http://repub.eur.nl/res/aut/1762/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/33248/ 2011-11-01T00:00:00Z http://repub.eur.nl/res/pub/31277/ 2011-07-28T00:00:00Z MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here, we show that ectopic expression of miR-17, -20,-93 and -106, all AAAGUGC seed-containing miRNAs, increases proliferation, colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1), an ubiquitin-binding protein and regulator of autophagy-mediated protein degradation, as a major target for these miRNAs in myeloid progenitors. In addition, we found increased expression of Sqstm1 transcripts during CSF3-induced neutrophil differentiation of 32D-CSF3R cells and an inverse correlation of SQSTM1 protein levels and miR-106 expression in AML samples. ShRNA-mediated silencing of Sqstm1 phenocopied the effects of ectopic miR-17/20/93/106 expression in hematopoietic progenitors in vitro and in mice. Further, SQSTM1 binds to the ligand-activated colony-stimulating factor 3 receptor (CSF3R) mainly in the late endosomal compartment, but not in LC3 positive autophagosomes. SQSTM1 regulates CSF3R stability and ligand-induced mitogen-activated protein kinase signaling. We demonstrate that AAAGUGC seed-containing miRNAs promote cell expansion, replating capacity and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways. http://repub.eur.nl/res/pub/24462/ 2009-10-01T00:00:00Z Thioredoxin-interacting protein (TXNIP) is involved in reactive oxygen species-induced stress responses. In a screen for novel disease genes in murine leukemia virus (MLV)-induced mouse leukemias, we identified Txnip as a frequent target for proviral integration. Ectopic TXNIP expression inhibited the proliferation of myeloid progenitor cells. TXNIP transcript and protein levels were significantly elevated in human AML blasts of certain patients, particularly those harboring translocation t(8;21). Nucleotide sequencing revealed no abnormalities in the TXNIP coding region in AML. These findings suggest that deregulated TXNIP expression contributes to MLV-induced murine leukemia as well as human AML. http://repub.eur.nl/res/pub/18464/ 2009-02-18T00:00:00Z Raf kinase inhibitory protein (RKIP) negatively regulates the MAP kinase (MAPK), G protein-coupled receptor kinase-2, and NF-κB signalling cascades. RKIP has been implicated as a metastasis suppressor for prostate cancer, but the mechanism is not known. Here, we show that RKIP inhibits invasion by metastatic breast cancer cells and represses breast tumour cell intravasation and bone metastasis in an orthotopic murine model. The mechanism involves inhibition of MAPK, leading to decreased transcription of LIN28 by Myc. Suppression of LIN28 enables enhanced let-7 processing in breast cancer cells. Elevated let-7 expression inhibits HMGA2, a chromatin remodelling protein that activates pro-invasive and pro-metastatic genes, including Snail. LIN28 depletion and let-7 expression suppress bone metastasis, and LIN28 restores bone metastasis in mice bearing RKIP-expressing breast tumour cells. These results indicate that RKIP suppresses invasion and metastasis in part through a signalling cascade involving MAPK, Myc, LIN28, let-7, and downstream let-7 targets. RKIP regulation of two pluripotent stem cell genes, Myc and LIN28, highlights the importance of RKIP as a key metastasis suppressor and potential therapeutic agent. http://repub.eur.nl/res/pub/14725/ 2008-09-16T00:00:00Z We report a system for Cre-regulated expression of RNA interference in vivo. Expression cassettes comprise selectable and FACS-sortable markers in tandem with additional marker genes and shRNAs in the antisense orientation. The cassettes are flanked by tandem LoxP sites arranged so that Cre expression inverts the marker-shRNA construct, allowing its regulated expression (and, at the same time, deletes the original selection/marker genes). The cassettes can be incorporated into retroviral or lentiviral vectors and delivered to cells in culture or used to generate transgenic mice. We describe cassettes incorporating various combinations of reporter genes, miRNA-based RNAi (including two shRNA constructs at once), and oncogenes and demonstrate the delivery of effective RNA interference in cells in culture, efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny, and rapid generation of regulated shRNA knockdown in transgenic mice. These vector systems allow regulated combinatorial manipulation (both overexpression and loss of function) of gene expression in multiple systems in vitro and in vivo. http://repub.eur.nl/res/pub/29190/ 2008-03-25T00:00:00Z Many microRNAs (miRNAs) target mRNAs involved in processes aberrant in tumorigenesis, such as proliferation, survival, and differentiation. In particular, the let-7 miRNA family has been proposed to function in tumor suppression, because reduced expression of let-7 family members is common in non-small cell lung cancer (NSCLC). Here, we show that let-7 functionally inhibits non-small cell tumor development. Ectopic expression of let-7g in K-RasG12D-expressing murine lung cancer cells induced both cell cycle arrest and cell death. In tumor xenografts, we observed significant growth reduction of both murine and human non-small cell lung tumors when overexpression of let-7g was induced from lentiviral vectors. In let-7g expressing tumors, reductions in Ras family and HMGA2 protein levels were detected. Importantly, let-7g-mediated tumor suppression was more potent in lung cancer cell lines harboring oncogenic K-Ras mutations than in lines with other mutations. Ectopic expression of K-RasG12D largely rescued let-7g mediated tumor suppression, whereas ectopic expression of HMGA2 was less effective. Finally, in an autochthonous model of NSCLC in the mouse, let-7g expression substantially reduced lung tumor burden. http://repub.eur.nl/res/pub/29039/ 2008-03-07T00:00:00Z miR-17∼92, miR-106b∼25, and miR-106a∼363 belong to a family of highly conserved miRNA clusters. Amplification and overexpression of miR-17∼92 is observed in human cancers, and its oncogenic properties have been confirmed in a mouse model of B cell lymphoma. Here we show that mice deficient for miR-17∼92 die shortly after birth with lung hypoplasia and a ventricular septal defect. The miR-17∼92 cluster is also essential for B cell development. Absence of miR-17∼92 leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B to pre-B transition. Furthermore, while ablation of miR-106b∼25 or miR-106a∼363 has no obvious phenotypic consequences, compound mutant embryos lacking both miR-106b∼25 and miR-17∼92 die at midgestation. These results provide key insights into the physiologic functions of this family of microRNAs and suggest a link between the oncogenic properties of miR-17∼92 and its functions during B lymphopoiesis and lung development. http://repub.eur.nl/res/pub/31786/ 2007-04-04T00:00:00Z The hematopoietic system provides an attractive model for studying growth factor-controlled expansion and differentiation of cells in relation to receptor routing and its consequences for signal transduction. Suppressor of cytokine signaling (SOCS) proteins regulate receptor signaling partly via their ubiquitin ligase (E3)-recruiting SOCS box domain. Whether SOCS proteins affect signaling through modulating intracellular trafficking of receptors is unknown. Here, we show that a juxtamembrane lysine residue (K632) of the granulocyte colony-stimulating factor receptor (G-CSFR) plays a key role in receptor routing and demonstrate that the effects of SOCS3 on G-CSF signaling to a major extent depend on this lysine. Mutation of K632 causes accumulation of G-CSFR in early endosomes and leads to sustained activation of signal transducer and activator of transcription 5 and ERK, but not protein kinase B. Myeloid progenitors expressing G-CSFR mutants lacking K632 show a perturbed proliferation/ differentiation balance in response to G-CSF. This is the first demonstration of SOCS-mediated ubiquitination and routing of a cytokine receptor and its impact on maintaining an appropriate signaling output. http://repub.eur.nl/res/pub/36493/ 2007-03-29T00:00:00Z Signals induced by granulocyte colony-stimulating factor (G-CSF), the major cytokine involved in neutrophil development, are tightly controlled by ligand-induced receptor internalization. Truncated G-CSF receptors (G-CSF-Rs) that fail to internalize show sustained proliferation and defective differentiation signaling. Steady-state forward routing also determines cell surface levels of cytokine receptors, but mechanisms controlling this are poorly understood. Here, we show that WD40 and suppressor of cytokine signaling (SOCS) box protein-2 (Wsb-2), an SOCS box-containing WD40 protein with currently unknown function, binds to the COOH-terminal region of G-CSF-R. Removal of this region did not affect internalization, yet resulted in increased membrane expression of G-CSF-R and enhanced proliferation signaling at the expense of differentiation induction. Conversely, Wsb-2 binding to the G-CSF-R reduced its cell surface expression and inhibited proliferation signaling. These effects depended on the SOCS box involved in ubiquitylation and on cytosolic lysines of G-CSF-R and imply a major role for ubiquitylation through the G-CSF-R C-terminus in forward routing of the receptor. Importantly, the Wsb-2 gene is commonly disrupted by virus integrations in mouse leukemia. We conclude that control of forward routing of G-CSF-R is essential for a balanced response of myeloid progenitors to G-CSF and suggest that disturbance of this balance may contribute to myeloid leukemia. http://repub.eur.nl/res/pub/37064/ 2007-01-01T00:00:00Z Retroviral insertion mutagenesis has recently received much attention because of its adverse effects in the application of retroviral vector-based gene therapy, resulting in leukemia in certain patients. At the same time, retroviral mutagenesis in mice is being considered a powerful forward genetic strategy to identify disease genes involved in cancer. The publication of the mouse genome sequence and the development of high-throughput genomic approaches have given a further boost to this rapidly evolving field. The increasing numbers of new potential oncogenes identified in retroviral screens have given a valuable basis for a better understanding of cancer related pathways in mice. Important challenges that now lie ahead of us are (i) to determine the relevance and causal relationship of these genes with various types of human cancer (ii) to develop strategies to identify tumor suppressor genes on a large scale, (iii) to place the disease genes into regulatory networks to better understand their role in the complex pathogenesis of cancer, and (iv) to determine their value for diagnosis refinement and therapeutic target intervention in human disease. In this review, we will give a brief update of the current state-of-the-art and thoughts concerning these issues. We will specifically focus on the value of employing retroviral insertion mutagenesis in mice and gene expression profiling in man in the context of acute myeloid leukemia. http://repub.eur.nl/res/pub/6751/ 2005-04-27T00:00:00Z Acute myeloïde leukemie (AML) wordt geclassificeerd in verschillende risico categorieën op basis van cytogenetische en moleculaire afwijkingen. De chromosomale translocaties t(8;21), t(15;17) en inv(16) bijvoorbeeld, resulteren in fusie-eiwitten met afwijkende transcriptioneel regulerende activiteiten en worden geassocieerd met een relatief gunstige prognose, terwijl 3q26, 5q, 7q en 11q23 afwijkingen correleren met een verminderde overlevingskans na behandeling. Mutaties in het gen dat codeert voor de transcriptiefactor CCAAT bindend eiwit alpha (CEBPA) en de goeifactor receptor FMS-like tyrosine kinase3 (FLT3) zijn tevens geassocieerd met respectievelijk een goede en slechte prognose. Ongeveer de helft van de AML patiënten heeft bekende (cyto-) genetische afwijkingen. De ontwikkeling van AML is een meerstapsproces waarbij defecten in meerdere genen betrokken zijn. Dit is vooral duidelijk geworden in studies in muizenmodellen. Zo ontwikkelen muizen die fusiegenen herbergen afkomstig van t(15;17), t(8;21) en inv(16) pas na langere tijd en met een lage frequentie leukemie, hetgeen aangeeft dat additionele afwijkingen nodig zijn voor volledige transformatie van hematopoietische voorlopercellen in AML cellen. Algemeen wordt aangenomen dat AML ontstaat door afwijkingen van combinaties van genen die hematopoietische stamcel proliferatie, vernieuwing en myeloïde differentiatie controleren. De beschikbaarheid van de sequentie van het muizengenoom heeft van retrovirale mutagenese een krachtige strategie gemaakt voor de identificatie van nieuwe ziektegenen die betrokken zijn bij diverse typen kanker, waaronder met name leukemie, lymfoom en borstkanker. Voor de identificatie van genen die voornamelijk een rol spelen in acute myeloïde leukemie (AML) hebben we Graffi-1.4 (Gr-1.4) muizen leukemievirus (MLV) gebruikt. Hiervoor zijn pasgeboren muizen subcutaan geïnjecteerd met Gr-1.4 virus. Al deze muizen ontwikkelden na 4-6 maanden een leukemie, voornamelijk van myeloïde origine (Hoofdstuk 2 en 4). Voor de identificatie van de virus-flankerende genen hebben we inverse PCR technologie gebruikt en gecombineerd met geautomatiseerde sequentie analyse. Met deze methode zijn meer dan 100 kandidaat ziektegenen geïdentificeerd die betrokken zijn in meerdere mechanismen, zoals bijvoorbeeld DNA herstel (Rad54/SNF2, Rad3­related), regulering van het niveau van reactieve zuurstof deeltjes (ROS)(Txnip, PrdxII) en geprogrammeerde celdood (Api-5) (Hoofdstuk 2). De meeste virus­flankerende genen maken deel uit van signaal transductie en transcriptionele netwerken (Hoofdstuk 2). Deze resultaten overlappen deels met die van andere MLV mutagenese studies. Zo werden bijvoorbeeld p53, Notch-1, Fli-1 en Evi-1 ook in eerdere screens gevonden. Deze genen zijn dus waarschijnlijk betrokken bij meerdere typen muizen leukemie. Daarnaast hebben we genen ontdekt die waarschijnlijk meer specifiek betrokken zijn bij myeloïde leukemie (Hoofdstuk 2). Genen die in meerdere onafhankelijke mutagenese screens zijn gevonden zijn zeer waarschijnlijk belangrijk voor het ontstaan van leukemie in de muis. Het belang van deze genen voor humane leukemie staat daarmee echter niet vast. Bovendien kan een retrovirus de expressie van een targetgen over een afstand van enkele honderden kilobasen beïnvloeden. Dit bemoeilijkt een strikte definitie van een virus targetgen op basis van de plek van integratie. In hoofdstuk 3 hebben we geprobeerd dit probleem te benaderen door verschillende groepen potentiële targetgenen, dichtbij of verder weg gelokaliseerd van de virusintegratieplek, te vergelijken met genen die differentieel tot expressie komen in subgroepen van AML. We vonden dat muizen leukemiegenen geïdentificeerd met retrovirale insertionele mutagenese differentieel tot expressie komen in subgroepen van AML bij zowel volwassenen als kinderen. Genen die direct grenzen aan de plek van virusintegratie correleerden significant met groepen van genen die verantwoordelijk waren voor classificatie van humane leukemie. Dit in tegenstelling tot genen die verderop liggen. Dit resultaat laat zien dat genen die het dichtst in de buurt liggen van virus integraties hoogst waarschijnlijk ook ziektegenen zijn met een rol in AML bij de mens. Deze genen konden geplaatst worden in vijf regulatoire netwerken. In dit proefschrift is de functionele rol van drie van dergelijke genen nader onderzocht; te weten de genen die coderen voor de transcriptiefactor Yin Yang 1 (Yy1), WD40-repeat domein en een SOCS box-2 (Wsb-2) en Thioredoxin bindend eiwit (Txnip). In hoofdstuk 4 wordt beschreven dat Gr-1.4 integraties in de Yin Yang 1 (YY1) promoter regio YY1 expressie dereguleren en het onafhankelijk maken van regulatie door Sp1. Bovendien had kunstmatig verhoogde YY1 expressie een negatief effect op de differentiatie van myeloïde 32D cellen en voorkwam de uitgroei van myeloïde voorlopercellen vanuit primaire beenmerg cellen. Verder hebben we gevonden dat in een bepaalde groep van AML patiënten de YY1 expressie significant verhoogd is ten opzichte van normale beenmergcellen. Deze resultaten maken aannemelijk dat verstoorde regulatie van YY1 expressie interfereren met normale differentiatie programma van myeloïde voorlopercellen en daardoor kan bijdragen aan de ontwikkeling van leukemie. Hoofdstuk 5 handelt over het gen dat voor een WD40-repeat domein en een SOCS box-2 (Wsb-2) codeert. Virusintegraties in Wsb-2 werden uitsluitend gevonden tussen exon 4 en exon 7, wat aangeeft dat het Wsb-2 gen waarschijnlijk geïnactiveerd wordt door de integraties. Ook bij een subgroep in humane AML werd gevonden dat het Wsb-2 mRNA niveau sterk verlaagd is. Wsb-2 blijkt te binden aan het distale domein van de G-CSF-R, een domein betrokken bij ernstige aangeboren neutropenie, myelodysplastisch syndroom (MDS) en AML. Verder vermindert Wsb­2 G-CSF-R membraan expressie en G-CSF-geïnduceerde STAT activatie en proliferatie signalen. Dit duidt erop dat Wsb-2 een tumoronderdrukkende werking uitoefent door remming van G-CSF geïnduceerde proliferatie van myeloïde voorlopercellen. In hoofdstuk 6 worden de gegevens van integraties in het Txnip gen beschreven. Integraties in Txnip vonden plaats in de 5' en 3' regio in 100% van de Gr-1.4 en 58% van de CasBrM geïnduceerde leukemie gevallen. Integraties in deze regio's resulteren in een verhoogd signaal in luciferase assays, vergeleken met een normale Txnip promoter. TXNIP mRNA niveaus blijken te zijn verhoogd in een groep van AML patiënten ten opzichte van CD34+ normale beenmergcellen. Kunstmatige verhoogde expressie van TXNIP resulteerde in een versnelde geprogrammeerde celdood en vertraagde celdeling. http://repub.eur.nl/res/pub/10305/ 2004-01-01T00:00:00Z Acute myeloid leukemia (AML) is a heterogeneous group of diseases in which chromosomal aberrations, small insertions or deletions, or point mutations in certain genes have profound consequences for prognosis. However, the majority of AML patients present without currently known genetic defects. Retroviral insertion mutagenesis in mice has become a powerful tool for identifying new disease genes involved in the pathogenesis of leukemia and lymphoma. Here we have used the Graffi-1.4 strain of murine leukemia virus, which causes predominantly AML, in a screen to identify novel genes involved in the pathogenesis of this disease. We report 79 candidate disease genes in common integration sites (CISs) and 15 genes whose family members previously were found to be affected in other studies. The majority of the identified sequences (60%) were not found in lymphomas and monocytic leukemias in previous screens, suggesting a specific involvement in AML. Although most of the virus integrations occurred in or near the 5' or 3' ends of the genes, suggesting deregulation of gene expression as a consequence of virus integration, 18 CISs were located exclusively within the genes, conceivably causing gene disruption. http://repub.eur.nl/res/pub/8224/ 2003-01-01T00:00:00Z The genetic defects underlying the pathogenesis of acute myeloid leukemia (AML) are still largely unknown. Retroviral insertion mutagenesis in mice has become a powerful tool to identify candidate genes involved in the development of leukemia and lymphoma. We have used this strategy with the 1.4 strain of Graffi murine leukemia virus (MuLV), which predominantly causes myeloid leukemias. Here, we report that Graffi-1.4-induced AML frequently harbors virus integrations in the gene encoding the transcription factor Yin Yang 1 (YY1). These integrations occurred in both orientations, and all were located in the 5' promoter region of the gene, 0.5 to 1.5 kb upstream of the major transcriptional start site. Luciferase reporter assays showed that virus integration in this region increases promoter activity and renders it independent of a functional binding site for Sp1, a major transcriptional regulator of YY1. We used the murine 32D model to study the consequence of perturbed YY1 expression for myelopoiesis. YY1 protein levels were high in 32D parental cells maintained in interleukin-3-containing medium, but they dropped when the cells were induced to differentiate by granulocyte-colony-stimulating factor (G-CSF). Strikingly, G-CSF-induced neutrophilic differentiation was reduced in 32D cell transfectants ectopically expressing YY1. In similar experiments on primary bone marrow cells, enforced YY1 expression blocked the outgrowth of CFU-GM colonies. Increased YY1 expression was seen in some cases of human AML. Collectively, these data imply a possible role of perturbed expression of YY1 in the development of AML through interference with the myeloid differentiation program in the leukemic progenitor cells.