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British Journal of Haematology 03/2013; · 4.94 Impact Factor
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Timothy R Hercus,
Urmi Dhagat,
Winnie L T Kan,
Sophie E Broughton,
Tracy L Nero,
Michelle Perugini,
Jarrod J Sandow, Richard J D'Andrea,
Paul G Ekert,
Timothy Hughes,
Michael W Parker,
Angel F Lopez
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ABSTRACT: The GM-CSF, IL-3 and IL-5 family of cytokines, also known as the ßc family due to their receptors sharing the signalling subunit ßc, regulates multiple biological processes such as native and adaptive immunity, inflammation, normal and malignant hemopoieis, and autoimmunity. Australian scientists played a major role in the discovery and biological characterisation of the ßc cytokines and their recent work is revealing unique features of cytokine receptor assembly and signalling. Furthermore, specific antibodies have been generated to modulate their function. Characterisation of the structural and dynamic requirements for the activation of the ßc receptor family and the molecular definition of downstream signalling pathways are providing new insights into cytokine receptor signalling as well as new therapeutic opportunities.
Cytokine & growth factor reviews 03/2013; · 6.49 Impact Factor
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British Journal of Haematology 11/2012; · 4.94 Impact Factor
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Lucia Gagliardi,
King-Hwa Ling,
Chung H Kok,
Joseph Carolan,
Peter Brautigan,
Rosalie Kenyon, Richard J D'Andrea,
Mark Van der Hoek,
Christopher N Hahn,
David J Torpy,
Hamish S Scott
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ABSTRACT: ACTH-independent macronodular adrenal hyperplasia (AIMAH) is a rare cause of sporadic or familial late-onset Cushing's syndrome. It is a cytologically benign disease, of unknown pathogenesis, and characterised by inefficient steroidogenesis, ascribed to differential cellular localisation of steroidogenic enzymes. The objectives were to determine the molecular mechanisms involved in the pathogenesis of familial AIMAH tumours and the mechanisms of their inefficient steroidogenesis. Using Affymetrix Human GeneChip® HumanGene 1.0 ST arrays, we compared the genome-wide gene expression profile of two AIMAH nodules from each of three affected siblings with normal adrenal cortex and analysed the data for differential expression and using Ingenuity Pathway Analysis, Gene Set Enrichment Analysis and Motif Activity Response Analysis. Expression profiling identified: (i) that amongst the most highly differentially expressed genes were ones known to have involvement in tumorigenesis and metastasis; (ii) enrichment for differentially expressed genes in sporadic AIMAH and other benign and malignant adrenocortical tumours and (iii) reduced activity of key transcriptional regulators (Steroidogenic factor-1, SF-1 and transcription factor Sp1, Sp1) of steroidogenic enzymes. Genome-wide gene expression studies of familial AIMAH nodules have identified overlap with malignant adrenocortical tumours, which is intriguing given the benign biological behaviour of these tumours. This requires further study. Novel mechanisms of inefficient steroidogenesis were also identified.
Endocrine Related Cancer 03/2012; 19(3):L19-23. · 4.36 Impact Factor
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Timothy R Hercus,
Sophie E Broughton,
Paul G Ekert,
Hayley S Ramshaw,
Michelle Perugini,
Michele Grimbaldeston,
Joanna M Woodcock,
Daniel Thomas,
Stuart Pitson,
Timothy Hughes, Richard J D'Andrea,
Michael W Parker,
Angel F Lopez
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ABSTRACT: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pluripotent cytokine produced by many cells in the body, which regulates normal and malignant hemopoiesis as well as innate and adaptive immunity. GM-CSF assembles and activates its heterodimeric receptor complex on the surface of myeloid cells, initiating multiple signaling pathways that control key functions such as cell survival, cell proliferation, and functional activation. Understanding the molecular composition of these pathways, the interaction of the various components as well as the kinetics and dose-dependent mechanics of receptor activation provides valuable insights into the function of GM-CSF as well as the related cytokines, interleukin-3 and interleukin-5. This knowledge provides opportunities for the development of new therapies to block the action of these cytokines in hematological malignancy and chronic inflammation.
Growth factors (Chur, Switzerland) 01/2012; 30(2):63-75. · 2.47 Impact Factor
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ABSTRACT: Krüppel-like factor 5 (KLF5) has been implicated as a tumor suppressor in various solid tumors such as breast and prostate, and recent studies have demonstrated a role for this protein in neutrophil differentiation of acute promyelocytic leukemia cells in response to ATRA. Here, we show that KLF5 expression increases during primary granulocyte differentiation and that expression of KLF5 is a requirement for granulocyte differentiation of 32D cells. In AML, we show that KLF5 mRNA expression levels are reduced in multiple French-American-British subtypes compared to normal controls, and also in leukemic stem cells relative to normal hematopoietic stem cells. We demonstrate that in selected AML cases, reduced expression is associated with hypermethylation of the KLF5 locus in the proximal promoter and/or intron 1, suggesting that this may represent a Class II genetic lesion in the development of AML.
Leukemia research 01/2012; 36(1):110-6. · 2.36 Impact Factor
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Anna L Brown,
Diana G Salerno,
Teresa Sadras,
Grant A Engler,
Chung H Kok,
Christopher R Wilkinson,
Saumya E Samaraweera,
Timothy J Sadlon,
Michelle Perugini,
Ian D Lewis,
Thomas J Gonda, Richard J D'Andrea
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ABSTRACT: Granulocyte-macrophage colony stimulating factor (GM-CSF) promotes the growth, survival, differentiation and activation of normal myeloid cells and is essential for fully functional macrophage differentiation in vivo. To better understand the mechanisms by which growth factors control the balance between proliferation and self-renewal versus growth-suppression and differentiation we have used the bi-potent FDB1 myeloid cell line, which proliferates in IL-3 and differentiates to granulocytes and macrophages in response to GM-CSF. This provides a manipulable model in which to dissect the switch between growth and differentiation. We show that, in the context of signaling from an activating mutant of the GM-CSF receptor β subunit, a single intracellular tyrosine residue (Y577) mediates the granulocyte fate decision. Loss of granulocyte differentiation in a Y577F second-site mutant is accompanied by enhanced macrophage differentiation and accumulation of β-catenin together with activation of Tcf4 and other Wnt target genes. These include the known macrophage lineage inducer, Egr1. We show that forced expression of Tcf4 or a stabilised β-catenin mutant is sufficient to promote macrophage differentiation in response to GM-CSF and that GM-CSF can regulate β-catenin stability, most likely via GSK3β. Consistent with this pathway being active in primary cells we show that inhibition of GSK3β activity promotes the formation of macrophage colonies at the expense of granulocyte colonies in response to GM-CSF. This study therefore identifies a novel pathway through which growth factor receptor signaling can interact with transcriptional regulators to influence lineage choice during myeloid differentiation.
Differentiation 01/2012; 83(1):47-59. · 2.81 Impact Factor
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Christopher N Hahn,
Chan-Eng Chong,
Catherine L Carmichael,
Ella J Wilkins,
Peter J Brautigan,
Xiao-Chun Li,
Milena Babic,
Ming Lin,
Amandine Carmagnac,
Young K Lee, [......],
Andrew E Timms,
Jan Storek,
Sarah Moore,
Meryl Altree,
Robert Escher,
Peter G Bardy,
Graeme K Suthers, Richard J D'Andrea,
Marshall S Horwitz,
Hamish S Scott
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ABSTRACT: We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies.
Nature Genetics 09/2011; 43(10):1012-7. · 35.53 Impact Factor
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British Journal of Haematology 08/2011; 156(2):268-70. · 4.94 Impact Factor
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British Journal of Haematology 02/2011; 153(5):672-5. · 4.94 Impact Factor
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Timothy J Sadlon,
Bridget G Wilkinson,
Stephen Pederson,
Cheryl Y Brown,
Suzanne Bresatz,
Tessa Gargett,
Elizabeth L Melville,
Kaimen Peng, Richard J D'Andrea,
Gary G Glonek,
Gregory J Goodall,
Heddy Zola,
M Frances Shannon,
Simon C Barry
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ABSTRACT: The transcription factor FOXP3 is essential for the formation and function of regulatory T cells (Tregs), and Tregs are essential for maintaining immune homeostasis and tolerance. This is demonstrated by a lethal autoimmune defect in mice lacking Foxp3 and in immunodysregulation polyendocrinopathy enteropathy X-linked syndrome patients. However, little is known about the molecular basis of human FOXP3 function or the relationship between direct and indirect targets of FOXP3 in human Tregs. To investigate this, we have performed a comprehensive genome-wide analysis for human FOXP3 target genes from cord blood Tregs using chromatin immunoprecipitation array profiling and expression profiling. We have identified 5579 human FOXP3 target genes and derived a core Treg gene signature conserved across species using mouse chromatin immunoprecipitation data sets. A total of 739 of the 5579 FOXP3 target genes were differentially regulated in Tregs compared with Th cells, thus allowing the identification of a number of pathways and biological functions overrepresented in Tregs. We have identified gene families including cell surface molecules and microRNAs that are differentially expressed in FOXP3(+) Tregs. In particular, we have identified a novel role for peptidase inhibitor 16, which is expressed on the cell surface of >80% of resting human CD25(+)FOXP3(+) Tregs, suggesting that in conjunction with CD25 peptidase inhibitor 16 may be a surrogate surface marker for Tregs with potential clinical application.
The Journal of Immunology 07/2010; 185(2):1071-81. · 5.79 Impact Factor
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ABSTRACT: Granulocyte/macrophage colony-stimulating factor promotes growth, survival, differentiation, and activation of normal myeloid cells and plays an important role in myeloid leukemias. The GM-CSF receptor (GMR) shares a signaling subunit, beta(c), with interleukin-3 and interleukin-5 receptors and has recently been shown to induce activation of Janus kinase 2 (JAK2) and downstream signaling via formation of a unique dodecameric receptor complex. In this study we use 2 activated beta(c) mutants that display distinct signaling capacity and have differential requirements for the GMR alpha-subunit (GMR-alpha) to dissect the signaling pathways associated with the GM-CSF response. The V449E transmembrane mutant selectively activates JAK2/signal transducer and activator of transcription 5 and extracellular signal-regulated kinase (ERK) pathways, resulting in a high level of sensitivity to JAK and ERK inhibitors, whereas the extracellular mutant (FIDelta) selectively activates the phosphoinositide 3-kinase/Akt and IkappaKbeta/nuclear factorkappaB pathways. We also demonstrate a novel and direct interaction between the SH3 domains of Lyn and Src with a conserved proline-rich motif in GMR-alpha and show a selective requirement for Src family kinases by the FIDelta mutant. We relate the nonoverlapping nature of signaling by the activated mutants to the structure of the unique GMR complex and propose alternative modes of receptor activation acting synergistically in the mature liganded receptor complex.
Blood 02/2010; 115(16):3346-53. · 9.90 Impact Factor
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Jason A Powell,
Daniel Thomas,
Emma F Barry,
Chung H Kok,
Barbara J McClure,
Anna Tsykin,
L Bik To,
Anna Brown,
Ian D Lewis,
Kirsten Herbert,
Gregory J Goodall,
Terence P Speed,
Norio Asou,
Bindya Jacob,
Motomi Osato,
David N Haylock,
Susan K Nilsson, Richard J D'Andrea,
Angel F Lopez,
Mark A Guthridge
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ABSTRACT: Deregulated cell survival programs are a classic hallmark of cancer. We have previously identified a serine residue (Ser585) in the betac subunit of the granulocyte-macrophage colony-stimulating factor receptor that selectively and independently promotes cell survival. We now show that Ser585 phosphorylation is constitutive in 20 (87%) of 23 acute myeloid leukemia (AML) patient samples, indicating that this survival-only pathway is frequently deregulated in leukemia. We performed a global expression screen to identify gene targets of this survival pathway and report a 138-gene betac Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI3-kinase target genes and a cluster of genes involved in cancer and cell survival. We show that one such gene, osteopontin (OPN), is a functionally relevant target of the Ser585-survival pathway as shown by siRNA-mediated knockdown of OPN expression that induces cell death in both AML blasts and CD34(+)CD38(-)CD123(+) leukemic progenitors. Increased expression of OPN at diagnosis is associated with poor prognosis with multivariate analysis indicating that it is an independent predictor of overall patient survival in normal karyotype AML (n = 60; HR = 2.2; P = .01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signaling network that is deregulated in AML and identify OPN as a potential prognostic and therapeutic target.
Blood 10/2009; 114(23):4859-70. · 9.90 Impact Factor
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ABSTRACT: Hematopoietic growth factor (HGF) mimetics offer a number of attractive advantages as therapeutic agents. Small chemical compounds, in particular, provide reduced cost and oral availability. As many of these mimetics are unrelated in structure to the normal cytokine the immunogenic response is not a significant issue. Isolation of small peptide agonists for erythropoietin (EPO) and thrombopoietin (TPO) receptors has been associated with significant translational challenges and here we summarize approaches used to achieve the potency and stability required for clinical utility. We also compare and contrast the initial screening approaches, and the translational and clinical issues associated with two recently approved TPO mimetics, romiplostim and the orally available eltrombopag. Finally we summarize the development and clinical findings for the EPO mimetic, Hematide, consider alternative approaches, and discuss the future potential for isolation of growth factor (GF) mimetics.
Cytokine & growth factor reviews 03/2009; 20(1):87-94. · 6.49 Impact Factor
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ABSTRACT: Adult stem cells are capable of generating all of the cells of the hematopoietic system, and this process is orchestrated in part by the interactions between these cells and the stroma. T cell progenitors emerge from the stem cell compartment and migrate to the thymus, where their terminal differentiation and maturation occur, and it is during this phase that selection shapes the immune repertoire. Notch ligands, including Delta-like 1 (DL1), play a critical role in this lymphoid differentiation. To mimic this in vitro, stroma-expressing DL1 have been used to generate CD4(+)CD8(+) double-positive and single-positive T cells from hematopoietic stem/progenitor cells. This system provides a robust tool to investigate thymopoiesis; however, its capacity to generate regulatory T cells (Tregs) has yet to be reported. Natural Tregs (nTregs) develop in the thymus and help maintain immune homeostasis and have potential clinical use as a cell therapy for modulation of autoimmune disease or for transplant tolerization. Here, we describe for the first time the development of a population of CD4(+)CD25(+) CD127(lo)FoxP3(+) cells that emerge in coculture of cord blood (CB) CD34(+) progenitors on OP9-DL1 stroma. These hematopoietic progenitor-derived CD4(+)CD25(+) Tregs have comparable suppressor function with CB nTregs in vitro. The addition of IL-2 to the coculture enhanced the expansion and survival of this population significantly. This manipulable culture system, therefore, generates functional Tregs and provides a system to elucidate the mechanism of Treg development.
Journal of leukocyte biology 01/2009; 85(3):445-51. · 4.99 Impact Factor
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ABSTRACT: The establishment of culture systems that promote haemopoietic stem cell (HSC) self-renewal and expansion ex vivo will increase the clinical potential of umbilical cord blood (CB) HSC transplantation. Studies defining key signalling pathways that regulate development and expansion of HSC in vivo have greatly facilitated development of protocols for expanding HSC in ex vivo culture. Recently a number of soluble factors with novel stem cell expansion activity have been identified as part of pathways associated with mesodermal induction, or as factors produced by supportive stroma. These have been reported to support, to varying degrees, HSC self-renewal under in vitro conditions. Here we review the activities of these new factors and consider their future potential as components in ex vivo expansion culture for CB HSC. Finally we discuss the challenges associated with applying these factors to clinically relevant culture systems.
Current Stem Cell Research & Therapy 10/2007; 2(3):229-37.
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Blood 02/2007; 109(1):386-7. · 9.90 Impact Factor
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ABSTRACT: Establishment of conditions supporting hematopoietic stem cell (HSC) maintenance and expansion ex vivo is critical for wider clinical application of cord blood (CB) transplantation. AFT024 is a murine fetal liver cell line that expands primitive hematopoietic cells via a process that is not understood. Here we show that bone morphogenic protein 4 (BMP4) is produced by AFT024 and contributes significantly to the maintenance of co-cultured CB-derived primitive cells. Significant amounts of BMP4 mRNA are produced by the supportive AFT024 stromal cell line, and secreted BMP4 protein accumulates in AFT024 conditioned medium. Blockade of BMP4 activity in this coculture model using neutralizing BMP4 monoclonal antibody reduced expansion of primitive CB cells on the basis of phenotypic (CD34(+)CD38(-)) and functional criteria [long-term culture initiating cells (LTC-IC)] and significantly reduced the capacity of the cultured CB stem cells to support repopulation in the nonobese diabetic-severe combined immunodeficiency (NOD-SCID) xenograft model. Therefore, BMP4 is a key growth factor for maintenance of HSC and contributes to the unique properties of the AFT024 stromal noncontact culture.
Stem Cells and Development 01/2007; 15(6):805-13. · 4.46 Impact Factor
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Anna L Brown,
Christopher R Wilkinson,
Scott R Waterman,
Chung H Kok,
Diana G Salerno,
Sonya M Diakiw,
Brenton Reynolds,
Hamish S Scott,
Anna Tsykin,
Gary F Glonek,
Gregory J Goodall,
Patty J Solomon,
Thomas J Gonda, Richard J D'Andrea
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ABSTRACT: Mechanisms controlling the balance between proliferation and self-renewal versus growth suppression and differentiation during normal and leukemic myelopoiesis are not understood. We have used the bi-potent FDB1 myeloid cell line model, which is responsive to myelopoietic cytokines and activated mutants of the granulocyte macrophage-colony stimulating factor (GM-CSF) receptor, having differential signaling and leukemogenic activity. This model is suited to large-scale gene-profiling, and we have used a factorial time-course design to generate a substantial and powerful data set. Linear modeling was used to identify gene-expression changes associated with continued proliferation, differentiation, or leukemic receptor signaling. We focused on the changing transcription factor profile, defined a set of novel genes with potential to regulate myeloid growth and differentiation, and demonstrated that the FDB1 cell line model is responsive to forced expression of oncogenes identified in this study. We also identified gene-expression changes associated specifically with the leukemic GM-CSF receptor mutant, V449E. Signaling from this receptor mutant down-regulates CCAAT/enhancer-binding protein alpha (C/EBPalpha) target genes and generates changes characteristic of a specific acute myeloid leukemia signature, defined previously by gene-expression profiling and associated with C/EBPalpha mutations.
Journal of Leukocyte Biology 09/2006; 80(2):433-47. · 4.99 Impact Factor
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ABSTRACT: Blood formation occurs throughout the life of an individual in a process driven by hematopoietic stem cells (HSCs). The ability of bone marrow (BM) and cord blood (CB) HSC to undergo self-renewal and develop into multiple blood lineages has made these cells an important clinical resource. Transplantation with BM- and CB-derived HSCs is now used extensively for treatment of hematological disorders, malignancies, and immunodeficiencies. An understanding of the embryonic origin of HSC and the factors regulating their generation and expansion in vivo will provide important information for the manipulation of these cells ex vivo. This is critical for the further development of CB transplantation, the potential of which is limited by small numbers of HSC in the donor population. Although the origins of HSCs have become clearer and progress has been made in identifying genes that are critical for the formation and maintenance of HSCs, less is known about the signals that commit specific populations of mesodermal precursors to hematopoietic cell fate. Critical signals acting on these precursor cells are likely to be derived from visceral endoderm in yolk sac and from underlying stroma in the aorta-gonad-mesonephros region. Here we summarize briefly the origin of yolk sac and embryonic HSCs before detailing evidence that bone morphogenic protein-4 (BMP4) has a crucial role in Xenopus and mammalian HSC development. We discuss evidence that BMP4 acts as a hematopoietic growth factor and review its potential to modulate HSC in ex vivo expansion cultures from cord blood.
Stem Cells 02/2004; 22(4):457-74. · 7.78 Impact Factor
