Molecular and cellular basis for the anti-proliferative effects of the HDAC inhibitor LAQ824.

We have developed a cinnamic hydroxamic class of histone deacetylase inhibitors of which a prototype was designated as NVP-LAQ824. NVP-LAQ824, inhibits histone deacetylase enzymatic activities in vitro and transcriptionally activated the p21 promoter in reporter gene assays. When tested on a variety of solid tumour cell lines, NVP-LAQ824 exhibited selective anti-proliferative effects, inducing cell growth inhibition in some, while inducing cell death in others. To induce cell death, a minimum of 16 h exposure to NVP-LAQ824 is required. Flow cytometry studies revealed that both tumour cell lines and normal diploid fibroblasts arrested in the G2/M phase of the cell cycle after compound treatment. However, an increased sub-G1 population at 48 h (reminiscent of apoptotic cells) was only observed in the cancer cell lines. Annexin V staining data confirmed that NVP-LAQ824 induced apoptosis in tumour cells, but not in normal cells. To relate HDAC inhibition to the anti-proliferative effects of NVP-LAQ824, expression of HDAC 1 was inhibited using antisense and this was sufficient to activate p21 expression, hypophosphorylate Rb and inhibit cell growth. Furthermore, tumour cells treated with NVP-LAQ824 caused acetylation of HSP90 and degradation of its cargo oncoproteins. Finally, NVP-LAQ824 exhibited antitumour effects in a xenograft animal model. To determine if NVP-LAQ824 inhibited histone deacetylases in vivo, tumours treated with the drug were immunoblotted with an antibody specific for acetylated histones H3 and H4 and the results indicated increased histone H3 and 114 acetylation levels in NVP-LAQ824 treated cancer cells. Together, our data indicated that the activity of NVP-LAQ824 was consistent with its intended mechanism of action. This novel HDAC inhibitor is currently in clinical trials as an anticancer agent.

Selective growth inhibition of tumor cells by a novel histone deacetylase inhibitor, NVP-LAQ824.

We have synthesized a histone deacetylase inhibitor, NVP-LAQ824, a cinnamic hydroxamic acid, that inhibited in vitro enzymatic activities and transcriptionally activated the p21 promoter in reporter gene assays. NVP-LAQ824 selectively inhibited growth of cancer cell lines at submicromolar levels after 48-72 h of exposure, whereas higher concentrations and longer exposure times were required to retard the growth of normal dermal human fibroblasts. Flow cytometry studies revealed that both tumor and normal cells arrested in the G(2)-M phase of the cell cycle after compound treatment. However, an increased sub-G(1) population at 48 h (reminiscent of apoptotic cells) was observed only in the cancer cell line. Annexin V staining data supported our hypothesis that NVP-LAQ824 induced apoptosis in tumor and transformed cells but not in normal cells. Western blotting experiments showed an increased histone H3 and H4 acetylation level in NVP-LAQ824-treated cancer cells, suggesting that the likely in vivo target of NVP-LAQ824 was histone deacetylase(s). Finally, NVP-LAQ824 exhibited antitumor effects in a xenograft animal model. Together, our data indicated that the activity of NVP-LAQ824 was consistent with its intended mechanism of action. This novel histone deacetylase inhibitor is currently in clinical trials as an anticancer agent.

N-hydroxy-3-phenyl-2-propenamides as novel inhibitors of human histone deacetylase with in vivo antitumor activity: discovery of (2E)-N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2-propenamide (NVP-LAQ824).

A series of N-hydroxy-3-phenyl-2-propenamides were prepared as novel inhibitors of human histone deacetylase (HDAC). These compounds were potent enzyme inhibitors, having IC(50)s < 400 nM in a partially purified enzyme assay. However, potency in cell growth inhibition assays ranged over 2 orders of magnitude in two human carcinoma cell lines. Selected compounds having cellular IC(50) < 750 nM were tested for maximum tolerated dose (MTD) and for efficacy in the HCT116 human colon tumor xenograft assay. Four compounds having an MTD > or = 100 mg/kg were selected for dose-response studies in the HCT116 xenograft model. One compound, 9 (NVP-LAQ824), had significant dose-related activity in the HCT116 colon and A549 lung tumor models, high MTD, and low gross toxicity. On the basis, in part, of these properties, 9 has entered human clinical trials in 2002.

Inhibitors of human histone deacetylase: synthesis and enzyme and cellular activity of straight chain hydroxamates.

Inhibitors of histone deacetylase (HDAC) have been shown to induce terminal differentiation of human tumor cell lines and to have antitumor effects in vivo. We have prepared analogues of suberoylanilide hydroxamic acid (SAHA) and trichostatin A and have evaluated them in a human HDAC enzyme inhibition assay, a p21(waf1) (p21) promoter assay, and in monolayer growth inhibition assays. One compound, 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]-benzamide, was found to affect the growth of a panel of eight human tumor cell lines differentially.

Isolation and characterization of a novel class II histone deacetylase, HDAC10.

A novel histone deacetylase, HDAC10, was isolated from a mixed tissue human cDNA library. HDAC10 was classified as a class II subfamily member based upon similarity to HDAC6. The genomic structure of HDAC10 was found to consist of 20 exons. HDAC10 has two sequence variants, HDAC10v1 and HDAC10v2, and two transcripts were detectable by Northern blot analysis. HDAC10v1 and HDAC10v2 were found to be identical through exon 17 but diverged after this exon. HDAC10v2 has an 82-bp alternate exon that generates a frameshift and shortens the sequence by 11 amino acids. In this study, the characterization of HDAC10v1 was performed. HDAC10v1 has an N-terminal catalytic domain, two putative C-terminal retinoblastoma protein binding domains, and a nuclear hormone receptor binding motif. The HDAC10v1 enzyme was found to be catalytically active based upon its ability to deacetylate a (3)H-acetylated histone H4 N-terminal peptide. Immunofluorescence detection of transfected HDAC10v1-FLAG indicated that the enzyme is a nuclear protein. Furthermore, coimmunoprecipitation experiments indicated that HDAC10v1 associated with HDAC2 and SMRT (silencing mediator for retinoid and thyroid hormone receptors). In addition, based upon the public data base, a single nucleotide polymorphism was found in the C terminus of HDAC10 which changes a Gly residue to Cys, suggesting that HDAC10 molecules containing these single nucleotide polymorphisms may be folded improperly. HDAC10 extends the HDAC superfamily and adds to a growing number of HDACs that have been found to have splice variants, suggesting that RNA processing may play a role in mediating the activity of HDACs.