Malignant Transformation Involving CXXC4 Mutations Identified in a Leukemic Progression Model of Severe Congenital Neutropenia

Olofsen et al. show that acquisition of a mutation in Cxxc4 results in increased CXXC4 protein levels, reduced TET2 protein, increased inflammatory signaling, and leukemic progression of a CSF3R/RUNX1 mutant mouse model of severe congenital neutropenia (SCN).Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the appearance of hematopoietic clones with CSF3 receptor (CSF3R) mutations in the neutropenic phase, followed by mutations in RUNX1 before AML becomes overt. To investigate how the combination of CSF3 therapy and CSF3R and RUNX1 mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in CSF3R/RUNX1 mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in Cxxc4 causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN

The role of STAT5 and KRAS in hematopoiesis and acute myeoloid leukemia

Chromosomal translocations or molecular abnormalities in hematopoietic stem cells can disrupt the normal process of self-renewal and differentiation, which can result in malignant transformation. A number of key transcription factors and signaling molecules have been identified that function aberrantly in leukemic cells. In this thesis we investigated the effects of STAT5 and KRAS on human hematopoietic stem cell selfrenewal and differentiation, and their potential role in leukemic transformation. Constitutive activation of STAT5 in CD34+ cord blood cells by retroviral transduction resulted in the upregulation of the membrane protein MUCIN1. Based on this information (chapter 2) we investigated the role of MUCIN1 in normal hematopoiesis as well as in primary AML cells. We demonstrated that 10% of the cord blood CD34+ cells were MUCIN1-positive. MUCIN1 mRNA expression was the highest in the CD34+/CD38-cell fraction. Experiments with cord blood CD34+/MUCIN1+ and CD34+/MUCIN1- cell populations revealed that stem/progenitor cells reside predominantly in the CD34+/MUCIN1+ fraction. To further study the role of MUCIN1 in hematopoiesis we stably overexpressed full-length MUCIN1 and a MUCIN1 isoform with a deleted extracellular domain (DTR) in cord blood CD34+ cells. In MS5 coculture assays a two-fold increase in expansion of suspension cells was observed, and LTC-IC frequencies and progenitor numbers were increased upon overexpression of MUCIN1 or DTR.

Endoplasmic Reticulum Stress–Induced Apoptosis Is Partly Mediated by Reduced Insulin Signaling Through Phosphatidylinositol 3-Kinase/Akt and Increased Glycogen Synthase Kinase-3β in Mouse Insulinoma Cells

Endoplasmic Reticulum Stress–Induced Apoptosis Is Partly Mediated by Reduced Insulin Signaling Through Phosphatidylinositol 3-Kinase/Akt and Increased Glycogen Synthase Kinase-3β in Mouse Insulinoma Cells Shanthi Srinivasan , Mitsuru Ohsugi , Zhonghao Liu , Szabolcs Fatrai , Ernesto Bernal-Mizrachi and M. Alan Permutt Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, St. Louis, Missouri Address correspondence and reprint requests to M. Alan Permutt MD, Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8127, St. Louis, MO 63110. E-mail: apermutt{at}im.wustl.edu Abstract An imbalance between the rate of protein synthesis and folding capacity of the endoplasmic reticulum (ER) results in stress that has been increasingly implicated in pancreatic islet β-cell apoptosis and diabetes. Because insulin/IGF/Akt signaling has been implicated in β-cell survival, we sought to determine whether this pathway is involved in ER stress–induced apoptosis. Mouse insulinoma cells treated with pharmacological agents commonly used to induce ER stress exhibited apoptosis within 48 h. ER stress–induced apoptosis was inhibited by cotreatment of the cells with IGF-1. Stable cell lines were created by small-interfering RNA (siRNA) with graded reduction of insulin receptor expression, and these cells had enhanced susceptibility to ER stress–induced apoptosis and reduced levels of phospho–glycogen synthase kinase 3β (GSK3β). In control cells, ER stress–induced apoptosis was associated with a reduction in phospho-Akt and phospho-GSK3β. To further assess the role of GSK3β in ER stress–induced apoptosis, stable cell lines were created by siRNA with up to 80% reduction in GSK3β expression. These cells were found to resist ER stress–induced apoptosis. These results illustrate that ER stress–induced apoptosis is mediated at least in part by signaling through the phosphatidylinositol 3-kinase/Akt/GSK3β pathway and that GSK3β represents a novel target for agents to promote β-cell survival. eIF2α, eukaryotic initiation factor 2α ER, endoplasmic reticulum GSK3β, glycogen synthase kinase 3β JNK, c-Jun NH2-terminal kinase IKRD, insulin receptor knock down PARP, poly-ADP-ribose polymerase PERK, PKR-like ER kinase/pancreatic eIF2α kinase PI3, phosphatidylinositol 3 Q-VD-OPh, N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone siRNA, small-interfering RNA Footnotes S.S. is currently affiliated with the Division of Digestive Diseases, Emory University School of Medicine, Atlanta, Georgia. S.S., M.O., and Z.L. contributed equally to this study. Accepted January 1, 2005. Received June 28, 2004. DIABETE

Mucin1 expression is enriched in the human stem cell fraction of cord blood and is upregulated in majority of the AML cases

Objective. Mucin1 is a membrane glycoprotein that is overexpressed in a variety of human cancers. Here, we analyzed the role of Mucin1 in human hematopoietic stem/progenitor cells as well as in acute myeloid leukemia (AML) cells. Materials and Methods. Mucin1 expression was determined within the normal stem cell and progenitor compartment, as well as in the AML CD34(+) and CD34(-) subfractions of patient samples. Stem cells were enumerated in long-term culture-initiating cell (LTC-IC) assays in limiting dilution and progenitor frequencies in colony-forming cell (CFC) assays in methylcellulose, and consequences of elevated Mucin I expression were studied using retroviral overexpression systems in cord blood (CB) CD34(+) cells. Results. Ten percent of CB and 5% of peripheral blood CD34(+) cells expressed Mucin1. Retroviral overexpression of Mucin1 in CB CD34(+) cells resulted in elevated stem cell and progenitor frequencies as determined in LTC-IC and CFC assays without affecting differentiation, which coincided with increased proliferation. Overexpression of intercellular adhesion molecule-1, a ligand for Mucin1, in MS5 stromal cells further increased LTC-IC frequencies. Mucin1 overexpression was associated with increased nuclear factor-kappa B p50 nuclear translocation, suggesting that Mucin1-induced phenotypes involve increased cell survival mechanisms. Finally, we observed increased Mucin1 expression in 70% of the AML cases (n = 24), suggesting that elevated Mucin1 levels might be involved in regulating the proliferative potential of the immature leukemic compartment as well. Conclusions. Our data indicate that hematopoietic stem cells as well as CD34(+) AML subfractions are enriched for Mucin1 expression, and that overexpression of Mucin1 in CB cells is sufficient to increase both progenitor and LTC-IC frequencies. (C) 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc

Identification of HIF2 alpha as an important STAT5 target gene in human hematopoietic stem cells

The transcription factor signal transducer and activator of transcription 5 (STAT5) fulfills essential roles in self-renewal in mouse and human hematopoietic stem cells (HSCs), and its persistent activation contributes to leukemic transformation, although little molecular insight into the underlying mechanisms has been obtained. In the present study, we show that STAT5 can impose long-term expansion exclusively on human HSCs, not on progenitors. This was associated with an enhanced cobblestone formation under bone marrow stromal cells of STAT5-transduced HSCs. Hypoxia-induced factor 2 alpha (HIF2 alpha) was identified as a STAT5 target gene in HSCs, and chromatin immunoprecipitation studies revealed STAT5 binding to a site 344 base pairs upstream of the start codon of HIF2 alpha. Lentiviral RNA interference (RNAi)-mediated down-modulation of HIF2 alpha impaired STAT5-induced long-term expansion and HSC frequencies, whereas differentiation was not affected. Glucose uptake was elevated in STAT5-activated HSCs, and several genes associated with glucose metabolism were up-regulated by STAT5 in an HIF2 alpha-dependent manner. Our studies indicate that pathways normally activated under hypoxia might be used by STAT5 under higher oxygen conditions to maintain and/or impose HSC self-renewal properties. (Blood. 2011; 117(12):3320-3330