Efficacy of selective histone deacetylase 6 inhibition in mouse models of Pseudomonas aeruginosa infection: A new glimpse for reducing inflammation and infection in cystic fibrosis.

The latest studies identified the histone deacetylase (HDAC) class of enzymes as strategic components of the complex molecular machinery underlying inflammation in cystic fibrosis (CF). Compelling new support has been provided for HDAC6 isoform as a key player in the generation of the dysregulated proinflammatory phenotype in CF, as well as in the immune response to the persistent bacterial infection accompanying CF patients. We herein provide in vivo proof-of-concept (PoC) of the efficacy of selective HDAC6 inhibition in contrasting the pro-inflammatory phenotype in a mouse model of chronic P. aeruginosa respiratory infection. Upon careful selection and in-house re-profiling (in vitro and cell-based assessment of acetylated tubulin level through Western blot analysis) of three potent and selective HDAC6 inhibitors as putative candidates for the PoC, we engaged the best performing compound 2 for pre-clinical studies. Compound 2 demonstrated no toxicity and robust anti-inflammatory profile in a mouse model of chronic P. aeruginosa respiratory infection upon repeated aerosol administration. A significant reduction of leukocyte recruitment in the airways, in particular neutrophils, was observed in compound 2-treated mice in comparison with the vehicle; moreover, quantitative immunoassays confirmed a significant reduction of chemokines and cytokines in lung homogenate. This effect was also associated with a modest reduced bacterial load after compound 2-treatment in mice compared to the vehicle. Our study is of particular significance since it demonstrates for the first time the utility of selective drug-like HDAC6 inhibitors in a relevant in vivo model of chronic P. aeruginosa infection, thus supporting their potential application for reverting CF phenotype.

MK-4101, a Potent Inhibitor of the Hedgehog Pathway, Is Highly Active against Medulloblastoma and Basal Cell Carcinoma.

Aberrant activation of the Hedgehog (Hh) signaling pathway is implicated in the pathogenesis of many cancers, including medulloblastoma and basal cell carcinoma (BCC). In this study, using neonatally irradiated Ptch1(+/-) mice as a model of Hh-dependent tumors, we investigated the in vivo effects of MK-4101, a novel SMO antagonist, for the treatment of medulloblastoma and BCC. Results clearly demonstrated a robust antitumor activity of MK-4101, achieved through the inhibition of proliferation and induction of extensive apoptosis in tumor cells. Of note, beside antitumor activity on transplanted tumors, MK-4101 was highly efficacious against primary medulloblastoma and BCC developing in the cerebellum and skin of Ptch1(+/-) mice. By identifying the changes induced by MK-4101 in gene expression profiles in tumors, we also elucidated the mechanism of action of this novel, orally administrable compound. MK-4101 targets the Hh pathway in tumor cells, showing the maximum inhibitory effect on Gli1 MK-4101 also induced deregulation of cell cycle and block of DNA replication in tumors. Members of the IGF and Wnt signaling pathways were among the most highly deregulated genes by MK-4101, suggesting that the interplay among Hh, IGF, and Wnt is crucial in Hh-dependent tumorigenesis. Altogether, the results of this preclinical study support a therapeutic opportunity for MK-4101 in the treatment of Hh-driven cancers, also providing useful information for combination therapy with drugs targeting pathways cooperating with Hh oncogenic activity. Mol Cancer Ther; 15(6); 1177-89. ©2016 AACR.

Critical role of zinc finger protein 521 in the control of growth, clonogenicity and tumorigenic potential of medulloblastoma cells.

The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of hematopoietic, osteo-adipogenic and neural progenitor cells. ZNF521 is highly expressed in cerebellum and in particular in the neonatal external granule layer that contains candidate medulloblastoma cells-of-origin, and in the majority of human medulloblastomas. Here we have explored its involvement in the control of human and murine medulloblastoma cells. The effect of ZNF521 on growth and tumorigenic potential of human medulloblastoma cell lines as well as primary Ptc1-/+ mouse medulloblastoma cells was investigated in a variety of in vitro and in vivo assays, by modulating its expression using lentiviral vectors carrying the ZNF521 cDNA, or shRNAs that silence its expression. Enforced overexpression of ZNF521 in DAOY medulloblastoma cells significantly increased their proliferation, growth as spheroids and ability to generate clones in single-cell cultures and semisolid media, and enhanced their migratory ability in wound-healing assays. Importantly, ZNF521-expressing cells displayed a greatly enhanced tumorigenic potential in nude mice. All these activities required the ZNF521 N-terminal motif that recruits the nucleosome remodeling and histone deacetylase complex, which might therefore represent an appealing therapeutic target. Conversely, silencing of ZNF521 in human UW228 medulloblastoma cells that display high baseline expression decreased their proliferation, clonogenicity, sphere formation and wound-healing ability. Similarly, Zfp521 silencing in mouse Ptc1-/+ medulloblastoma cells drastically reduced their growth and tumorigenic potential. Our data strongly support the notion that ZNF521, through the recruitment of the NuRD complex, contributes to the clonogenic growth, migration and tumorigenicity of medulloblastoma cells.

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclo- propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 2.

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclopropyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydroimid azo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 1.

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which controls patterning, growth and cell migration in most tissues, but evidence has accumulated showing that many human tumors aberrantly reactivate this pathway. Smoothened antagonists offer opportunities for the treatment of malignancies dependent on the Hh pathway, and the most advanced clinical candidates are demonstrating encourage initial results. A novel series of [6,5]-bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-dione smoothened antagonists has been identified, and the series has been extensively explored to ascertain the key detriments for activity, demonstrating that the trans-2-phenylcyclopropyl and hydantoin ring systems are critical for potency, while a variety of urea substituents can be tolerated. The combination of these optimal groups gives smoothened antagonists with activity in the low nanomolar range.

Loss of histone deacetylase 4 causes segregation defects during mitosis of p53-deficient human tumor cells.

We investigated the role of histone deacetylase 4 (HDAC4) using RNA interference (RNAi) and knockout cells to specifically address its role in cell cycle progression in tumor and normal cells. Ablation of HDAC4 led to growth inhibition in human tumor cells but not to detectable effects in normal human dermal fibroblasts (NHDF) or myelopoietic progenitors. HDAC4-/+ or HDAC4-/- murine embryonic fibroblasts showed no detectable growth defects. On the other hand, HDAC4 RNAi in HeLa cells produced mitotic arrest followed by caspase-dependent apoptosis. Mitotically arrested cells showed chromosome segregation defects. Even though the growth of both p53-wild-type and p53-null tumor cells were affected by HDAC4 ablation, segregation defects were observed only in p53-null cells. HDAC4 associates with the PP2A-B56 regulatory subunit, which is known to be involved in chromosome segregation, and RNAi of either the structural subunit A or the regulatory subunit B56 of PP2A also caused chromosome segregation defects. We conclude that HDAC4 is required for cell cycle progression of tumor cells by multiple mechanisms, one of which seems to be specific to p53-deficient cells through chromosome segregation defects. On the contrary, HDAC4 is not required for the progression of NHDF. We therefore suggest that systemic selective interference with the expression or function of HDAC4 is expected to have a significant therapeutic window, in particular, for p53-deficient tumors.

Smoothened antagonists: a promising new class of antitumor agents.

BACKGROUND: Hedgehog signaling is essential for the development of most metazoans. In recent years, evidence has accumulated showing that many human tumors aberrantly re-activate this developmental signaling pathway and that interfering with it may provide a new strategy for the development of novel anti-cancer therapeutics. Smoothened is a G-protein coupled receptor-like protein that is essentially involved in hedgehog signal transduction and small molecule antagonists of Smoothened have started to show antitumor activity in preclinical models and in clinical trials. OBJECTIVE: We critically review the role of hedgehog signaling in normal development and in human malignancies, the available drug discovery tools and the classes of small molecule inhibitors that are in development. We further aim to address the potential impact that pathway antagonists may have on the treatment options of cancer patients. METHODS: Literature, patents and clinical trial results from the past 5 years were analyzed. CONCLUSIONS: 1) A large body of evidence suggests a frequent reactivation of hedgehog signaling in human cancer. 2) Smoothened is an attractive, highly druggable target with extensive preclinical and initial clinical validation in basal cell carcinoma. Several promising novel classes of Smoothened antagonists have been discovered and are being developed as anticancer agents. 3) Our knowledge of the biology of hedgehog signaling in cancer is still very incomplete and significant efforts will be required to understand how to use the emerging novel agents in the clinic.

Dissecting the biological functions of Drosophila histone deacetylases by RNA interference and transcriptional profiling.

Zinc-dependent histone deacetylases (HDACs) are a family of hydrolases first identified as components of transcriptional repressor complexes, where they act by deacetylating lysine residues at the N-terminal extensions of core histones, thereby affecting transcription. To get more insight into the biological functions of the individual HDAC family members, we have used RNA interference in combination with microarray analysis in Drosophila S2 cells. Silencing of Drosophila HDAC1 (DHDAC1), but not of the other DHDAC family members, leads to increased histone acetylation. Silencing of either DHDAC1 or DHDAC3 leads to cell growth inhibition and deregulated transcription of both common and distinct groups of genes. Silencing DHDAC2 leads to increased tubulin acetylation levels but was not associated with a deregulation of gene expression. No growth of phenotype and no significant deregulation of gene expression was observed upon silencing of DHDAC4 and DHDACX. Loss of DHDAC1 or exposure of S2 cells to the small molecule HDAC inhibitor trichostatin both lead to a G(2) arrest and were associated with significantly overlapping gene expression signatures in which genes involved in nucleobase and lipid metabolism, DNA replication, cell cycle regulation, and signal transduction were over-represented. A large number of these genes were shown to also be deregulated upon loss of the co-repressor SIN3 (Pile, L. A., Spellman, P. T., Katzenberger, R. J., and Wassarman, D. A. (2003) J. Biol. Chem. 278, 37840-37848). We conclude the following. 1) DHDAC1 and -3 have distinct functions in the control of gene expression. 2) Under the tested conditions, DHDAC2, -4, and X have no detectable transcriptional functions in S2 cells. 3) The anti-proliferative and transcriptional effects of trichostatin are largely recapitulated by the loss of DHDAC1. 4) The deacetylase activity of DHDAC1 significantly contributes to the repressor function of SIN3.

Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human HDAC8, complexed with a hydroxamic acid inhibitor.

Histone deacetylases (HDACs) are a family of enzymes involved in the regulation of gene expression, DNA repair, and stress response. These processes often are altered in tumors, and HDAC inhibitors have had pronounced antitumor activity with promising results in clinical trials. Here, we report the crystal structure of human HDAC8 in complex with a hydroxamic acid inhibitor. Such a structure of a eukaryotic zinc-dependent HDAC has not be described previously. Similar to bacterial HDAC-like protein, HDAC8 folds in a single alpha/beta domain. The inhibitor and the zinc-binding sites are similar in both proteins. However, significant differences are observed in the length and structure of the loops surrounding the active site, including the presence of two potassium ions in HDAC8 structure, one of which interacts with key catalytic residues. CD data suggest a direct role of potassium in the fold stabilization of HDAC8. Knockdown of HDAC8 by RNA interference inhibits growth of human lung, colon, and cervical cancer cell lines, highlighting the importance of this HDAC subtype for tumor cell proliferation. Our findings open the way for the design and development of selective inhibitors of HDAC8 as possible antitumor agents.

The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus.

We discovered that the hepatitis C virus (HCV) envelope glycoprotein E2 binds to human hepatoma cell lines independently of the previously proposed HCV receptor CD81. Comparative binding studies using recombinant E2 from the most prevalent 1a and 1b genotypes revealed that E2 recognition by hepatoma cells is independent from the viral isolate, while E2-CD81 interaction is isolate specific. Binding of soluble E2 to human hepatoma cells was impaired by deletion of the hypervariable region 1 (HVR1), but the wild-type phenotype was recovered by introducing a compensatory mutation reported previously to rescue infectivity of an HVR1-deleted HCV infectious clone. We have identified the receptor responsible for E2 binding to human hepatic cells as the human scavenger receptor class B type I (SR-BI). E2-SR-BI interaction is very selective since neither mouse SR-BI nor the closely related human scavenger receptor CD36, were able to bind E2. Finally, E2 recognition by SR-BI was competed out in an isolate-specific manner both on the hepatoma cell line and on the human SR-BI-transfected cell line by an anti-HVR1 monoclonal antibody.