Histone Deacetylase Inhibitor LAQ824 Research Articles

Re: Rosato RR, Maggio SC, Almenara JA, Payne SG, Atadja P, Spiegel S, Dent P, and Grant S, "The Histone Deacetylase Inhibitor LAQ824 Induces Human Leukemia Cell Death through a Process Involving XIAP Down-Regulation, Oxidative Injury, and the Acid Sphingomyelinase-Dependent Generation of Ceramide." Mol Pharmacol January 2006 69:216-225; doi:10.1124/mol.105.017145.

Re: Rosato RR, Maggio SC, Almenara JA, Payne SG, Atadja P, Spiegel S, Dent P, and Grant S, "The Histone Deacetylase Inhibitor LAQ824 Induces Human Leukemia Cell Death through a Process Involving XIAP Down-Regulation, Oxidative Injury, and the Acid Sphingomyelinase-Dependent Generation of Ceramide." Mol Pharmacol January 2006 69:216-225; doi:10.1124/mol.105.017145.

Abstract 5277: Non-invasive metabolic biomarkers of histone deacetylase inhibition in human colon cancer cells and tumors

Abstract Introduction: Histone deacetylase (HDAC) inhibitors are targeted anti-cancer agents currently approved for cutaneous T-cell lymphoma and in mid-late stage trials for other cancers (Ma et al, Drugs 2009). Developing non-invasive biomarkers of drug activity could provide a useful tool to aid clinical development. We have previously shown that the HDAC inhibitor LAQ824 increases phosphocholine (PC) levels in human colon cancer cells and tumors as detected by magnetic resonance spectroscopy (MRS), a non-invasive method for monitoring tissue metabolism (Chung et al, Neoplasia 2008). Here we sought to confirm this effect using a different chemotype probe (PXD101, belinostat) and investigate the mechanism(s) underlying it. Methods: Human HT29 colon cancer cells were treated for 24h with DMSO or 2 M PXD101 in DMEM ± 28 µM [1,2-13C]-choline. For in vivo evaluation, female nude mice bearing HT29 xenografts were treated i.p. with vehicle (n=6) or 60mg/kg PXD101 (n=6) for 3 days. In vitro metabolism was assessed using 31P or 13C MRS on cell extracts, while in vivo metabolism was evaluated using 1H and 31P MRS pre- (d0) and 3 days post-treatment, followed by analysis of excised tumor extracts. Western blotting for acetyl histone-3 was used to verify inhibitor action. Results: PXD101 treatment in HT29 cells induced histone-3 acetylation and decreased cell counts to 55±6% of controls (p=0.005). In vivo, PXD101 treatment resulted in tumor stasis (grew by 2% from d0) while the control tumors grew by 20% from d0. 31P MRS showed increased cellular PC content to 156±13% (p=0.04) post-PXD101 treatment. In vivo 1H and 31P MRS of tumors revealed rises in total choline/water (169±25%; p=0.02) and phosphomonoester (i.e. PC+phosphoethanolamine)/total phosphate ratios (p=0.01). Ex-vivo analysis attributed this effect to a rise in PC up to 1.4-fold (p=0.03) in the PXD101-treated tumors relative to controls. PC is formed mainly through phosphorylation of choline via choline kinase (de novo route) or hydrolysis of phosphatidylcholine via phospholipases. 13C MRS analysis of cells grown in the presence of the tracer [1,2-13C]-choline showed increased 13C- PC formation post-PXD101 treatment (180±19% of controls, p=0.02) consistent with increased de novo PC synthesis. Conclusions: Our data show that HDAC inhibition with PXD101 increases PC levels in human colon cancer cells and tumors thus confirming our previous findings with the different chemotype agent LAQ824 (Chung et al, Neoplasia 2008). Importantly, we show that this effect is driven by a rise in de novo PC synthesis. These data further support the role of PC as a potential non-invasive biomarker for monitoring the action of HDAC inhibitors. We acknowledge the support received for the CRUK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England). We also acknowledge NHS funding to the NIHR Biomedical Research Centre and NCI for providing PXD101. 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 5277. doi:10.1158/1538-7445.AM2011-5277

Histone Deacetylase Inhibitor LAQ824 Augments Inflammatory Responses in Macrophages through Transcriptional Regulation of IL-10

APCs are important in the initiation of productive Ag-specific T cell responses and the induction of T cell anergy. The inflammatory status of the APC at the time of encounter with Ag-specific T cells plays a central role in determining such divergent T cell outcomes. A better understanding of the regulation of proinflammatory and anti-inflammatory genes in its natural setting, the chromatin substrate, might provide novel insights to overcome anergic mechanisms mediated by APCs. In this study, we show for the first time, to our knowledge, that treatment of BALB/c murine macrophages with the histone deacetylase inhibitor LAQ824 induces chromatin changes at the level of the IL-10 gene promoter that lead to enhanced recruitment of the transcriptional repressors HDAC11 and PU.1. Such an effect is associated with diminished IL-10 production and induction of inflammatory cells able of priming naive Ag-specific T cells, but more importantly, capable of restoring the responsiveness of anergized Ag-specific CD4(+) T cells.

Restoring the functional immunogenicity of chronic lymphocytic leukemia using epigenetic modifiers

Chronic lymphocytic leukemia (CLL) is a malignancy arising from immune cells (B-lymphocytes) endowed with intrinsic antigen-presenting capabilities. Such a function however is lost during malignant transformation and CLL cells are well known for their inability to process and present antigens to the T-cell arm of the immune system. Instead, malignant CLL cells elicit a vast array of immune regulatory mechanisms conducive to T-cell dysfunction and immunosuppression. Previously, we have shown that treatment of CLL cells with the demethylating agent 5-aza-2′-deoxycytidine unleashed target antigen expression. Here we show for the first time that combining two epigenetic modifiers, 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor LAQ824 effectively restores the immunogenicity of CLL cell lines as well as primary cells obtained from CLL patients. Indeed, such a combination induces the expression of novel and highly antigenic cancer-testis antigens (CTAs) and costimulatory molecules. These changes facilitate the formation of robust supramolecular activation complexes (SMAC) between CLL cells and responder T-cells leading to intracellular signaling, lytic granule mobilization, and polarization of functional and relevant T-cell responses. This cascade of T-cell activating events triggered by CLL cells with restored APC function, points to combined epigenetic modifier treatment as a potential immunotherapeutic strategy for CLL patients.

Enhanced Antitumor Activity Induced by Adoptive T-Cell Transfer and Adjunctive Use of the Histone Deacetylase Inhibitor LAQ824

Tumors grow in the presence of antigen-specific T cells, suggesting the existence of intrinsic cancer cell escape mechanisms. We hypothesized that a histone deacetylase (HDAC) inhibitor could sensitize tumor cells to immunotherapy because this class of agents has been reported to increase tumor antigen expression and shift gene expression to a proapoptotic milieu in cancer cells. To test this question, we treated B16 murine melanoma with the combination of the HDAC inhibitor LAQ824 and the adoptive transfer of gp100 melanoma antigen-specific pmel-1 T cells. The combined therapy significantly improved antitumor activity through several mechanisms: (a) increase in MHC and tumor-associated antigen expression by tumor cells; (b) decrease in competing endogenous lymphocytes in recipient mice, resulting in a proliferative advantage for the adoptively transferred cells; and (c) improvement in the functional activity of the adoptively transferred lymphocytes. We confirmed the beneficial effects of this HDAC inhibitor as a sensitizer to immunotherapy in a different model of prophylactic prime-boost vaccination with the melanoma antigen tyrosinase-related protein 2, which also showed a significant improvement in antitumor activity against B16 melanoma. In conclusion, the HDAC inhibitor LAQ824 significantly enhances tumor immunotherapy through effects on target tumor cells as well as improving the antitumor activity of tumor antigen-specific lymphocytes.

The histone deacetylase inhibitors LAQ824 and LBH589 do not require death receptor signaling or a functional apoptosome to mediate tumor cell death or therapeutic efficacy

AbstractLAQ824 and LBH589 (panobinostat) are histone deacetylase inhibitors (HDACi) developed as cancer therapeutics and we have used the Eμ-myc lymphoma model to identify the molecular events required for their antitumor effects. Induction of tumor cell death was necessary for these agents to mediate therapeutic responses in vivo and both HDACi engaged the intrinsic apoptotic cascade that did not require p53. Death receptor pathway blockade had no effect on the therapeutic activities of LAQ824 and LBH589; however, overexpression of Bcl-2 or Bcl-XL protected lymphoma cells from HDACi-induced killing and suppressed their therapeutic activities. Deletion of Apaf-1 or Caspase-9 delayed HDACi-induced lymphoma killing in vitro and in vivo, associated with suppression of many biochemical indicators of apoptosis, but did not provide long-term resistance to these agents and failed to inhibit their therapeutic activities. Eμ-myc lymphomas lacking a functional apoptosome displayed morphologic and biochemical features of autophagy after treatment with LAQ824 and LBH589, indicating that, in the absence of a complete intrinsic apoptosis pathway involving apoptosome formation, these HDACi can still mediate a therapeutic response. Our data indicate that damage to the mitochondria is the key event necessary for LAQ824 and LBH589 to mediate tumor cell death and a robust therapeutic response.

P53 Acetylation Is Crucial for Its Transcription-independent Proapoptotic Functions

Acetylation of p53 at carboxyl-terminal lysine residues enhances its transcriptional activity associated with cell cycle arrest and apoptosis. Here we demonstrate that p53 acetylation at Lys-320/Lys-373/Lys-382 is also required for its transcription-independent functions in BAX activation, reactive oxygen species production, and apoptosis in response to the histone deacetylase inhibitors (HDACi) suberoylanilide hydroxamic acid and LAQ824. Knock-out of p53 markedly reduced HDACi-induced apoptosis. Unexpectedly, expression of transactivation-deficient p53 variants sensitized p53-null cells to HDACi-mediated BAX-dependent apoptosis, whereas knockdown of endogenous mutant p53 in cancer cells reduced HDACi-mediated cytotoxicity. Evaluation of the mechanisms controlling this response led to the discovery of a novel interaction between p53 and Ku70. The association between these two proteins was acetylation-independent, but acetylation of p53 could prevent and disrupt the Ku70-BAX complex and enhance apoptosis. These results suggest a new mechanism of acetylated p53 transcription-independent regulation of apoptosis.

The Histone Deacetylase Inhibitor LAQ824 Maintains Normal Hematopoietic Progenitor cells (HPC) Associated with Induction of Notch Target Genes and Does Not Eliminate Leukemic HPC in Vitro.

Introduction: Histone deacetylase inhibitors (DACi) have shown promising antileukemic activity by overcoming the differentiation block and inducing apoptosis in AML blasts. Recent data demonstrating enhanced maintenance and functional capacity of normal, but also leukemic hematopoietic progenitor cells (HPC) by the selective class I DACi valproic acid (VPA) have raised concerns about VPA in AML therapy. As more potent pan-DACi have entered clinical trials, we analysed the impact of the hydroxamic acid LAQ824 on phenotype and function of normal and leukemic CD34+ HPC and studied LAQ824- induced gene expression in the most primitive CD34+CD38- population of normal HPC.Methods: Differentiation and proliferation of CD34+ cells of bone marrow of healthy donors and peripheral blood samples of newly diagnosed AML patients were evaluated after one week of culture in presence of SCF, FLT3 ligand, TPO, IL-3 +/− LAQ824. The effect of LAQ824 on gene expression profiles in normal CD34+CD38− cells was assessed in three independent cell samples following incubation with cytokines +/− LAQ824 for 48 hours using Affymetrix GeneChip Human Genome U133 Plus 2.0 and Gene Spring Software. Serial replating of murine Sca1+Lin- HPC was performed in the presence of SCF, G-CSF, GM-CSF, IL-3, IL-6 +/− LAQ824.Results: Treatment of murine Sca1+Lin- HPC with LAQ824 (10 nM) significantly augmented colony numbers (p<0.01; n=3), and supported colony growth after four cycles of replating whereas no colonies developed in its absence beyond the second plating indicating preservation of functionally active multipotent progenitor cells. LAQ824 (10–20 nM) mediated acetylation of histone H3 in human normal and leukemic HPC. In normal HPC, LAQ824 (0–20 nM) lead to a dose-dependent increase in the proportion of CD34+ cells (20% w/o LAQ824 vs. 36% with LAQ824 20nM, p=0.07) and a significant reduction of CD14+ monocytes (18% vs. 3%, p= 0.02; n=3). The total number of CD34+ cells remained stable up to 10 nM and decreased at 20 nM. Gene expression analysis showed, that LAQ824 (20 nM) lead to an at least 3-fold up-regulation of 221 genes in all three HPC samples tested including HDAC11 and the cell cycle inhibitor p21waf1/cip1 known to be induced by most DACi in HPC. We identified several members of the notch pathway such as mastermind-like protein 2 (MAML2, a component of the active notch transcriptional complex) and notch target genes including the transcription factors HES1, HEY1 and HOXA10 and confirmed increase of protein levels by Western blotting. Reduced gene expression of mini-chromosome-maintenance (MCM) protein family members was observed which - in addition to up-regulation of p21 - has previously been associated with notch-mediated cell cycle arrest. To compare the effect of LAQ824 (20 nM) with VPA (150 ng/ml) on leukemic HPC, cells were cultured for one week with or w/o DACi. Of note, LAQ824 resulted in a 0.8-fold reduction of CD34+ leukemic HPC, while VPA expanded this population 2.2-fold compared with cytokine-treated controls (p=0.03; n=12). CFU numbers growing from CD34+ leukemic HPC in presence of LAQ824 did not differ significantly from controls (n=9). Conclusion: LAQ824 seems to diminish, but not eliminate normal as well as leukemic HPC as determined by phenotypic and functional in vitro analyses. Our gene expression analysis suggested an association with coactivator and target genes of the notch pathway and cell cycle arrest-inducing genes. In contrast to VPA, LAQ824 does not seem to support growth of leukemic HPC which may contribute to its more potent antileukemic effect.

Role of histone deacetylase inhibitor-induced reactive oxygen species and DNA damage in LAQ-824/fludarabine antileukemic interactions

The role of reactive oxygen species (ROS) production on DNA damage and potentiation of fludarabine lethality by the histone deacetylase inhibitor (HDACI) LAQ-824 was investigated in human leukemia cells. Preexposure (24 h) of U937, HL-60, Jurkat, or K562 cells to LAQ-824 (40 nmol/L) followed by fludarabine (0.4 micromol/L) dramatically potentiated apoptosis (>or=75%). LAQ-824 triggered an early ROS peak (30 min-3 h), which declined by 6 h, following LAQ-824-induced manganese superoxide dismutase 2 (Mn-SOD2) upregulation. LAQ-824/fludarabine lethality was significantly diminished by either ROS scavengers N-acetylcysteine or manganese (III) tetrakis (4-benzoic acid) porphyrin or ectopic Mn-SOD2 expression and conversely increased by Mn-SOD2 antisense knockdown. During this interval, LAQ-824 induced early (4-8 h) increases in gamma-H2AX, which persisted (48 h) secondary to LAQ-824-mediated inhibition of DNA repair (e.g., down-regulation of Ku86 and Rad50, increased Ku70 acetylation, diminished Ku70 and Ku86 DNA-binding activity, and down-regulated DNA repair genes BRCA1, CHEK1, and RAD51). Addition of fludarabine further potentiated DNA damage, which was incompatible with cell survival, and triggered multiple proapoptotic signals including activation of nuclear caspase-2 and release of histone H1.2 into the cytoplasm. The latter event induced activation of Bak and culminated in pronounced mitochondrial injury and apoptosis. These findings provide a mechanistic basis for understanding the role of early HDACI-induced ROS generation and modulation of DNA repair processes in potentiation of nucleoside analogue-mediated DNA damage and lethality in leukemia. Moreover, they show for the first time the link between HDACI-mediated ROS generation and the recently reported DNA damage observed in cells exposed to these agents.

Effective Induction of Apoptosis in Chronic Myeloid Leukemia CD34+ Cells by the Histone Deacetylase Inhibitor LAQ824 in Combination with Imatinib.

Imatinib mesylate (IM) is highly effective in the treatment of chronic myeloid leukemia (CML). However IM does not eliminate quiescent, primitive CML hematopoietic cells, which persist through treatment and remain a potential source of relapse. Therefore additional interventions are required to more effectively target primitive CML cells. The hydroxamic acid analogue histone deacetylase (HDAC) inhibitors, LAQ824, and LBH589, are reported to induce apoptosis and inhibit cell cycle in CML cell lines and blast crisis (BC) CML cells. Although most pro-apoptotic agents preferentially target dividing cells, HDAC inhibitors are a rare class of drugs reported to induce apoptosis in non-dividing cells. We were therefore interested in investigating whether the HDAC inhibitor LAQ824 (LAQ) could induce apoptosis in CD34+ cells from chronic-phase (CP) CML patients. CML and normal CD34+ cells were cultured with LAQ (10–100nM) alone and in combination with IM (1μM) for 96 hours in growth factor-supplemented medium. Treatment with LAQ (25–100 nM) effectively enhanced Histone H3 acetylation in CML and normal CD34+ cells as detected by Western blotting with an anti-Histone H3 (Lys9/Lys14) antibody. Although LAQ treatment by itself induced significantly less apoptosis in CML compared to normal CD34+ cells, LAQ in combination with IM was highly effective in inducing apoptosis in CML CD34+ cells (11+/−3%, apoptosis with IM alone, 18+/−3% with IM+10 nM LAQ, 22+/−2% with IM+25 nM LAQ, and 32+/−3% with IM+50 nM LAQ824, n=5). Significantly higher levels of apoptosis were seen in CML compared with normal CD34+ cells at lower doses of LAQ (p=0.03 at 10 nM). LAQ combined with IM also resulted in enhanced apoptosis of non-dividing CML CD34+ cells, identified using CFSE labeling, with 43% apoptosis observed in cells treated with 50 nM LAQ [n=5]. Combined LAQ and IM treatment also resulted in significant inhibition of CML progenitor proliferation measured in CFSE assays [n=4] and in the number of CML progenitors measured in methylcellulose progenitor assays [n=5]. In contrast to what has been described for BCR-ABL expressing cell lines and BC CML cells, LAQ treatment did not result in significant change in BCR-ABL levels in CML CD34+ cells on Western blotting. Significant down-regulation of MCL-1, a critical apoptosis regulator in early hematopoietic cells, was observed on Western blotting of CML cells treated with the LAQ and IM combination [n=5]. Levels of other anti-apoptotic proteins BCL-xL and BCL-2 were not affected. MCL-1 inhibition was not observed in normal CD34+ cells [n=4]. In contrast, normal cells treated with LAQ or LAQ and IM demonstrated enhanced levels of the pro-apoptotic protein Bim, but Bim levels were not increased in CML CD34+ cells. Q-PCR analysis revealed changes in MCL-1 and BIM mRNA levels in CML and normal CD34+ cells with LAQ and IM treatment that were consistent with the results of Western blotting. We conclude that treatment with the HDAC inhibitor LAQ in combination with imatinib results in significantly enhanced apoptosis of CML CD34+ cells, including non-dividing cells, compared with IM alone. Different mechanisms appear to be responsible for HDAC induced apoptosis in CML and normal CD34+ cells. Our results provide a strong rationale for future clinical application of HDAC inhibitors to enhance elimination of CML progenitors in combination with BCR-ABL tyrosine kinase inhibitors.

Antitumor effect of the histone deacetylase inhibitor LAQ824 in combination with 13-cis-retinoic acid in human malignant melanoma

Resistance to chemotherapy is a major hurdle in the treatment of malignant melanoma. Histone deacetylase (HDAC) inhibitors have been shown to have antitumor activity in different tumor types, including melanoma, and to reverse epigenetic repression of tumor suppressor genes, such as retinoic acid receptor beta (RARbeta). In this study, we tested the antitumor effect of the HDAC inhibitor LAQ824 in combination with 13-cis-retinoic acid (CRA) on two human melanoma cell lines both in vitro and in vivo. Treatment of LAQ824 showed a dose-dependent inhibitory effect on A2058 and HMV-I cell lines in a clonogenic assay. These cell lines were relatively resistance to CRA. On treatment with combination of LAQ824 and CRA, a greater inhibitory effect (up to 98%) was achieved compared with single agents. Lack of RARbeta2 gene expression was associated with histone acetylation and gene methylation at the promoter level. Treatment with LAQ824 restored retinoid sensitivity by reverting RARbeta2 epigenetic silencing. The biological effect of LAQ824 was associated with p21 induction in both cell lines but G(2) cell cycle arrest in A2058 and apoptosis in HMV-I cell line. The induction of apoptosis by LAQ824 was associated with increased reactive oxygen species and induction of SM22 gene expression in HMV-I but not in A2058 cell line. Administration of the free radical scavenger l-N-acetylcysteine blocked LAQ824 + CRA-mediated apoptosis in HMV-I cells, suggesting a primary role for reactive oxygen species generation in LAQ824 + CRA-associated lethality. Combination treatment showed 61% and 82% growth inhibition in A2058 and HMV-I tumors, respectively. Greater induction of in vivo apoptosis was observed in the HMV-I but not in the A2058 tumors treated with combination therapy compared with single agents. These results suggest that the HDAC inhibitor LAQ824 has a greater antitumor activity in combination with CRA in melanoma tumors but the degree of induced apoptosis may vary. Combination of HDAC inhibitors and retinoids represents a novel therapeutic approach for malignant melanoma that warrants clinical testing.

Histone deacetylase inhibitors deplete enhancer of zeste 2 and associated polycomb repressive complex 2 proteins in human acute leukemia cells

Human enhancer of zeste 2 (EZH2) protein belongs to the multiprotein polycomb repressive complex 2, which also includes suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). The polycomb repressive complex 2 complex possesses histone methyltransferase activity mediated by the Su(var)3-9, enhancer of zeste, and trithorax domain of EZH2, which methylates histone H3 on lysine (K)-27 (H3K27). In the present studies, we determined that treatment with the hydroxamate histone deacetylase inhibitor LBH589 or LAQ824 depleted the protein levels of EZH2, SUZ12, and EED in the cultured (K562, U937, and HL-60) and primary human acute leukemia cells. This was associated with decreased levels of trimethylated and dimethylated H3K27, with concomitant depletion of the homeobox domain containing HOXA9 and of MEIS1 transcription factors. Knockdown of EZH2 by EZH2 small interfering RNA also depleted SUZ12 and EED, inhibited histone methyltransferase activity, and reduced trimethylated and dimethylated H3K27 levels, with a concomitant loss of clonogenic survival of the cultured acute myelogenous leukemia (AML) cells. EZH2 small interfering RNA sensitized the AML cells to LBH589-mediated depletion of EZH2, SUZ12, and EED; loss of clonogenic survival; and LBH589-induced differentiation of the AML cells. These findings support the rationale to test anti-EZH2 treatment combined with hydroxamate histone deacetylase inhibitors as an antileukemia epigenetic therapy, especially against AML with coexpression of EZH2, HOXA9, and MEIS1 genes.

Class II Histone Deacetylases Are Associated with VHL-Independent Regulation of Hypoxia-Inducible Factor 1α

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) plays a critical role in transcriptional gene activation involved in tumor angiogenesis. A novel class of agents, the histone deacetylase (HDAC) inhibitors, has been shown to inhibit tumor angiogenesis and HIF-1 alpha protein expression. However, the molecular mechanism responsible for this inhibition remains to be elucidated. In the current study, we investigated the molecular link between HIF-1 alpha inhibition and HDAC inhibition. Treatment of the VHL-deficient human renal cell carcinoma cell line UMRC2 with the hydroxamic HDAC inhibitor LAQ824 resulted in a dose-dependent inhibition of HIF-1 alpha protein via a VHL-independent mechanism and reduction of HIF-1 alpha transcriptional activity. HIF-1 alpha inhibition by LAQ824 was associated with HIF-1 alpha acetylation and polyubiquitination. HIF-1 alpha immunoprecipitates contained HDAC activity. Then, we tested different classes of HDAC inhibitors with diverse inhibitory activity of class I versus class II HDACs and assessed their capability of targeting HIF-1 alpha. Hydroxamic acid derivatives with known activity against both class I and class II HDACs were effective in inhibiting HIF-1 alpha at low nanomolar concentrations. In contrast, valproic acid and trapoxin were able to inhibit HIF-1 alpha only at concentrations that are effective against class II HDACs. Coimmunoprecipitation studies showed that class II HDAC4 and HDAC6 were associated with HIF-1 alpha protein. Inhibition by small interfering RNA of HDAC4 and HDAC6 reduced HIF-1 alpha protein expression and transcriptional activity. Taken together, these results suggest that class II HDACs are associated with HIF-1 alpha stability and provide a rationale for targeting HIF-1 alpha with HDAC inhibitors against class II isozymes.

Effect of histone deacetylase inhibitor LAQ824 on antineoplastic action of 5-Aza-2′-deoxycytidine (decitabine) on human breast carcinoma cells

Epigenetic silencing of tumor suppressor genes (TSGs) by aberrant DNA methylation and chromatin deacetylation provides interesting targets for chemotherapeutic intervention by inhibitors of these events. 5-Aza-2'-deoxycytidine (decitabine, 5AZA-CdR) is a potent demethylating agent, which can reactivate TSGs silenced by aberrant DNA methylation. LAQ824 (LAQ) is a novel inhibitor of histone deacetylase (HDAC) that shows antineoplastic activity and can activate genes that produce cell cycle arrest. Both 5AZA-CdR and LAQ as single agents are currently under clinical investigation in patients with cancer. Previous reports indicate that the "cross-talk" between inhibitors of DNA methylation and HDAC can result in a synergistic activation of silent TSGs. These observations suggest that combination of these inhibitors may be an effective form of epigenetic therapy for breast cancer. The objective of our study was to determine if the combination of 5AZA-CdR and LAQ would show additive or synergistic antineoplastic activity on human MDA-MB-231 and MCF-7 breast carcinoma cells. The antineoplastic activity of these agents was evaluated by clonogenic assay and inhibition of DNA synthesis. The combination produced greater antineoplastic activity for the MDA-MB-231 tumor cells than either agent alone. For the MCF-7 tumor cells, there were signs of antagonism between 5AZA-CdR and LAQ when administered simultaneously. When a sequential schedule (first 5AZA-CdR followed by LAQ) was used, there were no signs of antagonism of the antineoplastic action for the MCF-7 tumor cells. The mechanism of this interaction is probably due to the reduction of progression of MCF-7 tumor cells into S phase by LAQ. This would interfere with the antineoplastic action of 5AZA-CdR, since it is an S phase specific agent. These studies demonstrated the importance of the schedule of administration of 5AZA-CdR and LAQ and may have application for future clinical trials on the treatment of breast cancer with these agents.

Histone Deacetylase Inhibitors LBH589 and LAQ824 Deplete Ezh2 and Associated Polycomb Repressive Complex 2/3 Proteins Resulting in Downregulation of HOXA9 and MEIS1 Expression in Human Acute Leukemia Cells.

Human enhancer of Zeste homolog (Ezh2) protein belongs to Polycomb Repressive Complex (PRC) 2, which also includes Eed and Suz12. Ezh2 has been shown to promote cellular transformation, and increased Ezh2 expression has been strongly correlated with the invasiveness of prostate and breast cancers. The enzymatically-active Ezh2-containing, PRC2 complex possesses histone methyl transferase (HMTase) activity mediated by the SET domain of Ezh2, which is involved in the methylation of histone (H) 3, lysine (K)-27 and −9. Through this, the PRC2 complex regulates the expression of homeobox domain containing HOX family of transcription factors including HOXA9 and MEIS1, which have been shown to be involved in human leukemogenesis. Co-expression of HOXA9 and MEIS1 is common in acute myeloid leukemia and collaborates in the leukemogenesis. In the present studies, we determined the effect of hydroxamate histone deacetylase inhibitors LBH589 and LAQ824 on Ezh2 and PRC2 complex proteins and their activity in the cultured (K562, LAMA-84, U937 and HL-60) and primary human AML cells. Exposure to 10 to 100 nM of LBH589 or LAQ824 for 24 hours, in a dose dependent manner depleted the protein levels of Ezh2, as well as reduced Suz12 and Eed levels in the cultured and primary leukemia cells. This was associated with decreased levels of the tri- and dimethylated K27 and increased acetylation of K27, both on H3. Correspondingly, these H3 modifications were associated with a significant decline in the levels of HOXA9 and MEIS1. As has been previously reported, treatment with LBH589 and LAQ824 induced p21, as well as caused cell cycle growth arrest in the G1 phase and apoptosis of the cultured and primary AML cells. We next determined whether knockdown of Ezh2 by siRNA to Ezh2 also induces growth inhibitory and cytotoxic effects against the leukemia cells. In the cultured and primary acute leukemia cells, knockdown of Ezh2 expression (by ~80%) by Ezh2 siRNA, but not by the control siRNA, was associated with depletion of Suz12 levels and increased H3K27 acetylation. This was associated with increase in the % of cells in the G1 phase of the cell cycle and significant inhibition of their clonogenic survival in colony growth assays. Co-treatment with LBH589 and siRNA to Ezh2 caused further decline in the Ezh2 levels and increased loss of clonogenic survival of the leukemia cells. These findings suggest that down modulation of Ezh2 and PRC2 complex and its HMTase activity may inhibit clonogenic survival of human acute myeloid leukemia cells. Additionally, combined effect of the knockdown of Ezh2 and its activity along with treatment with LBH589 or LAQ824 may have an improved anti-leukemia efficacy, especially against human AML where HOXA9 and MEIS1 genes are deregulated.

Melphalan Resistance in Multiple Myeloma Can Be Overcome by Proteasome Inhibition or Histone Deacetylation: Lack of Syngergism of the Combination.

Multiple Myeloma (MM) is a disease of monoclonal plasma cells in the bone marrow which has a transient response to classic chemotherapy. At diagnosis, induction chemotherapy followed by high-dose melphalan (HDM) with stem cell support is used in most patients to achieve a clinical response. Because all patients will ultimately relapse, the treatment of melphalan-refractory disease represents a major clinical challenge and new agents are needed to overcome melphalan resistance. We have investigated the anti-myeloma efficacy of two new classes of targeted agents, i.e. proteasome inhibition and histone deacetylation inhibition alone or in combination in the melphalan sensitive MM1S and the Melphalan refractory MM1MEL2000 cell lines. The IC50 values of Bortezomib (B), Melphalan (M) and LAQ824 (L) in MM1S were 2.1 nM, 1.9 uM and 1.7 nM, respectively and in MM1MEL2000 3.9 nM, 50 uM and 4.0 nM. Using isobologram analysis a synergysm between B and L was observed in the sensitive, however not in the melphalan refractory cell line. These data indicate that B proteasome inhibition and histone deacetylation inhibition may be effective ways to overcome melphalan resistance. However, the previously reported synergism between these drugs does not seem to occur in melphalan resistant cells. The gene expression profiles of these cell lines were analysed using the Affymetrix U133plus 2.0 gene chip before and after treatment with melfaphalan or the proteasome inhibitor B or the histone deacetylation inhibitor L or the combination of B and L.Genes that were highly expressed in the melphalan refractory derivate cell line MM1MEL2000 as compared with wild-type MM1S included GP M6B, ADAM23 and HTPAP. Following melphalan exposure, TMF1, a CEBp glucocorticoid interaction factor, WHSC1L1, a MMSET homologue with EGF like domain and several transcription factors had highly increased expression as compared to MM1S. With exposure to B combined with L, increased expression in MM1MEL2000 over MM1S was observed for GTP exchange factor TIAM1 which interacts with RAS and JNK, and the lymphoid enhancer factor, a notch transcription factor. It is concluded that Bortezomib and the histone deacetylase inhibitor LAQ824 are effective agents to overcome melphalan resistance in multiple myeloma. However, the combination fails to show the synergism observed in melphalan sensitive cells. Gene analysis sofar does not provide a clear explanation for this lack of synergism. A comprehensive summary of the observed shifts of gene expression profiles in melphalan resistant cells following exposure to these agents, will be presented.

Potentiation of the lethality of the histone deacetylase inhibitor LAQ824 by the cyclin-dependent kinase inhibitor roscovitine in human leukemia cells

Interactions between the novel histone deacetylase inhibitor LAQ824 and the cyclin-dependent kinase inhibitor roscovitine were examined in human leukemia cells. Pretreatment (24 hours) with a subtoxic concentration of LAQ824 (30 nmol/L) followed by a minimally toxic concentration of roscovitine (10 micromol/L; 24 hours) resulted in greater than additive effects on apoptosis in U937, Jurkat, and HL-60 human leukemia cells and blasts from three patients with acute myelogenous leukemia. These events were associated with enhanced conformational changes in Bax; mitochondrial release of cytochrome c, Smac/DIABLO, and apoptosis-inducing factor; and a marked increase in caspase activation. LAQ824/roscovitine-treated cells displayed caspase-dependent down-regulation of p21(CIP1) and Mcl-1 and a pronounced caspase-independent reduction in X-linked inhibitor of apoptosis (XIAP) expression. The lethality of this regimen was significantly attenuated by ectopic expression of XIAP, a nuclear localization signal-defective p21(CIP1) mutant, Mcl-1, and Bcl-2. Combined exposure to LAQ824 and roscovitine resulted in a significant reduction in XIAP mRNA levels and diminished phosphorylation of the carboxyl-terminal domain of RNA polymerase II. Notably, roscovitine blocked LAQ824-mediated differentiation. Finally, LAQ824 and roscovitine individually and in combination triggered an increase in generation of reactive oxygen species; moreover, coadministration of the free radical scavenger N-acetylcysteine prevented LAQ824/roscovitine-mediated mitochondrial injury and apoptosis. Collectively, these findings suggest that combined treatment of human leukemia cells with LAQ824 and roscovitine disrupts maturation and synergistically induces apoptosis, lending further support for an antileukemic strategy combining novel histone deacetylase and cyclin-dependent kinase inhibitors.

RETRACTION: The Histone Deacetylase Inhibitor LAQ824 Induces Human Leukemia Cell Death through a Process Involving XIAP Down-Regulation, Oxidative Injury, and the Acid Sphingomyelinase-Dependent Generation of Ceramide

Determinants of differentiation and apoptosis induction by the novel histone deacetylase inhibitor (HDACI) LAQ824 were examined in human leukemia cells (U937 and Jurkat). Exposure of U937 cells to a low concentration of LAQ824 (30 nM) resulted in a delayed (2 h) increase in reactive oxygen species (ROS), induction of p21(WAF1/CIP1), pRb dephosphorylation, growth arrest of cells in G(0)/G(1) phase, and differentiation. On the other hand, exposure of cells to a higher concentration of LAQ824 (75 nM) resulted in the early (30 min) generation of ROS, arrest of cells in G(2)/M phase, down-regulation of XIAP (at the transcriptional level) and Mcl-1 (through a caspase-mediated process), the acid sphingomyelinase-dependent generation of ceramide, and profound mitochondrial injury, caspase activation, and apoptosis. LAQ824-induced lethality in U937 cells did not involve the extrinsic apoptotic pathway, nor was it associated with death receptor up-regulation; instead, it was markedly inhibited by ectopic expression of Bcl-2, Bcl-x(L), XIAP, and Mcl-1. The free radical scavenger N-acetyl cysteine blocked LAQ824-mediated ROS generation, mitochondrial injury, Mcl-1 down-regulation, ceramide generation, and apoptosis, suggesting a primary role for oxidative injury in LAQ824 lethality. Together, these findings indicate that LAQ824-induced lethality represents a multifactorial process in which LAQ824-mediated ROS generation is necessary but not sufficient to induce apoptosis, and that the degree of XIAP and Mcl-1 down-regulation and ceramide generation determines whether this agent engages a maturation rather than an apoptotic program.

Chemical ablation of androgen receptor in prostate cancer cells by the histone deacetylase inhibitor LAQ824

Androgen receptor plays a critical role in the development of primary as well as advanced hormone-refractory prostate cancer. Therefore, ablation of androgen receptor from prostate cancer cells is an interesting concept for developing a new therapy not only for androgen-dependent prostate cancer but also for metastatic hormone-refractory prostate cancer, for which there is no effective treatment available. We report here that LAQ824, a cinnamyl hydroxamatic acid histone deacetylase inhibitor currently in human clinical trials, effectively depleted androgen receptor in prostate cancer cells at nanomolar concentrations. LAQ824 seemed capable of depleting both the mutant and wild-type androgen receptors in either androgen-dependent and androgen-independent prostate cancer cells. Although LAQ824 may exert its effect through multiple mechanisms, several lines of evidence suggest that inactivation of the heat shock protein-90 (Hsp90) molecular chaperone is involved in LAQ824-induced androgen receptor depletion. Besides androgen receptor, LAQ824 reduced the level of Hsp90 client proteins HER-2 (ErbB2), Akt/PKB, and Raf-1 in LNCaP cells. Another Hsp90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), also induced androgen receptor diminution. LAQ824 induced Hsp90 acetylation in LNCaP cells, which resulted in inhibition of its ATP-binding activity, dissociation of Hsp90-androgen receptor complex, and proteasome-mediated degradation of androgen receptor. Consequently, LAQ824 blocked androgen-induced prostate-specific antigen production in LNCaP cells. LAQ824 effectively inhibited cell proliferation and induced apoptosis of these prostate cancer cells. These results reveal that LAQ824 is a potent agent for depletion of androgen receptor and a potential new drug for prostate cancer.

Cardiac monitoring in phase I trials of a novel histone deacetylase (HDAC) inhibitor LAQ824 in patients with advanced solid tumors and hematologic malignancies

3131 Introduction: LAQ824, a novel cinnamic acid hydroxamate, inhibits HDAC activity (IC50, 0.03 μM) and the hERG channel(IC50, 10 μM). Methods: In 3 phase I studies, LAQ824 was administered as a 3-hr intravenous infusion on several dosing schedules, principally days 1–3 of a 21-day cycle, to adult pts with advanced solid (studies 2102 and 2107) or hematologic (study 2101) malignancies. Pts with impaired cardiac function were excluded; drugs known to prolong QT interval were prohibited. Serial digital ECGs were performed at baseline (4 ECGs), on days of dosing and post-dose each cycle. ECG data were processed in a core lab by manual analysis. Cardiac enzymes were assessed on days of dosing. LVEF was assessed by MUGA scan or echocardiogram each cycle. Results: 77 pts (median age: 60 yrs; 48 males, 29 females) were treated at 11 dose levels, range 6 - 200 mg/m2/day. PK analysis showed dose proportionality, T1/2of 6 - 26 hrs, and 1.5 fold accumulation at steady state. There was considerable inter-patient var...