Overexpression of primary microRNA 221/222 in acute myeloid leukemia

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy with a dismal outcome in the majority of cases. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet, thus warranting further investigations. METHODS: The expression of 636 human miRNAs was compared between samples from 52 patients with AML and 13 healthy individuals by highly specific locked nucleic acid (LNA) based microarray technology. The levels of individual mature miRNAs and of primary miRNAs (pri-miRs) were determined by quantitative reverse transcriptase (qRT) PCR. Transfections and infections of human cell lines were performed using standard procedures. RESULTS: 64 miRNAs were significantly differentially expressed between AML and controls. Further studies on the clustered miRNAs 221 and 222, already known to act as oncogenes in other tumor types, revealed a deficiency of human myeloid cell lines to process vector derived precursor transcripts. Moreover, endogenous pri-miR-221/222 was overexpressed to a substantially higher extent than its mature products in most primary AML samples, indicating that its transcription was enhanced, but processing was rate limiting, in these cells. Comparison of samples from the times of diagnosis, remission, and relapse of AML demonstrated that pri-miR-221/222 levels faithfully reflected the stage of disease. CONCLUSIONS: Expression of some miRNAs is strongly regulated at the posttranscriptional level in AML. Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease

Interleukin-6 receptor blockade in treatment-refractory MOG-IgG–associated disease and neuromyelitis optica spectrum disorders

BACKGROUND AND OBJECTIVES: To evaluate the long-term safety and efficacy of tocilizumab (TCZ), a humanized anti–interleukin-6 receptor antibody in myelin oligodendrocyte glycoprotein–IgG–associated disease (MOGAD) and neuromyelitis optica spectrum disorders (NMOSD). METHODS: Annualized relapse rate (ARR), Expanded Disability Status Scale score, MRI, autoantibody titers, pain, and adverse events were retrospectively evaluated in 57 patients with MOGAD (n = 14), aquaporin-4 (AQP4)-IgG seropositive (n = 36), and seronegative NMOSD (n = 7; 12%), switched to TCZ from previous immunotherapies, particularly rituximab. RESULTS: Patients received TCZ for 23.8 months (median; interquartile range 13.0–51.1 months), with an IV dose of 8.0 mg/kg (median; range 6–12 mg/kg) every 31.6 days (mean; range 26–44 days). For MOGAD, the median ARR decreased from 1.75 (range 0.5–5) to 0 (range 0–0.9; p = 0.0011) under TCZ. A similar effect was seen for AQP4-IgG+ (ARR reduction from 1.5 [range 0–5] to 0 [range 0–4.2]; p < 0.001) and for seronegative NMOSD (from 3.0 [range 1.0–3.0] to 0.2 [range 0–2.0]; p = 0.031). During TCZ, 60% of all patients were relapse free (79% for MOGAD, 56% for AQP4-IgG+, and 43% for seronegative NMOSD). Disability follow-up indicated stabilization. MRI inflammatory activity decreased in MOGAD (p = 0.04; for the brain) and in AQP4-IgG+ NMOSD (p < 0.001; for the spinal cord). Chronic pain was unchanged. Regarding only patients treated with TCZ for at least 12 months (n = 44), ARR reductions were confirmed, including the subgroups of MOGAD (n = 11) and AQP4-IgG+ patients (n = 28). Similarly, in the group of patients treated with TCZ for at least 12 months, 59% of them were relapse free, with 73% for MOGAD, 57% for AQP4-IgG+, and 40% for patients with seronegative NMOSD. No severe or unexpected safety signals were observed. Add-on therapy showed no advantage compared with TCZ monotherapy. DISCUSSION: This study provides Class III evidence that long-term TCZ therapy is safe and reduces relapse probability in MOGAD and AQP4-IgG+ NMOSD

Biological properties and target genes of the myelodysplastic syndrome and acute myeloid leukaemia associated transcription factor Ecotropic Viral Integration Site 1

Die erhöhte Expression des Onkogens Ecotropic Viral integration Site 1 (EVI1) steht im Kontext mit der Pathogenese des Myelodysplastischen Syndroms (MDS) und der akuten myeloischen Leukämie (AML). Sie korreliert bei der AML mit einer schlechten Prognose. Obgleich der klinischen Relevanz ist wenig über den Funktionsmechanismus von EVI1 bekannt. Das Ziel dieser Arbeit war es, die biologischen Effekte von EVI1 zu erörtern und die zugrundeliegenden molekularen Mechanismen anhand der Suche nach Zielgenen zu untersuchen. Die Expression von EVI1 in humanen U937 Zellen wurde einerseits durch Transfektion eines Tetrazyklin-induzierbaren Systems (ergab U937T_EVI1 Zellen und Kontrollzelllinie U937T_vec), andererseits durch Transduktion eines konstitutiv exprimierenden Vektors (ergab U937_EVI1 Zellen und Kontrollzelllinie U937_vec) erreicht. In U937T_EVI1 Zellen wurden Zellzyklusarrest, mittelgradige spontane Apoptose sowie hochgradige Apoptose während der Differenzierung beobachtet. Der Phänotyp der U937_EVI1 Zellen beinhaltete: gestörtes Differenzierungsverhalten, beschleunigtes Tumorwachstum in vivo sowie partielle Zytostatikaresistenz. Microarrays der konstitutiven Zelllinien in An- und Abwesenheit von Etoposid ermittelten die Gene Cyclin Dependent Kinase Inhibitor p21 (p21 oder CDKN1A) und Cellular Adhesion Molecule 1 (CADM1) als dereguliert sowohl durch EVI1 als auch durch Etoposid. Ektope Expression von p21 in U937 Zellen führte zu einer analog zur EVI1 induzierten, partiellen Zytostatikaresistenz. Der Tumorsuppressor CADM1 war bei U937_EVI1 Zellen nicht induzierbar, dies korrelierte mit einer gesteigerten CADM1 Promotormethylierung. Unahbängig von Zytostatikabehandlung, wurde Membrane-Spanning 4-domain family, subfamily A member 3 (MS4A3) als Zielgen, welches in beiden EVI1 Zelllinien und gleichsam AML Patienten deutlich runterreguliert wurde, identifiziert. Reportengen Analysen und Chromatin-Immunopräzipitation zeigten, dass EVI1 direkt mit einem proximalen MS4A3 Promotorelement interagiert. Simultane Expression von EVI1 und MS4A3 in einem murinen Xenograft-Modell führte zu einer Reduktion des Tumorwachstums, verglichen mit nur durch EVI1 induzierten Tumoren. Unsere Ergebnisse zeigen, dass eine ektope EVI1 Expression in U937 Zellen phänotypisch an zentrale Krankheitsmerkmale von MDS und AML erinnert. Drei neue Zielgene, p21, CADM1 und MS4A3 wurden identifiziert und es bestätigte sich, dass diese Gene zu der EVI1 vermittelten Zytostatikaresistenz und zu einem aggressiven Krankheitsverlauf beitragen. Ein verbessertes Verständnis des molekularen Zusammenhangs von EVI1 und AML, zu dem diese Arbeit hoffentlich beitragen kann, ist der Grundstein für die Entwicklung gerichteter Therapien um die Prognose von AML Patienten, mit erhöhter EVI1 Expression, zu verbessern.Aberrant expression of the oncogene Ecotropic Viral Integration Site 1 (EVI1) has been implicated in the pathogenesis of myelodysplatic syndrome (MDS) and acute myeloid leukaemia (AML) and is correlated with poor outcome in AML patients. Despite of its prognostic importance, the molecular and cellular mechanisms of EVI1 action are still incompletely understood. This work aimed to investigate biological effects of EVI1 in haematopoietic cells as well as to unravel molecular mechanisms behind the observed effects by searching for target genes of EVI1. EVI1 overexpression in human U937 cells was accomplished by transfection with a tetracycline-inducible system on the one hand (resulting in U937T_EVI1 cells and U937T_vec as control cells) and by retroviral infection with a constitutively expressing vector on the other hand (resulting in U937_EVI1 cells and U937_vec as control cells). The phenotypes of the U937T_EVI1 cells included cell cycle arrest, moderate spontaneous apoptosis and massive apoptosis upon differentiation stimuli, while U937_EVI1 cells exhibited perturbed maturation, accelerated tumour growth in vivo, and partial resistance to cytostatic drugs. Microarray analysis of constitutively expressing cells in the presence and absence of etoposide identified the Cyclin Dependent Kinase Inhibitor p21 (p21 or CDKN1A) and Cellular Adhesion Molecule 1 (CADM1) genes as deregulated by both EVI1 and etoposide. Overexpression of CDKN1A, which was upregulated in response to both EVI1 and etoposide, in U937 cells, conferred partial drug resistance, thereby mimicking the phenotype of EVI1 expression. Upregulation of the tumour suppressor CADM1 was abolished in EVI1 overexpressing cells, and this correlated with increased promotor methylation. Independently of drug treatment, Membrane-Spanning 4-domain family, subfamily A member 3 (MS4A3) was strongly downregulated upon both inducible and constitutive EVI1 overexpression in U937 cells, and correlated with EVI1 expression in publicly available AML patient data sets. Reporter gene assays and chromatin immunoprecipitation proved that EVI1 directly regulated MS4A3 by binding to its promotor. Simultaneous expression of MS4A3 and EVI1 in murine xenografts antagonised the EVI1 mediated acceleration of tumour growth. In summary, our data show that ectopic expression of EVI1 in U937 cells mimics several key features of both MDS and AML. Three novel EVI1 target genes, p21, CADM1 and MS4A3 were identified, and shown to contribute to EVI1 mediated drug refractoriness and disease aggressiveness. The results of this work may contribute to a better understanding of the molecular crosstalk between EVI1 and AML, which again is indispensable for developing new therapy strategies, in order to eventually improve disease outcome in AML patients with EVI1 overexpression.submitted by Anna RommerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Diss., 2018(VLID)346357

Tetracycline Regulator Expression Alters the Transcriptional Program of Mammalian Cells

Background: Tetracycline regulated ectopic gene expression is a widely used tool to study gene function. However, the tetracycline regulator (tetR) itself has been reported to cause certain phenotypic changes in mammalian cells. We, therefore, asked whether human myeloid U937 cells expressing the tetR in an autoregulated manner would exhibit alterations in gene expression upon removal of tetracycline. Methodology/Principal Findings: Microarray analyses revealed that 172 and 774 unique genes were significantly differentially expressed by at least 2- or 1.5-fold, respectively, when tetR expressing U937 cells were maintained in media with or without the antibiotic. Conclusions/Significance: These alterations in gene expression are likely to contribute to the phenotypic consequences of tetR expression. In addition, they need to be taken into consideration when using the tetR system for the identification of target genes of transcription factors or other genes of interest

EVI1 inhibits apoptosis induced by antileukemic drugs via upregulation of CDKN1A/p21/WAF in human myeloid cells.

Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in acute myeloid leukemia (AML). Despite of its clinical importance, little is known about the mechanism through which EVI1 confers resistance to antileukemic drugs. Here, we show that a human myeloid cell line constitutively overexpressing EVI1 after infection with a retroviral vector (U937_EVI1) was partially resistant to etoposide and daunorubicin as compared to empty vector infected control cells (U937_vec). Similarly, inducible expression of EVI1 in HL-60 cells decreased their sensitivity to daunorubicin. Gene expression microarray analyses of U937_EVI1 and U937_vec cells cultured in the absence or presence of etoposide showed that 77 and 419 genes were regulated by EVI1 and etoposide, respectively. Notably, mRNA levels of 26 of these genes were altered by both stimuli, indicating that EVI1 regulated genes were strongly enriched among etoposide regulated genes and vice versa. One of the genes that were induced by both EVI1 and etoposide was CDKN1A/p21/WAF, which in addition to its function as a cell cycle regulator plays an important role in conferring chemotherapy resistance in various tumor types. Indeed, overexpression of CDKN1A in U937 cells mimicked the phenotype of EVI1 overexpression, similarly conferring partial resistance to antileukemic drugs

EVI1 promotes tumor growth via transcriptional repression of MS4A3

Background The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells. Methods U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice. Results Gene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis. Conclusions Our data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.(VLID)486732

Effects of <i>EVI1</i> on growth dynamics <i>in vitro</i> and <i>in vivo</i>.

<p>A) Examples of cell cycle profiles of U937_vec and U937_EVI1 cells. B) Cell cycle distribution of U937_vec and U937_EVI1 cells. Shown are the means+standard errors of the mean (SEM) from 3 independent experiments. None of the differences between the two cell lines are statistically significant (Student’s t-test). C) Tumor growth after subcutaneous injection of U937_vec and U937_EVI1 cells into the flanks of CB-17 scid/scid mice. The adjusted area under the curve (aAUC) was calculated for each tumor, and the two groups of tumors were compared by nonparametric bootstrap inference. *, p<0.05. D) Immunohistochemical (IHC) staining showing persistent expression of EVI1 in U937_EVI1 derived tumor xenografts. E) IHC revealing the presence of similar proportions of CD11b positive cells in U937_EVI1 and U937_vec derived tumors.</p

<i>EVI1</i> inhibits phorbol ester (TPA) induced differentiation of U937 cells <i>in vitro</i>.

<p>A) Percentage of CD11b positive U937_vec and U937_EVI1 cells after incubation with TPA (black bars) or solvent (ethanol; white bars) for five days. Shown are the means+SEMs from 3 independent experiments. *, p<0.05; **, p<0.01 (paired Student’s t-test). B) Morphology of Wright stained U937_vec and U937_EVI1 cells after incubation with TPA or vehicle for five days. Original magnification is 600-fold.</p

Regulation of the CDKN1A/p21/WAF mRNA and protein by <i>EVI1</i>, etoposide, and daunorubicin.

<p>A, B) qRT-PCR on RxNA from U937_vec and U937_EVI1 cells treated or not treated with 400 nM etoposide (A) or 30 nM daunorubicin (B) for 48 h. CDKN1A levels were normalized to those of the housekeeping gene B2M using the ΔΔct method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056308#pone.0056308-Livak1" target="_blank">[48]</a>, with untreated U937_vec cells as a calibrator. Shown are means+SEMs from 3 independent experiments. *, p<0.05; n.s., not significant (paired Student’s t-test). C) Immunoblot analysis of p21 protein in U937_vec and U937_EVI1 cells treated or not treated with 400 nM etoposide for 48 h. Hybridization with a β-tubulin antibody was used as a loading control. In the absence of etoposide, p21 is below detection level with the exposure time used. D) Immunoblot analysis of whole cell (WC), cytoplasmatic (cyto), and nuclear (nu) extracts from U937_vec and U937_EVI1 cells. The same amount of protein was loaded for cytoplasmatic and nuclear extracts, corresponding to up to twice as many cell equivalents for the latter. The cytoplasmatic protein β-tubulin and the nuclear protein RCC1 were used as loading controls.</p

Overexpression of <i>EVI1</i> decreases the sensitivity of U937 cells to drugs used in AML therapy.

<p>A–D) U937_vec and U937_EVI1 cells were treated with the indicated concentrations of etoposide (A, C) or daunorubicin (B, D) for 48 h. Cellular viability/metabolic activity was determined based on ATP content (A, B), and apoptosis was measured via caspase 3/7 activity (C, D). Data points represent the mean +/− SEM from at least three independent experiments. *, p<0.05 (paired Student’s t-test). E) Nuclear morphology of U937_vec and U937_EVI1 cells after treatment with or without 400 nM etoposide for 48 h. Apoptotic nuclei are marked by arrows. Please note difference in scale between etoposide and control treated cells. F) Quantitative assessment of nuclear morphology. Nuclei prepared as in E were counted as ‘intact’ or ‘apoptotic’ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056308#s2" target="_blank">Methods</a>) by an observer blinded to the identity of the samples. Data points represent the mean+SEM from 3 independent experiments. **, p<0.01; n.s., not significant (paired Student’s t-test).</p