Efficacy Of Histone Deacetylase Inhibitors Research Articles

Abstract 1044: Breast cancer subtype-specific regulation of gene transcription and therapeutic response by functional crosstalk between LSD1 and HDACs

Abstract The dysregulation of histone deacetylases (HDACs), which frequently leads to the silencing of gene expression, is linked to breast cancer progression. Studies in recent years have focused on reversing these changes in gene expression through inhibition of histone deacetylases (HDACs), and HDAC inhibitors (HDACi) have emerged as a potential new treatment option for cancer. However the current HDACi are less effective as monotherapy against solid tumors, highlighting the need to develop rational combinations of chemotherapeutic agents for the treatment of solid tumors. Our recent work has shown that combined inhibition of LSD1 and HDACs in breast cancer cell lines leads to re-expression of a unique subset of abnormally silenced genes and enhanced apoptosis in triple negative breast cancer (TNBC) cells, suggesting that crosstalk between lysine-specific demethylase 1 (LSD1) and HDACs is a novel and important epigenetic mechanism for aberrant gene silencing. These data also suggest that inhibition of LSD1 in combination with HDACi might enhance the therapeutic efficacy of HDAC inhibitors, and thus improve breast cancer treatment. Our further investigation showed that TNBC cells are overall more sensitive to combined treatment with LSD1 and HDAC inhibitors than other subtypes of breast tumors, or normal breast cells. LSD1-knockdown (KD) by shRNA sensitized TNBC MDA-MB-231 cells to HDACi-induced growth inhibition and apoptosis, and resulted in a striking synergistic mRNA induction of important growth control and apoptosis-related genes such as ERα, E-Cadherin, NR4A1, PCDH1, RGS16, BIK, CDKN1C, CRABP2, ING1, SQSTM1, TP53TG1, etc. In contrast, LSD1-KD exerted only minor effect on SAHA-induced gene re-expression in hormone receptor-positive or HER2 positive counterparts. Chromatin immunoprecipitation showed that re-expression of silenced genes was accompanied by concurrent increase of active chromatin marks H3K4me2 and AcetylH3K9 at gene promoters. ShRNA-mediated silencing of LSD1 significantly suppressed the mRNA expression of most of the class I (1-3, 8), II (6, 7, 10) and IV (11) HDAC isozymes in TNBC cells, but exerted marginal effect on transcription activities of HDAC isozymes in other subtypes of breast cancer cells. Moreover, siRNA-mediated silencing of the specific HDAC isozymes led to increase of H3K4me2 level in MDA-MB-231 cells. Taken together, our studies suggest that orchestrated interplay between LSD1 and HDACs is an important epigenetic signature contributing to aberrant gene silencing, and combination therapy targeting the crosstalk between histone demethylation and deacetylation may represent a novel therapeutic approach for aggressive TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1044. doi:1538-7445.AM2012-1044

Abstract 5048: Targeting regulators of Chk1 to enhance cytotoxic efficacy of HDAC inhibitors

Abstract Introduction: Predicting and overcoming drug resistance is a major difficulty facing the efficacy of drugs in clinical trials. Histone deacetylase inhibitors (HDACis) are known to induce cell death and have been used in clinic trials as anticancer drugs, however, the cytotoxic effects of HDACis remains to be limited with no clear mechanism of resistance has been elucidated to explain this limited efficacy. In this study we showed that downregulation of the G2 checkpoint kinase 1 (Chk1) plays an essential role in inducing cell death by HDACis. We identified E2F as a main regulator of Chk1 expression in cancer cells, suggesting that inhibition of Chk1 through E2F may enhance the cytotoxic effects of HDACis in clinical studies. Methods: A549, H1299, PC9, HCT116, HT1080, MNNG, H2172, Saos-2, MG63, U2OS, p21-/- and p21+/+ MEF cell lines were grown in appropriate medium and treated with the HDAC inhibitors, SaHa, LBH589 or MS275. Western blot and qRT-PCR experiments were performed to assess the effect of HDACis on Chk1/G2-M checkpoint pathways and on E2F-mediated gene expression profiles. Additionally, knock out and gain of function experiments were done to better understand the interaction between Chk1, E2F and p21Waf1 in HDACi mediated cell death. The results of the in vitro cell lines studies were corroborated in the ex vivo experiments with patient tumor tissue. Results: We showed that HDACi treatment leads to inhibition of Chk1 expression and consequently to increased activity of CDC25 and CDC2 (CDK1) enzymes causing premature mitotic entry and cell death. In the knock-down experiments we found that inhibition of E2F1 or E2F3 leads to reduction of Chk1 expression and potentiation of cell death by HDACis. Similar results were observed using patient-derived tumor samples treated with SaHa and an E2F inhibitor (HLM006474). Additionally, over-expression of E2F1 or E2F3 dramatically decreased HDACi-mediated cell death. In time course experiments, Chk1 as well as other E2F-responsive genes were down regulated after HDACi treatment accompanied by an expected p21Waf1 induction. In p21Waf1 +/+ and -/- MEFs we showed that lack of p21Waf1 results in increased apoptosis, suggesting that early induction of p21Waf1 by HDACis negatively regulates HDACi-induced cell death, which likely contributes to HDACi resistance in tumor cells by preventing cells to undergo mitotic cell death. Conclusions: Our results demonstrate that E2F-mediated downregulation of Chk1 plays a key role in HDACi-induced cell. We provide evidence that lack of Chk1 downregulation and activation of CDC2 is associated with resistance to HDACis indicating that Chk1 may represent a clinically relevant biomarker to assess the efficacy of HDACis in patient tumor samples. Taken together our data lends insight into the molecular mechanism of sensitivity and resistance to HDACi-induced cell death and will hopefully provide a stronger foundation for future clinical translation with HDACis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5048. doi:10.1158/1538-7445.AM2011-5048

ΔNp63, a Target of DEC1 and Histone Deacetylase 2, Modulates the Efficacy of Histone Deacetylase Inhibitors in Growth Suppression and Keratinocyte Differentiation

The p63 gene, a member of the p53 family, is expressed as TA and ΔN isoforms. ΔNp63 is the predominant isoform expressed in cells of epithelial origin and frequently overexpressed in cancers. However, what regulates p63 expression is uncertain. Here, we showed that ΔNp63 is regulated by the transcription factor DEC1, a p53 family target. We also showed that the ability of DEC1 to regulate ΔNp63 is enhanced by histone deacetylase (HDAC) inhibitors or knockdown of histone deacetylase 2 (HDAC2). Consistent with this, we found that DEC1 and HDAC2 physically interact and knockdown of HDAC2 leads to increased binding of DEC1 to the ΔNp63 promoter. Interestingly, we found that growth suppression induced by HDAC inhibitors is attenuated by ectopic expression of DEC1 in a ΔNp63-dependent manner. In addition, we showed that ectopic expression of DEC1 inhibits, whereas knockdown of DEC1 promotes, keratinocyte differentiation via modulating ΔNp63 expression. Finally, we showed that DEC1 cooperates with HDAC inhibitors to further decrease keratinocyte differentiation. Together, we conclude that ΔNp63 is a novel target of DEC1 and HDAC2 and modulates the efficacy of HDAC inhibitors in growth suppression and keratinocyte differentiation.

The Therapeutic Potential of HDAC Inhibitors in the Treatment of Multiple Sclerosis

Multiple sclerosis (MS) is a devastating autoimmune disorder of the central nervous system (CNS) for which there is no efficacious cure. Thanks to numerous preclinical and clinical studies, drugs able to mitigate the inexorable course of the disease have been made available recently. Still, there is a terrible need for compounds capable of reducing the severity of the autoimmune attack and of blocking progression of the disorder. Also, besides the classic immunosuppressive strategies, it is now appreciated that compounds directly targeting neuronal death can be of relevance to the treatment of MS patients. Acetylation homeostasis is a key regulator of both immune cell activation and neuronal survival. Of note, potent histone deacetylase inhibitors (HDACi) endowed with antiinflammatory and neuroprotective properties have been identified. Efficacy of HDACi in experimental models of MS has been reported consistently. In this review, we provide an appraisal of the literature on HDACi and MS, also discussing the mechanisms by which HDACi can suppress the autoimmune attack to the CNS.

Therapeutic Strategies to Enhance the Anticancer Efficacy of Histone Deacetylase Inhibitors

Histone acetylation is a posttranslational modification that plays a role in regulating gene expression. More recently, other nonhistone proteins have been identified to be acetylated which can regulate their function, stability, localization, or interaction with other molecules. Modulating acetylation with histone deacetylase inhibitors (HDACi) has been validated to have anticancer effects in preclinical and clinical cancer models. This has led to development and approval of the first HDACi, vorinostat, for the treatment of cutaneous T cell lymphoma. However, to date, targeting acetylation with HDACi as a monotherapy has shown modest activity against other cancers. To improve their efficacy, HDACi have been paired with other antitumor agents. Here, we discuss several combination therapies, highlighting various epigenetic drugs, ROS-generating agents, proteasome inhibitors, and DNA-damaging compounds that together may provide a therapeutic advantage over single-agent strategies.

Histone Deacetylase Inhibitors Downregulate Checkpoint Kinase 1 Expression to Induce Cell Death in Non-Small Cell Lung Cancer Cells

BackgroundHistone deacetylase inhibitors (HDACis) are promising anticancer drugs; however, the molecular mechanisms leading to HDACi-induced cell death have not been well understood and no clear mechanism of resistance has been elucidated to explain limited efficacy of HDACis in clinical trials.Methods and FindingsHere, we show that protein levels of checkpoint kinase 1 (Chk1), which has a major role in G2 cell cycle checkpoint regulation, was markedly reduced at the protein and transcriptional levels in lung cancer cells treated with pan-and selective HDACis LBH589, scriptaid, valproic acid, apicidin, and MS-275. In HDACi treated cells Chk1 function was impaired as determined by decreased inhibitory phosphorylation of cdc25c and its downstream target cdc2 and increased expression of cdc25A and phosphorylated histone H3, a marker of mitotic entry. In time course experiments, Chk1 downregulation occurred after HDACi treatment, preceding apoptosis. Ectopic expression of Chk1 overcame HDACi-induced cell death, and pretreating cells with the cdc2 inhibitor purvalanol A blocked entry into mitosis and prevented cell death by HDACis. Finally, pharmacological inhibition of Chk1 showed strong synergistic effect with LBH589 in lung cancer cells.ConclusionsThese results define a pathway through which Chk1 inhibition can mediate HDACi-induced mitotic entry and cell death and suggest that Chk1 could be an early pharmacodynamic marker to assess HDACi efficacy in clinical samples.

The Histone Deacetylase Inhibitor MGCD0103 Has Both Deacetylase and Microtubule Inhibitory Activity

Histone deacetylase inhibitors (HDACis) are currently in trial or are in clinical use for the treatment of a number of tumor types. The clinical efficacy of HDACis can be partly attributed to the modulation of the cell cycle by the HDACis. Here, we have examined the effects of N-(2-aminophenyl)-4-((4-pyridin-3-ylpyrimidin-2-ylamino)methyl)benzamide (MGCD0103), a class I-selective histone deacetylase inhibitor, on the cell cycle and cell killing. Surprisingly, MGCD0103 treatment failed to initiate a G(1)-phase arrest but caused marked accumulation of cells in G(2)/M at 6 and 12 h after treatment and was cytotoxic 24 h after treatment. These cell cycle effects were considerably distinct from the effects of suberic bishydroxamic acid, a representative of the pan-isoform HDACi used in this study. MGCD0103 shared the ability of the pan-isoform HDACi to trigger defective mitosis and promote mitotic slippage. Likewise, it also specifically targeted tumor cells and was nontoxic to normal nontransformed cells. However, MGDC0103 also seemed to disrupt normal microtubule spindle formation, whereas HDACis generally have only a minor effect on spindle formation. The effect of MGCD0103 on spindle formation was shown to be a consequence of microtubule destabilization. This is the first example of an HDACi with microtubule destabilizing activity, and the combined effects of this drug have advantages for its therapeutic use.

Targeting CHK1 pathway to induce cytotoxic response to histone deactylase inhibitors and evaluating the synergistic effects of CHK1 and HDAC inhibitors in human soft tissue sarcoma cell lines and primary tumor xenografts.

10049 Background: Histone deacetylase inhibitors (HDACis) are promising anticancer drugs; however, the molecular mechanisms leading to cell death by HDACis have not been well understood and no clear mechanism of resistance has been elucidated to explain limited efficacy of HDACis in clinical trials. Methods: In vitro treatment of cell lines with HDACi and Chk1 inhibitors, time course experiments, protein analysis by western blot, and ex vivo evaluation of sarcoma xenografts were performed. Results: In HDACi- treated sarcoma cell lines, Chk1 downregulation was functional as indicated by decreased inhibitory phosphorylation of cdc25c and its downstream target cdc2. Chk1 downregulation occurred upon HDACi treatment and preceded apoptosis, as assessed by poly(ADP-ribose) polymerase cleavage. Pharmacological inhibition of Chk1 showed strong synergistic effect with LBH589 in sarcoma cells in vitro. Serial sampling of mouse xenografts of these sarcoma cell lines using FNAB indicates that downregulation of Chk1 upon HDACi treatment occurs in vivo and can be used to assess and predict efficacy of treatment. Conclusions: Taken together these results suggest that Chk1 could be an early pharmacodynamic marker to assess HDACi efficacy in clinical samples. A combination of drugs targeting HDAC and Chk1 enzymes may be clinically useful for sarcoma treatment. We show that expression of checkpoint kinase 1 (Chk1), which has a major role in G2 cell cycle checkpoint regulation, was markedly reduced in multiple sarcoma cell lines both in vitro and in vivo as well as in human sarcoma samples tested ex vivo when cells were treated with the HDACi LBH589, scriptaid, valproic acid, or apicidin. No significant financial relationships to disclose.

Abstract 5415: Histone deacetylase inhibitors and 15-deoxy-Δ12,14-prostaglandin J2 synergistically induce apoptosis

Abstract The histone deacetylase inhibitors (HDIs) valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, vorinostat) are used clinically for the treatment of epilepsy and cutaneous T-cell lymphoma, respectively. HDIs have been shown to be more effectively utilized in combination with other agents despite the promising anti-tumor effect of HDIs alone. Therefore, additional mechanism-based therapeutic strategies for use of HDIs in combination with other agents should be investigated to overcome resistance to HDIs or to increase the efficacy of HDIs. The natural endogenous ligand of peroxisome proliferator-activated receptor γ (PPARγ) 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a member of cyclopentenone prostaglandins and known to be a potent anti-neoplastic agent. Thus, we examined whether VPA or SAHA in combination with 15d-PGJ2 could show synergistic anti-tumor activitiy in colon cancer DLD-1 cells. Cell viability was determined using a Cell Counting Kit-8 assay. Apoptosis and reactive oxygen species (ROS) generation were determined using flow cytometry analysis. Western blotting and real-time RT-PCR analysis were carried out to investigate the expressions of apoptosis-related molecules. Small interfering RNA or transient transfection with plasmids was used for gene silencing, gene overexpression or luciferase assays. Mice bearing DLD-1 xenograft were divided into 4 groups (n=5) and injected everyday (i.p.) with diluent, VPA (100 mg/kg), 15d-PGJ2 (5 mg/kg) or a combination for 25 days. Cotreatment with VPA or SAHA at clinically achievable concentrations and 15d-PGJ2 synergistically induced cell death and drastically caused caspase-dependent apoptosis in DLD-1 cells. The apoptosis induced by VPA/15d-PGJ2 cotreatment was due to alterations of apoptosis-related genes such as an antiapoptotic Bcl-2 family member Bcl-XL, X-chromosome-linked inhibitor of apoptosis (XIAP), C/EBP homologous protein (CHOP) and death receptor 5 (DR5) via ROS generation, subsequent endoplasmic reticulum stress and/or histone deacetylase inhibition. Moreover, VPA/15d-PGJ2 cotreatment induced ROS-dependent apoptosis in other malignant tumor cells and was more effective than a VPA or 15d-PGJ2 monotherapy in vivo. These results suggest that cotreatment with the clinically relevant HDIs and 15d-PGJ2 may warrant clinical investigation as a rational combination therapy against a broad spectrum of malignant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5415.

SB939, a Novel Potent and Orally Active Histone Deacetylase Inhibitor with High Tumor Exposure and Efficacy in Mouse Models of Colorectal Cancer

Although clinical responses in liquid tumors and certain lymphomas have been reported, the clinical efficacy of histone deacetylase inhibitors in solid tumors has been limited. This may be in part due to the poor pharmacokinetic of these drugs, resulting in inadequate tumor concentrations of the drug. SB939 is a new hydroxamic acid based histone deacetylase inhibitor with improved physicochemical, pharmaceutical, and pharmacokinetic properties. In vitro, SB939 inhibits class I, II, and IV HDACs, with no effects on other zinc binding enzymes, and shows significant antiproliferative activity against a wide variety of tumor cell lines. It has very favorable pharmacokinetic properties after oral dosing in mice, with >4-fold increased bioavailability and 3.3-fold increased half-life over suberoylanilide hydroxamic acid (SAHA). In contrast to SAHA, SB939 accumulates in tumor tissue and induces a sustained inhibition of histone acetylation in tumor tissue. These excellent pharmacokinetic properties translated into a dose-dependent antitumor efficacy in a xenograft model of human colorectal cancer (HCT-116), with a tumor growth inhibition of 94% versus 48% for SAHA (both at maximum tolerated dose), and was also effective when given in different intermittent schedules. Furthermore, in APC(min) mice, a genetic mouse model of early-stage colon cancer, SB939 inhibited adenoma formation, hemocult scores, and increased hematocrit values more effectively than 5-fluorouracil. Emerging clinical data from phase I trials in cancer patients indicate that the pharmacokinetic and pharmacologic advantages of SB939 are translated to the clinic. The efficacy of SB939 reported here in two very different models of colorectal cancer warrants further investigation in patients.

486 Intrathecal (IT) trastuzumab in leptomeningeal and central nervous system (CNS) metastases from HER2+ breast cancer (BC): What if we could bypass the blood–brain barrier (BBB)?

Histone deacetylases (HDACs) play an important role in the epigenetic regulation of gene expression by catalyzing the removal of acetyl groups, stimulating chromatin condensation and promoting transcriptional repression. Since aberrant epigenetic changes are a hallmark of cancer, HDACs are a promising target for pharmacological inhibition. HDAC inhibitors can induce cell-cycle arrest, promote differentiation, and stimulate tumor cell death. These properties have prompted numerous preclinical and clinical investigations evaluating the potential efficacy of HDAC inhibitors for a variety of malignancies. The preferential toxicity of HDAC inhibitors in transformed cells and their ability to synergistically enhance the anticancer activity of many chemotherapeutic agents has further generated interest in this novel class of drugs. Here we summarize the different mechanisms of HDAC inhibitor-induced apoptosis and discuss their use in combination with other anticancer agents.

Protein Acetylation in the Cardiorenal Axis

Acetylation of histone and nonhistone proteins provides a key mechanism for controlling signaling and gene expression in heart and kidney. Pharmacological inhibition of protein deacetylation with histone deacetylase (HDAC) inhibitors has shown promise in preclinical models of cardiovascular and renal disease. Efficacy of HDAC inhibitors appears to be governed by pleiotropic salutary actions on a variety of cell types and pathophysiological processes, including myocyte hypertrophy, fibrosis, inflammation and epithelial-to-mesenchymal transition, and occurs at compound concentrations below the threshold required to elicit toxic side effects. We review the roles of acetylation/deacetylation in the heart and kidney and provide rationale for extending HDAC inhibitors into clinical testing for indications involving these organs.

The Vk*MYC Mouse Model of Myeloma Accurately Reflects Clinical Efficacy of Established and Novel Drugs.

Abstract 1849Poster Board I-875With the increasing availability of novel compounds, a number of drugs that have shown promising anti-myeloma activity in extensive pre-clinical studies failed to demonstrate clear efficacy in subsequent clinical trials. One reason for that may be an intrinsic inadequacy of pre-clinical models to predict activity, since for the most part they do not represent the complexity of human MM in an endogenous micro-environment. Another reason may be that to be able to test novel compounds, only relapsed and refractory patients are studied, at a stage in their disease that is unlikely to respond to any drug. One solution to this problem is to treat patients earlier in the course of their disease, which has been done by using the novel drug in combination with an approved drug. Unfortunately, without a large randomized trial, it is often difficult to interpret the response rates observed in these patients, and determine the contribution of the novel compound to the response observed to the combination treatment.The immuno-competent Vk*MYC mouse model of myeloma has already demonstrated high biological fidelity to the human disease, making it a better model to study the behavior of myeloma cells in the context of a native microenvironment. Specifically, the Vk*MYC myeloma is indolent (with a very low proliferative index), confined to the bone marrow, and secretes a monoclonal protein detected by serum protein electrophoresis. The M-spike represents a very useful marker to monitor tumor burden by periodic bleeding, as is done in patients. We have re-evaluated the anti-myeloma activity of novel compounds that have passed extensive pre-clinical characterization and are currently in clinical trials. We have utilized “naïve‘, untreated Vk*MYC mice at early stages of myeloma disease. Response rates were calculated by comparing the M-spike levels after treatment to the levels observed at d0. We found that, in addition to the demonstrated single agent clinical activity of alkylating agents (melphalan), cortico-steroids (dexamethasone) and proteasome inhibitors (bortezomib and carfilzomib), the histone deacetylase (HDACs) inhibitors vorinistat and panobinostat showed strong anti-myeloma activity, inducing a dramatic reduction of the M-spike after only two weeks of treatment. In contrast, only a minor response was observed to a CDK inhibitor (SNS-032) or the AKT inhibitor perifosine. No response was observed to the HSP-90 inhibitor AUY-922, or to the PI-3K inhibitors BEZ-235 and GDC-0941, or statins. The summary of the results with all tested agents is given in Table 1. [Display omitted] Although the modest activity of SNS-032 and perifosine in the Vk*MYC mice mirrors the results of phase-I and –II trials, where disease stabilization was observed, the efficacy of HDACs inhibitors in the Vk*MYC mice is somewhat surprising, since no clear single agent activity had been reported in advanced MM patients. With all the limitations of a mouse model, our data, however, suggest that untreated, earlier stage myeloma patients may be more sensitive to HDAC inhibition.As more mice are treated and more compounds are screened to further validate the clinical predictive value of the Vk*MYC mice, we would like to propose a new approach to the introduction of novel compounds in the treatment of MM, in which they are tested alone in the Vk*MYC model, and the ones that clearly induce a complete response are then prioritized for testing in a small cohort of earlier stage/untreated patients for one cycle, accelerating the evaluation, and limiting the toxicity. For those compounds that generate a partial response, combination studies can be easily performed in the mouse to assess drug pair efficacy. Finally, drugs that do not show any response in the Vk*MYC mouse should be given a lower priority for further clinical development in MM. Disclosures:No relevant conflicts of interest to declare.

Dual Degradation of Aurora A and B Kinases by the Histone Deacetylase Inhibitor LBH589 Induces G2-M Arrest and Apoptosis of Renal Cancer Cells

This study is aimed at investigating antineoplastic efficacy of histone deacetylase inhibitor (HDACI) LBH589 on renal cell carcinoma (RCC) and elucidating the novel molecular mechanisms involved in growth arrest and apoptosis by targeting the important nonhistone molecules. We analyzed the growth-inhibitory effect of LBH589 on RCC by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in vitro and antitumor efficacy by xenograft experiments in vivo. To verify the associated molecular mechanisms involved in LBH589-mediated cell death and cell cycle progression by Western blotting and fluorescence-activated cell sorting analysis. HDACI LBH589 induced degradation of both Aurora A and B kinases through a proteasome-mediated pathway by targeting HDAC3 and HDAC6. The dual degradation of Aurora A and B kinases mediated by LBH589 resulted in inducing G2-M arrest and apoptosis of renal cancer cell lines and our results also showed that LBH589 potently inhibited renal cancer cell growth in vitro and suppressed tumor formation in vivo. The Aurora A and B kinases and HDAC3 are overexpressed in the human RCC tumor tissues examined, which make them perfect targets for HDACI LBH589 treatment. Our in vitro and in vivo data showed that LBH589 has potent anticancer effect of renal cancer cells. LBH589 and other HDACI treatment resulted in inducing G2-M arrest and apoptosis of renal cancer cells through degradation of Aurora A and B kinases by inhibition of HDAC3 and HDAC6. The clinical efficacy of LBH589 in the treatment of patients with metastatic RCC, especially those with high Aurora kinase and HDAC expression, is worthy of further investigation.

Glucose Metabolism as a Target of Histone Deacetylase Inhibitors

The therapeutic efficacy of histone deacetylase inhibitors (HDACI) is generally attributed to their ability to alter gene expression secondary to their effects on the acetylation status of transcription factors and histones. However, because HDACIs exhibit similar transcriptional effects in most cells, the molecular basis for their therapeutic selectivity toward malignant cells is largely unknown. In this study, we report that HDACI, of distinct chemotypes, quantitatively inhibit glucose transporter 1 (GLUT1)-mediated glucose transport into multiple myeloma cells through both down-regulation of GLUT1 and inhibition of hexokinase 1 (HXK1) enzymatic activity. Unexpectedly, however, this inhibition of glucose utilization is accompanied by an increase in amino acid catabolism with no increase in fatty acid oxidation. Our findings suggest that an HDACI-induced change in carbon source preference could contribute to the therapeutic efficacy of these drugs by creating a pattern of fuel utilization that is incompatible with rapid tumor growth and survival. Furthermore, these results, which implicate glucose metabolism as a target of HDACI, suggest that caution should be exercised in attributing effects of this class of drug to primary alterations in gene transcription.

Comparison between Therapeutic Efficacies of Histone Deacetylase Inhibitors and Established Drug Regimens Against Breast Cancer Cells using the Histoculture Drug Response Assay

Purpose: Histone deacetylase inhibitors (HDACIs) induce accumulation of acetylated histones in nucleosomes, which lead to reactivate gene expression and inhibit the growth and survival of tumor cells. This study evaluated the efficacy of HDACIs in breast cancer cells in comparison with other established drug regimens. Methods: Drug responses of tumor samples from mastectomy specimens of 78 breast cancer patients were evaluated using the histoculture drug response assay (HDRA). Tumor inhibition rates (IRs) of established drug regimens such as doxorubicin, cyclophosphamide, doxorubicin with cyclophosphamide (AC), paclitaxel, docetaxel and doxorubicin with docetaxel (AT), as well as those of three HDACIs (SAHA, PXD101, and a novel compound CG-2) were evaluate. Results: The percentages of chemosensitive tumors (chemoresponsiveness) were 26.9-60.3% with established regimens and 61.5-73.1% with HDACIs when the cutoff value for inhibition rate was set at 30%. Breast cancer cells appeared to be more chemoresponsive to HDACIs than to established drug regimens. Chemoresponsiveness to AT was the highest among the established drug regimens. A combination regimen offered higher activity than did a single drug (doxorubicin vs AT; p<0.001). HER2/Neu-overexpressing breast cancers were chemosensitive to SAHA and AT (p=0.031 and 0.04, respectively). Conclusion: Our findings show that breast cancer cells were sensitive to HDACIs, with therapeutic efficacies comparable to those of established drug regimens. Specific biological markers such as HER2/Neu could be assessed for effectiveness as HDACIs chemosensitivity markers in further clinical trials.

Glucose Metabolism as a Target of Histone Deacetylase Inhibitors

The therapeutic efficacy of histone deacetylase inhibitors (HDACI) is generally attributed to their ability to alter gene expression secondary to their effects on the acetylation status of transcription factors and histones. However, because HDACIs exhibit similar transcriptional effects in most cells, the molecular basis for their therapeutic selectivity toward malignant cells is largely unknown. In this study, we report that HDACI, of distinct chemotypes, quantitatively inhibit glucose transporter 1 (GLUT1)-mediated glucose transport into multiple myeloma cells through both down-regulation of GLUT1 and inhibition of hexokinase 1 (HXK1) enzymatic activity. Unexpectedly, however, this inhibition of glucose utilization is accompanied by an increase in amino acid catabolism with no increase in fatty acid oxidation. Our findings suggest that an HDACI-induced change in carbon source preference could contribute to the therapeutic efficacy of these drugs by creating a pattern of fuel utilization that is incompatible with rapid tumor growth and survival. Furthermore, these results, which implicate glucose metabolism as a target of HDACI, suggest that caution should be exercised in attributing effects of this class of drug to primary alterations in gene transcription.

HDAC inhibitors in models of inflammation-related tumorigenesis

Histone deacetylase (HDAC) inhibitors have been described in detail for their anti-proliferative potency. Recently, an anti-inflammatory property was characterized in vitro and in vivo. This dual efficacy of HDAC inhibitors is highly attractive, since chronic inflammations such as ulcerative colitis are associated with an increased risk of developing carcinomas. Additionally, in models of colitis and inflammation-induced tumorigenesis inflammation as well as tumor development was significantly inhibited by HDAC inhibitor treatment. The mechanisms involved reach beyond the simple regulation of histone acetylation and deacetylation. The currently known key target structures and mechanisms mediating this dual effect will be discussed in this review.

The Efficacy of Histone Deacetylase Inhibitor, Vorinostat against BCR-ABL Positive Leukemia Cells Include ABL Kinase Domain Mutation in Monotherapy and in Combination with Dasatinib

The development of imatinib are represented a major therapeutic advance in BCR-ABL positive diseases. However, a substantial number of patients are either primary refractory or acquire resistance to imatinib associated with ABL kinase domain mutation and this becomes a problematic in clinically. Histone acetylation and deacetylation is important for the accurate regulation of gene expression. Histone deacetylase (HDAC) inhibitors such as vorinostat (suberoylamide hydroxamic acid: SAHA) are the class of anticancer drugs currently in clinical trials for the treatment of hematological malignacies and solid tumors. Especially, vorinostat have shown efficacy in a wide range of cancers such as cutaneous T-cell lymphoma (CTCL). However, the molecular and functional consequences of vorinostat against BCR-ABL expressing cellswith Abl kinase mutation have not fully known. In this study, we investigated the vorinostat efficacy by using the murine Ba/F3 cell line which was transfected wild type (Wt) p210 BCR-ABL or imatinib resistant BCR-ABL mutants such as G250E, Q252H, Y253F, E255K, M294V, T315I, T315A, F317L, F317V, M351T and H396P. 48 hours treatment of vorinostat exhibits cell growth inhibition and proapoptotic activity murine Ba/F3 cells ectopically expressing wild type (Wt) p210 and imatinib resistant BCR-ABL mutants including T315I mutation in a dose dependent manner. IC50 of these cell lines are Wt(720nM), G250E(625nM), Q252H(220nM), Y253F(525nM), E255K(685nM), M294V(785nM), T315I(500nM), T315A(715nM), F317L(560nM), F317V(565nM), M351T(375nM) and H396P(485nM). Vorinostat is more effective in BCR-ABL mutant cells compared with Ba/F3 Wt BCR-ABL cells. We examined the intra cellular signaling by using these cell lines. We found that caspase 3, and poly (ADP-ribose) polymerase (PARP) were activated in a dose and a time dependent manner. Phosphorylation of Crk-L which is downstream target of BCRABL was reduced after vorinostat treatment. We also found the protein level of BCR-ABL was reduced after 48 hours vorinostat treatment in a dose dependent manner. BCR-ABL degradations in mutant cells were significantly enhanced compared with Ba/F3 Wt p210 BCR-ABL cells. Protein degradation of BCR-ABL was blocked by ubiquitin-proteasome inhibitor, Lactacystin suggested that this was involved ubiquitin-proteasome pathway. Dasatinib is a potent dual Src/BCR-ABL kinase inhibitor. Dasatinib is more than 100- fold more potent than imatinib against the ABL kinases and retains this low nanomolar inhibitory activity against the imatinib resistant mutations. Because dasatinib has shown efficacy against imatinib resistant BCR-ABL mutant, we investigated the efficacy between vorinostat and dasatinib by using these cell lines. We found that combination of vorinostat and dasatinib synergistically cell growth inhibition of Wt and BCR-ABL mutatants Ba/F3 cells in 48 hours treatment. Phosphorylation of Crk-L was reduced after vorinostat and dasatinib treatment in these cells. Caspase 3 and PARP activation were also synergistically increased after vorinostat and dasatinib treatment. Data from this study suggested that administration of the clinically available HDAC inhibitor vorinostat may be a powerful strategy against BCR-ABL mutant cells and enhance cytotoxic effects of dasatinib in those imatinib resistant BCR-ABL mutant cells and this approach warrants further examination in BCR-ABL related malignancies.

Histone deacetylase inhibitors: Mechanisms of cell death and promise in combination cancer therapy

Histone deacetylases (HDACs) play an important role in the epigenetic regulation of gene expression by catalyzing the removal of acetyl groups, stimulating chromatin condensation and promoting transcriptional repression. Since aberrant epigenetic changes are a hallmark of cancer, HDACs are a promising target for pharmacological inhibition. HDAC inhibitors can induce cell-cycle arrest, promote differentiation, and stimulate tumor cell death. These properties have prompted numerous preclinical and clinical investigations evaluating the potential efficacy of HDAC inhibitors for a variety of malignancies. The preferential toxicity of HDAC inhibitors in transformed cells and their ability to synergistically enhance the anticancer activity of many chemotherapeutic agents has further generated interest in this novel class of drugs. Here we summarize the different mechanisms of HDAC inhibitor-induced apoptosis and discuss their use in combination with other anticancer agents.