Roles of MOZ in Hematopoiesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2269-2269
Author(s):  
Takuo Katsumoto ◽  
Yukiko Aikawa ◽  
Takahiro Ochiya ◽  
Issay Kitabayashi
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fetal Liver ◽  
Liver Cells ◽  
Recipient Mouse ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Homozygous Mutant ◽  
Fetal Liver Cells ◽  
Acute Myeloid

Abstract The AML1-CBFβ transcription factor complex is the most frequent target of specific chromosome translocations in acute myeloid leukemia (AML). The monocytic leukemia zinc finger (MOZ) gene, which encodes a MYST-type histone acetyltransferase (HAT), is also involved in leukemia-associated translocations such as t(8;16), t(8;22) and inv(8), which are associated with acute myeloid leukemia with M4/5 subtypes. We previously found that MOZ functions as a potent coactivator for AML1. To investigate roles of MOZ in normal hematopoiesis, we generated MOZ-deficient mice using gene-targeting method. MOZ homozygous mutant is embryonic lethal and it died between days 14 and 15 of gestation. In fetal liver of MOZ-deficient E14.5 embryos, the total cell numbers and the colony-forming cells (CFCs) in a methylcellulose medium were remarkably reduced when compared with wild-type littermates. Flow cytometry analysis indicated that hematopoietic stem cells (HSCs) and progenitors of both myeloid and lymphoid lineages were severely reduced in MOZ-deficient embryos. Especially, the levels of c-kit expression were strongly reduced in lineage-negative cells. Differentiation arrest of erythroid progenitors at a terminal stage and increase in the numbers of Mac-1 and Gr-1 positive cells suggest that MOZ also plays roles in cell differentiation of erythroid, monocytic and granulocytic lineages. In E12.5 MOZ deficient fetal liver cells, expression profile analysis revealed decreases in expressions of thrombopoietin receptor c-mpl, Wnt related ligand dkk2 and HoxA9 and increase in HoxA5 expression. To further determine roles of MOZ in HSCs functions and their progenitors differentiation ability, competitive reconstitution assays were performed. Ly5.2+ fetal liver cells from wild-type, heterozygous or homozygous mutant embryos together with Ly5.1+ competitor fetal liver cells were transplanted into γ-irradiated Ly5.1+/Ly5.2+ recipient mouse. Ly5.2+ wild-type cells were observed in recipient mice after transplantation. However, cells derived from MOZ homozygous mutant embryos were not detected in peripheral blood, bone marrow, spleen and thymus. Reduced population of cells derived from heterozygous mutant embryos were observed. These data suggest that MOZ is required for lymphoid and myeloid development and for self-renewal of HSCs.

A Critical Role of YB-1 In NPMc-Induced Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3149-3149
Author(s):  
Yoko Ogawara ◽  
Takuo Katsumoto ◽  
Takeshi Uchiumi ◽  
Kimitoshi Kohno ◽  
Issay Kitabayashi
Keyword(s):  
Acute Myeloid Leukemia ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Embryonic Lethal ◽  
Hoxa Genes ◽  
Fetal Liver Cells ◽  
Acute Myeloid

Abstract Abstract 3149 Frameshift mutations in Nucleophosmin gene (NPM) are the most frequent abnormality in acute myeloid leukemia (AML), found in approximately 30% of all cases and 50% of patients with normal karyotype (NK) AML. NPM mutations result in an aberrant cytoplasmic localization of NPM protein (NPMc) through a loss of nucleolar localization signal accompanied by acquisition of new nuclear export signal. NPM mutations are heterozygous, so the other wild-type allele is consistently retained. NPMc binds to wild-type NPM through oligomerization domain and impairs its activity by delocalizing to the cytoplasm. It was reported that the NPM-null mice are early embryonic lethal and defective in primary hematopoiesis, suggesting important roles of NPM in early hematopoiesis. However, the molecular mechanism by which NPMc exerts its leukemogenic potential has never been established. Here we show that ectopic expression of NPMc, but not wild type (WT) NPM, in mouse bone marrow (BM) cells enhanced their colony formation activity in methylcellulose media. Increased expression of HoxA7, 9 and 10 genes were observed in cells expressing NPMc but not in those expressing WT NPM. It has been reported that the expression levels of HOXA genes are upregulated in various types of AML including NPMc+ AML. Since overexpression of HoxA9 immortalizes hematopoietic progenitor cells, our findings suggest that up-regulation of HoxA genes are involved in NPMc-mediated leukemogenesis. To clarify roles of NPMc in leukemogenesis, we purified the NPM protein complex and identified Y box-binding protein 1 (YB-1) as a binding partner for NPM. YB-1 belongs to the cold shock family and functions in gene transcription and RNA processing. YB-1 strongly bound to WT NPM but not to NPMc. In addition, interaction between YB-1 and NPM was impaired in the presence of NPMc. YB-1-deficient mice were embryonic lethal and their fetal liver were small. YB-1-deficient yolk sac cells showed decreased colony-forming activity, and decreased number of hematopoietic cells were observed when AGM region of YB-1-deficeint embryo were cultured on OP9 cells. Furthermore, expression of Hoxa9 was decreased in fetal liver cells derived from YB-1 knockout mice. To investigate the roles of YB-1 in NPMc-associated leukemogenesis, WT and YB-1-null E14.5 fetal liver cells were infected with retrovirus expressing NPMc. Analyses of colony-forming activity and mRNA expression showed that YB-1 was essential for NPMc-induced increases in colony formation activity as well as in expression of HoxA genes. However, YB-1 was not necessary for colony formation activity induced by other AML-associated fusion genes, such as AML1-MTG8 and MLL-AF10. These data indicate that YB-1 is specifically required for NPMc-induced leukemogenic transformation of hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Caspase-3 Can Promote Acute Myeloid Leukemia Development By Regulation of Autophagy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2714-2714
Author(s):  
Na Man ◽  
Yurong Tan ◽  
Fan Liu ◽  
Guoyan Cheng ◽  
Sarah Merin Greenblatt ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Fetal Liver ◽  
Liver Cells ◽  
Self Renewal ◽  
Leukemia Development ◽  
Fetal Liver Cells ◽  
Acute Myeloid

Abstract Background AML1-ETO (AE), a oncogenic protein generated by the t(8;21) translocation, causes acute myeloid leukemia (AML) in collaboration with other secondary events. The leukemogenicity of AE has been evaluated in multiple mouse models, such as expression of AE in Cdkn1a-null Hematopoietic stem cell (HSCs) and expression of AML1- ETO9a (AE9a), an alternatively spliced variant of AE, in WT HSCs. Both lead to the development of fully penetrant AML. Caspase-3 plays multiple roles in hematopoietic development and leukemia progression and treatment by affecting proliferation, self-renewal and differentiation. It has been shown that uncleaved caspase-3 levels are higher in the peripheral blood cells of AML patients compared to normal individuals, which suggests that the caspase pathway is dysregulated in AML. We and others have shown that Caspase-3 directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3 compromised background and accelerate leukemogenesis. Methods We developed a Caspase-3 knockout genetic mouse model of AML based on fetal liver cell transplantation. In brief, fetal liver cells from WT or Caspase3-/- mice were transduced to express AE9a in vitroand 100,000 AE9a+ transduced cells were transplanted into lethally irradiated recipient mice by tail-vein injection. Results We found loss of Caspase-3 impaired leukemia stem cell (LSC) self-renewal and delayed AE9a-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation or progression of AML. Moreover, we identified a new substrate of Caspase-3, ULK1, by in vitro cleavage assays and site-directed mutagenesis. ULK1 (serine/threonine UNC-51-like kinase) is the homology of Atg1 (the first autophagy related gene found in 1997) in mammalian cells, which is a direct target of mTOR and is responsible for initiation of the autophagic activity by forming a complex with mAtg13, FIP200 and Atg101. The induction of autophagy caused by upregulation of ULK1 in AE/AE9a-expressing Caspase-3-/- fetal liver cells acted to limit the leukemogenicity of AE9a in vivo. Inhibition of ULK1 by inhibitor or shRNAs could rescue the self-renewal capability induced by Caspase-3 deletion in serial replating assays. Unexpectedly, when we expressed AE/AE9a in fetal liver cells from WT and Caspase-3-/- mice, the protein levels were comparable suggesting the basal level of Caspase-3 didn't affect the expressing of AE/AE9a in fetal liver cells. Conclusion Autophagy may play a general role in the development and treatment of leukemia. In human AML, blasts display reduced expression of autophagy-related genes and decreased autophagic flux, indicating that low autophagy activity provides a general advantage for leukemia development. Beside this, a number of chemotherapy drugs have been reported to be able to induce leukemia cell death via activation of autophagy suggesting that autophagy plays critical roles in the leukemia treatment. Our study reveals that Caspase-3 regulates autophagy through its direct cleavage of ULK1 and this interaction dictates the pace of AE-driven leukemogenesis. Targeting this pathway may have therapeutic benefit for AML treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bclaf1 Promotes Maintenance and Self-Renewal of Fetal Hematopoietic Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1269-1269 ◽  
Author(s):  
Lynn S. White ◽  
Deepti Soodgupta ◽  
Rachel L. Johnston ◽  
Jeffrey A. Magee ◽  
Jeffrey J. Bednarski
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Liver Cells ◽  
Lower Percentage ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Abstract Hematopoietic stem cells (HSC) persist throughout life by undergoing extensive self-renewal divisions while maintaining an undifferentiated state. The mechanisms that support HSC self-renewal change throughout the course of development as temporal changes in transcriptional regulators coordinate distinct genetic programs in fetal, post-natal and adult HSCs. These self-renewal programs are often ectopically activated in leukemia cells to drive neoplastic proliferation and high expression of HSC-associated genes predicts a poor prognosis in acute myelogenous leukemia (AML). In this regard, it was recently shown that expression of the transcriptional regulator BCLAF1 (Bcl2 associated transcription factor 1) is increased in AML blasts relative to normal precursor populations and suppression of BCLAF1 causes reduced proliferation and induction of differentiation to a dendritic cell fate. These findings raise the question of whether BCLAF1 may regulate normal as well as neoplastic self-renewal programs. We find that Bclaf1 is highly expressed in HSCs versus committed bone marrow populations consistent with a potential role for this gene in HSC functions. To test whether BCLAF1 regulates HSC development and hematopoiesis, we used germline loss of function mice. Bclaf1-/- mice succumb to pulmonary failure shortly after birth due to poor lung development, so we assessed prenatal hematopoiesis. Bclaf1-deficient mice had significantly reduced HSC and hematopoietic progenitor cell (HPC) frequencies and numbers despite normal fetal liver cellularity. To determine if Bclaf1 is required for HSC function during fetal development, we performed competitive reconstitution assays. Fetal liver cells from Bclaf1+/+or Bclaf1-/-mice were transplanted along with wild-type competitor bone marrow cells into lethally irradiated recipient mice. Compared to recipients of Bclaf1+/+fetal liver cells, recipients of Bclaf1-/-cells had a significantly lower percentage of donor-derived leukocytes at all time points after transplantation as well as reduced percentage of donor HSCs at 16 weeks post-transplant. Notably, all leukocyte populations (B cells, T cells, granulocytes and macrophages) from Bclaf1-/-donors were reduced consistent with an abnormality in HSC repopulating activity rather than a defect in a specific differentiation pathway. Consistent with these findings, Bclaf-deficient cells did not engraft in competitive transplants with limiting numbers of sorted fetal liver HSCs whereas sorted wild-type Bclaf1+/+cells effectively reconstituted hematopoiesis in recipient mice. In addition, Vav-cre:Bclaf1flox/floxmice, which have selective deletion of Bclaf1 in hematopoietic cells, have reduced frequencies and numbers of fetal liver HSCs identical to the findings observed in germline Bclaf1-/-mice. These results show that loss of Bclaf1 leads to defective development and repopulating activity of fetal HSCs. Interestingly, when adult mice are successfully engrafted with Bclaf1-deficient HSCs, the donor HSCs suffer no additional functional impairment. Furthermore, in secondary transplant experiments Bclaf1-deficient HSCs maintain long-term repopulating activity. Thus, Bclaf1 may have distinct functions in fetal versus adult HSC self-renewal. Collectively, our findings reveal Bclaf1 is a novel regulator of fetal HSC function and suggest that it may have distinct functions in different developmental contexts. Disclosures No relevant conflicts of interest to declare.

Roles of Histone Acetyltransferase MORF and MOZ in Hematopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2525-2525
Author(s):  
Takuo Katsumoto ◽  
Issay Kitabayashi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Liver Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Abstract Abstract 2525 Poster Board II-502 MOZ (MOnocytic leukemia Zinc finger protein) and MORF (MOz Related Factor), Myst-type histone acetyltransferases, are involved in chromosome translocations associated with FAB-M4/5 subtypes of acute myeloid leukemia. We have reported that MOZ is essential for hematopoietic cell development and self-renewal of hematopoietic stem cells. To explore the possibility MORF also plays important roles in hematopoiesis, we generated Morf-deficient mice with homologous recombination methods. Morf−/− mice were smaller than their wildtype littermates and died within 4 weeks after birth on C57BL/6 background. In MORF−/− fetal liver, Flt3-negative KSL (c-Kit+ Sca-1+ Lineage-) cells containing hematopoietic stem cells were decreased. When fetal liver cells were transplanted into irradiated recipient mice, MORF−/− cells less efficiently reconstituted hematopoiesis than wild-type cells. Additionally, bone marrow cells reconstituted with MORF−/− cells rarely contributed to hematopoiesis in secondary transplants. To reveal relationship between MORF and MOZ in hematopoiesis, we generated double heterozygous (Moz+/− Morf+/−) mouse. Double heterozygous mice were smaller than wild-type littermates and died at least 4 weeks after birth. Numbers of KSL cells, especially Flt3- KSL cells and common myeloid progenitors were decreased in the double heterozygous embryos. The double heterozygous fetal liver cells also displayed less activity to reconstitute hematopoiesis than MOZ+/− or MORF+/− cells. Since MORF−/− mice and MOZ/MORF double heterozygous mice were alive at adult on a mixed C57BL/6/DBA2 genetic background, we investigated adult hematopoiesis in these mice. MORF−/− or MOZ/MORF double heterozygous mice were smaller than their wild-type littermates and had small numbers of thymocytes and splenocytes. However, there were no significant differences in number of bone marrow cells and hematopoietic lineage population in MORF−/− or MOZ/MORF double heterozygous mice. These results suggest that MORF as well as MOZ plays important roles in self-renewal of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

Impaired Repopulating Activity of Fus-Deficient Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2222-2222
Author(s):  
Takeaki Sugawara ◽  
Atsushi Iwama
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Chromosomal Translocation ◽  
Liver Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Fetal Liver Cells

Abstract RNA-binding protein FUS (also known as TLS) was originally identified in chromosomal translocation in human myxoid liposarcoma. The FUS gene is also translocated with the transcription factor gene ERG in human myeloid leukemia with recurrent chromosomal translocation t(16;21). Multiple data suggest that wild-type FUS is also involved in the development of leukemia as one of the downstream targets for oncoproteins including BCR-ABL. However, little is known about the role of FUS in the normal hematopoiesis. The previous report demonstrated that Fus-deficient (Fus−/−) newborn mice, which die shortly after birth because they cannot suckle, have a non-cell-autonomous defect in B lymphocyte development. No cell-autonomous defect of Fus−/− hematopoietic cells has been documented. Here we report the detailed analyses of the Fus−/− fetal liver hematopoietic stem cells (HSCs). Fus−/− fetal livers at embryonic day 14.5 were smaller in size and exhibited a significant reduction in hematopoietic cell numbers by 60% compared with the wild type (WT). Nonetheless, no significant difference was observed in the proportion of stem/progenitor cell fraction (lineage-marker-c-Kit+Sca-1+; KSL) as well as colony-forming cells between WT and Fus−/− fetal livers. Fus−/− KSL cells proliferated and differentiated almost normally in vitro. To examine in vivo repopulating activity, we transplanted fetal liver cells to lethally irradiated CD45.1 recipients with competitor bone marrow cells. Fus−/− fetal liver donor cells reconstituted recipients’ hematopoiesis for the long term and contributed to all cell lineages including B lymphocytes. In contrast to the in vitro results, however, the chimerism of donor-derived cells was significantly lower in recipients receiving Fus−/− fetal liver cells compared with WT controls (approximately 2-fold reduction). This trend was reproducible with both unfractionated and purified KSL fetal liver test cells. Our data demonstrated that the proto-oncogene Fus is involved in the maintenance of normal HSC functions. Detailed analyses on the underlying mechanisms are in progress.

Gi-Coupled GPCR Signaling Is Required at Multiple Levels and In Different Lineages In Hematopoiesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2635-2635
Author(s):  
Marie Terpager ◽  
Hiroshi Kataoka ◽  
Ivo Cornelissen ◽  
Shaun R. Coughlin
Keyword(s):  
Bone Marrow ◽  
Fetal Liver ◽  
Flow Cytometric Analysis ◽  
Liver Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells ◽  
Multiple Levels

Abstract Abstract 2635 G protein-coupled receptors (GPCRs) can regulate cell migration, survival, proliferation and differentiation –– key processes in hematopoiesis. The Gi-coupled receptor CXCR4 plays a key role in hematopoiesis, suggesting that related receptors might also contribute. Because all Gi family members except Gz are inhibited by pertussis toxin (PTX), we utilized a ROSA26-CreOnPTX mouse line that expresses PTX in a Cre-dependent manner to broadly probe the role of Gi signaling in hematopoiesis. Mice hemizygous for the hematopoietic lineage-specific Cre transgene Vav-iCre were crossed with ROSA26-CreOnPTX/CreOnPTX mice to generate offspring expressing PTX in hematopoietic lineages (Vav-PTX) and Cre-negative controls in which the PTX allele remained silent. Vav-PTX mice were born at the expected Mendelian rate, and except for a smaller thymus, were grossly normal, but all died with pneumonia between days 2 and 14. Bone marrow in 3 day-old Vav-PTX mice was hypocellular with significant underrepresentation of granulocytic and lymphocytic lineages as well as hematopoietic stem and progenitor cells (lin-, c-kit+, Sca1+). In bone marrow reconstitution studies, cells from Vav-PTX fetal livers (E14.5) showed impaired short-term and no long-term repopulating activity. Additionally, Vav-PTX fetal liver cells were significantly impaired in their ability to form granulocyte/macrophage and erythroid colonies in vitro. Interestingly, when wild-type E14.5 fetal liver cells were grown in vitro in presence of exogenous PTX, only erythroid colony formation was impaired, and flow cytometric analysis of the progenitor populations of Vav-PTX fetal liver revealed a significant decrease in granulocyte-macrophage progenitors (GMPs) as well as in common myeloid progenitors (CMPs) but not in megakaryocyte/erythroid progenitors (MEPs). Thus, reduced progenitor populations may account for reduced granulocyte/macrophage colony-forming activity in fetal liver cell cultures but does not account for reduced erythroid colony-forming activity. Indeed, normal MEP numbers in Vav-PTX livers and the ability of exogenous PTX to inhibit formation of erythroid colonies in wild-type fetal liver cultures suggests that Gi signaling in MEPs or their progeny may contribute to erythropoiesis in fetal liver. Several of the necessary roles of Gi signaling identified above are not accounted for by the function of CXCR4, and, taken together, our data suggest that Gi-coupled GPCRs likely contribute to hematopoiesis at multiple levels and in different lineages. An effort to identify GPCRs that contribute to erythropoiesis is underway. Disclosures: No relevant conflicts of interest to declare.

Long-Term Cell Autonomous Effect of Tet2 Loss in Hematopoietic Cells in Mice.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2416-2416
Author(s):  
Tomoya Muto ◽  
Goro Sashida ◽  
Motohiko Oshima ◽  
Chiaki Nakaseko ◽  
Kotaro Yokote ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Extramedullary Hematopoiesis ◽  
Liver Cells ◽  
Severe Anemia ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Myeloid Malignancies ◽  
Fetal Liver Cells

Abstract Abstract 2416 TET2 mutations are frequently observed in myeloid malignancies including myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) and MDS/MPN. Several groups have already reported that deletion of Tet2 in mice leads to dysregulated hematopoietic stem cell self-renewal and subsequent development of myeloid malignancies. Of note, mice hypomorphic or heterozygous for the Tet2 allele have been reported to show similar phenotypes as those of Tet2-null mice, suggesting that haploinsufficiency of Tet2 plays a role in the development of myeloid malignancies. However, little is known about long-term cell autonomous effects of Tet2 loss in hematopoietic cells: most of the reports were based on relatively short-term observations or did not exclude the influence of Tet2 loss in the niche cells. To study long-term cell autonomous effect of Tet2 loss in hematopoietic cells, we analyzed the hematopoiesis of wild-type recipient mice reconstituted with fetal liver cells from Tet2 hypomorphic mice for a longer period up to 1 year. Tet2 gene trap mice (Tet2trap/trap), in which the gene trap vector was inserted into the exon 2 of Tet2 just before the first coding exon, express Tet2 mRNA at the level approximately 20% of that of the wild-type (WT) mice (Shide et al. Leukemia 2012). We transplanted fetal liver cells from E14.5 WT or Tet2trap/trap mice into lethally irradiated recipient mice. At 4 months after transplantation, the recipient mice reconstituted with Tet2trap/trap cells showed a significantly increased proportion of monocytes in peripheral blood (PB) compared with those with WT cells (WT=5.59±2.57%, Tet2trap/trap=12.67±7.45%, p=0.01). While there were no significant differences between the two groups in the bone marrow (BM) compartments including the numbers of Lineage−Sca-1+c-Kit+ (LSK) hematopoietic stem/progenitor cells, extramedullary hematopoiesis in the spleen was markedly enhanced in the recipients with Tet2trap/trap cells. The proportion of LSK, granulocyte/macrophage progenitors (GMPs) and megakaryocyte/erythroid progenitors (MEPs) in the spleen of recipient mice reconstituted with WT and Tet2trap/trap cells were 0.002±0.001% vs 0.006±0.001% (p<0.01), 0.007±0.004% vs 0.029±0.01% (p=0.026) and 0.084±0.024% vs 0.25±0.044% (p<0.01), respectively. These findings were compatible with those reported previously and indicated that recipient mice reconstituted with Tet2trap/trapcells induce chronic myelomonocytic leukemia (CMML)-like disease. Of note, after a long observation period, particularly after 9 months post-transplantation, mice reconstituted with Tet2trap/trap cells developed advanced hematological disease and 53.3% (8 of 15) died or were killed because of their moribund condition by 11 months after transplantation. Detailed analysis on moribund as well as surviving mice reconstituted with Tet2trap/trap cells (n=3 each) revealed that the 2/3 of the mice developed CMML-like disease with monocytosis in PB and evident extramedullary hematopoiesis harboring increased number of LSK and GMPs in spleen, whereas the remaining 1/3 of mice developed MDS/MPN-like disease with severe anemia. The latter mice did not show monocytosis in PB and BM, but displayed dyserythropoiesis accompanied by massive extramedullary erythropoiesis, as we saw increased number of LSK and MEPs, but not GMPs, in spleen. The proportion of Annexin V+ cells in Ter119highCD71higherythroblasts in the BM of WT mice, CMML-like disease-carrying mice and MDS/MPN-like disease-carrying mice were 9.46±0.53%, 10.71±0.36%, and 19.04±0%, respectively, suggesting that the enhanced apoptosis led to the severe anemia seen in the MDS/MPN-like disease-carrying mice. Thus, decreased expression of Tet2 is sufficient to promote not only CMML-like disease, but also an MDS/MPN-like disease as well. Our findings confirmed a long-term cell autonomous effect from insufficient function of Tet2 in the development of hematological diseases. Interested in the molecular mechanism of disease progression, we have identified 1,642 differentially methylated regions (DMRs) in Tet2trap/trap GMPs compared with WT GMPs by ChIP-sequencing. We are now working to understand how these DMRs are involved in the development of the two distinct diseases associated with hypomorphic Tet2. Disclosures: No relevant conflicts of interest to declare.

hnRNP K Overexpression Synergizes with Mutant NPM1 to Drive Acute Myeloid Leukemia Progression

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2382-2382 ◽  
Author(s):  
Miguel Gallardo ◽  
Hun Ju Lee ◽  
Xiaorui Zhang ◽  
Laura R. Pageon ◽  
Asha Multani ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Genetic Alterations ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Hnrnp K ◽  
Differentiation And Proliferation ◽  
Acute Myeloid ◽  
Proliferation Potential

Abstract NPM1 mutations are one of the most common alterations observed in acute myeloid leukemia (AML). When coupled with wild type FLT3 status in cytogenetically normal (CN) patients, NPM1 mutations confer favorable prognoses compared with other alterations. However, a subset of CN NPM1Mut :FLT3Wt patients with AML have dismal outcomes, suggesting that uncharacterized alterations influence the outcomes in these patients. To address this, we performed reverse phase protein array (RPPA) analysis on CD34+ bone marrow cells isolated from 43 de novo CN NPM1Mut :FLT3Wt AML patient as well as healthy donor controls. Through these analyses, we observed that overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) associated with extremely poor outcomes within this a priori favorable prognostic group, as almost 90% of patients with increased hnRNP K expression died within 12 months of diagnosis while nearly 40% of individuals with normal hnRNP K expression survived seven years (Figure 1A). hnRNP K is a multifunctional RNA and DNA binding protein whose expression is often altered in cancers. To directly examine the functional relationship between hnRNP K overexpression and mutant NPM1 in hematologic malignancies, we generated tissue-specific transgenic mouse models with the ability to overexpress hnRNP K (hnRNP KTg) in the presence or absence of mutant Npm1 (Npm1Tg). By crossing these mice with Vav-Cre expressing mice, we specifically activated hnRNP K overexpression and mutant NPM1 expression in the hematological compartment. Using Lin-CD117+ hematopoietic stem cells (HSCs) from hnRNP KTg, Npm1Tg, and hnRNP KTg;Npm1Tg mice, we observed significant changes in differentiation and proliferation potential in colony formation assays. Overexpression of hnRNP K alone significantly increased the number of colonies compared to wild type and Npm1Tg HSCs while expression of mutant Npm1Tg resulted in increased numbers of cells compared to wild type and hnRNP KTg HSCs. Importantly, the combination of hnRNP K overexpression and mutant Npm1 resulted in a cumulative increase in both the number of colonies and number of cells, indicating that hnRNP K and mutant NPM1 cooperate to dictate differentiation and proliferation potential in HSCs (Figure 1B). Next, we examined the in vivo impact of hnRNP K overexpression and mutant Npm1 expression by analyzing the bone marrows of Npm Tg, hnRNP KTg, and Npm1Tg;hnRNP KTg mice. Within the first six months of life, these mice rapidly developed significant myeloid hyperplasias as determined by flow cytometry and pathologic analyses (Figure 1C). Together, our findings reveal that mutant Npm1 and hnRNP K overexpression result in similar myeloid phenotypes. However, these genetic alterations are also cooperative, suggesting both increased hnRNP K expression and mutant NPM1 synergize to impact hematopoietic phenotypes and drive AML progression through similar pathways but potentially via unique molecular processes. Currently, we are investigating the direct interaction and global relationship between hnRNP K and mutant Npm1 in regulating tumor suppressor and oncogenic programs (e.g.; p53- and c-Myc pathways). Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Oncogenic Role of N6-Methyladenosine Reader Protein IGF2BP3 in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1334-1334
Author(s):  
Huilin Huang ◽  
Hengyou Weng ◽  
Mingli Sun ◽  
Huizhe Wu ◽  
Zhenhua Chen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Viability ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Poor Prognosis ◽  
Expression Level ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract RNA N6-Methyladenosine (m6A) modification is an abundant modification of internal mRNAs in eukaryotes and some viruses, which is dynamically and reversibly fine-tuned during normal and pathological bioprocesses. Recent studies have shown that m6A methyltransferases, METTL3 and METTL14, play important roles in maintaining self-renewal capacity of hematopoietic stem/progenitor cells (HSPCs) and promoting acute myeloid leukemia (AML) development (Barbiori et al., Nature, 2017; Vu et al., Nature Method, 2017; Weng et al. Cell Stem Cell, 2018). The m6A demethylase, FTO, was also shown to promote leukemic cell transformation and leukemogenesis in various type of AML (Li et al., Cancer Cell, 2017). However, little is known about the functions of m6A readers in malignant hematopoiesis. We recently reported that Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is an specific m6A binding protein, which recognize m6A transcripts through the K Homology (KH) domains to stabilize and promote translation of its target mRNAs (Huang et al., Nature Cell Biology, 2018). In analysis of TCGA AML dataset (n=157), we found that a higher expression level of IGF2BP3 is significantly associated with a poor prognosis in AML patients (p<0.001; Median overall survival: IGF2BP3-High vs. IGF2BP3-Low = 11 months vs. 31 months). In addition, we analyzed our in-house microarray profiling of 113 AML patient samples and found that IGF2BP3 is highly expressed in mononuclear cells (MNC) from MLL-rearranged leukemia patients as compared to those from healthy donors (p<0.05) or non-MLL-rearranged leukemic patients (p<0.001). Consistent with the overexpression of IGF2BP3 in human MLL-rearranged AML, MLL-AF9 or MLL-AF10 transformed mouse hematopoietic stem/progenitor cells (HSPCs; herein mouse lineage negative (Lin-) bone marrow cells) showed a >10 fold increase in expression level of Igf2bp3, compare to the non-transformed counterpart HSPCs. Furthermore, in analysis of 562 samples from adult patients with AML (GSE37642), we found that within cytogenetically normal human AML, patients carrying FLT3-ITD mutation showed a significantly higher level of IGF2BP3 expression than those without FLT3-ITD mutation (p<0.01). To investigate the potential oncogenic role of IGF2BP3 in AML, we cotransduced mouse Lin- BM progenitor cells with MLL-AF9 and three individual shRNAs targeting Igf2bp3 or a scrambled control shRNA and performed colony-forming/replating assays. Knockdown of Igf2bp3 significantly (p<0.05) reduced the colony-forming capacity of MLL-AF9-transduced HSPCs to 20-50% of that of the control group. Conversely, forced expression of wild-type IGF2BP3 significantly (p<0.05) promoted colony formation of MLL-AF9-transduced Lin- BM progenitor cells. Such promotion was almost completely impaired when KH3-4 domain of IGF2BP3 was mutated or when Mettl14 was depleted, suggesting that IGF2BP3 exerts its oncogenic function as an m6A reader through an m6A-dependent mechanism. We further used human leukemia cell lines to investigate the function of IGF2BP3 in human AML cells. Silencing of IGF2BP3 by two shRNAs significantly inhibited cell viability and proliferation and induced cell apoptosis (p<0.01) in MonoMac6 AML cell line which harbors the t(9;11) translocation. In Molm13 and MV4-11 AML cells which are heterozygous and homozygous for the FLT3-ITD mutation, respectively, a further decrease of cell viability and increase of apoptotic cells upon IGF2BP3 knockdown was observed compared to MonoMac6 with wild-type FLT3. Mechanically, through cross-linking immunoprecipitation sequencing (CLIP-seq), we showed that IGF2BP3 targets mRNAs in cell cycle, DNA replication and protein synthesis pathways. Taken together, these results demonstrated the oncogenic role of the new m6A reader protein IGF2BP3 in AML. Given the fact that expression of IGF2BP3 correlates with an overall poor prognosis in AML, IGF2BP3 is likely a promising therapeutic target for AML treatment. Disclosures No relevant conflicts of interest to declare.

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