Proliferation Of Myeloid Progenitor Cells Research Articles

The transcription factor nuclear factor Y regulates the proliferation of myeloid progenitor cells

The transcription factor nuclear factor Y (NF-Y) consists of three subunits; NF-YA, -B and -C, and this complex binds DNA and the basal transcription machinery through binding of TBP.[1][1] NF-Y targets include cell cycle related genes and hematopoietic (stem) cell genes such as cd34 and hoxb4 .[2][

GABP Transcription Factor Is Required for Myeloid Cell Development In Vivo.

GABP is an ets transcription factor that regulates genes that are required for innate immunity, including CD18 (β2 leukocyte integrin), lysozyme, and neutrophil elastase. GABP consists of two distinct and unrelated proteins. GABPα binds to DNA through its ets domain and recruits GABPβ, which contains the transactivation domain; together, they form a functional tetrameric transcription factor complex. We recently showed that GABP is required for entry into S phase of the cell cycle through its regulation of genes that are required for DNA synthesis and cyclin dependent kinase inhibitors (Yang, et al. Nature Cell Biol 9:339, 2007). Furthermore, GABP is an essential component of a retinoic acid responsive myeloid enhanceosome (Resendes and Rosmarin Mol Cell Biol 26:3060, 2006). We cloned Gabpa (the gene that encodes mouse Gabpα) from a mouse genomic BAC library and prepared a targeting vector in which the ets domain is flanked by loxP recombination sites (floxed allele). Deletion of both floxed Gabpa alleles causes an early embryonic lethal defect. In order to define the role of Gabpα in myelopoiesis, we bred floxed Gabpa mice to mice that bear the Mx1-Cre transgene, which drives expression of Cre recombinase in response to injection of the synthetic polynucleotide, poly I-C. Deletion of Gabpa dramatically reduced granulocytes and monocytes in the peripheral blood, spleen, and bone marrow, but myeloid cells recovered within weeks. In vitro colony forming assays indicated that myeloid cells in these mice were derived only from Gabpa replete myeloid precursors (that failed to delete both Gabpa alleles), suggesting strong pressure to retain Gabpα in vivo. We used a novel competitive bone marrow transplantation approach to determine if Gabp is required for myeloid cell development in vivo. Sub-lethally irradiated wild-type recipient mice bearing leukocyte marker CD45.1 received equal proportions of bone marrow from wild type CD45.1 donor mice and floxed-Mx1-Cre donor mice that bear CD45.2. Both the CD45.2 (floxed-Mx1-Cre) and CD45.1 (wild type) bone marrow engrafted well. Mice were then injected with pI-pC to induce Cre-mediated deletion of floxed Gabpa. The mature myeloid and T cell compartments were derived almost entirely from wild type CD45.1 cells. This indicates that the proliferation and/or differentiation of myeloid and T cell lineages requires Gabp. In contrast, B cell development was not impaired. We conclude that Gabpa disruption causes a striking loss of myeloid cells in vivo and corroborates prior in vitro data that GABP plays a crucial role in proliferation of myeloid progenitor cells.

Chromosome 21-Encoded miR-125b and Its Role in the Development of Myeloid Leukemia in Children with Down's Syndrome.

Children with Down's syndrome (DS) are at high risk to develop transient leukemia (TL) or acute myeloid leukemia (ML-DS), primarily the megakaryoblastic subtype (AMKL). Recently, acquired mutations of the hematopoietic transcription factor GATA1 - encoded on chromosome X - have been identified in leukemic blasts from virtually all patients with ML-DS or TL leading to the expression of a shorter isoform of GATA1 (GATA1s). During embryonic/fetal development mice carrying the GATA1s mutation show a hyperproliferation of a transient population of yolk sac/fetal liver megakaryocytic progenitors. However, GATA1s mice as well as GATA1s mice bred to mouse DS models (Ts65Dn and Ts1Cje) have normal adult hematopoiesis and do not develop leukemia. Hence, the factors on chromosome 21 that predispose to TL or ML-DS in cooperation with oncogenic GATA1s remain largely unknown. To date, four miRNAs (miR-99a, miR-125b-2, miR-155 and let-7c) are known to be encoded on chromosome 21. Three out of four miRNAs (miR-99a, miR-125b-2, let-7c) are not trisomic in the DS mouse model. To elucidate their role in the development of TL and ML-DS, we first measured their expression level in sorted cells from patient samples [ML-DS (n = 4), TL (n = 4), non-DS AMKL (n = 4)], and in megakaryocytes from healthy donors (n = 3) by qRT-PCR. The expression of miR-125b was clearly elevated in ML-DS (10-fold), TL (5-fold) and non-DS AMKL (3-fold) compared to normal megakaryocytes (ppatients vs. control <0.01). Using Gene Set Enrichment Analysis (GSEA), we confirmed the downregulation of putative target genes of miR-125b - predicted by Targetscan and Pictar - in the gene expression profile of ML-DS in comparison to non-DS AMKL. By retroviral overexpression using the Mouse Stem Cell Virus (MSCV), we could show that ectopic expression of miR-125b markedly enhanced proliferation of myeloid progenitor cells (MPC, CD34+CD33+) (MPCmiR-125b) in vitro. In colony-forming assays, the number of MPCmiR-125b derived colonies exceeded the number of MPCvector derived colonies (empty vector) by 4-fold (χ2 < 0.01). All MPCmiR-125b derived colonies contained a mixture of immature cells (CFU-GEMM) indicating an affected differentiation. This could also be confirmed by immunophenotyping of MPC cultured in liquid medium containing various cytokines (percentage CD34+CD33+ 26% vs 9%). In GATA1s fetal liver progenitors retroviral overexpression of miR-125b-2 could further enhance the proliferation rate and mediate the formation of large megakaryocytic colonies. These data support the hypothesis that miR-125b is involved in development of trisomy 21 associated leukemia by mediating the proliferation of MPC while repressing their differentiation. Therefore, miR-125b might be potential target in the therapy of ML-DS.

Triad1 Regulates Myelopoiesis through Different Ubiquitin Ligase Activities.

The modification of cellular proteins with poly-ubiquitin chains plays an essential role in hematopoiesis. Different types of ubiquitin chains may have opposite effects on the marked proteins. Chains linked through lysine 48 of ubiquitin are recognized by the proteasome resulting in progressive degradation of the ubiquitylated proteins. Ubiquitin chains linked through lysine 63 are not recognized by the proteasome. Instead, these chains can bind proteins that regulate signal transduction and gene transcription. Although it is known that ubiquitylation is essential for hematopoiesis and that alterations in ubiquitylation have been implicated in malignant hematopoiesis the ubiquitin ligases that catalyze protein ubiquitylation remain largely unknown. Triad1 is an ubiquitin ligase that inhibits the proliferation of myeloid progenitor cells through its ligase activity. Triad1 belongs to a unique class of ligases that harbor two RING finger protein domains. This domain specifically binds ubiquitin conjugating enzymes (Ubcs). Together with the Ubcs the ligases determine which type of ubiquitin chain is catalyzed. To understand how Triad1 regulates myelopoiesis we screened a panel of Ubcs for Triad1 interaction and found that Triad1 binds UbcH7 through its N-terminal RING domain and Ubc13 through its C-terminal RING domain. UbcH7 catalyzes the formation of ubiquitin chains linked through lysine 48 that are recognized by the proteasome. Importantly, ubiquitin chains catalyzed by Ubc13 are linked through lysine 63 and are not recognized by the proteasome. In agreement with these interactions, in vitro ubiquitylation assays using different ubiquitin mutants containing only one lysine residue showed that Triad1 can catalyze the formation of both types of ubiquitin chains. The relevance of these findings in myelopoiesis was studied by generating a panel of Triad1 deletion mutants that lack the coiled coil, DRIL or RING domains. Next, the growth inhibitory effect of these mutants was tested in clonogenic assays by retroviral transduction of U937 cells. Like in primary cells, wild type Triad1 inhibited U937 colony formation by over 60% compared to empty vector transduced cells. Three Triad1 mutants lacking the DRIL domain or either one or two of the coiled coil domains inhibited clonogenic growth at a comparable rate as wild type Triad1. In contrast, deletion of either the N- or C-terminal RING finger completely abrogated the inhibitory effect of Triad1 in clonogenic growth. Thus, loss of either the UbcH7 or Ubc13 binding domain of Triad1 affects its inhibitory function in myeloid cell proliferation. Recently, we reported that Triad1 binds the transcription factor Gfi1. Gfi1 plays an important role during many hematopoietic developmental stages and is essential for neutrophilic differentiation. Remarkably, Triad1 inhibited Gfi1 ubiquitylation and proteasomal degradation. The finding here that Triad1 can catalyze the formation of different ubiquitin chains might suggest that Triad1 may modify Gfi1 with ubiquitin in a functional way, rather than marking it for proteasomal degradation. Indeed, in vivo ubiquitylation experiments showed that Gfi1 can be modified with ubiquitin chains not linked through lysine 48. The relevance of this finding and the role of Triad1 in this process is currently studied. Together, these data indicate that the dual ubiquitin ligase activity that results in the formation of different poly-ubiquitin chains is crucial to the central role of Triad1 in myelopoiesis.

G-CSF therapy for acute myocardial infarction

Granulocyte-colony-stimulating factor (G-CSF) has recently been shown to have various effects besides promoting the proliferation and differentiation of myeloid progenitor cells, including the mobilization of bone marrow stem cells and the regeneration infarcted hearts in mice. Recent animal studies have also revealed that G-CSF activates multiple signaling pathways, such as Akt and also the Janus family kinase-2 and signal transducer and activation of transcription-3 (Jak2-STAT3) pathway, in cardiac myocytes. It prevents left ventricular remodeling after myocardial infarction by decreasing cardiomyocyte death and by increasing the number of blood vessels, suggesting the importance of direct actions of G-CSF on the myocardium rather than through mobilization and differentiation of stem cells. Several clinical trials have been performed to study the efficacy of G-CSF therapy in patients with acute myocardial infarction but the results remain controversial because the protocols followed varied between the trials.

Granulocyte colony-stimulating factor and its receptor in normal myeloid cell development, leukemia and related blood cell disorders

Granulocyte colony-stimulating factor (G-CSF) is the major hematopoietic cytokine involved in the control of neutrophil production and thus serves as a critical regulator of the innate immunity against bacterial infections. G-CSF is applied on a routine basis in the clinic for treatment of congenital and acquired neutropenias, diseases characterized by a critical shortage of neutrophils, leading to severe opportunistic bacterial infections. Very recently, it has become clear that therapeutic application of G-CSF may not be limited to different types of neutropenia, but may extend to non-hematological conditions, in particular cardiac and brain infarctions. G-CSF drives the proliferation, survival and neutrophilic differentiation of myeloid progenitor cells by activation of a receptor of the hematopoietin receptor superfamily, which subsequently triggers multiple signaling mechanisms. These mechanisms exert positive as well as negative effects on the signaling function of the G-CSF receptor. The integrated output of these signaling pathways provide the appropriate balance needed for accurate production of neutrophils under both steady state and "emergency" conditions. Here we review how these mechanisms are thought to act in concert to meet with these demands and how perturbations in the function of the G-CSF receptor are implicated in various types of myeloid disease.

IRF-4 Functions as a Myeloid Tumor Suppressor.

Interferon regulatory factor 4 (IRF-4, also know as pip, LSIRF, ICSAT and MUM1) is a transcription factor with well-characterized functions in B and T cell development and immune response regulation. Though its expression has been demonstrated in macrophages, its function in the myeloid system is not as well characterized. The closely related IRF family member IRF-8 (ICSBP) has more clearly defined functions in the myeloid system. IRF-8 is a critical regulator of myelopoiesis, and its loss has been implicated in the pathogenesis of chronic myelogenous leukemia (CML). IRF-8 deficient mice manifest a CML-like syndrome, and we have shown previously that forced expression of IRF-8 in a BCR/ABL-induced murine model of CML represses the resulting myeloproliferative disease and prolongs survival. In the B-cell system, we have found that IRF-4 and IRF-8 function redundantly at the pre-B-to-B transition point of development. In this study we investigate whether IRF-4 and IRF-8 may also have overlapping function in myeloid cells. Our results show that mice deficient in both IRF-4 and IRF-8 develop from a very early age a more aggressive CML-like disease than mice deficient in IRF-8 alone. Bone marrow progenitors from IRF-4/8 DKO mice have increased proliferation, GM-CSF stimulated granulocytic differentiation, and colony-forming capacity compared to progenitors from WT or single KO mice. These data suggest that IRF-4 plays a role in myeloid lineage development and may act to suppress proliferation and granulocytic differentiation of myeloid progenitor cells. We further examined if, like IRF-8, IRF-4 is able to suppress BCR/ABL mediated transformation. We used a murine stem cell virus vector to co-express BCR/ABL and either IRF-4 or IRF-8 in BM progenitor cells and assessed colony forming capacity of infected cultures as well as the ability of IRF-4 to suppress BCR/ABL induced CML-like disease in a murine model. Our results demonstrate that IRF-4 potently suppresses BCR/ABL induced colony formation and that forced expression of IRF-4 suppresses BCR/ABL-induced CML-like disease in mice even more potently than IRF-8. These results provide direct evidence that IRF-4 is an important myeloid tumor suppressor and may allow elucidation of new molecular pathways significant to the pathogenesis of human CML.

Colony stimulating factors as adjunctive therapy in neonatal sepsis

Colony stimulating factors as adjunctive therapy in neonatal sepsis

Effect of Arachidonic Acid Reacylation on Leukotriene Biosynthesis in Human Neutrophils Stimulated with Granulocyte-macrophage Colony-stimulating Factor and Formyl-methionyl-leucyl-phenylalanine

Priming of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by treatment with formyl-methionyl-leucyl-phenylalanine (fMLP) stimulates cells in a physiologically relevant manner with modest 5-lipoxygenase activation and formation of leukotrienes. However, pretreatment of neutrophils with thimerosal, an organomercury thiosalicylic acid derivative, led to a dramatic increase (>50-fold) in the production of leukotriene B(4) and 5-hydroxyeicosatetraenoic acid, significantly higher than that observed after stimulation with calcium ionophore A23187. Little or no effect was observed with thimerosal alone or in combination with either GM-CSF or fMLP. Elevation of [Ca(2+)](i) induced by thimerosal in neutrophils stimulated with GM-CSF/fMLP was similar but more sustained compared with samples where thimerosal was absent. However, [Ca(2+)](i) was significantly lower compared with calcium ionophore-treated cells, suggesting that a sustained calcium rise was necessary but not sufficient to explain the effects of this compound on the GM-CSF/fMLP-stimulated neutrophil. Thimerosal was found to directly inhibit neutrophil lysophospholipid:acyl-CoA acyltransferase activity at the doses that stimulate leukotriene production, and analysis of lysates from neutrophil preparations stimulated in the presence of thimerosal showed a marked increase in free arachidonic acid, supporting the inhibition of the reincorporation of this fatty acid into the membrane phospholipids as a mechanism of action for this compound. The dramatic increase in production of leukotrienes by neutrophils when a physiological stimulus such as GM-CSF/fMLP is employed in the presence of thimerosal suggests a critical regulatory role of arachidonate reacylation that limits leukotriene biosynthesis in concert with 5-lipoxygenase and cytosolic phospholipase A(2)alpha activation.

362. Evaluation the Effects of MIP-1a on Ex Vivo Expansion of Cord Blood Hematopoietic Progenitor Cells in Different Culture Media

Introduction:The use of ex vivo expansion of hematopoietic progenitor cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood (CB) transplantation. Macrophage inflammatory protein-1 alpha (MIP-1a) has been shown to have a role in the control of myeloid stem and progenitor cell proliferation. So the aim of this study was to evaluate the effects of MIP-1a on ex vivo expansion of hematopoietic progenitor cells in different culture media for the propose of cord blood transplantation or gene therapy.

STAT3 Deletion after Birth Results in Marked Decreases in Absolute Numbers and Cell Cycling Status of Marrow and Spleen Myeloid Progenitor Cells, and STAT3 Is a Critical Molecule in Mediating Synergistically Stimulated Progenitor Cell Proliferation.

STAT3 is an important transcription factor involved in mediating intracellular signals initiated at the cell membrane by cytokines and growth factors. There is ample evidence that STAT3 acts as a positive regulator of cell growth, but most of this information derives from studies done with isolated cells in vitro. Because functional deletion of STAT3 in mice is lethal, it was difficult to evaluate a role for STAT3 in mediating hematopoietic effects in vivo in mice after birth. To address this problem, a unique strain of mice was developed with tissue specific disruption of STAT3 in bone marrow and hematopoietic cells (Welte et.al. PNAS 100: 1879, 2003). The availability of this conditional STAT3 −/− mouse model demonstrated a critical role for STAT3 in innate immunity. We have now utilized this conditional STAT3 −/− mouse model to evaluate a role for STAT3 in hematopoiesis after birth, with the hypothesis that STAT3 would be one critical factor involved in the proliferation of myeloid progenitor cells (MPC: CFU-GM, BFU-E, CFU-GEMM) in bone marrow and spleen. STAT3 −/− and their littermate control mice were evaluated at 4 weeks of age. STAT3 −/− mice manifested 40–44% decreases in absolute numbers of nucleated cells in the marrow (femur) and spleen. This was associated with decreased absolute numbers of CFU-GM (70%), BFU-E (70%) and CFU-GEMM (50%) per femur and CFU-GM (50%), BFU-E (30%), and CFU-GEMM (50%) per spleen for these MPC which are responsive in vitro to stimulation of colony formation by the combination of EPO, SCF, TPO and growth factors in PWMSCM. Moreover, MPC from STAT3 −/− mice were in a slow or non cycling state (0–4% MPC in S-phase) in marrow and spleen compared to 50–60% marrow and 32–48% spleen MPC from +/+ mice being in active cell cycle. There were also large decreases per femur in STAT3 −/− mice in terms of GM-CSF-, IL-3-, M-CSF-, GM-CSF plus SCF-, GM-CSF plus Flt3 ligand (FL)-, IL-3 plus SCF-, IL-3 plus FL-, M-CSF plus SCF-, and M-CSF plus FL- responsive CFU-GM. These decreases may in part reflect the finding that CFU-GM from STAT3 −/− mice did not respond to the synergistic proliferation effects of GM-CSF, IL-3, or M-CSF, each in combination with either SCF or FL. At best these cytokine combinations resulted in additive proliferative effects on MPC from marrow of STAT3 −/− mice in contrast to CFU-GM from +/+ mouse marrow where the effects were clearly synergistic. In terms of survival of MPC, there were no apparent differences between the survival of MPC from STAT3 −/− and +/+ mice after withdrawal of growth factors in vitro and their delayed addition to the cell cultures. MPC from STAT3 −/− and +/+ marrow responded similarly to the survival enhancing effects in vitro of SDF-1/CXCL12. Our results demonstrate that after birth STAT3 acts as a positive mediator of the proliferation of MPC in vivo, and STAT3 is a critical mediator of the synergistic proliferation effects of cytokines on MPC.

Genetic Pathways in Therapy-Related Leukemia: FHL2 Cooperates with del(5q).

Therapy-related myelodysplastic syndrome (t-MDS) and acute myeloid leukemia (t-AML) are late complications of cytotoxic therapy used in the treatment of both malignant and non-malignant diseases. The most common subtype of t-MDS/t-AML (~75% of cases) develops after exposure to alkylating agents, and is characterized by loss of a whole chromosome 5 and/or 7, or a deletion of the long arms of these chromosomes [-5/del(5q), -7/del(7q)]. Patients who develop t-MDS/t-AML in this setting typically show a latency of 5 years from alkylating agent exposure, and present with cytopenias and myelodysplasia. In the University of Chicago's series of 306 patients with t-MDS/t-AML, 64 (21%) patients had abnormalities of chromosome 5, 85 (28%) patients had abnormalities of chromosome 7, and 65 (21%) patients had abnormalities of both chromosomes 5 and 7 (Smith et al. Blood 102:43, 2003). Survival times of these t-AML patients are short (median 8 mos), and new therapeutic approaches are needed.Genetic pathways in t-MDS/t-AML are poorly-understood. Monosomy 7/del(7q) has been associated with activating mutations of the RAS pathway and methylation silencing of the CDKN2B (p15INK4B) gene, whereas -5/del(5q) is associated with TP53 mutations and a complex karyotype. To identify novel molecular pathways involved in the pathogenesis of t-MDS/t-AML, we previously performed gene expression profiling of CD34+ hematopoietic progenitor cells from t-AML patients, and identified two major groups, characterized by a -7/del(7q) or normal karyotype (Group A) or -5/del(5q) (Group B) (Qian et al., Proc Natl Acad Sci USA 99:14925, 2002). Patients with a -5/del(5q) have a higher expression of genes involved in cell cycle control (CCNA2, CCNE2, CDC2), checkpoints (BUB1), or growth (MYC), and loss of expression of ICSBP. In addition, FHL2 is upregulated in this subgroup. FHL2 is a multi-functional LIM domain protein that has been implicated as a transcriptional co-activator or co-repressor mediating proliferation and apoptosis in a tissue-specific fashion. Interacting partners of FHL2 include WT1, b-catenin, PLZF, and the androgen receptor. In subsequent studies, we determined that hematopoietic cells express a novel isoform of FHL2 (termed B-FHL2), that contains a unique 5′-UTR, but encodes the same peptide, suggesting that FHL2 is regulated by a different promoter in hematopoietic cells. To examine the effects of enhanced expression of FHL2 on hematopoiesis, we transduced primary bone marrow cells from mice treated with 5-FU with retroviral vectors expressing FHL2 (MIGR1-FHL2, MSCV-FHL2). Up-regulation of FHL2 enhanced proliferation of myeloid progenitor cells, and self-renewal capacity of hematopoietic cells in vitro. To determine if up-regulation of FHL2 in vivo leads to alterations in lineage-specific differentiation, we used retroviral transduction/transplantation techniques. Expression of FHL2 resulted in increased numbers of colony-forming cells and GM colonies, but not BFU-E or GEMM colonies. Recipient mice followed for up to 12 months were characterized by enhanced myelopoiesis and partially blocked B lymphopoiesis, with no detectable effect on T cell development. Expression of FHL2 did not induce leukemia in transplanted mice. These results suggest that FHL2 is involved in hematopoiesis. Furthermore, upregulation of FHL2 results in increased stem cell and progenitor cell self-renewal; however, leukemogenesis may require the acquisition of secondary cooperating mutations.

G-CSF Receptor Lysine Residues Are Involved in Lysosomal Degradation and Attenuation of G-CSF Signaling Via Distinct Effector Mechanisms.

The granulocyte colony-stimulating factor receptor (G-CSF-R) induces proliferation, survival and differentiation of myeloid progenitor cells in a tightly controlled temporal fashion. These responses depend on multiple signaling mechanisms that are activated via distinct regions in the cytoplasmic domain of wild type (WT) G-CSF-R. About 20% of severe congenital neutropenia patients acquire mutations that truncate the C-terminus of G-CSF-R, which is often associated with disease progression to acute myeloid leukemia. Myeloid cells expressing these truncated G-CSF-R hyperproliferate and are hampered in differentiation in response to G-CSF. Multiple mechanisms have been linked to perturbed signaling of truncated G-CSF-R. Specifically, defective internalization has been associated with a prolonged activation status of the truncated G-CSF-R, which is e.g. reflected by strongly increased and sustained activation of STAT5. We found that internalized WT G-CSF-R are rapidly targeted to lysosomes, suggesting that lysosomal degradation is a major mechanism for signal attenuation. Lysine (K) residues in the cytoplasmic tail of transmembrane receptors are often determinants for ubiquitin-mediated sorting into multi-vesicular bodies and lysosomes. We observed that G-CSF-R is ubiquitinated and subsequently assessed the contribution of K residues in G-CSF-R routing and signaling. To this end, we generated mutant G-CSF-R-K5R, in which all five conserved cytoplasmic K residues were replaced by arginine (R). To study differential localization of internalized K5R and WT G-CSF-R, we introduced constitutively active Rab5 (Rab5-Q79L) to enlarge early endosomes. Under these conditions, internalized WT G-CSF-R colocalized extensively with endosomal microdomains containing the endosome-to-lysosome sorting protein Hrs. In contrast, internalized G-CSF-R-K5R was mostly found outside these domains. Consequently, lysosomal routing of G-CSF-R-K5R was severely impaired, which resulted in strongly increased receptor protein levels. Upon removal of G-CSF, downregulation of STAT5 activity in 32D/K5R cells was significantly delayed compared to 32D/WT cells, establishing that the lysine residues are not only crucial for receptor stability, but also for duration of signaling. Moreover, in the continuous presence of G-CSF, 32D/K5R cells showed strongly increased STAT5 activity and proliferation and reduced granulocytic differentiation compared to 32D/WT. Similar results were obtained in colony assays with g-csfr deficient primary bone marrow progenitors transduced with K5R or WT G-CSF-R. To substantiate a relationship between signal duration and receptor degradation, we inhibited lysosomal degradation by pharmacological inhibitors and by knockdown of Hrs and the related protein Tsg101 by RNA interference. As expected, receptor stability was significantly increased by these treatments. Surprisingly however, this did not influence STAT5 activity, suggesting that signal attenuation predominantly occurred in a pre-lysosomal compartment. We conclude from these data that ubiquitinated lysine residues of G-CSF-R are required to target internalized receptors for lysosomal degradation. However, they also appear to be involved in contracting an as yet unidentified inhibitor, perhaps a phosphatase, to a specific Hrs-positive subendosomal compartment, thereby switching off G-CSF-R already prior to its degradation in lysosomes.

Gfi-1 Represses C/EBPe and Is Required for the Survival and Proliferation of Myeloid Cells Stimulated by G-CSF.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that plays a major role in granulopoiesis. G-CSF supports the proliferation, differentiation and survival of myeloid progenitor cells. Treatment of cells with G-CSF activates multiple intracellular signal transduction pathways leading to alterations in the activities of transcription factors involved in myeloid development. Gfi-1 is a zinc-finger transcriptional repressor that is required for granulopoiesis. Mutations in Gfi-1 have been associated with severe congenital neutropenia (SCN). How Gfi-1 acts in myeloid cells is still poorly understood. Expression of Gfi-1 was upregulated during granulocytic differentiation induced by G-CSF in myeloid 32D cells. Truncation of the carboxy terminus of the G-CSF receptor, as seen in patients with acute myeloid leukemia (AML) evolving from SCN, disrupted the ability of the G-CSF receptor to upregulate Gfi-1. Ectopic expression of a naturally occurring dominant negative Gfi-1 mutant, N382S, identified in patients with SCN, resulted in premature apoptosis and reduced proliferation of cells induced to differentiate with G-CSF. The expression of neutrophil elastase (NE) and C/EBPe was significantly increased in 32D cells expressing N382S. In contrast, overexpression of wild type Gfi-1 abolished G-CSF-induced upregulation of C/EBPe, but had no apparent effect on NE upregulation by G-CSF. Notably, G-CSF-dependent proliferation and survival were inhibited upon overexpression of C/EBPe, but not NE. These data suggest that a key role of Gfi-1 in granulopoiesis is to repress C/EBPe and that overt C/EBPe overexpression may be deleterious to the proliferation and survival of myeloid cells. Our data may explain why loss of Gfi-1 function, either as a result of gene knockout in mice or gene mutations in human, results in a block of granulocytic differentiation and severe neutropenia.

An Important Role for CD1d and NKT Cells in the Suppression of Hematopoiesis in Mice Induced by Infection with Cytomegalovirus.

Cytomegalovirus (CMV) is a serious problem in the context of immunocompromised hosts after conditioning for stem cell transplantation. Infection with CMV impacts hematopoiesis, and inhibits colony formation by myeloid progenitor cells (MPC) in vitro and myelopoiesis in vivo. It is not clear how these effects are mediated. Murine (M)CMV has been used as a model for the study of viral pathogenesis and persistent infections. Little is known how CD1d or Natural Killer T (NKT) cells influence effects of CMV infections on hematopoiesis. We hypothesized that CD1d and NKT cells may play a protective role in this effect. We first assessed a role for CD1d on hematopoiesis and found that absolute numbers of MPC (CFU-GM, BFU-E, CFU-GEMM) in marrow and spleen of CD1d −/− mice were significantly higher than in control mice, an observation reproducible in CD1d −/− mice on either a C57Bl/6 or Balb/c background, suggesting that the effects were not due to mouse strain differences. This increased number of MPC was due to increased proliferation of MPC in marrow and spleen since the percentage of MPC in S phase of the cell cycle in CD1d −/− mice, as determined by a high specific activity tritiated thymidine kill assay, was at least two fold higher than in control (+/+) mice. CD1d −/− mice are deficient in NKT cells. We also used Jα18 −/− (C57Bl/6 and Balb/c) mice, which are deficient in NKT cells but express normal cell surface levels of CD1d expression, to demonstrate that the enhanced hematopoiesis in CD1d −/− mice was not a reflection of NKT cell effects; Jα18 −/− mice had normal numbers and cycling status of MPC in marrow and spleen. We then assessed the effects of MCMV (5 x 104 plaque forming units per mouse) on absolute numbers and cycling of MPC in marrow and spleen of +/+, CD1d −/−, and Jα18 −/− mice. Mice were evaluated 4 days post MCMV infection when viral titers are high. While MCMV had some suppressive effects on MPC numbers and cycling of MPC in +/+ C57Bl/6 and Balb/c mice, this suppression was much more pronounced in CD1d −/− and Jα18 −/− mice, with the most potent MCMV effects manifested on these −/− mice on a Balb/c background strain, suggesting that lack of CD1d and/or NKT cells are involved and likely protective in these suppressive viral effects on hematopoiesis. In order to confirm that NKT cells were responsible for this MCMV-induced decrease in hematopoiesis, we utilized Jα18 −/− mice on a Balb/c background to evaluate effects of adding back to these mice (which don't contain NKT cells) purified Vα 14 Jα18 T cell receptor positive cells isolated from murine liver using CD1d dimers loaded with αGalCer. Mice were given these NKT cells at 2.5 x 105 cells i.v./mouse and immediately after injected with MCMV i.p. The NKT cells blocked MCMV-induced suppression of absolute numbers and cycling of marrow and spleen CFU-GM, BFU-E, and CFU-GEMM, demonstrating that NKT cells play a role in and can block MCMV effects on murine hematopoiesis. This information may thus be relevant to treatment of CMV infections during stem cell transplantation.

A functional single-nucleotide polymorphism of the G-CSF receptor gene predisposes individuals to high-risk myelodysplastic syndrome

AbstractThe granulocyte colony-stimulating factor receptor (G-CSF-R) transmits signals for proliferation and differentiation of myeloid progenitor cells. Here we report on the identification of a rare single nucleotide polymorphism within its intracellular domain (G-CSF-R_Glu785Lys). Screening a cohort of 116 patients with primary myelodysplastic syndromes (MDS), de novo acute myeloid leukemia (AML) (84 patients), as well as 232 age- and sex-matched controls revealed a highly significant association of the G-CSF-R_785Lys allele with the development of high-risk MDS as defined by more than 5% bone marrow blasts (9.7% versus 0.9% in controls; P = .001; odds ratio [OR], 12.5; 95% confidence interval [CI], 2.4-58.9) or an International Prognostic Scoring System score of intermediate-2 or high (13.0% versus 0.9%; P < .001; OR, 14.0; 95% CI, 3.4-85.0). Functional analysis by retroviral transfer of G-CSF-R_785Lys into myeloid progenitor cells of G-CSF-R–deficient mice showed a significantly diminished colony-formation capacity after G-CSF stimulation as compared with cells transduced with the wild-type receptor. These results suggest that lifelong altered G-CSF response by the G-CSF-R_785Lys may render individuals susceptible to development of high-risk MDS.

Impact of Granulocyte Colony-Stimulating Factor during Induction Therapy in Children with Acute Myelogenous Leukemia: Results from the Prospective and Randomized Trial AML-BFM 98.

Background: Treatment for acute myelogenous leukemia (AML) is one of the most intensive treatment modalities in pediatric oncology. Infection-related mortality is particularly high among these patients during chemotherapy-induced neutropenia. Granulocyte colony-stimulating factor (G-CSF) expands the circulating pool of neutrophils by stimulating proliferation and maturation of myeloid progenitor cells. Although G-CSF is widely used for the prevention of infectious complications in cancer patients, no larger randomized study has evaluated the effect of G-CSF in pediatric cancer patients undergoing treatment for AML.Patients and Methods: The impact of G-CSF on hematopoetic recovery, infectious complications and five-year event-free survival (5-EFS) was prospectively evaluated in the multicenter clinical trial AML-BFM 98. Patients (inclusion criteria: primary diagnosis of AML, age 0–18 years) were randomized to receive or not to receive G-CSF (5 μg/kg/day) after induction 1 (cytarabine, idarubicin, etoposide; AIE) and induction 2 (high-dose cytarabine and mitoxantrone; HAM). Patients who had more than 5% blasts in the bone marrow on day 15 were excluded from randomization. Patients with Down Syndrome were included in the clinical trial, but received only 60% of idarubicin in induction 1 and no second induction (HAM).Results: Between July 1998 and June 2003, 546 patients entered the clinical trial AML-BFM 98 (Down Syndome: n=45). Three-hundred-and-twenty-seven patients were eligible for randomization. Hundred-fifty eight of these patients were randomized to receive G-CSF, and 169 patients not to receive G-CSF. Both groups did not differ in their clinical characteristics. The G-CSF group had a significantly shorter duration of neutropenia (ANC<500/μl) both after induction 1 (16.3 vs 20.0 days, P=.019) and after induction 2 (12.4 vs 16.3 days, P=.0024). The time of thrombocytopenia (<20,000/μl) was similar in both groups (9.3, 9.4, 5.5, and 6.3 days, respectively). No impact of G-CSF was found on the incidence of grade II infections (fever without an identified pathogen) and grade III/IV infections (fever due to an identified pathogen/sepsis syndrome). Five-year event-free survival (5-EFS) did not significantly differ between the groups with and without G-CSF (65.6% + 3.9% vs 65.7% + 3.8%, P-logrank=.86). No significant difference was found when comparing the results of children randomized for G-CSF (intended-to treat) and of children who actually received the hematopoietic growth factor. Children with Down Syndrome showed also similar 5-EFS with and without G-CSF (P-logrank=.72).Conclusion: G-CSF significantly shortens the duration of neutropenia in children undergoing induction therapy for AML, but does not influence the incidence of infectious complications nor outcome (5-EFS probability). Whether there is a subgroup of children with AML who benefit from the administration of the hematopoetic growth factor has to be evaluated.

Cyclin A Is a Target Gene of Activated CREB Downstream of GM-CSF Signaling That Regulates Normal and Malignant Myelopoiesis.

Granulocyte-Macrophage Colony-Stimulating Factor stimulates the proliferation and survival of myeloid progenitor cells in vitro and in vivo. Primary AML blast cells express high affinity GM-CSF receptors. We previously reported that the basic leucine zipper transcription factor, CREB (cAMP Responsive Element Binding Protein), is phosphorylated at serine 133 in response to GM-CSF stimulation through a MEKK- and pp90RSK-dependent pathway. CREB protein and mRNA levels are elevated in the bone marrow of over 60% of AML patients at diagnosis. Furthermore, CREB is activated in primary AML blast cells that overexpress CREB. CREB overexpression in myeloid leukemia cell lines results in increased cell proliferation and survival, and decreased myeloid cell differentiation. Cells overexpressing CREB have increased numbers of cells in S phase in Brdu incorporation experiments in comparison to control cells. In addition, myeloid progenitor cells from CREB transgenic mice proliferate more and are growth-factor independent compared to cells from littermate controls. In colony assays, progenitor cells from CREB overexpressing mice are immortalized and acquire a blast-like phenotype. To understand the downstream pathways that regulate CREB-induced proliferation and survival in normal myeloid and leukemic cells, we examined the expression of two known CREB target genes, BCL-2 and Cyclin A, in CREB overexpressing myeloid leukemia cell lines. Cyclin A, but not BCL-2, was upregulated in cells overexpressing CREB compared to control cells. Cyclin A regulates G1 to S transition through activation of the cyclin-dependent kinase, CDK2. Myeloid progenitor cells from transgenic mice that overexpress CREB also overexpressed Cyclin A. We next investigated Cyclin A expression in primary blast cells from AML patients. Cyclin A protein levels were elevated in bone marrow from AML patients in which CREB was overexpressed but not in normal bone marrow. CREB has been shown to directly bind the CRE in the cyclin A promoter and increase transcription of cyclin A upon activation in response to growth factor stimulation. To study the possibility of cyclin A upregulation downstream of CREB, we examined transcriptional activation of Cyclin A in CREB overexpressing cells. A luciferase reporter construct containing the cyclin A promoter with the CRE was transfected into NFS60 cells that overexpressed CREB. A 4-fold increase in relative luciferase activity was observed in CREB overexpressing cells compared to control cells (p<0.05). Microarray analysis was performed with RNA from CREB overexpressing and non-overexpressing cells. In addition to cyclin A, several other target genes such as the pim-1 oncogene were found to be upregulated in CREB overexpressing cells. Our results suggest that aberrant cell cycle progression and increased proliferation from overexpression and activation of CREB downstream of GM-CSF signaling is a consequence of critical target genes that control myelopoiesis.

The IMPDH Inhibitor VX-944 Demonstrates In Vivo Efficacy in an Aggressive Leukemia Model.

VX-944 is a small molecule, orally bioavailable, specific inhibitor of inosine monophosphate dehydrogenase (IMPDH), an essential rate-limiting enzyme in de novo guanine nucleotide synthesis. We have previously shown that VX-944 has broad anti-cancer properties in vitro and that its potency is not affected by MDR pumps (Jain et al, Blood 2002, 100:572a; ibid 2003, 102: 622a). Here, we describe studies that demonstrate its oncolytic activity in blast cells derived from patients with acute myeloid leukemia (AML). We also report the efficacy of VX-944 in an aggressive murine model of myeloproliferative disease.The anti-leukemic activity of VX-944 was established both in colony formation assays and in viability assays using primary cells from AML patients. VX-944 inhibited the clonogenic proliferation of acute myeloid progenitor cells in a dose-dependent manner. The mean and median IC50 values were 218±92 and 199 nM respectively (n=8), indicating that no samples were resistant to VX-944. In another study, VX-944 reduced the viability of AML blasts from 4 additional patient samples, with IC50 values ranging from 20–200 nM. VX-944 was observed to be 3–40-fold more potent than mycophenolic acid (MPA, MMF), another IMPDH inhibitor. Importantly, genotyping of these samples revealed that VX-944 is active against cancer cells with wild type and point- or ITD-mutations in Flt3 that are implicated in approx 30% of AML patients. The anti-proliferative activity of VX-944 was at least additive, and in some patient samples, synergistic, when tested in combination with Daunorubicin, a standard chemotherapy drug for AML.To determine the therapeutic potential of VX-944 in vivo, VX-944 was tested in a leukemia model using Ba/F3 cells transduced with an activating human Flt-3 mutation injected into Balb/c mice. The anti-proliferative activity of VX-944 was first established in vitro in the cell lines used in the model. VX-944 inhibited the proliferation of the human MV-4-11 and murine Ba/F3-Flt3-ITD-dependent cell lines with IC50 values of 26 and 30 nM, respectively. In PK studies, a dose-dependent increase in Cmax and AUC values were observed in Balb/c mice. In the leukemia model, VX-944 was administered orally at 75 or 150 mg/kg BID. Doxorubicin (3 mg/kg, weekly), a standard AML therapy, and MLN518 (60 mg/kg BID), a selective Flt3 inhibitor, were administered as reference compounds. All treatments began 5 days following cell implantation. Both dose groups of VX-944 provided a significant increase in median survival time as compared with the vehicle treated group (p <0.001). Three of the 12 mice treated with 150 mg/kg VX-944 were still alive on Day 35 when the study was terminated. Further studies are underway to optimize the dosing regimen of VX-944 in xenograft models.In conclusion, we have demonstrated both potency and efficacy of VX-944 in in vitro and in vivo leukemia models. These preclinical results support further clinical development of VX-944 for the treatment of patients with leukemias and other rapidly proliferating hematological malignancies. VX-944 may provide significant therapeutic benefit when used alone, or in combination with approved chemotherapy agents such as Daunorubicin.

Constitutive Activation of Akt and mTOR by Flt3 Internal Tandem Duplications Mediates Myeloid Leukemogenesis and Can Be Inhibited by Rapamycin.

Up to 30% of patients with acute myeloid leukemia (AML) harbor internal tandem duplications (ITD) within the FLT3 gene, a tyrosine kinase receptor. The signalling pathways and downstream targets are only partly understood. We demonstrated that the presence of Flt3-ITD constitutively activated Akt (PKB), a key serine-threonine kinase within the PI3-kinase pathway. The constitutive activation of Akt induced phosphorylation and transcriptional inhibition of the transcription factor Foxo3a. Also, forced Foxo3a activity reversed Flt3-ITD-mediated growth properties. Using a novel Akt inhibitor, we showed that Flt3-ITD-mediated Foxo3a phosphorylation was dependent on Akt. Conditional Akt activation targeted to the cell membrane conferred cytokine-independent survival, proliferation and transformation of myeloid progenitor cells. Induction of Akt activity in vivo promoted the development of a myeloproliferative disease in a syngeneic mouse model. Importantly, phosphorylation of Akt was frequently found in primary myeloid blasts, supporting that this signalling pathway may be important in the development of AML. Furthermore, activation of Akt constitutively activated the downstream kinase mammalian target of rapamycin (mTOR). mTOR has several important biological functions, notably in nutrient sensing and protein translation. Treatment with rapamycin, a specific inhibitor of mTOR, inhibited mTOR kinase activity at 1nM in all examined cell systems. Rapamycin caused cell cycle arrest, apoptosis and completely inhibited transformation in FLT3-ITD-positive cells. These effects were most pronounced in the low nanomolar range, which are therapeutic concentrations of rapamycin-treated patients. The biological advantage in Akt-expressing cells was fully reversed by treatment with rapamycin. In all examined primary blasts from AML patients, mTOR was inhibited by rapamycin. In short term proliferation assays, twelve out of 18 (67%) examined showed marked sensitivity with an IC50 below 10nM rapamycin, while one third were found to be more resistant.Taken together, increased survival, proliferation and leukemic transformation by Flt3-ITD is mediated by Akt and mTOR, and can be inhibited by rapamycin. We conclude that rapamycin may be an attractive substance for the treatment of patients with AML.