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Mutations in acute myeloid leukemia (AML)-associated oncogenes often arise in hematopoietic stem cells (HSCs) and promote acquisition of leukemia stem cell (LSC) phenotypes. However, as LSCs often share features of lineage-restricted progenitors, the relative contribution of differentiation status to LSC transformation is unclear. Using murine MLL-AF9 and MOZ-TIF2 AML models, we show that myeloid differentiation to granulocyte macrophage progenitors (GMPs) is critical for LSC generation. Disrupting GMP formation by deleting the lineage-restricted transcription factor C/EBPa blocked normal granulocyte formation and prevented initiation of AML. However, restoring myeloid differentiation in C/EBPa mutants with inflammatory cytokines reestablished AML transformation capacity. Genomic analyses of GMPs, including gene expression and H3K79me2 profiling in conjunction with ATAC-seq, revealed a permissive genomic environment for activation of a minimal transcription program shared by GMPs and LSCs. Together, these findings show that myeloid differentiation is a prerequisite for LSC formation and AML development, providing insights for therapeutic development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory, and development of novel therapeutics is required. CCAAT/enhancer binding protein α (C/EBPα) is one of the crucial transcription factors that induce granulocytic differentiation, and its activity is perturbed in human myeloid leukemias. As its reexpression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBPα expression and/or activity would lead to myeloid differentiation of leukemic cells. Using a cell-based high-throughput screening, we identified 2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone as a potent inducer of C/EBPα and myeloid differentiation. Leukemia cell lines and primary blast cells isolated from human patients with AML treated with ICCB280 demonstrated evidence of morphological and functional differentiation, as well as massive apoptosis. We performed conformational analyses of the high-throughput screening hit compounds to postulate the spatial requirements for high potency. Our results warrant a development of novel differentiation therapies and significantly affect care of patients with AML with unfavorable prognosis in the near future., (© 2015 Society for Laboratory Automation and Screening.)

Dendritic cells (DCs) are master regulators of the immune system, but molecular regulation of early DC differentiation has been poorly understood. Here, we report that the transcription factor C/EBPα coordinates the development of progenitor cells required for production of multiple categories of DCs. C/EBPα was needed for differentiation from stem/progenitor cells to common DC progenitors (CDPs), but not for transition of CDP to mature DCs. C/EBPα deletion in mature DCs did not affect their numbers or function, suggesting that this transcription factor is not needed for maintenance of DCs in lymphoid tissues. ChIP-seq and microarrays were used to identify candidate genes regulated by C/EBPα and required for DC formation. Genes previously shown to be critical for DC formation were bound by C/EBPα, and their expression was decreased in the earliest hematopoietic compartments in the absence of C/EBPα. These data indicate that C/EBPα is important for the earliest stages of steady-state DC differentiation.

In blood, the transcription factor C/EBPa is essential for myeloid differentiation and has been implicated in regulating self-renewal of fetal liver haematopoietic stem cells (HSCs). However, its function in adult HSCs has remained unknown. Here, using an inducible knockout model we found that C/EBPa-deficient adult HSCs underwent a pronounced increase in number with enhanced proliferation, characteristics resembling fetal liver HSCs. Consistently, transcription profiling of C/EBPa-deficient HSCs revealed a gene expression program similar to fetal liver HSCs. Moreover, we observed that age-specific Cebpa expression correlated with its inhibitory effect on the HSC cell cycle. Mechanistically we identified N-Myc as a downstream target of C/EBPa, and loss of C/EBPa resulted in de-repression of N-Myc. Our data establish C/EBPa as a central determinant in the switch from fetal to adult HSCs.

To provide a lifelong supply of blood cells, hematopoietic stem cells (HSCs) need to carefully balance both self-renewing cell divisions and quiescence. Although several regulators that control this mechanism have been identified, we demonstrate that the transcription factor PU.1 acts upstream of these regulators. So far, attempts to uncover PU.1's role in HSC biology have failed because of the technical limitations of complete loss-of-function models. With the use of hypomorphic mice with decreased PU.1 levels specifically in phenotypic HSCs, we found reduced HSC long-term repopulation potential that could be rescued completely by restoring PU.1 levels. PU.1 prevented excessive HSC division and exhaustion by controlling the transcription of multiple cell-cycle regulators. Levels of PU.1 were sustained through autoregulatory PU.1 binding to an upstream enhancer that formed an active looped chromosome architecture in HSCs. These results establish that PU.1 mediates chromosome looping and functions as a master regulator of HSC proliferation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

C/EBPs are a family of transcription factors that regulate growth control and differentiation of various tissues. We found that C/EBPγ is highly upregulated in a subset of acute myeloid leukemia (AML) samples characterized by C/EBPα hypermethylation/silencing. Similarly, C/EBPγ was upregulated in murine hematopoietic stem/progenitor cells lacking C/EBPα, as C/EBPα mediates C/EBPγ suppression. Studies in myeloid cells demonstrated that CEBPG overexpression blocked neutrophilic differentiation. Further, downregulation of Cebpg in murine Cebpa-deficient stem/progenitor cells or in human CEBPA-silenced AML samples restored granulocytic differentiation. In addition, treatment of these leukemias with demethylating agents restored the C/EBPα-C/EBPγ balance and upregulated the expression of myeloid differentiation markers. Our results indicate that C/EBPγ mediates the myeloid differentiation arrest induced by C/EBPα deficiency and that targeting the C/EBPα-C/EBPγ axis rescues neutrophilic differentiation in this unique subset of AMLs.

The transcription factor C/EBPα is a critical mediator of myeloid differentiation and is often functionally impaired in acute myeloid leukemia. Recent studies have suggested that oncogenic FLT3 activity disrupts wild-type C/EBPα function via phosphorylation on serine 21 (S21). Despite the apparent role of pS21 as a negative regulator of C/EBPα transcription activity, the mechanism by which phosphorylation tips the balance between transcriptionally competent and inhibited forms remains unresolved. In the present study, we used immuno-affinity purification combined with quantitative mass spectrometry to delineate the proteins associated with C/EBPα on chromatin. We identified DEK, a protein with genetic links to leukemia, as a member of the C/EBPα complexes, and demonstrate that this association is disrupted by S21 phosphorylation. We confirmed that DEK is recruited specifically to chromatin with C/EBPα to enhance GCSFR3 promoter activation. In addition, we demonstrated that genetic depletion of DEK reduces the ability of C/EBPα to drive the expression of granulocytic target genes in vitro and disrupts G-CSF-mediated granulocytic differentiation of fresh human BM-derived CD34(+) cells. Our data suggest that C/EBPα and DEK coordinately activate myeloid gene expression and that S21 phosphorylation on wild-type C/EBPα mediates protein interactions that regulate the differentiation capacity of hematopoietic progenitors.

In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL(+) Lin(-)Sca-1(+)c-kit(+) (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin(-)Sca-1(-)c-kit(+)), nor mature granulocytes (CD11b(+)Gr-1(+)), nor potential stem cell niche cells (CD45(-)Ter119(-)) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL(+) LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity.

2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation and apoptosis of tumor cells in vitro and in vivo. Here we assessed the effects of CDDO on CCAAT enhancer-binding protein alpha (CEBPA), a transcription factor critical for granulocytic differentiation. In HL60 acute myeloid leukemia (AML) cells, CDDO (0.01 to 2 muM) induces apoptosis in a dose-dependent manner. Conversely, subapoptotic doses of CDDO promote phagocytic activity and granulocytic-monocytic differentiation of HL60 cells through increased de novo synthesis of p42 CEBPA protein. CEBPA translational up-regulation is required for CDDO-induced granulocytic differentiation and depends on the integrity of the CEBPA upstream open reading frame (uORF). Moreover, CDDO increases the ratio of transcriptionally active p42 and the inactive p30 CEBPA isoform, which, in turn, leads to transcriptional activation of CEBPA-regulated genes (eg, GSCFR) and is associated with dephosphorylation of eIF2alpha and phosphorylation of eIF4E. In concordance with these results, CDDO induces a CEBPA ratio change and differentiation of primary blasts from patients with acute myeloid leukemia (AML). Because AML is characterized by arrested differentiation, our data suggest the inclusion of CDDO in the therapy of AML characterized by dysfunctional CEBPA expression.

Monocyte differentiation involves the participation of lineage-restricted transcription factors, although the mechanisms by which this process occurs are incompletely defined. Within the hematopoietic system, members of the Kruppel-like family of factors (KLFs) play essential roles in erythrocyte and T lymphocyte development. Here we show that KLF4/GKLF is expressed in a monocyte-restricted and stage-specific pattern during myelopoiesis and functions to promote monocyte differentiation. Overexpression of KLF4 in HL-60 cells confers the characteristics of mature monocytes. Conversely, KLF4 knockdown blocked phorbol ester-induced monocyte differentiation. Forced expression of KLF4 in primary common myeloid progenitors (CMPs) or hematopoietic stem cells (HSCs) induced exclusive monocyte differentiation in clonogenic assays, whereas KLF4 deficiency inhibited monocyte but increased granulocyte differentiation. Mechanistic studies demonstrate that KLF4 is a target gene of PU.1. Consistently, KLF4 can rescue PU.1-/- fetal liver cells along the monocytic lineage and can activate the monocytic-specific CD14 promoter. Thus, KLF4 is a critical regulator in the transcriptional network controlling monocyte differentiation.

In recent years, great progress has been made in elucidating the progenitor-cell hierarchy of the myeloid lineage. Transcription factors have been shown to be key determinants in the orchestration of myeloid identity and differentiation fates. Most transcription factors show cell-lineage-restricted and stage-restricted expression patterns, indicating the requirement for tight regulation of their activities. Moreover, if dysregulated or mutated, these transcription factors cause the differentiation block observed in many myeloid leukaemias. Consequently, therapies designed to restore defective transcription factor functions are an attractive option in the treatment of myeloid and other human cancers.

Tightly regulated expression of the transcription factor PU.1 is crucial for normal hematopoiesis. PU.1 knockdown mice develop acute myeloid leukemia (AML), and PU.1 mutations have been observed in some populations of patients with AML. Here we found that conditional expression of promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA), the protein encoded by the t(15;17) translocation found in acute promyelocytic leukemia (APL), suppressed PU.1 expression, while treatment of APL cell lines and primary cells with all-trans retinoic acid (ATRA) restored PU.1 expression and induced neutrophil differentiation. ATRA-induced activation was mediated by a region in the PU.1 promoter to which CEBPB and OCT-1 binding were induced. Finally, conditional expression of PU.1 in human APL cells was sufficient to trigger neutrophil differentiation, whereas reduction of PU.1 by small interfering RNA (siRNA) blocked ATRA-induced neutrophil differentiation. This is the first report to show that PU.1 is suppressed in acute promyelocytic leukemia, and that ATRA restores PU.1 expression in cells harboring t(15;17).

Basophils and mast cells, which are selectively endowed with the high-affinity IgE receptor and mediate a range of adaptive and innate immune responses, have an unknown developmental relationship. Here, by evaluating the expression of the beta7 integrin, a molecule that is required for selective homing of mast cell progenitors (MCPs) to the periphery, we identified bipotent progenitors that are capable of differentiating into either cell type in the mouse spleen. These basophil/mast cell progenitors (BMCPs) gave rise to basophils and mast cells at the single-cell level and reconstituted both mucosal and connective tissue mast cells. We also identified the basophil progenitor (BaP) and the MCP in the bone marrow and the gastrointestinal mucosa, respectively. We further show that the granulocyte-related transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) plays a primary role in the fate decision of BMCPs, being expressed in BaPs but not in MCPs. Thus, circulating basophils and tissue mast cells share a common developmental stage at which their fate decision might be controlled principally by C/EBPalpha.

The PU.1 transcription factor is a key regulator of hematopoietic development, but its role at each hematopoietic stage remains unclear. In particular, the expression of PU.1 in hematopoietic stem cells (HSCs) could simply represent "priming" of genes related to downstream myelolymphoid lineages. By using a conditional PU.1 knock-out model, we here show that HSCs express PU.1, and its constitutive expression is necessary for maintenance of the HSC pool in the bone marrow. Bone marrow HSCs disrupted with PU.1 in situ could not maintain hematopoiesis and were outcompeted by normal HSCs. PU.1-deficient HSCs also failed to generate the earliest myeloid and lymphoid progenitors. PU.1 disruption in granulocyte/monocyte-committed progenitors blocked their maturation but not proliferation, resulting in myeloblast colony formation. PU.1 disruption in common lymphoid progenitors, however, did not prevent their B-cell maturation. In vivo disruption of PU.1 in mature B cells by the CD19-Cre locus did not affect B-cell maturation, and PU.1-deficient mature B cells displayed normal proliferation in response to mitogenic signals including the cross-linking of surface immunoglobulin M (IgM). Thus, PU.1 plays indispensable and distinct roles in hematopoietic development through supporting HSC self-renewal as well as commitment and maturation of myeloid and lymphoid lineages.

The transcription factor C/EBP alpha is required for granulopoiesis and frequently disrupted in human acute myeloid leukemia (AML). Here, we show disruption of C/EBP alpha blocks the transition from the common myeloid to the granulocyte/monocyte progenitor but is not required beyond this stage for terminal granulocyte maturation. C/EBP alpha-deficient hematopoietic stem cells (HSCs) have increased expression of Bmi-1 and enhanced competitive repopulating activity. Bone marrow in adult C/EBP alpha-deficient mice was filled with myeloblasts, similar to human AML, supporting the notion that disruption of C/EBP alpha cooperates with other events in the development of leukemia. Therefore, C/EBP alpha is not only essential for granulocyte development but, in addition, is a regulator of hematopoietic stem cell activity.

Although much is understood about the ways in which transcription factors regulate various differentiation systems, and one of the hallmarks of many human cancers is a lack of cellular differentiation, relatively few reports have linked these two processes. Recent studies of acute myeloid leukaemia (AML), however, have indicated how disruption of transcription-factor function can disrupt normal cellular differentiation and lead to cancer. This model involves lineage-specific transcription factors, which are involved in normal haematopoietic differentiation. These factors are often targeted in AML--either by direct mutation or by interference from translocation proteins. Uncovering these underlying pathways will improve the diagnosis and treatment of AML, and provide a working model for other types of human cancer, including solid tumours.

The transcription factor C/EBPalpha is crucial for the differentiation of granulocytes. Conditional expression of C/EBPalpha triggers neutrophilic differentiation, and C/EBPalpha can block 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation of bipotential myeloid cells. In C/EBPalpha knockout mice, no mature granulocytes are present. A dramatic increase of c-Jun mRNA in C/EBPalpha knockout mouse fetal liver was observed. c-Jun, a component of the AP-1 transcription factor complex and a coactivator of the transcription factor PU.1, is important for monocytic differentiation. Here we report that C/EBPalpha downregulates c-Jun expression to drive granulocytic differentiation. An ectopic increase of C/EBPalpha expression decreases the c-Jun mRNA level, and the human c-Jun promoter activity is downregulated eightfold in the presence of C/EBPalpha. C/EBPalpha and c-Jun interact through their leucine zipper domains, and this interaction prevents c-Jun from binding to DNA. This results in downregulation of c-Jun's capacity to autoregulate its own promoter through the proximal AP-1 site. Overexpression of c-Jun prevents C/EBPalpha-induced granulocytic differentiation. Thus, we propose a model in which C/EBPalpha needs to downregulate c-Jun expression and transactivation capacity for promoting granulocytic differentiation.

The article discusses the application of the micro-RNA molecule miR-22 in Hematopoiesis. It discusses its application with regard to it being an oncogenic tumor suppressor or a tumor-suppressive oncogene. Topics discussed include research studies with regard to the matter, discoveries of micro-RNAs and approval of drugs based on micro-RNA.

In contrast to the definitive role of the transcription factor, CCAAT/Enhancer binding protein a (C/EBPa), in steady-state granulopoiesis, previous findings have suggested that granulopoiesis during emergency situations, such as infection, is dependent on C/EBPb. In this study, a novel lentivirus-based reporter system was developed to elucidate the molecular switch required for C/EBPb-dependency. The results demonstrated that two cyclic AMP responsive elements (CREs) in the proximal promoter region of C/EBPb were involved in the positive regulation of C/EBPβ transcription during granulocyte- macrophage colony-stimulating factor (GM-CSF)-induced differentiation of bone marrow cells. In addition, the transcripts of CRE binding (CREB) family proteins were readily detected in hematopoietic stem/progenitor cells. CREB was upregulated, phosphorylated and bound to the CREs in response to GM-CSF stimulation. Retroviral transduction of a dominant negative CREB mutant reduced C/EBPb mRNA levels and significantly impaired the proliferation/differentiation of granulocyte precursors, while a constitutively active form of CREB facilitated C/EBPb transcription. These data suggest that CREB proteins are involved in the regulation of granulopoiesis via C/EBPb upregulation. [ABSTRACT FROM AUTHOR]

The family of cyclin D proteins plays a crucial role in the early events of the mammalian cell cycle. Recent studies have revealed the involvement of AML1 transactivation activity in promoting cell cycle progression through the induction of cyclin D proteins. This information in combination with our previous observation that a region in AML1 between amino acids 213 and 289 is important for its function led us to investigate prospective proteins associating with this region. We identified cyclin D3 by a yeast two-hybrid screen and detected AML1 interaction with the cyclin D family by both in vitro pull-down and in vivo coimmunoprecipitation assays. Furthermore, we demonstrate that cyclin D3 negatively regulates the transactivation activity of AML1 in a dose-dependent manner by competing with CBFIβ for AML1 association, leading to a decreased binding affinity of AML1 for its target DNA sequence. AML1 and its fusion protein AML1-ETO have been shown to shorten and prolong the mammalian cell cycle, respectively. In addition, AML1 promotes myeloid cell differentiation. Thus, our observations suggest that the direct association of cyclin D3 with AML1 functions as a putative feedback mechanism to regulate cell cycle progression and differentiation. [ABSTRACT FROM AUTHOR]

Coactivator-associated arginine methyltransferase I (CARM1; PRMT4) regulates gene expression by multiple mechanisms including methylation of histones and coactivation of steroid receptor transcription. Mice lacking CARM1 are small, fail to breathe and die shortly after birth, demonstrating the crucial role of CARM1 in development. In adults, CARM1 is overexpressed in human grade-III breast tumors and prostate adenocarcinomas, and knockdown of CARM1 inhibits proliferation of breast and prostate cancer cell lines. Based on these observations, we hypothesized that loss of CARM1 in mouse embryos would inhibit pulmonary cell proliferation, resulting in respiratory distress. By contrast, we report here that loss of CARM1 results in hyperproliferation of pulmonary epithelial cells during embryonic development. The lungs of newborn mice lacking CARM1 have substantially reduced airspace compared with their wild-type littermates. In the absence of CARM1, alveolar type II cells show increased proliferation. Electron microscopic analyses demonstrate that lungs from mice lacking CARM1 have immature alveolar type II cells and an absence of alveolar type I cells. Gene expression analysis reveals a dysregulation of cell cycle genes and markers of differentiation in the Carm1 knockout lung. Furthermore, there is an overlap in gene expression in the Carm1 knockout and the glucocorticoid receptor knockout lung, suggesting that hyperproliferation and lack of maturation of the alveolar cells are at least in part caused by attenuation of glucocorticoid-mediated signaling. These results demonstrate for the first time that CARM1 inhibits pulmonary cell proliferation and is required for proper differentiation of alveolar cells. [ABSTRACT FROM AUTHOR]

The leucine zipper family transcription factor CCAAT enhancer binding protein alpha (C/EBP α) inhibits proliferation and promotes differentiation in various cell types. In this study, we show, using a lung-specific conditional mouse model of C/EBP α deletion, that loss of C/EBP α in the respiratory epithelium leads to respiratory failure at birth due to an arrest in the type II alveolar cell differentiation program. This differentiation arrest results in the lack of type I alveolar cells and differentiated surfactant-secreting type II alveolar cells. In addition to showing a block in type II cell differentiation, the neonatal lungs display increased numbers of proliferating cells and decreased numbers of apoptotic cells, leading to epithelial expansion and loss of airspace. Consistent with the phenotype observed, genes associated with alveolar maturation, survival, and proliferation were differentially expressed. Taken together, these results identify C/EBP αas a master regulator of airway epithelial maturation and suggest that the loss of C/EBP α could also be an important event in the multistep process of lung tumorigenesis. Furthermore, this study indicates that exploring the C/EBP α pathway might have therapeutic benefits for patients with respiratory distress syndromes [ABSTRACT FROM AUTHOR]

The tumor suppressor transcription factor CCAAT enhancer-binding protein α (C/EBPα) expression is downregulated in myeloid leukemias and enhancement of C/EBPα expression induces granulocytic differentiation in leukemic cells. Previously we reported that Styryl quinazolinones induce myeloid differentiation in HL-60 cells by upregulating C/EBPα expression. To identify more potent molecule that can induce leukemic cell differentiation we synthesized and evaluated new series of styryl quinazolinones, ethynyl styryl quinazolinones, styryl quinolinones and thienopyrimidinones. Thienopyrimidinones were found toxic and styryl quinolinones were found inactive. Ethynyl styryl quinazolinone 39 and styryl quinazolinone 5 were found active on par with the earlier reported analogues 1 and 2 suggesting that the 5-nitro furan-2-yl styryl quinazolinones find a real promise in leukemic cell differentiation. The improved potency of 5 suggested that further modifications in the 5-nitro furan-2-yl styryl quinazolinones can be at the phenyl substitution at the 3-position of the quinazolinone ring apart from the 5-position of the heteroaryl ring. [ABSTRACT FROM AUTHOR]