Publications by authors named "Toshihiko Oki"

40 Publications

Imaging dynamic mTORC1 pathway activity in vivo reveals marked shifts that support time-specific inhibitor therapy in AML.

Nat Commun 2021 01 11;12(1):245. Epub 2021 Jan 11.

Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA.

Acute myeloid leukemia (AML) is a high remission, high relapse fatal blood cancer. Although mTORC1 is a master regulator of cell proliferation and survival, its inhibitors have not performed well as AML treatments. To uncover the dynamics of mTORC1 activity in vivo, fluorescent probes are developed to track single cell proliferation, apoptosis and mTORC1 activity of AML cells in the bone marrow of live animals and to quantify these activities in the context of microanatomical localization and intra-tumoral heterogeneity. When chemotherapy drugs commonly used clinically are given to mice with AML, apoptosis is rapid, diffuse and not preferentially restricted to anatomic sites. Dynamic measurement of mTORC1 activity indicated a decline in mTORC1 activity with AML progression. However, at the time of maximal chemotherapy response, mTORC1 signaling is high and positively correlated with a leukemia stemness transcriptional profile. Cell barcoding reveals the induction of mTORC1 activity rather than selection of mTORC1 high cells and timed inhibition of mTORC1 improved the killing of AML cells. These data define the real-time dynamics of AML and the mTORC1 pathway in association with AML growth, response to and relapse after chemotherapy. They provide guidance for timed intervention with pathway-specific inhibitors.
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http://dx.doi.org/10.1038/s41467-020-20491-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801403PMC
January 2021

Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal.

J Exp Med 2021 Jan;218(1)

Laboratory of Lymphocyte Differentiation, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan.

A still unanswered question is what drives the small fraction of activated germinal center (GC) B cells to become long-lived quiescent memory B cells. We found here that a small population of GC-derived CD38intBcl6hi/intEfnb1+ cells with lower mTORC1 activity favored the memory B cell fate. Constitutively high mTORC1 activity led to defects in formation of the CD38intBcl6hi/intEfnb1+ cells; conversely, decreasing mTORC1 activity resulted in relative enrichment of this memory-prone population over the recycling-prone one. Furthermore, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR that, in turn, contributed to their survival and development. We also found that downregulation of Bcl6 resulted in increased expression of both Bcl2 and BCR. Given the positive correlation between the strength of T cell help and mTORC1 activity, our data suggest a model in which weak help from T cells together with provision of an increased survival signal are key for GC B cells to adopt a memory B cell fate.
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http://dx.doi.org/10.1084/jem.20200866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555411PMC
January 2021

Induction of a Timed Metabolic Collapse to Overcome Cancer Chemoresistance.

Cell Metab 2020 Sep 6;32(3):391-403.e6. Epub 2020 Aug 6.

Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA; Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA. Electronic address:

Cancer relapse begins when malignant cells pass through the extreme metabolic bottleneck of stress from chemotherapy and the byproducts of the massive cell death in the surrounding region. In acute myeloid leukemia, complete remissions are common, but few are cured. We tracked leukemia cells in vivo, defined the moment of maximal response following chemotherapy, captured persisting cells, and conducted unbiased metabolomics, revealing a metabolite profile distinct from the pre-chemo growth or post-chemo relapse phase. Persisting cells used glutamine in a distinctive manner, preferentially fueling pyrimidine and glutathione generation, but not the mitochondrial tricarboxylic acid cycle. Notably, malignant cell pyrimidine synthesis also required aspartate provided by specific bone marrow stromal cells. Blunting glutamine metabolism or pyrimidine synthesis selected against residual leukemia-initiating cells and improved survival in leukemia mouse models and patient-derived xenografts. We propose that timed cell-intrinsic or niche-focused metabolic disruption can exploit a transient vulnerability and induce metabolic collapse in cancer cells to overcome chemoresistance.
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http://dx.doi.org/10.1016/j.cmet.2020.07.009DOI Listing
September 2020

Discrimination of Dormant and Active Hematopoietic Stem Cells by G Marker Reveals Dormancy Regulation by Cytoplasmic Calcium.

Cell Rep 2019 12;29(12):4144-4158.e7

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between "dormant" and "active" HSCs remains unresolved. Here we generate a G marker (GM) mouse line that visualizes quiescent cells and identify a small population of active HSCs (GM), which are distinct from dormant HSCs (GM), within the conventional quiescent HSC fraction. Single-cell RNA-seq analyses show that the gene expression profiles of these populations are nearly identical but differ in their Cdk4/6 activity. Furthermore, high-throughput small-molecule screening reveals that high concentrations of cytoplasmic calcium ([Ca]) are linked to dormancy of HSCs. These findings indicate that GM separates dormant and active adult HSCs, which are regulated by Cdk4/6 and [Ca]. This GM mouse line represents a useful resource for investigating physiologically important stem cell subpopulations.
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http://dx.doi.org/10.1016/j.celrep.2019.11.061DOI Listing
December 2019

A new bioavailable fenretinide formulation with antiproliferative, antimetabolic, and cytotoxic effects on solid tumors.

Cell Death Dis 2019 07 23;10(7):529. Epub 2019 Jul 23.

Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy.

Fenretinide is a synthetic retinoid characterized by anticancer activity in preclinical models and favorable toxicological profile, but also by a low bioavailability that hindered its clinical efficacy in former clinical trials. We developed a new formulation of fenretinide complexed with 2-hydroxypropyl-beta-cyclodextrin (nanofenretinide) characterized by an increased bioavailability and therapeutic efficacy. Nanofenretinide was active in cell lines derived from multiple solid tumors, in primary spheroid cultures and in xenografts of lung and colorectal cancer, where it inhibited tumor growth independently from the mutational status of tumor cells. A global profiling of pathways activated by nanofenretinide was performed by reverse-phase proteomic arrays and lipid analysis, revealing widespread repression of the mTOR pathway, activation of apoptotic, autophagic and DNA damage signals and massive production of dihydroceramide, a bioactive lipid with pleiotropic effects on several biological processes. In cells that survived nanofenretinide treatment there was a decrease of factors involved in cell cycle progression and an increase in the levels of p16 and phosphorylated p38 MAPK with consequent block in G0 and early G1. The capacity of nanofenretinide to induce cancer cell death and quiescence, together with its elevated bioavailability and broad antitumor activity indicate its potential use in cancer treatment and chemoprevention.
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http://dx.doi.org/10.1038/s41419-019-1775-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6646369PMC
July 2019

Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8)/CLM-6 is an FcRγ-coupled receptor selectively expressed in mouse tissue plasmacytoid dendritic cells.

Sci Rep 2018 05 29;8(1):8259. Epub 2018 May 29.

Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.

Plasmacytoid dendritic cells (pDCs) produce large amounts of type-I interferon (IFN) in response to viral infection or self nucleic acids. Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8), also called CMRF-35-like molecule-6 (CLM-6), is a putative activating receptor among mouse LMIR/CLM/CD300 members; however, the expression and function of LMIR8 remain unclear. Here, we characterize mouse LMIR8 as a pDC receptor. Analysis of Flag-tagged LMIR8-transduced bone marrow (BM)-derived mast cells demonstrated that LMIR8 can transmit an activating signal by interacting with immunoreceptor tyrosine-based activating motif (ITAM)-containing FcRγ. Flow cytometric analysis using a specific antibody for LMIR8 showed that LMIR8 expression was restricted to mouse pDCs residing in BM, spleen, or lymph node. FcRγ deficiency dampened surface expression of LMIR8 in mouse pDCs. Notably, LMIR8 was detected only in pDCs, irrespective of TLR9 stimulation, suggesting that LMIR8 is a suitable marker for pDCs in mouse tissues; LMIR8 is weakly expressed in Flt3 ligand-induced BM-derived pDCs (BMpDCs). Crosslinking of transduced LMIR8 in BMpDCs with anti-LMIR8 antibody did not induce IFN-α production, but rather suppressed TLR9-mediated production of IFN-α. Taken together, these observations indicate that LMIR8 is an FcRγ-coupled receptor selectively expressed in mouse tissue pDCs, which might suppress pDC activation through the recognition of its ligands.
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http://dx.doi.org/10.1038/s41598-018-25646-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5974347PMC
May 2018

The CD300e molecule in mice is an immune-activating receptor.

J Biol Chem 2018 03 22;293(10):3793-3805. Epub 2018 Jan 22.

From the Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421,

CD300 molecules (CD300s) belong to paired activating and inhibitory receptor families, which mediate immune responses. Human CD300e (hCD300e) is expressed in monocytes and myeloid dendritic cells and transmits an immune-activating signal by interacting with DNAX-activating protein 12 (DAP12). However, the CD300e ortholog in mice (mCD300e) is poorly characterized. Here, we found that mCD300e is also an immune-activating receptor. We found that mCD300e engagement triggers cytokine production in mCD300e-transduced bone marrow-derived mast cells (BMMCs). Loss of DAP12 and another signaling protein, FcRγ, did not affect surface expression of transduced mCD300e, but abrogated mCD300e-mediated cytokine production in the BMMCs. Co-immunoprecipitation experiments revealed that mCD300e physically interacts with both FcRγ and DAP12, suggesting that mCD300e delivers an activating signal via these two proteins. Binding and reporter assays with the mCD300e extracellular domain identified sphingomyelin as a ligand of both mCD300e and hCD300e. Notably, the binding of sphingomyelin to mCD300e stimulated cytokine production in the transduced BMMCs in an FcRγ- and DAP12-dependent manner. Flow cytometric analysis with an mCD300e-specific Ab disclosed that mCD300e expression is highly restricted to CD115Ly-6C peripheral blood monocytes, corresponding to CD14CD16 human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115Ly-6C monocytes. Stimulation with sphingomyelin failed to activate the CD115Ly-6C mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14CD16 human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12.
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http://dx.doi.org/10.1074/jbc.RA117.000696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846139PMC
March 2018

Disrupting ceramide-CD300f interaction prevents septic peritonitis by stimulating neutrophil recruitment.

Sci Rep 2017 06 27;7(1):4298. Epub 2017 Jun 27.

Atopy Research Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.

Sepsis is a serious clinical problem. Negative regulation of innate immunity is associated with sepsis progression, but the underlying mechanisms remains unclear. Here we show that the receptor CD300f promotes disease progression in sepsis. CD300f mice were protected from death after cecal ligation and puncture (CLP), a murine model of septic peritonitis. CD300f was highly expressed in mast cells and recruited neutrophils in the peritoneal cavity. Analysis of mice (e.g., mast cell-deficient mice) receiving transplants of wild-type or CD300f mast cells or neutrophils indicated that CD300f deficiency did not influence intrinsic migratory abilities of neutrophils, but enhanced neutrophil chemoattractant production (from mast cells and neutrophils) in the peritoneal cavity of CLP-operated mice, leading to robust accumulation of neutrophils which efficiently eliminated Escherichia coli. Ceramide-CD300f interaction suppressed the release of neutrophil chemoattractants from Escherichia coli-stimulated mast cells and neutrophils. Administration of the reagents that disrupted the ceramide-CD300f interaction prevented CLP-induced sepsis by stimulating neutrophil recruitment, whereas that of ceramide-containing vesicles aggravated sepsis. Extracellular concentrations of ceramides increased in the peritoneal cavity after CLP, suggesting a possible role of extracellular ceramides, CD300f ligands, in the negative-feedback suppression of innate immune responses. Thus, CD300f is an attractive target for the treatment of sepsis.
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http://dx.doi.org/10.1038/s41598-017-04647-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487349PMC
June 2017

Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells.

Cell 2016 11;167(5):1310-1322.e17

Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address:

Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.
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http://dx.doi.org/10.1016/j.cell.2016.10.045DOI Listing
November 2016

Distinctive Mesenchymal-Parenchymal Cell Pairings Govern B Cell Differentiation in the Bone Marrow.

Stem Cell Reports 2016 08 21;7(2):220-35. Epub 2016 Jul 21.

Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Boston, MA 02114, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02139, USA; Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Stem Cell Institute, 185 Cambridge Street, Boston, MA 02115, USA. Electronic address:

Bone marrow niches for hematopoietic progenitor cells are not well defined despite their critical role in blood homeostasis. We previously found that cells expressing osteocalcin, a marker of mature osteolineage cells, regulate the production of thymic-seeding T lymphoid progenitors. Here, using a selective cell deletion strategy, we demonstrate that a subset of mesenchymal cells expressing osterix, a marker of bone precursors in the adult, serve to regulate the maturation of early B lymphoid precursors by promoting pro-B to pre-B cell transition through insulin-like growth factor 1 (IGF-1) production. Loss of Osx(+) cells or Osx-specific deletion of IGF-1 led to a failure of B cell maturation and the impaired adaptive immune response. These data highlight the notion that bone marrow is a composite of specialized niches formed by pairings of specific mesenchymal cells with parenchymal stem or lineage committed progenitor cells, thereby providing distinctive functional units to regulate hematopoiesis.
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http://dx.doi.org/10.1016/j.stemcr.2016.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982987PMC
August 2016

Novel working hypothesis for pathogenesis of hematological malignancies: combination of mutations-induced cellular phenotypes determines the disease (cMIP-DD).

J Biochem 2016 Jan 20;159(1):17-25. Epub 2015 Nov 20.

Division of Cellular Therapy/Division of Stem Cell Signaling, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Recent progress in high-speed sequencing technology has revealed that tumors harbor novel mutations in a variety of genes including those for molecules involved in epigenetics and splicing, some of which were not categorized to previously thought malignancy-related genes. However, despite thorough identification of mutations in solid tumors and hematological malignancies, how these mutations induce cell transformation still remains elusive. In addition, each tumor usually contains multiple mutations or sometimes consists of multiple clones, which makes functional analysis difficult. Fifteen years ago, it was proposed that combination of two types of mutations induce acute leukemia; Class I mutations induce cell growth or inhibit apoptosis while class II mutations block differentiation, co-operating in inducing acute leukemia. This notion has been proven using a variety of mouse models, however most of recently found mutations are not typical class I/II mutations. Although some novel mutations have been found to functionally work as class I or II mutation in leukemogenesis, the classical class I/II theory seems to be too simple to explain the whole story. We here overview the molecular basis of hematological malignancies based on clinical and experimental results, and propose a new working hypothesis for leukemogenesis.
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http://dx.doi.org/10.1093/jb/mvv114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882650PMC
January 2016

A C-terminal mutant of CCAAT-enhancer-binding protein α (C/EBPα-Cm) downregulates Csf1r, a potent accelerator in the progression of acute myeloid leukemia with C/EBPα-Cm.

Exp Hematol 2015 Apr 20;43(4):300-8.e1. Epub 2014 Dec 20.

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Division of Stem Cell Signaling, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. Electronic address:

Two types of CCAAT-enhancer-binding protein α (C/EBPα) mutants are found in acute myeloid leukemia (AML) patients: N-terminal frame-shift mutants (C/EBPα-N(m)) generating p30 as a dominant form and C-terminal basic leucine zipper domain mutants (C/EBPα-C(m)). We have previously shown that C/EBPα-K304_R323dup belonging to C/EBPα-C(m), but not C/EBPα-T60fsX159 belonging to C/EBPα-N(m), alone induced AML in mouse bone marrow transplantation (BMT) models. Here we show that various C/EBPα-C(m) mutations have a similar, but not identical, potential in myeloid leukemogenesis. Notably, like C/EBPα-K304_R323dup, any type of C/EBPα-C(m) tested (C/EBPα-S299_K304dup, K313dup, or N321D) by itself induced AML, albeit with different latencies after BMT; C/EBPα-N321D induced AML with the shortest latency. By analyzing the gene expression profiles of C/EBPα-N321D- and mock-transduced c-kit(+)Sca-1(+)Lin(-) cells, we identified Csf1r as a gene downregulated by C/EBPα-N321D. In addition, leukemic cells expressing C/EBPα-C(m) exhibited low levels of colony stimulating factor 1 receptor in mice. On the other hand, transduction with C/EBPα-N(m) did not influence Csf1r expression in c-kit(+)Sca-1(+)Lin(-) cells, implying a unique role for C/EBPα-C(m) in downregulating Csf1r. Importantly, Csf1r overexpression collaborated with C/EBPα-N321D to induce fulminant AML with leukocytosis in mouse BMT models to a greater extent than did C/EBPα-N321D alone. Collectively, these results suggest that C/EBPα-C(m)-mediated downregulation of Csf1r has a negative, rather than a positive, impact on the progression of AML involving C/EBPα-C(m), which might possibly be accelerated by additional genetic and/or epigenetic alterations inducing Csf1r upregulation.
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http://dx.doi.org/10.1016/j.exphem.2014.11.011DOI Listing
April 2015

The molecular basis of myeloid malignancies.

Proc Jpn Acad Ser B Phys Biol Sci 2014 ;90(10):389-404

Division of Cellular Therapy/Division of Stem Cell Signaling, The Institute of Medical Science, The University of Tokyo.

Myeloid malignancies consist of acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and myeloproliferative neoplasm (MPN). The latter two diseases have preleukemic features and frequently evolve to AML. As with solid tumors, multiple mutations are required for leukemogenesis. A decade ago, these gene alterations were subdivided into two categories: class I mutations stimulating cell growth or inhibiting apoptosis; and class II mutations that hamper differentiation of hematopoietic cells. In mouse models, class I mutations such as the Bcr-Abl fusion kinase induce MPN by themselves and some class II mutations such as Runx1 mutations induce MDS. Combinations of class I and class II mutations induce AML in a variety of mouse models. Thus, it was postulated that hematopoietic cells whose differentiation is blocked by class II mutations would autonomously proliferate with class I mutations leading to the development of leukemia. Recent progress in high-speed sequencing has enabled efficient identification of novel mutations in a variety of molecules including epigenetic factors, splicing factors, signaling molecules and proteins in the cohesin complex; most of these are not categorized as either class I or class II mutations. The functional consequences of these mutations are now being extensively investigated. In this article, we will review the molecular basis of hematological malignancies, focusing on mouse models and the interfaces between these models and clinical findings, and revisit the classical class I/II hypothesis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335136PMC
http://dx.doi.org/10.2183/pjab.90.389DOI Listing
August 2015

Hes1 promotes blast crisis in chronic myelogenous leukemia through MMP-9 upregulation in leukemic cells.

Blood 2014 Jun 13;123(25):3932-42. Epub 2014 May 13.

Division of Cellular Therapy, Advanced Clinical Research Center, Division of Stem Cell Signaling, Center for Stem Cell Therapy, Institute of Medical Science, The University of Tokyo, Tokyo, Japan;

High levels of HES1 expression are frequently found in BCR-ABL(+) 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-κB. 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, CMPs expressing BCR-ABL and Hes1 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.
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http://dx.doi.org/10.1182/blood-2013-01-476747DOI Listing
June 2014

A novel cell-cycle-indicator, mVenus-p27K-, identifies quiescent cells and visualizes G0-G1 transition.

Sci Rep 2014 Feb 6;4:4012. Epub 2014 Feb 6.

1] Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan [2] Division of Stem Cell Signaling, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

The quiescent (G0) phase of the cell cycle is the reversible phase from which the cells exit from the cell cycle. Due to the difficulty of defining the G0 phase, quiescent cells have not been well characterized. In this study, a fusion protein consisting of mVenus and a defective mutant of CDK inhibitor, p27 (p27K(-)) was shown to be able to identify and isolate a population of quiescent cells and to effectively visualize the G0 to G1 transition. By comparing the expression profiles of the G0 and G1 cells defined by mVenus-p27K(-), we have identified molecular features of quiescent cells. Quiescence is also an important feature of many types of stem cells, and mVenus-p27K(-)-transgenic mice enabled the detection of the quiescent cells with muscle stem cell markers in muscle in vivo. The mVenus-p27K(-) probe could be useful in investigating stem cells as well as quiescent cells.
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http://dx.doi.org/10.1038/srep04012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3915272PMC
February 2014

Hes1 upregulation contributes to the development of FIP1L1-PDGRA-positive leukemia in blast crisis.

Exp Hematol 2014 May 31;42(5):369-379.e3. Epub 2014 Jan 31.

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Division of Stem Cell Signaling, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. Electronic address:

We have previously shown that elevated expression of Hairy enhancer of split 1 (Hes1) contributes to blast crisis transition in Bcr-Abl-positive chronic myelogenous leukemia. Here we investigate whether Hes1 is involved in the development of other myeloid neoplasms. Notably, Hes1 expression was elevated in only a few cases of 65 samples with different types of myeloid neoplasms. Interestingly, elevated expression of Hes1 was found in two of five samples of Fip1-like1 platelet-derived growth factor receptor-α (FIP1L1-PDGFA)-positive myeloid neoplasms associated with eosinophilia. Whereas FIP1L1-PDGFRα alone induced acute T-cell leukemia or myeloproliferative neoplasms in mouse bone marrow transplantation models, mice transplanted with bone marrow cells expressing both Hes1 and FIP1L1-PDGFRα developed acute leukemia characterized by an expansion of myeloid blasts and leukemic cells without eosinophilic granules. FIP1L1-PDGFRα conferred cytokine-independent growth to Hes1-transduced common myeloid progenitors, interleukin-3-dependent cells. Imatinib inhibited the growth of common myeloid progenitors expressing Hes1 with FIP1L1-PDGFRα, but not with imatinib-resistant FIP1L1-PDGFRα mutants harboring T674I or D842V. In contrast, ponatinib efficiently eradicated leukemic cells expressing Hes1 and the imatinib-resistant FLP1L1-PDGFRΑ mutant in vitro and in vivo. Thus, we have established mouse models of FIP1L1-PDGFRA-positive leukemia in myeloid blast crisis, which will help elucidate the pathogenesis of the disease and develop a new treatment for it.
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http://dx.doi.org/10.1016/j.exphem.2014.01.009DOI Listing
May 2014

Myelodysplastic syndromes are induced by histone methylation–altering ASXL1 mutations.

J Clin Invest 2013 Nov;123(11):4627-40

Recurrent mutations in the gene encoding additional sex combs-like 1 (ASXL1) are found in various hematologic malignancies and associated with poor prognosis. In particular, ASXL1 mutations are common in patients with hematologic malignancies associated with myelodysplasia, including myelodysplastic syndromes (MDSs), and chronic myelomonocytic leukemia. Although loss-of-function ASXL1 mutations promote myeloid transformation, a large subset of ASXL1 mutations is thought to result in stable truncation of ASXL1. Here we demonstrate that C-terminal–truncating Asxl1 mutations (ASXL1-MTs) inhibited myeloid differentiation and induced MDS-like disease in mice. ASXL1-MT mice displayed features of human-associated MDS, including multi-lineage myelodysplasia, pancytopenia, and occasional progression to overt leukemia. ASXL1-MT resulted in derepression of homeobox A9 (Hoxa9) and microRNA-125a (miR-125a) expression through inhibition of polycomb repressive complex 2–mediated (PRC2-mediated) methylation of histone H3K27. miR-125a reduced expression of C-type lectin domain family 5, member a (Clec5a), which is involved in myeloid differentiation. In addition, HOXA9 expression was high in MDS patients with ASXL1-MT, while CLEC5A expression was generally low. Thus, ASXL1-MT–induced MDS-like disease in mice is associated with derepression of Hoxa9 and miR-125a and with Clec5a dysregulation. Our data provide evidence for an axis of MDS pathogenesis that implicates both ASXL1 mutations and miR-125a as therapeutic targets in MDS.
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http://dx.doi.org/10.1172/JCI70739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3809801PMC
November 2013

Sphingomyelin and ceramide are physiological ligands for human LMIR3/CD300f, inhibiting FcεRI-mediated mast cell activation.

J Allergy Clin Immunol 2014 Jan 12;133(1):270-3.e1-7. Epub 2013 Sep 12.

Division of Cellular Therapy, The Institute of Medical Science, University of Tokyo, Tokyo, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2013.08.008DOI Listing
January 2014

APC(CDH1) targets MgcRacGAP for destruction in the late M phase.

PLoS One 2013 16;8(5):e63001. Epub 2013 May 16.

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.

Background: Male germ cell RacGTPase activating protein (MgcRacGAP) is an important regulator of the Rho family GTPases--RhoA, Rac1, and Cdc42--and is indispensable in cytokinesis and cell cycle progression. Inactivation of RhoA by phosphorylated MgcRacGAP is an essential step in cytokinesis. MgcRacGAP is also involved in G1-S transition and nuclear transport of signal transducer and activator of transcription 3/5 (STAT3/5). Expression of MgcRacGAP is strictly controlled in a cell cycle-dependent manner. However, the underlying mechanisms have not been elucidated.

Methodology/principal Findings: Using MgcRacGAP deletion mutants and the fusion proteins of full-length or partial fragments of MgcRacGAP to mVenus fluorescent protein, we demonstrated that MgcRacGAP is degraded by the ubiquitin-proteasome pathway in the late M to G1 phase via APC(CDH1). We also identified the critical region for destruction located in the C-terminus of MgcRacGAP, AA537-570, which is necessary and sufficient for CDH1-mediated MgcRacGAP destruction. In addition, we identified a PEST domain-like structure with charged residues in MgcRacGAP and implicate it in effective ubiquitination of MgcRacGAP.

Conclusions/significance: Our findings not only reveal a novel mechanism for controlling the expression level of MgcRacGAP but also identify a new target of APC(CDH1). Moreover our results identify a C-terminal region AA537-570 of MgcRacGAP as its degron.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0063001PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656054PMC
December 2013

Human CD300C delivers an Fc receptor-γ-dependent activating signal in mast cells and monocytes and differs from CD300A in ligand recognition.

J Biol Chem 2013 Mar 31;288(11):7662-7675. Epub 2013 Jan 31.

Division of Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Electronic address:

CD300C is highly homologous with an inhibitory receptor CD300A in an immunoglobulin-like domain among the human CD300 family of paired immune receptors. To clarify the precise expression and function of CD300C, we generated antibodies discriminating between CD300A and CD300C, which recognized a unique epitope involving amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Notably, CD300C was highly expressed in human monocytes and mast cells. Cross-linking of CD300C by its specific antibody caused cytokine/chemokine production of human monocytes and mast cells. Fc receptor γ was indispensable for both efficient surface expression and activating functions of CD300C. To identify a ligand for CD300A or CD300C, we used reporter cell lines expressing a chimera receptor harboring extracellular CD300A or CD300C and intracellular CD3ζ, in which its unknown ligand induced GFP expression. Our results indicated that phosphatidylethanolamine (PE) among the lipids tested and apoptotic cells were possible ligands for both CD300C and CD300A. PE and apoptotic cells more strongly induced GFP expression in the reporter cells through binding to extracellular CD300A as compared with CD300C. Differential recognition of PE by extracellular CD300A and CD300C depended on different amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Interestingly, GFP expression induced by extracellular CD300C-PE binding in the reporter cells was dampened by co-expression of full-length CD300A, indicating the predominance of CD300A over CD300C in PE recognition/signaling. PE consistently failed to stimulate cytokine production in monocytes expressing CD300C with CD300A. In conclusion, specific engagement of CD300C led to Fc receptor γ-dependent activation of mast cells and monocytes.
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http://dx.doi.org/10.1074/jbc.M112.434746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597807PMC
March 2013

The receptor LMIR3 negatively regulates mast cell activation and allergic responses by binding to extracellular ceramide.

Immunity 2012 Nov 1;37(5):827-39. Epub 2012 Nov 1.

Division of Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.

Mast cells (MCs) are key effector cells in allergic reactions. However, the inhibitory mechanism that prevents excessive activation of MCs remains elusive. Here we show that leukocyte mono-immunoglobulin-like receptor 3 (LMIR3; also called CD300f) is a negative regulator of MC activation in vivo. LMIR3 deficiency exacerbated MC-dependent allergic responses in mice, including anaphylaxis, airway inflammation, and dermatitis. Both physical binding and functional reporter assays via an extracellular domain of LMIR3 showed that several extracellular lipids (including ceramide) and lipoproteins were possible ligands for LMIR3. Importantly, MCs were frequently surrounded by extracellular ceramide in vivo. Upon engagement of high-affinity immunoglobulin E receptor, extracellular ceramide-LMIR3 binding inhibited MC activation via immunoreceptor tyrosine-based inhibitory and switch motifs of LMIR3. Moreover, pretreatment with LMIR3-Fc fusion protein or antibody against either ceramide or LMIR3 interfered with this binding in vivo, thereby exacerbating passive cutaneous anaphylaxis. Thus, the interaction between extracellular ceramide and LMIR3 suppressed MC-dependent allergic responses.
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http://dx.doi.org/10.1016/j.immuni.2012.08.018DOI Listing
November 2012

Upregulation of CD200R1 in lineage-negative leukemic cells is characteristic of AML1-ETO-positive leukemia in mice.

Int J Hematol 2012 Nov 25;96(5):638-48. Epub 2012 Oct 25.

Division of Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Activating mutations of c-Kit are frequently found in acute myeloid leukemia (AML) patients harboring t(8;21) chromosomal translocation generating a fusion protein AML1-ETO. Here we show that an active mutant of c-Kit cooperates with AML1-ETO to induce AML in mouse bone marrow transplantation models. Leukemic cells expressing AML1-ETO with c-Kit(D814V) were serially transplantable. Transplantation experiments indicated that lineage(-)c-Kit(+)Sca-1(+) (KSL) leukemic cells, but not lineage(+) leukemic cells, were enriched for leukemia stem cells (LSCs). Comparison of gene expression profiles between KSL leukemic and normal cells delineated that CD200R1 was highly expressed in KSL leukemic cells as compared with KSL normal cells. Upregulation of CD200R1 was verified in lineage(-) leukemic cells, but not in lineage(+) leukemic cells. CD200R1 expression in the lineage(-) leukemic cells was not correlated with the frequency of LSCs, indicating that CD200R1 is not a useful marker for LSCs in these models. Interestingly, CD200R1 was upregulated in KSL cells transduced with AML1-ETO, but not with other leukemogenic mutants, including c-Kit(D814V), AML1(D171N), and AML1(S291fsX300). Consistently, upregulation of CD200R1 in lineage(-) leukemic cells was observed only in the BM of mice suffering from AML1-ETO-positive leukemia. In conclusion, AML1-ETO upregulated CD200R1 in lineage(-) cells, which was characteristic of AML1-ETO-positive leukemia in mice.
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http://dx.doi.org/10.1007/s12185-012-1207-6DOI Listing
November 2012

A soluble form of LMIR5/CD300b amplifies lipopolysaccharide-induced lethal inflammation in sepsis.

J Immunol 2012 Aug 6;189(4):1773-9. Epub 2012 Jul 6.

Division of Cellular Therapy, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan;

Leukocyte mono-Ig-like receptor 5 (LMIR5, also called CD300b) is an activating receptor expressed in myeloid cells. We have previously demonstrated that T cell Ig mucin 1 works as a ligand for LMIR5 in mouse ischemia/reperfusion injury of the kidneys. In this article, we show that LMIR5 is implicated in LPS-induced sepsis in mice. Notably, neutrophils constitutively released a soluble form of LMIR5 (sLMIR5) through proteolytic cleavage of surface LMIR5. Stimulation with TLR agonists augmented the release of sLMIR5. LPS administration or peritonitis induction increased serum levels of sLMIR5 in mice, which was substantially inhibited by neutrophil depletion. Thus, neutrophils were the main source of LPS-induced sLMIR5 in vivo. On the other hand, i.p. administration of LMIR5-Fc, a surrogate of sLMIR5, bound to resident macrophages (M) and stimulated transient inflammation in mice. Consistently, LMIR5-Fc induced in vitro cytokine production of peritoneal M via its unknown ligand. Interestingly, LMIR5 deficiency profoundly reduced systemic cytokine production and septic mortality in LPS-administered mice, although it did not affect in vitro cytokine production of LPS-stimulated peritoneal M. Importantly, the resistance of LMIR5-deficient mice to LPS- or peritonitis-induced septic death was decreased by LMIR5-Fc administration, implicating sLMIR5 in LPS responses in vivo. Collectively, neutrophil-derived sLMIR5 amplifies LPS-induced lethal inflammation.
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http://dx.doi.org/10.4049/jimmunol.1201139DOI Listing
August 2012

Fyn is not essential for Bcr-Abl-induced leukemogenesis in mouse bone marrow transplantation models.

Int J Hematol 2012 Feb 22;95(2):167-75. Epub 2011 Dec 22.

Division of Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.

The Bcr-Abl oncogene causes human Philadelphia chromosome-positive (Ph(+)) leukemias, including B-cell acute lymphoblastic leukemia (B-ALL) and chronic myeloid leukemia (CML) with chronic phase (CML-CP) to blast crisis (CML-BC). Previous studies have demonstrated that Src family kinases are required for the induction of B-ALL, but not for CML, which is induced by Bcr-Abl in mice. In contrast, it has been reported that Fyn is up-regulated in human CML-BC compared with CML-CP, implicating Fyn in the blast crisis transition. Here, we aimed to delineate the exact role of Fyn in the induction/progression of Ph(+) leukemias. We found that Fyn is expressed in mouse hematopoietic cells at varying stages of development, including c-kit(+)Sca-1(+)Lin(-) cells. Notably, Fyn is highly expressed in some of human lymphomas, but not in human Ph(+) leukemias including CML-BC. In mouse bone marrow transplantation models, mice transplanted with wild-type or Fyn-deficient bone marrow cells transduced with Bcr-Abl showed no differences in the development of B-ALL or CML-like diseases. Similarly, Fyn deficiency failed to impact the development of myeloid CML-BC induced by Bcr-Abl and Hes1. Elevated expression of Fyn was not found in mouse samples of Bcr-Abl-mediated CML- and CML-BC-like diseases. Thus, Fyn is not required for the pathogenesis of Bcr-Abl-mediated leukemias.
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http://dx.doi.org/10.1007/s12185-011-0994-5DOI Listing
February 2012

Transforming growth factor-β-stimulated clone-22 is a negative-feedback regulator of Ras / Raf signaling: Implications for tumorigenesis.

Cancer Sci 2012 Jan 1;103(1):26-33. Epub 2011 Nov 1.

Division of Cellular Therapy, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Transforming growth factor-β (TGF-β)-stimulated clone-22 (TSC-22), also called TSC22D1-2, is a putative tumor suppressor. We previously identified TSC-22 downstream of an active mutant of fms-like tyrosine kinase-3 (Flt3). Here, we show that TSC-22 works as a tumor suppressor through inhibiting Ras/Raf signaling. Notably, TSC-22 was upregulated by Ras/Raf activation, whereas its upregulation was inhibited by concurrent STAT5 activation. Although TSC-22 was normally retained in the cytoplasm by its nuclear export signal (NES), Ras/Raf activation caused nuclear translocation of TSC-22, but not TSC22D1-1. Unlike glucocorticoid-induced leucine zipper (GILZ/TSC22D3-2) previously characterized as a negative regulator of Ras/Raf signaling, TSC-22 failed to interact physically with Ras/Raf. Importantly, transduction with TSC-22, but not TSC22D1-1, suppressed the growth, transformation and tumorigenesis of NIH3T3 cells expressing oncogenic H-Ras: this suppression was enhanced by transduction with a TSC-22 mutant lacking NES that had accumulated in the nucleus. Collectively, upregulation and nuclear translocation of TSC-22 played an important role in the feedback suppression of Ras/Raf signaling. Consistently, TSC22D1-deficient mice were susceptible to tumorigenesis in a mouse model of chemically-induced liver tumors bearing active mutations of Ras/Raf. Thus, TSC-22 negatively regulated Ras/Raf signaling through a mechanism different from GILZ, implicating TSC-22 as a novel suppressor of oncogenic Ras/Raf-induced tumors.
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http://dx.doi.org/10.1111/j.1349-7006.2011.02108.xDOI Listing
January 2012

Identification of RHOXF2 (PEPP2) as a cancer-promoting gene by expression cloning.

Int J Oncol 2012 Jan 19;40(1):93-8. Epub 2011 Aug 19.

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

Multiple mutations contribute to establish cancers. We have searched for potential oncogenes by screening cDNA libraries derived from gastric cancer cell lines, pancreatic cancer cell lines and glioma cell lines, using retrovirus-mediated expression cloning. Two types of interleukin-3 (IL-3)-dependent cell lines, Ba/F3 and HF6, were transduced with the cDNA libraries and several genes that render these cells factor-independent were identified including PIM-1, PIM-2, PIM-3, GADD45B and reproductive homeobox genes on the X chromosome gene F2 (RHOXF2). Although no mutation in these genes was found, these molecules were highly expressed in cancer cell lines and they may play important roles in cell transformation. Among them, we focused on a transcriptional repressor RHOXF2. Transduction of RHOXF2 rendered HF6 cells factor-independent, while knockdown of RHOXF2 inhibited growth of the HGC27 gastric cancer cell line which highly expresses RHOXF2. In addition, RHOXF2-transduced HF6 cells quickly induced leukemia when transplanted into sublethally irradiated mice. Moreover, RHOXF2 is highly expressed in some leukemia cell lines and a variety of human cancer samples including colon and lung cancers. Thus, these results indicate that RHOXF2 is involved in carcinogenesis.
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http://dx.doi.org/10.3892/ijo.2011.1173DOI Listing
January 2012

Redefining the concept of standardization for pluripotent stem cells.

Stem Cell Rev Rep 2011 Jun;7(2):221-6

The Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Yoshida-Ushinomiyacho, Sakyo-ku, Kyoto, 606-8501, Japan.

In this report we review the concept of standardization and propose an exhaustive framework for the proper management of technology on pluripotent stem cells based on studies of global and regional initiatives. We demonstrated detailed observational analysis on global initiatives for the standardization of related technologies as well as regional attempts with particular interest in the cases of the UK, the US and Japan. Consequently, we came up two fundamental issues: first, these initiatives and attempts tend to be limited to each of currently existing categories of pluripotent stem cells, whereas the technological opportunity to enable clinical/commercial application is equally open to all stem cell types. Second, the subject to be examined for standardization is set to a quite narrow range compared to precedent practices in other industrial sectors. To address these issues, we propose a strategic framework for standardization with an emphasis on comprehensiveness covering various technological opportunities and consistency to learning in the management science. By utilizing this framework development of intellectual property rights not only through patents but through taking leadership in standardization can be considered as means for improving research and development competence. Of particular concerns is the formation of quality standards for final products/services and core elemental technology, especially specific pluripotent stem cell lines. Furthermore, we inferred two stages of the standardization process, individualization where a particular product/service is qualified by the market, then standardization where the elemental technology is subsequently established as compatibility standards.
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http://dx.doi.org/10.1007/s12015-010-9204-8DOI Listing
June 2011

Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.

Blood 2011 Jan 30;117(1):221-33. Epub 2010 Sep 30.

Division of Cellular Therapy, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Two types of mutations of a transcription factor CCAAT-enhancer binding protein α (C/EBPα) are found in leukemic cells of 5%-14% of acute myeloid leukemia (AML) patients: N-terminal mutations expressing dominant negative p30 and C-terminal mutations in the basic leucine zipper domain. Our results showed that a mutation of C/EBPα in one allele was observed in AML after myelodysplastic syndrome, while the 2 alleles are mutated in de novo AML. Unlike an N-terminal frame-shift mutant (C/EBPα-N(m))-transduced cells, a C-terminal mutant (C/EBPα-C(m))-transduced cells alone induced AML with leukopenia in mice 4-12 months after bone marrow transplantation. Coexpression of both mutants induced AML with marked leukocytosis with shorter latencies. Interestingly, C/EBPα-C(m) collaborated with an Flt3-activating mutant Flt3-ITD in inducing AML. Moreover, C/EBPα-C(m) strongly blocked myeloid differentiation of 32Dcl3 cells, suggesting its class II mutation-like role in leukemogenesis. Although C/EBPα-C(m) failed to inhibit transcriptional activity of wild-type C/EBPα, it suppressed the synergistic effect between C/EBPα and PU.1. On the other hand, C/EBPα-N(m) inhibited C/EBPα activation in the absence of PU.1, despite low expression levels of p30 protein generated by C/EBPα-N(m). Thus, 2 types of C/EBPα mutations are implicated in leukemo-genesis, involving different and cooperating molecular mechanisms.
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http://dx.doi.org/10.1182/blood-2010-02-270181DOI Listing
January 2011