Spatiotemporal dynamics of fetal liver hematopoietic niches.

Embryonic hematopoietic cells develop in the fetal liver (FL), surrounded by diverse non-hematopoietic stromal cells. However, the spatial organization and cytokine production patterns of the stroma during FL development remain poorly understood. Here, we characterized and mapped the hematopoietic and stromal cell populations at early (E12.

Highly efficient Runx1 enhancer eR1-mediated genetic engineering for fetal, child and adult hematopoietic stem cells.

A cis-regulatory genetic element which targets gene expression to stem cells, termed stem cell enhancer, serves as a molecular handle for stem cell-specific genetic engineering. Here we show the generation and characterization of a tamoxifen-inducible CreER transgenic (Tg) mouse employing previously identified hematopoietic stem cell (HSC) enhancer for Runx1, eR1 (+24 m). Kinetic analysis of labeled cells after tamoxifen injection and transplantation assays revealed that eR1-driven CreER activity marks dormant adult HSCs which slowly but steadily contribute to unperturbed hematopoiesis.

Prdm3 and Prdm16 cooperatively maintain hematopoiesis and clonogenic potential.

Mds1-Evi1 (also known as Prdm3) and Prdm16 are two highly related zinc finger transcription factors that, within the hematopoietic system, are both expressed primarily in hematopoietic stem cells (HSCs). Our laboratory previously found that constitutive Mds1-Evi1 knockout mice are viable, but their HSCs are unable to withstand myeloablative chemotherapy or effectively transplant irradiated recipient mice. A similar phenotype has been observed for Prdm16, except that the Prdm16 constitutive knockout is lethal.

Live-animal imaging of native haematopoietic stem and progenitor cells.

The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow.

Aged marrow macrophages expand platelet-biased hematopoietic stem cells via Interleukin1B.

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function, though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mφs) directs HSC platelet-bias. Mφs from the marrow of aged mice and humans exhibited an activated phenotype, with increased expression of inflammatory signals.

Next-Generation-Sequencing-Based Hospital Outbreak Investigation Yields Insight into Klebsiella aerogenes Population Structure and Determinants of Carbapenem Resistance and Pathogenicity.

is a nosocomial pathogen associated with drug resistance and outbreaks in intensive care units. In a 5-month period in 2017, we experienced an increased incidence of cultures for carbapenem-resistant (CR-KA) from an adult cardiothoracic intensive care unit (CICU) involving 15 patients. Phylogenomic analysis following whole-genome sequencing (WGS) identified the outbreak CR-KA isolates to group together as a tight monoclonal cluster (with no more than six single nucleotide polymorphisms [SNPs]), suggestive of a protracted intraward transmission event.

EVI1 overexpression reprograms hematopoiesis via upregulation of Spi1 transcription.

Inv(3q26) and t(3:3)(q21;q26) are specific to poor-prognosis myeloid malignancies, and result in marked overexpression of EVI1, a zinc-finger transcription factor and myeloid-specific oncoprotein. Despite extensive study, the mechanism by which EVI1 contributes to myeloid malignancy remains unclear. Here we describe a new mouse model that mimics the transcriptional effects of 3q26 rearrangement.

The creatine kinase pathway is a metabolic vulnerability in EVI1-positive acute myeloid leukemia.

Expression of the MECOM (also known as EVI1) proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A screen using pooled short hairpin RNAs (shRNAs) identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as necessary for survival of EVI1-expressing cells in subjects with EVI1-positive AML.

EVI1 Interferes with Myeloid Maturation via Transcriptional Repression of Cebpa, via Binding to Two Far Downstream Regulatory Elements.

One mechanism by which oncoproteins work is through perturbation of cellular maturation; understanding the mechanisms by which this occurs can lead to the development of targeted therapies. EVI1 is a zinc finger oncoprotein involved in the development of acute myeloid leukemia; previous work has shown it to interfere with the maturation of granulocytes from immature precursors. Here we investigate the mechanism by which that occurs, using an immortalized hematopoietic progenitor cell line, EML-C1, as a model system.

Mice carrying a hypomorphic Evi1 allele are embryonic viable but exhibit severe congenital heart defects.

The ecotropic viral integration site 1 (Evi1) oncogenic transcription factor is one of a number of alternative transcripts encoded by the Mds1 and Evi1 complex locus (Mecom). Overexpression of Evi1 has been observed in a number of myeloid disorders and is associated with poor patient survival. It is also amplified and/or overexpressed in many epithelial cancers including nasopharyngeal carcinoma, ovarian carcinoma, ependymomas, and lung and colorectal cancers.

The role of EVI1 in myeloid malignancies.

The EVI1 oncogene at human chr 3q26 is rearranged and/or overexpressed in a subset of acute myeloid leukemias and myelodysplasias. The EVI1 protein is a 135 kDa transcriptional regulator with DNA-binding zinc finger domains. Here we provide a critical review of the current state of research into the molecular mechanisms by which this gene plays a role in myeloid malignancies.

Deletion of Mecom in mouse results in early-onset spinal deformity and osteopenia.

Recent studies have indicated a role for a MECOM allele in susceptibility to osteoporotic fractures in humans. We have generated a mutation in Mecom in mouse (termed ME(m1)) via lacZ knock-in into the upstream transcription start site for the gene, resulting in disruption of Mds1 and Mds1-Evi1 transcripts, but not of Evi1 transcripts. We demonstrate that ME(m1/m1) mice have severe kyphoscoliosis that is reminiscent of human congenital or primary kyphoscoliosis.

Essential role of PR-domain protein MDS1-EVI1 in MLL-AF9 leukemia.

A subgroup of leukemogenic mixed-lineage leukemia (MLL) fusion proteins (MFPs) including MLL-AF9 activates the Mecom locus and exhibits extremely poor clinical prognosis. Mecom encodes EVI1 and MDS1-EVI1 (ME) proteins via alternative transcription start sites; these differ by the presence of a PRDI-BF1-RIZ1 (PR) domain with histone methyltransferase activity in the ME isoform. Using an ME-deficient mouse, we show that ME is required for MLL-AF9-induced transformation both in vitro and in vivo.

Global Identification of EVI1 Target Genes in Acute Myeloid Leukemia.

The ecotropic virus integration site 1 (EVI1) transcription factor is associated with human myeloid malignancy of poor prognosis and is overexpressed in 8-10% of adult AML and strikingly up to 27% of pediatric MLL-rearranged leukemias. For the first time, we report comprehensive genomewide EVI1 binding and whole transcriptome gene deregulation in leukemic cells using a combination of ChIP-Seq and RNA-Seq expression profiling. We found disruption of terminal myeloid differentiation and cell cycle regulation to be prominent in EVI-induced leukemogenesis.

Master regulatory GATA transcription factors: mechanistic principles and emerging links to hematologic malignancies.

Numerous examples exist of how disrupting the actions of physiological regulators of blood cell development yields hematologic malignancies. The master regulator of hematopoietic stem/progenitor cells GATA-2 was cloned almost 20 years ago, and elegant genetic analyses demonstrated its essential function to promote hematopoiesis. While certain GATA-2 target genes are implicated in leukemogenesis, only recently have definitive insights emerged linking GATA-2 to human hematologic pathophysiologies.

Overexpression of Evi-1 oncoprotein represses TGF-β signaling in colorectal cancer.

Human colorectal cancer (CRC) cells are resistant to the anti-proliferative effect of transforming growth factor-β (TGF-β), suggesting that disruption of TGF-β signaling plays an important role in colorectal carcinogenesis. Ecotropic virus integration site-1 (Evi-1) oncoprotein represses TGF-β signaling by interacting with Smads, but its role in CRC has not been established. The purpose of this study is to determine whether Evi-1 plays role(s) in CRCs and to characterize Evi-1 transcript(s) in CRCs.

Targeting a DNA binding motif of the EVI1 protein by a pyrrole-imidazole polyamide.

The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole-imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure-function studies on both EVI1 and the t(3;21)(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1-7) is essential transforming activity.

PR-domain-containing Mds1-Evi1 is critical for long-term hematopoietic stem cell function.

The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed ME(m1), which eliminates Mds1-Evi1 (ME), the longer, PR-domain-containing isoform produced by the gene (also known as PRDM3).

C/EBPepsilon directs granulocytic-vs-monocytic lineage determination and confers chemotactic function via Hlx.

Objective: Mutations in the CCAAT enhancer binding protein epsilon (C/EBPepsilon) gene have been identified in the cells of patients with neutrophil specific granule deficiency, a rare congenital disorder marked by recurrent bacterial infections. Their neutrophils, in addition to lacking specific granules required for normal respiratory burst activity, also lack normal phagocytosis and chemotaxis. Although the specific granule deficiency phenotype has been replicated in C/EBPepsilon(-/-) (knockout [KO]) mice, the mechanisms by which C/EBPepsilon mutations act to decrease neutrophil function are not entirely clear.

Burkitt lymphoma in adults.

This review will begin with a detail of the revision of the WHO classification, and pathological definitions of Burkitt lymphoma. Over the past several years, molecular understanding of Burkitt lymphoma has improved significantly. Using gene expression profiling, a genomic "signature" of Burkitt lymphoma may be identified, that has fidelity beyond c-myc expression, and the presence of the classical t(8;14).

Regulation of the calreticulin gene by GATA6 and Evi-1 transcription factors.

Calreticulin is a Ca (2+)-buffering chaperone of the endoplasmic reticulum. The protein is highly expressed in embryonic heart but downregulated in postnatal heart, indicating that expression of calreticulin in the heart is highly regulated. In this study we identify GATA6 and Evi-1 transcription factors as new regulators of the calreticulin gene.

Sox4 cooperates with Evi1 in AKXD-23 myeloid tumors via transactivation of proviral LTR.

Myeloid leukemias in AKXD23 mice contain proviral insertions at Evi1, resulting in transcriptional activation. Although Evi1 is clearly involved in leukemia, gene transfer studies in mice with Evi1 fail to cause leukemia, arguing that cooperating events are necessary. We reanalyzed AKXD-23 tumors for cooperating proviral insertion and found that each tumor had a proviral insertion in Sox4, which encodes an HMG-box transcription factor.

Identification of binding sites of EVI1 in mammalian cells.

The leukemia-associated protein EVI1 possesses seven zinc fingers within an N-terminal domain (amino acids 1-250) that binds to GACAAGATA. Single amino acid missense mutants of EVI1 were developed that failed to bind DNA either in vitro, as assessed by gel shift assay, or in vivo, as shown by transactivation studies. Specifically, mutation R205N lacks high affinity binding to the GACAAGATA motif.