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• W2024697617 abstract "•C/EBPα-Cm mutations have a similar, but not identical, potential in leukemogenesis•Csf1r was identified as a gene downregulated by C/EBPα-Cm•Csf1r signals collaborated with C/EBPα-N321D to induce aggressive AML in mouse model•C/EBPα-Cm-mediated downregulation of Csf1r negatively regulated the AML progression 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-Nm) generating p30 as a dominant form and C-terminal basic leucine zipper domain mutants (C/EBPα-Cm). We have previously shown that C/EBPα-K304_R323dup belonging to C/EBPα-Cm, but not C/EBPα-T60fsX159 belonging to C/EBPα-Nm, alone induced AML in mouse bone marrow transplantation (BMT) models. Here we show that various C/EBPα-Cm mutations have a similar, but not identical, potential in myeloid leukemogenesis. Notably, like C/EBPα-K304_R323dup, any type of C/EBPα-Cm 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-Cm exhibited low levels of colony stimulating factor 1 receptor in mice. On the other hand, transduction with C/EBPα-Nm did not influence Csf1r expression in c-kit+Sca-1+Lin− cells, implying a unique role for C/EBPα-Cm 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-Cm-mediated downregulation of Csf1r has a negative, rather than a positive, impact on the progression of AML involving C/EBPα-Cm, which might possibly be accelerated by additional genetic and/or epigenetic alterations inducing Csf1r upregulation. 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-Nm) generating p30 as a dominant form and C-terminal basic leucine zipper domain mutants (C/EBPα-Cm). We have previously shown that C/EBPα-K304_R323dup belonging to C/EBPα-Cm, but not C/EBPα-T60fsX159 belonging to C/EBPα-Nm, alone induced AML in mouse bone marrow transplantation (BMT) models. Here we show that various C/EBPα-Cm mutations have a similar, but not identical, potential in myeloid leukemogenesis. Notably, like C/EBPα-K304_R323dup, any type of C/EBPα-Cm 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-Cm exhibited low levels of colony stimulating factor 1 receptor in mice. On the other hand, transduction with C/EBPα-Nm did not influence Csf1r expression in c-kit+Sca-1+Lin− cells, implying a unique role for C/EBPα-Cm 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-Cm-mediated downregulation of Csf1r has a negative, rather than a positive, impact on the progression of AML involving C/EBPα-Cm, which might possibly be accelerated by additional genetic and/or epigenetic alterations inducing Csf1r upregulation. The transcription factor CCAAT-enhancer-binding protein α (C/EBPα) regulates proliferation and differentiation in myeloid cells. The C/EBPα factor has an N-terminal transactivation domain and a C-terminal basic leucine zipper (bZIP) domain essential for DNA binding and dimerization [1Zhang P. Iwasaki-Arai J. Iwasaki H. et al.Enhancement of hematopoietic stem cell repopulating capacity and self-renewal in the absence of the transcription factor C/EBP alpha.Immunity. 2004; 2: 853-863Abstract Full Text Full Text PDF Scopus (410) Google Scholar, 2Tenen D.G. Hromas R. Licht J.D. Zhang D.-E. Transcription factors, normal myeloid development, and leukemia.Blood. 1997; 90: 489-519Crossref PubMed Google Scholar, 3Friedman A.D. McKnight S.L. Identification of two polypeptide segments of CCAAT/enhancer-binding protein required for transcriptional activation of the serum albumin gene.Genes Dev. 1990; 4: 1416-1426Crossref PubMed Scopus (128) Google Scholar, 4Landschulz W.H. Johnson P.F. McKnight S.L. The DNA binding domain of the rat liver nuclear protein C/EBP is bipartite.Science. 1989; 243: 1681-1688Crossref PubMed Scopus (426) Google Scholar, 5Nerlov C. Ziff E.B. CCAAT/enhancer binding protein-alpha amino acid motifs with dual TBP and TFIIB binding ability co-operate to activate transcription in both yeast and mammalian cells.EMBO J. 1995; 14: 4318-4328Crossref PubMed Scopus (134) Google Scholar]. Two types of C/EBPα mutations are frequently found on different alleles of de novo acute myeloid leukemia (AML) patients. One is an N-terminal frame-shift mutation (C/EBPα-Nm; e.g., C/EBPα-T60fsX159) resulting in the shorter form (p30) of C/EBPα, and the other is a C-terminal in-frame mutation in the bZIP domain (C/EBPα-Cm; e.g., C/EBPα-K304_R323dup, C/EBPα-S299_K304dup, C/EBPα-K313dup, and C/EBPα-N321D) [6Lin F.T. MacDougald O.A. Diehl A.M. Lane M.D. A 30-kDa alternative translation product of the CCAAT/enhancer binding protein alpha message: transcriptional activator lacking antimitotic activity.Proc Natl Acad Sci U S A. 1993; 90: 9606-9610Crossref PubMed Scopus (259) Google Scholar, 7Pabst T. Mueller B.U. Zhang P. et al.Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia.Nat Genet. 2001; 27: 263-270Crossref PubMed Scopus (750) Google Scholar, 8Renneville A. Boissel N. Gachard N. et al.The favorable impact of CEBPA mutations in patients with acute myeloid leukemia is only observed in the absence of associated cytogenetic abnormalities and FLT3 internal duplication.Blood. 2009; 113: 5090-5093Crossref PubMed Scopus (78) Google Scholar, 9Wouters B.J. Lowenberg B. Erpelinck-Verschueren C.A. van Putten W.L. Valk P.J. Delwel R. Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome.Blood. 2009; 113: 3088-3091Crossref PubMed Scopus (462) Google Scholar, 10Nerlov C. C/EBPalpha mutations in acute myeloid leukaemias.Nat Rev Cancer. 2004; 4: 394-400Crossref PubMed Scopus (225) Google Scholar, 11Kirstetter P. Schuster M.B. Bereshchenko O. et al.Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells.Cancer Cell. 2008; 13: 299-310Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 12Bereshchenko O. Mancini E. Moore S. et al.Hematopoietic stem cell expansion precedes the generation of committed myeloid leukemia-initiating cells in C/EBPalpha mutant AML.Cancer Cell. 2009; 16: 390-400Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar]. Using knock-in mice, Kirstetter et al. demonstrated that a C/EBPα-Cm and a C/EBPα-Nm collaborated in inducing leukemia [12Bereshchenko O. Mancini E. Moore S. et al.Hematopoietic stem cell expansion precedes the generation of committed myeloid leukemia-initiating cells in C/EBPalpha mutant AML.Cancer Cell. 2009; 16: 390-400Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar]. We also found synergism between C/EBPα-K304_R323dup and C/EBPα-T60fsX159 in rapidly inducing more aggressive AML in a mouse bone marrow transplantation (BMT) model [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar]. Thus, two types of C/EBPα mutants were shown to play distinct but collaborative roles in myeloid leukemogenesis [11Kirstetter P. Schuster M.B. Bereshchenko O. et al.Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells.Cancer Cell. 2008; 13: 299-310Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 12Bereshchenko O. Mancini E. Moore S. et al.Hematopoietic stem cell expansion precedes the generation of committed myeloid leukemia-initiating cells in C/EBPalpha mutant AML.Cancer Cell. 2009; 16: 390-400Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar]. Any type of C/EBPα-Nm uniformly generates p30, which retains an intact bZIP domain. Accordingly, C/EBPα-Nm acts as a dominant-negative regulator of a full-length form (p42) of C/EBPα. On the other hand, the precise mechanisms by which C/EBPα-Cm impaired the function of C/EBPα-p42 remained elusive. One possible explanation is that C/EBPα-Cm and C/EBPα-p42 compete for interaction with other molecules, including transcription factors, which regulate myeloid differentiation and proliferation [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar, 14Reddy V.A. Iwama A. Iotzova G. et al.Granulocyte inducer C/EBPalpha inactivates the myeloid master regulator PU. 1: possible role in lineage commitment decisions.Blood. 2002; 100: 483-490Crossref PubMed Scopus (129) Google Scholar, 15Iwama A. Osawa M. Hirasawa R. et al.Reciprocal roles for CCAAT/enhancer binding protein (C/EBP) and PU. 1 transcription factors in Langerhans cell commitment.J Exp Med. 2002; 195: 547-558Crossref PubMed Scopus (94) Google Scholar]. In addition, we have previously shown that C/EBPα-K304_R323dup, but not C/EBPα-T60fsX159, alone induced AML in mouse BMT models [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar]. It remained unclear whether each individual mutant of C/EBPα-Cm played the same role in myeloid leukemogenesis. In this study, we analyze C/EBPα-S299_K304dup, C/EBPα-K313dup, and C/EBPα-N321D as an additional mutant of C/EBPα-Cm, and we demonstrate that these types of C/EBPα-Cm have an essentially similar function in myeloid leukemogenesis. Colony stimulating factor 1 receptor (Csf1r; also known as macrophage colony–stimulating factor receptor [m-csfr] or CD115) is a receptor for colony stimulating factor 1 (CSF-1; also known as macrophage colony–stimulating factor [M-CSF]) that stimulates the proliferation and differentiation of monocyte/macrophage precursors. Several recent reports suggest the association of Csf1r upregulation with the development of AML [16Aikawa Y. Katsumoto T. Zhang P. et al.PU.1-mediated upregulation of CSF1R is crucial for leukemia stem cell potential induced by MOZ-TIF2.Nat Med. 2010; 16: 580-585Crossref PubMed Scopus (75) Google Scholar, 17Dai X.M. Ryan G.R. Hapel A.J. et al.Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects.Blood. 2002; 99: 111-120Crossref PubMed Scopus (849) Google Scholar, 18Wang C. Kelleher C.A. Cheng G.Y. et al.Expression of the CSF-1 gene in the blast cells of acute myeloblastic leukemia: association with reduced growth capacity.J Cell Physiol. 1988; 135: 133-138Crossref PubMed Scopus (12) Google Scholar, 19Rambaldi A. Wakamiya N. Vellenga E. et al.Expression of the macrophage colony-stimulating factor and c-fms genes in human acute myeloblastic leukemia cells.J Clin Invest. 1988; 81: 1030-1035Crossref PubMed Scopus (98) Google Scholar, 20Preisler H.D. Kinniburgh A.J. Wei-Dong G. Khan S. Expression of the protooncogenes c-myc, c-fos, and c-fms in acute myelocytic leukemia at diagnosis and in remission.Cancer Res. 1987; 47: 874-880PubMed Google Scholar, 21Gisselbrecht S. Fichelson S. Sola B. et al.Frequent c-fms activation by proviral insertion in mouse myeloblastic leukaemias.Nature. 1987; 329: 259-261Crossref PubMed Scopus (71) Google Scholar, 22Heard J.M. Roussel M.F. Rettenmier C.W. Sherr C.J. Multilineage hematopoietic disorders induced by transplantation of bone marrow cells expressing the v-fms oncogene.Cell. 1987; 51: 663-673Abstract Full Text PDF PubMed Scopus (45) Google Scholar]: Csf1r upregulation is critical for leukemic stem cell potential induced by MOZ-TIF2 in mice [16Aikawa Y. Katsumoto T. Zhang P. et al.PU.1-mediated upregulation of CSF1R is crucial for leukemia stem cell potential induced by MOZ-TIF2.Nat Med. 2010; 16: 580-585Crossref PubMed Scopus (75) Google Scholar]. In the present study, we demonstrate that Csf1r is downregulated by C/EBPα-Cm, but not by C/EBPα-Nm, and aim to delineate the impact of Csf1r expression on the development of AML with C/EBPα-Cm. Our results suggest that enhancement of Csf1r signaling drives the progression of AML with C/EBPα-Cm. We used samples derived from AML patients who had been diagnosed at Hiroshima University Hospital [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar, 23Ding Y. Harada Y. Imagawa J. Kimura A. Harada H. AML1/RUNX1 point mutation possibly promotes leukemic transformation in myeloproliferative neoplasms.Blood. 2009; 114: 5201-5205Crossref PubMed Scopus (54) Google Scholar]. All studies were approved by the Institutional Review Board at Hiroshima University as well as by the Ethics Committee of the University of Tokyo (approval no. 20-10-0727). Patients' informed consents were obtained in accordance with the Declaration of Helsinki. All animal studies using C57BL/6 mice (Charles River Laboratories, Yokohama, Japan) were approved by the Animal Care Committee of the Institute of Medical Science, The University of Tokyo (approval no. PA13-19). Phycoerythrin (PE)-conjugated anti-Ly5.1, Gr-1, CD11b, c-Kit, CD115, B220, and human nerve growth factor receptor (NGFR) antibodies (Abs), fluorescein isothiocyanate (FITC)-conjugated anti-CD34 and CD11b Abs, allophycocyanin (APC)-conjugated anti-c-Kit Ab, PE-Cy7-conjugated anti-Sca-1 Ab, and peridinin chlorophyll protein (PerCP)-conjugated streptavidin (BioLegend, San Diego, CA) were used [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar, 24Watanabe-Okochi N. Kitaura J. Ono R. et al.AML1 mutations induced MDS and MDS/AML in a mouse BMT model.Blood. 2008; 111: 4297-4308Crossref PubMed Scopus (131) Google Scholar, 25Nakahara F. Sakata-Yanagimoto M. Komeno Y. et al.Hes1 immortalizes committed progenitors and plays a role in blast crisis transition in chronic myelogenous leukemia.Blood. 2010; 115: 2872-2881Crossref PubMed Scopus (59) Google Scholar, 26Kagiyama Y. Kitaura J. Togami K. et al.Upregulation of CD200R1 in lineage-negative leukemic cells is characteristic of AML1-ETO-positive leukemia in mice.Int J Hematol. 2012; 96: 638-648Crossref PubMed Scopus (7) Google Scholar]. The lineage-negative (Lin−) fraction derived from bone marrow (BM) cells was incubated with FITC-anti-CD34, PE-anti-CD16/32, APC-anti–c-Kit, PE/Cy7-anti–Sca-1, and PerCP-conjugated streptavidin. c-Kit+Sca-1+Lin− (KSL) cells were sorted with a FACSAria cell sorter (BD Biosciences, San Jose, CA), as previously described. Alternatively, Ter119−CD3−B220− BM cells were sorted as control myeloid cells in BM [25Nakahara F. Sakata-Yanagimoto M. Komeno Y. et al.Hes1 immortalizes committed progenitors and plays a role in blast crisis transition in chronic myelogenous leukemia.Blood. 2010; 115: 2872-2881Crossref PubMed Scopus (59) Google Scholar, 26Kagiyama Y. Kitaura J. Togami K. et al.Upregulation of CD200R1 in lineage-negative leukemic cells is characteristic of AML1-ETO-positive leukemia in mice.Int J Hematol. 2012; 96: 638-648Crossref PubMed Scopus (7) Google Scholar]. Retrovirus vectors pMYs-IG, pMYs-INGFR, and pMYs-IP were used as previously described. C/EBPα-304_323dup, C/EBPα-299_304dup, C/EBPα-K313dup, or C/EBPα-N321D, which was tagged with a Myc epitope at the N terminus, was subcloned into pMYs-IP or IG. We tagged C/EBPα-T60fsX159 with a Myc epitope at the C terminus and subcloned it into pMYs-INGFR. Mouse Csf1r cDNA (provided by Dr. I. Kitabayashi, National Cancer Center Research Institute, Tokyo, Japan) was subcloned into pMYs-INGFR [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar, 24Watanabe-Okochi N. Kitaura J. Ono R. et al.AML1 mutations induced MDS and MDS/AML in a mouse BMT model.Blood. 2008; 111: 4297-4308Crossref PubMed Scopus (131) Google Scholar, 25Nakahara F. Sakata-Yanagimoto M. Komeno Y. et al.Hes1 immortalizes committed progenitors and plays a role in blast crisis transition in chronic myelogenous leukemia.Blood. 2010; 115: 2872-2881Crossref PubMed Scopus (59) Google Scholar, 26Kagiyama Y. Kitaura J. Togami K. et al.Upregulation of CD200R1 in lineage-negative leukemic cells is characteristic of AML1-ETO-positive leukemia in mice.Int J Hematol. 2012; 96: 638-648Crossref PubMed Scopus (7) Google Scholar]. Retroviral infection was done as described previously [27Morita S. Kojima T. Kitamura T. Plat-E: an efficient and stable system for transient packaging of retroviruses.Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1377) Google Scholar, 28Kitamura T. Koshino Y. Shibata F. et al.Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics.Exp Hematol. 2003; 31: 1007-1014Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar]. 32Dcl3 cells were cultured in the presence of 1 ng/mL interleukin (IL) 3 for cell growth assay or in the presence of 50 ng/mL granulocyte colony–stimulating factor (G-CSF) for cell differentiation assay, as previously described [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar, 29Lu Y. Kitaura J. Oki T. et al.Identification of TSC-22 as a potential tumor suppressor that is upregulated by Flt3-D835V but not Flt3-ITD.Leukemia. 2007; 21: 2246-2257Crossref PubMed Scopus (21) Google Scholar]. Colony-forming assay procedure was previously described [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar, 25Nakahara F. Sakata-Yanagimoto M. Komeno Y. et al.Hes1 immortalizes committed progenitors and plays a role in blast crisis transition in chronic myelogenous leukemia.Blood. 2010; 115: 2872-2881Crossref PubMed Scopus (59) Google Scholar]. Briefly, equal numbers of infected BM mononuclear cells were plated in methylcellulose medium (STEMCELL Technologies, Vancouver, Canada) including 50 ng/mL each of IL-3, IL-6, stem cell factor, and granulocyte/macrophage colony–stimulating factor (GM-CSF). Colonies were counted, and single-cell suspensions of colonies were replated. All cytokines were from R&D Systems (Minneapolis, MN). Mouse BMT was performed as described previously [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar, 24Watanabe-Okochi N. Kitaura J. Ono R. et al.AML1 mutations induced MDS and MDS/AML in a mouse BMT model.Blood. 2008; 111: 4297-4308Crossref PubMed Scopus (131) Google Scholar, 25Nakahara F. Sakata-Yanagimoto M. Komeno Y. et al.Hes1 immortalizes committed progenitors and plays a role in blast crisis transition in chronic myelogenous leukemia.Blood. 2010; 115: 2872-2881Crossref PubMed Scopus (59) Google Scholar, 26Kagiyama Y. Kitaura J. Togami K. et al.Upregulation of CD200R1 in lineage-negative leukemic cells is characteristic of AML1-ETO-positive leukemia in mice.Int J Hematol. 2012; 96: 638-648Crossref PubMed Scopus (7) Google Scholar]. Briefly, BM mononuclear cells were infected with retroviruses harboring Myc-S299_L304dup-IG, Myc-K313dup-IG, Myc-C/EBPα(N321D)-IG, or the empty vector (pMYs-IG), or with a combination of retroviruses harboring C/EBPα(N321D)-IG or the empty vector (pMYs-IG) with Csf1r-INGFR or the empty vector (pMYs-INGFR). Then, sublethally γ-irradiated recipient mice were given an injection of 1 × 105 of the infected BM cells. Overall survival of transplanted mice was estimated using the Kaplan-Meier method. Immunoblotting, using anti-α-tublin, extracellular signal-regulated kinase 1/2 (ERK 1/2), Signal transducer and activator of transcription (Stat) 3, or Stat5 Ab (Santa Cruz Biotechnology, Santa Cruz, CA), anti-c-Myc Ab (Sigma-Aldrich, St Louis, MO), anti-phospho-Erk1/2 Ab (Cell Signaling), or anti-phospho-Stat3 or Stat5 Ab (BD Biosciences), was performed as described previously [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar, 29Lu Y. Kitaura J. Oki T. et al.Identification of TSC-22 as a potential tumor suppressor that is upregulated by Flt3-D835V but not Flt3-ITD.Leukemia. 2007; 21: 2246-2257Crossref PubMed Scopus (21) Google Scholar]. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed as described previously [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar]. The reaction was subject to 95°C for 30 seconds followed by 40 cycles of PCR at 95°C for 5 seconds, 58°C for 10 seconds, and 72°C for 10 seconds. The following primer pairs were used: 5′-TGTCATCGAGCCTAGTGGC-3′ (forward) and 5′- CGGGAGATTCAGGGTCCAAG-3′ (reverse) for mouse Csf1r, 5′-GCATTGTGGAAGGGCTCATG-3′ (forward) and 5′-TTGCTGTTGAAGTCGCAGGAG-3′ (reverse) for mouse Gapdh, 5′-CCCCGACTTCAGTCCTTGC-3′ (forward) and 5′- GATGCACGTAGGGGTGGTG-3′ (reverse) for mouse Hoxa9, 5′-GCAAAGTATGCCAGGGGAGTA-3′ (forward) and 5′-TCCTGTGTTAAGAACCGAGGG-3′ (reverse) for mouse Mesi1, 5′-GCCGATCAGCTGGAGATGA-3′ (forward) and 5′-GTCGTCAGGATCGCAGATGAAG-3′ (reverse) for mouse cMyc, 5′-ACCTTCATCGGAAACTCCAAAG-3′ (forward) and 5′-ACTGTTAGGCTCAGGTGAACT-3′ (reverse) for mouse Hif1a, 5′-CCGGAGGCCCTTTACTACCA-3′ (forward) and 5′-GGAGTAGGGGTGAGTAGGCAG-3′ (reverse) for mouse Bcl3, 5′-CAAGCGCATGACGTATCAGAA-3′ (forward) and 5′-GCTGTCAAACTGGTAGGTGAG-3′ (reverse) for mouse Spib, 5′-AGTTGTGCTGAGCTGTATGGA -3′ (forward) and 5′-CGGCTGCTTGAAGTAAAACAGG-3′ (reverse) for mouse Mpo, 5′-CCCTCACACTCAGATCATCTTCT-3′ (forward) and 5′-GCTACGACGTGGGCTACAG-3′ (reverse) for mouse Tnf, 5′-CTGCAAGAGACTTCCATCCAG-3′ (forward) and 5′-AGTGGTATAGACAGGTCTGTTGG-3′ (reverse) for mouse Il6. Product quality was checked by melting curve analysis via LightCycler software (Roche Diagnostics, Basel, Switzerland). Expression levels were normalized by a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression level. Microarray analysis was performed as described previously [26Kagiyama Y. Kitaura J. Togami K. et al.Upregulation of CD200R1 in lineage-negative leukemic cells is characteristic of AML1-ETO-positive leukemia in mice.Int J Hematol. 2012; 96: 638-648Crossref PubMed Scopus (7) Google Scholar]. Briefly, double-stranded cDNA was synthesized, amplified, and hybridized to Affymetrix Mouse Expression array 430A, which contains 45,000 probe sets for 39,000 transcripts and variants from over 34,000 well characterized mouse genes (Affymetrix, Santa Clara, CA). The arrays were scanned on the GeneChip system confocal scanner (Affymetrix). The intensity for each feature of the array was captured with Affymetrix Microarray Suite Version 5.0 software. Statistical significance was calculated using the Student's t test (p values < .05 were considered statistically significant). We previously clarified the role of C/EBPα-304_323dup mutant, which had been identified as a type of C/EBPα-Cm, in myeloid leukemogenesis [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar]. To ascertain whether different types of C/EBPα-Cm exerted similar effects on myeloid leukemogenesis, we performed functional analysis of C/EBPα-S299_K304dup, C/EBPα-K313dup, and C/EBPα-N321D (Fig. 1A) [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar]. First, 32Dcl3 cells were retrovirally transduced with these mutants or mock. We found equivalent expression levels of each C/EBPα mutant in the 32Dcl3 transfectants (data not shown). All the transfectants exhibited comparable growth speed in the presence of IL-3 (Fig. 1B). On the other hand, after 6 days of G-CSF treatment, the mock-transduced 32Dcl3 cells differentiated into polymorphonucleated neutrophils expressing high levels of CD11b on the surface (Figs. 1C and 1D). With the same treatment, however, most 32Dcl3 cells expressing any type of C/EBPα-Cm remained immature myeloid cells expressing low levels of surface CD11b, demonstrating that C/EBPα-Cm blocked neutrophilic differentiation in 32Dcl3 cells (Figs. 1C and 1D). We observed that G-CSF-induced neutrophilic differentiation of 32Dcl3 cells was inhibited most strongly by C/EBPα-K313dup (Figs. 1C and 1D). We then performed serial colony-forming assays using BM mononuclear cells transduced with different types of C/EBPα-Cm. The results showed that BM cells transduced with any type of C/EBPα-Cm, but not with mock, were serially transplantable in the presence of cytokine cocktail (Fig. 1E). After four rounds of replating, BM cells transduced with any type of C/EBPα-Cm remained immature, exhibiting similar morphology and equivalent growth speed in the presence of IL-3 (data not shown). Thus, all types of C/EBPα-Cm tested inhibited myeloid differentiation in 32Dcl3 cells and immortalized BM cells. We then asked whether different types of C/EBPα-Cm induced similar phenotypes of myeloid malignancies in mouse BMT models. Sublethally irradiated Ly-5.2 mice were transplanted with Ly-5.1 BM mononuclear cells infected with retroviruses harboring C/EBPα-S299_K304dup, C/EBPα-K313dup, C/EBPα-N321D, or mock (pMYs-IG). We confirmed that 40%–60% of the transduced BM cells were positive for green fluorescent protein (GFP) expression before transplantation. Mice transplanted with the mock-transduced BM cells remained healthy over the observation period (Fig. 2A). On the other hand, mice that received BM cells expressing any type of C/EBPα-Cm developed AML after transplantation, although the disease latency for leukemia development was different among the groups: the median survival time was 107 days, 151 days, or 298 days in C/EBPα-N321D-, C/EBPα-K313dup-, or C/EBPα-S299_K304dup-induced AML, respectively (Fig. 2A). The morbid mice with AML induced by any type of C/EBPα-Cm exhibited a similar phenotype characterized by splenomegaly, anemia, and hypocellular BM with 40%–60% myeloid blasts (Fig. 2B and data not shown). As revealed by Western blot analysis, BM leukemic cells expressing any type of C/EBPα-Cm displayed comparable expression levels of the transduced C/EBPα-Cm (Fig. 2C). Flow cytometric analysis displayed a similar surface marker profile (Gr-1+CD11b+c-Kitlow/+) of leukemic cells expressing C/EBPα-S299_K304dup, C/EBPα-K313dup, or C/EBPα-N321D (Fig. 2D). Real-time RT-PCR analysis displayed no significant difference for expression of leukemia-associated genes tested among leukemic BM cells expressing C/EBPα-S299_K304dup, C/EBPα-K313dup, and C/EBPα-N321D (Supplementary Figure E1A, online only, available at www.exphem.org). Thus, any type of C/EBPα-Cm alone induced AML in mouse BMT models. For further analysis of C/EBPα-Cm, we used C/EBPα-N321D, which was able to reproducibly and quickly induce AML in mouse BMT models. To next explore the regulatory mechanisms underlying C/EBPα-Cm-induced AML, we compared the gene expression profiles between C/EBPα-N321D- and mock-transduced KSL cells. The results showed that four genes (Spib, Tnf, Csf1r, and Bcl3) regulating cell proliferation and/or differentiation were downregulated at least threefold between C/EBPα-N321D-transduced KSL cells and mock-transduced KSL cells (Table 1; Supplementary Table E1, online only, available at www.exphem.org). We then turned our attention to Csf1r as a gene downregulated by C/EBPα-N321D, because Csf1r is known to play a role in myeloid differentiation and proliferation. Real-time RT-PCR analysis verified lower levels of Csf1r transcript in C/EBPα-N321D-transduced KSL cells as compared with C/EBPα-Nm- or mock-transduced KSL cells (Fig. 3A). Consistently, surface expression levels of Csf1r were lower in C/EBPα-N321D-transduced KSL cells than in C/EBPα-Nm- or mock-transduced KSL cells (Fig. 3B). In addition, leukemic BM cells expressing C/EBPα-S299_K304dup, C/EBPα-K313dup, or C/EBPα-N321D exhibited lower levels of Csf1r transcript than those displayed by control BM myeloid cells (Fig. 3C). On the other hand, transduction with C/EBPα-Nm did not influence Csf1r expression in C/EBPα-N321D-expressing KSL cells or BM leukemic cells (Fig. 3A–3D). Thus, C/EBPα-Cm was shown to downregulate Csf1r in the transduced cells.Table 1Genes associated with cell proliferation and/or differentiation downregulated by transduction with C/EBPα-N321D in KSL cellsGene symbolGene titleFold changeSpibSpi-B transcription factor (Spi-1/PU.1 related)-5.99Tnftumor necrosis factor-5.44Csf1rcolony stimulating factor 1 receptor-3.46Bcl3B-cell leukemia/lymphoma 3-3.41 Open table in a new tab We next examined the effect of Csf1r expression on the development of C/EBPα-Cm-induced AML in mouse BMT models. Notably, all mice receiving BM cells transduced with both Csf1r and C/EBPα-N321D died of fulminant AML within 37.5 days after transplantation (Fig. 4A). Like the morbid mice with AML induced by C/EBPα-N321D alone, those with AML induced by C/EBPα-N321D with Csf1r overexpression exhibited splenomegaly and hypocellular BM with 40–80% myeloid blasts (Fig. 4B). On the other hand, unlike the former mice, the latter displayed prominent leukocytosis (Fig. 4B). Real-time RT-PCR analysis verified that leukemic BM cells from the latter mice expressed higher levels of Csf1r than those from the former (Supplementary Figure E1A, online only, available at www.exphem.org). Transduction with Csf1r failed to significantly alter C/EBPα-N321D-induced downregulation of Spi-B, TNFα, or Bcl3 in KSL cells or in leukemic BM cells (Supplementary Figures E1A and E1B, online only, available at www.exphem.org). In addition, transduction with Csf1r did not influence expression of several leukemia-associated genes tested, including Hoxa9 and Meis1, in C/EBPα-N321D-expressing KSL cells or BM leukemic cells (Supplementary Figures E1A and E1B, online only, available at www.exphem.org). Flow cytometric analysis demonstrated that leukemic BM cells transduced with both Csf1r and C/EBPα-N321D were Gr-1low/+CD11b+c-kit−/lintB220−/lintCsf1rlow/+ (Fig. 4C). It should be noted that mice transplanted with Csf1r-transduced BM cells remained healthy during the observation period (Fig. 4A). Western blot analysis displayed a prominent activation of ERK, but not of Stat3 or Stat5, in BM leukemic cells expressing C/EBPα-N321D with Csf1r compared with those expressing C/EBPα-N321D alone (Fig. 4D). Thus, ectopic expression of Csf1r accelerated the development of C/EBPα-N321D-induced AML, suggesting that C/EBPα-N321D-mediated downregulation of Csf1r negatively regulated C/EBPα-N321D-induced AML in mouse BMT models. A series of experiments demonstrated that any type of C/EBPα-Cm by itself inhibited myeloid differentiation in 32Dcl3 cells, immortalized BM cells, and induced AML of similar phenotypes in mouse BMT models. However, it was interesting that, among the types of C/EBPα-Cm tested, C/EBPα-K313dup was the strongest inhibitor of G-CSF-induced neutrophilic differentiation in 32Dcl3 cells, whereas C/EBPα-N321D was the most powerful inducer of AML in mouse BMT models. It was therefore plausible to speculate that C/EBPα-Cm had the potential not only to inhibit myeloid differentiation but also to promote self-renewal and/or proliferation of leukemic cells. Considering that C/EBPα-K313dup was reported to induce premalignant hematopoietic stem cell expansion in knock-in mice [11Kirstetter P. Schuster M.B. Bereshchenko O. et al.Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells.Cancer Cell. 2008; 13: 299-310Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 12Bereshchenko O. Mancini E. Moore S. et al.Hematopoietic stem cell expansion precedes the generation of committed myeloid leukemia-initiating cells in C/EBPalpha mutant AML.Cancer Cell. 2009; 16: 390-400Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar], C/EBPα-N321D might be superior to C/EBPα-K313dup in this ability. However, we were not able to identify specific downstream targets of individual C/EBPα-Cm. Given that C/EBPα-Cm inhibited C/EBPα-dependent transcription by interfering with the interaction between C/EBPα and other transcriptional factors [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar], the degree of such transcriptional inhibition associated with myeloid leukemogenesis might be influenced by different mutations of C/EBPα-Cm. In any case, a set of genes regulated specifically by C/EBPα-Cm could be involved in the development of AML induced by C/EBPα-Cm alone. Accordingly, we compared gene expression profiles between C/EBPα-N321D- and mock-transduced KSL cells, identifying Csf1r as a gene downregulated by C/EBPα-Cm, but not by C/EBPα-Nm. In fact, leukemic cells expressing any type of C/EBPα-Cm exhibited low levels of Csf1r expression in mice, whereas Csf1r overexpression cooperated with C/EBPα-N321D to rapidly induce AML with leukocytosis in mouse BMT models. These results suggested that C/EBPα-Cm-mediated downregulation of Csf1r inhibited, rather than promoted, the progression of AML with C/EBPα-Cm. However, the finding that C/EBPα-321D, the strongest inhibitor of Csf1r expression, induced AML with the shortest latency implied a limited role for Csf1r downregulation in the development of C/EBPα-Cm-induced AML. On the other hand, marked leukocytosis induced by C/EBPα-N321D plus ectopic expression of Csf1r suggested that, like a class I mutant Flt3-ITD [13Kato N. Kitaura J. Doki N. et al.Two types of C/EBPα mutations play distinct but collaborative roles in leukemogenesis: lessons from clinical data and BMT models.Blood. 2011; 117: 221-233Crossref PubMed Scopus (55) Google Scholar], Csf1r signaling promoted proliferation of leukemic cells expressing C/EBPα-Cm. Consistent with this finding, we found constitutive activation of ERK in leukemic cells expressing C/EBPα-N321D and Csf1r. A previous finding, that Csf1r upregulation was essential for leukemia stem cell potential induced by MOZ-TIF2, aligns with this hypothesis [16Aikawa Y. Katsumoto T. Zhang P. et al.PU.1-mediated upregulation of CSF1R is crucial for leukemia stem cell potential induced by MOZ-TIF2.Nat Med. 2010; 16: 580-585Crossref PubMed Scopus (75) Google Scholar]. It is therefore possible to assume that additional genetic and epigenetic alterations upregulating Csf1r would accelerate AML involving C/EBPα-Cm. To investigate this possibility, it will be necessary to measure Csf1r expression in samples derived from AML patients harboring C/EBPα-Cm. Although PU.1 had been found to activate Csf1r promoter [16Aikawa Y. Katsumoto T. Zhang P. et al.PU.1-mediated upregulation of CSF1R is crucial for leukemia stem cell potential induced by MOZ-TIF2.Nat Med. 2010; 16: 580-585Crossref PubMed Scopus (75) Google Scholar], C/EBPα-Cm failed to influence PU.1-mediated luciferase activation of Csf1r promoter (Togami et al., unpublished data). Further examination will be required to understand the mechanisms underlying C/EBPα-Cm-mediated downregulation of Csf1r. In the present study, we were not able to identify a gene downregulated by C/EBPα-Cm having a negative impact on C/EBPα-Cm-induced AML. However, similar approaches using BMT models will help us understand the relevant mechanisms. In conclusion, we have demonstrated that all types of C/EBPα-Cm tested had a similar potential to induce myeloid leukemogenesis and that C/EBPα-Cm-mediated downregulation of Csf1r had a negative rather than a positive impact on the progression of AML. We thank Dr. I. Kitabayashi for providing the cDNA of Csf1r. We also thank R&D Systems for providing cytokines. We thank Dr. Dovie Wylie for her excellent language assistance. This work was supported by a grant (no. 23249051) from the Ministry of Education, Science, Technology, Sports and Culture, Japan. No financial interest/relationships with financial interest relating to the topic of this article have been declared. Supplementary Table E1Genes down-regulated by transduction with C/EBPα-N321D in KSL cellsGene symbolFold changeGene symbolFold changeLyz1-6.04Ctsh-3.46Spib-5.99Csf1r-3.46Cngb1-5.66Capg-3.45Siglech-5.63S100a9-3.42Tnf-5.44Cybb-3.41Ly6d-5.31Bcl3-3.41H2-Aa-4.95Lpl-3.37Cd22-4.68Cfp-3.37Kmo-4.40Slfn2-3.30Rnd3-4.39Gm11428-3.28Clec4a2-4.31Cdh17-3.27H2-Aa-4.208430408G22Rik-3.26Hnmt-4.15Pacsin1-3.25Adrb2-4.03Upb1-3.21Tbc1d8-3.99Rnase6-3.15Ngp-3.92Olfm1-3.15Klrd1-3.92Elane-3.11Ceacam1-3.89Ttf2-3.09Lpl-3.85Hp-3.08Cd74-3.72Arhgap6-3.06Ifitm6-3.72Rhbdd2-3.06Gzmb-3.68Cx3cr1-3.06H2-Eb1-3.64Ctsh-3.04Dhcr24-3.64Gpr183-3.01Cybb-3.57Ppbp-3.01Mmp11-3.54Thbs1-3.00Cd300a-3.47 Open table in a new tab" @default.
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