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Transcriptional and Epigenetic Regulation in the Development of Myeloid Cells: Normal and Diseased Myelopoiesis

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Abstract

Myeloid cells constitute the innate arm of the vertebrate immune system and arise from haematopoietic stem cells being committed to their cell fate through a series of lineage restrictions regulated by a gene regulatory network. This gene network consists of transcription factors as well as components of the epigenetic machinery that, in cooperation with one another, will programme progenitors to adopt and differentiate along a certain lineage programme. By virtue of their obligatory function, dysregulation in the activity of these regulatory factors can contribute to the pathogenesis of myeloid leukaemias. To understand the molecular aetiology of myeloid dysplasias it is imperative to first study and model the network that regulates normal development. Equipped with this crucial understanding we can then begin to decipher what, how and why things have gone wrong in the pathology of myeloid leukaemias.

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Twenty-four patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (45 to 100 mg/m2/day). Of these, eight cases had been either nonresponsive or resistant to previous chemotherapy; the other 16 cases were previously untreated. All patients attained complete remission without developing bone marrow hypoplasia. Bone marrow suspension cultures were studied in 15 of the 24 patients. Fourteen of these patients had morphological maturation in response to the retinoic acid (1 mumol/L). Chloroacetate esterase and alpha-naphthyl acetate esterase staining as well as electronmicroscopic examination confirmed that retinoic acid-induced cells differentiated to granulocytes with increased functional maturation (as measured by nitroblue tetrazolium reduction, NBT). The single nonresponder to retinoic acid in vitro was resistant to treatment with retinoic acid but attained complete remission after addition of low-dose cytosine arabinoside (ara-C). During the course of therapy, none of the patients showed any abnormalities in the coagulation parameters we measured, suggesting an absence of any subclinical disseminated intravascular coagulation. The only side effects consisted of mild dryness of the lips and skin, with occasional headaches and digestive symptoms. Eight patients have relapsed after 2 to 5 months of complete remission. The others remain in complete remission at 1+ to 11+ months and are still being followed up. We conclude that all-trans retinoic acid is an effective inducer for attaining complete remission in APL.
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The AML1 gene encoding the DNA-binding alpha-subunit in the Runt domain family of heterodimeric transcription factors has been noted for its frequent involvement in chromosomal translocations associated with leukemia. Using reverse transcriptase-polymerase chain reaction (RT-PCR) combined with nonisotopic RNase cleavage assay (NIRCA), we found point mutations of the AML1 gene in 8 of 160 leukemia patients: silent mutations, heterozygous missense mutations, and biallelic nonsense or frameshift mutations in 2, 4, and 2 cases, respectively. The mutations were all clustered within the punt domain. Missense mutations identified in 3 patients showed neither DNA binding nor transactivation, although being active in heterodimerization. These defective missense mutants may be relevant to the predisposition or progression of leukemia. On the other hand, the biallelic nonsense mutants encoding truncated AML1 proteins lost almost all functions examined and may play a role in leukemogenesis leading to acute myeloblastic leukemia. (C) 1999 by The American Society of Hematology.
Article
Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by the proliferation and accumulation of immature myeloid cells in the bone marrow and blood. The World Health Organization classification has changed the criteria for the diagnosis and classification of AML. Cytogenetics, age of the patient, and molecular markers are important prognostic variables. Treatment decisions should be based on risk assessment and biology of the AML.
Article
The transcription factor PU.1 is required for normal blood cell development. PU.1 regulates the expression of a number of crucial myeloid genes, such as the macrophage colony-stimulating factor (M-CSF) receptor, the granulocyte colony-stimulating factor (G-CSF) receptor, and the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor. Myeloid cells derived from PU.1−/− mice are blocked at the earliest stage of myeloid differentiation, similar to the blast cells that are the hallmark of human acute myeloid leukemia (AML). These facts led us to hypothesize that molecular abnormalities involving the PU.1 gene could contribute to the development of AML. We identified 10 mutant alleles of the PU.1 gene in 9 of 126 AML patients. The PU.1 mutations comprised 5 deletions affecting the DNA-binding domain, and 5 point mutations in 1) the DNA-binding domain (2 patients), 2) the PEST domain (2 patients), and 3) the transactivation domain (one patient). DNA binding to and transactivation of the M-CSF receptor promoter, a direct PU.1 target gene, were deficient in the 7 PU.1 mutants that affected the DNA-binding domain. In addition, these mutations decreased the ability of PU.1 to synergize with PU.1-interacting proteins such as AML1 or c-Jun in the activation of PU.1 target genes. This is the first report of mutations in the PU.1 gene in human neoplasia and suggests that disruption of PU.1 function contributes to the block in differentiation found in AML patients.
Article
Mutations in the enzyme cytosolic isocitrate dehydrogenase 1 (IDH1) are a common feature of a major subset of primary human brain cancers. These mutations occur at a single amino acid residue of the IDH1 active site, resulting in loss of the enzyme's ability to catalyse ...
Article
Mutations in the enzyme cytosolic isocitrate dehydrogenase 1 (IDH1) are a common feature of a major subset of primary human brain cancers. These mutations occur at a single amino acid residue of the IDH1 active site, resulting in loss of the enzyme's ability to catalyse conversion of isocitrate to {alpha}-ketoglutarate. However, only a single copy of the gene is mutated in tumours, raising the possibility that the mutations do not result in a simple loss of function. Here we show that cancer-associated IDH1 mutations result in a new ability of the enzyme to catalyse the NADPH-dependent reduction of {alpha}-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). Structural studies demonstrate that when arginine 132 is mutated to histidine, residues in the active site are shifted to produce structural changes consistent with reduced oxidative decarboxylation of isocitrate and acquisition of the ability to convert {alpha}-ketoglutarate to 2HG. Excess accumulation of 2HG has been shown to lead to an elevated risk of malignant brain tumours in patients with inborn errors of 2HG metabolism. Similarly, in human malignant gliomas harbouring IDH1 mutations, we find markedly elevated levels of 2HG. These data demonstrate that the IDH1 mutations result in production of the onco-metabolite 2HG, and indicate that the excess 2HG which accumulates in vivo contributes to the formation and malignant progression of gliomas.
Article
Starting with multipotent progenitors, hematopoietic lineages are specified by lineage-restricted transcription factors. The transcription factors that determine the decision between lymphoid and myeloid cell fates, and the underlying mechanisms, remain largely unknown. Here, we report that enforced expression of C/EBPalpha and C/EBPbeta in differentiated B cells leads to their rapid and efficient reprogramming into macrophages. C/EBPs induce these changes by inhibiting the B cell commitment transcription factor Pax5, leading to the downregulation of its target CD19, and synergizing with endogenous PU.1, an ETS family factor, leading to the upregulation of its target Mac-1 and other myeloid markers. The two processes can be uncoupled, since, in PU.1-deficient pre-B cells, C/EBPs induce CD19 downregulation but not Mac-1 activation. Our observations indicate that C/EBPalpha and beta remodel the transcription network of B cells into that of macrophages through a series of parallel and sequential changes that require endogenous PU.1.
Article
The transcription factor C/EBPα (for CCAAT/enhancer binding protein-α; encoded by the gene CEBPA) is crucial for the differentiation of granulocytes. Conditional expression of C/EBPα triggers neutrophilic differentiation, and no mature granulocytes are observed in Cebpa-mutant mice. Here we identify heterozygous mutations in CEBPA in ten patients with acute myeloid leukemia (AML). We found that five mutations in the amino terminus truncate the full-length protein, but did not affect a 30-kD protein initiated further downstream. The mutant proteins block wild-type C/EBPα DNA binding and transactivation of granulocyte target genes in a dominant-negative manner, and fails to induce granulocytic differentiation. Ours is the first report of CEBPA mutations in human neoplasia, and such mutations are likely to induce the differentiation block found in AML.
Article
A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). These mutations alter residues in the enzyme active sites and confer a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite (R)-2-hydroxyglutarate (2HG). We developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia cells in vitro. These data provide proof-of-concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.
Article
Epigenetic 5-azacitidine (AZA) therapy of high-risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) represents a promising, albeit not fully understood, approach. Hematopoietic transcription factor PU.1 is dynamically regulated by upstream regulatory element (URE), whose deletion causes downregulation of PU.1 leading to AML in mouse. In this study a significant group of the high-risk MDS patients, as well as MDS cell lines, displayed downregulation of PU.1 expression within CD34+ cells, which was associated with DNA methylation of the URE. AZA treatment in vitro significantly demethylated URE, leading to upregulation of PU.1 followed by derepression of its transcriptional targets and onset of myeloid differentiation. Addition of colony-stimulating factors (CSFs; granulocyte-CSF, granulocyte–macrophage-CSF and macrophage-CSF) modulated AZA-mediated effects on reprogramming of histone modifications at the URE and cell differentiation outcome. Our data collectively support the importance of modifying the URE chromatin structure as a regulatory mechanism of AZA-mediated activation of PU.1 and induction of the myeloid program in MDS.Keywords: PU.1; 5-azacitidine; MDS; AML; cytokines; differentiation
Article
Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.
Article
Somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) gene occur in a significant proportion of patients with myeloid malignancies. Although there are extensive genetic data implicating TET2 mutations in myeloid transformation, the consequences of Tet2 loss in hematopoietic development have not been delineated. We report here an animal model of conditional Tet2 loss in the hematopoietic compartment that leads to increased stem cell self-renewal in vivo as assessed by competitive transplant assays. Tet2 loss leads to a progressive enlargement of the hematopoietic stem cell compartment and eventual myeloproliferation in vivo, including splenomegaly, monocytosis, and extramedullary hematopoiesis. In addition, Tet2(+/-) mice also displayed increased stem cell self-renewal and extramedullary hematopoiesis, suggesting that Tet2 haploinsufficiency contributes to hematopoietic transformation in vivo.
Article
The relationships between normal and leukemic stem/progenitor cells are unclear. We show that in ∼80% of primary human CD34+ acute myeloid leukemia (AML), two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. Both populations have leukemic stem cell (LSC) activity and are hierarchically ordered; one LSC population gives rise to the other. Global gene expression profiling shows the LSC populations are molecularly distinct and resemble normal progenitors but not stem cells. The more mature LSC population most closely mirrors normal granulocyte-macrophage progenitors (GMP) and the immature LSC population a previously uncharacterized progenitor functionally similar to lymphoid-primed multipotential progenitors (LMPPs). This suggests that in most cases primary CD34+ AML is a progenitor disease where LSCs acquire abnormal self-renewal potential.
Article
Stem cells are defined as cells that have the ability to perpetuate themselves through self-renewal and to generate functional mature cells by differentiation. During each stage, coordinated gene expression is crucial to maintain the balance between self-renewal and differentiation. Disturbance of this accurately balanced system can lead to a variety of malignant disorders. In mammals, DNA cytosine-5 methylation is a well-studied epigenetic pathway that is catalyzed by DNA methyltransferases and is implicated in the control of balanced gene expression, but also in hematological malignancies. In this review, we focus on the TET (ten-eleven-translocation) genes, which recently were identified to catalyze the conversion of cytosine-5 methylation to 5-hydroxymethyl-cytosine, an intermediate form potentially involved in demethylation. In addition, members of the TET family are playing a role in ES cell maintenance and inner cell mass cell specification and were demonstrated to be involved in hematological malignancies. Recently, a correlation between low genomic 5-hydroxymethyl-cytosine and TET2 mutation status was shown in patients with myeloid malignancies.
Article
Cancer-associated IDH mutations are characterized by neomorphic enzyme activity and resultant 2-hydroxyglutarate (2HG) production. Mutational and epigenetic profiling of a large acute myeloid leukemia (AML) patient cohort revealed that IDH1/2-mutant AMLs display global DNA hypermethylation and a specific hypermethylation signature. Furthermore, expression of 2HG-producing IDH alleles in cells induced global DNA hypermethylation. In the AML cohort, IDH1/2 mutations were mutually exclusive with mutations in the α-ketoglutarate-dependent enzyme TET2, and TET2 loss-of-function mutations were associated with similar epigenetic defects as IDH1/2 mutants. Consistent with these genetic and epigenetic data, expression of IDH mutants impaired TET2 catalytic function in cells. Finally, either expression of mutant IDH1/2 or Tet2 depletion impaired hematopoietic differentiation and increased stem/progenitor cell marker expression, suggesting a shared proleukemogenic effect.
Article
In myelodysplastic syndromes (MDS), deletions of chromosome 7 or 7q are common and correlate with a poor prognosis. The relevant genes on chromosome 7 are unknown. We report here that EZH2, located at 7q36.1, is frequently targeted in MDS. Analysis of EZH2 deletions, missense and frameshift mutations strongly suggests that EZH2 is a tumor suppressor. As EZH2 functions as a histone methyltransferase, abnormal histone modification may contribute to epigenetic deregulation in MDS.
Article
Abnormalities of chromosome 7q are common in myeloid malignancies, but no specific target genes have yet been identified. Here, we describe the finding of homozygous EZH2 mutations in 9 of 12 individuals with 7q acquired uniparental disomy. Screening of a total of 614 individuals with myeloid disorders revealed 49 monoallelic or biallelic EZH2 mutations in 42 individuals; the mutations were found most commonly in those with myelodysplastic/myeloproliferative neoplasms (27 out of 219 individuals, or 12%) and in those with myelofibrosis (4 out of 30 individuals, or 13%). EZH2 encodes the catalytic subunit of the polycomb repressive complex 2 (PRC2), a highly conserved histone H3 lysine 27 (H3K27) methyltransferase that influences stem cell renewal by epigenetic repression of genes involved in cell fate decisions. EZH2 has oncogenic activity, and its overexpression has previously been causally linked to differentiation blocks in epithelial tumors. Notably, the mutations we identified resulted in premature chain termination or direct abrogation of histone methyltransferase activity, suggesting that EZH2 acts as a tumor suppressor for myeloid malignancies.
Article
Genome-scale studies have revealed extensive, cell type-specific colocalization of transcription factors, but the mechanisms underlying this phenomenon remain poorly understood. Here, we demonstrate in macrophages and B cells that collaborative interactions of the common factor PU.1 with small sets of macrophage- or B cell lineage-determining transcription factors establish cell-specific binding sites that are associated with the majority of promoter-distal H3K4me1-marked genomic regions. PU.1 binding initiates nucleosome remodeling, followed by H3K4 monomethylation at large numbers of genomic regions associated with both broadly and specifically expressed genes. These locations serve as beacons for additional factors, exemplified by liver X receptors, which drive both cell-specific gene expression and signal-dependent responses. Together with analyses of transcription factor binding and H3K4me1 patterns in other cell types, these studies suggest that simple combinations of lineage-determining transcription factors can specify the genomic sites ultimately responsible for both cell identity and cell type-specific responses to diverse signaling inputs.
Article
Enhancers determine tissue-specific gene expression programs. Enhancers are marked by high histone H3 lysine 4 mono-methylation (H3K4me1) and by the acetyl-transferase p300, which has allowed genome-wide enhancer identification. However, the regulatory principles by which subsets of enhancers become active in specific developmental and/or environmental contexts are unknown. We exploited inducible p300 binding to chromatin to identify, and then mechanistically dissect, enhancers controlling endotoxin-stimulated gene expression in macrophages. In these enhancers, binding sites for the lineage-restricted and constitutive Ets protein PU.1 coexisted with those for ubiquitous stress-inducible transcription factors such as NF-kappaB, IRF, and AP-1. PU.1 was required for maintaining H3K4me1 at macrophage-specific enhancers. Reciprocally, ectopic expression of PU.1 reactivated these enhancers in fibroblasts. Thus, the combinatorial assembly of tissue- and signal-specific transcription factors determines the activity of a distinct group of enhancers. We suggest that this may represent a general paradigm in tissue-restricted and stimulus-responsive gene regulation.
Article
PML/RARalpha is of crucial importance in acute promyelocytic leukemia (APL) both pathologically and therapeutically. Using a genome-wide approach, we identified in vivo PML/RARalpha binding sites in a PML/RARalpha-inducible cell model. Of the 2979 targeted regions, >62% contained canonical PU.1 motifs and >84% of these PU.1 motifs coexisted with one or more RARE half (RAREh) sites in nearby regions. Promoters with such PU.1-RAREh binding sites were transactivated by PU.1. PU.1-mediated transactivation was repressed by PML/RARalpha and restored by the addition of all-trans retinoic acid (ATRA). Genes containing such promoters were significantly represented by genes transcriptionally suppressed in APL and/or reactivated upon treatment with ATRA. Thus, selective targeting of PU.1-regulated genes by PML/RARalpha is a critical mechanism for the pathogenesis of APL.
Article
Many different molecular mechanisms have been associated with PML-RARalpha-dependent transformation of hematopoietic progenitors. Here, we identified high confidence PML-RARalpha binding sites in an acute promyelocytic leukemia (APL) cell line and in two APL primary blasts. We found colocalization of PML-RARalpha with RXR to the vast majority of these binding regions. Genome-wide epigenetic studies revealed that treatment with pharmacological doses of all-trans retinoic acid induces changes in H3 acetylation, but not H3K27me3, H3K9me3, or DNA methylation at the PML-RARalpha/RXR binding sites or at nearby target genes. Our results suggest that PML-RARalpha/RXR functions as a local chromatin modulator and that specific recruitment of histone deacetylase activities to genes important for hematopoietic differentiation, RAR signaling, and epigenetic control is crucial to its transforming potential.