Regulation of gene expression by translation factor eIF5A: Hypusine-modified eIF5A enhances nonsense-mediated mRNA decay in human cells.

Nonsense-mediated mRNA decay (NMD) couples protein synthesis to mRNA turnover. It eliminates defective transcripts and controls the abundance of certain normal mRNAs. Our study establishes a connection between NMD and the translation factor eIF5A (eukaryotic initiation factor 5A) in human cells. eIF5A modulates the synthesis of groups of proteins (the eIF5A regulon), and undergoes a distinctive two-step post-translational modification (hypusination) catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. We show that expression of NMD-susceptible constructs was increased by depletion of the major eIF5A isoform, eIF5A1. NMD was also attenuated when hypusination was inhibited by RNA interference with either of the two eIF5A modifying enzymes, or by treatment with the drugs ciclopirox or deferiprone which inhibit deoxyhypusine hydroxylase. Transcriptome analysis by RNA-Seq identified human genes whose expression is coordinately regulated by eIF5A1, its modifying enzymes, and the pivotal NMD factor, Upf1. Transcripts encoding components of the translation system were highly represented, including some encoding ribosomal proteins controlled by alternative splicing coupled to NMD (AS-NMD). Our findings extend and strengthen the association of eIF5A with NMD, previously inferred in yeast, and show that hypusination is important for this function of human eIF5A. In addition, they advance drug-mediated NMD suppression as a therapeutic opportunity for nonsense-associated diseases. We propose that regulation of mRNA stability contributes to eIF5A's role in selective gene expression.

Drug-Based Lead Discovery: The Novel Ablative Antiretroviral Profile of Deferiprone in HIV-1-Infected Cells and in HIV-Infected Treatment-Naive Subjects of a Double-Blind, Placebo-Controlled, Randomized Exploratory Trial.

UNLABELLED: Antiretrovirals suppress HIV-1 production yet spare the sites of HIV-1 production, the HIV-1 DNA-harboring cells that evade immune detection and enable viral resistance on-drug and viral rebound off-drug. Therapeutic ablation of pathogenic cells markedly improves the outcome of many diseases. We extend this strategy to HIV-1 infection. Using drug-based lead discovery, we report the concentration threshold-dependent antiretroviral action of the medicinal chelator deferiprone and validate preclinical findings by a proof-of-concept double-blind trial. In isolate-infected primary cultures, supra-threshold concentrations during deferiprone monotherapy caused decline of HIV-1 RNA and HIV-1 DNA; did not allow viral breakthrough for up to 35 days on-drug, indicating resiliency against viral resistance; and prevented, for at least 87 days off-drug, viral rebound. Displaying a steep dose-effect curve, deferiprone produced infection-independent deficiency of hydroxylated hypusyl-eIF5A. However, unhydroxylated deoxyhypusyl-eIF5A accumulated particularly in HIV-infected cells; they preferentially underwent apoptotic DNA fragmentation. Since the threshold, ascertained at about 150 µM, is achievable in deferiprone-treated patients, we proceeded from cell culture directly to an exploratory trial. HIV-1 RNA was measured after 7 days on-drug and after 28 and 56 days off-drug. Subjects who attained supra-threshold concentrations in serum and completed the protocol of 17 oral doses, experienced a zidovudine-like decline of HIV-1 RNA on-drug that was maintained off-drug without statistically significant rebound for 8 weeks, over 670 times the drug's half-life and thus clearance from circulation. The uniform deferiprone threshold is in agreement with mapping of, and crystallographic 3D-data on, the active site of deoxyhypusyl hydroxylase (DOHH), the eIF5A-hydroxylating enzyme. We propose that deficiency of hypusine-containing eIF5A impedes the translation of mRNAs encoding proline cluster ('polyproline')-containing proteins, exemplified by Gag/p24, and facilitated by the excess of deoxyhypusine-containing eIF5A, releases the innate apoptotic defense of HIV-infected cells from viral blockade, thus depleting the cellular reservoir of HIV-1 DNA that drives breakthrough and rebound. TRIAL REGISTRATION: ClinicalTrial.gov NCT02191657.

Blocking eIF5A modification in cervical cancer cells alters the expression of cancer-related genes and suppresses cell proliferation.

Cancer etiology is influenced by alterations in protein synthesis that are not fully understood. In this study, we took a novel approach to investigate the role of the eukaryotic translation initiation factor eIF5A in human cervical cancers, where it is widely overexpressed. eIF5A contains the distinctive amino acid hypusine, which is formed by a posttranslational modification event requiring deoxyhypusine hydroxylase (DOHH), an enzyme that can be inhibited by the drugs ciclopirox and deferiprone. We found that proliferation of cervical cancer cells can be blocked by DOHH inhibition with either of these pharmacologic agents, as well as by RNA interference-mediated silencing of eIF5A, DOHH, or another enzyme in the hypusine pathway. Proteomic and RNA analyses in HeLa cervical cancer cells identified two groups of proteins in addition to eIF5A that were coordinately affected by ciclopirox and deferiprone. Group 1 proteins (Hsp27, NM23, and DJ-1) were downregulated at the translational level, whereas group 2 proteins (TrpRS and PRDX2) were upregulated at the mRNA level. Further investigations confirmed that eIF5A and DOHH are required for Hsp27 expression in cervical cancer cells and for regulation of its key target IκB and hence NF-κB. Our results argue that mature eIF5A controls a translational network of cancer-driving genes, termed the eIF5A regulon, at the levels of mRNA abundance and translation. In coordinating cell proliferation, the eIF5A regulon can be modulated by drugs such as ciclopirox or deferiprone, which might be repositioned to control cancer cell growth.

Drug-induced reactivation of apoptosis abrogates HIV-1 infection.

HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal antivirals that eliminate viral infection by destroying infected cells. A drug-based drug discovery program, based on these compounds, is warranted to determine the potential of such agents in clinical trials of HIV-infected patients.

Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5A.

BACKGROUND: Eukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture. RESULTS: Ciclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression. CONCLUSION: At clinically relevant concentrations, two widely used drugs blocked HIV-1 replication ex vivo. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs.

Correlations of mRNA expression and in vitro chemosensitivity to enzastaurin in freshly explanted human tumor cells.

PURPOSE: Enzastaurin (LY317615) is a novel serine/threonine kinase inhibitor, targeting Protein Kinase C-beta (PKC-beta), and PI3K/AKT pathways to inhibit angiogenesis and tumor cell proliferation. The aims of this study were to determine whether Enzastaurin has direct antitumor activity against freshly explanted tumor cells and to correlate mRNA expression of genes related to the proposed mechanism of action of enzastaurin with in vitro chemosensitivity. EXPERIMENTAL DESIGN: Freshly biopsied tumor cells were studied using soft-agar cell cloning experiments (SACCE) to determine the in vitro chemosensitivity to enzastaurin. An aliquot of the same tumor specimens was shock-frozen and total RNA was isolated for standardized multiplex rt-PCR experiments for gene expression of PKC-beta1, PKC-beta2, IL-8, IL-8RA, IL-8RB, Glycogen Synthase Kinase 3 beta (GSK-3beta) and TGF-beta1. Correlations, threshold optimization, sensitivity, specificity, and efficiency were analyzed using the appropriate statistical methodologies. RESULTS: Seventy-two tumor samples were collected and 63 were fully evaluable. Low levels of mRNA expression of GSK-3beta and high levels of mRNA expression of IL-8 were highly significantly correlated with chemosensitivity to enzastaurin. Optimization analyses demonstrated threshold values of 4,000 copies for IL-8 and three copies for GSK-3beta relative to 10(4) copies of beta-actin. However, no correlation between mRNA expression of PKC-beta1, PKC-beta2, IL-8RA, IL-8RB and chemosensitivity to enzastaurin was observed. Expression of TGF-beta1 mRNA was not detectable in the specimens investigated. CONCLUSIONS: mRNA expression levels of IL-8 and GSK-3beta correlate with antitumor activity of enzastaurin. These results form a rational basis for clinical trials to evaluate the expression of these genes as potential predictors for treatment outcome after enzastaurin chemotherapy.

Eukaryotic initiation factor 5A-1 (eIF5A-1) as a diagnostic marker for aberrant proliferation in intraepithelial neoplasia of the vulva.

OBJECTIVE: The mature eukaryotic translation initiation factor 5A contains the unusual amino acid hypusine, formed post-translationally from a specific lysine residue and essential for proliferation of eukaryotic cells. We hypothesized that the major eIF5A isoform, eIF5A-1, is an in situ biomarker for proliferation. NIH-353, a polyclonal immunoreagent specific for hypusine-containing eIF5A-1, was used to test this proposal in biopsies of vulvar high-grade intraepithelial neoplasia (VIN), characterized by the presence of proliferating cells throughout the thickness of the epithelium. Methods. Formalin-fixed and paraffin-embedded archival samples with an independently established diagnosis of VIN 3 were stained immunohistochemically after antigen retrieval, employing NIH-353 and, for comparison, the standard Ki-67 antibody. RESULTS: NIH-353 labeled neoplastic keratinocytes throughout the thickness of the epithelium in all VIN 3 samples. Malignant cells in a case of focally invasive squamous cell carcinoma also stained strongly for mature, hypusine-containing eIF5A-1. Epithelium adjacent to these lesions, though still of apparently normal morphology, was immunoreactive throughout its full thickness. At inflammatory foci of lesional sites, solitary reactive lymphocytes were positive, as were individual proliferating cells within dermal appendages. The submucosal stroma lacked reactive cells. CONCLUSION: NIH-353 identifies mature eIF5A-1 as an in situ biomarker for proliferation. Like Ki-67, this immunoreagent promises broad applicability in histopathological diagnosis and may be helpful in outcome prediction. In contrast to Ki-67, NIH-353 visualizes a molecular target for antineoplastic therapy, and thus may guide the development and clinical testing of drugs that, like the fungicide ciclopirox, inhibit hypusine formation and cell proliferation.

The HAG mechanism: a molecular rationale for the therapeutic application of iron chelators in human diseases involving the 2-oxoacid utilizing dioxygenases.

'Iron chelation' is widely understood as synonymous with non-specificity and viewed as a purely physicochemical mode of action, without any defined biomolecular target, broadly interfering with metalloenzymes. The 2-oxoacid-utilizing dioxygenases challenge this preconception. A family of non-heme iron enzymes that rely on chelation-dependent catalysis, they employ common molecules like Krebs cycle intermediates as endogenous iron chelators and consume atmospheric oxygen, inserting one of its atoms into cellular components. These enzymes control the adaptation of cells to hypoxia; the reversal of mutagenic DNA alkylations, the initiation of DNA replication, the translation of mRNAs; the production of extracellular matrix proteins like collagens and fibrillins; and numerous metabolic pathways: from the synthesis of the gibberellin growth hormones of plants, and the formation of carnitine, atropine, endotoxins, and cephalosporin antibiotics, to the breakdown of amino acids. Their pivotal roles in human pathology encompass oncogenesis and cancer angiogenesis, scarring and organ fibrosis, inherited diseases, and retroviral infections. Their unique catalysis, termed earlier the 'HAG mechanism' and known in subatomic detail, requires at least three different substrates to form three different products, and proceeds as a ligand reaction at the non-heme iron atom inside the active site pocket, without any direct involvement of apoenzyme residues. The apoenzyme sterically controls ligand access to the metal. The HAG mechanism-based concept of catalytic chelation directed by an apoenzyme, not merely by complexation parameters, has enabled knowledge-guided design of systemic and tissue-selective inhibitors, and of clinical trials. The HAG mechanism also lends itself to the development of novel, man-made biocatalysts.

Procollagen-derived biomarkers in malignant ascites of ovarian cancer. Independent prognosticators for progression-free interval and survival.

OBJECTIVE: Matrix formation is a hallmark of solid tumor biology. Circulating antigens of structural matrix proteins should reflect this fact, yet are subject to systemic variables. We propose that if measured regionally, in a cancer-induced extravascular fluid pool such as malignant ascites of ovarian cancer, the same antigens retain their conceptual advantage as surrogate markers for tumor biology. METHODS: In malignant ascites obtained at staging laparatomy of 35 women with ovarian cancer, the protein-normalized levels of the C-terminal propeptide of procollagen type I (pnPICP) and the N-terminal propeptide of procollagen type III (pnPIIINP) were determined. Using univariate and multivariate analysis, we examined these parameters, their (pnPIIINP/pnPICP) quotient, and clinical criteria (FIGO stage, age, residual tumor, histology, and tumor grade) for impact on progression-free interval and survival. RESULTS: The absolute level of pnPIIINP was the single most powerful independent factor impacting on survival, its P value being distinctly below (P = 0.0005 vs 0.003) and its risk ratio distinctly above (15 vs 2.5) residual tumor after debulking surgery. The relative level of pnPIIINP, i.e. (pnPIIINP / pnPICP), impacted on the likelihood of recurrence even more than residual tumor. By Kaplan-Meier analysis, cutoff values for the absolute or relative pnPIIINP level significantly discriminated patients with shortened survival or progression-free interval, respectively. CONCLUSIONS: Since malignant ovarian epithelium itself forms collagen type III, and since collagen type III is a solid-phase regulator of angiogenesis, we propose that ascitic pnPIIINP is a fluid-phase indicator for angiogenic activity in ovarian cancer and thus represents a tumor virulence index.

Tyrosinemia I, a model for human diseases mediated by 2-oxoacid-utilizing dioxygenases: hepatotoxin suppression by NTBC does not normalize hepatic collagen metabolism.

OBJECTIVES: Medical treatment of tyrosinemia I relies on the herbicide NTBC [Orfadin 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione], an inhibitor of plant and mammalian 2-oxoacid-utilizing dioxygenases with a collective catalytic cycle ('HAG' mechanism). We hypothesize that NTBC-treated tyrosinemia I is a human model for the pathogenic role of two major enzymes in this class, 4-hydroxyphenylpyruvate dioxygenase (4-HPPD; EC 1.13.11.27) and prolyl 4-hydroxylase (P4-H; E.C. 1.14.11.2), essential for tyrosine and collagen metabolism, respectively. METHODS: In a patient with established tyrosinemia I, we monitored the in vivo activities of 4-HPPD and P4-H via five biomarkers before and during NTBC medication. Hypothesis testing at the molecular level was performed by computational modeling of NTBC binding to the crystal structure-derived active site of 4-HPPD, and then relating these findings to our experimental results and to known P4-H data. RESULTS: NTBC rapidly normalized the biomarkers for 4-HPPD activity. However, those for P4-H activity remained uniformly elevated after one hundred days on NTBC, the PIIINP biomarker even increasing above its grossly abnormal, initial level. This selective enzyme inhibition despite a collective catalytic cycle is attributed to the conformation of NTBC, which only fits the active site of 4-HPPD, as confirmed by its crystal structure. CONCLUSIONS: Normalization of hepatic collagen formation, highly desirable in all fibrotic liver diseases, is not achieved by NTBC in tyrosinemia I. By establishing the molecular cause for this failure, our results also establish a rational approach to identify inhibitors that achieve that goal, either by joint 4-HPPD / P-4H inhibition, or by inhibition of only P-4H.

The antifungal drug ciclopirox inhibits deoxyhypusine and proline hydroxylation, endothelial cell growth and angiogenesis in vitro.

The hypusine biosynthetic steps represent novel targets for intervention in cell proliferation. Hypusine is a rare amino acid, formed posttranslationally in one cellular protein, eIF5A, and is essential for cell proliferation. Deoxyhypusine hydroxylase, the metalloenzyme catalyzing the final step in hypusine biosynthesis, and prolyl 4-hydroxylase, a non-heme iron enzyme critical for collagen processing, can be inhibited by small chelating molecules that target their essential metal atom. We examined the effects of 5 compounds (ciclopirox, deferiprone, deferoxamine, mimosine and 2,2'-dipyridyl) on these protein hydroxylases in HUVECs, on cell proliferation and on angiogenesis using 2 model assays: tube-like vessel formation on Matrigel and the chick aortic arch sprouting assay. These compounds inhibited cellular deoxyhypusine hydroxylase in a concentration-dependent manner, but their efficacy varied widely in the following order: ciclopirox--> deferoxamine-->2,2'-dipyridyl-->deferiprone-->mimosine (IC(50) 5-200 microM). Inhibition of DNA synthesis, following the same order (IC(50) 10-450 microM), correlated with G(1) arrest of the cell cycle. These compounds also inhibited proline hydroxylation and maturation of collagen in HUVECs and caused inhibition of angiogenesis in vitro. Of the compounds tested, ciclopirox was by far the most effective inhibitor of HUVEC proliferation and angiogenesis. The strong antiangiogenic activity of this readily available antifungal drug along with its antiproliferative effects suggests a new potential application for ciclopirox in the treatment of solid tumors.

Basement membrane biomarkers in very low birth weight premature infants. Association with length of NICU stay and bronchopulmonary dysplasia.

Basement membranes, critical for vital organs like the lungs, consist of two interwoven homopolymers, one assembled by type IV collagens and one by laminins. We hypothesized their serum antigens C-IV and P1, respectively, to be global measures for the maturity of these organs. In 39 very low birth weight premature neonates (means: gestational age, 25.8 weeks; birth weight, 779 g) requiring intensive care, we analyzed these biomarkers during the first two months post partum. Median C-IV and P1 exceeded adult levels by one order of magnitude. The individuals with the lowest first week C-IV values (mean: 667 ng/ml) required significantly longer neonatal intensive care unit stays than those with the highest values (mean: 2,467 ng/ml), on average 109 vs. 80 days (p = 0.008) irrespective of gestational age. Patients diagnosed with bronchopulmonary dysplasia (BPD) at 36 weeks postconceptional age, already in their first week of life displayed C-IV levels lower than in controls, suggesting a defect in pulmonary basement membrane remodeling. This is the first identification by a matrix biomarker of a BPD-antecedent state.