Unknown,Transcriptomics,Genomics,Proteomics

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Identification of differentially regulated genes upon overexpression of HLX in wild-type hematopoietic stem cells

ABSTRACT: The goal was to study the role of Hlx in hematopoiesis. Sorted Lin-Kit+Sca-1+ cells from wild-type FVB/nJ bone marrow were infected with control (pCAD-IRES-GFP) or Hlx lentivirus (pCAD-IRES-GFP-Hlx) and cultured for 2 days in Iscove’s modified Dulbecco’s medium (IMDM) containing FBS, mIL-3, mIL-6 and mSCF with lentiviral supernatants in the presence of 8ug/ml polybrene. Subsequently, GFP+ cells were sorted by FACS and RNA was prepared. Three replicates of each, control vector transduced and HLX-transduced cells were used.

ORGANISM(S): Mus musculus

SUBMITTER: Boris Bartholdy

PROVIDER: E-GEOD-27938 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

H2.0-like homeobox regulates early hematopoiesis and promotes acute myeloid leukemia.

Kawahara Masahiro M Pandolfi Ashley A Bartholdy Boris B Barreyro Laura L Will Britta B Roth Michael M Okoye-Okafor Ujunwa C UC Todorova Tihomira I TI Figueroa Maria E ME Melnick Ari A Mitsiades Constantine S CS Steidl Ulrich U

Cancer cell 20120801 2

Homeobox domain-containing transcription factors are important regulators of hematopoiesis. Here, we report that increased levels of nonclustered H2.0-like homeobox (HLX) lead to loss of functional hematopoietic stem cells and formation of aberrant progenitors with unlimited serial clonogenicity and blocked differentiation. Inhibition of HLX reduces proliferation and clonogenicity of leukemia cells, overcomes the differentiation block, and leads to prolonged survival. HLX regulates a transcripti ...[more]

PMID: 22897850

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Project description:High levels of Hes1 expression are frequently found in BCR-ABL-positive chronic myelogenous leukemia in blast crisis (CML-BC). In mouse bone marrow transplantation (BMT) models, co-expression of BCR-ABL and Hes1 induces CML-BC–like disease; however the underlying mechanism remained elusive. Here, based on gene expression analysis, we show that MMP-9 is upregulated by Hes1 in common myeloid progenitors (CMPs). Analysis of promoter activity demonstrated that Hes1 upregulated MMP-9 by activating NF-kB. Analysis of 20 samples from CML-BC patients showed that MMP-9 was highly expressed in three, with two exhibiting high levels of Hes1 expression. Interestingly, MMP-9 deficiency impaired the cobblestone area-forming ability of CMPs expressing BCR-ABL and Hes1 that were in conjunction with a stromal cell layer. In addition, these CMPs secreted MMP-9, promoting the release of soluble Kit-ligand (sKitL) from stromal cells, thereby enhancing proliferation of the leukemic cells. In accordance, mice transplanted with CMPs expressing BCR-ABL and Hes1 exhibited high levels of sKitL as well as MMP-9 in the serum. Importantly, MMP-9 deficiency impaired the development of CML-BC–like disease induced by BCR-ABL and Hes1 in mouse BMT models. The present results suggest that Hes1 promotes the development of CML-BC, partly through MMP-9 upregulation in leukemic cells. Common myeloid progenitors (CMPs; Lineage negative, c-Kit positive, Sca-1 negative, Fc-gamma-receptor low, CD34 positive fraction) were sorted with a FACSAria cell sorter (Becton Dickinson). Retroviruses were generated by transfecting Plat-E packaging cells with retrovirus vector pMYs-Hes1-IRES-GFP or empty vector (pMYs-IRES-GFP) using FuGENE 6 (Roche Diagnostics). Infection of retrovirus harboring Hes1 (pMYs-Hes1-IRES-GFP) or empty vector (pMYs-IRES-GFP) into progenitors was performed using RetroNectin (Takara Bio). Hes1-transfected CMPs and Mock-transduced CMPs were isolated 36 hours after infection with a FACSAria cell sorter. One sample of Hes1-transfected CMPs and one sample of mock-transduced CMPs were analyzed with GeneChip Mouse Genome 430 2.0 Array.

Project description:Aberrant expression of the homeodomain transcription factor CDX2 occurs in most cases of acute myeloid leukemia (AML) and promotes leukemogenesis, making CDX2, in principle, an attractive therapeutic target. Conversely, CDX2 acts as a tumor suppressor in colonic epithelium. The effectors mediating the leukemogenic activity of CDX2 and the mechanism underlying its context-dependent properties are poorly characterized, and strategies for interfering with CDX2 function in AML remain elusive. We report data implicating repression of the transcription factor KLF4 as important for the oncogenic activity of CDX2, and demonstrate that CDX2 differentially regulates KLF4 in AML versus colon cancer cells through a mechanism that involves tissue-specific patterns of promoter binding and epigenetic modifications. Furthermore, we identified deregulation of the PPARγ signaling pathway as a feature of AML expressing CDX2, and observed that PPARγ agonists derepress KLF4 and are preferentially toxic to CDX2-positive leukemic cells. These data delineate transcriptional programs associated with CDX2 expression in hematopoietic cells; provide insight into the antagonistic duality of CDX2 function in AML versus colon cancer; and suggest reactivation of KLF4 expression, through modulation of PPARγ signaling, as a new therapeutic modality in a large proportion of AML patients. Experiment 1 (Samples 1-6): The transcriptional changes induced by Cdx2 were assessed ex vivo in primary murine hematopoietic stem and progenitor cells. Bone marrow cells were harvested from 3x15 C57BL/6 mice, and differentiated cells were removed by incubation with rat antibodies against lineage antigens (CD3, CD4, CD8, Gr-1, B220, CD19, IL-7R, Ter119) followed by depletion with magnetic beads (Dynabeads, Invitrogen). Lineage-depleted cells were stained with APC-conjugated anti-c-Kit and PE-Cy5-conjugated goat anti-rat antibodies, and c-Kit+ Lin– cells were sorted using a BD FACSAria cell sorter (BD Biosciences). Cells were plated in RetroNectin-coated tissue culture dishes (Takara Bio) and transduced twice with pMSCV-Cdx2-IRES-GFP or pMSCV-IRES-GFP retroviral constructs. After 48 hours, GFP-positive cells were sorted using a BD FACSAria cell sorter. Experiment 2 (Samples 7-10): The transcriptional changes induced by Cdx2 were assessed in vivo in Cdx2-initiated murine leukemias and compared with those of leukemias initiated by 5 different MLL fusion oncogenes (previously published data, available at GSE13690). Bone marrow cells isolated from C57BL/6 mice were transduced with pMSCV-Cdx2-IRES-GFP, and 8x10e5 GFP-positive cells were injected into lethally irradiated syngeneic recipient mice after 48 hours, followed by injection of 1x10e6 spleen cells from primary leukemic animals into sublethally irradiated secondary recipients. Spleen cells from secondary leukemic animals were harvested.

Dataset Information

Identification of differentially regulated genes upon overexpression of HLX in wild-type hematopoietic stem cells

Publications

H2.0-like homeobox regulates early hematopoiesis and promotes acute myeloid leukemia.

Similar Datasets

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