Development of histone deacetylase inhibitors for cancer treatment

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1 Review Development of histone deacetylase inhibitors for cancer treatment Douglas Marchion and Pamela Münster CONTENTS HDAC enzymes & cancer HDAC inhibitors Biological effects of HDAC inhibitors Clinical experience FDA-approved HDAC inhibitors Reported clinical studies involving select HDAC inhibitors Other HDAC inhibitors in clinical development Rationally designed combination of HDAC inhibitors & other therapies Potential targets & future directions Expert commentary Five-year view References Affiliations Author for correspondence H Lee Moffitt Cancer Center, Experimental Therapeutics and Breast Medical Oncology Programs, Department of Interdisciplinary Oncology, Magnolia Dr., Tampa, FL 33612, USA Tel.: Fax: munstepn@moffitt.usf.edu KEYWORDS: depsipeptide, HDAC, HDAC inhibitor, LBH589, MGCD0103, MS-275, PXD101, sodium butyrate, valproic acid, vorinostat, zolinza Histone deacetylase (HDAC) inhibitors are an exciting new addition to the arsenal of cancer therapeutics. The inhibition of HDAC enzymes by HDAC inhibitors shifts the balance between the deacetylation activity of HDAC enzymes and the acetylation activity of histone acetyltransferases, resulting in hyperacetylation of core histones. Exposure of cancer cells to HDAC inhibitors has been associated with a multitude of molecular and biological effects, ranging from transcriptional control, chromatin plasticity, protein DNA interaction to cellular differentiation, growth arrest and apoptosis. In addition to the antitumor effects seen with HDAC inhibitors alone, these compounds may also potentiate cytotoxic agents or synergize with other targeted anticancer agents. The exact mechanism by which HDAC inhibitors cause cell death is still unclear and the specific roles of individual HDAC enzymes as therapeutic targets has not been established. However, emerging evidence suggests that the effects of HDAC inhibitors on tumor cells may not only depend on the specificity and selectivity of the HDAC inhibitor, but also on the expression patterns of HDAC enzymes in the tumor tissue. In this review, the recent advances in the understanding and clinical development of HDAC inhibitors, as well as their current role in cancer therapy, will be discussed. Expert Rev. Anticancer Ther. 7(4), (2007) Aberrant gene transcription is a hallmark of cancer and anomalies in the regulation of transcription may lead to cellular transformation [1,2]. Transcriptional regulation is accomplished by a complex interaction of several mechanisms including histone tail modifications [3,4], DNA methylation [5 7] and the activity of regulatory proteins that interact with both histones and DNA [8]. The first level of control over gene activity is the addition or removal of chemical groups to histone tail moieties, such as acetyl groups [1]. The acetylation and deacetylation of histone tails is accomplished by the competing activity of histone acetyl transferases (HAT) and histone deacetylases (HDACs), respectively [4]. Generally speaking, histone acetylation by HATs masks the positive charges of lysine residues on histone tails, thereby reducing the tight interaction between the histones and the negatively charged DNA. In contrast, HDACs remove the acetyl groups from the lysine residues of histone tails, reinstating the positive charge and subsequent interaction with the negatively charged DNA [3,4]. In this respect, the acetylation status of histones may determine the access of transcription factors to targets on the DNA promoters. Deregulation in the expression or activity of HATs and notably HDACs may lead to alterations in gene expression profiles and has been linked to the development of cancers [9,10]. HDAC enzymes & cancer Several recent studies have implicated individual HDAC enzymes in the development of cancer and as potential therapeutic targets. HDACs can be divided into at least three different classes, I III. Each class contains several structurally and functionally variable HDACs (TABLE 1) [11,12]. Class I HDAC enzymes include HDAC 1 3 and 8 and are typically localized to the nucleus. Class II HDAC enzymes include HDAC 4 7, 9 and 10, and may exist in both the nucleus and the cytoplasm. In / Future Drugs Ltd ISSN

2 Marchion & Münster addition, HDAC11, which shows similarities to both class I and II HDACs, is distinctive enough to be categorized by some into a class IV [13]. Class III HDAC enzymes comprise nicotinamide adenosine dinucleotide (NAD)-dependent deacetylases termed sirtuins. The clinical relevance of sirtuins and their corresponding inhibitors remains unknown. A more in-depth understanding of their preclinical roles may be derived from recent reviews by the groups of Longo, Denu and Buck [14 16]. Currently, there are at least 17 known HDAC enzyme family members. Given their global effect on histone modulation, it is not surprising that the HDAC enzymes are involved in many biological functions, ranging from transcriptional control, chromatin plasticity and protein DNA interaction to cellular differentiation (FIGURE 1). An extensive body of literature proposes many relevant downstream effects involved in cell biology and, in particular, in the development and proliferation of tumors, and has been reviewed extensively [17 23]. A link between HDAC1 and HDAC3 expression with specific tumor features has been evaluated in breast cancer tumor samples by several investigators. HDAC1 mrna levels by real-time (RT)-PCR, as well as HDAC1 and HDAC3 protein expression by immunohistochemistry, were statistically associated with smaller, estrogen- and progesterone-positive tumors, while HDAC1 mrna, but not HDAC1 protein expression was linked to node-negative tumors [24,25]. In addition, increased HDAC1 mrna and protein expression were linked to better outcome, while HDAC3 expression did not appear to impact overall or disease-free survival [24,25]. However, the reports were equivocal as to whether HDAC1 mrna was an independent prognostic indicator or linked to other features [24]. HDAC1 protein overexpression has also been reported in gastric cancer; however, its role as a predictor has not been assessed [26]. In patients with esophageal cancer, low HDAC1 expression was associated with a more invasive phenotype [27]. Table 1. Histone deacetylase enzymes. Class I Class II Class III Class IV Members HDAC1 HDAC4 (Sirtuins) HDAC11 HDAC2 HDAC5 Sir2 HDAC3 HDAC6 Sirtuin homologs HDAC8 HDAC7 HDAC9 HDAC10 Function Related to Rpd3 protein Similarity to the yeast Hda1 protein Related to Rpd3 protein Location Ubiquitously expressed HDAC: Histone deacetylase. Tissue-restricted patterns of expression Ubiquitously expressed A retrospective analysis of banked tumor tissues suggested that increased expression of HDAC6 was associated with improved disease-free and overall survival in patients with hormone-sensitive breast tumors treated with tamoxifen [28]. Furthermore, a direct interaction of HDAC1 and the estrogen receptor (ER) may be involved in the response to antiestrogen therapy [29]. Expression of HDAC5 and HDAC10 were associated with poor prognosis in lung cancer [30]. HDAC2 overexpression has been found in precancerous lesions and colon cancers associated with the adenomatosis polyposis coli (APC) tumor-suppressor gene, suggesting that HDAC inhibitors may not only have an important role in the treatment, but also in the prevention of colon cancer in family members affected by an APC gene mutation [31]. Specific under- and overexpression patterns of HDAC enzymes have been shown in acute myeloblastic leukemia (AML). These patterns have been further correlated with response to therapy; however, these studies will require further validation in clinical trials [32] and many more studies on the prognostic role of select HDAC enzymes in cancer are currently ongoing. These studies suggest that individual HDAC enzymes may play a role in the development and progression of cancer. However, while these studies point toward individual HDAC enzymes as prognostic factors and potential selective therapeutic targets, most were small and retrospective, and require further validation. Furthermore, little is known regarding the relevance of specific HDAC-expression patterns as therapeutic targets. HDAC inhibitors The association of HDAC enzymes and carcinogenesis has increased interest in the use of HDAC inhibitors as antitumor agents [33 36]. HDAC inhibitors have been shown to induce cellcycle arrest, growth inhibition, chromatin decondensation, differentiation and apoptosis in several cancer cell types [18,37 41]. An example of mammary differentiation in MCF-7 cells is shown in FIGURE 2. HDAC inhibitors are classified by structure and include the short-chain fatty acids, valproic acid (VPA) and sodium butyrate (NaB); the cyclic tetrapeptides, depsipeptide; the hydroxamic acids, suberoylanilide hydroxamic acid (SAHA), trichostatin A (TSA), LAQ824, LBH529 and PXD101; and the benzamides, MS-275 (TABLE 2) [18]. Although these HDAC inhibitors differ in structure, potency and possibly HDAC enzyme selectivity, they target primarily class I and II HDAC enzymes and do not affect the activity of the class III sirtuins (TABLE 1) [11,12]. The downstream effects of HDAC inhibition, which ultimately lead to growth inhibition and apoptosis in different tumor types, are currently under review and it is becoming increasingly clear that these effects may depend upon the HDAC inhibitor as well as the cell type [23,42,43]. HDAC inhibitors may affect the epigenetic regulation of chromatin by shifting the balance between HDAC and HAT enzyme activity, resulting in the hyperacetylation of histones. While the mechanism of action of 584 Expert Rev. Anticancer Ther. 7(4), (2007)

3 Histone deacetylase inhibitors and cancer HDAC inhibitors should favor chromatin decondensation and a global increase in gene transcription, only a small percentage The HDACs and of genes are affected [44,45] and it appears The HDACs and transcriptional control that as many genes are transcriptionally transcriptional control Histone code HDAC:protein downregulated as are transcriptionally HDAC protein Regulation of transcription factor access interactions interactions Histone code of HDAC inhibition upregulated [9,23,37,46]. Transcriptional consequences Regulation of transcription factor access Multiprotein While the induction of gene expression Kinetics of histone acetylation/deacetylation Transcriptional consequences of HDAC inhibition Multiprotein HDAC corepressor Nonhistone (de)acetylation by HDAC inhibitors seems to occur by a Kinetics of histone acetylation/deacetylation HDAC -corepressor complexes Long-range repression histone (de)acetylation Noncommon mechanism, the HDAC inhibicomplexes HDAC complexes Long-range repression tor-induced repression of gene expression Heterochromatin HDAC complexes may occur through multiple pathways Heterochromatin employing several accessory proteins. For Post-translational Biological Biological consequences consequences example, a commonality between several modifications ofofhdac HDACinhibition inhibition HDAC inhibitors is the induced exprescell differentiation Phosphorylation Cell differentiation sion of the cell-cycle regulator, p21 [47,48]. Cell-cycle arrest Dephosphorylation Cell cycle arrest Apoptosis SUMOylation Treatment of cancer cells with the HDAC Apoptosis Cytoskeletal alterations inhibitor SAHA resulted in the association Cytoskeletal alterations Angiogenesis Angiogenesis of the p21 promoter with acetylated histones and a decrease in association with HDAC1-containing repressor complexes Figure 1. Biological functions of HDAC enzymes. [47,49,50]. Similarly, TSA treatment of HDAC: Histone deacetylase; SUMO: Small ubiquitin modifier. MCF-7 cells resulted in a reduced association of the p100 resulted in increased acetylation of p52 and subsequent Sin3a HDAC repressor complex from the luteinizing hormone sequestration and inhibition of p65 binding to the cyclin receptor promoter and, when coupled with a demethylation D1 promoter. agent, resulted in gene expression [51]. By contrast, the reduced expression of ERα in MCF-7 Biological effects of HDAC inhibitors breast cancer cells in response to the HDAC inhibitor TSA Cell growth arrest, differentiation & apoptosis may involve a member of the sirtuin family of deacetylases in HDAC inhibitors have antitumor activity against several cancer combination with local promoter methylation [52]. However, cell types [56 69]. A common sequelae of HDAC inhibitor treatthe repression of cb1 expression in HT-29 colon cancer cells ment is upregulation of p21 and downregulation of cyclin D, after treatment with the HDAC inhibitor NaB was dependent resulting in reduced cancer cell proliferation and cell-cycle arrest upon the induction of p21 [53,54]. In a more complex scenario, [47,70,71]. In breast cancer cell lines, growth arrest was associated TSA was shown to downregulate the expression of cyclin D1 with morphological changes characteristic of differentiation, in mouse JB6 cells through the activation of p100 processing including the production of milk fat proteins and a decrease in of p52 [55]. These data suggest that TSA-induced activation of the nuclear cytoplasmic ratio (FIGURE 2) [40,72 74]. Furthermore, A B C Figure 2. Mammary differentiation in breast cancer (MCF)-7 cells. Suberoylanilide hydroxamic acid (SAHA)-induced differentiation of MCF-7 cells required continuous presence of drug. Cells were grown on Lab Tek cover culture chambers in the presence of 5 µm SAHA for 48 h. The SAHA-containing media was then removed and the cells were cultured for an additional 48 h in complete media without drug. Cells were evaluated for the production of milk fat globulin (green) and milk fat globule membrane protein (red). Nuclear DNA was stained with bisbenzimide. (A) 0 h, (B) 48 h of SAHA treatment, (C) 48 h after the removal of SAHA. Images were acquired by confocal microscopy

4 Marchion & Münster HDAC inhibitor treatment may also induce apoptosis through both the intrinsic and extrinsic pathways. HDAC inhibitors have been shown to upregulate the expression of the proapoptotic proteins Bax, Bak, Bim, Bad, Noxa, Puma, Bid and Apaf1 [75 84]; facilitate the translocation of Bax to the mitochondria [75,85]; and decrease the expression of the antiapoptotic proteins Bcl-2, Bcl-xl, Mcl-1 and survivin [86 89]. In addition, HDAC inhibitors have been shown to upregulate the expression of Fas and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors [90,91]. with the downregulation of structural maintenance of chromatin (SMC) proteins 1 5, SMC-associated proteins, HCAP-H and HCAP-G, DNA methyltransferase (DNMT)1 and heterochromatin protein (HP)1 (FIGURE 3) [39]. SMC proteins [92,93], DNMT1 [5 7] and HP1 [8,94,95] play specific roles in the condensation of chromatin and are required for the proper formation of heterochromatin. These findings suggest that not only the HDAC inhibitor-induced hyperacetylation of histones but rather the downstream effects of HDAC inhibition leading to changes in gene expression are required for the onset of chromatin decondensation. Chromatin structure Angiogenesis HDAC inhibitors compete for the active site within HDAC enzymes leading to the accumulation of acetyl groups on histone tails [18]. This negates the charge interaction between histones and the DNA, resulting in a loosened chromatin structure [3,4]. While histone acetylation has been thought to be solely indicative of chromatin decondensation, more recent data suggest a correlation between HDAC inhibitor-induced modulation of gene expression and HDAC inhibitor-induced chromatin decondensation [39]. While hyperacetylation of core histone is a rapid process induced by HDAC inhibitors, structural changes to the chromatin required prolonged exposure times to the HDAC inhibitors and correlated An important aspect in tumor progression is the formation of blood vessels or angiogenesis. Angiogenesis is an inducible response, which may be triggered by hypoxia and serum starvation and is characterized by the expression of hypoxia-inducible factor (HIF)1α and vascular endothelial growth factor (VEGF) [96,97]. The HDAC inhibitors TSA [98 100], SAHA [99], MS-275 [100], FK228 [101], phenylbutyrate [102], LBH589 [103] and LAQ824 [104] have been shown to inhibit angiogenesis. The mechanism by which this occurs seems to be through the ability of HDAC inhibitors to modulate gene expression, resulting in the repression of the proangiogenic factors such as HIF1α, VEGF, VEGF receptor and endothelial nitric oxide synthase and the cytokines IL-2 and IL-8 [98,100,101, ] and the induction of antiangiogenic factors, such as p53 and von Hippel Lindau (VHL) [98]. The clinical applicability of these findings remains to be tested. A Immunity/inflammation Relative mrna expression There is significant evidence suggesting that HDAC inhibitors may modulate inflammation and innate immune system responses [108,109]. However, many reports are in conflict as to whether HDAC 0h inhibitors may suppress or enhance these h responses. In animal models, HDAC B 100 inhibitors, such as TSA and SAHA, may reduce graft-versus-host disease in experi80 mental bone marrow transplantation and 60 reduce symptoms of adjuvant-induced rheumatoid arthritis and multiple sclero40 sis [ ], presumably through a sup20 pression of the proinflammatory 0 cytokines, including IL-12, TNFα, intersmc1 SMC2 SMC3 SMC4 SMC5 HCAP-HHCAP-G DNMT1 HP1 feron (IFN)γ and IL-23 [ ]. In addition, the HDAC inhibitor LAQ824 was Figure 3. Histone deacetylase inhibitor-induced chromatin decondensation. (A) Electron micrographs showing chromatin dispersion in MCF-7 cells after a 48-h treatment with valproic acid 2 mm reported to reduce the migration of antigen-presenting cells as well as the activacompared with treatment with saline (vehicle; x32,000). (B) Microarray analysis showing a decrease in heterochromatin maintenance protein mrna expression in MCF-7 cells after a 48-h treatment with VPA tion and chemotaxis of the T-helper 2 mm. Reduced expression of SMC protein 1-5, SMC-associated proteins, HCAP-H and HCAP-G, DNMT-1 (Th)1 subset of CD4+ T cells [113]. By and HP-1 correlate with the onset of chromatin dispersion as shown in (A). contrast, other groups have reported that DNMT: DNA methyltransferase; HP: Heterochromatin protein; SMC: Structural maintenance of chromatin. it is the activity of HDAC enzymes that 586 Expert Rev. Anticancer Ther. 7(4), (2007)

5 Histone deacetylase inhibitors and cancer suppresses the expression of inflammatory genes [117] and HDAC inhibitors may actually increase the cellular response to the proinflammatory cytokine IL-6 [118]. However, it was suggested that these apparent contradictions may be due to the timing of HDAC inhibitor treatment [116]. TSA and SAHA were shown to enhance lipopolysaccharide (LPS)-induced IL-6 secretion and inflammation in mouse and rat microglial cells when given after LPS stimulation; however, anti-inflammatory effects were noted when given prior to LPS stimulation [116]. These findings are currently being evaluated in clinical studies. Nonhistone targets It has been suggested that the primary target of HATs and HDACs may not be histones but nonhistone proteins [34] and that acetylation may rival phosphorylation in substrate availability and substrate diversity [119,120]. Acetylation may affect the activity and stability of proteins, as well as the ability of proteins to interact with DNA and other proteins [120]. An example of this is signal transducer and activator of transcription (STAT)3 where acetylation may direct STAT3 dimerization and cellular localization [121,122]. Acetylation may also affect the activity of E2F, p53, STAT1, c-jun and many others [ ]. The sheer number of transcription factors known to be acetylated suggests that the acetylation of these nonhistone proteins may have as much regulatory effect on transcription as the acetylation of histone proteins. A more comprehensive description of nonhistone targets of acetylation has been reviewed recently [34,120,130]. Combinations of HDAC inhibitors & chemotherapy Although HDAC inhibitors have shown antitumor activity as single agents, promising interactions between HDAC inhibitors and cytotoxic agents have been described in select cancer cell types for platinum salts [131,132], taxanes [133,134], topoisomerase inhibitors [38,39,56,67, ] and nucleoside analogs [133,139,140]. HDAC inhibitors may further enhance the activity of proteosome inhibitors [ ], retinoic acid [ ], methylation inhibitors [ ] or radiation therapy [ ]. However, the mechanisms by which HDAC inhibitors potentiate these treatment modalities in specific diseases are largely unknown with the exception of a subset of acute promyelocytic leukemia (APL). Promyelocytic leukemia (PML) is characterized by the expression of the PML retinoic acid receptor (RAR)α and the PML zinc finger (PLZF) RARα fusion proteins [ ]. Cells expressing the PML RARα fusion protein are sensitive to treatment with retinoic acid, while cells expressing the PLZF RARα fusion protein are not sensitive to retinoic acid [161]. In cells expressing the PLZF RARα fusion protein, it was suggested that HDAC1 was recruited to repress the retinoic acid response genes [162,163]. HDAC inhibitors were found to restore retinoic acid sensitivity by reversing the transcriptional repression of HDAC1 [160,164,165]. In this situation, it was the direct activity of HDAC inhibitors that proved beneficial to the enhancement of a therapeutic agent. Table 2. Histone deacetylase inhibitors. Class Drugs Clinical development (manufacturer) Short-chain fatty acids Cyclic tetrapeptides Hydroxamic acids Butyrate/phenyl butyrate Valproic acid Phase I Phase I/II (approved for seizures; Abbott Laboratories) Depsipeptide (FK228) Phase III (Gloucester Pharmaceuticals, Inc.) CHAPs TSA Vorinostat (Zolinza TM, SAHA) LBH589/LAQ824 PXD101 Phase III (approved; Merck) Phase I/II (Novartis Corp.) Phase I/II (CuraGen Corp., TopoTarget USA, Inc.) Amides MS-275 Phase III (Bayer Schering Pharma AG) MGCD0103 Phase II (Methylgene, Inc.) CI-994 Phase II (Pfizer, Inc.) CHAP: Cyclic hydroxamic acid-containing peptide; SAHA: Suberoylanilide hydroxamic acid; TSA: Trichostatin A. In addition, the indirect effects of HDAC inhibitors to induce chromatin decondensation may be successfully exploited by combining them with DNA-targeting agents. The HDAC inhibitor-induced chromatin decondensation may facilitate the access of DNA-targeting agents to their DNA substrate, thereby enhancing the ensuing DNA damage. The proposed mechanism and the kinetics of the HDAC inhibitorinduced chromatin decondensation suggest that the sequence of drug administration may be relevant. This was studied by several investigators. Johnson and coworkers reported an antagonistic effect when the HDAC inhibitor TSA was combined with the topoisomerase II inhibitor etoposide in leukemia cells [137], whereas the groups of Tsai and Kurz reported increased sensitivity [56,67]. These diametrically opposing reports may be explained by differences in the sequence of drug administration used in these studies. In the first study, Johnson and coworkers pre-exposed leukemia cells to the HDAC inhibitor for 0.5 h prior to the topoisomerase inhibitor, while Kurz and coworkers used h HDAC inhibitor pre-exposures. The relevance of drug sequencing and timing was further reported by Marchion and coworkers [38,135]. Prolonged preexposure (24 48 h) to an HDAC inhibitor sensitized breast cancer cell lines to the cytotoxic effects of topoisomerase inhibitors. A potentiation of the topoisomerase inhibitor by a HDAC inhibitor was not observed with shorter pre-exposure times (4 12 h) or with the administration of the topoisomerase 587

6 Marchion & Münster inhibitor before the HDAC inhibitor [38,39]. A similar observation was reported by Kim and coworkers using the HDAC inhibitors, SAHA (vorinostat) and TSA, in combination with the topoisomerase inhibitor etoposide [136]. A cooperative activity between HDAC inhibitors and topoisomerase I inhibitors has been noted by a number of investigators; however, a review of the reported data suggests that the benefits of adding a HDAC inhibitor to a topoisomerase I inhibitor may depend not only on the schedule of drug administration but also on specific tumor characteristics and the type of topoisomerase I inhibitor used [38, ]. Clinical experience HDAC inhibitors have been evaluated for their clinical feasibilities in different settings. Given the wide-ranging biological functions of HDACs, their inhibitors may have roles in different stages of cancer development, ranging from cancer prevention to treatment of metastatic disease. However, as with many other novel compounds, the initial clinical testing of the activity of HDAC inhibitors typically involves studies in patients who have advanced solid tumors or hematological malignancies; many of these patients will have treatment-refractory tumors associated with extensive prior therapy exposure. Furthermore, the absence of a clearly defined target or a predictive marker does not allow the enrichment of a target population of patients who are more likely to respond to therapy, with the exception of patients with cutaneous T-cell lymphoma (CTCL). Furthermore, while many of the preclinical studies postulate a potential class or compound specificity of HDAC inhibitors in their actions, its relevance has yet to be determined in the clinical setting. The following sections will describe the current state of the clinical development of several HDAC inhibitors, with the caveat that these reports will change rapidly as the clinical trials with HDAC inhibitors mature. FDA-approved HDAC inhibitors At the time of this review only vorinostat (Zolinza, Merck Inc.) has been approved as a HDAC inhibitor for clinical use. Vorinostat (400 mg, administered orally daily) has received approval for the treatment of refractory CTCL. The main adverse effects associated with vorinostat include fatigue, nausea, dehydration, diarrhea and thrombocytopenia. Other potentially linked adverse effects include thromboembolic events. However, as these events are not uncommon in cancer patients, their relevance may be difficult to elucidate. In Phase II studies in patients with CTLC or peripheral T-cell lymphoma (PTCL), vorinostat resulted in an objective antitumor response in eight of 33 patients and 14 patients had an improvement in their symptoms [166]. Currently, vorinostat is being further evaluated in this disease and in other hematological malignancies. Reported clinical studies involving select HDAC inhibitors Vorinostat (Zolinza) In the first clinical trial, vorinostat (previously studied under the name, SAHA) was initially administered intravenously for 3 days every 3 weeks and dose escalated to 5 days per week for 21 days. Out of 37 patients, four had an objective response; two of these patients had lymphomas and two had bladder cancer [167]. While the responses were encouraging, the number of intravenous injections required in this regimen prompted a change in formulation. Furthermore, emerging in vitro and in vivo studies suggested a greater benefit of more continuous rather than intermittent exposure to this HDAC inhibitor [40,168]. A subsequent Phase I trial with an oral formulation of vorinostat in 73 patients showed responses in six patients, occurring at doses of 400 mg twice daily or 600 mg once daily. Tumor histologies of responding patients included lymphoma, laryngeal cancer, papillary thyroid cancer and mesotheliomas [169]. In particular, the number of responses in patients with lymphomas seen in these two Phase I trials suggested a potential role of vorinostat in this disease [170]. Acetylation of histones induced by vorinostat was seen in peripheral blood mononuclear cells as well as in tumor cells. The observed histone acetylation persisted beyond the pharmacological half-life of the drug [167]. Based on these findings, there are at least 37 ongoing clinical trials for solid tumors as well as hematological malignancies including leukemias and lymphomas, head and neck, prostate, breast, kidney, thyroid and lung cancer, as well sarcomas, melanomas and primary brain cancer. Vorinostat is being evaluated alone and in combination with targeted therapies or chemotherapeutic agents [301]. Depsipeptide The clinical development of depsipeptide included Phase I and II trials in solid tumor malignancies as well as a Phase II trial in patients with CTCL. In the initial Phase I trial evaluating depsipeptide as an intravenous infusion on days 1 and 5 every 21 days, this compound was associated with nausea, vomiting, fatigue, thrombocytopenia and cardiac arrhythmias manifesting as changes in the electrocardiogram (ECG), as well as a case of atrial fibrillation [171]. Histone acetylation was seen in peripheral blood mononuclear cells [171]. Of 37 patients, one had an objective response [171]. A second Phase I trial performed in the USA evaluated a weekly administration (3 out of 4 weeks) of depsipeptide in 33 patients with advanced cancer. Similar to the findings from the Canadian study [171], ECG changes were also reported in this trial [172]. A Phase II trial in patients with CTCL and PTCL demonstrated further efficacy in this disease. Given as a weekly infusion for 3 out of 4 weeks, antitumor activity was noted with depsipeptide in 10 of 27 patients with PTCL [173]. As seen in the Phase I trials, the adverse effects associated with this drug included fatigue, nausea and vomiting, as well as myelosuppression. This trial showed no adverse effects on cardiac ejection fractions; however, there were further reports of conduction abnormalities, in particular prolongation of the QTc-interval [173]. In solid tumors, a Phase II trial in 29 patients with refractory renal cell cancer showed an objective response rate in 7% of the patients [174] and minimal activity in patients with neuroendocrine tumors [175]. Both of these trials were associated with QT prolongation, tachycardia and atrial fibrillation. Adverse cardiac effects were not only seen in the adult population but also in children enrolled 588 Expert Rev. Anticancer Ther. 7(4), (2007)

7 Histone deacetylase inhibitors and cancer in a trial for pediatric tumors [176]. While this drug may have activity in hematological malignancies, the clinical relevance of the adverse cardiac effects reported in several trials may have to be carefully considered for its relationship to dose, formulation or particular patient population. Currently, depsipeptide is being evaluated in more than ten studies, either alone or in combination [301]. Valproic acid VPA is currently marketed as an antiseizure drug and has been available for over 30 years. Over the last few years, VPA has also been used as a mood stabilizer and to treat migraine headaches. Therapeutic doses typically range from 15 to 60 mg/kg/day resulting in plasma concentrations of µg/ml ( mm). It has recently being found to act as a HDAC inhibitor [ ]. Concentrations necessary for histone acetylation range from 0.25 to 5 mm. While VPA may not be a potent HDAC inhibitor, concentrations required for in vitro efficacy are achievable in patients. VPA has been evaluated as a HDAC inhibitor in patients with leukemia either as a single agent or in combination with all-trans retinoic acid (ATRA) in two clinical trials. While VPA was well tolerated, the response rate for AML was less than 10% [146,148]. By contrast, when VPA was combined with the demethylating agent 5-aza-2 deoxycitabine [182] in 54 patients with refractory leukemia, the response rate was 22%, with a significant number of patients achieving complete remission [183]. The maximally tolerated dose for VPA was 50 mg/kg/day administered daily for 10 days. The main toxicities were transient confusion and somnolence. Histone acetylation was evaluated in peripheral blood mononuclear cells and tumor cells. While VPA-induced histone acetylation was noted in only a few patients treated with the lower dose of VPA (20 and 35 mg/kg/day), histone acetylation was more commonly seen in patients treated with higher doses of VPA (50 mg/kg/day). However, this trial did not suggest a correlation between histone acetylation and response [183]. LBH589 & LAQ824 These two HDAC inhibitors of the hydroxamic class were found to be highly potent and showed both in vitro and in vivo antitumor activity [ ]. A Phase I trial involving 15 patients with acute leukemia and myelodysplastic syndrome (MDS) evaluated an intravenous infusion of LBH589. While this trial did not report objective responses, toxicities were similar to those reported with other HDAC inhibitors with regards to nausea, vomiting, fatigue and thrombocytopenia. In addition, the intravenous administration of this drug was associated with QTc prolongations. Further studies with an oral formulation of LBH589, either alone or in combination, are currently underway. Other HDAC inhibitors in clinical development MS-275 MS-275 was initially studied as an intravenous formulation; however, a recently reported PhaseI trial in patients with advanced solid tumor malignancies or lymphomas suggested that an oral formulation afforded some clinical benefits. Toxicities were similar to those seen with other HDAC inhibitors, however there were no reported cardiac toxicities [187]. Unlike other HDAC inhibitors, the half-life of this compound was found to be fairly long (39 80 h), rendering daily dosing too toxic. While a once-a-fortnight dosing at 10mg/m 2 was feasible, histone acetylation could not be maintained with this schedule. A weekly dosing of this compound is currently being evaluated. MGCD0103 MGCD0103, a class I-specific HDAC inhibitor, has passed Phase I trials and is currently in Phase II trials either alone or in combination with the demethylating agent, 2 -deoxy-5-azacytidine (Vidaza ), for AML and MDS. In addition, Phase II monotherapy trials have been initiated for refractory Hodgkin s lymphoma and B-cell lymphoma, as well as trials evaluating MGCD0103 in combination with gemcitabine. The outcomes of these trials have not yet been published [301]. Sodium butyrate Sodium butyrate, a HDAC inhibitor of the fatty acid group, was studied in an oral formulation. Toxicities were similar to other HDAC inhibitors, including fatigue, nausea and vomiting. However, no objective responses were seen with this compound [188]. No trials are currently listed evaluating this compound for patients with cancer [301]. PXD101 PXD101 is a novel hydroxamate-type HDAC inhibitor that exhibits in vitro cytotoxicity at low micromolar concentrations [189]. Based on preclinical data suggesting single-agent efficacy of this agent in ovarian cancer and a synergistic interaction with carboplatin or a taxane [190], several clinical trials were initiated and are currently ongoing. Areas of interest in these trials include the use of PXD101 in patients with CTCL, non-hodgkin s lymphoma, AML and MDS either alone or in combination with 2 -deoxy-5-azacytidine, bortezomib, isotretinoin or 17-N-allylamino-17-demethoxygeldanamycin. Clinical trials for patients with solid tumors are focused on ovarian and liver cancer, mesotheliomas and sarcomas [301]. Rationally designed combination of HDAC inhibitors & other therapies Several investigators have focused on the preclinical and clinical development of HDAC inhibitors in combination with other targeted therapies or cytotoxic agents. Our group has mainly focused on HDAC inhibitors in combination with DNA-targeting agents. In vitro and in vivo studies suggested that pre-exposure of cells to an HDAC inhibitor resulted in chromatin decondensation, thus facilitating the interaction between the DNA-targeting agents and their DNA substrate and topoisomerase II target recruitment. This was associated with an increase in the topoisomerase inhibitor-induced DNA damage and cell death. While these findings were not limited to a certain class of HDAC inhibitor, 589

8 Marchion & Münster the preclinical in vivo efficacy and the well-established clinical toxicity profile had rendered VPA an ideal candidate for a combination study. In particular, the absence of any known cardiac toxicity (as initially suspected with depsipeptide) was crucial for the combination with an anthracycline. Based on extensive preclinical studies suggesting a synergistic interaction, the efficacy of a sequence-specific administration of VPA followed by the topoisomerase II inhibitor epirubicin was evaluated. A total of 44 patients with advanced solid tumor malignancies were treated with increasing doses of VPA for 3 days and epirubicin on day 3, cycles were repeated every 3 weeks. Beginning at doses typically administered for seizure disorders, the VPA doses were escalated to maximally tolerated doses of 140 mg/kg/day VPA. The dose-limiting toxicities of this combination were transient neurovestibular (somnolence, dizziness, confusion) associated with VPA and neutropenia associated predominantly with epirubicin. An exacerbation of the epirubicin-induced myelosuppression or cardiac toxicity was not observed. As seen with other VPA trials, the neurotoxicities were exacerbated by other neurotropic agents [183]. Despite a median of three prior regimens and prior anthracycline exposure in 25% of the patients, objective responses were seen in 22% patients and 39% of patients had a clinical benefit for at least 12 weeks. This combination is currently being explored in a Phase II trial as primary therapy in women with locally advanced breast cancer [191]. Other studies exploring HDAC inhibitors in combination with DNA-damaging agents include a Phase I/II trial of VPA and the topoisomerase I inhibitor karenitecin in advanced melanoma, a combination of vorinostat and doxorubicin in solid tumors and a combination of VPA and temozolomide in patients with brain metastasis undergoing whole-brain radiation therapy. Potential targets & future directions The treatment of cancer cells with HDAC inhibitors may result in cell-cycle arrest, growth inhibition, differentiation, chromatin decondensation and apoptosis. However, these effects may vary with dose and between HDAC inhibitors, cell type and even cell line. For example, treatment of MCF-7 breast cancer cells with 0.5 µm SAHA resulted in histone acetylation within 1 h of exposure and chromatin decondensation after 48 h of exposure [38]. In contrast, at concentrations five- and tenfold higher, treatment of MCF-7 cells with SAHA resulted in a cell-cycle arrest in G1 and G2/M, respectively [40]. In addition, treatment of MCF-7 cells with VPA resulted in G1 cell-cycle arrest at concentrations above 3 mm; however, cell-cycle arrest in G2 was not achieved even with VPA doses that exceeded 5 mm (MUNSTER, UNPUB. DATA). These observations suggest that the various biological effects observed in response to HDAC inhibitors may be mediated through the inhibition of different HDAC enzymes. Similarly, varying effects of HDAC inhibitors the on cell cycle have been reported with other HDAC inhibitors [138]. Therefore, the questions become: Which HDAC enzyme is important in each of the observed biological consequences of HDAC inhibitors? Does more than one HDAC enzyme, or inhibition thereof, contribute to an individual effect? Are HDAC enzymes redundant in function? The answers to these questions are dependent on the elucidation of the biological function of individual HDAC enzymes. It is well documented that HDAC1 and HDAC2 coexist in several multiprotein, corepressor complexes, such as NuRD, Sin3 and CoREST [ ]. Although this may suggest that these enzymes may have redundancy, several reports indicate that HDAC enzymes have individual functions and are differentially regulated [46,197,198]. HDAC1 has been associated with proliferation in several tumor cell types, as well as differentiation in MCF-7 breast cancer cells and zebrafish retina [74, ], whereas HDAC2 may be associated with cell survival [31,202] but may also be involved in differentiation of endometrial stromal sarcoma cells [203]. HDAC3 may be involved in colon cell maturation [204] and may be required for the activity of HDAC 4, 5 and 7 [205,206]. HDAC4 may be involved in bone development [207] and may play a role in muscle differentiation in conjunction with HDAC5 and 7 [ ]. HDAC6 may play a role in the regulation of the cytoskeleton [ ], whereas HDAC7 may also be involved in maintaining vascular integrity [214], while HDAC8 was reportedly associated with smooth muscle cell differentiation and contractility [215,216]. The biological function of the remaining class I and II HDAC enzymes remains largely unknown. These studies elucidating the biological function and regulation of HDAC enzymes are imperative as they may not only answer the previous questions, but may guide the future development of HDAC inhibitors. Currently, most of the available HDAC inhibitors are nonspecific, targeting multiple class I and II HDAC enzymes [217]. For example, TSA is a pan-histone inhibitor but may have the highest potency toward HDAC1, 3 and 8, while MS-275 may preferentially inhibit HDAC1 [218]. In addition, VPA may have the highest activity against HDAC1 and 2, but also affects HDAC3, 4, 5 and 7 in higher concentrations [179]. These findings suggest that different HDAC inhibitors may be most effective in tumors that express the target enzymes of those inhibitors. Furthermore, it is well known that not all cell types or even cell lines within the same disease type, respond the same to a particular HDAC inhibitor. This would, therefore, suggest that HDAC enzyme expression may dictate sensitivity differentially to various HDAC inhibitors. This idea is supported by Ropero and coworkers study evaluating colorectal cell line sensitivity to different HDAC inhibitors [219]. This study found that cells that express mutated HDAC2 were resistant to histone acetylation and cell-cycle arrest induced by the hydroxamic acid TSA but not by the short-chain fatty acids butyrate or VPA. Expert commentary Several preclinical and clinical studies have indicated the value of HDAC inhibitors as monotherapy and in combination with either more standard chemotherapy or targeted therapy. The 590 Expert Rev. Anticancer Ther. 7(4), (2007)

9 Histone deacetylase inhibitors and cancer first HDAC inhibitor has recently been approved for the treatment of CTCL. Many trials involving a number of different HDAC inhibitors are currently ongoing, either as monotherapy or in combination. However, the mechanism(s) by which HDAC inhibitor treatment leads to these biological sequelae is not yet known and may differ between the HDAC inhibitor used, and may ultimately depend on the differential expression patterns of HDAC enzymes in tumors. Furthermore, the mode of action leading to cell growth arrest and cell death may differ from those resulting in potentiation of other antitumor therapies. As cellular markers that may predict response to HDAC inhibitors alone or in combination with chemotherapy are not yet defined, the selection of patients remains arbitrary and may not include those most likely to benefit from these agents. Further studies are required to determine whether potency and/or class specificity of the HDAC inhibitor are clinically relevant. To achieve the optimal therapeutic potential of these novel and exciting compounds, HDAC inhibitors, it is imperative that future studies focus on the biological roles of select HDAC enzymes and the cellular consequences of the specific inhibition of individual HDAC inhibitors. Five-year view The first HDAC inhibitor has been approved for use in CTCL. Currently, it is not entirely clear why this compound is active against this type of malignancy. Many other HDAC inhibitors are currently being tested in this disease and appear to be active. Further studies will show whether one representative within the same class or from a different class will be superior to the currently approved vorinostat. There is a suggestion of activity of HDAC inhibitors in other malignancies; however, there are currently no markers to foretell response. The roles of individual HDAC enzymes in tumor development or proliferation as well as therapeutic targets will be essential for progression in the field. HDAC inhibitors are currently being evaluated in combination with chemotherapy, antihormonal therapies or therapies targeting specific pathways. The mechanism of synergy may differ from the mechanism of action leading to cell death in CTCL. Different HDAC enzymes may need to be inhibited. Furthermore, while most of the currently used HDAC inhibitors are administered in a more chronic form as single agents, this may need to be re-evaluated when combining HDAC inhibitors with other anticancer agents. Higher doses at shorter intervals may be more applicable when combining these agents with chemotherapy. Key issues Histone deacetylase (HDAC) enzymes have been linked to the development and progression of cancers. HDAC inhibitors have antitumor activity in certain cancer types but may also act as sensitizers to chemotherapy. The sensitivity of cancer cells to HDAC inhibitors may depend on the class of HDAC inhibitor, dose of administration and the cell type. The biological effects of HDAC inhibition, such as cell-cycle arrest and differentiation, may be mediated through different HDAC enzymes. The first HDAC inhibitor (vorinostat) has been approved for the treatment of cutaneous T-cell lymphoma. For the optimal use of HDAC inhibitors as anticancer agents, future experiments must: Define cellular markers that predict response to HDAC inhibitors as monotherapy or as sensitizers to chemotherapy in individual diseases. Determine whether selective versus nonselective HDAC inhibitors are more efficacious. Determine whether high short-term doses or chronic administration of lower doses are optimal. References Papers of special note have been highlighted as: of interest of considerable interest 1 Santos-Rosa H, Caldas C. Chromatin modifier enzymes, the histone code and cancer. Eur. J. 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16 Marchion & Münster 219 Ropero S, Fraga MF, Ballestar E et al. A truncating mutation of HDAC2 in human cancers confers resistance to histone deacetylase inhibition. Nat. Genet. 38(5), Website 301 US National Institutes of Health Clinical Trials Affiliations Douglas Marchion, PhD H Lee Moffitt Cancer Center, Experimental Therapeutics and Breast Medical Oncology Programs, Department of Interdisciplinary Oncology, H Lee Moffitt Cancer Center, Magnolia Dr., Tampa, FL 33612, USA Pamela Münster, MD H Lee Moffitt Cancer Center, Experimental Therapeutics and Breast Medical Oncology Programs, Department of Interdisciplinary Oncology, H Lee Moffitt Cancer Center, Magnolia Dr., Tampa, FL 33612, USA Tel.: Fax: munstepn@moffitt.usf.edu 598 Expert Rev. Anticancer Ther. 7(4), (2007)