Rapid in vitro generation of bona fide exhausted CD8+ T cells is accompanied by Tcf7 promotor methylation.

Exhaustion is a dysfunctional state of cytotoxic CD8+ T cells (CTL) observed in chronic infection and cancer. Current in vivo models of CTL exhaustion using chronic viral infections or cancer yield very few exhausted CTL, limiting the analysis that can be done on these cells. Establishing an in vitro system that rapidly induces CTL exhaustion would therefore greatly facilitate the study of this phenotype, identify the truly exhaustion-associated changes and allow the testing of novel approaches to reverse or prevent exhaustion. Here we show that repeat stimulation of purified TCR transgenic OT-I CTL with their specific peptide induces all the functional (reduced cytokine production and polyfunctionality, decreased in vivo expansion capacity) and phenotypic (increased inhibitory receptors expression and transcription factor changes) characteristics of exhaustion. Importantly, in vitro exhausted cells shared the transcriptomic characteristics of the gold standard of exhaustion, CTL from LCMV cl13 infections. Gene expression of both in vitro and in vivo exhausted CTL was distinct from T cell anergy. Using this system, we show that Tcf7 promoter DNA methylation contributes to TCF1 downregulation in exhausted CTL. Thus this novel in vitro system can be used to identify genes and signaling pathways involved in exhaustion and will facilitate the screening of reagents that prevent/reverse CTL exhaustion.

The miR-200c/141-ZEB2-TGFß axis is aberrant in human T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is mostly characterized by aberrant expansion of small- to medium-sized prolymphocytes with a mature post-thymic phenotype, high aggressiveness of the disease and poor prognosis. However, T-PLL is more heterogeneous with a wide range of clinical, morphological, and molecular features, which occasionally impedes the diagnosis. We hypothesized that T-PLL consists of phenotypic and/or genotypic subgroups that may explain the heterogeneity of the disease. Multi-dimensional immuno-phenotyping and gene expression profiling did not reveal clear T-PLL subgroups, and no clear T-cell receptor a or ß CDR3 skewing was observed between different T-PLL cases. We revealed that the expression of microRNA (miRNA) is aberrant and often heterogeneous in T-PLL. We identified 35 miRNA that were aberrantly expressed in T-PLL with miR-200c/141 as the most differentially expressed cluster. High miR- 200c/141 and miR-181a/181b expression was significantly correlated with increased white blood cell counts and poor survival. Furthermore, we found that overexpression of miR-200c/141 correlated with downregulation of their targets ZEB2 and TGFßR3 and aberrant TGFß1- induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFß pathway is affected in T-PLL. Our results thus highlight the potential role for aberrantly expressed oncogenic miRNA in T-PLL and pave the way for new therapeutic targets in this disease.

Lung transcriptional unresponsiveness and loss of early influenza virus control in infected neonates is prevented by intranasal Lactobacillus rhamnosus GG.

Respiratory viral infections contribute substantially to global infant losses and disproportionately affect preterm neonates. Using our previously established neonatal murine model of influenza infection, we demonstrate that three-day old mice are exceptionally sensitive to influenza virus infection and exhibit high mortality and viral load. Intranasal pre- and post-treatment of neonatal mice with Lactobacillus rhamnosus GG (LGG), an immune modulator in respiratory viral infection of adult mice and human preterm neonates, considerably improves neonatal mice survival after influenza virus infection. We determine that both live and heat-killed intranasal LGG are equally efficacious in protection of neonates. Early in influenza infection, neonatal transcriptional responses in the lung are delayed compared to adults. These responses increase by 24 hours post-infection, demonstrating a delay in the kinetics of the neonatal anti-viral response. LGG pretreatment improves immune gene transcriptional responses during early infection and specifically upregulates type I IFN pathways. This is critical for protection, as neonatal mice intranasally pre-treated with IFNß before influenza virus infection are also protected. Using transgenic mice, we demonstrate that the protective effect of LGG is mediated through a MyD88-dependent mechanism, specifically via TLR4. LGG can improve both early control of virus and transcriptional responsiveness and could serve as a simple and safe intervention to protect neonates.

Proteomic Analysis of Mesenchymal Stromal Cell-Derived Extracellular Vesicles and Reconstructed Membrane Particles.

Extracellular vesicles (EV) derived from mesenchymal stromal cells (MSC) are a potential therapy for immunological and degenerative diseases. However, large-scale production of EV free from contamination by soluble proteins is a major challenge. The generation of particles from isolated membranes of MSC, membrane particles (MP), may be an alternative to EV. In the present study we generated MP from the membranes of lysed MSC after removal of the nuclei. The yield of MP per MSC was 1 × 105 times higher than EV derived from the same number of MSC. To compare the proteome of MP and EV, proteomic analysis of MP and EV was performed. MP contained over 20 times more proteins than EV. The proteins present in MP evidenced a multi-organelle origin of MP. The projected function of the proteins in EV and MP was very different. Whilst proteins in EV mainly play a role in extracellular matrix organization, proteins in MP were interconnected in diverse molecular pathways, including protein synthesis and degradation pathways and demonstrated enzymatic activity. Treatment of MSC with IFNγ led to a profound effect on the protein make up of EV and MP, demonstrating the possibility to modify the phenotype of EV and MP through modification of parent MSC. These results demonstrate that MP are an attractive alternative to EV for the development of potential therapies. Functional studies will have to demonstrate therapeutic efficacy of MP in preclinical disease models.

Responsiveness of chronic lymphocytic leukemia cells to B-cell receptor stimulation is associated with low expression of regulatory molecules of the nuclear factor-κB pathway.

Chronic lymphocytic leukemia (CLL) is a disease with heterogeneous clinical and biological characteristics. Differences in Ca2+ levels among cases, both basal and upon B-cell receptor (BCR) stimulation, may reflect heterogeneity in the pathogenesis due to cell-intrinsic factors. Our aim was to elucidate cell-intrinsic differences between BCR-responsive and -unresponsive cases. We therefore determined BCR responsiveness ex vivo based on Ca2+ influx upon α-IgM stimulation of purified CLL cell fractions from 52 patients. Phosphorylation levels of various BCR signaling molecules, and expression of activation markers were assessed by flow cytometry. Transcription profiling of responsive (n=6) and unresponsive cases (n=6) was performed by RNA sequencing. Real-time quantitative polymerase chain reaction analysis was used to validate transcript level differences in a larger cohort. In 24 cases an α-IgM response was visible by Ca2+ influx which was accompanied by higher phosphorylation of PLCγ2 and Akt after α-IgM stimulation in combination with higher surface expression of IgM, IgD, CD19, CD38 and CD43 compared to the unresponsive cases (n=28). Based on RNA sequencing analysis several components of the canonical nuclear factor (NF)-κB pathway, especially those related to NF-κB inhibition, were expressed more highly in unresponsive cases. Moreover, upon α-IgM stimulation, the expression of these NF-κB pathway genes (especially genes coding for NF-κB pathway inhibitors but also NF-κB subunit REL) was upregulated in BCR-responsive cases while the level did not change, compared to basal level, in the unresponsive cases. These findings suggest that cells from CLL cases with enhanced NF-κB signaling have a lesser capacity to respond to BCR stimulation.

Molecular bases of anorexia nervosa, bulimia nervosa and binge eating disorder: shedding light on the darkness.

Eating-disorders (EDs) consequences to human health are devastating, involving social, mental, emotional, physical and life-threatening aspects, concluding on impairment and death in cases of extreme anorexia nervosa. It also implies that people suffering an ED need to find psychiatric and psychological help as soon as possible to achieve a fully physical and emotional recovery. Unfortunately, to date, there is a crucial lack of efficient clinical treatment to these disorders. In this review, we present an overview concerning the actual pharmacological and psychological treatments, the knowledge of cells, circuits, neuropeptides, neuromodulators and hormones in the human brain- and other organs- underlying these disorders, the studies in animal models and, finally, the genetic approaches devoted to face this challenge. We will also discuss the need for new perspectives, avenues and strategies to be developed in order to pave the way to novel and more efficient therapeutics.

FireDB: a compendium of biological and pharmacologically relevant ligands.

FireDB (http://firedb.bioinfo.cnio.es) is a curated inventory of catalytic and biologically relevant small ligand-binding residues culled from the protein structures in the Protein Data Bank. Here we present the important new additions since the publication of FireDB in 2007. The database now contains an extensive list of manually curated biologically relevant compounds. Biologically relevant compounds are informative because of their role in protein function, but they are only a small fraction of the entire ligand set. For the remaining ligands, the FireDB provides cross-references to the annotations from publicly available biological, chemical and pharmacological compound databases. FireDB now has external references for 95% of contacting small ligands, making FireDB a more complete database and providing the scientific community with easy access to the pharmacological annotations of PDB ligands. In addition to the manual curation of ligands, FireDB also provides insights into the biological relevance of individual binding sites. Here, biological relevance is calculated from the multiple sequence alignments of related binding sites that are generated from all-against-all comparison of each FireDB binding site. The database can be accessed by RESTful web services and is available for download via MySQL.

Antiproliferative and Structure Activity Relationships of Amaryllidaceae Alkaloids.

The antiproliferative activity of a set of seven natural Amaryllidaceae alkaloids and 32 derivatives against four cancer cell lines (A2780, SW1573, T47-D and WiDr) was determined. The best antiproliferative activities were achieved with alkaloids derived from pancracine (2), haemanthamine (6) and haemantidine (7). For each skeleton, some structure-activity relationships were outlined.

Risk Prediction of Metachronous Colorectal Cancer from Molecular Features of Adenomas: A Nested Case-Control Study.

Current morphologic features defining advanced adenomas (size ≥10 mm, high-grade dysplasia or ≥25% villous component) cannot optimally distinguish individuals at high risk or low risk of metachronous colorectal cancer (me-CRC), which may result in suboptimal surveillance. Certain DNA copy-number alterations (CNAs) are associated with adenoma-to-carcinoma progression. We aimed to evaluate whether these molecular features can better predict an individual's risk of me-CRC than the morphologic advanced adenoma features.In this nested case-control study, 529 individuals with a single adenoma at first colonoscopy were selected from a Norwegian adenoma cohort. DNA copy-number profiles were determined, by low-coverage whole-genome sequencing. Prevalence of CNAs in advanced and non-advanced adenomas and its association (OR) with me-CRC was assessed. For the latter, cases (with me-CRC) were matched to controls (without me-CRC) on follow-up, age and sex.CNAs associated with adenoma-to-carcinoma progression were observed in 85/267 (32%) of advanced adenomas and in 27/262 (10%) of non-advanced adenomas. me-CRC was statistically significantly associated, also after adjustment for other variables, with age at baseline [OR, 1.14; 95% confidence interval CI), 1.03-1.26; P = 0.012], advanced adenomas (OR, 2.46; 95% CI, 1.50-4.01; P < 0.001) and with the presence of ≥3 DNA copy-number losses (OR, 1.90; 95% CI. 1.02-3.54; P = 0.043).Molecularly-defined high-risk adenomas were associated with me-CRC, but the association of advanced adenoma with me-CRC was stronger. SIGNIFICANCE: Identifying new biomarkers may improve prediction of me-CRC for individuals with adenomas and optimize surveillance intervals to reduce risk of colorectal cancer and reduce oversurveillance of patients with low risk of colorectal cancer. Use of DNA CNAs alone does not improve prediction of me-CRC. Further research to improve risk classification is required.

Reactivity and biological properties of a series of cytotoxic PtI2(amine)2 complexes, either cis or trans configured.

Six diiodido-diamine platinum(II) complexes, either cis or trans configured, were prepared, differing only in the nature of the amine ligand (isopropylamine, dimethylamine, or methylamine), and their antiproliferative properties were evaluated against a panel of human tumor cell lines. Both series of complexes manifested pronounced cytotoxic effects, with the trans isomers being, generally, more effective than their cis counterparts. Cell cycle analysis revealed different modes of action for these new Pt(II) complexes with respect to cisplatin. The reactivity of these platinum compounds with a number of biomolecules, including cytochrome c, two sulfur containing modified amino acids, 9-ethylguanine, and a single strand oligonucleotide, was analyzed in depth by mass spectrometry and NMR spectroscopy. Interestingly, significant differences in the reactivity of the investigated compounds toward the various model biomolecules were observed: in particular we observed that trans complexes preferentially release their iodide ligands upon biomolecule binding, while the cis isomers may release the amine ligands with retention of iodides. Such differences in reactivity may have important mechanistic implications and a relevant impact on the respective pharmacological profiles.

miR-181a is a novel player in the STAT3-mediated survival network of TCRαß+ CD8+ T large granular lymphocyte leukemia.

T-LGL cells arise as a consequence of chronic antigenic stimulation and inflammation and thrive because of constitutive activation of the STAT3 and ERK pathway. Notably, in 40% of patients, constitutive STAT3 activation is due to STAT3 activating mutations, whereas in 60% this is unknown. As miRNAs are amongst the most potent regulators in health and disease, we hypothesized that aberrant miRNA expression could contribute to dysregulation of these pathways. miRNA sequencing in T-LGL leukemia cases and aged-matched healthy control TEMRA cells revealed overexpression of miR-181a. Furthermore, geneset enrichment analysis (GSEA) of downregulated targets of miR-181a implicated involvement in regulating STAT3 and ERK1/2 pathways. Flow cytometric analyses showed increased SOCS3+ and DUSP6+ T-LGL cells upon miR-181a inhibition. In addition, miR-181a-transfected human CD8+ T cells showed increased basal STAT3 and ERK1/2 phosphorylation. By using TL1, a human T-LGL cell line, we could show that miR-181a is an actor in T-LGL leukemia, driving STAT3 activation by SOCS3 inhibition and ERK1/2 phosphorylation by DUSP6 inhibition and verified this mechanism in an independent cell line. In addition, miR-181a inhibition resulted in a higher sensitivity to FAS-mediated apoptosis. Collectively, our data show that miR-181a could be the missing link to explain why STAT3-unmutated patients show hyperactive STAT3.

The tumor suppressor MIR139 is silenced by POLR2M to promote AML oncogenesis.

MIR139 is a tumor suppressor and is commonly silenced in acute myeloid leukemia (AML). However, the tumor-suppressing activities of miR-139 and molecular mechanisms of MIR139-silencing remain largely unknown. Here, we studied the poorly prognostic MLL-AF9 fusion protein-expressing AML. We show that MLL-AF9 expression in hematopoietic precursors caused epigenetic silencing of MIR139, whereas overexpression of MIR139 inhibited in vitro and in vivo AML outgrowth. We identified novel miR-139 targets that mediate the tumor-suppressing activities of miR-139 in MLL-AF9 AML. We revealed that two enhancer regions control MIR139 expression and found that the polycomb repressive complex 2 (PRC2) downstream of MLL-AF9 epigenetically silenced MIR139 in AML. Finally, a genome-wide CRISPR-Cas9 knockout screen revealed RNA Polymerase 2 Subunit M (POLR2M) as a novel MIR139-regulatory factor. Our findings elucidate the molecular control of tumor suppressor MIR139 and reveal a role for POLR2M in the MIR139-silencing mechanism, downstream of MLL-AF9 and PRC2 in AML. In addition, we confirmed these findings in human AML cell lines with different oncogenic aberrations, suggesting that this is a more common oncogenic mechanism in AML. Our results may pave the way for new targeted therapy in AML.

Antiproliferative activity of dmoPTA-Ru(II) complexes against human solid tumor cells.

The biological evaluation of new Ru(II) complexes carrying dmoPTA (dmoPTA=3,7-dimethyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) ligands is reported. The results on the biological activity revealed that the organometallic complexes are active against all cell lines with GI(50) values in the range 1.1-2.6 µM. When compared to the standard anticancer drug cisplatin, the bimetallic Ru(II) complexes showed a greater activity profile. The cell cycle analysis revealed that the new compounds induced arrest in G(1) phase. Contrary to cisplatin, these Ru(II) complexes do not interact with DNA. This result suggests that DNA might not be the key pharmacological target.

A modular approach to trim cellular targets in anticancer drug discovery.

A Phenotypic Drug Discovery strategy was applied to study a set of pyrimidine analogs prepared by means of intramolecular oxidation-reduction reactions of N-substituted-N-(2,6-disubstituted-5-nitro-4-pyrimidinyl)aminoacetic acid methyl esters in basic media. The combined and rational use of specific assays allowed in short time reducing from all possible cellular targets to those involved in metaphase to anaphase transition.

Staurosporine increases toxicity of gemcitabine in non-small cell lung cancer cells: role of protein kinase C, deoxycytidine kinase and ribonucleotide reductase.

Gemcitabine, a deoxycytidine analog, active against non-small cell lung cancer, is phosphorylated by deoxycytidine kinase (dCK) to active nucleotides. Earlier, we found increased sensitivity to gemcitabine in P-glycoprotein (SW-2R160) and multidrug resistance-associated protein (SW-2R120), overexpressing variants of the human SW1573 non-small cell lung cancer cells. This was related to increased dCK activity. As protein kinase C (PKC) is higher in 2R120 and 2R160 cells and may control the dCK activity, we investigated whether gemcitabine sensitivity was affected by the protein kinase C inhibitor, staurosporine, which also modulates the cell cycle. Ten nmol/l staurosporine enhanced the sensitivity of SW1573, 2R120 and 2R160 cells 10-fold, 50-fold and 270-fold, respectively. Staurosporine increased dCK activity about two-fold and the activity of thymidine kinase 2, which may also activate gemcitabine. Staurosporine also directly increased dCK in cell free extracts. Staurosporine decreased expression of the free transcription factor E2F and of ribonucleotide reductase (RNR), a target for gemcitabine inhibition. In conclusion, staurosporine may potentiate gemcitabine by increasing dCK and decreasing E2F and RNR, which will lead to a more pronounced RNR inhibition.

Antiproliferative activity of synthetic naphthoquinones related to lapachol. First synthesis of 5-hydroxylapachol.

A series of 5-hydroxy-1,4-naphthoquinones analogues was synthesized from juglone (6) and their antiproliferative activity against a representative panel of six human solid tumor cell lines has been investigated. The 2,5-dihydroxy-3-(3-methylbut-2-enyl)naphthalene-1,4-dione (4) and 2,3-dihydro-5-hydroxy-2-(prop-1-en-2-yl)naphtho[2,3-b]furan-4,9-dione (27) were the most potent antiproliferative agents with GI(50) values of 0.42-8.1 and 0.80-2.2microM, respectively. The results provide insight into the correlation between some structural properties of 5-hydroxynaphthoquinones and their antiproliferative activity.

Enhancement of antiproliferative activity by molecular simplification of catalpol.

Two iridoid scaffolds were synthesized enantioselectively using as key step an l-proline-catalyzed alpha-formyl oxidation. The in vitro antiproliferative activities were evaluated against a representative panel of human solid tumor cell lines. Both iridoids induced considerably growth inhibition in the range 0.38-1.86muM. Cell cycle studies for these compounds showed the induction of cell cycle arrest at the G(1) phase. This result was consistent with a decrease in the expression of cyclin D1. Damaged cells underwent apoptosis as indicated by specific Annexin V staining.

Heatr9 is an infection responsive gene that affects cytokine production in alveolar epithelial cells.

During infection, viruses enter susceptible host cells in order to replicate their components for production of new virions. In the process of infection, the gene expression of infected cells undergoes changes because of the production of viral components and due to the host response from detection of viral products. In the advent of RNA sequencing, the discovery of new genes and their functions in the host response generates new avenues for interventions in the host-pathogen interaction. We have identified a novel gene, Heatr9, as a virus and cytokine inducible viral responsive gene. We confirm Heatr9's expression in vitro and in vivo during virus infection and correlate it with viral burden. Heatr9 is induced by influenza virus and RSV. Heatr9 knockdown during viral infection was shown to affect chemokine expression. Our studies identify Heatr9 as a novel inflammatory and virus infection induced gene that can regulate the induction of specific cytokines.

Glucocorticoid Resistant Pediatric Acute Lymphoblastic Leukemia Samples Display Altered Splicing Profile and Vulnerability to Spliceosome Modulation.

Glucocorticoid (GC) resistance is a crucial determinant of inferior response to chemotherapy in pediatric acute lymphoblastic leukemia (ALL); however, molecular mechanisms underlying this phenomenon are poorly understood. Deregulated splicing is a common feature of many cancers, which impacts drug response and constitutes an attractive therapeutic target. Therefore, the aim of the current study was to characterize global splicing profiles associated with GC resistance and determine whether splicing modulation could serve as a novel therapeutic option for GC-resistant patients. To this end, 38 primary ALL samples were profiled using RNA-seq-based differential splicing analysis. The impact of splicing modulators was investigated in GC-resistant leukemia cell lines and primary leukemic specimens. Our findings revealed, for the first time, markedly distinct splicing landscapes in ALL samples of B-cell precursor (BCP)-ALL and T-ALL lineages. Differential splicing events associated with GC resistance were involved in RNA processing, a direct response to GCs, survival signaling, apoptosis, cell cycle regulation and energy metabolism. Furthermore, our analyses showed that GC-resistant ALL cell lines and primary samples are sensitive to splicing modulation, alone and in combination with GC. Together, these findings suggest that aberrant splicing is associated with GC resistance and splicing modulators deserve further interest as a novel treatment option for GC-resistant patients.

Synthesis and antiproliferative activity of 2,4-disubstituted 6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxides.

A series of 2,4-disubstituted 6-aryl-7H-pyrrolo[3,2-d]pyrimidin-7-one 5-oxides were synthesized and in vitro antiproliferative activities were examined in the human solid tumor cell lines A2780, HBL-100, HeLa, SW1573, T-47D, and WiDr. The most potent analog induced considerably growth inhibition in the range 0.35-2.0microM. Cell cycle studies in the breast and lung cancer cells revealed arrest in the G(2)/M compartment. The results showed that the title compounds bearing alkylamino or dialkylamino moieties in position 2 of the pyrimidine ring are more active than those bearing hydrogen or methylthio groups.