Specific MicroRNA Deregulated in Myleoproliferative Neoplasm Are Regulated by JAK2V617F and May Contribute to Aberrant Hematopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 965-965
Author(s):  
Xiaoqing Lin ◽  
Monica Buzzai ◽  
Martin Carroll ◽  
Elizabeth Hexner ◽  
Fabricio F Costa ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Mirna Expression ◽  
Colony Formation ◽  
Lentiviral Vector ◽  
Myeloproliferative Neoplasms ◽  
Rna Extraction ◽  
Differentially Expressed ◽  
Loss Of Function ◽  
Cd34 Cells

Abstract Abstract 965 The myeloproliferative neoplasms (MPN), PV, ET and IMF, harbor the same gain-of-function mutation JAK2V617F at a high frequency (∼100%, 70% and 50% respectively). Accumulating evidence suggest that JAK2V617F may not be the initiating event in MPN, and other genetic anomalies play an important role in MPN pathogenesis. We hypothesized that miRNA deregulation contributes to the development of MPN. To test this idea, miRNA expression in CD34+ cells isolated from 8 patient samples (4 PV with JAK2V617F, 3 ET with wild-type JAK2 and 1 IMF with unknown JAK2 status) and 4 healthy controls was determined using a Taqman Low Density Array (TLDA) representing 667 known miRNAs. PV (JAK2V617F) and ET cases (JAK2WT) showed 14 and 78 differentially expressed miRNAs, respectively, when compared to controls. 6 miRNAs were commonly deregulated in PV and ET, while the majority were unique to each disease type. When all MPN patients were grouped and compared to controls, 28 miRNAs were significantly deregulated (p<0.05). These miRNAs differ from those previously reported to be differentially expressed in the peripheral blood of PV patients. Among these 28, mir-214 was down-regulated and mir-410, mir-22* and mir-505* were up-regulated most consistently. Several miRNAs, including mir-135b, mir-542-5p, mir-149, mir-133b and mir-134 were undetectable in normal CD34+ cells and activated in MPN patients. We further hypothesized that some miRNAs are regulated through the action of the mutant JAK2V617F kinase. To test this, miRNA levels were assessed by TaqMan array in HEL and UKE-1 cells (harboring JAK2V617F) treated with 2 μM JAK inhibitor I (Calbiochem) for 20h before RNA extraction. In parallel, miRNA expression as determined by TLDA in TF-1 cells rendered cytokine independent by stable expression of JAK2V617F was compared to that of control TF-1 cells, both cultured overnight in the absence of cytokines. A total of 24 miRNAs were significantly deregulated (>2 fold) in at least two cell line systems. To test which deregulated miRNAs in MPN patients were JAK2 responsive, JAK2 activity was manipulated in HEL and TF-1 cells as described above, and the expression of miRNAs was determined by individual Taqman miRNA assays. mir-1, mir-200a, mir-9, mir-133b, mir-22* and mir-155 were responsive to manipulation of JAK2 activity. miR-155 expression was repressed 50% with the inhibition of JAK2 in HEL cells and stimulated almost 2 fold with the overexpression of JAK2V617F in TF-1 cells. By contrast, mir-214 (downregulated in MPN) and mir-134 (upregulated in MPN) were not responsive to manipulation of JAK2V617F activity in either the gain or loss-of-function systems. To further confirm the ability of JAK2V617F to regulate specific miRNAs, lineage negative (lin-) murine marrow progenitor cells were transduced with JAK2V617F or empty vector, allowed to form colonies for 7 days and miRNA levels in the colonies were determined. Again miR-200a, miR-9 and miR-22* and miR-155 were responsive to JAKV617F overexpression, while mir-134 was not. Transduction of lineage negative murine marrow progenitor cells with a lentiviral vector harboring mir-155 yielded a 30% increase in a myeloid colony formation in vitro. The effect is consistent with the reported ability of mir-155 to induce myeloproliferation in mice. Transduction of marrow progenitors with miR-133b, which is activated in MPN patients, responsive to JAK2V617F manipulation and not previously reported to have a role in hematopoiesis, led to an increase in both erythroid and myeloid colony formation. Taken together we conclude that at least 4 miRNAs are deregulated in CD34+ cells of MPN patients as a result of aberrant JAK2 activity. Two of these tested so far have a role in hematopoiesis. Part of the action of JAK2V617F in myeloproliferation may be mediated by specific miRNA, thus representing alternative therapeutic targets in MPN. Disclosures: Carroll: Sanofi Aventis Corp: Research Funding; Cephalon Oncoloy: Consultancy.

Combined Inhibition of JAK2 and mTOR Signaling Results in Enhanced Efficacy in in-Vitro and Preclinical Mouse Models of JAK2V617F-Driven Myeloproliferative Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 708-708
Author(s):  
Alessandro M. Vannucchi ◽  
Niccolò Bartalucci ◽  
Costanza Bogani ◽  
Serena Martinelli ◽  
Lorenzo Tozzi ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Mouse Models ◽  
Drug Combination ◽  
Colony Formation ◽  
Myeloproliferative Neoplasms ◽  
Solid Cancer ◽  
Synergistic Activity ◽  
Inhibition Of Proliferation ◽  
Cd34 Cells

Abstract Abstract 708 Background and Aims. The JAK1/JAK2 inhibitor Ruxolitinib (ruxo) produced rapid and sustained responses in splenomegaly and symptomatic improvement in patients (pts) with myelofibrosis (MF), supporting the central role of dysregulated JAK2 signaling in myeloproliferative neoplasms (MPN). Splenomegaly and constitutional symptoms improved also after treatment with Everolimus (RAD001), a rapamycin-derivative inhibitor of the serine/threonine kinase mTOR, in a phase I/II study, pointing to mTOR pathway as a novel target for MPN therapy. BEZ235 is a dual phosphatidylinositol-3-kinase (PI3K) and mTOR inhibitor currently in clinical trials in solid cancer. Aim of the study was to investigate the efficacy of BEZ235, alone and in combination with ruxo, against MPN cells in vitro and in preclinical mouse models. Methods. We used mouse (Ba/F3 and Ba/F3-EPOR expressing wild-type (WT) or V617F(VF) mutated JAK2) and human (VF HEL and SET2 or WT K562) cell lines and primary MPN CD34+ cells from pts with MF or polycythemia vera (PV); cell proliferation, colony formation, apoptosis, cell cycle and protein phosphorylation status were evaluated. Effects of drug combination were analyzed according to Chou and Talalay to calculate the combination index (CI); a CI <1.0 indicates synergistic activity. For in vivo studies, two mouse models were used. (1) SCID mice receiving iv Ba/F3-EPOR VF-luciferase (luc) cells (gift of T. Radimerski) were randomized on d6 to treatment groups (either drugs alone and in combination or vehicle (VE)) based on baseline luminescence. Bioluminescence measurement was done at week intervals until death. (2) A C57Bl6/J JAK2 V617F Knock-in mouse model was generated by the flex switch strategy with insertion of the reversed JAK2V617F exon 13 sequence; mating with Vav-Cre transgenic mice activates the VF allele producing a MPN phenotype in progenies from VF heterozygous expression. Mice were treated for 5d, then blood, spleen and bone marrow cells were analyzed. Results. We found that BEZ235 inhibited proliferation of Ba/F3 VF and Ba/F3-EPOR VF cells at concentrations significantly lower than the wt counterparts (IC50 was 64±10nM vs 10,000±500nM and 87±50nM vs 676±200nM; P<0.01); similar preferential inhibition was observed in HEL and SET2 cells compared to K562 cells (IC50, 387±90nM and 334±40nM vs 5,000±1,000nM; P<0.01). BEZ235 dose-dependently increased the percentage of cells in G0/G1 and induced apoptosis. Western blot analysis showed marked reduction of the mTOR target p4EBP1 as well as appreciable downregulation of pSTAT5 and pSTAT3 at 6 to 24h of treatment. BEZ235 impaired the proliferation of CD34+ cells from MF pts with an IC50= 43±20nM vs 780±150nM in healthy donors (P<0.01), and reduced colony formation of MF and PV hematopoietic progenitors at doses statistically lower (2 to 15-fold) than normal cells. The growth of EPO-independent colonies (EEC) in PV pts was potently inhibited (IC50=20±10nM). Co-treatment of BEZ235+ruxo resulted in synergistic inhibition of proliferation in SET2 (median CI=0.37) and BaF3-EPOR VF (CI=0.77) cells and increased apoptosis rate in SET2 (CI=0.25); drug combination was highly effective also in the EEC assay (CI=0.17). In the Ba/F3VF luc model, the median bioluminescence index (No. of pixel) on day 7 of treatment was 4.7×106 in VE animals, 7.2×106 in ruxo (P=ns), 1.1×106 in BEZ235 (P=0.04) and 4.6×105 in animals receiving the BEZ+ruxo combination (P<0.01). The median survival in mice treated with the combination of BEZ+ruxo was 30d, significantly longer than either ruxo (18.5d; P<0.01) or BEZ (24d; P<0.04) alone (15.0d in VE animals). In JAK2V617F KI mice treated for 5d, we found that drug combination was significantly more effective in reducing enlarged spleen (median spleen index (spleen weight/body weightx100): 38, 35, 27 and 7 for VE, BEZ, Ruxo and BEZ+Ruxo) and reticulocyte count (median No. per HPF: 48, 50, 44, and 3 for VE, BEZ, Ruxo and BEZ+Ruxo) than either drugs alone. The phosho levels of STAT5 and 4EBP1 in the spleen were significantly reduced in mice receiving BEZ+Ruxo as compared to single treatment. Conclusions. Combined inhibition of PI3K/mTOR and JAK2 signaling resulted in enhanced activity in preclinical models of MPN compared with either treatment alone, providing a rationale for the development of combination clinical trials. Disclosures: Vannucchi: Novartis: Membership on an entity's Board of Directors or advisory committees.

The PI3K Specific Inhibitor BKM120 Results Effective and Synergizes With Ruxolitinib In Preclinical Models Of Myeloproliferative Neoplasms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1599-1599 ◽  
Author(s):  
Niccolò Bartalucci ◽  
Costanza Bogani ◽  
Serena Martinelli ◽  
Carmela Mannarelli ◽  
Jean-Luc Villeval ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Colony Formation ◽  
Myeloproliferative Neoplasms ◽  
Janus Kinase ◽  
Preclinical Models ◽  
Mtor Inhibition ◽  
Jak2 Inhibitor ◽  
Cd34 Cells

Abstract Background and Aims A gain-of-function mutation in Janus kinase 2 (JAK2V617F) is at the basis of the majority of chronic myeloproliferative neoplasms (MPN). The dual JAK1/JAK2 inhibitor ruxolitinib (ruxo) determined rapid and sustained responses in splenomegaly and symptomatic improvement in patients with myelofibrosis (MF), supporting the central role of dysregulated JAK2 signaling. Enhanced activation of other downstream pathways including the PI3K/mTOR pathway has been documented as well. We previously reported (Bogani et al, PlosOne 2013;8:54828) that targeting mTOR by the allosteric inhibitor RAD001 resulted in inhibition of JAK2VF mutated cells and produced clinical benefits in a phase I/II trial (Guglielmelli et al, Blood 2011;118:2069). In this study we evaluated the effects of BKM120, a specific PI3K inhibitor, alone and in combination with ruxolitinib, in in-vitro and in-vivo MPN models. Methods To evaluate cell proliferation, colony formation, apoptosis, cell cycle and protein phosphorylation status we used mouse BaF3 and BaF3-EPOR cells expressing wild type (WT) or VF mutated JAK2, the human VF-mutated HEL and SET2 cell lines, and primary MPN CD34+ cells from patients with MF or polycythemia vera (PV). Effect of drug combination was analyzed according to Chou and Talalay calculating the combination index (CI); a CI <1 indicates synergistic activity. For in vivo studies we used two mouse models: (1) SCID mice receiving iv BaF3-EPOR VF-luciferase (luc) cells (gift of T. Radimerski) were randomized on day 6 to different treatment groups based on baseline luminescence. (2) C57Bl6/J JAK2 VF Knock-in mice were generated by insertion of the reversed JAK2V617F exon 13 sequence; mating with Vav-Cre transgenic mice activates the VF allele producing a MPN phenotype in progenies with VF heterozygous expression (Hasan et al, Blood 2013;Epub). Mice were treated for 15 days, then blood, spleen and bone marrow cells were analyzed. Results We found that BKM120 preferential inhibited BAF3 VF and BaF3-EpoR VF cells (IC50: 364±200nM and 1100±207nM, respectively) compared to their respective WT counterpart (5300±800nM and 3122±1000nM: p<.05). HEL and SET2 cells resulted also sensitive to BKM120 (2000±500nM and 1000±300nM). Interestingly we found that BKM120 significantly increased G2/M phase and decreased S phase of cell cycle (p<.01) and induced apoptosis (IC50, SET2=10µM, BaF3-EPOR VF=1.8 µM). Western blot analysis showed marked reduction of phospho-mTOR and its target phospho-4EBP1 as well as downregulation of phospho-STAT5 at 6 and 24h of treatment. BKM120 impaired colony formation from MF and PV CD34+ cells at doses 2 to 8-fold lower than healthy controls (p<.01). BKM120 strongly inhibited EEC colony growth from PV pts (IC50, 9±4nM). Co-treatment of BKM120+ruxo resulted in synergistic inhibition of proliferation of SET2 (median CI=0.45) and BaF3-EPOR VF (median CI=0.8) cells. Triple combinations including BKM120/ruxo plus either RAD001 (Torc1 inhibitor) or PP242 (Torc1/2 inhibitor) resulted highly synergistic (median CI=0.27 and 0.52) to indicate the importance of complete mTOR inhibition. BKM120 at 45mpk and 60mpk increased mean lifespan of BaF3 VF luc mouse model from 21d in control mice to 27.2d and 28d in BMK120 treated mice. In KI mice, co-treatment with 60mpk BKM120 + 60mpk ruxo resulted in improvement of splenomegaly (median spleen weight: 1.4, 0.82, 0.8 and 0.6 g respectively for controls, 60mpk BKM120, 60mpk ruxo and 60mpk BKM120+60mpk ruxo) and reduction of leukocytosis and reticulocyte count. The level of phosho-STAT5 and -4EBP1 in the spleen was significantly reduced in mice receiving BKM120+ruxo as compared to single drug treatment. We finally analyzed the effects of BKM120+/-ruxo on the in-vitro clonogenic growth of BM cells from VF and WT KI mice mixed in a 1:1 ratio. The proportion of VF-positive colonies resulted reduced in a dose dependent manner by 19%, 33% and 44% (p<.03) compared to controls with 50nM, 100nM and 300nM BKM120 respectively. A 25% and 39% of VF-positive colonies reduction was achieved with 50nM and 100nM ruxolitinib. The combined treatment with 100nM BKM120 + 50nM ruxo resulted in a 50% decrease of the number of mutated colonies (p<.02). Conclusions Inhibition of PI3K by BKM120 alone and combined with JAK2 inhibitor ruxolitinib resulted in enhanced activity in preclinical models of MPN, providing a rationale for the ongoing combination clinical trial. Disclosures: Vannucchi: Novartis: Membership on an entity’s Board of Directors or advisory committees.

Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4107-4107
Author(s):  
Susan Hilgendorf ◽  
Hendrik Folkerts ◽  
Jan Jacob Schuringa ◽  
Edo Vellenga
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Erythroid Differentiation ◽  
Lentiviral Vectors ◽  
Apoptotic Response ◽  
Double Positive ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract In recent clinical studies, it has been shown that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). The majority of ASXL1 mutations leads to truncation of the protein and thereby to loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in the hematopoietic system in vitro mimics the changes observed in MDS-patients. We downregulated ASXL1 in CD34+ cord blood (CB) cells using lentiviral vectors containing several independent shRNAs and obtained a 40-50% reduction of ASXL1 expression. Colony Forming Cell (CFC) assays revealed that erythroid colony formation was significantly impaired (p<0.01) and, to some extent, granulocytic and macrophage colony formation as well (p<0.09, p<0.05 respectively). In myeloid suspension culture assays, we observed a modest reduction in expansion (two-fold at week 1) upon ASXL1 knockdown under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p<0.02). The increase in cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. In addition, we tested whether HSCs were affected by ASXL1 loss. Long-term culture-initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, transduced cells were cultured on MS5 bone marrow stromal cells with or without additional cytokines. shASXL1 CB CD34+ cells cultured on MS5 showed a modest two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. ASXL1 has been proposed to be an epigenetic modifier by recruiting/stabilizing the polycomb repressive complex 2 (PRC2). Active PRC2 can lead to trimethylation of H3K27 and silencing of certain loci. It has been proposed that perturbed ASXL1 activity may disturb PRC2 function, leading to reduced H3K27me3 and increased gene expression. Using an erythroid leukemic cell line, we downregulated ASXL1 and as a positive control EZH2, one of the core subunits of PRC2. We then performed ChIP and did PCR for several loci. Upon knockdown of ASXL1, we did not observe changes in H3K27me3 on any of he investigated loci. However, upon knockdown of EZH2 we observed more than 50% loss of the H3k27m3 mark for many of the loci. This implies that our observed phenotypes may not be conveyed via the PRC2 complex but maybe via an alternative pathway. Preliminary data revealed an increase in H2AK119ub, suggesting that the BAP1-ASXL1 complex may be involved. In patients, mutations in ASXL1 are frequently accompanied by a mutation of TP53. Possibly, this additional mutation is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, shASXL1/shTP53 transduced cells grown in erythroid liquid conditions revealed a decrease in apoptosis compared to the ASXL1 single mutation and an outgrowth of these double positive cells. Nevertheless, no transformation occurred in vitro. We therefore injected shASXL/TP53 transduced CB CD34+ in a humanized scaffold model in mice to determine whether transformation can occur in vivo. In conclusion, our data indicate that mutations in ASXL1 trigger an apoptotic response in CB CD34+ cells with a delay in differentiation, which leads to reduced stem and progenitor output in vitro without affecting H3K27me3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Loss-of-Function and Gain-of-Function Consequences of GATA2 Disease Mutations

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2519-2519
Author(s):  
Koichi Ricardo Katsumura ◽  
Peng Liu ◽  
Charu Mehta ◽  
Kyle J Hewitt ◽  
Alexandra Soukup ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Molecular Mechanisms ◽  
Differentially Expressed ◽  
Homozygous Mutation ◽  
Loss Of Function ◽  
Granulocytic Differentiation ◽  
Gain Of Function ◽  
Hematopoietic Stem ◽  
Disease Mutations

The master regulator of hematopoiesis GATA2 controls generation and function of hematopoietic stem and progenitor cells, and heterozygous GATA2 mutations create a predisposition to develop immunodeficiency, myelodysplasia, and acute myeloid leukemia (Spinner et al. Blood, 2014; Dickinson et al. Blood, 2014; Churpek and Bresnick J. Clin. Invest. 2019). Although mechanisms that trigger the transition of a non-pathogenic GATA2 mutation into overt pathology are enigmatic, a paradigm has arisen in which GATA2 mutations are considered to be loss-of-function. We developed a genetic rescue assay to quantify the function of wild type GATA2 and GATA2 disease mutants when expressed at near-physiological levels in primary progenitor cells and demonstrated that GATA2 disease mutations abrogate certain biological and molecular activities, while enabling others (Katsumura et al., 2018, PNAS). We isolated lineage-negative (Lin-) or Lin-Kit+ cells from fetal liver of mice with a homozygous mutation of the Gata2 -77 enhancer, which downregulates Gata2 expression by ~80%. The mutant progenitor cells are largely defective in erythroid, megakaryocytic and granulocytic differentiation and exhibit a predominant monocytic differentiation fate (Johnson et al., 2015, Science Adv.). We compared GATA2 and GATA2 disease mutant activities in the rescue system using a colony formation assay. GATA2, R307W mutant (in N-finger) and T354M mutant (in DNA-binding C-finger) rescued myeloid colony formation and promoted granulocyte proliferation. Surprisingly, R307W and T354M induced more CFU-GM than GATA2. GATA2 and R307W, but not T354M, rescued BFU-E. These data indicated that GATA2 disease mutations were not strictly inhibitory, and in certain contexts, mutant activities exceeded that of GATA2. To extend these results, we subjected -77+/+ or -77-/- Lin- cells to a short-term ex vivo liquid culture, expressed GATA2, R307W, or T354M and used RNA-seq to elucidate progenitor cell transcriptomes. While -77+/+ Lin- cells generate erythroid and myeloid cells, -77-/- Lin- cells are competent for myeloid, but not erythroid, differentiation. Comparison of -77+/+ and -77-/- cell transcriptomes revealed 3064 differentially expressed genes (>2-fold). 1824 genes were >2-fold higher in -77+/+ cells, and 1240 genes were >2-fold higher in -77-/- cells. GATA2 expression in -77-/- cells activated 834 genes >2-fold and repressed 503 genes >2-fold. 60-65% of these genes overlapped with genes differentially expressed between -77+/+ cells and -77-/- cells. R307W expression activated 661 genes >2-fold and repressed 523 genes >2-fold. T354M expression activated 468 genes >2-fold and repressed 575 genes >2-fold. The genes regulated by mutants included GATA2-regulated genes and certain genes that were not GATA2-regulated. Multiple genes were hypersensitive to the mutants, relative to GATA2, and the mutants ectopically regulated certain genes. However, R307W and T354M did not universally regulate an identical gene cohort. For example, both R307W and T354M activated Ncam1, Nrg4, and Mpo more strongly than GATA2. R307W, but not T354M, activated Ear2 and Ces1d more strongly than GATA2. By contrast, T354M, but not R307W, activated Ctsg, Epx, and Rab38 more strongly than GATA2. Both R307W and T354M repressed macrophage genes similarly to GATA2, but they lacked the capacity to activate mast cell genes, differing from GATA2. To elucidate molecular mechanisms underlying GATA2 mutant activities, we leveraged our prior discovery that p38 or ERK kinases induce multi-site GATA2 phosphorylation (Katsumura et al. Blood. 2017). We tested whether these kinases mediate the ectopic transcriptional regulatory activity of GATA2 disease mutants. p38 inhibition attenuated aberrant regulation of Ear2 and Ces1d by R307W (p < 0.05), and mutation of S192 to S192A decreased R307W-induced CFU-GM formation by 49% (p < 0.05). In aggregate, these results indicate that GATA2 disease mutants exert context-dependent activities to regulate transcription and differentiation, activities can be signal-dependent and certain activities are distinct from GATA2. It is attractive to consider the pathogenic consequences of GATA2 disease mutant gain-of-function activities, and an important implication is GATA2 mutation-associated hematologic diseases might not solely reflect haploinsufficiency. Disclosures No relevant conflicts of interest to declare.

scholarly journals Growth-Supporting Activities of Fibronectin on Hematopoietic Stem/Progenitor Cells In Vitro and In Vivo: Structural Requirement for Fibronectin Activities of CS1 and Cell-Binding Domains

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3263-3272 ◽  
Author(s):  
Takafumi Yokota ◽  
Kenji Oritani ◽  
Hideki Mitsui ◽  
Keisuke Aoyama ◽  
Jun Ishikawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Cell Binding ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Colony Forming Units

Abstract Fibronectin (FN) is supposed to play important roles in various aspects of hematopoiesis through binding to very late antigen 4 (VLA4) and VLA5. However, effects of FN on hematopoietic stem cells are largely unknown. In an effort to determine if FN had a growth-supporting activity on hematopoietic stem cells, human CD34+/VLA4bright/VLA5dullhematopoietic stem cells and a murine stem cell factor (SCF)-dependent multipotent cell line, EML-C1, were treated with or without FN in a serum and growth-factor–deprived medium, and then subjected to clonogenic assay in the presence of hematopoietic growth factors. The pretreatment of the CD34+ cells with FN gave rise to significantly increased numbers of granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst colony-forming units, and mixed erythroid-myeloid colony-forming units. In addition, the numbers of blast colony-forming units and CFU-GM that developed after culture of EML-C1 cells with SCF and the combination of SCF and interleukin-3, respectively, were augmented by the pretreatment with FN. The augmented colony formation by FN was completely abrogated by the addition of CS1 fragment, but not of GRGDSP peptide, suggesting an essential role of FN-VLA4 interaction in the FN effects. Furthermore, the effects of various FN fragments consisting of RGDS-containing cell-binding domain (CBD), heparin-binding domain (HBD), and/or CS1 portion were tested on clonogenic growth of CD34+ cells. Increased colony formation was induced by CBD-CS1 and CBD-HBD-CS1 fragments, but not with other fragments lacking CBD or CS1 domains, suggesting that both CS1 and CBD of FN were required for the augmentation of clonogenic growth of hematopoietic stem/progenitor cells in vitro. In addition to the in vitro effects, the in vivo administration of CBD-CS1 fragment into mice was found to increase the numbers of hematopoietic progenitor cells in bone marrow and spleen in a dose-dependent manner. Thus, FN may function on hematopoietic stem/progenitor cells as a growth-supporting factor in vitro and in vivo.

Selective FLT3 Inhibition Uncovers the Role of FL and FLT3 in Normal CD34+ Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1327-1327 ◽  
Author(s):  
Katja C. Weisel ◽  
Sedat Yildirim ◽  
Xingkui Xue ◽  
Lothar Kanz ◽  
Robert Mohle
Keyword(s):  
Cell Culture ◽  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Progenitor Cell ◽  
Cell Function ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Several cytokines in the bone marrow act through stem cell or progenitor cell specific receptors to regulate the capacity of immature hematopoietic cells to potentiate downstream multilineage expansion. Ligand-mediated activation of the FMS-like tyrosine kinase-3 (FLT3) receptor is important for normal proliferation of primitive hematopoietic cells. FLT3 expression in the bone marrow is restricted to CD34+ cells and a subset of dendritic precursors. FLT3 as a member of the type III RTK subfamily is closely related to c-KIT, c-FMS and PDGFRα/β. Activating mutations of FLT3 play an important role in leukemogenesis and their presence is associated with poor prognosis in acute myeloid leukemia (AML). Targeting the mutation by inhibiting the tyrosine kinase activity of FLT3 is a promising therapeutic option in treatment of AML patients. CEP-701, a potent FLT3 tyrosine kinase inhibitor is known to be cytotoxic to cell lines and primary AML cells harbouring FLT3 mutations and shows biological and clinical activity in patients with relapsed or refractory AML. In this study, we investigated the effect of FLT3 kinase inhibition in normal hematopoietic stem and progenitor cells in-vitro. Peripheral blood mobilized CD34+ cells were cultured in serum-free conditions supplemented with various cytokine combinations. Flt-3 inhibition was performed with addition of CEP-701 in different concentrations. FLT3 inhibition resulted in dose-dependent growth inhibition of CD34+ cells in in-vitro culture. This effect was independent of cytokine combinations chosen. A tetrazolium-based MTT assay was used to quantify 50% growth inhibition after 48h of exposure to CEP-701. The mean IC50 (± SD) for CEP-701 on normal CD34+ progenitor cells was 56 ± 19 nM. Immunophenotypic analysis of cell cultures showed a markedly decrease of CD34+ expressing cells under FLT3 inhibiting conditions. Surprisingly, inhibition of cell growth was even present, when cell culture was performed in absence of FL. In addition, the effect of FLT3 inhibition could be restored by addition of a neutralizing FL-antibody to cell culture even in conditions without FL substitution. Expression of FLT3 and FL under cytokine-supplemented culture conditions and FLT-3 inhibition were monitored by Western Blot analysis. In order to evaluate the effect of FLT-3 inhibition on the progenitor cell function, colony formation was analysed. Addition of CEP-701 into cell culture resulted in decrease of absolute colony production, however, relative colony formation per input cells was not significantly decreased showing that progenitor cell function of surviving cells was not markedly affected. These data demonstrate a significant inhibitory effect of CEP-701 on normal CD34+ cells. We could demonstrate for the first time, that FL might act as an autocrine mediator activating FLT3 in CD34+ cells as CD34+ cells similarily express both, FLT3 and FL. Finally, these findings might also explain hematotoxicity of various tyrosine kinase inhibitors, e.g. imatinib, which also show unspecific targeting of FLT3.

scholarly journals Growth-Supporting Activities of Fibronectin on Hematopoietic Stem/Progenitor Cells In Vitro and In Vivo: Structural Requirement for Fibronectin Activities of CS1 and Cell-Binding Domains

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3263-3272 ◽  
Author(s):  
Takafumi Yokota ◽  
Kenji Oritani ◽  
Hideki Mitsui ◽  
Keisuke Aoyama ◽  
Jun Ishikawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Cell Binding ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Colony Forming Units

Fibronectin (FN) is supposed to play important roles in various aspects of hematopoiesis through binding to very late antigen 4 (VLA4) and VLA5. However, effects of FN on hematopoietic stem cells are largely unknown. In an effort to determine if FN had a growth-supporting activity on hematopoietic stem cells, human CD34+/VLA4bright/VLA5dullhematopoietic stem cells and a murine stem cell factor (SCF)-dependent multipotent cell line, EML-C1, were treated with or without FN in a serum and growth-factor–deprived medium, and then subjected to clonogenic assay in the presence of hematopoietic growth factors. The pretreatment of the CD34+ cells with FN gave rise to significantly increased numbers of granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst colony-forming units, and mixed erythroid-myeloid colony-forming units. In addition, the numbers of blast colony-forming units and CFU-GM that developed after culture of EML-C1 cells with SCF and the combination of SCF and interleukin-3, respectively, were augmented by the pretreatment with FN. The augmented colony formation by FN was completely abrogated by the addition of CS1 fragment, but not of GRGDSP peptide, suggesting an essential role of FN-VLA4 interaction in the FN effects. Furthermore, the effects of various FN fragments consisting of RGDS-containing cell-binding domain (CBD), heparin-binding domain (HBD), and/or CS1 portion were tested on clonogenic growth of CD34+ cells. Increased colony formation was induced by CBD-CS1 and CBD-HBD-CS1 fragments, but not with other fragments lacking CBD or CS1 domains, suggesting that both CS1 and CBD of FN were required for the augmentation of clonogenic growth of hematopoietic stem/progenitor cells in vitro. In addition to the in vitro effects, the in vivo administration of CBD-CS1 fragment into mice was found to increase the numbers of hematopoietic progenitor cells in bone marrow and spleen in a dose-dependent manner. Thus, FN may function on hematopoietic stem/progenitor cells as a growth-supporting factor in vitro and in vivo.

scholarly journals Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2812-2821 ◽  
Author(s):  
Fabiana Perna ◽  
Nadia Gurvich ◽  
Ruben Hoya-Arias ◽  
Omar Abdel-Wahab ◽  
Ross L. Levine ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Myeloproliferative Neoplasms ◽  
Erythroid Differentiation ◽  
Polycomb Group ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Cd34 Cells

Abstract L3MBTL1, the human homolog of the Drosophila L(3)MBT polycomb group tumor suppressor gene, is located on chromosome 20q12, within the common deleted region identified in patients with 20q deletion-associated polycythemia vera, myelodysplastic syndrome, and acute myeloid leukemia. L3MBTL1 is expressed within hematopoietic CD34+ cells; thus, it may contribute to the pathogenesis of these disorders. To define its role in hematopoiesis, we knocked down L3MBTL1 expression in primary hematopoietic stem/progenitor (ie, CD34+) cells isolated from human cord blood (using short hairpin RNAs) and observed an enhanced commitment to and acceleration of erythroid differentiation. Consistent with this effect, overexpression of L3MBTL1 in primary hematopoietic CD34+ cells as well as in 20q− cell lines restricted erythroid differentiation. Furthermore, L3MBTL1 levels decrease during hemin-induced erythroid differentiation or erythropoietin exposure, suggesting a specific role for L3MBTL1 down-regulation in enforcing cell fate decisions toward the erythroid lineage. Indeed, L3MBTL1 knockdown enhanced the sensitivity of hematopoietic stem/progenitor cells to erythropoietin (Epo), with increased Epo-induced phosphorylation of STAT5, AKT, and MAPK as well as detectable phosphorylation in the absence of Epo. Our data suggest that haploinsufficiency of L3MBTL1 contributes to some (20q−) myeloproliferative neoplasms, especially polycythemia vera, by promoting erythroid differentiation.

scholarly journals The influence in vivo of murine colony-stimulating factor-1 on myeloid progenitor cells in mice recovering from sublethal dosages of cyclophosphamide

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 913-918 ◽  
Author(s):  
HE Broxmeyer ◽  
DE Williams ◽  
S Cooper ◽  
A Waheed ◽  
RK Shadduck
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Colony Stimulating Factor ◽  
Myeloid Progenitor ◽  
Accessory Cells ◽  
Myeloid Progenitor Cells ◽  
In Vivo Administration ◽  
Stimulating Factor

Abstract Pure murine colony-stimulating factor-1 (CSF-1) was assessed for its effects in vivo in mice pretreated seven days earlier with a sublethal dosage of cyclophosphamide. The multipotential (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells in these mice were in a slowly cycling or noncycling state. Intravenous administration of 20,000 units of CSF-1 to these mice stimulated the hematopoietic progenitors into a rapidly cycling state in the marrow and spleen within three hours. Significant increases in absolute numbers of marrow and spleen CFU-GM and spleen BFU-E and CFU-GEMM were also detected. No endotoxin was detected in the CSF-1 preparation by Limulus lysate assay, and treatment of CSF-1 at 100 degrees C for 20 to 30 minutes completely inactivated the in vitro and in vivo stimulating effects. The effects of CSF-1 were not mimicked by the in vivo administration of 0.1 to 10 ng Escherichia coli lipopolysaccharide. These results suggest that the effects of CSF-1 in vivo were not due to contaminating endotoxin or to a nonspecific protein effect. CSF-1 did not enhance colony formation by BFU-E or stimulate colony formation by CFU-GEMM in vitro, thus suggesting that at least some of the effects of CSF-1 noted in vivo are probably indirect and mediated by accessory cells.

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