Touw, I.P.

Prevalence of a new auto-activating colony stimulating factor 3 receptor mutation (CSF3R-T595I) in acute myeloid leukemia and severe congenital neutropenia (Article)

2013-05-01T00:00:00Z

Granulocyte Colony-Stimulating Factor Receptor Signaling. Implications for G-CSF Responses and Leukemic Progression in Severe Congenital Neutropenia. (Article)

2013-02-01T00:00:00Z

Following activation by their cognate ligands, cytokine receptors undergo intracellular routing toward lysosomes, where they are degraded. This review focuses on the signaling function of the G-CSFR in relation to the dynamics of endosomal routing of the G-CSFR. Mechanisms involving receptor lysine ubiquitination and redox-controlled phosphatase activities are discussed. Specific attention is paid to the consequences of G-CSFR mutations, acquired in patients with severe congenital neutropenias who receive G-CSF therapy, particularly in the context of leukemic transformation, a major clinical complication of the disease.

Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts (Article)

2012-01-05T00:00:00Z

Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (eg, RPS19) or large (eg, RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site-mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.

Editorial: Scrambling for a response to G-CSF (Article)

2011-08-01T00:00:00Z

MiR-17/20/93/106 promote hematopoietic cell expansion by targeting sequestosome 1-regulated pathways in mice (Article)

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.

Analysis of jak2 catalytic function by peptide microarrays: The role of the JH2 domain and V617F mutation (Article)

2011-04-29T00:00:00Z

Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the Kmfor ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or Kmfor ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches.

The deubiquitinating enzyme DUB2A enhances CSF3 signalling by attenuating lysosomal routing of the CSF3 receptor (Article)

2011-03-01T00:00:00Z

Ubiquitination of the CSF3R [CSF3 (colony-stimulating factor 3) receptor] occurs after activated CSF3Rs are internalized and reside in early endosomes. CSF3R ubiquitination is crucial for lysosomal routing and degradation. The E3 ligase SOCS3 (suppressor of cytokine signalling 3) has been shown to play a major role in this process. Deubiquitinating enzymes remove ubiquitin moieties from target proteins by proteolytic cleavage. Two of these enzymes, AMSH [associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)] and UBPY (ubiquitin isopeptidase Y), interact with the general endosomal sortingmachinery. Whether deubiquitinating enzymes control CSF3R trafficking from early towards late endosomes is unknown. In the present study, we asked whether AMSH, UBPY or a murine family of deubiquitinating enzymes could fulfil such a role. This DUB family (deubiquitin enzyme family) comprises four members (DUB1, DUB1A, DUB2 and DUB2A), which were originally described as being haematopoietic-specific and cytokine-inducible, but their function in cytokine receptor routing and signalling has remained largely unknown. We show that DUB2A expression is induced by CSF3 in myeloid 32D cells and that DUB2 decreases ubiquitination and lysosomal degradation of the CSF3R, leading to prolonged signalling. These results support a model in which CSF3R ubiquitination is dynamically controlled at the early endosome by feedback mechanisms involving CSF3-induced E3 ligase (SOCS3) and deubiquitinase (DUB2A) activities.

Hematopoietic stem cell transplantation in severe congenital neutropenia (Article)

2011-03-01T00:00:00Z

Background: Severe congenital neutropenia (SCN) is an immunodeficiency characterized by disturbed myelopoiesis and an absolute neutrophil count (ANC) <0.5 × 109/L. SCN is also a premalignant condition; a significant proportion of patients develop myelodysplastic syndrome or leukemia (MDS/L). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for SCN. Procedure: Since 2004, eight HSCT have been performed in seven patients at our center. The indications were transformation to MDS/L (n = 2), granulocyte colony-stimulating factor receptor (CSF3R) mutation(s) (n = 2), granulocyte colony-stimulating factor (G-CSF) resistance (n = 2), and at the patient's own request (n = 1). Results: The mean age at transplantation was 13 years (2.8-28 years) (mean follow-up 32 months, range 21-60). Three patients harbored ELANE mutations, three HAX1 mutations, and in one patient no causative mutation was identified. Two of the ELANE mutations were novel mutations. Three patients initially received myeloablative conditioning and four had reduced intensity conditioning (RIC). Three grafts were from HLA-identical siblings, three from matched unrelated donors and two were cord blood units. Engraftment occurred in all patients. Two of seven (29%) patients died; both had MDS/L and both were among the three that underwent myeloablative conditioning. One patient has chronic GVHD 2 years post-transplant. Conclusions: The role of HSCT should be explored further in patients with SCN. In particular, the influence of the conditioning regime needs to be evaluated in a larger cohort of patients.

The Antioxidant Protein Peroxiredoxin 4 Is Epigenetically Down Regulated in Acute Promyelocytic Leukemia (Article)

2011-02-03T00:00:00Z

The antioxidant peroxiredoxin (PRDX) protein family comprises 6 members, which are implicated in a variety of cellular responses, including growth factor signal transduction. PRDX4 resides in the endoplasmic reticulum (ER), where it locally controls oxidative stress by reducing H2O2levels. We recently provided evidence for a regulatory function of PRDX4 in signal transduction from a myeloid growth factor receptor, the granulocyte colony-stimulating factor receptor (G-CSFR). Upon activation, the ligand-induced G-CSFR undergoes endocytosis and routes via the early endosomes where it physically interacts with ER-resident PRDX4. PRDX4 negatively regulates G-CSFR mediated signaling. Here, we investigated whether PRDX4 is affected in acute myeloid leukemia (AML); genomic alterations and expression levels of PRDX4 were investigated. We show that genomic abnormalities involving PRDX4 are rare in AML. However, we find a strong reduction in PRDX4 expression levels in acute promyelocytic leukemia (APL) compared to normal promyelocytes and different molecular subtypes of AML. Subsequently, the possible role of DNA methylation and histone modifications in silencing of PRDX4 in APLs was investigated. We show that the reduced expression is not due to methylation of the CpG island in the promoter region of PRDX4 but correlates with increased trimethylation of histone 3 lysine residue 27 (H3K27me3) and lysine residue 4 (H3K4me3) at the transcriptional start site (TSS) of PRDX4, indicative of a bivalent histone code involved in transcriptional silencing. These findings suggest that the control of G-CSF responses by the antioxidant protein PRDX4 may be perturbed in APL.

The deubiquitinating enzyme DUB2A enhances CSF3 signalling by attenuating lysosomal routing of the CSF3 receptor (Article)

2011-01-01T00:00:00Z

Lineage-instructive function of C/EBPα in multipotent hematopoietic cells and early thymic progenitors (Article)

2010-11-18T00:00:00Z

Hematopoiesis is tightly controlled by transcription regulatory networks, but how and when specific transcription factors control lineage commitment are still largely unknown. Within the hematopoietic stem cell (Lin -Sca-1+c-Kit+) compartment these lineage-specific transcription factors are expressed at low levels but are up-regulated with the process of lineage specification. CCAAT/enhancer binding protein α (C/EBPα) represents one of these factors and is involved in myeloid development and indispensable for formation of granulocytes. To track the cellular fate of stem and progenitor cells, which express C/EBPα, we developed a mouse model expressing Cre recombinase from the Cebpa promoter and a conditional EYFP allele. We show that Cebpa/EYFP+ cells represent a significant subset of multipotent hematopoietic progenitors, which predominantly give rise to myeloid cells in steady-state hematopoiesis. C/EBPα induced a strong myeloid gene expression signature and down-regulated E2A-induced regulators of early lymphoid development. In addition, Cebpa/EYFP+ cells compose a fraction of early thymic progenitors with robust myeloid potential. However, Cebpa/EYFP+ multipotent hematopoietic progenitors and early thymic progenitors retained the ability to develop into erythroid and T-lymphoid lineages, respectively. These findings support an instructive but argue against a lineage-restrictive role of C/EBPα in multipotent hematopoietic and thymic progenitors.

G-CSF and its receptor in myeloid malignancy. (Article)

2010-06-01T00:00:00Z

Granulocyte colony-stimulating factor (G-CSF) has been used in the clinic for more than 2 decades to treat congenital and acquired neutropenias and to reduce febrile neutropenia before or during courses of intensive cytoreductive therapy. In addition, healthy stem cell donors receive short-term treatment with G-CSF for mobilization of hematopoietic stem cells. G-CSF has also been applied in priming strategies designed to enhance the sensitivity of leukemia stem cells to cytotoxic agents, in protocols aimed to induce their differentiation and accompanying growth arrest and cell death, and in severe aplastic anemia and myelodysplastic syndrome (MDS) to alleviate anemia. The potential adverse effects of G-CSF administration, particularly the risk of malignant transformation, have fueled ongoing debates, some of which can only be settled in follow-up studies extending over several decades. This specifically applies to children with severe congenital neutropenia who receive lifelong treatment with G-CSF and in which the high susceptibility to develop MDS and acute myeloid leukemia (AML) has now become a major clinical concern. Here, we will highlight some of the controversies and challenges regarding the clinical application of G-CSF and discuss a possible role of G-CSF in malignant transformation, particularly in patients with neutropenia harboring mutations in the gene encoding the G-CSF receptor.

G-CSF and its receptor in myeloid malignancy. (Article)

2010-06-01T00:00:00Z

HAT: Hypergeometric Analysis of Tiling-arrays with application to promoter-GeneChip data (Article)

2010-05-21T00:00:00Z

Background: Tiling-arrays are applicable to multiple types of biological research questions. Due to its advantages (high sensitivity, resolution, unbiased), the technology is often employed in genome-wide investigations. A major challenge in the analysis of tiling-array data is to define regions-of-interest, i.e., contiguous probes with increased signal intensity (as a result of hybridization of labeled DNA) in a region. Currently, no standard criteria are available to define these regions-of-interest as there is no single probe intensity cut-off level, different regions-of-interest can contain various numbers of probes, and can vary in genomic width. Furthermore, the chromosomal distance between neighboring probes can vary across the genome among different arrays.Results: We have developed Hypergeometric Analysis of Tiling-arrays (HAT), and first evaluated its performance for tiling-array datasets from a Chromatin Immunoprecipitation study on chip (ChIP-on-chip) for the identification of genome-wide DNA binding profiles of transcription factor Cebpa (used for method comparison). Using this assay, we can refine the detection of regions-of-interest by illustrating that regions detected by HAT are more highly enriched for expected motifs in comparison with an alternative detection method (MAT). Subsequently, data from a retroviral insertional mutagenesis screen were used to examine the performance of HAT among different applications of tiling-array datasets. In both studies, detected regions-of-interest have been validated with (q)PCR.Conclusions: We demonstrate that HAT has increased specificity for analysis of tiling-array data in comparison with the alternative method, and that it accurately detects regions-of-interest in two different applications of tiling-arrays. HAT has several advantages over previous methods: i) as there is no single cut-off level for probe-intensity, HAT can detect regions-of-interest at various thresholds, ii) it can detect regions-of-interest of any size, iii) it is independent of probe-resolution across the genome, and across tiling-array platforms and iv) it employs a single user defined parameter: the significance level. Regions-of-interest are detected by computing the hypergeometric-probability, while controlling the Family Wise Error. Furthermore, the method does not require experimental replicates, common regions-of-interest are indicated, a sequence-of-interest can be examined for every detected region-of-interest, and flanking genes can be reported.

The gene encoding thioredoxin-interacting protein (TXNIP) is a frequent virus integration site in virus-induced mouse leukemia and is overexpressed in a subset of AML patients (Article)

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.

Site-specific ubiquitination determines lysosomal sorting and signal attenuation of the granulocyte colony-stimulating factor receptor (Article)

2009-07-10T00:00:00Z

Ubiquitination of cytokine receptors controls intracellular receptor routing and signal duration, but the underlying molecular determinants are unclear. The suppressor of cytokine signaling protein SOCS3 drives lysosomal degradation of the granulocyte colony-stimulating factor receptor (G-CSFR), depending on SOCS3-mediated ubiquitination of a specific lysine located in a conserved juxtamembrane motif. Here, we show that, despite ubiquitination of other lysines, positioning of a lysine within the membrane-proximal region is indispensable for this process. Neither reallocation of the motif nor fusion of ubiquitin to the C-terminus of the G-CSFR could drive lysosomal routing. However, within this region, the lysine could be shifted 12 amino acids toward the C-terminus without losing its function, arguing against the existence of a linear sorting motif and demonstrating that positioning of the lysine relative to the SOCS3 docking site is flexible. G-CSFR ubiquitination peaked after endocytosis, was inhibited by methyl-β-cyclodextrin as well as hyperosmotic sucrose and severely reduced in internalization-defective G-CSFR mutants, indicating that ubiquitination mainly occurs at endosomes. Apart from elucidating structural and spatio-temporal aspects of SOCS3-mediated ubiquitination, these findings have implications for the abnormal signaling function of G-CSFR mutants found in severe congenital neutropenia, a hematopoietic disorder with a high leukemia risk.

Janus kinases promote cell-surface expression and provoke autonomous signalling from routing-defective G-CSF receptors. (Article)

2009-02-01T00:00:00Z

CSF3R [G-CSF (granulocyte colony-stimulating factor) receptor] controls survival, proliferation and differentiation of myeloid progenitor cells via activation of multiple JAKs (Janus kinases). In addition to their role in phosphorylation of receptor tyrosine residues and downstream signalling substrates, JAKs have recently been implicated in controlling expression of cytokine receptors, predominantly by masking critical motifs involved in endocytosis and lysosomal targeting. In the present study, we show that increasing the levels of JAK1, JAK2 and TYK2 (tyrosine kinase 2) elevated steady-state CSF3R cell-surface expression and enhanced CSF3R protein stability in haematopoietic cells. This effect was not due to inhibition of endocytotic routing, since JAKs did not functionally interfere with the dileucine-based internalization motif or lysine-mediated lysosomal degradation of CSF3R. Rather, JAKs appeared to act on CSF3R in the biosynthetic pathway at the level of the ER (endoplasmic reticulum). Strikingly, increased JAK levels synergized with internalization- or lysosomal-routing-defective CSF3R mutants to confer growth-factor independent STAT3 (signal transducer and activator of transcription 3) activation and cell survival, providing a model for how increased JAK expression and disturbed intracellular routing of CSF3R synergize in the transformation of haematopoietic cells.

Suppressor of cytokine signaling 3 controls lysosomal routing of G-CSF receptor (Article)

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.

Novel role of WD40 and SOCS box protein-2 in steady-state distribution of granulocyte colony-stimulating factor receptor and G-CSF-controlled proliferation and differentiation signaling (Article)

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.

Granulocyte colony-stimulating factor: Key (F)actor or innocent bystander in the development of secondary myeloid malignancy? (Article)

2007-02-01T00:00:00Z

Retroviral insertion mutagenesis in mice as a comparative oncogenomics tool to identify disease genes in human leukemia (Article)

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.

Reduced hematopoietic reserves in DNA interstrand crosslink repair- deficient Ercc1-/- mice. (Article)

2005-02-23T00:00:00Z

The ERCC1-XPF heterodimer is a structure-specific endonuclease involved in both nucleotide excision repair and interstrand crosslink repair. Mice carrying a genetic defect in Ercc1 display symptoms suggestive of a progressive, segmental progeria, indicating that disruption of one or both of these DNA damage repair pathways accelerates aging. In the hematopoietic system, there are defined age-associated changes for which the cause is unknown. To determine if DNA repair is critical to prolonged hematopoietic function, hematopoiesis in Ercc1-/- mice was compared to that in young and old wild-type mice. Ercc1-/- mice (3-week-old) exhibited multilineage cytopenia and fatty replacement of bone marrow, similar to old wild-type mice. In addition, the proliferative reserves of hematopoietic progenitors and stress erythropoiesis were significantly reduced in Ercc1-/- mice compared to age-matched controls. These features were not seen in nucleotide excision repair-deficient Xpa-/- mice, but are characteristic of Fanconi anemia, a human cancer syndrome caused by defects in interstrand crosslink repair. These data support the hypothesis that spontaneous interstrand crosslink damage contributes to the functional decline of the hematopoietic system associated with aging.

Large-scale identification of disease genes involved in acute myeloid leukemia (Article)

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.

G-CSF receptor truncations found in SCN/AML relieve SOCS3-controlled inhibition of STAT5 but leave suppression of STAT3 intact (Article)

2004-01-01T00:00:00Z

Truncated granulocyte colony-stimulating factor receptors (G-CSF-Rs) are implicated in severe congenital neutropenia (SCN) and the consecutive development of acute myeloid leukemia (AML). Mice expressing G-CSF-R truncation mutants (gcsfr-d715) show defective receptor internalization, an increased signal transducer and activator of transcription 5 (STAT5)/STAT3 activation ratio, and hyperproliferative responses to G-CSF treatment. We determined whether a lack of negative feedback by suppressor of cytokine signaling (SOCS) proteins contributes to the signaling abnormalities of G-CSF-R-d715. Expression of SOCS3 transcripts in bone marrow cells from G-CSF-treated gcsfr-d715 mice was approximately 60% lower than in wild-type (WT) littermates. SOCS3 efficiently suppressed STAT3 and STAT5 activation by WT G-CSF-R in luciferase reporter assays. In contrast, while SOCS3 still inhibited STAT3 activation by G-CSF-R-d715, STAT5 activation was no longer affected. This was due mainly to loss of the SOCS3 recruitment site Tyr729, with an additional contribution of the internalization defects of G-CSF-R-d715. Because Tyr729 is also a docking site for the Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2), which binds to and inactivates STAT5, we suggest a model in which reduced SOCS3 expression, combined with the loss of recruitment of both SOCS3 and SHP-2 to the activated receptor complex, determine the increased STAT5/STAT3 activation ratio and the resulting signaling abnormalities projected by truncated G-CSF-R mutants.

Receptor activation and 2 distinct COOH-terminal motifs control G-CSF receptor distribution and internalization kinetics (Article)

2004-01-01T00:00:00Z

We have studied the intracellular distribution and internalization kinetics of the granulocyte colony-stimulating factor receptor (G-CSF-R) in living cells using fusion constructs of wild-type or mutant G-CSF-R and enhanced green fluorescent protein (EGFP). Under steady-state conditions the G-CSF-R localized predominantly to the Golgi apparatus, late endosomes, and lysosomes, with only low expression on the plasma membrane, resulting from spontaneous internalization. Internalization of the G-CSF-R was significantly accelerated by addition of G-CSF. This ligand-induced switch from slow to rapid internalization required the presence of G-CSF-R residue Trp650, previously shown to be essential for its signaling ability. Both spontaneous and ligand-induced internalization depended on 2 distinct amino acid stretches in the G-CSF-R COOH-terminus: 749-755, containing a dileucine internalization motif, and 756-769. Mutation of Ser749 at position -4 of the dileucine motif to Ala significantly reduced the rate of ligand-induced internalization. In contrast, mutation of Ser749 did not affect spontaneous G-CSF-R internalization, suggesting the involvement of a serine-threonine kinase specifically in ligand-accelerated internalization of the G-CSF-R. COOH-terminal truncation mutants of G-CSF-R, found in severe congenital neutropenia, lack the internalization motifs and were completely defective in both spontaneous and ligand-induced internalization. As a result, these mutants showed constitutively high cell-surface expression.

Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells (Article)

2003-01-01T00:00:00Z

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Tyr704, Tyr729, Tyr744, Tyr764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation, and cell survival. However, it is unclear whether these tyrosines are equally important under more physiologic conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated G-CSF-R-deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (m0), single tyrosine "add-back" mutants, or wild-type (WT) receptors. G-CSF-induced responses were determined in primary colony assays, serial replatings, and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Tyr764 strongly enhanced proliferative responses, which was reverted by inhibition of ERK activity. Tyr729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing m0 gradually dropped compared with WT. The presence of Tyr729, but also Tyr704 and Tyr744, both involved in activation of signal transducer and activator of transcription 3 (STAT3), further reduced replating efficiencies. Conversely, Tyr764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a more than 10(4)-fold increase of colony-forming cells over m0 after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells

The gene encoding the transcriptional regulator Yin Yang 1 (YY1) is a myeloid transforming gene interfering with neutrophilic differentiation (Article)

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.

The Jak-Stat pathway in normal and perturbed hematopoiesis (Article)

2000-01-01T00:00:00Z

Somatostatin receptors in the haematopoietic system (Article)

2000-01-01T00:00:00Z

Van het rechte pad (Inaugural Lecture)

1999-11-05T00:00:00Z

Sustained receptor activation and hyperproliferation in response to granulocyte colony-stimulating factor (G-CSF) in mice with a severe congenital neutropenia/acute myeloid leukemia-derived mutation in the G-CSF receptor gene (Article)

1999-01-01T00:00:00Z

In approximately 20% of cases of severe congenital neutropenia (SCN), mutations are found in the gene encoding the granulocyte colony-stimulating factor receptor (G-CSF-R). These mutations introduce premature stop codons, which result in truncation of 82-98 COOH-terminal amino acids of the receptor. SCN patients who develop secondary myelodysplastic syndrome and acute myeloid leukemia almost invariably acquired a GCSFR mutation, suggesting that this genetic alteration represents a key step in leukemogenesis. Here we show that an equivalent mutation targeted in mice (gcsfr-Delta715) results in the selective expansion of the G-CSF- responsive progenitor (G-CFC) compartment in the bone marrow. In addition, in vivo treatment of gcsfr-Delta715 mice with G-CSF results in increased production of neutrophils leading to a sustained neutrophilia. This hyperproliferative response to G-CSF is accompanied by prolonged activation of signal transducer and activator of transcription (STAT) complexes and extended cell surface expression of mutant receptors due to defective internalization. In view of the continuous G-CSF treatment of SCN patients, these data provide insight into why progenitor cells expressing truncated receptors clonally expand in vivo, and why these cells may be targets for additional genetic events leading to leukemia.

Multiple signals mediate proliferation, differentiation, and survival from the granulocyte colony-stimulating factor receptor in myeloid 32D cells (Article)

1999-01-01T00:00:00Z

Granulocyte colony-stimulating factor (G-CSF) regulates neutrophil production through activation of its cognate receptor, the G-CSF-R. Previous studies with deletion mutants have shown that the membrane-proximal cytoplasmic domain of the receptor is sufficient for mitogenic signaling, whereas the membrane-distal domain is required for differentiation signaling. However, the function of the four cytoplasmic tyrosines of the G-CSF-R in the control of proliferation, differentiation, and survival has remained unclear. Here we investigated the role of these tyrosines by expressing a tyrosine "null" mutant and single tyrosine "add back" mutants in maturation-competent myeloid 32D cells. Clones expressing the null mutant showed only minimal proliferation and differentiation, with survival also reduced at low G-CSF concentrations. Analysis of clones expressing the add-back mutants revealed that multiple tyrosines contribute to proliferation, differentiation, and survival signals from the G-CSF-R. Analysis of signaling pathways downstream of these tyrosines suggested a positive role for STAT3 activation in both differentiation and survival signaling, whereas SHP-2, Grb2 and Shc appear important for proliferation signaling. In addition, we show that a tyrosine-independent "differentiation domain" in the membrane-distal region of the G-CSF-R appears necessary but not sufficient for mediating neutrophilic differentiation in these cells.

Novel point mutation in the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor in a case of severe congenital neutropenia hyporesponsive to G-CSF treatment (Article)

1999-01-01T00:00:00Z

Severe congenital neutropenia (SCN) is a heterogeneous condition characterized by a drastic reduction in circulating neutrophils and a maturation arrest of myeloid progenitor cells in the bone marrow. Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF). Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment. When this mutant G-CSF-R was expressed in myeloid cells, it was defective in both proliferation and survival signaling. This correlated with diminished activation of the receptor complex as determined by signal transducer and activator of transcription (STAT) activation, although activation of STAT5 was more affected than STAT3. Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor. This suggests that the mutation in the extracellular domain affects ligand-receptor complex formation with severe consequences for intracellular signal transduction. Together these data add to our understanding of the mechanisms of cytokine receptor signaling, emphasize the role of GCSFR mutations in the etiology of SCN, and implicate such mutations in G-CSF hyporesponsiveness.

Mutations in the gene for the granulocyte colony-stimulating-factor receptor in patients with acute myeloid leukemia preceded by severe congenital neutropenia (Article)

1995-01-01T00:00:00Z

BACKGROUND. In severe congenital neutropenia the maturation of myeloid progenitor cells is arrested. The myelodysplastic syndrome and acute myeloid leukemia develop in some patients with severe congenital neutropenia. Abnormalities in the signal-transduction pathways for granulocyte colony-stimulating factor (G-CSF) may play a part in the progression to acute myeloid leukemia. METHODS. We isolated genomic DNA and RNA from hematopoietic cells obtained from two patients with acute myeloid leukemia and histories of severe congenital neutropenia. The nucleotide sequences encoding the cytoplasmic domain of the G-CSF receptor were amplified by means of the polymerase chain reaction and sequenced. Murine myeloid 32D.C10 cells were transfected with complementary DNA encoding the wild-type or mutant G-CSF receptors and tested for their responses to G-CSF. RESULTS. Point mutations in the gene for the G-CSF receptor were identified in both patients. The mutations, a substitution of thymine for cytosine at the codon for glutamine at position 718 (Gln718) in one patient and at the codon for glutamine at position 731(Gln731) in the other, caused a truncation of the C-terminal cytoplasmic region of the receptor. Both mutant and wild-type genes for the G-CSF receptor were present in leukemic cells from the two patients. In one patient, the mutation was also found in the neutropenic stage, before the progression to acute myeloid leukemia. The 32D.C10 cells expressing mutant receptors had abnormally high proliferative responses but failed to mature when cultured in G-CSF. The mutant G-CSF receptors also interfered with terminal maturation mediated by the wild-type G-CSF receptor in the 32D.C10 cells that coexpressed the wild-type and mutant receptors. CONCLUSIONS. Mutations in the gene for the G-CSF receptor that interrupt signals required for the maturation of myeloid cells are involved in the pathogenesis of severe congenital neutropenia and associated with the progression to acute myeloid leukemia.

Identification of a nonsense mutation in the granulocyte-colony-stimulating factor receptor in severe congenital neutropenia (Article)

1994-01-01T00:00:00Z

Severe congenital neutropenia (Kostmann syndrome) is characterized by profound absolute neutropenia and a maturation arrest of marrow progenitor cells at the promyelocyte-myelocyte stage. Marrow cells from such patients frequently display a reduced responsiveness to granulocyte-colony-stimulating factor (G-CSF). G-CSF binds to and activates a specific receptor which transduces signals critical for the proliferation and maturation of granulocytic progenitor cells. Here we report the identification of a somatic point mutation in one allele of the G-CSF receptor gene in a patient with severe congenital neutropenia. The mutation results in a cytoplasmic truncation of the receptor. When expressed in murine myeloid cells, the mutant receptor transduced a strong growth signal but, in contrast to the wild-type G-CSF receptor, was defective in maturation induction. The mutant receptor chain may act in a dominant negative manner to block granulocytic maturation.

Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation (Article)

1993-01-01T00:00:00Z

The granulocyte colony-stimulating factor receptor (G-CSF-R) transduces signals important for the proliferation and maturation of myeloid progenitor cells. To identify functionally important regions in the cytoplasmic domain of the G-CSF-R, we compared the actions of the wild-type receptor, two mutants, and a natural splice variant in transfectants of the mouse pro-B cell line BAF3 and two myeloid cell lines, 32D and L-GM. A region of 55 amino acids adjacent to the transmembrane domain was found to be sufficient for generating a growth signal. The immediate downstream sequence of 30 amino acids substantially enhanced the growth signaling in the three cell lines. In contrast, the carboxy-terminal part of 98 amino acids strongly inhibited growth signaling in the two myeloid cell lines but not in BAF3 cells. Truncation of this region lead to an inability of the G-CSF-R to transduce maturation signals in L-GM cells. An alternative carboxy tail present in a splice variant of the G-CSF-R also inhibited growth signaling, notably in both the myeloid cells and BAF3 cells, but appeared not to be involved in maturation.

In vitro growth characteristics of human lymphoid malignancies in primary cell culture (Doctoral Thesis)

1986-10-29T00:00:00Z

The experimental work presented in this thesis deals with the analysis of the in vitro growth and differentiation characteristics of acute lymphoblastic leukemia ( T end non-T), T cell non Hodgkin's lymphoma and B cell chronic lymphocytic leukemia in primary cell culture. For these studies, reproducible cell culture assays for the growth of these lymphoid tumours first needed to be developed. Considerable attention has been paid to the response of these neoplasms to the polypeptide hormone interleukin 2. The experiments described in chapters 2 and 3 deal with the in vitro colony growth of non-T acute lymphoblastic leukemia (ALL) and with the effect of interleukin 2 on these cells in combination with a factor elaborated by feeder leukocytes. The requirements of the ALL cells for activation with a lectin (PHA) or a phorbol ester (TPA) for colony growth have also been investigated. To assess whether non-T ALL cells differentiate toward more mature cell types during in vitro growth, the morphological and immunological phenotypes of colony cells were determined. To compare the differentiation capacities of ALL with those of acute leukemia of the myeloid differentiation lineage (AML) the abilities of AML to produce more mature progeny under comparable in vitro conditions were studied (chapter 4). Chapter 5 deals with an analysis of growth requirements of 8 cell type chronic lymphocytic leukemia (CLL) in colony culture and specifically with the role of IL2 in the proliferation of B CLL cells. This analysis is extended in chapter 6, in which the results of binding experiments with radiolabeled IL2 are presented. These experiments were carried out to determine numbers and affinity of IL2 receptors expressed by B CLL cells- In addition, the hypothesis that certain CLL cells might be capable of self-stimulation via the autocrine production of IL2 is approached in this chapter. In chapter 7, culture characteristics of acute lymphoblastic leukemia and non Hodgkin's lymphoma (NHL) of the T cell differentiation lineage are presented. The studies described in chapter 8 are our first attempts toward the characterization of membrane phenotypes and growth requirements of normal T -lymphocytic precursor cells in the human bone marrow. For this purpose, we applied the human long-term bone marrow culture system. Knowledge of the growth end differentiation features of normal lymphoid progenitors is essential for our understanding of whether or how the leukemic counter parts of these cells reflect a modified response to growth and differentiation stimuli. In chapter 9 a brief overview of our current understanding of the role of IL2 in the proliferation of neoplastic T and B cells is presented. Moreover, the results of this thesis are discussed in this chapter with reference to their implications for our insight into the control of proliferation and differentiation of the different types of lymphoid leukemia/lymphoma