled manner to either exemestane (25 mg QD) + entinostat (5 mg once weekly) or to exemestane (25 mg QD) + placebo. The study is designed to evaluate the hypothesis that the addition of entinostat to exemestane will increase the PFS by 2.3 months. (i.e. 3.7 months with exemestane versus 6 months with combination; 1-sided=10%, power= 80%). Correlative studies include analysis of changes in acetylation and gene expression in peripheral blood monocytes, and levels of circulating endothelial cells before and after initiation of treatment. The study is open in the United States, Hungary, Czech Republic, and Russia and current enrollment is 70% complete. No significant financial relationships to disclose.

Randomized, double-blind, placebo-controlled phase II study of erlotinib with and without entinostat, a class 1 isoform selective histone deacetylase inhibitor (HDAC), in patients with advanced non-small cell lung cancer (NSCLC; ENCORE-401).

LBA7532 The full, final text of this abstract will be available in Part II of the 2010 ASCO Annual Meeting Proceedings, distributed onsite at the Meeting on June 5, 2010, and as a supplement to the...

The Isoform Selective Histone Deacetylase Inhibitor Entinostat Is Active in Primary Human Triple-Negative Breast Cancer Models.

Abstract Background: Triple-negative (ER, PR, HER2 negative) breast cancer represents an unmet need for which novel agents and approaches are essential. Entinostat is an oral, class 1 isoform selective histone deacetylase inhibitor currently in multiple phase 2 clinical studies including advanced NSCLC and breast cancer. Previous studies have a) established the anti-proliferative activity of entinostat in the triple negative cell line model MDA-MB-231 b) shown that entinostat induces expression of ERα and c) that entinostat is synergistic with aromatase inhibitors to inhibit tumor growth of ER negative cancer cells. The aim of these studies was to validate the activity of entinostat in highly predictive primary human tumor models of triple negative breast cancer.Methods: Two approaches have been taken. First, entinostat was tested in the Oncotech Extreme Drug Resistance (EDR) proliferation assay using ten cryopreserved breast tumor explants known to lack ER, PR and HER2/neu expression by immunohistochemistry (IHC). Second, entinostat was tested at 5, 15, 30 mg/kg/d in two xenograft models using tissue derived directly from patient tumor resections. The breast cancer models MV4151 (from a postmenopausal patient with ductal invasive breast carcinoma) and MV4586 (from a premenopausal patient with relapsed invasive lobular breast carcinoma) were negative for ERa protein, however still positive for ERa mRNA. Both xenograft tumors did not express ERb. The tumor doubling times in nude mice were between 10 and 30 days. Both tumors are HER-2 negative (determined by IHC).Results: Dose response curves of entinostat were analyzed and compared to paclitaxel at 2.45 µM in ten triple negative breast tumor explants in the EDR assay. Entinostat was broadly effective in all of the tumors tested with IC50's ranging from 10nM to 270nM which are within the clinically achievable concentrations of entinostat. Two of the breast tumors that were most sensitive to entinostat (IC50 30- 100nM) were also sensitive to paclitaxel. In contrast, entinostat inhibited (IC50 10-270nM) the growth of eight of the tumors that were resistant to paclitaxel (i.e. < 50% growth inhibition at 2.45 µM). For the xenograft studies, at the time of writing, dose response data in the MV4151 model has demonstrated tumor growth inhibition by entinostat at the 15 and 30 mg/kg/d administered orally with no apparent weight loss. Ongoing experiments for which data will be presented include dose response in MV4586 as well as combinations with tamoxifen and fulvestrant in both xenograft models.Conclusions: Entinostat is an effective agent at inhibiting the growth of triple negative breast tumors in human primary tumor models. A pre-surgical clinical study to assess the activity of entinostat in triple negative breast cancer patients is planned. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1146.

Abstract B196: Entinostat: A novel class 1 isoform selective histone deacetylase inhibitor (HDACi) with unique pharmacological properties

Abstract Several distinct HDACi have been characterized. While we do not fully understand how structure and selectivity correlate with efficacy and toxicity, targeting selective isoforms involved in maintaining tumor proliferation may be sufficient for antitumor activity. Entinostat (ENT) is a benzamide Class I selective compound and has low nM MEC50 values against HDAC1, and low µM to HDAC 2 , 3 and 9, with no inhibition of Class II HDAC 4,6,7 and 8 (Khan 2008). In contrast, the hydroxamate, vorinostat (VOR) has µM inhibition of Class II HDACs. HDACi kinetic properties also influence activity of both histones and other proteins involved in diverse biologic processes. VOR has a fast-on/fast-off rate to HDAC 1 and 3. In contrast, ENT has a slow tight binding that translates into sustained inhibition of HDAC enabling prolonged activation (or repression) of genes even after elimination of the inhibitor. To better understand differences and similarities of PK/PD profiles of ENT and VOR data from the Ph 1 studies were reviewed. In the NCI Ph 1 study (Ryan 2005, Kummar 2007) 71 patients (68 solid tumors) were treated either daily (n=2) every 2 weeks (wks) (n=28) or weekly for 4 wks with 2 wks off (n=41). Ph 1 data for VOR has been previously summarized (Kelly 2005). ENT displayed both intra-and inter-patient variability as seen with most oral cancer agents. Daily dosing was not tolerated and PK data suggested that ENT had a 30–50x longer t1/2 than predicted from animal models. Subsequently doses were tested every 2 wks or weekly and up to 10 mg/m2 were tolerated. ENT absorption was variable with a median Tmax of 2 hours (range 0.5 to 60 hrs), the elimination is bi-exponential and the terminal halflife was 30 to 80 hours (hrs). ENT is detectable within 8 hrs and remains above baseline throughout the treatment cycle even after 5 days (Gojo 2007). In contrast VOR has a t1/2 ranging from 91–127 minutes. The AUC with both agents appears to have a linear increase with dose. PK parameters have led to once wkly or every 2 wk dosing with ENT and qD dosing with VOR. Pharmacodynamic analysis on histone acetylation in peripheral blood mononuclear cells is evident at 8-12 hs with ENT and 4–10 with VOR, and acetylation increases with time and continued treatment with both agents, however is not dose related with ENT. Acetylation persists for at least 2 to 3 wks following the last ENT administration in contrast to 10 hrs with VOR. Antitumor activity in Ph 1 was demonstrated with both agents; with SD (for > 90 days) in 30–40% of patients with multiple tumor types. PRs were seen with ENT in melanoma and sarcoma, and mesothelioma and thyroid cancer with VOR. Responses were not dose related. The most common toxicities with both drugs are primarily gastrointestinal, asthenia, and electrolyte abnormalities. Grade 3/4 cytopenias occur in 10–16% of solid tumor patients with both agents and is dose dependent with ENT. With every 2 wks or weekly dosing continuous target inhibition (up to 3 wks without interruption) can be achieved with ENT. Normal cells appear to be relatively resistant to prolonged exposure and therapy is well tolerated. Potent Class I HDACi (and/or related proteins) targeted by ENT is sufficient to induce antitumor activity in a broad spectrum of tumors. Structural differences, prolonged PD effects and impact on other cellular processes may provide important antitumor activity with ENT not achieved with other HDACi. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B196.

Ex-vivo analysis of the isoform selective histone deacetylase inhibitor entinostat in triple-negative breast cancer tumors

e14597 Background: Triple-negative (ER, PR, HER2 negative) breast cancer represents an unmet need for which novel agents and approaches are essential. Entinostat is an orally available, class 1 isoform selective histone deacetylase inhibitor currently in multiple phase 2 clinical studies including advanced NSCLC and breast cancer. Studies in vitro and in vivo in the triple negative cell line model MDA-MB-231 have established single agent activity of entinostat in inhibiting tumor growth as well preventing bone metastases. In addition, entinostat induces expression of ERα in MDA-MB-231 in vivo and is synergistic with endocrine therapy agents to inhibit tumor growth of ER negative cancer cells. The aim of these studies was to confirm the activity of entinostat in triple negative breast cancers using human breast tumor explants. Methods: Cytotoxicity of entinostat was determined by the Oncotech Extreme Drug Resistance (EDR) proliferation assay using ten cryopreserved breast tumor explants known to lack estrogen receptor, progesterone receptor and HER2/neu expression (i.e. Triple negative) by immunohistochemistry. Results: Dose response curves of entinostat were analyzed and compared to paclitaxel at 2.45 μM in ten triple negative breast tumor explants in the EDR assay. Entinostat was broadly effective in all of the tumors tested, with most samples showing dose-dependent response for the range of concentrations analyzed (0.003 - 10 μM). Two of the breast tumors that were most sensitive to entinostat (IC50 30- 100nM) were also sensitive to paclitaxel. In contrast, entinostat effectively inhibited (IC50 10–270nM) the growth of eight of the tumors that were resistant to paclitaxel (i.e. < 50% growth inhibition). Overall, all breast tumors tested were sensitive to entinostat at clinically achievable concentrations regardless of paclitaxel resistance indicating entinostat may improve the treatment outcome of triple negative breast cancer patients. Conclusions: Entinostat is an effective agent at inhibiting the growth of triple negative breast tumors with clinically relevant IC50's ranging from 10nM to 270nM. A pre-surgical clinical study to assess the activity of entinostat in triple negative breast cancer patients is planned. [Table: see text]

Novel HDAC6 isoform selective chiral small molecule histone deacetylase inhibitors

In an effort to identify HDAC isoform selective inhibitors, we designed and synthesized novel, chiral 3,4-dihydroquinoxalin-2(1 H)-one and piperazine-2,5-dione aryl hydroxamates showing selectivity (up to 40-fold) for human HDAC6 over other class I/IIa HDACs. The observed selectivity and potency (IC 50 values 10–200 nM against HDAC6) is markedly dependent on the absolute configuration of the chiral moiety, and suggests new possibilities for use of chiral compounds in selective HDAC isoform inhibition.

Residues in the 11 Å Channel of Histone Deacetylase 1 Promote Catalytic Activity: Implications for Designing Isoform-Selective Histone Deacetylase Inhibitors

Histone deacetylase 1 (HDAC1) has been linked to cell growth and cell cycle regulation, which makes it a widely recognized target for anticancer drugs. Whereas variations of the metal-binding and capping groups of HDAC inhibitors have been studied extensively, the role of the linker region is less well known, despite the potency of inhibitors with diverse linkers, such as MS-275. To facilitate a drug design that targets HDAC1, we assessed the influence of residues in the 11 A channel of the HDAC1 active site on activity by using an alanine scan. The mutation of eight channel residues to alanine resulted in a substantial reduction in deacetylase activity. Molecular dynamics simulations indicated that alanine mutation results in significant movement of the active-site channel, which suggests that channel residues promote HDAC1 activity by influencing substrate interactions. With little characterization of HDAC1 available, the combined experimental and computational results define the active-site residues of HDAC1 that are critical for substrate/inhibitor binding and provide important insight into drug design.

Structural Origin of Selectivity in Class II-Selective Histone Deacetylase Inhibitors

The development of class- and isoform-selective histone deacetylase (HDAC) inhibitors is highly desirable for the study of the complex interactions of these proteins central to transcription regulation as well as for the development of selective HDAC inhibitors as drugs in epigenetics. To provide a structural basis for the rational design of such inhibitors, a combined computational and experimental study of inhibition of three different histone deacetylase isoforms, HDAC1, -6, and -8, with three different hydroxamate inhibitors is reported. While SAHA was found to be unselective for the inhibition of class I and class II HDACs, the other inhibitors were found to be selective toward class II HDACs. Molecular dynamics simulations indicate that this selectivity is caused by both the overall shape of the protein surface leading to the active site and specific interactions of an aspartate residue in a polar loop and two phenylalanines and a methionine in a nonpolar loop. Monitoring the specific interactions as a function of the simulation time identifies a key sulfur-pi interaction. The implications of the structural motifs for the design of class II-selective HDAC inhibitors are discussed.

Polyaminohydroxamic Acids and Polyaminobenzamides as Isoform Selective Histone Deacetylase Inhibitors

A series of polyaminohydroxamic acids (PAHAs) and polyaminobenzamides (PABAs) were synthesized and evaluated as isoform-selective histone deacetylase (HDAC) inhibitors. These analogues contain a polyamine chain to increase affinity for chromatin and facilitate cellular import. Seven PAHAs inhibited HDAC >50% (1 microM), and two PABAs inhibited HDAC >50% (5 microM). Compound 17 increased acetylated alpha-tubulin in HCT116 colon tumor cells 253-fold but only modestly increased p21 (waf1) and acetylated histones 3 and 4, suggesting that 17 selectively inhibits HDAC 6. PABA 22 alone minimally increased p21 (waf1) and acetylated histones 3 and 4 but caused dose-dependent increases in p21 (waf1) in combination with 0.1 microM 5-azadeoxycytidine. Finally, 22 appeared to be a substrate for the polyamine transport system. None of these compounds were cytotoxic at 100 microM. PAHAs and PABAs exhibit strikingly different cellular effects from SAHA and have the potential for use in combination antitumor therapies with reduced toxicity.

Isoform-selective histone deacetylase inhibitors

Histone deacetylase (HDAC) proteins are transcription regulators linked to cancer. As a result, multiple small molecule HDAC inhibitors are in various phases of clinical trials as anti-cancer drugs. The majority of HDAC inhibitors non-selectively influence the activities of eleven human HDAC isoforms, which are divided into distinct classes. This tutorial review focuses on the recent progress toward the identification of class-selective and isoform-selective HDAC inhibitors. The emerging trends suggest that subtle differences in the active sites of the HDAC isoforms can be exploited to dictate selectivity.

351 POSTER Activity of the histone deacetylase (HDAC) inhibitor PXD101 in preclinical prostate cancer studies

Cytokine-induced beta-cell apoptosis is important to the etiology of type-1 diabetes. Although previous reports have shown that general inhibitors of histone deacetylase (HDAC) activity, such as suberoylanilide hydroxamic acid and trichostatin A, can partially prevent beta-cell death, they do not fully restore beta-cell function. To understand HDAC isoform selectivity in beta cells, we measured the cellular effects of 11 structurally diverse HDAC inhibitors on cytokine-induced apoptosis in the rat INS-1E cell line. All 11 compounds restored ATP levels and reduced nitrite secretion. However, caspase-3 activity was reduced only by MS-275 and CI-994, both of which target HDAC1, 2, and 3. Importantly, both MS-275 and genetic knockdown of Hdac3 alone were sufficient to restore glucose-stimulated insulin secretion in the presence of cytokines. These results suggest that HDAC3-selective inhibitors may be effective in preventing cytokine-induced beta-cell apoptosis.