Interpreting clinical assays for histone deacetylase inhibitors.

Introduction

The transcriptional state of a eukaryotic gene is determined by the surrounding chromatin architecture, the state of DNA cytosine methylation in the promoter/first exons and the associated regulating microRNAs (miRNAs).

In the cell nucleus, the genome is packaged into a superstructure, the chromatin, whose elementary dynamic units are the nucleosomes. Each is made up of four associated dimers of core histones (H2A, H2B, H3, and H4) around which 147 base pairs of DNA are wrapped, the nucleosomes being finally linked together via the linker histone H1 ( Figure 1 ). Chromatin is compacted around the DNA into a so-called “closed state” when cells are resting. It is opened into an “active state” to allow for gene transcription by adenosine triphosphate (ATP)-dependent protein complexes, which remodel the chromatin architecture. Theses complexes modify the accessibility of DNA regulatory sites through both repositioning (sliding) and ejecting nucleosomes. Modeling complexes include transcription co-activators, transcription factors, and epi-enzymes. 1 Histone acetyltransferases (HATs), for instance, acetylate specific lysine residues of histones and convert them into an amide form, loosening histone contacts with DNA, resulting in exposed binding sites for the transcription machinery. On the other hand, other complexes function as gene silencers and deny the same machinery access to DNA. Repressive complexes include histone deacetylases (HDACs), which deacetylate specific lysine residues of the histones tails to induce tighter interactions between the now positively charged lysine (Nε protonated form) and the negatively-charged DNA phosphate groups. 2 Beside acetylation, several other post-translational lysine modifications in histones have been described: methylation, phosphorylation, SUMOylation, and ATP-ribosylation. 3 , 4

All the modifications incurred by histones form the “histone code”. For example, K9 in H3 turns the chromatin into inactivity when methylated. Phosphorylation of serine 10 in the same H3 is required for methylation of K4 and acetylation of K9 and K14. Similar enzyme crosstalk has also been described, for ubiquitination of K120 in H2B prior to methylation of K79 in H3. 5 Several epi-enzyme families are involved in the histones modifications. HATs and HDACs 2 have balancing actions for histones acetylation. Methylation of histones 6 is controlled by histones methyltransferases (HMTs) and histones demethylases (HDMs), while histones arginine methylation is catalyzed by the protein arginine N-methyltransferases family of enzymes. 7 – 9 One, two, or three methylations are possible, with impact on gene expression/repression. 10 The analysis of epigenetic marks at the genome-wide scale has shown that monomethylated H3K4 is associated with transcription factors binding to enhancers, trimethylated H3K4 with transcription start sites, and dimethylated H3K4 with both transcription start sites and enhancers. 11

DNA methyltransferases (DNMTs) repress gene expression via DNA cytosine methylation, unfavorable to transcription factor binding. DNMTs are recruited and stabilized, on DNA, by HMTs and HDMs. Both are also able to recruit HDACs, methyl-binding proteins like methyl CpG binding protein 2 (MECP2), and several co-factors to further tune gene expression. 12 , 13 Since DNMT co-factors are lacking in normal tissues, gene re-expression induced by DNMT inhibitors could be limited to tumor tissues to reduce “off-target” effects. DNA methylation in the epigenomes of human embryonic stem cells is an important field of research. The roles of DNA methylation in cancer genesis have also been extensively studied. 12 , 13

Catalyzed by the ten-eleven translocation 1 (TET) family of enzymes, DNA hydroxymethylcytosine has been recently described as a step towards cytosine demethylation. 14 Mutations and translocations of TET are present in myeloid malignancies. 15 The role of hydroxymethylcytosine, if any, is not yet understood, but its existence questions all the results obtained so far when determining the cytosine methylation status.

In a further step of complexity, specialized miRNAs read the epi-code and target effectors genes to modulate their expression. MiRNAs are small non-coding RNAs of 20–22 nucleotides that inhibit gene expression when they engage either in imperfect base-pairing with their target mRNA 3′-untranslated region or affect its stability. MiRNA 29-a, -b and -c target DNMT3a and b directly and cell-specifically. HDAC4 is targeted by both miR-1 and miR-140, while miR-449-a targets HDAC1 in prostate malignant cells. 16 Onco-proteins like promyelocyte leukemia retinoic acid receptor-α (PML-RARα) in promyelocytic leukemia and B-cell lymphoma 6 in non-Hodgkin’s lymphoma result from translocations. It is near the premiR-223 region that the t(8;21) translocation juxtaposes the Runt-related transcription factor 1 gene on chromosome 21 with the Cytochrome B Termination 1 gene on chromosome 8, generating the acute myeloid leukemia (AML)1-Eight Twenty One fusion gene. 17 The recruitment, on this chimerical site, of DNMT, MeCP2, and HDAC1 repressor complexes, promotes leukemogenesis. Epi-miRNAs write their own epi-code when their cytosines are methylated. Downregulation of miR-124a induces an up regulation of its target, cyclin-dependent kinase 6 (CDK6), as well as phosphorylation of retinoblastoma, and contributes to the abnormal proliferation of acute lymphoblastic leukemia (ALL) cells both in vitro and in vivo.

Most epigenetic changes translate into either up regulation or silencing of gene expression. 18 When inappropriate, they predispose the organism to more mutational events via increased genomic instability and aberrant cellular signaling. The field of epigenetic being extremely prolific, we have restricted our reference list to the essentials.

HDACs

HDACs remove the acetyl group from an N-ε-acetyl lysine located near the amino terminus of a core histone, cleaving an amide bond and increasing the positive charge of the histone. The removal of acetyl groups from the histones tails stabilizes nucleosomal DNA-histones interactions by its subsequent change in electrostatic charges. It is the basis for HDAC-mediated transcriptional repression via chromatin condensation. 19 HDACs have been categorized into four classes. Class I HDACs (HDAC1, 2, 3, and 8) are nuclear proteins with ubiquitous expression involved in regulating cell proliferation. 20 HDAC2 has been shown to suppress apoptosis in tumor cells not only via both the intrinsic/mitochondrial and the extrinsic/death-receptor pathways, but also via mitotic failure and autophagic cell death, while HDAC3 is involved in bone structure and S-phase check point. 21 , 22 Class II HDACs have a tissue-specific expression and can shuttle between the nucleus and the cytoplasm. They are divided into two subclasses: IIa with HDAC4, 5, 7, and 9. HDAC4 represses chondrocyte hypertrophy. HDAC7 functions in the down regulation and apoptosis of T-cells. 20 HDAC9 is involved in cardiomyocyte differentiation. 23 Class IIb includes HDAC10 and HDAC6. The latter contains two tandem catalytic domains: one is for histones deacetylation and the other for deacetylation of α-tubulin. HDAC6 has also the capacity to bind directly to ubiquitinated proteins through an ubiquitin-binding domain, to target cargo proteins for subsequent processing. HDAC6-specific effects on cell motility and the proteasome are thought to be responsible for much of the toxicity of HDACs inhibitors (HDACi). HDAC 10 and 9 are required for chromosome homologous recombination. 24 Class III HDACs include 7 different members of the sirtuin (SIRT) family. They are dependent on nicotinamine adenine dinucleotide (NAD+) to remove the acetyl group from lysine residues in histones and nonhistone substrates. Resveratrol from grapes and red wine is a SIRT1 activator. 25 HDAC11 is the only member of Class IV.

Thus, it appears that HDAC activity depends on isoform types, sub cellular localization, association into multi-protein complexes and even post-translational modifications. HDACs are also able to deacetylate nonhistone proteins such as transcription factors, chaperone proteins and effectors of DNA repair, cell-signaling and metabolism. The ongoing concept is that deacetylation stabilizes these proteins. HDACs have different developmental functions, as shown by the different phenotypes obtained in knockout mice. 26 Disruption of HDAC1 causes early embryonic lethality. HDAC2 knockout mice are viable but present fatal multiple cardiac defects. Germline HDAC3-deficiency causes embryonic lethality. HDAC3 conditional knockout mice gave severe deficits in membranous and endochondral bone formation. Germline deletion of HDAC4 causes premature ossification of the developing bones. HDAC6-deficiency slightly enhances trabecular bone formation. HDAC7 knockout gave vascular defects. HDAC8 is essential for neural crest progenitor cell differentiation and skull bone formation. HDAC9 knockout mice are viable at birth but have a myocardial hypertrophy.

HDACs and their inhibitors

In tumor cells, deletion of a single HDAC is not sufficient to induce cell death but leads to nuclear bridging and fragmentation, and ‘in fine’ to cell mitotic catastrophe. This suggests that inhibition of HDAC may be sufficient for anticancer activity and provides a rational incentive for the development of HDACi. 27 In the 1970s, seminal experiments showed that treatment of cells with the short-chain fatty acid NaB (sodium butyrate) caused hyperacetylation of histone octamers and led to the discovery of HDACs. 28 The zinc-dependent HDACs of classes I, II, and IV are now known to have a common active site made of a tubular hydrophobic channel with a zinc atom (Zn 2+ ) at its end, forming the enzyme catalytic pocket. 29 The acetyl part of the lysine substrate in histones/proteins bind to the zinc atom while the protein four carbon lysine chains fits into the catalytic pocket, and deacetylation then follows. An HDACi is designed to block the HDAC catalytic activity. Several possibilities exist: irreversible or reversible binding to the enzyme catalytic site, competition with the enzymatic substrate, and deformation of the enzyme. Accordingly and as shown in Figure 2 , the pharmacophore model for HDACi includes a zinc-binding group, competing somehow with the natural acetyl lysine substrate, a hydrophobic cap interacting with the external surface of the active site (generally aromatic) generating specificity, and a short linker connecting these two elements, which fits in the catalytic pocket. The zinc-binding groups can be a carboxylic acid in valproic acid (VPA), a hydroxamic acid in Vorinostat, benzamide in Entinostat, sulfhydryl in Romidepsin, 30 and a ketone in Trapoxin. The linkers can be simple carbon chains, like in Vorinostat, or aromatic groups, like in Entinostat. HDACi in clinical trials are reported in Figure 3 . Trichostatine (TSA) from Streptomyces hygroscopius was isolated as an antifungal antibiotic and was incidentally shown to have an anti-proliferative activity on murine leukemia cells. Further studies demonstrated that it was a pan-HDACi. The hydroxamate portion at the end of the molecule acts as a zinc-binding group. Because of toxic side effects, it is not used clinically but participates in the rational conception of HDACi via molecular modeling, as shown in Figure 2 .

Besides its HDAC inhibition activity, Vorinostat ( Figure 2 , left), deriving from TSA, has a complex and not yet fully characterized activity leading to the accumulation of acetylated histones and non histone proteins. First generation HDACi are not selective except for a partial selectivity achieved 31 , 32 in rare cases using bulk chemical groups to generate specific interactions with the external surface of the active site of the enzyme, like in tubacin. 33 Sulfur-based zinc-binding groups also showed some selectivity in compounds like Largazole, a potent and selective HDACi for HDAC1 and 2. It is a densely functionalized macrocyclic peptide isolated from the Cyanobacterium symploca sp. by Luesch and coworkers. 34 Entinostat and Mocetinostat have selectivity for HDAC1-3 and also against HDAC11 for the latter. Valproate and sodium butyrate (NaB) better target HDAC I and IIa. For sirtuins 35 inhibition is based on NAD+competitive binding with attempts to propose a pharmacophore, according to the various inhibitor structures described. 36

SIRT1 activation is the novel therapeutic approach to treat chronic inflammatory diseases, and enzyme activators are therefore sought. Many screening tests to search for HDACi use short histone peptides, capturing baits and engineered cells. All have their limitations because, in vivo, HDAC are parts of mega Daltons modeling chromatin complexes that may change within each cell type.

HDAC inhibitors metabolism

The metabolism of HDACi is an important concern during clinical assays. It is studied to determine the correlation between HDACi blood concentration, effective biological effects and eventual drug interactions. The known metabolisms of some HDACi are reported in Figure 4 . TSA is metabolized as the inactive trichostatic acid, which is further demethylated 37 for rapid clearance ( Figure 4 ). Phenyl butyrate (PB) metabolism has been described in several contexts. 38 PB is β-oxidized to phenylacetate, and cleared out upon glutamine addition. Vorinostat is also oxidized to 4-anilino-4-oxobutanoic acid and glucuronylated. 39 , 40 Romidepsin is a disulfide prodrug. The real active form corresponds to the free thiol metabolite, 41 produced in vivo; the butenthiol part being thought to be the zinc-binding group. A glutathione conjugate has also been described, 42 which is metabolized in vivo by the cytochrome P450s 43 with slow and high acetylating subjects. Other HDACi stabilities have been investigated. 44 , 45

In-vitro effects of HDAC inhibitors

DNA chips studied the transcriptome of cells treated with Vorinostat and Romidepsin, 46 revealing that the expression of 40% of all genes was affected over a period of 16 hours. A Belinostat mRNA signature of 25 genes was sufficient to assess the overall gene modulation. Panobinostat modulated cell cycles and angiogenic genes. 47 Tumor antigen expression modulation and major histo-compatibility antigen (MHC) molecule induction 48 have been observed with Dacinostat. 49 Mice bearing human tumor xenografts treated with Belinostat showed a modulation of the expression of genes active in the cellular G2/M phase. This was different from what was seen with 5-fluorouracil (5-FU), Cisplatin, Paclitaxel, or Thiotepa. Synergistic effects were obtained when combining HDACi and DNA demethylating agents, 50 or HDACi and all-trans retinoic acid (ATRA), a cell-differentiating agent used to treat acute promyelocytic leukemia (APL). 51 The influence of epigenetic modulators to modify stem cell fate and its relevance for curing diseases has been reviewed. 52 Successful therapeutic use of HDACi may thus depend on the cellular environment, the specific HDAC targeted, and the relative dependence of the tumor on the unique set of pathways influenced by a specific HDAC. Results are summarized in Table 1 . 53

Clinical trials with zinc-dependent HDACi

This part of the review describes the HDACi that have been or are being investigated in clinical trials. In Table 2 , all current trials are recapitulated. In Table 3 , and for each molecule, some data related to epigenetic measurements are summarized.

PB or its sodium salt

PB or its corresponding sodium salt (NaPB) is a short chain fatty acid approved by the Food and Drug Administration (FDA) for the treatment of hyperammonemia. It stops the cell cycle in its G1–G0 phase. PB is an efficient HDACi at about 0.5 mM. 54 , 55 PB induces apoptosis – probably via c-jun N-terminal kinase (JNK) – in lung carcinoma cells, 56 p21waf1-mediated growth arrest in MCF-7 cells, 57 tumor necrosis factor (TNF)-α 58 or peroxisome proliferator-activated receptor (PPAR)λ-mediated 59 cell differentiation, and is more potent than phenylacetate in prostate cancer cells, 60 while increasing MHC class I expression. PB is converted in vivo into the active metabolite phenylacetate (PA) by β-oxidation in the liver and kidney mitochondria. 61 Most dose-limiting toxicities (DLTs) are fatigue, nausea, and somnolence. Preliminary studies have been conducted in patients with recurrent glioblastoma multiform (GBM) 62 ( Table 3, A ). Phase I studies have been conducted in patients with hormone refractory prostate cancers, 63 refractory solid tumor malignancies 64 like colon carcinoma, non small cell lung cancer (NSCLC), anaplastic astrocytoma, GBM, bladder carcinoma, sarcoma, ovarian carcinoma, rectal hemangiopericytoma, and pancreatic carcinoma, 65 mainly as intravenous infusions but also in AML and myelodysplastic syndrome (MDS). 66 DLTs were neuro-cortical with milder fatigue and nausea/vomiting, light-headedness, short-term memory loss, sedation, confusion, and hypocalcemia. Although central nervous system (CNS) toxicity was observed, infusions were well tolerated ( Table 3, B ). The active metabolite PA accumulated.

In the AML/MDS study, 67 with sequential administration of 5-aza-cytidine (5-aza) ( Table 3, C ), partial remissions or stable diseases were obtained. Targeting different biological mechanisms is feasible with acceptable toxicity. Phase I trials in combination with several drugs have been reported. Prostate, colorectal, leiomyosarcoma, and esophageal cancers were treated in combination with 5-aza ( Table 3, C ), 68 metastatic colorectal cancer with fluorouracil 5-FU as a 24-hour continuous intravenous infusion (CIV). 69 With 5-aza, no re-expression of E-cadherin, endothelin B, and glutathione S transferase (GST) pi was observed, a result explained by the lack of dose effect or by the fact that DNA methylation is an S-phase-dependent process while in-vivo prostatic cells may be in S-phase at any given time. Stable disease was the best response. Combining 5-FU appeared also feasible.

Pivaloyloxymethyl butyrate

Pivaloyloxymethyl butyrate (AN)-9, is an ester prodrug of butyric acid (BA) 70 but with a greater potency at inducing malignant cell differentiation and tumor growth inhibition. It showed more favorable toxicological, pharmacological, and pharmaceutical properties than BA in preclinical studies. BA itself induces p16 expression and growth arrest of colon cancer cells, 71 and modifies caspase distribution during apoptosis. 72 AN-9 down regulates c-jun and c-myc and induces differentiation in leukemia cells. 73 It is decomposed by esterases in vivo to yield butyric and pivaloyl acids and a formaldehyde molecule, responsible for toxicity resulting in visual acuity disorders. It has demonstrated a synergistic effect with other anticancer agents by reducing bcl-2 levels. 74 Initial study 75 with I.V. of AN-9 in advanced solid malignancies ( Table 3, E ) gave partial responses, and stable diseases as best responses. Later, a multicenter trial of pivaloyloxymethyl butyrate 76 in refractory NSCLC ( Table 3, F ), administered as a continuous I.V. infusion, gave partial responses.

VPA

VPA is a nontoxic short-chain carboxylic acid used for the treatment of epilepsy with a long clinical history and well known pharmacokinetics (PKs) and pharmacodynamics (PDs). 77 , 78 VPA induces chromatin decondensation, 79 and differentiation in neural progenitor cells, 80 and inhibits HDAC activity 81 in the mM range (preferentially HDAC1, 2). 82 The antiproliferative activity was associated with aberrant cyclin D3 functionality during the C6 glioma G1 phase. 83 Activation of PPARδ was present in F9 cells. 84 VPA induces caspase-dependent and -independent apoptosis in leukemia cells, 85 and in AML cells expressing P-gp and multidrug resistance protein 1 (MRP1), 86 inhibits production of TNF-α and interleukin (IL)-6 and activates nuclear factor kappa B (NF-κB). 87 VPA has been evaluated in combination with other anticancer compounds. For AML, increased 5-aza cytotoxicity was associated to cyclin D1 and p27(Kip1) expression, 88 while sequential VPA/ATRA treatment reprograms differentiation. 89 VPA induces p16INK4A upregulation and apoptosis and sensitizes melanoma cells to chemotherapy. 90 Interestingly, most of the clinical trials reported are for combination therapies.

A Phase I was conducted 91 for refractory advanced cancer (colorectal, melanoma, NSCLC, and others) ( Table 3, G ). VPA/ATRA combination was evaluated for several diseases. Poor risk AML 92 ( Table 3, H ), MDS and relapsed or refractory AML 93 ( Table 3, I ) have also been investigated. A 52% response rate was observed in MDS patients. ATRA exerted no additional effect in patients receiving the combination, but could be used to induce a second response in relapsing VPA-treated patients. In recurrent or refractory AML or MDS in a Phase II protocol 94 ( Table 3, J ), ATRA was administered when VPA reached the target serum concentration. The differentiation therapy with VPA was effective in 30% of patients. In 11 elderly patients, de novo AML 95 ( Table 3, K ) was also treated with theophyllin to increase cAMP levels and major cell differentiation. 96 Complete marrow response was observed in three patients, including one complete remission. Two additional patients had hematologic improvement. Patients with AML-M6 were found particularly 97 responsive, probably due to T-cell acute lymphocytic leukemia 1 (TAL1) 98 and GATA1 99 interactions with HDACi, inducing differentiation in murine erythroleukemia (MEL) cells. Siitonen et al 100 reported a negative study trying VPA, in combination with 13-cis-retinoic acid (13-cis-RA) 101 and 1,25-dihydroxyvitamin D3, in 19 naive patients with MDS or chronic myelomonocytic leukemia (CMML) ( Table 3, L ). Combinations with demethylating agents have been reported. Phase I/II 102 study with 5-aza-2′-deoxycytidine (5-azaDc) in leukemia ( Table 3, M ) included gene expression analysis (p57kip2, p15, p73, MDR1 and THBS2). Initial DNMT1 levels were too low to be informative. A Phase I study 103 with 5-azaDc in AML ( Table 3, N ) gave partial to complete remissions, warranting further studies of 5-azaDc alone or with alternative HDACi. A Phase I 104 study of epigenetic modulation with 5-aza for advanced cancers ( Table 3, O , colon, skin melanoma, breast, other) gave stable diseases. A Phase I/II study 105 with 5-aza and ATRA for AML and MDS ( Table 3, P ) gave 42% positive overall responses.

Other combinations were investigated: a Phase I dose escalation combination trial with epirubicin, 5-FU, and cyclophosphamide 106 in breast cancer ( Table 3, Q ), and a Phase I trial 107 with epirubicin for solid tumors ( Table 3, R ). The rationale for the combination was to facilitate epirubicin access to DNA to potentiate its strand breaks activity as a topoisomerase II inhibitor. Intrinsic epirubicin toxicity was not exacerbated. Reverse combination was found inadequate by the same group. The same group investigated combination with the topoisomerase I inhibitor karenitecin (KTN) ( Table 3, S ) for treating melanoma with both Phase I/II trials. 108 No VPA/KTN synergistic toxicity was observed. The best response was disease stabilization. VPA plus chemoimmunotherapy was investigated in a Phase II study 109 for advanced inoperable or metastatic melanoma ( Table 3, T ), HDACi having been previously found to have a tumorigenic potential in melanoma. 110 Some patients then received dacarbazine plus interferon-α with VPA.

The magnesium salt of VPA has been tested in phase I 111 for cervical cancer (squamous and in adenocarcinoma) and Phase II 112 clinical trials. In the Phase I study ( Table 3, U ), VPA was given per os, and the authors emphasized the requirement for new endpoint trials based on biomarker analysis 113 , 114 with, in this particular case, H3 and H4 acetylation and in vivo HDAC inhibition detection. The Phase II study was conducted with hydralazine, a demethylating agent, 115 ( Table 3, V ) to overcome chemotherapy resistance in refractory solid tumors (cervix, breast, ovarian, and others). Partial responses and disease stabilization were the best responses.

Vorinostat

Vorinostat (suberoylanilide hydroxamicacid [SAHA], Zolinza ® ) has probably been the most studied compound in clinical trials on several cancer types. SAHA induces differentiation, 116 growth arrest, 117 or apoptosis at micromolar concentrations. Vorinostat is an unselective zinc-binding 118 hydroxamic-acid-type inhibitor of HDAC1, 2, 3, 6, and 8. In glioma cells, SAHA induced expression of DR5, TNFα, p21Waf1, and p27Kip1 and reduced expression of CDK2, CDK4, cyclin D1, and cyclin D2. 119 SAHA can induce thyroid cancer cell death by caspase-mediated pathways, 120 and induces G1 and G2-M arrest and apoptosis in several types of breast cancer cell lines, 121 , 122 NSCLC, 123 and prostate cancer cells. 124 It potentiates the activity of other molecules like Paclitaxel in ovarian cancers. 125

Phase I trials have been described for both oral and I.V administrations. Escalating I.V. administration 126 in solid tumors and hematological malignancies ( Table 3, W ) gave hypotension for one schedule. In mesothelioma, with I.V. or oral formulations, 127 the best responses were partial ( Table 3, X ). An oral formulation for hematologic malignancies (Hodgkin’s and others) ( Table 3, Y ) and solid tumors (mainly mesothelioma, prostate, urothelial, thyroid, and renal) 128 gave one complete response while others were incomplete. Oral twice- or thrice-daily administrations in advanced leukemias and MDS (AML, CLL [chronic lymphocytic leukemia], MDS, ALL and CML [chronic myelocytic leukemia]) ( Table 3, Z ) 129 gave two complete responses and two complete responses with incomplete blood-count recovery (all with AML treated at/below maximum tolerated dose [MTD]).

Phase II clinical trials were mainly proposed with oral formulations. A multi-institutional trial 130 in women with recurrent or persistent epithelial ovarian ( Table 3, AA ) or primary peritoneal carcinoma platinum-resistant/refractory gave one partial response. Another multicenter open-label oral trial 131 investigated measurable, relapsed, or refractory breast cancer, NSCLC, or colorectal cancer ( Table 3, AB ). Disease stabilization was observed in eight patients. SAHA is tolerated at 200 mg only, in a daily oral schedule for 14 days–3 weeks. In recurrent and/or metastatic head and neck cancer (400 mg every day) ( Table AC ) 132 no confirmed responses have been observed. In patients with metastatic breast cancer, 133 there were no complete or partial responses, and the heterogeneity of the recruited patients did not allow production of significant statistical results. Eight patients were positive for estrogen and/or progesterone receptors, four had amplified CerB-2. Fatigue, nausea, diarrhea, and lymphopenia were the most frequent clinically significant adverse effects. In GBM 134 ( Table 3 AD ), an oral dose of 200 mg followed by a 7-day rest period showed that SAHA monotherapy is well tolerated with modest single-agent activity. Although HDACi were shown to induce cell death and sensitize cells to cytotoxic chemotherapy in thyroid cancer cell lines, Woyach et al 135 described the lack of therapeutic effect of SAHA in patients with metastatic radioiodine-refractory thyroid carcinoma in a Phase II study ( Table 3, AE ). A Phase II oral combination therapy was proposed with carboplatin (I.V.) and Paclitaxel (I.V.) for advanced solid malignancies 136 ( Table 3, AF ). Eleven partial responses occurred and seven disease stabilizations. The regimen requires drug–drug interaction to be determined. Encouraging results were obtained in patients with previously untreated NSCLC.

Belinostat

Belinostat (PXD101) is a recent hydroxamic acid HDACi that has growth-inhibitory and pro-apoptotic activity in several cancer types at submicromolar concentrations, 137 , 138 and that has been investigated in ovarian cancers. 139 It down regulates thymidilate synthase, vascular endothelial growth factor (VEGF), aurora kinase, and epidermal growth factor receptor (EGFR), and up regulates cyclin A. It has been used in combination. A gene expression-signature profiling has been reported for Belinostat. 140 According to publications 141 PKs gave a general 1–2-hour half-life. In early trials, DNA fragmentation increased with a combination of 5-FU in HCT116 colon cancer cells in vitro and in both HT-29 and HCT116 in xenograft models, 142 and also a poly(ADP-ribose) polymerase (PARP) cleavage and down regulation of thymidylate synthase expression in HCT116. Improved tumor reduction was obtained in vivo with mouse HT29 and HCT116 xenograft models compared with single compounds, validating a rationale for the clinical schedule.

In Phase I treatment of refractory solid tumors by I.V. administration ( Table 3, AG ) 143 the caspase-dependent cleavage of cytokeratin-18 was determined to measure the level of apoptosis. 144 Heavily pre-treated patients with advanced hematological neoplasia 145 ( Table 3, AH ) were also treated. In Phase II trials, investigations of I.V. administration of relapsed malignant pleural mesothelioma 146 ( Table 3, AI ) indicated that combination strategies or alternate dosing schedules might be necessary. In resistant micro-papillary ovarian tumors (low malignant potential [LMP]) and epithelial ovarian cancers (EOC) ( Table 3, AJ ), 147 a Phase II trial with Belinostat gave partial responses or stable diseases for LMP, and stabilized diseases for EOC.

Givinostat

Givinostat (ITF2357) belongs to the hydroxamic acid family of HDACi which is very similar to SAHA. It inhibits IL-6 and VEGF production in stromal cells. 148

Two Phase II studies were described for relapsed/refractory Hodgkin lymphoma (HL) ( Table 3, AK ). A first oral one, gave stable diseases by computed tomography (CT) scan that have been associated with a significant reduction in fluorodeoxyglucose-positron emission tomography scan uptake. 149 Galli et al 150 developed a Phase II multicenter trial in 19 heavily treated patients that were relapsing from progressive multiple myeloma (MM) ( Table 3, AL ). The best responses were disease stabilization. This regimen appears as unlikely to play a significant role for advanced MM, and other combinations with currently used drugs should be investigated. A combination with the alkylating agent mechlorethamine 151 ( Table 3, AM ) was investigated in relapsed/refractory HL.

Panobinostat

Panobisnostat (LBH589) is a hydroxamic acid HDACi, which has demonstrated anti-angiogenic and anti-proliferative activities in human prostate carcinoma cell PC-3 xenografts in vivo, inducing H3 and tubulin acetylation 152 in human umbilical vascular endothelial cells (HUVEC), which corresponded to G2-M cell cycle arrest and inhibition of HUVEC cell proliferation and viability. Non cytotoxic concentrations of Panobinostat inhibited endothelial tube formation, matrigel invasion, AKT, extracellular signal-regulated kinase 1/2 phosphorylation, and chemokine receptor CXCR4 expression. Association with anti-VEGF therapies should be considered. Prince et al have discussed preclinical data on Panobinostat and emerging data from Phase I and II studies in cancer patients. 153

A Phase I study in refractory hematologic malignancies (AML, ALL, and MDS) ( Table 3, AN ) 154 with I.V. administration appeared convenient to obtain anti-leukemic and biological effects. In cutaneous T-cell lymphoma (CTCL) 47 with oral formulation ( Table 3, AO ), the responses ranged from disease stabilization to complete remission, showing the potential of this molecule in CTCL. In combination with Docetaxel 155 ( Table 3, AP ), a microtubule interacting agent for castration-resistant prostate cancer, Panobinostat inhibited LnCAP androgen receptor positive prostate cancer cell proliferation, potentiated by Docetaxel. Single or combined treatments were administered with oral Panobinostat.

Dacinostat

Dacinostat (NVPLAQ824, LAQ) is a hydroxamic acid derivative similar to Panobinostat. 156 It showed anti-neoplastic activity and can activate genes that produce cell cycle arrest. It acetylates hsp90, inducing proteosomal degradation of Bcr-Abl and HER-2. Combination of Dacinostat with 5-azaDc 157 in human MDA-MB-231 and MCF-7 breast carcinoma cells showed a synergic anti-neoplastic activity for the MDA-MB-231. For the MCF-7 tumor cells, simultaneous 5-azaDc and Dacinostat administration were antagonistic, unseen when used in a sequential schedule (5-azaDc first). This is probably due to interference in the S-phase of Dacinostat since 5-azaDc is a S-phase specific interfering molecule. Dacinostat appeared to be well tolerated in clinical trials. Phase I investigations 158 in advanced solid tumors included measure of HSP72 levels and was consistent with HSP90 inhibition ( Table 3, AQ ). Another group 159 reported the same results with increased expression of Hsp70 and decreased c-Raf levels. The biological importance of these non histones mediated effects requires further study. I.V. administration for ALL, AML, CLL, CML 160 ( Table 3, AR ) in blast crisis or advanced MDS gave some stable diseases.

PCI-24781

PCI-24781 is a broad-spectrum hydroxamic acid-based HDACi. PCI-24781 reverses drug resistance in four multidrug resistant sarcoma cell lines and synergizes with chemotherapeutic agents to enhance caspase-3/7 activity. 161

In refractory advanced solid tumors 162 ( Table 3, AS ), I.V. administration followed by dose escalation was well tolerated. Electro cardiac monitoring revealed grade ≤1 QTcF (QT interval corrected for heart rate using Fridericia’s formula) prolongation and asymptomatic nonspecific ST and T wave changes leading to discontinuation.

Entinostat

Entinostat (MS-275, SNX-275) is a benzamide HDACi, which promotes expression of genes involved in growth arrest and differentiation, like p21 and the maturation marker: gelsolin, 163 inducing caspase-dependant apoptosis in CLL B-cells, 164 p21CIP1/WAF1 differentiation or apoptosis in human leukemia cells, 165 and also tissue growth factor (TGF)βII receptor expression in human breast cancer. 166 Reported half-life in animals is about 1 hour, and species-variable protein binding was reported. 167 Half-life in human plasma was higher than in animals, which is supposedly to be linked to protein binding, as Entinostat was found to be 80% bound. 167

Phase I study in advanced solid tumors or lymphoma by oral route 168 ( Table 3, AT ) was reasonably well tolerated. In refractory solid tumors and human lymphoid malignancies 169 ( Table 3, AU ), drug exposure increases linearly with dose. In AML 170 ( Table 3, AV ), results showed that Entinostat effectively inhibits HDAC in vivo in patients with AML and should be further tested, preferably in patients with less-advanced disease. Several protocols were designed for patients with advanced solid malignancies and lymphomas 171 ( Table 3, AW ). PKs revealed dose-dependent and dose-proportional increases. Responses were partial remissions and prolonged disease stabilization. In a Phase II study for metastatic melanoma ( Table 3, AX ) with low efficacy treatment, 172 long-term tumor stabilizations have been observed, but no objective responses was assessed.

Mocetinostat

Mocetinostat (MGCD0103) 173 is a benzamide selective class I/IV HDACi (1, 2, 3, and 11). It inhibits neoplastic growth in multiple human tumor xenograft models including colon (HCT116, SW48, and Colo205), NSCLC (A549), prostate (DU145), pancreatic (PANC1), and vulval epidermal (A431) cancer models and does not interact with the potassium voltage-gated channel, subfamily H (eag-related), member 2 (HERG) channel. Gene expression induced by Mocetinostat is modest compared with other hydroxamic HDACi. 174

In patients with advanced solid tumors 175 ( Table 3, AY ), a phase I study gave disease stabilization as the best response. IL-6 induction related to HDACi activity has been postulated but not confirmed. At the tested doses, Mocetinostat appeared tolerable and exhibited favorable PK and PD profiles, as well as evidence of target inhibition in surrogate tissues. Cytogenetically analyzed patients with AML, MDS, ALL, and CML 176 ( Table 3, AZ ) were treated orally. A total of 18 of the 29 patients had abnormal cytogenetics. PK analyses indicated rapid absorption of Mocetinostat. Several administration schedules have been proposed for advanced leukemias or MDS 177 ( Table 3, BA ). A Phase II study for HL 178 ( Table 3, BB ) demonstrated significant anti-tumor activity in relapsed/refractory post-transplant HL. For 437 patients 179 ( Table 3, BC ), partial responses were obtained. Extended studies are ongoing.

Tacedinaline

The long-known molecule Tacedinaline (CI-994) is a benzamide HDACi similar to MS-275 180 with anti-tumor activities in HCT-8 colon carcinoma. 181 Following Tacedinaline administration, inhibition of both histone deacetylation and cellular proliferation at the G1 to S transition phase of the cell cycle are observed.

Oral administration with food intake for solid tumors 182 ( Table 3, BD ) did not affect the rate or extent of the drug absorption. Best responses were partial or stable diseases. Advanced solid malignancies (mainly colorectal, pancreatic and mesothelioma) were treated in combination with Capecitabine 183 ( Table 3, BE ), an FDA-approved compound used to treat a variety of cancer. Three treatment protocols were implemented. A combination Phase II study with Gemcitabine 184 for advanced pancreatic carcinoma ( Table 3, BF ) gave no improvement.

Depsipeptide

Depsipeptide (Romidepsin, FK228, FR901228 ) is a cyclic tetrapeptide isolated from Chromobacterium violaceum which has demonstrated anti tumor activities (A549 lung adenocarcinoma, MCF-7 and ZR-75-1 breast adenocarcinoma, and LOX IMVI melanoma cell lines) and is postulated as a Pg-p substrate. 185 , 186 It is considered as a natural HDACi prodrug, as its disulfide bond is reduced in vivo to give the active species 40 and is the only reported sulfur-based HDACi used in clinical trials. It received FDA approval for cutaneous T-cell lymphoma in 2009. Romidepsin induces growth arrest and apoptosis in lung cancer cells. 187 Romidepsin induces p21-dependent G1 arrest and p21-independent G2 arrest 188 by downregulating cyclin D1 and upregulating cyclin E. 189 It inhibits c-Myc and Fas ligand expression, 190 modulates p53, ErbB1, HER2, and Raf-1 expression in lung cancer cells, 191 increases p21, phosphorylation of Bcl2, and apoptosis in human breast cancer cells, 192 increases expression of a NaI symporter in thyroid carcinoma cells for possible resensitization of radio resistant thyroid cancer, 193 and activates the caspase 8-mediated apoptosis and down regulates the c-FLIP protein. 194 Sequential treatments with 5-azaDc facilitates cancer cell recognition by T lymphocytes specific for cancer/testis antigen 1B (NY-ESO-1) as a possible option for immunotherapy 195 or induces tissue factor pathway inhibitor (TFPI)-2 expression in cancer cells. 196 Initial cardiac toxicity was resolved by convenient administration schedules but cardiac monitoring is most of the time implemented during clinical investigations. Concentrations studies in CLL and AML have correlated apoptosis induction and HDAC inhibition. Combination of Romidepsin and DNA demethylating agents is potentiated in ETO positive leukemia cells. 197 A gene signature specific for Romidepsin sensitivity has been reported. 198 Due to the recent approval of Romidepsin in CTCL, all early published clinical trials for this disease are not discussed. 199 , 200 Romidepsin induced MDR-1 gene expression in several cancer cell lines.

A Phase I study in advanced, incurable cancers 201 ( Table 3, BG ) indicated that further clinical trials are warranted. Used for advanced or refractory neoplasms 202 ( Table 3, BH ), elimination half-life was 8.1 hours. In CCL and AML 203 ( Table 3, BI ), intravenous treatment gave no responses. Romidepsin effectively inhibits HDAC in vivo in patients with CLL and AML, but future studies should examine alternative administration routes. In refractory or recurrent solid tumors 204 ( Table 3, BJ ), DLTs were not associated with changes in troponin levels or evidence of ventricular dysfunction, transient asymptomatic sick sinus syndrome and hypocalcemia. For MDS and AML 205 ( Table 3, BK ), intravenous administration gave no significant cardiac toxicity. Romidepsin therapy can be administered with acceptable short-term toxicity. Gastrointestinal symptoms and fatigue seemed to be treatment-limiting after multiple cycles. Phase II performed on refractory renal cell cancer 206 ( Table 3, BL ), I.V., gave classical but serious toxicities. Two patients developed a prolonged QT interval, one patient developed grade 3 atrial fibrillation and tachycardia, and there was one sudden death. In metastatic neuroendocrine tumors 207 ( Table 3, BM ), adverse events were ventricular tachycardia and prolonged QT, possibly resulting in a sudden death, terminating the study prematurely. Romidepsin has serious cardiac adverse events, and risks need to be comprehensively evaluated. In lung cancers (NSCLC and SCLC) ( Table 3, BN ), 208 Romidepsin was not appropriate. This study presented an in depth gene expression profiling.

Conclusion

A number of clinical trials have been completed and many others are ongoing using HDACi as single agents and in combination with radiotherapy and/or chemotherapy for the treatment of various hematological and solid malignancies with some promising early results. Vorinostat is the most established HDACi, and was approved in October 2006 by the FDA for the treatment of advanced forms of cutaneous T-cell lymphoma that have failed multiple other systemic treatment options. Significant single agent activity for Romidepsin has also been demonstrated in peripheral cutaneous T-cell lymphoma, and encouraging results have also been seen in HL with Mocetinostat. From the trials conducted, it is also clear that a major clinical advantage is that HDACi are well tolerated in the majority of patients. The future of HDACi lies in designing rational combination therapies. The sequence of drug administration may be of paramount importance to avoid antagonistic effects. The possibility of drug–drug interactions and enhanced toxicities is to be considered. HDACi are also evaluated in non cancerous pathologies like AIDS, graft versus host diseases, and polycythemia verae. Very soon, SIRT activators could find therapeutic applications in lung interstitial diseases. Like for the kinase inhibitors, more selective third generation HDACi are sought, yet specific tests remain to be designed to screen for bioactivity in vitro and in vivo. 211

This work was supported in part by the COST action TD0905 Epigenetics: bench to bedside and by the Agence Nationale de la Recherche (France).

Disclosure

The authors report no conflicts of interest in this work.

References

1. Choudhuri S Cui Y Klaassen CD Molecular targets of epigenetic regulation and effectors of environmental influences Toxicol Appl Pharmacol 2010 245 3 378 393 20381512 10.1016/j.taap.2010.03.022 PMC2911443

2. Santos-Rosa H Caldas C Chromatin modifier enzymes, the histone code and cancer Eur J Cancer 2005 41 16 2381 2402 16226460 10.1016/j.ejca.2005.08.010

3. Oliver SS Denu JM Dynamic interplay between histone H3 modifications and protein interpreters: emerging evidence for a “histone language” Chembiochem 2011 12 2 299 307 21243717 10.1002/cbic.201000474 PMC3073147

4. Zee BM Levin RS Dimaggio PA Global turnover of histone post-translational modifications and variants in human cells Epigenetics Chromatin 2010 3 22 33 21134274 10.1186/1756-8935-3-22 PMC3004898

5. Dekker F Haisma HJ Histone acetyl transferases as emerging drug targets Drug Discovery Today 2009 14 19/20 942 948 19577000 10.1016/j.drudis.2009.06.008

6. Lim S Metzger E Schüle R Epigenetic regulation of cancer growth by histone demethylases Int J Cancer 2010 127 9 1991 1998 20607829 10.1002/ijc.25538

7. Nicholson TB Chen T Richard S The physiological and pathophysiological role of PRMT1-mediated protein arginine methylation Pharmacol Res 2009 60 6 466 474 19643181 10.1016/j.phrs.2009.07.006

8. Fackelmayer FO Protein arginine methyltransferases: guardians of the Arg? Trends Biochem Sci 2005 30 12 666 671 16257219 10.1016/j.tibs.2005.10.002

9. Tini M Naeem H Torchia J Biochemical analysis of arginine methylation in transcription Methods Mol Biol 2009 523 235 247 19381935 10.1007/978-1-59745-190-1_16

10. Spannhoff AT Hauser R Heinke W The emerging therapeutic potential of histone methyltransferase and demethylase inhibitors ChemMedChem 2009 4 10 1568 1582 19739196 10.1002/cmdc.200900301

11. Zhang Y Xu K Ni M Nucleosome dynamics define transcriptional enhancers Nature Genetics 2010 42 4 343 347 20208536 10.1038/ng.545 PMC2932437

12. Copeland RA Olhava EJ Scott MP Targeting epigenetic enzymes for drug discovery Curr Opin Chem Biol 2010 14 4 505 510 20621549 10.1016/j.cbpa.2010.06.174

13. Jones PA Baylin SB The fundamental role of epigenetic events in cancer Nat Rev Genet 2002 3 6 415 428 12042769 10.1038/nrg816

14. Kriaucionis S Heintz N The nuclear DNA base 5-hydroxymethylcytosine is present in purkinje neurons and the brain Science 2009 324 5929 929 930 19372393 10.1126/science.1169786 PMC3263819

15. Abdel-Wahab O Mullally A Hedvat C Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies Blood 2009 114 1 144 147 19420352 10.1182/blood-2009-03-210039 PMC2710942

16. Fabbri M Calin GA Epigenetics and miRNAs in human cancer Adv Genet 2010 70 87 99 20920746 10.1016/B978-0-12-380866-0.60004-6

17. Zain J Kaminetzki D O’Connor OA Emerging role of epigenetic therapies in cutaneous T-cell lymphomas Expert Rev Hematol 2010 3 2 187 203 21083462 10.1586/ehm.10.9

18. Graham JS Kaye SB Brown R The promises and pitfalls of epigenetic therapies in solid tumors Eur J Cancer 2009 45 7 1129 1136 19211243 10.1016/j.ejca.2009.01.003

19. Bolden JE Peart MJ Johnstone RW Anticancer activities of histone deacetylase inhibitors Nat Rev Drug Discov 2006 5 9 5769 5784 10.1038/nrd2133 16955068

20. Haberland M Montgomery RL Olson EN The many roles of histone deacetylases in development and physiology: implications for disease and therapy Nat Rev Genet 2009 10 1 32 42 19065135 10.1038/nrg2485 PMC3215088

21. Razidlo DF Whitney TJ Casper ME Histone deacetylase 3 depletion in osteo/chondroprogenitor cells decreases bone density and increases marrow fat PLoS One 2010 5 7 e11492 20628553 10.1371/journal.pone.0011492 PMC2901996

22. Bhaskara S Chyla BJ Amann JM Deletion of histone deacetylase 3 reveals critical roles in S phase progression and DNA damage control Mol Cell 2008 30 1 61 72 18406327 10.1016/j.molcel.2008.02.030 PMC2373760

23. De Zoeten EF Wang L Sai H Inhibition of HDAC9 increases T regulatory cell function and prevents colitis in mice Gastroenterology 2010 138 2 583 594 19879272 10.1053/j.gastro.2009.10.037 PMC3369426

24. Kotian S Liyanarachchi S Zelent A Histone deacetylases 9 and 10 are required for homologous recombination J Biol Chem. Epub 2011 Jan 18. 10.1074/jbc.C110.194233 PMC3048658 21247901

25. Kelly G A review of the sirtuin system, its clinical implications, and the potential role of dietary activators like resveratrol: part 1 Altern Med Rev 2010 15 3 245 263 21155626

26. Zhang Y Kwon S Yamaguchi T Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally Mol Cell Biol 2008 28 5 1688 1701 18180281 10.1128/MCB.01154-06 PMC2258784

27. Mai A Altucci L Epi-drugs to fight cancer: from chemistry to cancer treatment, the road ahead Int J Biochem Cell Biol 2009 41 1 199 213 18790076 10.1016/j.biocel.2008.08.020

28. Marks PA Dokmanovic M Histone deacetylase inhibitors: discovery and development as anticancer agents Expert Opin Investig Drugs 2005 14 12 1497 1511 10.1517/13543784.14.12.1497 16307490

29. Bertrand P Inside HDAC with HDAC inhibitors Eur J Med Chem 2010 45 6 2095 2116 20223566 10.1016/j.ejmech.2010.02.030

30. Yurek-George A Cecil AR Mo AH The first biologically active synthetic analogues of FK228, the depsipeptide histone deacetylase inhibitor J Med Chem 2007 50 23 5720 5726 17958342 10.1021/jm0703800

31. Khan N Jeffers M Kumar S Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors Biochem J 2008 409 2 581 589 17868033 10.1042/BJ20070779

32. KrennHrubec K Marshall BL Hedglin M Design and evaluation of ‘linkerless’ hydroxamic acids as selective HDAC8 inhibitors Bioorg Med Chem Lett 2007 17 10 2874 2878 17346959 10.1016/j.bmcl.2007.02.064

33. Estiu G Greenberg E Harrison CB Structural origin of selectivity in class II-selective histone deacetylase inhibitors J Med Chem 2008 51 10 2898 2906 18412327 10.1021/jm7015254

34. Taori K Paul VJ Luesch H Structure and activity of largazole, a potent antiproliferative agent from the Floridian marine cyanobacterium Symploca sp J Am Chem Soc 2008 130 6 1806 1807 18205365 10.1021/ja7110064

35. Huhtiniemi T Suuronen T Lahtela-Kakkonen M Nε-Modified lysine containing inhibitors for SIRT1 and SIRT2 Bioorg Med Chem 2010 18 15 5616 5625 20630764 10.1016/j.bmc.2010.06.035

36. Huhtiniemi T Wittekindt C Laitinen T Comparative and pharmacophore model for deacetylase SIRT1 J Comput Aided Mol Des 2006 20 9 589 599 17103016 10.1007/s10822-006-9084-9

37. Elaut G Török G Vinken M Major phase I biotransformation pathways of Trichostatin A in rat hepatocytes and in rat and human liver microsomes Drug Metab Dispos 2002 30 12 1320 1328 12433798 10.1124/dmd.30.12.1320

38. Ebbel EN Leymarie N Schiavo S Identification of phenylbutyrate-generated metabolites in Huntington disease patients using parallel liquid chromatography/electrochemical array/mass spectrometry and off-line tandem mass spectrometry Analytical Biochem 2010 399 2 152 161 10.1016/j.ab.2010.01.010 PMC3568495 20074541

39. Parise RA Holleran JL Beuner JH A liquid chromatography–electrospray ionization tandem mass spectrometric assay for quantitation of the histone deacetylase inhibitor, vorinostat (suberoylanilide hydroxamicacid, SAHA), and its metabolites in human serum J Chrom B 2006 840 2 108 115 10.1016/j.jchromb.2006.04.044 16725386

40. Du L Musson DG Wang AQ Stability studies of vorinostat and its two metabolites in human plasma, serum and urine J Pharm Biomed Anal 2006 42 5 556 564 16824724 10.1016/j.jpba.2006.05.005

41. Furumai R Matsuyama A Kobashi N FK228 (depsipeptide) as a natural prodrug that inhibits class I histone deacetylases Cancer Res 2002 62 17 4916 4921 12208741

42. Xiao JJ Byrd J Marcucci G Identification of thiols and glutathione conjugates of depsipeptide FK228 (FR901228), a novel histone protein deacetylase inhibitor, in the blood Rapid Commun Mass Spectrom 2003 17 8 757 766 12672127 10.1002/rcm.976

43. Shiraga T Tozuka Z Ishimura R Identification of cytochrome P450 enzymes involved in the metabolism of FK228, a potent histone deacetylase inhibitor, in human liver microsomes Biol Pharm Bull 2005 28 1 124 129 15635176 10.1248/bpb.28.124

44. Kim HM Oh SJ Park SK In vitro metabolism of KBH-A40, a novel delta-lactam-based histone deacetylase (HDAC) inhibitor, in human liver microsomes and serum Xenobiotica 2008 38 3 281 293 18274957 10.1080/00498250701813222

45. Fonsi M Fiore F Jones P Metabolism-related liabilities of a potent histone deacetylase (HDAC) inhibitor and relevance of the route of administration on its metabolic fate Xenobiotica 2009 39 10 722 737 19569735 10.1080/00498250903082279

46. Peart MJ Smyth GK van Laar RK Identification and functional significance of genes regulated by structurally different histone deacetylase inhibitors PNAS 2005 102 10 3697 3702 15738394 10.1073/pnas.0500369102 PMC552783

47. Ellis L Pan Y Smyth GK Histone deacetylase inhibitor panobinostat induces clinical responses with associated alterations in gene expression profiles in cutaneous T-cell lymphoma Clin Cancer Res 2008 14 14 4500 4510 18628465 10.1158/1078-0432.CCR-07-4262

48. Kato N Tanaka J Sugita J Regulation of the expression of MHC class I-related chain A, B (MICA, MICB) via chromatin remodeling and its impact on the susceptibility of leukemic cells to the cytotoxicity of NKG2D-expressing cells Leukemia 2007 21 10 2103 2108 17625602 10.1038/sj.leu.2404862

49. Vo DD Prins RM Begley JL Enhanced antitumor activity induced by adoptive T-cell transfer and adjunctive use of the histone deacetylase inhibitor LAQ824 Cancer Res 2009 69 22 8693 8699 19861533 10.1158/0008-5472.CAN-09-1456 PMC2779578

50. Cameron EE Bachman KE Myöhänen S Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer Nat Genet 1999 21 1 103 107 9916800 10.1038/5047

51. Appelbaum FR Baer MR Carabasi MH NCCN Practice guidelines for acute myelogenous leukemia Oncology 2000 14 11A 53 61 11195419

52. Snykers S Vinken M Rogiers V Differential role of epigenetic modulators in malignant and normal stem cells: a novel tool in preclinical in vitro toxicology and clinical therapy Arch Toxicol 2007 81 8 533 544 17387455 10.1007/s00204-007-0195-4

53. Marks PA Miller T Richon VM Histone deacetylases Curr Opin Pharmacol 2003 3 4 344 351 12901942 10.1016/s1471-4892(03)00084-5

54. DiGiuseppe JA Weng LJ Yu KH Phenylbutyrate-induced G1 arrest and apoptosis in myeloid leukemia cells: structure-function analysis Leukemia 1999 13 8 1243 1253 10450753 10.1038/sj.leu.2401471

55. Carducci MA Nelson JB Chan-Tack KM Phenylbutyrate induces apoptosis in human prostate cancer and is more potent than phenylacetate Clin Cancer Res 1996 2 2 379 387 9816181

56. Zhang X Wei L Yang Y Yu Q Sodium 4-phenylbutyrate induces apoptosis of human lung carcinoma cells through activating JNK pathway J Cell Biochem 2004 93 4 819 829 15389886 10.1002/jcb.20173

57. Gorospe M Shack S Guyton KZ Up-regulation and functional role of p21Waf1/Cip1 during growth arrest of human breast carcinoma MCF-7 cells by phenylacetate Cell Growth Differ 1996 7 12 1609 1615 8959328

58. Liu L Hudgins WR Miller AC Transcriptional upregulation of TGF-α by phenylacetate and phenylbuytrate is associated with differentiation of human melanoma cells Cytokine 1995 7 5 449 456 7578983 10.1006/cyto.1995.0061

59. Han S Wada RK Sidell N Differentiation of human neuroblastoma by phenylacetate is mediated by peroxisome proliferatoractivated receptor gamma Cancer Res 2001 61 10 3998 4002 11358817

60. Carducci MA Nelson JB Chan-Tack KM Phenylbutyrate induces apoptosis in human prostate cancer and is more potent than phenylacetate Clin Cancer Res 1996 2 2 379 387 9816181

61. Brusilow SW Phenylacetylglutamine may replace urea as a vehicle for waste nitrogen excretion Pediatr Res 1991 29 2 147 150 2014149 10.1203/00006450-199102000-00009

62. Phuphanich S Baker SD Grossman SA Oral sodium phenylbutyrate in patients with recurrent malignant gliomas: a dose escalation and pharmacologic study Neuro Oncol 2005 7 2 177 182 15831235 10.1215/S1152851704000183 PMC1871887

63. Carducci MA Gilbert J Bowling MK A phase I clinical and pharmacological evaluation of sodium phenylbutyrate on an 120-h infusion schedule Clin Cancer Res 2001 7 10 3047 3055 11595694

64. Gilbert J Baker SD Bowling MK A phase I dose escalation and bioavailability study of oral sodium phenylbutyrate in patients with refractory solid tumor malignancies Clin Cancer Res 2001 7 8 2292 2300 11489804

65. Camacho LH Olson J Tong WP Phase I dose escalation clinical trial of phenylbutyrate sodium administered twice daily to patients with advanced solid tumors Invest New Drugs 2007 25 2 131 138 17053987 10.1007/s10637-006-9017-4

66. Gore SD Weng LJ Zhai S Impact of prolonged infusions of the putative differentiating agent sodium phenylbutyrate on myelodysplastic syndromes and acute myeloid leukemia Clin Cancer Res 2002 8 4 963 970 11948101

67. Maslak P Chanel S Camacho LH Pilot study of combination transcriptional modulation therapy with sodium phenylbutyrate and 5-azacytidine in patients with acute myeloid leukemia or myelodysplastic syndrome Leukemia 2006 20 2 212 217 16357841 10.1038/sj.leu.2404050

68. Gilbert J Baker SD Donehower R Methytransferase (MT) activity and gene expression in tumor biopsies from patients enrolled in a Phase I study of the MT inhibitor, 5-azacytidine (5AC), and the histone deacetylase inhibitor, phenylbutyrate (PB), in refractory solid tumors Proc Am Soc Clin Onc. 2001 87a

69. Sung MW Waxman S Combination of cytotoxicdifferentiation therapy with 5-fluorouracil and phenylbutyrate in patients with advanced colorectal cancer Anticancer Res 2007 27 2 995 1001 17465233

70. Rephaeli A Rabizadeh E Aviram A Derivatives of butyric acid as potential anti-neoplastic agents Int J Cancer 1991 49 1 66 72 1874573 10.1002/ijc.2910490113

71. Schwartz B Avivi-Green C Polak-Charcon S Sodium butyrate induces retinoblastoma protein dephosphorylation, p16 expression and growth arrest of colon cancer cells Mol Cell Biochem 1998 188 1–2 21 30 9823007

72. Mandal M Adam L Kumar R Redistribution of activated caspase-3 to the nucleus during butyric acid-induced apoptosis Biochem Biophys Res Commun 1999 260 3 775 780 10403841 10.1006/bbrc.1999.0966

73. Rabizadeh E Shaklai M Nudelman A Rapid alteration of c-myc and c-jun expression in leukemic cells induced to differentiate by a butyric acid prodrug FEBS Lett 1993 328 3 225 229 8348968 10.1016/0014-5793(93)80932-k

74. Rabizadeh E Bairey O Aviram A Doxorubicin and a butyric acid derivative effectively reduce levels of Bcl-2 protein in the cells of chronic lymphocytic leukemia patient Eur J Haematol 2001 66 4 263 271 11380606 10.1034/j.1600-0609.2001.066004263.x

75. Patnaik A Rowinsky EK Villalona MA A phase I study of pivaloyloxymethyl butyrate, a prodrug of the differentiating agent butyric acid, in patients with advanced solid malignancies Clin Cancer Res 2002 8 7 2142 2148 12114414

76. Reid T Valone F Lipera W Phase II trial of the histone deacetylase inhibitor pivaloyloxymethyl butyrate (Pivanex, AN-9) in advanced non-small cell lung cancer Lung Cancer 2004 45 3 381 386 15301879 10.1016/j.lungcan.2004.03.002

77. Loscher W Basic pharmacology of valproate: a review after 35 years of clinical use for the treatment of epilepsy CNS Drugs 2002 16 10 669 694 12269861 10.2165/00023210-200216100-00003

78. Perucca E Pharmacological and therapeutic properties of valproate: a summary after 35 years of clinical experience CNS Drugs 2002 16 10 695 714 12269862 10.2165/00023210-200216100-00004

79. Marchion DC Bicaku E Daud AI Valproic acid alters chromatin structure by regulation of chromatin modulation proteins Cancer Res 2005 65 9 3815 3822 15867379 10.1158/0008-5472.CAN-04-2478

80. Jung GA Yoon JY Moon BS Valproic acid induces differentiation and inhibition of proliferation in neural progenitor cells via the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway BMC Cell Biol 2008 9 66 78 19068119 10.1186/1471-2121-9-66 PMC2639384

81. Gottlicher M Minucci S Zhu J Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells EMBO J 2001 20 24 6969 6978 11742974 10.1093/emboj/20.24.6969 PMC125788

82. Gurvich N Tsygankova OM Meinkoth JL Histone deacetylase is a target of valproic acid-mediated cellular differentiation Cancer Res 2004 64 3 1079 1086 14871841 10.1158/0008-5472.can-03-0799

83. Bacon CL Gallagher HC Haughey JC Antiproliferative action of valproate is associated with aberrant expression and nuclear translocation of cyclin D3 during the C6 glioma G1 phase J Neurochem 2002 83 1 12 19 12358724 10.1046/j.1471-4159.2002.01081.x

84. Werling U Siehler S Litfin M Induction of differentiation in F9 cells and activation of peroxisome proliferator-activated receptor delta by valproic acid and its teratogenic derivatives Mol Pharmacol 2001 59 5 1269 1276 11306712 10.1124/mol.59.5.1269

85. Kawagoe R Kawagoe H Sano K Valproic acid induces apoptosis in human leukemia cells by stimulating both caspase dependent and -independent apoptotic signaling pathways Leuk Res 2002 26 5 495 502 11916526 10.1016/s0145-2126(01)00151-5

86. Tang R Faussat AM Majdak P Valproic acid inhibits proliferation and induces apoptosis in acute myeloid leukemia cells expressing P-gp and MRP1 Leukemia 2004 18 7 1246 1251 15116123 10.1038/sj.leu.2403390

87. Ichiyama T Okada K Lipton JM Sodium valproate inhibits production of TNF-α and IL-6 and activation of NF-κB Brain Res 2000 857 1–2 246 251 10700573 10.1016/s0006-8993(99)02439-7

88. Siitonen T Koistinen P Savolainen ER Increase in Ara-C cytotoxicity in the presence of valproate, a histone deacetylase inhibitor, is associated with the concurrent expression of cyclin D1 and p27(Kip 1) in acute myeloblastic leukemia cells Leuk Res 2005 29 11 1335 1342 15936818 10.1016/j.leukres.2005.04.018

89. Cimino G Lo-Coco F Fenu S Sequential valproic acid/Alltrans retinoic acid treatment reprograms differentiation in refractory and highrisk acute myeloid leukemia Cancer Res 2006 66 17 8903 8911 16951208 10.1158/0008-5472.CAN-05-2726

90. Valentini A Gravina P Federici G Valproic acid induces apoptosis, p16INK4A upregulation and sensitization to chemotherapy in human melanoma cells Cancer Biol Ther 2007 6 2 185 191 17218782 10.4161/cbt.6.2.3578

91. Atmaca A Al-Batran SE Maurer A Valproic acid (VPA) in patients with refractory advanced cancer: a dose escalating Phase I clinical trial British J Cancer 2007 97 2 177 182 10.1038/sj.bjc.6603851 PMC2360302 17579623

92. Bug G Ritter M Wassmann B Clinical trial of valproic acid and all-trans retinoic acid in patients with poor-risk acute myeloid leukemia Cancer 2005 104 12 2717 2725 16294345 10.1002/cncr.21589

93. Kuendgen A Knipp S Fox F Results of a Phase 2 study of valproic acid alone or in combination with alltrans retinoic acid in 75 patients with myelodysplastic syndrome and relapsed or refractory acute myeloid leukemia Ann Hematol 2005 84 Suppl 1 61 66 16270213 10.1007/s00277-005-0026-8

94. Pilatrino C Cilloni D Messa E Increase in platelet count in older, poor-risk patients with acute myeloid leukemia or myelodysplastic syndrome treated with valproic acid and all-trans retinoic acid Cancer 2005 104 1 101 109 15895376 10.1002/cncr.21132

95. Raffoux E Chaibi P Dombret H Valproic acid and all-trans retinoic acid for the treatment of elderly patients with acute myeloid leukemia Haematologica 2005 90 7 986 988 15996941

96. Duprez E Lillehaug JR Naoe T cAMP signalling is decisive for recovery of nuclear bodies (PODs) during maturation of RA-resistant t(15;17) promyelocytic leukemia NB4 cells expressing PML-RAR alpha Oncogene 1996 12 11 2451 2459 8649787

97. Kuendgen A Schmid M Schlenk R The histone deacetylase (HDAC) inhibitor valproic acid as monotherapy or in combination with all-trans retinoic acid in patients with acute myeloid leukemia Cancer 2006 106 1 112 119 16323176 10.1002/cncr.21552

98. Huang S Brandt SJ mSin3A regulates murine erythroleukemia cell differentiation through association with the TAL1 (or SCL) transcription factor Mol Cell Biol 2000 20 6 2248 2259 10688671 10.1128/mcb.20.6.2248-2259.2000 PMC110841

99. Watamoto K Towatari M Ozawa Y Altered interaction of HDAC5 with GATA-1 during MEL cell differentiation Oncogene 2003 22 57 9176 9184 14668799 10.1038/sj.onc.1206902

100. Siitonen T Timonen T Juvonen E Valproic acid combined with 13-cis retinoic acid and 1,25-dihydroxyvitamin D3 in the treatment of patients with myelodysplastic syndromes Haematologica 2007 92 8 1119 1122 17650442 10.3324/haematol.11262

101. Santini V Ferrini PR Differentiation therapy of myelodysplastic syndromes: fact or fiction? Br J Heamatol 1998 102 5 1124 1138 10.1046/j.1365-2141.1998.00881.x 9753034

102. Garcia-Manero G Kantarjian HM Sanchez-Gonzalez B Phase 1/2 study of the combination of 5-aza-2′-deoxycytidine with valproic acid in patients with leukemia Blood 2006 108 10 3271 3279 16882711 10.1182/blood-2006-03-009142 PMC1895437

103. Blum W Klisovic RB Hackanson B Phase I study of decitabine alone or in combination with valproic acid in acute myeloid leukemia J Clin Oncol 2007 25 25 3884 3891 17679729 10.1200/JCO.2006.09.4169

104. Braiteh F Soriano AO Garcia-Manero G Phase I study of epigenetic modulation with 5-azacytidine and valproic acid in patients with advanced cancers Clin Cancer Res 2008 14 19 6296 6301 18829512 10.1158/1078-0432.CCR-08-1247 PMC2582814

105. Soriano AO Yang H Faderl S Safety and clinical activity of the combination of 5-azacytidine, valproic acid, and all-trans retinoic acid in acute myeloid leukemia and myelodysplastic syndrome Blood 2007 110 7 2302 2308 17596541 10.1182/blood-2007-03-078576

106. Münster P Marchion D Bicaku E Clinical and biological effects of valproic acid as a histone deacetylase inhibitor on tumor and surrogate tissues: Phase I/II trial of valproic acid and epirubicin/FEC Clin Cancer Res 2009 15 7 2488 2496 19318486 10.1158/1078-0432.CCR-08-1930

107. Münster P Marchion D Bicaku E Phase I trial of histone deacetylase inhibition by valproic acid followed by the topoisomerase II inhibitor epirubicin in advanced solid tumors: a clinical and translational study J Clin Oncology 2007 25 15 1979 1985 10.1200/JCO.2006.08.6165 17513804

108. Daud AI Dawson J DeConti RC Potentiation of a topoisomerase I inhibitor, karenitecin, by the histone deacetylase inhibitor valproic acid in melanoma: translational and Phase I/II clinical trial Clin Cancer Res 2009 15 7 2479 2489 19318485 10.1158/1078-0432.CCR-08-1931

109. Rocca A Minucci S Tosti G A Phase I–II study of the histone deacetylase inhibitor valproic acid plus chemoimmunotherapy in patients with advanced melanoma British J Cancer 2009 100 1 28 36 10.1038/sj.bjc.6604817 PMC2634690 19127265

110. Fachetti F Previdi S Ballarini M Modulation of pro- and anti-apoptotic factors in human melanoma cells exposed to histone deacetylase inhibitors Apoptosis 2004 9 5 573 582 15314285 10.1023/B:APPT.0000038036.31271.50

111. Chavez-Blanco A Segura-Pacheco B Perez-Cardenas E Histone acetylation and histone deacetylase activity of magnesium valproate in tumor and peripheral blood of patients with cervical cancer. A Phase I study Mol Cancer 2005 4 1 22 16001982 10.1186/1476-4598-4-22 PMC1198251

112. Candelaria M Gallardo-Rincon D Arce C A Phase II study of epigenetic therapy with hydralazine and magnesium valproate to overcome chemotherapy resistance in refractory solid tumors Ann Oncol 2007 18 9 1529 1538 17761710 10.1093/annonc/mdm204

113. Parulekar WR Einsenhauer EA Novel endpoints and design of early clinical trials Annal Oncol 2002 13 Suppl 4 139 143 10.1093/annonc/mdf651 12401680

114. Hunsberger S Rubinstein LV Dancey J Dose escalation trial designs based on a molecularly targeted endpoint Stat Med 2005 24 14 2171 2181 15909289 10.1002/sim.2102

115. Segura-Pacheco B Perez-Cardenas E Taja-Chayeb L Global DNA hypermethylation-associated cancer chemotherapy resistance and its reversion with the demethylating agent hydralazine J Transl Med 2006 4 32 16893460 10.1186/1479-5876-4-32 PMC1563479

116. Richon VM Webb Y Merger R Second generation hybrid polar compounds are potent inducers of transformed cell differentiation Proc Natl Acad Sci U S A 1996 93 12 5705 5708 8650156 10.1073/pnas.93.12.5705 PMC39124

117. Butler LM Agus DB Scher HL Suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase, suppresses the growth of prostate cancer cells in vitro and in vivo Cancer Res 2000 60 78 5165 5170 11016644

118. Finnin MS Donigian JR Cohen A Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors Nature 1999 401 6749 188 193 10490031 10.1038/43710

119. Yin D Ong JM Hu J Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor: effects on gene expression and growth of glioma cells in vitro and in vivo Clin Cancer Res 2007 13 3 1045 1052 17289901 10.1158/1078-0432.CCR-06-1261

120. Mitsiades CS Poulaki V McMullan C Novel histone deacetylase inhibitors in the treatment of thyroid cancer Clin Cancer Res 2005 11 10 3958 3965 15897598 10.1158/1078-0432.CCR-03-0776

121. Munster PN Troso-Sandoval T Rosen N The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces differentiation of human breast cancer cells Cancer Res 2001 61 23 8492 8497 11731433

122. Bali P Pranpat M Swaby R Activity of suberoylanilide hydroxamic acid against human breast cancer cells with amplification of her-2 Clin Cancer Res 2005 11 17 6382 6389 16144943 10.1158/1078-0432.CCR-05-0344

123. Komatsu N Kawamata N Takeuchi S SAHA, a HDAC inhibitor, has profound anti-growth activity against non-small cell lung cancer cells Oncol Rep 2006 15 1 187 191 16328054

124. Butler LM Agus DB Scher HI Suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase, suppresses the growth of prostate cancer cells in vitro and in vivo Cancer Res 2000 60 18 5165 5170 11016644

125. Dietrich C Greenberg VL Desimone CP Suberoylanilide hydroxamic acid potentiates paclitaxel-induced apoptosis in ovarian cancer cell lines Gynecol Oncol 2006 116 1 126 130 19875160 10.1016/j.ygyno.2009.09.039

126. Kelly WK Richon VM O’Connor O Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously Clin Cancer Res. 2003 9 10 Pt 1 3578 3588 14506144

127. Krug LM Curley T Schwartz L Potential role of histone deacetylase inhibitors in mesothelioma: clinical experience with suberoylanilide hydroxamic acid Clin Lung Cancer 2006 7 4 257 261 16512979 10.3816/CLC.2006.n.003

128. Kelly WK O’Connor OA Krug LM Phase I study of an oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced cancer J Clin Oncol 2005 23 17 3923 3931 15897550 10.1200/JCO.2005.14.167 PMC1855284

129. Garcia-Manero G Yangn H Bueso-Ramos C Phase 1 study of the histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid [SAHA]) in patients with advanced leukemias andmyelodysplastic syndromes Blood 2008 111 3 1060 1066 17962510 10.1182/blood-2007-06-098061

130. Modesitt SC Sill M Hoffman JS A Phase II study of vorinostat in the treatment of persistent or recurrent epithelial ovarian or primary peritoneal carcinoma: a Gynecologic Oncology Group study Gynecol Oncol 2008 109 2 182 186 18295319 10.1016/j.ygyno.2008.01.009

131. Vansteenkiste J Van Cutsem E Dumez H Early Phase II trial of oral vorinostat in relapsed or refractory breast, colorectal, or nonsmall cell lung cancer Invest New Drugs 2008 26 5 483 488 18425418 10.1007/s10637-008-9131-6

132. Blumenschein GR Jr Kies MS Papadimitrakopoulou VA Phase II trial of the histone deacetylase inhibitor vorinostat (Zolinza, suberoylanilide hydroxamic acid, SAHA) in patients with recurrent and/or metastatic head and neck cancer Invest New Drugs 2008 26 1 81 87 17960324 10.1007/s10637-007-9075-2

133. Luu TH Morgan RJ Leong L A phase II trial of vorinostat (suberoylanilide hydroxamic acid) in metastatic breast cancer: a California Cancer Consortium study Clin Cancer Res 2008 14 21 7138 7142 18981013 10.1158/1078-0432.CCR-08-0122 PMC3543872

134. Galanis E Jaeckle KA Maurer MJ Phase II trial of vorinostat in recurrent glioblastoma multiforme: a north central cancer treatment group study J Clin Oncol 2009 27 12 2052 2058 19307505 10.1200/JCO.2008.19.0694 PMC2669764

135. Woyach JA Kloos RT Ringel MD Lack of therapeutic effect of the histone deacetylase inhibitor vorinostat in patients with metastatic radioiodine-refractory thyroid carcinoma J Clin Endocrinol Metab 2009 94 1 164 170 18854394 10.1210/jc.2008-1631 PMC2630867

136. Ramalingam SS Parise RA Ramanathan RK Phase I and pharmacokinetic study of vorinostat, a histone deacetylase inhibitor, in combination with carboplatin and paclitaxel for advanced solid malignancies Clin Cancer Res 2007 13 12 3605 3610 17510206 10.1158/1078-0432.CCR-07-0162

137. Plumb JA Finn PW Williams RJ Pharmacodynamic response and inhibition of growth of human tumor xenografts by the novel histone deacetylase inhibitor PXD101 Mol Cancer Ther 2003 2 8 721 728 12939461

138. Gimsing P Belinostat: a new broad acting antineoplastic histone deacetylase inhibitor Expert Opin Investig Drugs 2009 18 4 501 508 10.1517/13543780902852560 19335278

139. Gian X LaRochelle WJ Ara G Activity of PXD101, a histone deacetylase inhibitor, in preclinical ovarian cancer studies Mol Cancer Ther 2006 5 8 2086 2095 16928830 10.1158/1535-7163.MCT-06-0111

140. Monks A Hose CD Pezzoli P Gene expression-signature of belinostat in cell lines is specific for histone deacetylase inhibitor treatment, with a corresponding signature in xenografts Anticancer Drugs 2009 20 8 682 692 19606018 10.1097/CAD.0b013e32832e14e1

141. Warren KE McCully C Dvinge H Plasma and cerebrospinal fluid pharmacokinetics of the histone deacetylase inhibitor, belinostat (PXD101), in non-human primates Cancer Chemother Pharmacol 2008 62 3 433 437 17960383 10.1007/s00280-007-0622-5

142. Tumber A Collins LS Petersen K The histone deacetylase inhibitor PXD101 synergises with 5-Xuorouracil to inhibit colon cancer cell growth in vitro and in vivo Cancer Chemother Pharmacol 2007 60 275 283 17124594 10.1007/s00280-006-0374-7

143. Steele NL Plumb JA Vidal L A Phase 1 pharmacokinetic and pharmacodynamic study of the histone deacetylase inhibitor belinostat in patients with advanced solid tumors Clin Cancer Res 2008 14 3 804 810 18245542 10.1158/1078-0432.CCR-07-1786

144. Takada M Kataoka A Toi M A close association between alteration in growth kinetics by neoadjuvant chemotherapy and survival outcome in primary breast cancer Int J Oncol 2004 25 2 397 405 15254737

145. Gimsing P Hansen M Knudsen LM A phase I clinical trial of the histone deacetylase inhibitor belinostat in patients with advanced hematological neoplasia Eur J Hematol 2008 81 3 170 176 10.1111/j.1600-0609.2008.01102.x 18510700

146. Ramalingam SS Belani CP Ruel C Phase II study of belinostat (PXD101), a histone deacetylase inhibitor, for second line therapy of advanced malignant pleural mesothelioma J Thorac Oncol 2009 4 1 97 101 19096314 10.1097/JTO.0b013e318191520c PMC3263397

147. Mackay HJ Hirte H Colgan T Phase II trial of the histone deacetylase inhibitor belinostat in women with platinum resistant epithelial ovarian cancer and micropapillary (LMP) ovarian tumours Eur J Cancer 2010 46 9 1573 1579 20304628 10.1016/j.ejca.2010.02.047 PMC3244274

148. Golay J Cuppini L Leoni F The histone deacetylase inhibitor ITF2357 has anti-leukemic activity in vitro and in vivo and inhibits IL-6 and VEGF production by stromal cells Leukemia 2007 21 9 1892 1900 17637810 10.1038/sj.leu.2404860

149. Viviani S Bonfante V Fasola C Phase II study of the histonedeacetylase inhibitor ITF2357 in relapsed/refractory Hodgkin’s lymphoma patients J Clin Oncol. 2008 26 abstract 8532.

150. Galli M Salmoiraghi S Golay J A Phase II multiple dose clinical trial of histone deacetylase inhibitor ITF2357 in patients with relapsed or progressive multiple myeloma Ann Hematol 2010 89 2 185 190 19633847 10.1007/s00277-009-0793-8

151. Carlo-Stella C Guidetti A Viviani S Safety and clinical activity of the histone deacetylase inhibitor givinostat in combination with meclorethamine in relapsed/refractory Hodgkin lymphoma (HL) J Clin Oncol. 2010 28 abstract 3068.

152. Qian DZ Kato Y Shabbeer S Targeting tumor angiogenesis with histone deacetylase inhibitors: the hydroxamic acid derivative LBH589 Clin Cancer Res 2006 12 2 634 642 16428510 10.1158/1078-0432.CCR-05-1132

153. Prince HM Bishton MJ Johnstone RW Panobinostat (LBH589): a potent pan-deacetylase inhibitor with promising activity against hematologic and solid tumors Future Oncol 2009 5 5 601 612 19519200 10.2217/fon.09.36

154. Giles F Fischer T Cortes J Phase I Study of Intravenous LBH589, a novel cinnamic hydroxamic acid analogue histone deacetylase inhibitor, in patients with refractory hematologic malignancies Clin Cancer Res 2006 12 15 4628 4635 16899611 10.1158/1078-0432.CCR-06-0511

155. Rathkopf D Wong BY Ross RW A Phase I study of oral panobinostat alone and in combination with docetaxel in patients with castration-resistant prostate cancer Cancer Chemother Pharmacol 2010 66 1 181 189 20217089 10.1007/s00280-010-1289-x

156. Remiszewski SW The discovery of NVPLAQ824: from concept to clinic Curr Med Chem 2003 10 22 2393 2402 14529481 10.2174/0929867033456675

157. Hurtubise A Momparler RL Effect of histone deacetylase inhibitor LAQ824 on antineoplastic action of 5-Aza-2-deoxycytidine (decitabine) on human breast carcinoma cells Cancer Chemother Pharmacol 2006 58 5 618 625 16783580 10.1007/s00280-006-0225-6

158. De Bono JS Kristeleit R Tolcher A Phase I pharmacokinetic and pharmacodynamic study of LAQ824, a hydroxamate histone deacetylase inhibitor with a heat shock protein-90 inhibitory profile, in patients with advanced solid tumors Clin Cancer Res 2008 14 20 6663 6673 18927309 10.1158/1078-0432.CCR-08-0376

159. Kristeleit RS Tandy D Atadja P Effects of the histone deacetylase inhibitor (HDACI) LAQ824 on histone acetylation, Hsp70 and c-Raf in peripheral blood lymphocytes from patients with advanced solid tumours enrolled in a phase I clinical trial J Clin Oncol. 2004 22 abstract 3023.

160. Ottmann OG Deangelo DJ Stone RM A Phase I, pharmacokinetic (PK) and pharmacodynamic (PD) study of a novel histone deacetylase inhibitor LAQ824 in patients with hematologic malignancies J Clin Oncol. 2004 22 abstract 3024.

161. Choy E Cao Y Hornicek F Effect of histone deacetylase inhibitor (HDACI) PCI-24781 on chemotherapy-induced apoptosis in multidrug-resistant sarcoma cell lines J Clin Oncol. 2010 28 abstract 10089. PMC3744220 21508354

162. Undevia SD Janisch L Schilsky RL Phase I study of the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of the histone deacetylase inhibitor (HDACi) PCI-24781 J Clin Oncol. 2008 26 abstract 14514.

163. Jaboin J Wild J Hamidi H MS-27-275, an inhibitor of histone deacetylase, has marked in vitro and in vivo antitumor activity against pediatric solid tumors Cancer Res 2002 62 21 6108 6115 12414635

164. Lucas DM Davis ME Parthun MR The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells Leukemia 2004 18 7 1207 1214 15116122 10.1038/sj.leu.2403388

165. Rosato RR Almenara JA Grant S The histone deacetylase inhibitor MS-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21CIP1/WAF1 1 Cancer Res 2003 63 13 3637 3645 12839953

166. Lee BI Park SH Kim JW MS-275, a histone deacetylase inhibitor, selectively induces transforming growth factor beta type II receptor expression in human breast cancer cells Cancer Res 2001 61 3 931 934 11221885

167. Acharya MR Sparreboom A Sausville EA Interspecies differences in plasma protein binding of MS-275, a novel histone deacetylase inhibitor Cancer Chemother Pharmacol 2006 57 3 275 281 16028097 10.1007/s00280-005-0058-8

168. Ryan QC Headlee D Acharya M Phase I and pharmacokinetic study of MS-275, a histone deacetylase inhibitor, in patients with advanced and refractory solid tumors or lymphoma J Clin Oncol 2005 23 17 3912 3922 15851766 10.1200/JCO.2005.02.188

169. Kummar S Gutierrez M Gardner ER Phase I trial of MS-275, a histone deacetylase inhibitor, administered weekly in refractory solid tumors and lymphoid malignancies Clin Cancer Res 2007 13 18 Pt 1 5411 5417 17875771 10.1158/1078-0432.CCR-07-0791

170. Gojo I Jiemjit A Trepel JB Phase 1 and pharmacologic study of MS-275, a histone deacetylase inhibitor, in adults with refractory and relapsed acute leukemias Blood 2007 109 7 2781 2790 17179232 10.1182/blood-2006-05-021873 PMC1852211

171. Gore L Rothenberg ML O’Bryant CL A phase I and pharmacokinetic study of the oral histone deacetylase inhibitor, MS-275, in patients with refractory solid tumors and lymphomas Clin Cancer Res 2008 14 14 4517 4525 18579665 10.1158/1078-0432.CCR-07-1461 PMC2813676

172. Hauschild A Trefzer U Garbe C Multicenter phase II trial of the histone deacetylase inhibitor pyridylmethyl-N-{4-[(2-aminophenyl)-carbamoyl]-benzyl}-carbamate in pretreated metastatic melanoma Melanoma Res 2008 18 4 274 278 18626312 10.1097/CMR.0b013e328307c248

173. Zhou N Moradei O Raeppel S Discovery of N-(2-aminophenyl)-4-[(4-pyridin-3-ylpyrimidin-2-ylamino)methyl]benzamide (MGCD0103), an orally active histone deacethylase inhibitor J Med Chem 2008 51 14 4072 4075 18570366 10.1021/jm800251w

174. Fournel M Bonfils C Hou Y MGCD0103, a novel isotype-selective histone deacetylase inhibitor, has broad spectrum antitumor activity in vitro and in vivo Mol Cancer Ther 2008 7 4 759 768 18413790 10.1158/1535-7163.MCT-07-2026

175. Siu LL Pili R Duran I Phase I study of MGCD0103 given as a three-times-per-week oral dose in patients with advanced solid tumors J Clin Oncol 2008 26 12 1940 1947 18421048 10.1200/JCO.2007.14.5730 PMC3501257

176. Garcia-Manero G Assouline S Cortes J Phase 1 study of the oral isotype specific histone deacetylase inhibitor MGCD0103 in leukemia Blood 2008 112 4 981 989 18495956 10.1182/blood-2007-10-115873 PMC4081529

177. Lancet JE Nichols G Assouline S A Phase I study of MGCD0103 given as a twice weekly oral dose in patients with advanced leukemias or myelodysplastic syndromes (MDS) J Clin Oncol. 2007 25 abstract 2516.

178. Bociek RG Kuruvilla GJ Pro B Isotype-selective histone deacetylase (HDAC) inhibitor MGCD0103 demonstrates clinical activity and safety in patients with relapsed/refractory classical Hodgkin Lymphoma (HL) J Clin Oncol. 2008 26 abstract 8507.

179. Martell RE Younes A Assouline SE Phase II study of MGCD0103 in patients with relapsed follicular lymphoma (FL): Study reinitiation and update of clinical efficacy and safety J Clin Oncol. 2010 28 abstract 8086.

180. Berger MR Bischoff H Fritschi E Synthesis, toxicity, and therapeutic efficacy of 4-amino-N-(2-aminophenyl) benzamide: a new compound preferentially active in slowly growing tumors Cancer Treat Rep 1985 69 12 1415 1424 3841025

181. Kraker AJ Mizzen CA Hartl BG Modulation of histone acetylation by [4-(acetylamino)-N-(2-amino-phenyl) benzamide] in HCT-8 colon carcinoma Mol Cancer Ther 2003 2 4 401 408 12700284

182. Prakash S Foster BJ Meyer M Chronic oral administration of CI-994: a Phase 1 study Invest New Drugs 2001 19 1 1 11 11291827 10.1023/a:1006489328324

183. Undevia SD Kindler HL Janisch L A Phase I study of the oral combination of CI-994, a putative histone deacetylase inhibitor, and capecitabine Ann Oncol 2004 15 11 1705 1711 15520075 10.1093/annonc/mdh438

184. Richards DA Boehm KA Waterhouse DM Gemcitabine plus CI-994 offers no advantage over gemcitabine alone in the treatment of patients with advanced pancreatic cancer: results of a phase II randomized, double-blind, placebo-controlled, multicenter study Ann Oncol 2006 17 7 1096 1102 16641168 10.1093/annonc/mdl081

185. Ueda H Manda T Matsumoto S FR901228, a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum No. 968, III: antitumor activities on experimental tumors in mice J Antibiot 1994 47 3 315 323 8175484 10.7164/antibiotics.47.315

186. Ueda H Nakajima H Hori Y FR901228, a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum No. 968. I. Taxonomy, fermentation, isolation, physicochemical and biological properties, and antitumor activity J Antibiot 1994 47 3 301 310 7513682 10.7164/antibiotics.47.301

187. Yu XD Wang SY Chen GA Apoptosis induced by depsipeptide FK228 coincides with inhibition of survival signaling in lung cancer cells Cancer J 2007 13 2 105 113 17476138 10.1097/PPO.0b013e318046eedc

188. Sandor V Robbins AR Robey R FR901228 causes mitotic arrest but does not alter microtubule polymerization Anticancer Drugs 2000 11 11 445 454 11001385 10.1097/00001813-200007000-00005

189. Sandor V Senderowicz A Mertins S P21-dependent G(1) arrest with downregulation of cyclin D1 and upregulation of cyclin E by the histone deacetylase inhibitor FR901228 Br J Cancer 2000 83 6 817 825 10952788 10.1054/bjoc.2000.1327 PMC2363539

190. Wang R Brunner T Zhang L Fungal metabolite FR901228 inhibits c-Myc and Fas ligand expression Oncogene 1998 17 12 1503 1508 9794227 10.1038/sj.onc.1202059

191. Yu X Guo ZS Marcu MG Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228 J Natl Cancer Inst 2002 94 7 504 513 11929951 10.1093/jnci/94.7.504

192. Rajgolikar G Chan KK Wang HC Effects of a novel antitumor depsipeptide, FR901228, on human breast cancer cells Breast Cancer Res Treat 1998 51 1 29 38 9877027 10.1023/a:1006091014092

193. Kitazono M Robey R Zhan Z Low concentrations of the histone deacetylase inhibitor, depsipeptide (FR901228), increase expression of the Na/I symporter and iodine accumulation in poorly differentiated thyroid carcinoma cells J Clin Endocrinol Metab 2001 86 7 3430 3435 11443220 10.1210/jcem.86.7.7621

194. Aron JL Parthun MR Marcucci G Depsipeptide (FR901228) induces histone acetylation and inhibition of histone deacetylase in chronic lymphocytic leukemia cells concurrent with activation of caspase 8-mediated apoptosis and down-regulation of c-FLIP protein Blood 2003 102 2 652 658 12649137 10.1182/blood-2002-12-3794

195. Weiser TS Guo ZS Ohnmacht GA Sequential 5-aza-2 deoxycytidine-depsipeptide FR901228 treatment induces apoptosis preferentially in cancer cells and facilitates their recognition by cytolytic T lymphocytes specific for NY-ESO-1 J Immunother 2001 24 2 151 161 11265773 10.1097/00002371-200103000-00010

196. Steiner FA Hong JA Fischette MR Sequential 5-Aza 2-deoxycytidine/depsipeptide FK228 treatment induces tissue factor pathway inhibitor 2 (TFPI-2) expression in cancer cells Oncogene 2005 24 14 2386 2397 15735751 10.1038/sj.onc.1208376

197. Klisovic MI Maghraby EA Parthun MR Depsipeptide (FR 901228) promotes histone acetylation, gene transcription, apoptosis and its activity is enhanced by DNA methyltransferase inhibitors in AML1/ETO-positive leukemic cells Leukemia 2003 17 2 350 358 12592335 10.1038/sj.leu.2402776

198. Sasakawa Y Naoe Y Sogo N Marker genes to predict sensitivityto FK228, a histone deacetylase inhibitor Biochem Pharmacol 2005 69 4 603 616 15670579 10.1016/j.bcp.2004.11.008

199. Piekarz RL Frye R Turner M Phase II multi-institutional trial of the histone deacetylase inhibitor romidepsin as monotherapy for patients with cutaneous T-cell lymphoma J Clin Oncol 2009 27 32 5410 5417 19826128 10.1200/JCO.2008.21.6150 PMC2773225

200. Woo S Gardner ER Chen X Population pharmacokinetics of romidepsin in patients with cutaneous T-cell lymphoma and relapsed peripheral T-cell lymphoma Clin Cancer Res 2009 15 4 1496 1503 19228751 10.1158/1078-0432.CCR-08-1215 PMC2707030

201. Marshall JL Rizvi N Kauh J A Phase I trial of depsipeptide (FR901228) in patients with advanced cancer J Exp Ther Oncol 2002 2 6 325 332 12440223 10.1046/j.1359-4117.2002.01039.x

202. Sandor V Bakkes S Robey RW Phase I trial of the histone deacetylase inhibitor, depsipeptide (FR901228, NSC 630176), in patients with refractory neoplasms Clin Cancer Res 2002 8 3 718 728 11895901

203. Byrd JC Marcucci G Parthun MR A phase 1 and pharmacodynamic study of depsipeptide (FK228) in chronic lymphocytic leukemia and acute myeloid leukemia Blood 2005 105 3 959 967 15466934 10.1182/blood-2004-05-1693

204. Fouladi M Furman WL Chin T Phase I study of depsipeptide in pediatric patients with refractory solid tumors: a Children’s Oncology Group report J Clin Oncol 2006 24 22 3678 3685 16877737 10.1200/JCO.2006.06.4964

205. Klimek VM Fircanis S Maslak P Tolerability, pharmacodynamics, and pharmacokinetics studies of depsipeptide (romidepsin) in patients with acute myelogenous leukemia or advanced myelodysplastic syndromes Clin Cancer Res 2008 14 3 826 832 18245545 10.1158/1078-0432.CCR-07-0318

206. Stadler WM Margolin K Ferber S A Phase II study of depsipeptide in refractory metastatic renal cell cancer Clin Genitourin Cancer 2006 5 1 57 60 16859580 10.3816/CGC.2006.n.018

207. Shah MH Binkley P Chan K Cardiotoxicity of histone deacetylase inhibitor depsipeptide in patients with metastatic neuroendocrine tumors Clin Cancer Res 2006 12 13 3997 4003 16818698 10.1158/1078-0432.CCR-05-2689

208. Schrump DS Fischette MR Nguyen DM Clinical and molecular responses in lung cancer patients receiving Romidepsin Clin Cancer Res 2008 14 1 188 198 18172270 10.1158/1078-0432.CCR-07-0135

209. Sosman JA Aronson FR Sznol M Concurrent phase I trials of intravenous interleukin 6 in solid tumor patients: reversible dose-limiting neurological toxicity Clin Cancer Res 1997 3 1 39 46 9815535

210. Maloney A Workman P HSP90 as a new therapeutic target for cancer therapy: the story unfolds Expert Opin Biol Ther 2002 2 1 3 24 11772336 10.1517/14712598.2.1.3

211. Bantscheff M Hopf C Savitski MM Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes. Nat Biotechnol. 2011 In press. 10.1038/nbt.1759 21258344