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  • 1
    ISSN: 1546-170X
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Medicine
    Notes: [Auszug] Embryonic development of multilineage hematopoiesis requires the precisely regulated expression of lineage-specific transcription factors, including AML-1 (encoded by Runx1; also known as CBFA-2 or PEBP-2αB). In vitro studies and findings in human diseases, including leukemias, ...
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  • 2
  • 3
    Publication Date: 2007-11-16
    Description: AML1/Runx1 is one of the most frequent targets of gene aberrations associated with human acute myelogenous leukemia and myelodysplastic syndrome. Furthermore, gene-targeting studies in mice demonstrated that AML1 is a critical regulator for T-cell differentiation and early development of definitive hematopoiesis. It has been shown that phosphorylation of AML1 at specific serine/threonine residues (276, 293, 300, 303) controls both transcriptional activity and rate of degradation. However, the biological consequences of phosphorylation with respect to AML1 function are unclear. In this study, we evaluated hematopoietic activities of AML1 mutants which harbor serine/threonine-to-alanine (A) or serine/threonine-to-asparatic acid (D) mutations at these phosphorylation sites using primary culture systems. We first evaluated the biologic effects of AML1 phosphorylation on T-cell differentiation using the fetal liver (FL)/OP9-DL1 coculture system. In this system, AML1-excised FL cells failed to undergo normal T-cell differentiation, which is successfully restored by the reintroduction of wild-type AML1. AML1-4A, which cannot be phosphorylated at any of the four serine/threonine residues, showed a severely impaired capacity to rescue the defective T-cell differentiation of AML1-deficient cells. We also demonstrate that blocking the ERK-mediated phosphorylation of AML1 by the MEK inhibitor significantly suppressed the AML1-induced T-cell differentiation. Next, we assessed the effect of AML1 phosphorylation on early hematopoietic development using para-aortic splanchnopleural (P-Sp) region/OP9 coculture system. In this system, AML1-deficient P-Sp cells failed to produce any hematopoietic cells and this hematopoietic defect can be rescued by retrovirally transferred AML1. AML1–4A could rescue the hematopoietic defect of AML1-deficient P-Sp cells, but the emergence of hematopoietic cells in cultures infected with AML1–4A was reproducibly delayed two or more days compared with those infected with wild-type AML1. Because it was shown that serine 462 is phosphorylated upon phorbol ester treatment together with the four phosphorylation sites, we then generated AML1 mutants in which serine 462 is substituted with alanine (A) or aspartic acid (D) in AML1–4A or AML1–4D respectively (AML1–5A or AML1-5D). Interestingly, introduction of AML1–5A into AML1-deficient P-Sp cells did not generate any hematopoietic cells. Moreover, AML1–5A completely lost its T-cell differentiation activity. In contrast, the phospho-mimic protein, AML1–5D rescued the hematopoietic defects of AML1-deficient cells as efficiently as wild-type AML1. Taken together, these results suggest that phosphorylation of AML1 at the five serine/threonine residues (276, 293, 300, 303 and 462) is essential for T cell differentiation and early hematopoietic development.
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    Electronic ISSN: 1528-0020
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  • 4
    Publication Date: 2011-12-15
    Description: Functional deregulation of transcription factors has been found in many types of tumors. Transcription factor AML1/RUNX1 is one of the most frequent targets of chromosomal abnormalities in human leukemia and altered function of AML1 is closely associated with malignant transformation of hematopoietic cells. However, the molecular basis and therapeutic targets of AML1-related leukemia are still elusive. Here, we explored immediate target pathways of AML1 by in vitro synchronous inactivation in hematopoietic cells. We found that AML1 inhibits NF-κB signaling through interaction with IκB kinase complex in the cytoplasm. Remarkably, AML1 mutants found in myeloid tumors lack the ability to inhibit NF-κB signaling, and human cases with AML1-related leukemia exhibits distinctly activated NF-κB signaling. Furthermore, inhibition of NF-κB signaling in leukemic cells with mutated AML1 efficiently blocks their growth and development of leukemia. These findings reveal a novel role for AML1 as a cytoplasmic attenuator of NF-κB signaling and indicate that NF-κB signaling is one of the promising therapeutic targets of hematologic malignancies with AML1 abnormality.
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  • 5
    Publication Date: 2011-09-01
    Description: Dysfunction of AML1/Runx1, a transcription factor, plays a crucial role in the development of many types of leukemia. Additional events are often required for AML1 dysfunction to induce full-blown leukemia; however, a mechanistic basis of their cooperation is still elusive. Here, we investigated the effect of AML1 deficiency on the development of MLL-ENL leukemia in mice. Aml1 excised bone marrow cells lead to MLL-ENL leukemia with shorter duration than Aml1 intact cells in vivo. Although the number of MLL-ENL leukemia-initiating cells is not affected by loss of AML1, the proliferation of leukemic cells is enhanced in Aml1-excised MLL-ENL leukemic mice. We found that the enhanced proliferation is the result of repression of p19ARF that is directly regulated by AML1 in MLL-ENL leukemic cells. We also found that down-regulation of p19ARF induces the accelerated onset of MLL-ENL leukemia, suggesting that p19ARF is a major target of AML1 in MLL-ENL leukemia. These results provide a new insight into a role for AML1 in the progression of leukemia.
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  • 6
    Publication Date: 2009-11-20
    Description: Abstract 1962 Poster Board I-985 Introduction: AML1/Runx1 is one of the most frequent targets of chromosomal abnormalities in human leukemia. Functional impairment of AML1 caused by point mutation is also reported in patients with leukemia or myelodysplastic syndrome (MDS). However, molecular basis for leukemogenesis caused by functional impairment of AML1 is still elusive. In this study, we clarified the deregulated signaling pathway induced by loss of AML1. Results: To find the direct target of AML1, we compared gene expression profile between AML1-conditionally deleted and normal KSL cells using Cre-ER system. Gene set enrichment analysis (GSEA) using molecular signature database (MSigDB) clarified enhanced expression of NF-kB target genes in AML1 deficient cells. In addition, NF-kB inhibitor attenuated the enhanced colony forming activity of bone marrow cells from AML1 conditional knockout (cKO) mice. These data indicate the aberrant activation of NF-kB signaling pathway in stem/progenitor cells of AML1 deficient mice. NF-kB is a transcription factor which is involved in many physiological phenomena including proliferation, survival, and inflammation. Because deregulated activation of NF-kB signaling has been reported to be responsible for many types of tumors including hematological malignancies, we assumed that lack of AML1-mediated suppression of NF-kB signaling lead to malignant transformation of hematopoietic cells. p65, one of the major components of NF-kB stays in cytoplasm with IkB in a steady state. Once receiving stimulating signals from cell surface receptors such as TNF-a receptor, IkB is phosphorylated by IKK complex and subsequently degraded through the ubiquitin-proteasome pathway, resulting in nuclear translocation of p65 and transactivation of NF-kB target genes. First, we found that AML1 inhibits nuclear translocation of p65 and that nuclear localization of p65 is enhanced in AML1 deficient cells, which is cancelled by NF-kB inhibitors. In addition, AML1 inhibited p65 phosphorylation at serine 536, which is important for its activation. We found that AML1 physically interacts with IKK complex and thus suppresses its kinase activity, which accounts for a mechanistic basis for inhibition of NF-kB signaling by AML1. Suppression of IKK kinase activity by AML1 results in inhibition of both nuclear translocation of p65 and activation of NF-kB target genes. Next, we examined how leukemia-related AML1 mutants affect NF-kB signaling. Remarkably, AML1 D171N mutant found in MDS neither inhibited nuclear translocation of p65 nor attenuated the kinase activity of IKK complex. Similar results were obtained with AML1/ETO generated in leukemia with t(8;21). Mouse bone marrow cells immortalized by AML1/ETO showed enhanced nuclear localization of p65 compared with those immortalized by MLL/ENL, another leukemia-related fusion protein. Indeed, AML1/ETO immortalized cells are more sensitive to NF-kB inhibitor-mediated growth suppression, indicating a critical role of NF-kB signaling in transformation by AML1/ETO. To verify the activation of NF-kB signaling by AML1/ETO in human hematopoietic cells, we analyzed the gene expression data reported by Valk et al. in silico. We found that NF-kB signaling is distinctly activated in AML1-related leukemia patients. These results suggest that aberrant activation of NF-kB signaling induced by functional impairment of AML1 may contribute to the development of leukemia via proliferation signals. Conclusions: We found that AML1 is a cytoplasmic attenuator of NF-kB signaling pathway. Functional impairment of AML1 caused by genetic disruption results in distinct activation of NF-kB signaling by altering IKK kinetic activity. This aberrant activation may play a central role in pathogenesis of AML1-related leukemia and MDS. Therefore, NF-kB signaling is one of the attractive candidates for molecular targeted therapy against AML1-related hematological disorders. Disclosures: No relevant conflicts of interest to declare.
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  • 7
    Publication Date: 2012-11-16
    Description: Abstract 3033 In hematopoietic stem cell transplantation (HSCT), oral intake is severely impaired by preparative regimen-induced mucosal toxicity and nausea. Therefore, parenteral nutrition (PN) is usually administered. The vitamin preparation accompanying PN therapy is based on the FDA recommendation proposed in 2000. However, it remains unknown whether this preparation retains adequateness for HSCT recipients who undergo strenuous conditioning regimens and immunological reactions, which put them in a hyper catabolic state. Additionally, almost no study has been conducted to examine the relevance of standard regimen of mineral microelements supplementation during HSCT. In this prospective observational study, we measured the blood level of selected vitamins (vitamin B1, B6, C, K, E, and folate) and mineral microelements (iron, zinc, copper, and selenium) during the peri-HSCT period (measured weekly from 1 week before until 4 weeks after HSCT). Oral and parenteral total energy amounts, administration of multivitamin preparation, and mineral supplementation, were recorded. The subjects were 15 adult patients who received allogeneic HSCT for hematological malignancies (AML: 7, ALL: 1, CML: 1, MPN: 2, MDS: 2, NHL: 2) at The University of Tokyo Hospital. Eight of them received CY/TBI-based full conditioning and the others received fludarabine-based reduced intensity regimens. All the patients received PN with vitamin and mineral preparations when their oral intake decreased to less than 700 Kcal/day. PN was modified adequately in case of organ dysfunctions. Engraftment was observed in all but one patient, and one died before day 28 due to sepsis. The most striking finding was the marked deficiency of vitamin C, a major antioxidant vitamin. Vitamin C levels dropped just after conditioning regimens started, and reached a nadir (2.1+-1.49 microgram/mL, normal range: 5.5–16.8) at day 14. The deficiency levels at day 7 and day 14 were significantly correlated with increase of acute phase inflammatory proteins (p=0.03 for C-reactive protein and p=0.004 for ferritin, at day 7), which suggests that the current vitamin C complement regimen is insufficient for patients who suffer from intense inflammation. On the other hand, vitamin E, another antioxidant vitamin, remained within the normal level during the surveillance period. Selenium is a microelement that assists antioxidative effect and has attracted attention because emerging evidence suggests its relevance for critically ill condition. Although it is not contained in the standard PN preparations, our results indicated no sign of shortage. Selenium is abundant in normal diet, and short suspension of oral intake does not induce selenium depletion. Marked excess of vitamin K was another point of note. The average vitamin K level continued to rise after the start of the conditioning regimen, and exceeded 10 times of the normal upper level (0.15 – 1.25 ng/mL) at day14 (12.8 +- 5.6 ng/mL), day 21 (17.4 +- 17.9), and day 28 (12.1+- 25.0). These abnormal rises were observed especially in patients with prolonged administration of PN, suggesting overdosing of vitamin K in the standard vitamin preparations. Vitamin K excess would be problematic because it causes hypercoagulability; however, it was not clinically correlated with the onset of thrombotic microangiopathy in our cohort. Vitamin B1 was slightly but significantly below the normal lower limit. Its deficiency started before the conditioning regimen and persisted through day14. Considering increased requirement of vitamin B1 during a debilitating state, this shortage should be corrected with active supplementation before the patients undergo a stressful conditioning process. Other substances (folate, zinc, iron, and copper) remained almost within a normal range. In conclusion, the nourishment status during peri-HSCT period is characterized by marked depletion of vitamin C and excess of vitamin K, and other aberrations with less degree of deviation. These results indicate that normal PN is not adequate for HSCT patients. Development of vitamin and microelement preparation that is optimized for HSCT setting should be discussed. Disclosures: Nannya: Otsuka Seiyaku Koujyou: Research Funding.
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  • 8
    Publication Date: 2011-11-18
    Description: Abstract 48 Elevated expression of BAALC (brain and acute leukemia, cytoplasmic) is an adverse prognostic factor in patients with cytogenetically normal acute myeloid leukemia (CNAML). However, its precise role in normal hematopoiesis and leukemogenesis remains to be elucidated. To address this issue, we evaluated the effect of BAALC overexpression upon hematopoiesis and leukemogenesis and generated BAALC-deficient mice. First, we examined the effect of BAALC overexpression upon hematopoiesis. BAALC-transduced bone marrow (BM) cells had decreased colony-forming capacity. Bone marrow transplantation (BMT) assay demonstrated that the donor chimerism was lower in transplanted mice with BAALC-overexpressed cells than with control cells. To examine the mechanism, we performed gene expression analysis. This revealed that p53 pathway is up-regulated in BAALC-overexpressed cells. Colony-forming assay and BMT assay using p53-deficient BM cells demonstrated that the blockage of proliferation by BAALC overexpression is canceled in p53-deficient cells in vitro and in vivo, suggesting that the inhibition of proliferation by BAALC overexpression is dependent on p53 pathway. Next, we generated BAALC-conventionally deficient mice. Most BAALC-deficient female mice died between E11.5 and E12.5 because of defects of placental development. BAALC-deficient E11.5 yolk sac cells had increased colony-forming capacity. Transplantation assay showed that the donor chimerism at 4 weeks after transplantation was higher in transplanted mice with BAALC-deficient E12.5 fetal liver cells than with wild fetal liver cells. Moreover, the frequency of G0/G1 phase cells was decreased in BAALC-deficient yolk sac. Thus, the proliferation is activated and the frequency of quiescent cells is reduced in BAALC-deficient embryonic hematopoietic cells. In addition, we examined the role of BAALC in adult hematopoiesis using BAALC-conditionally deficient mice with Mx-Cre system. In BAALC-deficient (Mx-Cre+BAALC flox/flox) BM, long-term hematopoietic stem cells (HSCs), identified as CD34−Flt3−cKit+Sca1+Lin− cells, were reduced as compared with control (Mx-Cre−BAALC flox/flox) BM. BAALC-deficient adult BM had impaired long-term repopulating ability in serial competitive BMT. Moreover, BAALC-deficient mice showed higher mortality rates after weekly 5-FU injections. On the other hand, the ability of colony forming unit spleen (CFU-S) was enhanced and the recovery after myelosuppression induced by single 5-FU injection was faster in BAALC-deficient mice, indicating that BAALC-deficient HSCs have enhanced short-term repopulating ability. Next, to address the relationship between BAALC and p53 protein levels, we performed intracellular flow cytometric analysis and immunocytochemical staining. These experiments revealed that p53 protein is reduced in BAALC-deficient HSCs. Immunoprecipitation revealed that BAALC interferes with the binding of MDM2 to p53, which leads to stabilization of p53 protein. Finally, we tested the effect of BAALC overexpression in leukemia mouse models. BAALC overexpression promoted leukemia in mice in cooperation with TEL/PDGFBR-AML1/ETO (TPAE). BM cells transduced with TPAE and BAALC had increased colony-forming capacity as compared with TPAE and mock-transduced cells. By contrast, BAALC cannot accelerate myc/BCL2-induced leukemia. Western blot showed that p53 is inactivated in TPAE leukemic cells but not in myc/BCL2 leukemic cells. BM cells transduced with myc/BCL2 and BAALC had decreased colony-forming capacity as compared with myc/BCL2 and mock-transduced cells. However, BAALC overexpression promoted colony-forming capacity of p53-deficient cells transduced with myc/BCL2. Similarly, BAALC overexpression promoted the colony-forming capacity of BM cells transduced with BCR/ABL which is known to inactivate p53. Promoted proliferation of BCR/ABL-transduced cells by BAALC overexpression was canceled by the MDM2 antagonist, Nutlin-3. Taken together, BAALC overexpression accelerates leukemia with impaired p53 pathway. In conclusion, these data suggest that BAALC activates p53 pathway and maintains the stemness in normal HSCs. Once p53 pathway is impaired, however, BAALC promotes proliferation of leukemic cells. Our data for the first time uncovered a novel function of BAALC, which may lead to novel therapeutic strategy for CNAML with poor prognosis. Disclosures: No relevant conflicts of interest to declare.
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  • 9
    Publication Date: 2007-11-16
    Description: Transcription factor AML1/RUNX1, initially isolated from the t(8;21) chromosomal translocation in human leukemia, is essential for the development of multilineage hematopoiesis in mouse embryos. AML1 negatively regulates the number of immature hematopoietic cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. However, it remains yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population fraction and c-Kit-positive cells in the G0 cell cycle status were increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We also found an increase in HSC number within the AML1-deficient bone marrow using limiting dilution bone marrow transplantation assays. Thus, the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia. To identify mechanisms through which functional loss of AML1 exerts leukemogenic potential, we focused on the AML1-Evi-1 chimeric protein, which is generated by the t(3;21) chromosomal translocation and disturbs the normal function of AML1. We introduced AML1-Evi-1 and its mutants into murine bone marrow cells, and evaluated hematopoietic cell transformation by colony replating assays. The transforming activity of AML1-Evi-1 was impaired when any of the major functional domains of AML1-Evi-1 was lost. Moreover, overexpression of Evi-1 could not transform AML1-deleted bone marrow cells, suggesting that fusion of AML1 and Evi-1, rather than AML1 suppression and Evi-1 overexpression, is essential for AML1-Evi-1 leukemogenesis. Intriguingly, among the hematopoietic progenitor cell fractions, AML1-Evi-1 could transform only the uncommitted, immature hematopoietic cells, which contrasts with MLL-ENL, a chimeric protein generated in t(11;19) leukemia. AML1-Evi-1 transformed cells show a surface marker profile different from that of the cells transformed by AML1-MTG8/ETO, another leukemic gene product that also perturbs AML1 function. These results provide a valuable clue to a distinct mechanism determined by the Evi-1 moiety in the AML1-Evi-1 leukemogenesis and to a role of AML1 loss in the self-renewal of leukemic stem cells.
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  • 10
    Publication Date: 2010-11-19
    Description: Abstract 1605 Reactive oxygen species (ROS) are small molecules containing oxygen with unpaired electron. ROS are always generated as the products of cellular metabolism, and cells have several antioxidant systems to avoid their harmful effect induced by their high chemical reactivity. While studies about biological effects of ROS have been mainly focused on the harmful aspects, a growing body of evidence suggests that ROS are critical mediators of several signaling pathways in cellular homeostasis. In hematopoiesis, for example, it was reported that the generation of intracellular ROS functions as the initiation signal in the development of Drosophila hematopoietic cells and that ROS control self-renewal of hematopoietic stem cells. However, little is known about the functions of ROS in regulating hematopoietic progenitors. In this study, we show a critical role of ROS in lineage decision of myeloid progenitor cells. Firstly, we measured the intracellular ROS level of hematopoietic cells from murine bone marrow by flow cytometry with H2-DCFDA (2’,7’-dichlorofluorescin diacetate) staining. It was found that intracellular ROS level of megakaryocyte-erythrocyte progenitor cells (MEP) was kept equal to or lower than that of lineage marker negative, Sca-1 positive, c-Kit positive (KSL) cells. On the other hand, that of granulocyte-monocyte progenitor cells (GMP) was significantly elevated. Additionally, mRNA expression of NADPH oxidase 2 (cytochrome b-245) and NOXA2 (neutrophil cytosolic factor 2), both of which are major components of the membrane-bound oxidase complex generating ROS in cells, was significantly suppressed in MEP and KSL cells, whereas it was up-regulated in GMP. Thus, intracellular ROS level of MEP is kept lowest in hematopoietic cells. Next, we investigated the effect of ROS on the differentiation of myeloid progenitors. In liquid culture assay, loading of ROS with low dose hydrogen peroxide inhibited the differentiation of progenitor cells into MEP, whereas removal of ROS with catalase accelerated the differentiation of those into MEP. Similarly, in the colony-forming assay with semisolid culture medium, we observed that loading of ROS inhibited the formation of megakaryocyte-erythrocyte colonies. To investigate the effect of intrinsic ROS on the colony-forming capacity, we sorted ROS-high and low common myeloid progenitor cells (CMP) individually and cultured them. It was shown that ROS-low CMP had high colony-forming capacity of megakaryocyte-erythrocyte, whereas ROS-high CMP had high colony-forming capacity of granulocyte-monocyte. There is inverse correlation between intracellular ROS level of CMP and colony-forming capacity of megakaryocyte-erythrocyte. In order to confirm that ROS can control the differentiation of CMP into MEP or GMP in vivo, we injected lipopolysaccharide (LPS), which can increase intracellular ROS level of bone marrow cells, into mice. We found that MEP were decreased in LPS-injected mice. Finally, we performed gene expression microarray analysis to compare gene expression profiles between ROS-low and high CMP. In terms of gene expression profiles, ROS-low CMP were similar to MEP and magakaryocyte whereas ROS-high CMP were similar to GMP. In conclusion, these findings suggest that intracellular ROS play a critical role in lineage decision of myeloid progenitor cells, especially in the generation of MEP. In several diseases with anemia or thrombocytopenia, such as myelodysplastic syndrome, Fanconi anemia and autoimmune diseases with chronic inflammation, it was reported that intracellular ROS level of hematopoietic cells was up-regulated. Thus, up-regulated intracellular ROS may be involved in their symptoms via differentiation disorder of MEP. Disclosures: No relevant conflicts of interest to declare.
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