The AML1-MTG8 Leukemic Fusion Protein Forms a Complex with a Novel Member of the MTG8(ETO/CDR) Family, MTGR1

ABSTRACT The AML1-CBFβ transcription factor complex is essential for the definitive hematopoiesis of all lineages and is the most frequent target of chromosomal rearrangements in human leukemia. In the t(8;21) translocation associated with acute myeloid leukemia (AML), theAML1(CBFA2/PEBP2αB) gene is juxtaposed to theMTG8(ETO/CDR) gene. We show here that the resultant AML1-MTG8 gene product specifically and strongly interacts with an 85-kDa phosphoprotein. Molecular cloning of cDNA indicated that the AML1-MTG8-binding protein (MTGR1) is highly related to MTG8 and similar to Drosophila Nervy. Comparison of amino acid sequences among MTGR1, MTG8, and Nervy revealed four evolutionarily conserved regions (NHR1 to NHR4). Ectopic expression of AML1-MTG8 in L-G murine myeloid progenitor cells inhibits differentiation to mature neutrophils and induces cell proliferation in response to granulocyte colony-stimulating factor (G-CSF). Analysis with C-terminal deletion mutants of AML1-MTG8 indicated that the region of 51 residues (488 to 538), which contains NHR2, is essential for the induction of G-CSF-dependent cell proliferation. Immunoprecipitation analysis indicates that this region is required for AML1-MTG8 to form a stable complex with MTGR1. Overexpression of MTGR1 stimulates AML1-MTG8 to induce G-CSF-dependent proliferation of L-G cells and to interfere with AML1-dependent transcription. These results suggest that AML1-MTG8 could function as a complex with MTGR1 and that the complex might be important in promoting leukemogenesis.

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Myc Induces Acute Myeloid Leukemia by Conferring Self-Renewal Activity to Committed Myeloid Progenitor Cells That Resist Apoptosis Via Endogenous Mcl-1.

Blood ◽

10.1182/blood.v110.11.2634.2634 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2634-2634

Author(s):

Hui Luo ◽

Jennifer A. Cain ◽

AnnaLynn Molitoris ◽

Joseph Opferman ◽

Michael H. Tomasson

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Median Survival ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Ectopic Expression ◽

Hematopoietic Stem ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells ◽

Induced Apoptosis

Abstract Ectopic expression of Myc in most primary cell types induces apoptosis, and cancer development typically requires additional, anti-apoptotic mutations. We reported previously that ectopic expression of Myc in unfractionated murine bone marrow cells induced rapid onset acute myeloid leukemia (AML) without detectable anti-apoptotic mutations. We hypothesized that AML developed in our model because a subset of normal primary bone marrow cells were inherently resistant to Myc-induced apoptosis. Consistent with this model, seven days of Myc activation in the bone marrow of mice caused the reduction of B-lineage cells while at the same time inducing the expansion of myeloid lineage cells. We sought to determine the mechanism by which myeloid progenitor cells evaded Myc-induced apoptosis, and found that Myc-induced AML cells exhibited a distinct profile of pro- and anti-apoptotic proteins, including high levels of the anti-apoptotic Bcl-2 family member Mcl-1. To prioritize apoptosis genes, we examined AML patient microarray data and found MCL1 to be uniformly expressed at high levels in human AML (94/94, 100%). We used Mcl1 heterozygous mice (Mcl1F/null) as bone marrow donors for transduction-transplantation experiments and found that, compared with Mcl1 wild-type (median survival=60 days), haploinsufficiency for Mcl1 completely protected mice from Myc-induced AML (median survival not reached). Mice transplanted with Mcl1F/null cells co-expressing Myc and Bcl2 succumbed rapidly to disease (median survival 25 days). In wild-type mice, defined hematopoietic stem and myeloid progenitor cell populations were not significantly increased by Myc activation. However, Myc transduction conferred serial replating ability to sorted hematopoietic stem and progenitor cells including lineage-committed (Lin+Kit+) progenitors cells. These data demonstrate a critical role for Mcl1 in our AML model and suggest that dysregulation of MYC in MCL1-expressing progenitor cells may mediate AML pathogenesis in humans.

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Identification of functional cooperative mutations of GNAO1 in human acute lymphoblastic leukemia

Blood ◽

10.1182/blood.2020005622 ◽

2020 ◽

Cited By ~ 1

Author(s):

Lili Song ◽

Bo Yu ◽

Yi Yang ◽

Jianwei Liang ◽

Yingwen Zhang ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Chromosomal Rearrangements ◽

Ectopic Expression ◽

Monozygotic Twins ◽

Human Leukemia ◽

Missense Mutations ◽

Protein Subunit ◽

Whole Exome ◽

Human Acute Lymphoblastic Leukemia

Leukemogenesis is characterized by chromosomal rearrangements with additional molecular disruptions, yet the cooperative mechanisms are still unclear. Using whole-exome sequencing of a pair of monozygotic twins discordant for childhood acute lymphoblastic leukemia (ALL) with ETV6-RUNX1 (E/R) gene fusion successively after birth, we identified the R209C mutation of G protein subunit alpha o1 (GNAO1) as a new ALL risk loci. Moreover, GNAO1 missense mutations are only recurrent in ALL patients and are associated with E/R fusion. Ectopic expression of the GNAO1 R209C mutant increased its GTPase activity and promoted cell proliferation and cell neoplastic transformation. Combined with the E/R fusion, the GNAO1 R209C mutant promoted leukemogenesis through activating PI3K/Akt/mTOR signaling. Reciprocally, activated mTORC1 phosphorylated p300 acetyltransferase, which acetylated E/R and thereby enhanced the E/R transcriptional activity of GNAO1 R209C. Thus, our study provides clinical evidence for the functional cooperation of GNAO1 mutants and E/R fusion, suggesting GNAO1 as a potential therapeutic target in human leukemia.

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AML1-MTG8 leukemic protein induces the expression of granulocyte colony-stimulating factor (G-CSF) receptor through the up-regulation of CCAAT/enhancer binding protein epsilon

Blood ◽

10.1182/blood.v96.1.288.013k05_288_296 ◽

2000 ◽

Vol 96 (1) ◽

pp. 288-296 ◽

Cited By ~ 6

Author(s):

Kimiko Shimizu ◽

Issay Kitabayashi ◽

Nanao Kamada ◽

Tatsuo Abe ◽

Nobuo Maseki ◽

...

Keyword(s):

Cell Proliferation ◽

Binding Protein ◽

Ectopic Expression ◽

Precursor Cells ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

G Cells ◽

Enhancer Binding Protein ◽

Dependent Cell ◽

Stimulating Factor

The t(8;21) translocation is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). In this translocation, the AML1 (CBFA2/PEBP2aB) gene is disrupted and fused to the MTG8 (ETO) gene. The ectopic expression of the resulting AML1-MTG8 fusion gene product in L-G and 32Dcl3 murine myeloid precursor cells stimulates cell proliferation without inducing morphologic terminal differentiation into mature granulocytes in response to granulocyte-colony stimulating factor (G-CSF). This study found that the ectopic expression of AML1-MTG8 elevates the expression of the G-CSF receptor (G-CSFR). Analysis of the promoter region of the G-CSFR gene revealed that up-regulation of G-CSFR expression by AML1-MTG8 does not depend on the AML1-binding sequence, but on the C/EBP (CCAAT/enhancer binding protein) binding site. The results suggest that the overproduction of G-CSFR is at least partly mediated by C/EBPɛ, whose expression is activated by AML1-MTG8. The ectopic expression of G-CSFR in L-G cells induced cell proliferation in response to G-CSF, but did not inhibit cell differentiation into mature neutrophils. Overexpression of C/EBPɛ in L-G cells also stimulated G-CSF–dependent cell proliferation. High expression levels of G-CSFR were also found in the leukemic cells of AML patients with t(8;21). Therefore, G-CSF–dependent cell proliferation of myeloid precursor cells may be implicated in leukemogenesis.

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The cadherins: cell-cell adhesion molecules controlling animal morphogenesis

Development ◽

10.1242/dev.102.4.639 ◽

1988 ◽

Vol 102 (4) ◽

pp. 639-655 ◽

Cited By ~ 51

Author(s):

M. Takeichi

Keyword(s):

Cell Adhesion ◽

Ectopic Expression ◽

Amino Acid Sequences ◽

Single Animal ◽

Cell Layers ◽

Dependent Cell ◽

Cell Collective ◽

Cell Cell

Cadherins are a family of glycoproteins involved in the Ca2+-dependent cell-cell adhesion mechanism which is detected in most kinds of tissues. Inhibition of the cadherin activity with antibodies induces dissociation of cell layers, indicating a fundamental importance of these molecules in maintaining the multicellular structure. Cadherins are divided into subclasses, including E-, N- and P-cadherins. While all subclasses are similar in molecular weight, Ca2+- and protease-sensitivity, each subclass is characterized by a unique tissue distribution pattern and immunological specificity. Analysis of amino acid sequences deduced from cDNA encoding these molecules showed that they are integral membrane proteins of 723–748 amino acids long and share common sequences; similarity in the sequences between subclasses is in a range of 50–60% when compared within a single animal species. L cells, with very little endogenous cadherin activity, transfected with the cadherin cDNA acquired high cadherin-mediated aggregating activity. Their colony morphology was altered by the ectopic expression of cadherins from the dispersed type to the compact type, providing direct evidence for a key role of cadherins in cell-cell adhesion. It has been suggested that cadherins bind cells by their homophilic interactions at the extracellular domain and are associated with actin bundles at the cytoplasmic domain. It appears that each cadherin subclass has binding specificity and this molecular family is involved in selective cell-cell adhesion. In development, the expression of each cadherin subclass is spatiotemporally regulated and associated with a variety of morphogenetic events; e.g. the termination or initiation of expression of a cadherin subclass in a given cell collective is correlated with its segregation from or connection with other cell collectives. Antibodies to cadherins were shown to perturb the morphogenesis of some embryonic organs in vitro. These observations suggest that cadherins play a crucial role in construction of tissues and the whole animal body.

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Oxidation state governs structural transitions in peroxiredoxin II that correlate with cell cycle arrest and recovery

The Journal of Cell Biology ◽

10.1083/jcb.200606005 ◽

2006 ◽

Vol 175 (5) ◽

pp. 779-789 ◽

Cited By ~ 101

Author(s):

Timothy J. Phalen ◽

Kelly Weirather ◽

Paula B. Deming ◽

Vikas Anathy ◽

Alan K. Howe ◽

...

Keyword(s):

Cell Cycle ◽

Hydrogen Peroxide ◽

Cell Proliferation ◽

Cell Cycle Arrest ◽

Ectopic Expression ◽

Structural Transitions ◽

Mitogenic Signaling ◽

Cycle Arrest ◽

Subcellular Processes ◽

Dependent Cell

Inactivation of eukaryotic 2-Cys peroxiredoxins (Prxs) by hyperoxidation has been proposed to promote accumulation of hydrogen peroxide (H2O2) for redox-dependent signaling events. We examined the oxidation and oligomeric states of PrxI and -II in epithelial cells during mitogenic signaling and in response to fluxes of H2O2. During normal mitogenic signaling, hyperoxidation of PrxI and -II was not detected. In contrast, H2O2-dependent cell cycle arrest was correlated with hyperoxidation of PrxII, which resulted in quantitative recruitment of ∼66- and ∼140-kD PrxII complexes into large filamentous oligomers. Expression of cyclin D1 and cell proliferation did not resume until PrxII-SO2H was reduced and native PrxII complexes were regenerated. Ectopic expression of PrxI or -II increased Prx-SO2H levels in response to oxidant exposure and failed to protect cells from arrest. We propose a model in which Prxs function as peroxide dosimeters in subcellular processes that involve redox cycling, with hyperoxidation controlling structural transitions that alert cells of perturbations in peroxide homeostasis.

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AML1-MTG8 leukemic protein induces the expression of granulocyte colony-stimulating factor (G-CSF) receptor through the up-regulation of CCAAT/enhancer binding protein epsilon

Blood ◽

10.1182/blood.v96.1.288 ◽

2000 ◽

Vol 96 (1) ◽

pp. 288-296 ◽

Cited By ~ 44

Author(s):

Kimiko Shimizu ◽

Issay Kitabayashi ◽

Nanao Kamada ◽

Tatsuo Abe ◽

Nobuo Maseki ◽

...

Keyword(s):

Cell Proliferation ◽

Binding Protein ◽

Ectopic Expression ◽

Precursor Cells ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

G Cells ◽

Enhancer Binding Protein ◽

Dependent Cell ◽

Stimulating Factor

Abstract The t(8;21) translocation is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). In this translocation, the AML1 (CBFA2/PEBP2aB) gene is disrupted and fused to the MTG8 (ETO) gene. The ectopic expression of the resulting AML1-MTG8 fusion gene product in L-G and 32Dcl3 murine myeloid precursor cells stimulates cell proliferation without inducing morphologic terminal differentiation into mature granulocytes in response to granulocyte-colony stimulating factor (G-CSF). This study found that the ectopic expression of AML1-MTG8 elevates the expression of the G-CSF receptor (G-CSFR). Analysis of the promoter region of the G-CSFR gene revealed that up-regulation of G-CSFR expression by AML1-MTG8 does not depend on the AML1-binding sequence, but on the C/EBP (CCAAT/enhancer binding protein) binding site. The results suggest that the overproduction of G-CSFR is at least partly mediated by C/EBPɛ, whose expression is activated by AML1-MTG8. The ectopic expression of G-CSFR in L-G cells induced cell proliferation in response to G-CSF, but did not inhibit cell differentiation into mature neutrophils. Overexpression of C/EBPɛ in L-G cells also stimulated G-CSF–dependent cell proliferation. High expression levels of G-CSFR were also found in the leukemic cells of AML patients with t(8;21). Therefore, G-CSF–dependent cell proliferation of myeloid precursor cells may be implicated in leukemogenesis.

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Regulated Hox Gene Expression Reveals a Novel Role for Hox Genes in Myeloid Cell Proliferation and Survival

Blood ◽

10.1182/blood.v112.11.5323.5323 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5323-5323

Author(s):

Marika Salmanidis ◽

Gabi Brumatti ◽

Anissa M Jabbour ◽

Benjamin D Green ◽

John Silke ◽

...

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Cell Death ◽

Growth Factor ◽

Myeloid Leukaemia ◽

Cell Lines ◽

Hox Genes ◽

Hox Gene ◽

Myeloid Progenitor ◽

Dependent Cell

Abstract The Hox family of homeodomain transcription factors are essential for the regulation of hematopoiesis and deregulated expression of some Hox gene is associated with the development of myeloproliferative disorders and leukaemia. In mammals, 39 Hox genes are organized into four clusters (A, B, C or D). The expression of these genes is tightly regulated at particular differentiation points in haematopoiesis. Importantly, the over-expression of Hox genes, HoxB4, HoxA9 and HoxA10 is frequent in acute myeloid leukaemia, and may arise as a result of MLL rearrangements or from translocations fusing Hox genes to the nucleoporin Nup98. Overexpression of murine HoxB8 together with IL-3 results in myeloid leukaemia in mice. Primary myeloid progenitor cells can be immortalised using retroviral expression of Homeobox genes HoxB8 or HoxA9 in the presence of exogenous growth factors Interleukin-3 (IL-3) or GM-CSF. We have exploited this observation to generate IL-3 dependent cell lines from gene-deleted mice to identify which members of the Bcl-2 family of apoptosis regulators are required for apoptosis provoked by IL-3 deprivation (Blood, 2006 108:1461-8). Using a unique lentiviral expression system we have now generated IL-3 dependent myeloid progenitor cell lines in which we can regulate the expression of wild-type (untagged) HoxB8 or HoxA9 using 4-hydroxy tamoxifen (4HT), to determine how these genes immortalise myeloid cells. The mechanisms of action of Hox proteins in leukaemiagenesis remain to be determined but are thought, in part at least, to result from a block in myeloid differentiation. Conditional (growth-factor dependent) immortalisation of myeloid progenitors was possible only in the presence of induced Hox gene expression and surprisingly, withdrawal of HoxB8 expression did not result in terminal differentiation of all cells. Instead, loss of Hox expression, even in the presence of IL-3, induced Go/G1 cell cycle arrest and caspase-dependent cell death. This death was substantially slower that that induced by IL-3 deprivation, indicating that for some time at least, survival signals transduced by IL-3 remained intact. Thus whilst the IL-3 survival signal persisted, the proliferative signal was inhibited. We also show that HoxB8 regulates expression of the pro-apoptotic Bcl-2 family member Bim, since loss of HoxB8 resulted in substantially increased Bim expression and the cell death induced by loss of HoxB8 expression was inhibited in Bim-deficient cells. Importantly, re-addition of 4HT to cell cultures after various periods of no HoxB8 expression restored HoxB8 expression and resulted in an increase in cell viability, cell proliferation and decrease of Bim expression, indicating that at least some cells without HoxB8 expression have not terminally differentiated and retain the ability to proliferate. Our results suggest that overexpression of Hox genes such as HoxB8 (or HoxA9) contribute to myeloid transformation by coupling a growth factor signal to proliferation and also regulate the apoptotic machinery. Using this system will be able to provide proof of principal that leukemia-associated Hox genes are valid therapeutic targets.

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