Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations, invariably involving the retinoic acid receptor alpha (RAR alpha) gene fused to one of several distinct loci, including the PML or PLZF genes, involved in t(15;17) or t(11;17), respectively. Patients with t(15;17) APL respond well to retinoic acid (RA) and other treatments, whereas those with t(11;17) APL do not. The PML-RAR alpha and PLZF-RAR alpha fusion oncoproteins function as aberrant transcriptional repressors, in part by recruiting nuclear receptor-transcriptional corepressors and histone deacetylases (HDACs). Transgenic mice harboring the RAR alpha fusion genes develop forms of leukemia that faithfully recapitulate both the clinical features and the response to RA observed in humans with the corresponding translocations. Here, we investigated the effects of HDAC inhibitors (HDACIs) in vitro and in these animal models. In cells from PLZF-RAR alpha/RAR alpha-PLZF transgenic mice and cells harboring t(15;17), HDACIs induced apoptosis and dramatic growth inhibition, effects that could be potentiated by RA. HDACIs also increased RA-induced differentiation. HDACIs, but not RA, induced accumulation of acetylated histones. Using microarray analysis, we identified genes induced by RA, HDACIs, or both together. In combination with RA, all HDACIs tested overcame the transcriptional repression exerted by the RAR alpha fusion oncoproteins. In vivo, HDACIs induced accumulation of acetylated histones in target organs. Strikingly, this combination of agents induced leukemia remission and prolonged survival, without apparent toxic side effects.

Histone deacetylase inhibitors as new cancer drugs.

Histone deacetylase inhibitors are potent inducers of growth arrest, differentiation, or apoptotic cell death in a variety of transformed cells in culture and in tumor bearing animals. Histone deacetylases and the family of histone acetyl transferases are involved in determining the acetylation of histones, which play a role in regulation of gene expression. Radiograph crystallographic studies reveal that the histone deacetylase inhibitors, suberoylanilide hydroxamic acid and trichostatin A, fit into the catalytic site of histone deacetylase, which has a tubular structure with a zinc atom at its base. The hydroxamic acid moiety of the inhibitor binds to the zinc. Histone deacetylase inhibitors cause acetylated histones to accumulate in both tumor and peripheral circulating mononuclear cells. Accumulation of acetylated histones has been used as a marker of the biologic activity of the agents. Hydroxamic acid-based histone deacetylase inhibitors limit tumor cell growth in animals with little or no toxicity. These compounds act selectively on genes, altering the transcription of only approximately 2% of expressed genes in cultured tumor cells. A number of proteins other than histones are substrates for histone deacetylases. The role that these other targets play in histone deacetylase inducement of cell growth arrest, differentiation, or apoptotic cell death is not known. This review summarizes the characteristics of a variety of inhibitors of histone deacetylases and their effects on transformed cells in culture and tumor growth in animal models. Several structurally different histone deacetylase inhibitors are in phase I or II clinical trials in patients with cancers.

Cloning and characterization of a histone deacetylase, HDAC9.

Histone deacetylase (HDAC) catalyzes the removal of the acetyl group from the lysine residues in the N-terminal tails of nucleosomal core histones. Eight human HDACs have been identified so far. Here, we report the identification of a ninth member of the HDAC family, designated HDAC9. HDAC9 is a class II HDAC and its gene resides on human chromosome 7. HDAC9 has several alternatively spliced isoforms. One of these isoforms is histone deacetylase-related protein or myocyte enhancer-binding factor 2-interacting transcriptional repressor that we and others have previously reported and which does not possess an HDAC catalytic domain. The longest of the HDAC9 isoforms contains 1,011 aa. The isoform, designated HDAC9a, is 132 aa shorter at the C terminus than HDAC9. Also, we have identified isoforms of HDAC9 that lack the nuclear localization signal. Similar to histone deacetylase-related protein, HDAC9 transcripts are expressed at high levels in brain and skeletal muscle. The ratio of HDAC9 and HDAC9a transcripts differs among the tissues examined. HDAC9 and HDAC9a contain the HDAC catalytic domain, and Flag-tagged HDAC9 and HDAC9a possess deacetylase activity. HDAC9 and HDAC9a also repress myocyte enhancer-binding factor 2-mediated transcription. In the present study, we have identified HDAC9 and a number of alternatively spliced isoforms of HDAC9 with potentially different biological activities.

The histone deacetylase inhibitor, CBHA, inhibits growth of human neuroblastoma xenografts in vivo, alone and synergistically with all-trans retinoic acid.

Histone deacetylase inhibitors (HDACIs) inhibit the growth of a variety of transformed cells in culture. We demonstrated previously that the hybrid-polar HDACI m-carboxycinnamic acid bis-hydroxamide (CBHA) induces apoptosis of human neuroblastoma in vitro and is effective in lower doses when combined with retinoids. The current study investigates the effect of CBHA on the growth of human neuroblastoma in vivo, both alone and in combination with all-trans retinoic acid (atRA), using a severe combined immunodeficiency-mouse xenograft model. CBHA (50, 100, and 200 mg/kg/day) inhibited growth of SMS-KCN-69n tumor xenografts in a dose-dependent fashion, with 200 mg/kg CBHA resulting in a complete suppression of tumor growth. The efficacy of 50 and 100 mg/kg CBHA was enhanced by the addition of 2.5 mg/kg atRA. This dose of atRA was ineffective when administered alone. Treatment was accompanied by mild weight loss in all groups except the lowest dose of CBHA. Our results suggest HDACIs alone or combined with retinoids may have therapeutic utility for neuroblastoma.

Inhibition of transformed cell growth and induction of cellular differentiation by pyroxamide, an inhibitor of histone deacetylase.

PURPOSE: We have synthesized a series of hybrid polar compounds that induce differentiation and/or apoptosis of various transformed cells. These agents are also potent inhibitors of histone deacetylases (HDACs). Pyroxamide (suberoyl-3-aminopyridineamide hydroxamic acid) is a new member of this class of compounds that is currently under development as an anticancer agent. We investigated the activity of pyroxamide as an inducer of differentiation and/or apoptosis in transformed cells. EXPERIMENTAL DESIGN AND RESULTS: Pyroxamide, at micromolar concentrations, induced terminal differentiation in murine erythroleukemia (MEL) cells and caused growth inhibition by cell cycle arrest and/or apoptosis in MEL, prostate carcinoma, bladder carcinoma, and neuroblastoma cells. Administration of pyroxamide (100 or 200 mg/kg/day) to nude mice at doses that caused little evident toxicity significantly suppressed the growth of s.c. CWR22 prostate cancer xenografts. Despite the potent growth-inhibitory effects of pyroxamide in this tumor model, serum prostate-specific antigen levels in control versus pyroxamide-treated mice were not significantly different. Pyroxamide is a potent inhibitor of affinity-purified HDAC1 (ID(50) = 100 nM) and causes the accumulation of acetylated core histones in MEL cells cultured with the agent. Human CWR22 prostate tumor xenografts from mice treated with pyroxamide (100 or 200 mg/kg/day) showed increased levels of histone acetylation and increased expression of the cell cycle regulator p21/WAF1, compared with tumors from vehicle-treated control animals. CONCLUSIONS: The findings suggest that pyroxamide may be a useful agent for the treatment of malignancy and that induction of p21/WAF1 in transformed cells by pyroxamide may contribute to the antitumor effects of this agent.

Histone deacetylases and cancer: causes and therapies.

Together, histone acetyltransferases and histone deacetylases (HDACs) determine the acetylation status of histones. This acetylation affects the regulation of gene expression, and inhibitors of HDACs have been found to cause growth arrest, differentiation and/or apoptosis of many tumours cells by altering the transcription of a small number of genes. HDAC inhibitors are proving to be an exciting therapeutic approach to cancer, but how do they exert this effect?

Histone deacetylase 4 associates with extracellular signal-regulated kinases 1 and 2, and its cellular localization is regulated by oncogenic Ras.

Histone deacetylase 4 (HDAC4) is a member of a family of enzymes that catalyze the removal of acetyl groups from core histones, resulting in a compact chromatin structure that is generally associated with repressed gene transcription. Protein phosphorylation has been implicated in the regulation of the corepressor activity of the deacetylase. Here we report that serine/threonine kinases are found in association with HDAC4 and phosphorylate HDAC4 in vitro, and HDAC4 is phosphorylated in cells. The extracellular signal-regulated kinases 1 and 2 (ERK1/2), also known as p44(MAPK) and p42(MAPK), respectively, are two of the kinases associated with HDAC4. ERK1/2 are components of the Ras-mitogen-activated protein kinase (MAPK) signal transduction pathway. Activation of the Ras-MAPK pathway by expression of oncogenic Ras or constitutively active MAPK/ERK kinase 1 results in an increased percentage of cells (from approximately 10% to approximately 70%) that express HDAC4 in the nucleus in C2C12 myoblast cells. In cells transfected with oncogenic Ras, nuclear HDAC4 is associated with kinase activity. Our results provide evidence that protein kinase activity is present in a protein complex with HDAC4 and directly links the Ras-MAPK signal transduction pathway to a mechanism for chromatin remodeling (i.e., histone deacetylation).

Suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase, suppresses the growth of prostate cancer cells in vitro and in vivo.

Suberoylanilide hydroxamic acid (SAHA) is the prototype of a family of hybrid polar compounds that induce growth arrest in transformed cells and show promise for the treatment of cancer. SAHA induces differentiation and/or apoptosis in certain transformed cells in culture and is a potent inhibitor of histone deacetylases. In this study, we examined the effects of SAHA on the growth of human prostate cancer cells in culture and on the growth of the CWR22 human prostate xenograft in nude mice. SAHA suppressed the growth of the LNCaP, PC-3, and TSU-Pr1 cell lines at micromolar concentrations (2.5-7.5 microM). SAHA induced dose-dependent cell death in the LNCaP cells. In mice with transplanted CWR222 human prostate tumors, SAHA (25, 50, and 100 mg/kg/day) caused significant suppression of tumor growth compared with mice receiving vehicle alone; treatment with 50 mg/kg/day resulted in a 97% reduction in the mean final tumor volume compared with controls. At this dose, there was no detectable toxicity as evaluated by weight gain and necropsy examination. Increased accumulation of acetylated core histones was detected in the CWR22 tumors within 6 h of SAHA administration. SAHA induced prostate-specific antigen mRNA expression in CWR22 prostate cancer cells, resulting in higher levels of serum prostate-specific antigen than predicted from tumor volume alone. The results suggest that hydroxamic acid-based hybrid polar compounds inhibit prostate cancer cell growth and may be useful, relatively nontoxic agents for the treatment of prostate carcinoma.

Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells.

Histone deacetylase (HDAC) inhibitors have been shown to be potent inducers of growth arrest, differentiation, and/or apoptotic cell death of transformed cells in vitro and in vivo. One class of HDAC inhibitors, hydroxamic acid-based hybrid polar compounds (HPCs), induce differentiation at micromolar or lower concentrations. Studies (x-ray crystallographic) showed that the catalytic site of HDAC has a tubular structure with a zinc atom at its base and that these HDAC inhibitors, such as suberoylanilide hydroxamic acid and trichostatin A, fit into this structure with the hydroxamic moiety of the inhibitor binding to the zinc. HDAC inhibitors cause acetylated histones to accumulate in both tumor and normal tissues, and this accumulation can be used as a marker of the biologic activity of the HDAC inhibitors. Hydroxamic acid-based HPCs act selectively to inhibit tumor cell growth at levels that have little or no toxicity for normal cells. These compounds also act selectively on gene expression, altering the expression of only about 2% of the genes expressed in cultured tumor cells. In general, chromatin fractions enriched in actively transcribed genes are also enriched in highly acetylated core histones, whereas silent genes are associated with nucleosomes with a low level of acetylation. However, HDACs can also acetylate proteins other than histones in nucleosomes. The role that these other targets play in the induction of cell growth arrest, differentiation, and/or apoptotic cell death has not been determined. Our working hypothesis is that inhibition of HDAC activity leads to the modulation of expression of a specific set of genes that, in turn, result in growth arrest, differentiation, and/or apoptotic cell death. The hydroxamic acid-based HPCs are potentially effective agents for cancer therapy and, possibly, cancer chemoprevention.

Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation.

Histone deacetylases (HDACs) catalyze the removal of acetyl groups on the amino-terminal lysine residues of core nucleosomal histones. This activity is associated generally with transcriptional repression. We have reported previously that inhibition of HDAC activity by hydroxamic acid-based hybrid polar compounds, such as suberoylanilide hydroxamic acid (SAHA), induces differentiation and/or apoptosis of transformed cells in vitro and inhibits tumor growth in vivo. SAHA is a potentially new therapeutic approach to cancer treatment and is in Phase I clinical trials. In several tumor cell lines examined, HDAC inhibitors alter the expression of less than 1% of expressed genes, including the cell cycle kinase inhibitor p21(WAF1). In T24 bladder carcinoma cells, SAHA induces up to a 9-fold increase in p21(WAF1) mRNA and protein, which is, at least in part, because of an increase in the rate of transcription of the gene. SAHA causes an accumulation of acetylated histones H3 and H4 in total cellular chromatin by 2 h, which is maintained through 24 h of culture. An increase in the accumulation of acetylated H3 and H4 was detected throughout the p21(WAF1) promoter and the structural gene after culture with SAHA. The level of histone acetylation did not change in chromatin associated with the actin and p27 genes, and their mRNA expression was not altered during culture of T24 cells with SAHA. Thus, the present findings indicate that the induction of p21(WAF1) by SAHA is regulated, at least in part, by the degree of acetylation of the gene-associated histones and that this induced increase in acetylation is gene selective.

Identification of a transcriptional repressor related to the noncatalytic domain of histone deacetylases 4 and 5.

Histone deacetylases (HDACs) are involved in regulating transcription by modifying the core histones of the nucleosome. To date, six HDACs have been identified in mammalian cells: the yeast RPD3 homologs HDAC1, 2, and 3 and the yeast HDA1 homologs HDAC4, 5, and 6. HDAC4 and HDAC5 contain a noncatalytic N-terminal domain. Herein, we report the identification of a protein HDRP (HDAC-related protein) that shares 50% identity in deduced amino acid sequence to the noncatalytic N-terminal domain of HDAC4 and 5. The steady-state levels of HDRP mRNA are high in human brain, heart, and skeletal muscle and low in the several other tissues. HDRP has an apparent molecular mass of approximately 75 kDa. HDRP does not possess intrinsic HDAC activity but forms complexes with both HDAC1 and HDAC3. HDRP represses both basal and activated transcription in transient transfection assays when tethered to DNA as a Gal4-fusion protein. HDAC inhibitors do not reverse transcriptional repression mediated by Gal4-HDRP. Thus, HDRP is a transcriptional repressor and can repress transcription in the presence of HDAC inhibitors.

Hybrid polar histone deacetylase inhibitor induces apoptosis and CD95/CD95 ligand expression in human neuroblastoma.

Inhibitors of histone deacetylase (HDAC) have been shown to have both apoptotic and differentiating effects on various tumor cells. M-carboxycinnamic acid bishydroxamide (CBHA) is a recently developed hybrid polar compound structurally related to hexamethylene bisacetamide. CBHA is a potent inhibitor of HDAC activity. CBHA induces cellular growth arrest and differentiation in model tumor systems. We undertook an investigation of the effects of CBHA on human neuroblastoma cell lines in vitro. When added to cultures of a panel of neuroblastoma cell lines, CBHA induced the accumulation of acetylated histones H3 and H4, consistent with the inhibition of HDAC. Concentrations of CBHA between 0.5 microM and 4 microM led to apoptosis in nine of nine neuroblastoma cell lines. Apoptosis was assessed by DNA fragmentation analysis and the appearance of a sub-G1 (<2N ploidy) population by flow cytometric analysis. The addition of a caspase inhibitor (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) completely abrogated CBHA-induced apoptosis in three of three cell lines. The addition of cycloheximide greatly reduced CBHA-induced apoptosis, suggesting that apoptotic induction was dependent on de novo protein synthesis. In addition, CBHA induced the expression of both CD95 (APO-1/Fas) and CD95 ligand within 12 h. The effect of CBHA on human neuroblastoma cells suggests that this agent and structurally related synthetic hybrid polar compounds have therapeutic potential for the treatment of this malignancy.

Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors.

Histone deacetylases (HDACs) mediate changes in nucleosome conformation and are important in the regulation of gene expression. HDACs are involved in cell-cycle progression and differentiation, and their deregulation is associated with several cancers. HDAC inhibitors, such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), have anti-tumour effects, as they can inhibit cell growth, induce terminal differentiation and prevent the formation of tumours in mice models, and they are effective in the treatment of promyelocytic leukemia. Here we describe the structure of the histone deacetylase catalytic core, as revealed by the crystal structure of a homologue from the hyperthermophilic bacterium Aquifex aeolicus, that shares 35.2% identity with human HDAC1 over 375 residues, deacetylates histones in vitro and is inhibited by TSA and SAHA. The deacetylase, deacetylase-TSA and deacetylase-SAHA structures reveal an active site consisting of a tubular pocket, a zinc-binding site and two Asp-His charge-relay systems, and establish the mechanism of HDAC inhibition. The residues that make up the active site and contact the inhibitors are conserved across the HDAC family. These structures also suggest a mechanism for the deacetylation reaction and provide a framework for the further development of HDAC inhibitors as antitumour agents.

Cloning of the cDNA encoding phenylalanyl tRNA synthetase regulatory alpha-subunit-like protein whose expression is down-regulated during differentiation.

Hybrid polar compounds (HPCs), such as suberoylanilide hydroxamic acid (SAHA), induce differentiation of transformed cells. Differential display of RNA was used to identify genes whose expression is changed during SAHA-induced differentiation of murine erythroleukemia (MEL) cells. One such cDNA was identified whose mRNA level decreased by 50% after 8h of SAHA treatment as determined by Northern blot analysis. The full-length cDNA (1944bp in length) was cloned by sequencing of an EST clone and rapid amplification of 5' cDNA ends (5'-RACE). The predicted amino acid sequence is 589 amino acids and shares 45% identity with the yeast cytoplasmic phenylalanyl tRNA synthetase (PheRS) regulatory alpha-subunit. Human EST clones which share over 90% identity of predicted amino acid sequence with this cDNA map to chromosome 2 near the paired box homeotic gene 3 (PAX3) locus, a region syngenic to mouse chromosome 1. This is the first report of the cloning of the full-length cDNA for the murine PheRS regulatory alpha-subunit-like protein. The level of PheRS alpha-subunit-like mRNA is regulated during differentiation but not during cell cycle progression.

Photoaffinity labeling and mass spectrometry identify ribosomal protein S3 as a potential target for hybrid polar cytodifferentiation agents.

The ability of a novel class of hybrid polar compounds (HPCs) to induce differentiation and consequent cessation of proliferation of transformed cells has led to their development as potential chemotherapeutic agents in the treatment of cancer. Suberoylanilide hydroxamic acid (SAHA) is a prototype of a family of hydroxamic acid based compounds (SAHA-like HPCs) that can, at micromolar concentrations, induce a variety of transformed cell lines to differentiate. The mechanism of action of the HPCs is not entirely understood. Searching for a cellular target of the SAHA-like HPCs, we synthesized a photoaffinity labeling reagent structurally based on SAHA, and probed for SAHA-binding proteins in murine erythroleukemia (MEL) cells. Photoaffinity labeling in cell free extracts identified a 32-kDa protein (p32) that was specifically labeled by the photoaffinity reagent. Cell fractionation assays localized p32 to the P100 fraction. p32 was partially purified and identified by mass spectrometry as the 40 S ribosomal protein S3. Expression of epitope-tagged S3 in bacterial lysates followed by photoaffinity labeling confirmed its specific labeling. Identification of a cytodifferentiation agent target may shed light on the mechanism by which the SAHA-like HPCs exert their antitumor effects.

Chemoprevention of carcinogen-induced mammary tumorigenesis by the hybrid polar cytodifferentiation agent, suberanilohydroxamic acid (SAHA).

Hybrid Polar Cytodifferentiation (HPC) agents represent a novel class of anticancer compounds which act by inducing terminal differentiation and/or apoptosis rather than by cytotoxic action. Among these are HPC agents such as hexamethylenebisacetamide (HMBA) and more potent 2nd generation hybrid/polar compounds such as suberanilohydroxamic acid (SAHA). As of the present, most studies on HPC agents have focused on cancers of the hematopoietic system rather than solid epithelial tumors. The objective of the present study therefore was to assess the chemopreventive action of these two related compounds in the N-methylnitrosourea (NMU)-induced rat mammary tumor model. Female Sprague-Dawley rats were fed diets containing 450 and 900 ppm, SAHA and 1000 and 2000 ppm HMBA, starting one week prior to NMU administration and continued for a period of 18 weeks. Mammary tumor development was monitored by palpation throughout the study, and at termination tumor incidence, number, multiplicity, latency and volume were determined. Weight gain was measured biweekly throughout the study. The salient results were as follows: SAHA at 900 ppm reduced NMU-induced mammary tumor incidence by 40%, total tumors by 66%, mean tumor multiplicity by 43% and mean tumor volume by 78%, with no detectable toxic side effects. HMBA exerted no tumor inhibiting effects at either concentration. This study represents the first demonstration that an HPC agent, namely SAHA, can inhibit the development of a chemically-induced, solid, epithelial tumor, at a relatively low dose (approximately 13 mgs/rat/day) without untoward side effects.

A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases.

Hybrid polar compounds (HPCs) have been synthesized that induce terminal differentiation and/or apoptosis in various transformed cells. We have previously reported on the development of the second-generation HPCs suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bishydroxamide (CBHA) that are 2,000-fold more potent inducers on a molar basis than the prototype HPC hexamethylene bisacetamide (HMBA). Herein we report that CBHA and SAHA inhibit histone deacetylase 1 (HDAC1) and histone deacetylase 3 (HDAC3) activity in vitro. Treatment of cells in culture with SAHA results in a marked hyperacetylation of histone H4, but culture with HMBA does not. Murine erythroleukemia cells developed for resistance to SAHA are cross-resistant to trichostatin A, a known deacetylase inhibitor and differentiation inducer, but are not cross-resistant to HMBA. These studies show that the second-generation HPCs, unlike HMBA, are potent inhibitors of HDAC activity. In this sense, HMBA and the second-generation HPCs appear to induce differentiation by different pathways.