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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.

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 β 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 TGFβR3 and aberrant TGFβ1- induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFβ 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.

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 demon-strated 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.

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.

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., Author summary In this manuscript, we describe an in vitro method that rapidly establishes large numbers of exhausted CD8+ T cells. The exhaustion of CTL induced by this method has been fully validated by multiple approaches (cytokine production, polyfunctionality, cytotoxicity, in vivo proliferation, inhibitory receptors, transcription factors, RNAseq and DNA methylation). This method will facilitate not only the study of T cell exhaustion but also the screening of drugs. As proof of point, we use this method to show that TCF-1 downregulation in terminally exhausted T cells is accompanied by Tcf7 DNA promoter methylation and show that a transmethylase inhibitor can prevent TCF-1 downregulation. Our method presents a critical resource for the study of CTL exhaustion and the screening of drugs and interventions.

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.

Background Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder caused by expansion of the polyglutamine repeat in the ataxin-3 protein. Expression of mutant ataxin-3 is known to result in transcriptional dysregulation, which can contribute to the cellular toxicity and neurodegeneration. Since the exact causative mechanisms underlying this process have not been fully elucidated, gene expression analyses in brains of transgenic SCA3 mouse models may provide useful insights. Methods Here we characterised the MJD84.2 SCA3 mouse model expressing the mutant human ataxin-3 gene using a multi-omics approach on brain and blood. Gene expression changes in brainstem, cerebellum, striatum and cortex were used to study pathological changes in brain, while blood gene expression and metabolites/lipids levels were examined as potential biomarkers for disease. Results Despite normal motor performance at 17.5 months of age, transcriptional changes in brain tissue of the SCA3 mice were observed. Most transcriptional changes occurred in brainstem and striatum, whilst cerebellum and cortex were only modestly affected. The most significantly altered genes in SCA3 mouse brain were Tmc3, Zfp488, Car2, and Chdh. Based on the transcriptional changes, α-adrenergic and CREB pathways were most consistently altered for combined analysis of the four brain regions. When examining individual brain regions, axon guidance and synaptic transmission pathways were most strongly altered in striatum, whilst brainstem presented with strongest alterations in the pi-3 k cascade and cholesterol biosynthesis pathways. Similar to other neurodegenerative diseases, reduced levels of tryptophan and increased levels of ceramides, di- and triglycerides were observed in SCA3 mouse blood. Conclusions The observed transcriptional changes in SCA3 mouse brain reveal parallels with previous reported neuropathology in patients, but also shows brain region specific effects as well as involvement of adrenergic signalling and CREB pathway changes in SCA3. Importantly, the transcriptional changes occur prior to onset of motor- and coordination deficits. Electronic supplementary material The online version of this article (10.1186/s13024-018-0261-9) contains supplementary material, which is available to authorized users.

5-Fluorouracil (5FU) is still a major drug in combinations regimens for the treatment of colorectal cancer (CRC) both in the adjuvant and palliative setting. 5FU or its oral prodrug capecitabine is usually combined with irinotecan/oxaliplatin and the novel agents bevacizumab/cetuximab. Although this improved the outcome, the overall prognosis in patients with metastasized disease is still relatively poor. Although the target for 5FU, thymidylate synthase was shown to have a predictive value, this could only predict response in a subset of patients. Given the heterogeneous and complex nature of CRC, it is likely that other aberrations can affect therapeutic response. As an alternative, we investigated Copy number alterations using oligonucleotide-based high-throughput array-comparative-genomic-hybridization (aCGH) to obtain an unbiased screening of the whole genetic spectrum. Chromosomal aberrations have been identified in 85% of CRC patients and include genomic regions harboring copy number alterations in the DNA. These alterations may change the expression of many genes and might explain the differential response to therapy as shown in recent studies with several 5FU combinations. In order to clarify new predictive parameters for 5FU, we used aCGH in a historical cohort of patients, which received treatment with single agent 5FU, and an unsupervised clustering analysis showed a statistical (p0.05) difference between responding and nonresponding patients. We also find that several regions showed differences between responders/non-responders, such as losses in 12p12.3-12q15 and in 18p (where TS is located) in responding patients. Genome-wide analysis may provide an additional tool to discriminate between responders and nonresponders.

The aim of the present study was to develop chick-embryo chorioallantoic membrane (CAM) bioluminescent tumor models employing low passage cell cultures obtained from primary pancreatic ductal adenocarcinoma (PDAC) cells. Primary PDAC cells transduced with lentivirus expressing Firefly-luciferase (Fluc) were established and inoculated onto the CAM membrane, with >80% engraftment. Fluc signal reliably correlated with tumor growth. Tumor features were evaluated by immunohistochemistry and genetic analyses, including analysis of mutations and mRNA expression of PDAC pivotal genes, as well as microRNA (miRNA) profiling. These studies showed that CAM tumors had histopathological and genetic characteristic comparable to the original tumors. We subsequently tested the modulation of key miRNAs and the activity of gemcitabine and crizotinib on CAM tumors, showing that combination treatment resulted in 63% inhibition of tumor growth as compared to control (p

We have read with interest the article by Vena and colleagues ([1][1]), on the combination of the MEK1/2 inhibitor pimasertib with gemcitabine in pancreatic ductal adenocarcinoma (PDAC). MEK emerged as a promising target to sensitize gemcitabine therapy through downregulation of the ribonucleotide

Introduction. Despite many clinical efforts, non-small-cell lung cancer (NSCLC) has a dismal 5-year survival rate of 16%, and high incidence of recurrence. The success of biologically targeted agents, as well as the activity of well-established chemotherapeutic regimens, has been limited by inherited/acquired resistance, and biomarkers to adapt the prescription of anticancer drugs to patients' features are urgently warranted. Areas covered. In oncology, pharmacogenetics should provide the way to select patients who may benefit from a specific therapy that best match the individual and tumor genetic profile, thus allowing maximum activity and minimal toxicity. The present review summarizes the main findings on NSCLC pharmacogenetics, critically reappraising the most important studies on polymorphisms correlated with outcome of pemetrexed and EGFR-inhibitors, and provides perspective on clinical application of genomic tests for treatment decision-making. Expert Opinion. A major challenge in NSCLC is the identification of subgroups of diseases/patients that will truly benefit from specific treatments. Ideally, convenient and minimally invasive tests to decipher biomarkers of chemosensitivity/resistance and toxicity should be developed alongside novel anticancer treatments. Integration with the latest generation of whole-genome analyses and liquid biopsies as well as prospective validation in large cohorts of patients will overcome the limitations of the traditional pharmacogenetic approaches.

Neopterin is produced by monocytes and a useful biomarker of inflammatory responses. We found recently in mice that neopterin suppressed erythropoiesis but enhanced myelopoiesis possibly by activating stromal cells, the same phenomenon observed in inflammation. In this study, we investigated whether neopterin affects the proliferation and differentiation of immature erythroid progenitor cells (BFU-E) by stimulating bone-marrow stromal cells in culture. Neopterin decreased the number of colonies of BFU-E formed by unfractionated bone-marrow cells in a semisolid culture system, but increased the number of granulocyte-macrophage progenitor cells (CFUGM). In contrast, it did not affect numbers of BFU-E or CFU-GM among a lineage-negative (fractionated) bonemarrow- cell population cultured without stromal cells. These results suggest that neopterin decreased the number of colonies of BFU-E by activating bone-marrow stromal-cell function.

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.

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.

BackgroundOnly a subset of radically resected pancreatic ductal adenocarcinoma (PDAC) patients benefit from chemotherapy, and identification of prognostic factors is warranted. Recently miRNAs emerged as diagnostic biomarkers and innovative therapeutic targets, while high-throughput arrays are opening new opportunities to evaluate whether they can predict clinical outcome. The present study evaluated whether comprehensive miRNA expression profiling correlated with overall survival (OS) in resected PDAC patients.Methodology/principal findingsHigh-resolution miRNA profiles were obtained with the Toray's 3D-Gene™-miRNA-chip, detecting more than 1200 human miRNAs. RNA was successfully isolated from paraffin-embedded primary tumors of 19 out of 26 stage-pT3N1 homogeneously treated patients (adjuvant gemcitabine 1000 mg/m(2)/day, days-1/8/15, every 28 days), carefully selected according to their outcome (OS30 months (N = 6), i.e. short/long-OS). Highly stringent statistics included t-test, distance matrix with Spearman-ranked correlation, and iterative approaches. Unsupervised hierarchical analysis revealed that PDACs clustered according to their short/long-OS classification, while the feature selection algorithm RELIEF identified the top 4 discriminating miRNAs between the two groups. These miRNAs target more than 1500 transcripts, including 169 targeted by two or more. MiR-211 emerged as the best discriminating miRNA, with significantly higher expression in long- vs. short-OS patients. The expression of this miRNA was subsequently assessed by quantitative-PCR in an independent cohort of laser-microdissected PDACs from 60 resected patients treated with the same gemcitabine regimen. Patients with low miR-211 expression according to median value had a significantly shorter median OS (14.8, 95%CI = 13.1-16.5, vs. 25.7 months, 95%CI = 16.2-35.1, log-rank-P = 0.004). Multivariate analysis demonstrated that low miR-211 expression was an independent factor of poor prognosis (hazard ratio 2.3, P = 0.03) after adjusting for all the factors influencing outcome.Conclusions/significanceThrough comprehensive microarray analysis and PCR validation we identified miR-211 as a prognostic factor in resected PDAC. These results prompt further prospective studies and research on the biological role of miR-211 in PDAC.

Background Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The high risk of recurrence following surgical resection provides the rationale for adjuvant therapy. However, only a subset of patients benefit from adjuvant therapy. Identification of molecular markers to predict treatment outcome is therefore warranted. The aim of the present study was to evaluate whether expression of novel candidate biomarkers, including microRNAs, can predict clinical outcome in PDAC patients treated with adjuvant therapy. Methodology/Principal Findings Formalin-fixed paraffin embedded specimens from a cohort of 82 resected Korean PDAC cases were analyzed for protein expression by immunohistochemistry and for microRNA expression using quantitative Real-Time PCR. Cox proportional hazards model analysis in the subgroup of patients treated with adjuvant therapy (N = 52) showed that lower than median miR-21 expression was associated with a significantly lower hazard ratio (HR) for death (HR = 0.316; 95%CI = 0.166–0.600; P = 0.0004) and recurrence (HR = 0.521; 95%CI = 0.280–0.967; P = 0.04). MiR-21 expression status emerged as the single most predictive biomarker for treatment outcome among all 27 biological and 9 clinicopathological factors evaluated. No significant association was detected in patients not treated with adjuvant therapy. In an independent validation cohort of 45 frozen PDAC tissues from Italian cases, all treated with adjuvant therapy, lower than median miR-21 expression was confirmed to be correlated with longer overall as well as disease-free survival. Furthermore, transfection with anti-miR-21 enhanced the chemosensitivity of PDAC cells. Conclusions Significance Low miR-21 expression was associated with benefit from adjuvant treatment in two independent cohorts of PDAC cases, and anti-miR-21 increased anticancer drug activity in vitro. These data provide evidence that miR-21 may allow stratification for adjuvant therapy, and represents a new potential target for therapy in PDAC.

4 pages, 2 figures, 1 table.-- PMID: 17110107 [PubMed].-- Available online Oct 27, 2006., In this study, we synthesized a series of enantiomerically pure (2R,3R)- and (2R,3S)-disubstituted tetrahydropyrans bearing a CH2O spacer group on the side chain at position 2 of the heterocyclic ring. The in vitro antiproliferative activities of the compounds were examined in the human solid tumor cell lines A2780 (ovarian cancer), SW1573 (non-small cell lung cancer), and WiDr (colon cancer). Overall, the results point out the relevance for antiproliferative activity of the distance between the heterocycle and the unsaturated group., This research was supported by the EU INTERREG IIIB-MAC initiative (05/MAC/2.5/C14 BIOPOLIS), the Ministerio de Educación y Ciencia of Spain, cofinanced by the European Regional Development Fund (CTQ2005-09074-C02-01/BQU), and the Canary Islands Government. R.C. thanks the Spanish MEC for an FPU fellowship.

// Elena Galvani 1 , Jing Sun 2 , Leticia G. Leon 3 , Rocco Sciarrillo 1,4 , Ravi S. Narayan 5 , Robert Tjin Tham Sjin 6 , Kwangho Lee 6 , Kadoaki Ohashi 2 , Danielle A.M. Heideman 7 , Roberta R. Alfieri 8 , Guus J. Heynen 9 , Rene Bernards 9 , Egbert F. Smit 10 , William Pao 2 , Godefridus J. Peters 1 and Elisa Giovannetti 1,11 1 Department Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands 2 Division of Hematology/Oncology, Department of Medicine, Vanderbilt University School of Medicine and Vanderbilt-Ingram Cancer Center, Nashville, TN, USA 3 Instituto de Tecnologias Biomedicas, Center for Biomedical Research of the Canary Islands, University of La Laguna, Tenerife, Spain 4 Department Hematology, VU University Medical Center, Amsterdam, The Netherlands 5 Department Radiation Oncology, VU University Medical Center, Amsterdam, The Netherlands 6 Celgene Avilomics Research, Bedford, MA, USA 7 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands 8 Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy 9 Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam, The Netherlands 10 Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands 11 Cancer Pharmacology Lab, AIRC Start-Up Unit, DIPINT, University of Pisa, Pisa, Italy Correspondence to: Elisa Giovannetti, email: // Keywords : drug-resistance, EGFR-T790M, NSCLC, NF-κB, EMT Received : January 10, 2015 Accepted : April 08, 2015 Published : April 29, 2015 Abstract The clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations is limited by the emergence of acquired resistance, mostly ascribed to the secondary EGFR-T790M mutation. Selective EGFR-T790M inhibitors have been proposed as a new, extremely relevant therapeutic approach. Here, we demonstrate that the novel irreversible EGFR-TKI CNX-2006, a structural analog of CO-1686, currently tested in a phase-1/2 trial, is active against in vitro and in vivo NSCLC models expressing mutant EGFR, with minimal effect on the wild-type receptor. By integration of genetic and functional analyses in isogenic cell pairs we provide evidence of the crucial role played by NF-κB1 in driving CNX-2006 acquired resistance and show that NF-κB activation may replace the oncogenic EGFR signaling in NSCLC when effective and persistent inhibition of the target is achieved in the presence of the T790M mutation. In this context, we demonstrate that the sole, either genetic or pharmacologic, inhibition of NF-κB is sufficient to reduce the viability of cells that adapted to EGFR-TKIs. Overall, our findings support the rational inhibition of members of the NF-κB pathway as a promising therapeutic option for patients who progress after treatment with novel mutant-selective EGFR-TKIs.