Expansion Of Hematopoietic Progenitors Research Articles

Molecular synergy underlies the co-occurrence patterns and phenotype of NPM1-mutant acute myeloid leukemia

AbstractNPM1 mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with FLT3 internal tandem duplications (ITD) or, less commonly, NRAS or KRAS mutations. Co-occurrence of mutant NPM1 with FLT3-ITD carries a significantly worse prognosis than NPM1-RAS combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis show that compound Npm1cA/+;NrasG12D/+ or Npm1cA;Flt3ITD share a number of features: Hox gene overexpression, enhanced self-renewal, expansion of hematopoietic progenitors, and myeloid differentiation bias. However, Npm1cA;Flt3ITD mutants displayed significantly higher peripheral leukocyte counts, early depletion of common lymphoid progenitors, and a monocytic bias in comparison with the granulocytic bias in Npm1cA/+;NrasG12D/+ mutants. Underlying this was a striking molecular synergy manifested as a dramatically altered gene expression profile in Npm1cA;Flt3ITD, but not Npm1cA/+;NrasG12D/+, progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with Npm1cA/+;NrasG12D/+. During AML evolution, both models acquired additional copies of the mutant Flt3 or Nras alleles, but only Npm1cA/+;NrasG12D/+ mice showed acquisition of other human AML mutations, including IDH1 R132Q. We also find, using primary Cas9-expressing AMLs, that Hoxa genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with NPM1c/FLT3-ITD vs NPM1/NRAS-G12D-mutant AML and functionally confirm the role of HOXA genes in NPM1c-driven AML.

Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling

Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered invivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells invitro and invivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

Stroma-free, serum-free differentiation and expansion of hematopoietic progenitors to the T cell lineage

Stroma-free, serum-free differentiation and expansion of hematopoietic progenitors to the T cell lineage

Aryl Hydrocarbon Receptor (AhR) Pathway Directly Controls Hematopoietic Proliferation in Chronic Myeloid Leukemia (CML) and Its Inhibition Allows Massive Expansion of Leukemic Progenitors and Stem Cells

AhR is an ubiquitous basic helix-loop-helix protein, mediating cell responses to external ligands and involved in cell division, cell quiescence and inflammatory responses. After ligand binding, AhR, which is an intracytoplasmic receptor associated to HSP60, dissociates from it and translocates to nucleus where it binds to aryl hydrocarbon nuclear translator (ARNT), this dimeric complex activating transcription. It has been shown that AhR is involved in normal hematopoietic progenitor proliferation in human cells. In addition, AhR K/O mice exhibit a myeloproliferative syndrome-like disease, suggesting a role of AhR in hematopoietic stem cell (HSC) quiescence. The goal of this study was to study the potential role of AhR and its partners in CML progenitors and stem cells. We have first evaluated the expression of AhR in UT7 cell line and UT7-cells expressing BCR-ABL. AhR expression was highly reduced in UT7 cell expressing BCR-ABL as compared to control. AhR transcript was then quantified in primary peripheral blood CML cells at diagnosis (n=31 patients) and were found to be very significantly reduced as compared to normal controls (n=15) (p < 0.05). In 8 of these patients, AhR mRNA was quantified during TKI-induced major molecular response (MMR) and were found to increase towards levels found in controls. To determine if AhR expression is differentially expressed in HSC as compared to progenitors, we have analyzed AhR expression in CD34+ cells (n=11) as well as in highly purified CD34+ CD38- cell populations (n=3). Interestingly, AhR expression was reduced in leukemic CD34+ progenitors (n=11) but not in more primitive leukemic CD34+ CD38- cells (n=3) as compared to normal controls (n=15). These data suggested that AhR, known to be involved in HSC quiescence, could be amenable to pharmacological manipulation in CML cells. We first evaluated the potential effects of StemRegenin (SR1), which is an AhR antagonist on the growth of leukemic progenitors and stem cells. CD34+ CML cells treated with SR-1 had a reduced expression of AHHR, a repressor of AhR. In 7-day liquid cultures, the use of SR-1 at 0.75 µM induced a major expansion of leukemic cells by 60-fold as compared to conditions without SR-1 (p < 0.05). Similarly, SR-1 allowed expansion of leukemic CD34+ cells by 6-fold in 7 days, as compared to controls. CD34+ cells expanded in SR-1, were then used in CFC and LTC-IC assays to determine if SR-1 could allow maintenance and/or expansion of CML progenitors and stem cells. CML CD34+ cells cultured with SR-1 for 7 and 14 days exhibited a massive expansion of leukemic CFC (> 3 fold increase). Similarly, CD34+ cells expanded with SR-1 for 4 and 7 days followed by long-term culture initiating cells (LTC-IC) assays, revealed a massive expansion ( > 10-Fold) of LTC-IC-derived progenitors. We then used the 6-Formylindolo (3,2-b) carbazole (FICZ), a natural agonist of AhR, to determine its effects on leukemic progenitors and stem cells. The agonistic effect of FICZ was validated by the increase of the downstream target of AHR CYP1A1 transcripts after 7 days of culture. As expected, liquid culture of CML CD34+ cells induced a 2-fold decrease of CD34+ cells at day+7 in the presence of 100 nM of FICZ. Day-4 FICZ cultured CD34+ cells showed a significant reduction of leukemic CFC potential with a synergistic profile with Imatinib and Dasatinib ( p < 0.05). Similarly, in three patients, in LTC-IC assays started after 3-day FICZ-treated cells, 5-week LTC-IC-derived progenitors showed a significant growth inhibition with a synergistic profile with Imatinib. In conclusion, these findings demonstrate for the first time that down-regulation of AhR expression, a major cell cycle regulator, is involved in the MPD phenotype associated with CML. The further inhibition of AhR expression by its antagonist allowed massive expansion of hematopoietic progenitors and stem cells. In contrast, AhR agonists inhibit clonogenic and LTC-IC-derived stem cell growth and could be used in LSC targeting in CML. DisclosuresTurhan:Bristol Myers Squibb: Consultancy; Novartis: Research Funding.

Exploring the role of kit ligand at the onset of hematopoiesis

Hematopoietic stem cells (HSCs) are generated during mammalian development from hemogenic endothelium in the major arteries through a process called endothelial-to-hematopoietic transition. The extrinsic and intrinsic factors that orchestrate this process are poorly understood. Kit ligand (KitL; also known as Stem Cell Factor/SCF) is a cytokine that plays a pivotal role in the adult bone marrow HSC niche, and it is thought to act on several cell types during embryogenesis. It is known that the absence of KitL causes a significant reduction in the HSC pool and leads to death in utero with severe anaemia. However, it is currently unclear how and when the hematopoietic defect originates in embryos lacking KitL, and what is the precise role of this cytokine in the first steps of hematopoiesis. Using a novel KitL transgenic reporter line, we show that KitL expressing cells are found in the microenvironment of all hematopoietic sites of the E8.5-E10.5 mouse embryo. We set up to investigate in detail the hematopoietic phenotype in Steel mutants at early stages. We show for the first time that Sl/Sl embryos display smaller and less proliferative hematopoietic clusters in the dorsal aorta and in the yolk sac. Already at E10.5, homozygous Steel mutants show a reduction in Pre-HSC numbers, fetal liver cellularity and yolk sac erythropoiesis. At E11.5 these mutants display decreased progenitor numbers in all hematopoietic sites. These data suggest that, contrary to earlier reports, Kit signalling plays a role in early hematopoiesis. Moreover, our data identify a previously unrecognized role for KitL in the maturation and/or expansion of hematopoietic progenitors belonging to the second yolk sac wave, that could contribute to the anaemia observed later in development. Our ongoing studies aim at elucidating the mechanism by which KitL exerts its role in early hematopoiesis, and identifying other candidate cytokines with possible synergistic effects. Hematopoietic stem cells (HSCs) are generated during mammalian development from hemogenic endothelium in the major arteries through a process called endothelial-to-hematopoietic transition. The extrinsic and intrinsic factors that orchestrate this process are poorly understood. Kit ligand (KitL; also known as Stem Cell Factor/SCF) is a cytokine that plays a pivotal role in the adult bone marrow HSC niche, and it is thought to act on several cell types during embryogenesis. It is known that the absence of KitL causes a significant reduction in the HSC pool and leads to death in utero with severe anaemia. However, it is currently unclear how and when the hematopoietic defect originates in embryos lacking KitL, and what is the precise role of this cytokine in the first steps of hematopoiesis. Using a novel KitL transgenic reporter line, we show that KitL expressing cells are found in the microenvironment of all hematopoietic sites of the E8.5-E10.5 mouse embryo. We set up to investigate in detail the hematopoietic phenotype in Steel mutants at early stages. We show for the first time that Sl/Sl embryos display smaller and less proliferative hematopoietic clusters in the dorsal aorta and in the yolk sac. Already at E10.5, homozygous Steel mutants show a reduction in Pre-HSC numbers, fetal liver cellularity and yolk sac erythropoiesis. At E11.5 these mutants display decreased progenitor numbers in all hematopoietic sites. These data suggest that, contrary to earlier reports, Kit signalling plays a role in early hematopoiesis. Moreover, our data identify a previously unrecognized role for KitL in the maturation and/or expansion of hematopoietic progenitors belonging to the second yolk sac wave, that could contribute to the anaemia observed later in development. Our ongoing studies aim at elucidating the mechanism by which KitL exerts its role in early hematopoiesis, and identifying other candidate cytokines with possible synergistic effects.

TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis.

In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.

Disruption of the 5S RNP-Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2(C305F) knock-in mice that have a disrupted 5S RNP-Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2(C305F) reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP-Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP-Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

Early B-cell Factor 1 Regulates the Expansion of B-cell Progenitors in a Dose-dependent Manner

Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2Rα-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1(+/-) pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development.

MYBL2 is a sub-haploinsufficient tumor suppressor gene in myeloid malignancy.

A common deleted region (CDR) in both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) affects the long arm of chromosome 20 and has been predicted to harbor a tumor suppressor gene. Here we show that MYBL2, a gene within the 20q CDR, is expressed at sharply reduced levels in CD34+ cells from most MDS cases (65%; n = 26), whether or not they harbor 20q abnormalities. In a murine competitive reconstitution model, Mybl2 knockdown by RNAi to 20-30% of normal levels in multipotent hematopoietic progenitors resulted in clonal dominance of these 'sub-haploinsufficient' cells, which was reflected in all blood cell lineages. By 6 months post-transplantation, the reconstituted mice had developed a clonal myeloproliferative/myelodysplastic disorder originating from the cells with aberrantly reduced Mybl2 expression. We conclude that downregulation of MYBL2 activity below levels predicted by classical haploinsufficiency underlies the clonal expansion of hematopoietic progenitors in a large fraction of human myeloid malignancies. DOI:http://dx.doi.org/10.7554/eLife.00825.001.

Fetal hepatic progenitors support long-term expansion of hematopoietic stem cells

We have developed a coculture system that establishes DLK(+) fetal hepatic progenitors as the authentic supportive cells for expansion of hematopoietic stem (HSCs) and progenitor cells. In 1-week cultures supplemented with serum and supportive cytokines, both cocultured DLK(+) fetal hepatic progenitors and their conditioned medium supported rapid expansion of hematopoietic progenitors and a small increase in HSC numbers. In 2- and 3-week cultures DLK(+) cells, but not their conditioned medium, continuously and significantly (>20-fold) expanded both hematopoietic stem and progenitor cells. Physical contact between HSCs and DLK(+) cells was crucial to maintaining this long-term expansion. Similar HSC expansion (approximately sevenfold) was achieved in cocultures using a serum-free, low cytokine- containing medium. In contrast, DLK(-) cells are incapable of expanding hematopoietic cells, demonstrating that hepatic progenitors are the principle supportive cells for HSC expansion in the fetal liver.

Insulin-like growth factor binding protein-2 and neurotrophin 3 synergize together to promote the expansion of hematopoietic cells ex vivo

Co-culture of Umbilical Cord Blood (UCB) CD34+ cells with irradiated Mesenchymal Stem Cells (MSCs) without contact increase the expansion of Hematopoietic Progenitor Cells (HPC). Neurotrophin-3 (NT-3) and insulin-like growth factor binding protein-2 (IGFBP-2) are two factors whose expressions were significantly elevated in conditioned media derived from irradiated MSCs. To determine whether these factors are partly responsible for the growth promoting potential of MSCs, we investigated their impact on the growth and differentiation of UCB-CD34+ cells. Addition of either factor alone had little impact on cell growth, however both factors synergized together to increase the expansion of total nucleated cells, erythroids, megakaryocytes (Mk) and CD34+ cells. However, in contrast to MSCs they failed to significantly improve the expansion of hematopoietic progenitors. Consistent with the impact of these factors on hematopoietic cells, both synergized to activate ERK1/2 and AKT in primary human UCB cells. In conclusion, the study demonstrates for the first time that a neurotrophin factor can synergize with IGFBP-2 to promote hematopoietic cell expansion.

Infection-Induced Hematopoiesis: A Zebrafish Perspective

In response to infection, the bone marrow adjusts production of leukocyte cell types to fight off disease. In this issue of Cell Stem Cell, Hall etal. (2012) use the zebrafish model to show that nitric oxide (NO) production drives expansion of hematopoietic progenitors to produce more granulocytes.

The Triterpenoid CDDO-Me Promotes Hematopoietic Progenitor Expansion and Myelopoiesis in Mice

The synthetic triterpenoid CDDO-Me has been shown to directly inhibit the growth of myeloid leukemias and lends itself to a wide array of therapeutic indications, including inflammatory conditions, because of its inhibition of NF-κB. We have previously demonstrated protection from acute graft-versus-host disease after CDDO-Me administration in an allogeneic bone marrow transplantation model. In the current study, we observed that CDDO-Me promoted myelopoiesis in both naive and transplanted mice. This effect was dose dependent, as high doses of CDDO-Me inhibited myeloid growth in vitro. All lineages (granulocyte macrophage colony-forming unit, BFU-E) were promoted by CDDO-Me. We then compared the effects with granulocyte colony-stimulating factor, a known inducer of myeloid expansion and mobilization from the bone marrow. Whereas both drugs induced terminal myeloid expansion in the spleen, peripheral blood, and bone marrow, granulocyte colony-stimulating factor only induced granulocyte macrophage colony-forming unit precursors in the spleen, while CDDO-Me increased these precursors in the spleen and bone marrow. After sublethal total-body irradiation, mice pretreated with CDDO-Me further displayed an accelerated recovery of myeloid progenitors and total nucleated cells in the spleen. A similar expansion of myeloid and myeloid progenitors was noted with CDDO-Me treatment after syngeneic bone marrow transplantation. Combined, these data suggest that CDDO-Me may be of use posttransplantation to accelerate myeloid recovery in addition to the prevention of graft-versus-host disease.

Effects of extracellular matrix proteins on the growth of haematopoietic progenitor cells

Umbilical cord blood (UCB) transplantation and haematological recovery are currently limited by the amount of haematopoietic progenitor cells (HPCs) present in each unit. HPCs and haematopoietic stem cells (HSCs) normally interact with cells and extracellular matrix (ECM) proteins present within the endosteal and vascular niches. Hence, we investigated whether coating of culture surfaces with ECM proteins normally present in the marrow microenvironment could benefit the ex vivo expansion of HPCs. Towards this, collagen types I and IV (COL I and IV), laminin (LN) and fibronectin (FN) were tested individually or as component of two ECM-mix complexes. Individually, ECM proteins had both common and unique properties on the growth and differentiation of UCB CD34+ cells; some ECM proteins favoured the differentiation of some lineages over that of others (e.g. FN for erythroids), some the expansion of HPCs (e.g. LN and megakaryocyte (MK) progenitor) while others had less effects. Next, two ECM-mix complexes were tested; the first one contained all four ECM proteins (4ECMp), while the second ‘basement membrane-like structure’ was without COL I (3ECMp). Removal of COL I led to strong reductions in cell growth and HPCs expansion. Interestingly, the 4ECMp-mix complex reproducibly increased CD34+ (1.3-fold) and CD41+ (1.2-fold) cell expansions at day 6 (P < 0.05) versus control, and induced greater myeloid progenitor expansion (P < 0.05) than 3ECMp. In conclusion, these results suggest that optimization of BM ECM protein complexes could provide a better environment for the ex vivo expansion of haematopoietic progenitors than individual ECM protein.

Nanoengineering life: from cell to tissue

Nanoengineering, which brings several traditional disciplines including physics, engineering, biology and medicine together, is an emerging tool for manipulating major constituents of living organisms such as cells and tissues. Generally, the scopes of nanoengineering techniques include

Irradiated mesenchymal stem cells improve the ex vivo expansion of hematopoietic progenitors by partly mimicking the bone marrow endosteal environment

Mesenchymal Stem Cells (MSCs) regulate the growth and differentiation of Hematopoietic Progenitor cells (HPCs) through the release of soluble factors or through their differentiation into osteoblasts. We recently demonstrated that expansion of megakaryocyte (MK) progenitors ex vivo had reached a plateau when CD34 + cells were grown with two optimized cytokine cocktails developed for the growth of MK. Hence, we sought to determine whether co-culture of CD34 + cells with Bone Marrow (BM) MSCs could further increase the expansion of myeloid and MK progenitors. First, we tested the impact of cell–cell contact and pre-irradiation treatment of the MSCs to identify the condition that best supports HPC expansion. This screen revealed that HPC expansions were generally greater in the non-contact conditions, and that pre-irradiation of the MSCs appeared to be of added benefits. Improved expansion of both myeloid and MK progenitors in co-culture with irradiated MSCs without contact was subsequently confirmed. Next, cytokine array profiling was carried out to investigate why irradiation promoted progenitor expansion. This revealed that the levels of as many as 33 factors were potentially altered. ELISA confirmed the significant up regulation of NT-3 and IGFBP-2. Since, these factors are known to be released by and important for osteogenic and endothelial cells, we investigated and confirmed that irradiation of MSCs induced their rapid differentiation into osteogenic-like cells, but not into endothelial-like cells. Supporting this finding, expansions of myeloid and MK progenitors were increased when CD34 + cells were co-culture with MSCs-derived osteoblasts. Altogether, these results indicate that the improved expansion of HPCs obtained with irradiated MSCs is due in part to their differentiation into osteoblast-like cells, thereby recreating an endosteal-like environment that provides improved support for HPCs expansion.

Identification of a High-Affinity Ligand That Exhibits Complete Aryl Hydrocarbon Receptor Antagonism

The biological functions of the aryl hydrocarbon receptor (AHR) can be delineated into dioxin response element (DRE)-dependent or -independent activities. Ligands exhibiting either full or partial agonist activity, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin and α-naphthoflavone, have been demonstrated to potentiate both DRE-dependent and -independent AHR function. In contrast, the recently identified selective AHR modulators (SAhRMs), e.g., 1-allyl-3-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-1H-indazole (SGA360), bias AHR toward DRE-independent functionality while displaying antagonism with regard to ligand-induced DRE-dependent transcription. Recent studies have expanded the physiological role of AHR to include modulation of hematopoietic progenitor expansion and immunoregulation. It remains to be established whether such physiological roles are mediated through DRE-dependent or -independent pathways. Here, we present evidence for a third class of AHR ligand, "pure" or complete antagonists with the capacity to suppress both DRE-dependent and -independent AHR functions, which may facilitate dissection of physiological AHR function with regard to DRE or non-DRE-mediated signaling. Competitive ligand binding assays together with in silico modeling identify N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine (GNF351) as a high-affinity AHR ligand. DRE-dependent reporter assays, in conjunction with quantitative polymerase chain reaction analysis of AHR targets, reveal GNF351 as a potent AHR antagonist that demonstrates efficacy in the nanomolar range. Furthermore, unlike many currently used AHR antagonists, e.g., α-naphthoflavone, GNF351 is devoid of partial agonist potential. It is noteworthy that in a model of AHR-mediated DRE-independent function, i.e., suppression of cytokine-induced acute-phase gene expression, GNF351 has the capacity to antagonize agonist and SAhRM-mediated suppression of SAA1. Such data indicate that GNF351 is a pure antagonist with the capacity to inhibit both DRE-dependent and -independent activity.

Individual and synergistic cytokine effects controlling the expansion of cord blood CD34+ cells and megakaryocyte progenitors in culture

Expansion of hematopoietic progenitors ex vivo is currently investigated as a means of reducing cytopenia following stem cell transplantation. The principal objective of this study was to develop a new cytokine cocktail that would maximize the expansion of megakaryocyte (Mk) progenitors that could be used to reduce periods of thrombocytopenia. We measured the individual and synergistic effects of six cytokines [stem cell factor (SCF), FLT-3 ligand (FL), interleukin (IL)-3, IL-6, IL-9 and IL-11] commonly used to expand cord blood (CB) CD34(+) cells on the expansion of CB Mk progenitors and major myeloid populations by factorial design. These results revealed an elaborate array of cytokine individual effects complemented by a large number of synergistic and antagonistic interaction effects. Notably, strong interactions with SCF were observed with most cytokines and its concentration level was the most influential factor for the expansion and differentiation kinetics of CB CD34(+) cells. A response surface methodology was then applied to optimize the concentrations of the selected cytokines. The newly developed cocktail composed of SCF, thrombopoietin (TPO) and FL increased the expansion of Mk progenitors and maintained efficient expansion of clonogenic progenitors and CD34(+) cells. CB cells expanded with the new cocktail were shown to provide good short- and long-term human platelet recovery and lymphomyeloid reconstitution in NOD/SCID mice. Collectively, these results define a complex cytokine network that regulates the growth and differentiation of immature and committed hematopoietic cells in culture, and confirm that cytokine interactions have major influences on the fate of hematopoietic cells.

The expansion of T-cells and hematopoietic progenitors as a result of overexpression of the lymphoblastic leukemia gene, Lyl1 can support leukemia formation

This study investigates the function of the lymphoblastic leukemia gene, Lyl1 in the hematopoietic system and its oncogenic potential in the development of leukemia. Overexpression of Lyl1 in mouse bone marrow cells caused T-cell increase in the peripheral blood and expansion of the hematopoietic progenitors in culture and in the bone marrow. These observations were the result of increased proliferation and suppressed apoptosis of the progenitor cells caused by the Lyl1-overexpression. Our studies present substantial evidence supporting the secondary, pro-leukemic effect of Lyl1 in early hematopoietic progenitors with the potential to cause expansion of malignant cells with a stem/early progenitor-like phenotype.

Accelerated DNA replication in E2F1- and E2F2-deficient macrophages leads to induction of the DNA damage response and p21CIP1-dependent senescence

E2F1-3 proteins appear to have distinct roles in progenitor cells and in differentiating cells undergoing cell cycle exit. However, the function of these proteins in paradigms of terminal differentiation that involve continued cell division has not been examined. Using compound E2F1/E2F2-deficient mice, we have examined the effects of E2F1 and E2F2 loss on the differentiation and simultaneous proliferation of bone-marrow-derived cells toward the macrophage lineage. We show that E2F1/E2F2 deficiency results in accelerated DNA replication and cellular division during the initial cell division cycles of bone-marrow-derived cells, arguing that E2F1/E2F2 are required to restrain proliferation of pro-monocyte progenitors during their differentiation into macrophages, without promoting their cell cycle exit. Accelerated proliferation is accompanied by early expression of DNA replication and cell cycle regulators. Remarkably, rapid proliferation of E2F1/E2F2 compound mutant cultures is temporally followed by induction of a DNA damage response and the implementation of a p21(CIP1)-dependent senescence. We further show that differentiating E2F1/E2F2-knockout macrophages do not trigger a DNA damage response pathway in the absence of DNA replication. These findings underscore the relevance of E2F1 and E2F2 as suppressors of hematopoietic progenitor expansion. Our data indicate that their absence in differentiating macrophages initiates a senescence program that results from enforcement of a DNA damage response triggered by DNA hyper-replication.