Transcription factor C/EBPα is a master regulator of myelopoiesis and its inactivation is associated with acute myeloid leukemia. Deregulation of C/EBPα by microRNAs during granulopoiesis or acute ...myeloid leukemia development has not been studied. Here we show that oncogenic miR-182 is a strong regulator of C/EBPα. Moreover, we identify a regulatory loop between C/EBPα and miR-182. While C/EBPα blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBPα protein level and impairs granulopoiesis in vitro and in vivo. In addition, miR-182 expression is highly elevated particularly in acute myeloid leukemia patients with C-terminal CEBPA mutations, thereby depicting a mechanism by which C/EBPα blocks miR-182 expression. Furthermore, we present miR-182 expression as a prognostic marker in cytogenetically high-risk acute myeloid leukemia patients. Our data demonstrate the importance of a controlled balance between C/EBPα and miR-182 for the maintenance of healthy granulopoiesis.C/EBPα is a critical transcription factor involved in myelopoiesis and its inactivation is associated with acute myeloid leukemia (AML). Here the authors show a negative feedback loop between C/EBPα and miR-182 and identify this miRNA as a marker of high-risk AML.
The canonical Wnt signaling pathway is mediated by interaction of β-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and subsequent transcription ...activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of β-catenin that yielded contradictory results. Here, we used a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) that specifically abrogates β-catenin-TCF/LEF interaction. Disruption of the β-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced granulocyte colony-stimulating factor (G-CSF) receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of β-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candida albicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of β-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the β-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the β-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.
•Disruption of the β-catenin-TCF/LEF interaction compromises steady-state and emergency granulopoiesis.•TCF/LEF factors regulate G-CSF receptor expression by directly interacting with CSF3R promoter and enhancer regions.
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The canonical Wnt signaling pathway is mediated by interaction of β-catenin with the Tcf/Lef transcription factors and subsequent transcriptional activation of Wnt-target genes. This pathway acts as ...an essential regulator of differentiation and cell fate decisions in various tissues. In the hematopoietic system, the function of the pathway has been investigated mainly by genetic manipulation of β-catenin. However, this approach does not allow to discriminate between Tcf/Lef dependent or independent β-catenin activity.
In order to specifically identify the function of β-catenin-Tcf/Lef signaling in hematopoietic cells, we employed a transgenic mouse model expressing a dominant negative form of the human TCF4 transcription factor (dnTCF4). dnTCF4, a truncated protein lacking the β-catenin binding domain, abrogates activation of Wnt target genes, even when β-catenin is stabilized and translocated into the nucleus. In our model, expression of dnTCF4 is activated from the Rosa26 locus only in cells producing Cre recombinase (driven by Vav-iCre). Importantly, all components of Wnt signaling, including endogenous Tcf/Lef proteins and β-catenin, are intact in Cre-expressing cells.
We observed that dnTCF4 transgenic mice have reduced numbers of granulocytes together with accumulation of short-term hematopoietic stem cells (ST-HSC) and common myeloid progenitors (CMPs) in bone marrow. Accordingly, dnTCF4-expressing bone marrow consistently showed a block of granulocytic differentiation and retention of an immature phenotype in colony forming assays. This differentiation arrest and accumulation of immature cells was also observed when wild type cells were cultured in semi-solid medium in the presence of a cell-penetrating peptide that disrupts β-catenin-Tcf/Lef interaction. Together, these results indicate that disruption of the β-catenin/Tcf-Lef interaction, either by genetic manipulation or a drug based approach, alters steady-state hematopoiesis. To identify a mechanism through which β-catenin-Tcf/Lef signaling affects granulopoiesis, wild type and dnTCF4 expressing ST-HSCs were subjected to RNA sequencing. Several genes related to myeloid development were differentially expressed in dnTCF4 expressing cells, including downregulation of Csf3r, the gene encoding for the G-CSF receptor. Publicly available datasets from ChIP-seq experiments on human cell lines confirmed TCF4 enrichment in the distal promoter of the CSF3R gene, suggesting that CSF3R is directly regulated by canonical Wnt signaling. Using flow cytometry we verified reduced levels of G-CSF receptor on the cell surface of dnTCF4 progenitor cells, and attenuation of downstream Stat3 phosphorylation after G-CSF treatment. Finally, when grown in the presence of G-CSF, dnTCF4-expressing bone marrow cells showed impaired differentiation abilities and reduced granulocytic counts compared to wild type bone marrow cells. These results encouraged us to investigate the role of the β-catenin-Tcf/Lef signaling pathway during emergency granulopoiesis by challenging mice with lipopolysaccharide (LPS). Remarkably, dnTCF4 mice showed defects upon LPS stimulation, and completely failed to maintain and expand myeloid progenitor populations, a critical step during emergency granulopoiesis.
Altogether, we showed that β-catenin-Tcf/Lef signaling axis is crucial for proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Mechanistically, we demonstrated that the β-catenin-Tcf/Lef interaction controls expression of genes involved in myeloid differentiation, and directly enhances expression of the G-CSF receptor, a crucial molecule for proper development of granulocytes.
No relevant conflicts of interest to declare.
Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data ...that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.
The DLX homeodomain genes are part of the Drosophila distal-less family, originally identified in the forebrain of the developing mouse embryo. DLX1 gene is expressed also in the hematopoietic cells. ...Our previous data showed that patients with FLT3/ITD (internal tandem duplication) mutation representing about 35% of all acute myeloid leukemia (AML) cases have higher expression of DLX1 compared to non-FLT3/ITD AML patients. Further, FLT3 signaling was described to regulate DLX1 gene expression.
In the current study we found an association between DLX1 expression level and overall survival using GEP data from TCGA database within the group of FLT3/ITD-positive patients. Worse overall survival was linked to lower expression of DLX1 (p=0.003, n=46) even though generally this group of patients is characterized by their higher DLX1 level. This association was also observed when all AML patients were analyzed (p=0.01, n=197). Since the role of DLX1 in the leukemogenic process is not yet described we aimed to characterize the phenotype of leukemic cells with different expression levels of DLX1 gene.
We designed two functional shRNAs (pGhU6-sh1_DLX1 and pGhU6-sh2_DLX1, referred as sh1 and sh2) to downregulate DLX1 expression in MV4;11 (FLT3/ITD positive) leukemic cells, which present high endogenous level of DLX1, and a non-silenced control (pGhU6-NSC). In vitro studies showed that cells with silenced DLX1 were arrested in G0 phase of cell cycle (35%±4.8 (NSC) to 67.5%±2.2 (sh1; p <0.01) and 65.5%±2.5 (sh2; p <0.01); flow cytometry - Pyronin Y/ Hoechst 33342 staining) and had lower proliferative activity (trypan blue over the period of 10 passages). Moreover, cells with silenced DLX1 were less apoptotic (Annexin V/PI staining).
Next, we studied the impact of DLX1 downregulation on leukemic cell infiltration in vivo using sub-lethally irradiated NSG (NOD SCID gamma) mice. We injected 1x106 silenced or non-silenced MV4;11 cells via tail vein (n=20, 6 mice/group, 2 control mice). After two weeks we measured the absolute number of MV4;11 cells by flow cytometry (CD33+/GFP+/DAPI-) in bone marrow (BM) and spleen (SP) of recipient mice. Absolute cell counts of leukemic cells with silenced DLX1 were 1.26x106 (sh1; p=0.015) and 2.89x106 (sh2; p<0.0001) vs NSC cells 0.52x106 in BM and 1.59x106 (p=0.036) and 14.2x106 (p=0.025) vs 0.34x106 in SP. To ensure that the increased cell numbers were not the result of enhanced homing of DLX1 silenced cells, we performed homing experiments. Control and DLX1 silenced cells were transplanted and the number of cells in BM and SP was determined 16 hours after transplantation. We observed no differences between the studied groups, indicating that DLX1 silencing does not affect the homing ability of MV4;11 cells. Moreover, we analyzed cell cycle in leukemic cells isolated from recipient mice three weeks after transplantation. In agreement with our in vitro results, leukemic cells with silenced DLX1 had higher percentage of cells arrested in G0 phase (48.6%±6.3 (sh1; p <0.001) and 80.9%±9.3 (sh2; p <0.001) vs 26.4%±4.8 (NSC) in SP). Overall, mice with silenced DLX1 presented worse fitness and bigger splenomegaly.
Further, we investigated the signaling pathways which could lead to G0 arrest. Since DLX1 inhibits TGF-β pathway through direct interaction with SMAD4, a key downstream effector of TGF-β/BMP signaling, we studied the changes in target genes in our model cell lines. Expression of CUTL-1 (sh1 - 1.5-fold, sh2 - 2-fold change to NSC), PAI-1 (3.5-fold, 4.2-fold) and CDKN1C (1.3-fold, 2.3-fold) were significantly increased in DLX1 silenced cells. PAI-1 was shown to induce replicative senescence and CDKN1C is an inhibitor of cell cycle progression. While both targets disturb cell cycle and could be responsible for the phenotype we have observed, this hypothesis needs to be elucidated in future experiments.
Altogether, our data demonstrate that dysregulation of DLX1 gene in leukemic cells changes the cell phenotype. Lower level of DLX1 gene leads to arrest in G0 phase which in vitro slows down the proliferation whereas in vivo it allows the cells to persist in spleen and BM. We hypothesize that the DLX1 silenced cells become more resistant to external effects which could then be reflected as a reduced survival observed in patients with low levels of DLX1.
Supported by P304/12/2214.
No relevant conflicts of interest to declare.
Development of hematopoietic populations through the process of differentiation is critical for proper hematopoiesis. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is a ...master regulator of myeloid differentiation, and the identification of C/EBPα target genes is key to understand this process. Here we identified the Ecotropic Viral Integration Site 2B (EVI2B) gene as a direct target of C/EBPα. We showed that the product of the gene, the transmembrane glycoprotein EVI2B (CD361), is abundantly expressed on the surface of primary hematopoietic cells, the highest levels of expression being reached in mature granulocytes. Using shRNA-mediated downregulation of EVI2B in human and murine cell lines and in primary hematopoietic stem and progenitor cells, we demonstrated impaired myeloid lineage development and altered progenitor functions in EVI2B-silenced cells. We showed that the compromised progenitor functionality in Evi2b-depleted cells can be in part explained by deregulation of cell proliferation and apoptosis. In addition, we generated an Evi2b knockout murine model and demonstrated altered properties of hematopoietic progenitors, as well as impaired G-CSF dependent myeloid colony formation in the knockout cells. Remarkably, we found that EVI2B is significantly downregulated in human acute myeloid leukemia samples characterized by defects in CEBPA. Altogether, our data demonstrate that EVI2B is a downstream target of C/EBPα, which regulates myeloid differentiation and functionality of hematopoietic progenitors.
Natural killer (NK) cells participate in innate and adaptive immune responses, and upon activation rapidly produce cytokines, chemokines, and growth factors, including IFNγ, TNFα, TGFβ, GM-CSF, ...MIP1α, MIP1β, IL-10, and others, which can affect the function of other hematopoietic cells. Considering the recent evidences that hematopoietic stem cells (HSCs) respond to cytokine signaling, we hypothesized that NK cell-mediated cytokine production could mediate HSC function.
By the use of co-cultures of purified Ly5.1 murine NK cells and congenic Ly5.2 HSCs, we concluded that NK activity affects HSC frequency in vitro as well as hematopoietic reconstitution in vivo. Sorted NK cells (CD3- NK1.1+) and HSCs (Lin-, Sca1+, ckithi, CD48-, CD150+) were co-cultured in the presence or absence of IL2 over an OP9 stromal cells layer for 14 to 28 days. After 14 days, the addition of NK cells to HSC cultures resulted in an approximate 2-fold reduction of lineage negative cells (Lin-) recovered cells, as compared to control HSC cultures, as determined by flow cytometry analysis. Lin- counts were even lower in HSC+NK long-term cultures when compared to HSC only cultures. Ly5.1 HSCs and/or Ly5.2 NK cells were injected into sublethally irradiated Ly5.1/2 chimeric mice in a ratio of 105 NK to 103 HSCs per mouse. The addition of IL2-stimulated NK to injected HSCs reduced engraftment from 15.7% to 1.82% when the 16 weeks bone marrow (BM) chimerism was analyzed. In agreement, donor CD45.1 cells contribution to the LSK and HSC subpopulations was reduced in the HSC+NK transplanted mice. To test whether NK depletion from BM grafts would affect HSC function, we performed limiting dilution transplantation assays where whole BM from Ly5.2 mice was submitted to immunonagnetic NK1.1 or IgG depletion and injected into lethally irradiated Ly5.1 animals. Donor chimerism after 8 and 16 weeks of transplant showed that depleting NK cells improves the engraftment ability of HSC in a cell dose-dependent manner. When 25 x104 BM cells were injected, chimerism increased from 40 to more than 90% in NK depleted group. Of note, HSC frequency was 1 in 1595 in the control and 1 in 95 in the NK depleted group.
In order to understand the mechanisms by which NK cells could regulate HSCs, we took advantage of a CCAAT/enhancer-binding protein gamma (C/ebpg) knockout (KO) conditional mouse model generated in our laboratory, considering that C/ebpg had been previously shown to regulate NK cytotoxicity. Using similar culture conditions, HSCs and NK cells isolated from control (CT) or Cebpg KO mice were injected into congenic sublethally irradiated recipients. Results showed that Cebpg-deficient NK cells do not harm HSC engraftment as CT NK cells do. For instance, after 8 weeks, the addition of CT non-stimulated and IL-2-stimulated NK cells to normal transplanted HSCs reduced the engraftment from 40% to 20% and 10%, respectively. In contrast, chimerism was not different when HSCs only or HSCs + stimulated KO NK cells were transplanted. Gene expression and cytokine profiles of deficient and normal NK cells revealed the potential players of this HSC-NK regulation. Of these, interferon gamma (IFNg), was lower produced by the C/ebpg deficient NK cells. Therefore, besides controlling NK cytotoxicity, we showed here that C/ebpg also plays a role in the regulation of HSCs by NK-mediated cytokine production.
Next, we investigated whether depletion of NK cells from human BM samples would improve transplantation efficiency. NK cells were removed using CD56 antibody and transplanted into sublethally irradiated NSG mice. Sixteen weeks after transplantation, animals were sacrificed and the percentage of human CD45 cells in blood, BM, and spleen demonstrated that NK depletion from human BM favors engraftment.
Altogether, these findings provide new insights to the knowledge of HSC regulation by NK cells, which are present in BM transplantation (BMT) grafts. Although the alloreactive effect of NK cells against non-identical tumor cells from BMT recipients is well known, its cytokine-mediated effects over identical progenitor cells from the graft were not previously explored. We show that NK-secreted cytokines harm stem cell function, thus suggesting that depletion of NK cells from BM donor cells preparations can improve stem cell engraftment, particularly in the setting of alternative transplants with limiting cell numbers or non-myeloablative conditioning regimens.
No relevant conflicts of interest to declare.
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