pci-34051 and Neuroblastoma

Histone deacetylase 8 (HDAC8) is overexpressed in multiple cancers and lack of effective chemical probes which could detect and visualize HDAC8 in tumor cells and tissues remains unsolved. In this work, three novel turn-on HDAC8 fluorescent probes 17-19 derived from solvatochromic fluorophore 4-sulfamonyl-7-aminobenzoxadiazole (SBD) conjugating with a potent HDAC8 inhibitor PCI-34051 (IC

Topics: Fluorescent Dyes; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Neuroblastoma; Percutaneous Coronary Intervention; Repressor Proteins

The prognosis of advanced stage neuroblastoma patients remains poor and, despite intensive therapy, the 5-year survival rate remains less than 50%. We previously identified histone deacetylase (HDAC) 8 as an indicator of poor clinical outcome and a selective drug target for differentiation therapy in vitro and in vivo. Here, we performed kinome-wide RNAi screening to identify genes that are synthetically lethal with HDAC8 inhibitors. These experiments identified the neuroblastoma predisposition gene ALK as a candidate gene. Accordingly, the combination of the ALK/MET inhibitor crizotinib and selective HDAC8 inhibitors (3-6 µM PCI-34051 or 10 µM 20a) efficiently killed neuroblastoma cell lines carrying wildtype ALK (SK-N-BE(2)-C, IMR5/75), amplified ALK (NB-1), and those carrying the activating ALK F1174L mutation (Kelly), and, in cells carrying the activating R1275Q mutation (LAN-5), combination treatment decreased viable cell count. The effective dose of crizotinib in neuroblastoma cell lines ranged from 0.05 µM (ALK-amplified) to 0.8 µM (wildtype ALK). The combinatorial inhibition of ALK and HDAC8 also decreased tumor growth in an in vivo zebrafish xenograft model. Bioinformatic analyses revealed that the mRNA expression level of HDAC8 was significantly correlated with that of ALK in two independent patient cohorts, the Academic Medical Center cohort (n = 88) and the German Neuroblastoma Trial cohort (n = 649), and co-expression of both target genes identified patients with very poor outcome. Mechanistically, HDAC8 and ALK converge at the level of receptor tyrosine kinase (RTK) signaling and their downstream survival pathways, such as ERK signaling. Combination treatment of HDAC8 inhibitor with crizotinib efficiently blocked the activation of growth receptor survival signaling and shifted the cell cycle arrest and differentiation phenotype toward effective cell death of neuroblastoma cell lines, including sensitization of resistant models, but not of normal cells. These findings reveal combined targeting of ALK and HDAC8 as a novel strategy for the treatment of neuroblastoma.

Topics: Anaplastic Lymphoma Kinase; Animals; Antineoplastic Agents; Cell Cycle Checkpoints; Cell Differentiation; Cell Proliferation; Crizotinib; Drug Screening Assays, Antitumor; Histone Deacetylases; Humans; Hydroxamic Acids; Indoles; Neuroblastoma; Protein Kinase Inhibitors; Repressor Proteins; RNA Interference; Tumor Cells, Cultured; Zebrafish

Histone deacetylases (HDACs) are important modulators of epigenetic gene regulation and additionally control the activity of non-histone protein substrates. While for HDACs 1-3 and 6 many potent selective inhibitors have been obtained, for other subtypes much less is known on selective inhibitors and the consequences of their inhibition. The present report describes the development of substituted benzhydroxamic acids as potent and selective HDAC8 inhibitors. Docking studies using available crystal structures have been used for structure-based optimization of this series of compounds. Within this study, we have investigated the role of HDAC8 in the proliferation of cancer cells and optimized hits for potency and selectivity, both in vitro and in cell culture. The combination of structure-based design, synthesis, and in vitro screening to cellular testing resulted in potent and selective HDAC8 inhibitors that showed anti-neuroblastoma activity in cellular testing.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Cell Line, Tumor; Drug Design; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; HEK293 Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Molecular Docking Simulation; Neuroblastoma; Repressor Proteins; Structure-Activity Relationship

For differentiation-defective malignancies, compounds that modulate transcription, such as retinoic acid and histone deacetylase (HDAC) inhibitors, are of particular interest. HDAC inhibitors are currently under investigation for the treatment of a broad spectrum of cancer diseases. However, one clinical drawback is class-specific toxicity of unselective inhibitors, limiting their full anticancer potential. Selective targeting of individual HDAC isozymes in defined tumor entities may therefore be an attractive alternative treatment approach. We have previously identified HDAC family member 8 (HDAC8) as a novel target in childhood neuroblastoma. Using small-molecule inhibitors, we now demonstrate that selective inhibition of HDAC8 exhibits antineuroblastoma activity without toxicity in two xenograft mouse models of MYCN oncogene-amplified neuroblastoma. In contrast, the unselective HDAC inhibitor vorinostat was more toxic in the same models. HDAC8-selective inhibition induced cell cycle arrest and differentiation in vitro and in vivo. Upon combination with retinoic acid, differentiation was significantly enhanced, as demonstrated by elongated neurofilament-positive neurites and upregulation of NTRK1. Additionally, MYCN oncogene expression was downregulated in vitro and tumor cell growth was markedly reduced in vivo. Mechanistic studies suggest that cAMP-response element-binding protein (CREB) links HDAC8- and retinoic acid-mediated gene transcription. In conclusion, HDAC-selective targeting can be effective in tumors exhibiting HDAC isozyme-dependent tumor growth in vivo and can be combined with differentiation-inducing agents.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cell Survival; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Indoles; Mice; Mice, Nude; Neuroblastoma; Repressor Proteins; Tretinoin; Xenograft Model Antitumor Assays

To find HDAC8-selective inhibitors, we designed a library of HDAC inhibitor candidates, each containing a zinc-binding group that coordinates with the active-site zinc ion, linked via a triazole moiety to a capping structure that interacts with residues on the rim of the active site. These compounds were synthesized by using click chemistry. Screening identified HDAC8-selective inhibitors including C149 (IC(50) = 0.070 μM), which was more potent than PCI-34058 (6) (IC(50) = 0.31 μM), a known HDAC8 inhibitor. Molecular modeling suggested that the phenylthiomethyl group of C149 binds to a unique hydrophobic pocket of HDAC8, and the orientation of the phenylthiomethyl and hydroxamate moieties (fixed by the triazole moiety) is important for the potency and selectivity. The inhibitors caused selective acetylation of cohesin in cells and exerted growth-inhibitory effects on T-cell lymphoma and neuroblastoma cells (GI(50) = 3-80 μM). These findings suggest that HDAC8-selective inhibitors have potential as anticancer agents.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Proliferation; Click Chemistry; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Lymphoma, T-Cell; Models, Molecular; Neuroblastoma; Repressor Proteins; Small Molecule Libraries; Tumor Cells, Cultured