- Email: [email protected]
M-CSF instructs both cell division and cell identity in HSC through independent transcription factor circuits
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3168 - OVEREXPRESSION OF RUNX1 SHORT ISOFORM PLAYS A PIVOTAL ROLE IN THE DEVELOPMENT OF MYELODYSPLASTIC SYNDROMES/MYELOPROLIFERATIVE NEOPLASMS Hiroko Sakurai1, Yuka Harada1,5, Hirotaka Matsui2, Hideaki Nakajima3, Toshio Kitamura4, Norio Komatsu1, and Hironori Harada1 1 Juntendo University School of Medicine, Tokyo, Japan; 2Kumamoto University, Kumamoto, Japan; 3Keio University School of Medicine, Tokyo, Japan; 4Institute of Medical Science, The University of Tokyo, Tokyo, Japan; 5Bunkyo Gakuin University, Tokyo, Japan RUNX1 mutations have been shown to contribute to the development of myeloid neoplasms. Although the mutations have been analyzed, its expression level has not been investigated. Therefore, we attempt to clarify the expression of RUNX1 in the pathogenesis of myeloid neoplasms. Expression levels of full length isoform (RUNX1b) and short isoform (RUNX1a which has a dominant negative effect on RUNX1b) in CD34+ cells from patients with myeloid neoplasms were examined. A part of patients with myelodysplasitc syndromes (MDS) or myelodysplastic syndrome / myeloproliferative neoplasms (MDS/MPN) showed RUNX1a overexpression. During the disease progression, the expression of RUNX1a became higher. We next analyzed the mechanism of RUNX1a overexpression. Splicing factor mutations, mainly SRSF2 and U2AF1, were detected frequently in MDS and MDS/MPN. Patients with splicing factor mutations showed higher RUNX1a expression than patients without the mutations. To confirm that the splicing factor mutations affect the expression of RUNX1a, we performed enforced expression of SRSF2 p.P95H mutant in a MDS-derived cell line, TF-1. After a single cell sorting, expanding clones demonstrated higher expression of RUNX1a than mock cells. Moreover, higher SRSF2 p.P95H intensity among the clones seemed to induce higher expression of RUNX1a. On the other hand, RUNX1b expression was reduced in all clones. Furthermore, intronic mutations including splice sites were detected, which may be another mechanism of RUNX1a overexpression. These results suggest that overexpression of RUNX1a may play a pivotal role in the development of MDS and MDS/MPN, in addition to RUNX1 mutations.
3169 - LYMPH NODE-ASSOCIATED STROMAL FIBROBLASTS AS A POTENTIAL SOURCE OF HUMORAL FACTORS TO SUPPORT B-CELL LYMPHOMA CELLS VIA THE PHOSPHATIDYLINOSITOL 3-KINASE PATHWAY Akihiko Sakamoto1, Kazuyuki Shimada1,2, Miyuki Katayama3, Fumihiko Hayakawa1, Akihiro Tomita1, and Hitoshi Kiyoi1 1 Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Institute for Advanced Research, Nagoya University, Nagoya, Japan; 3Division of Molecular Medicine, Aichi Cancer Center Research Institute, Nagoya, Japan Although various malignant lymphomas arise from lymph nodes (LNs), microenvironmental support of lymphoma cells remains to be understood. Here we demonstrate LN-associated stromal fibroblasts as a potential source of various humoral factors to support B-cell lymphoma cells via the phosphatidylinositol 3-kinase (PI3K) pathway. All procedures were approved by the institutional review board. We first successfully expanded stromal cells in vitro from primary LN samples with various hematological diseases, which were identified as fibroblasts by the marker expression. These fibroblasts supported the survival of LN samples with various hematological diseases (P 5 9.8 10 4; n 5 11). Furthermore, stromal fibroblasts supported the survival of primary B-cell lymphoma cells, which was seemingly correlated with their vulnerability. We then examined the underlying mechanism using patient-derived B-cell lymphoma cells, which were successfully expanded in vitro when cocultured with stromal fibroblasts. Fibroblast-conditioned media promoted the viability of B-cell lymphoma cells in a dose-dependent manner, suggesting a contribution of humoral factors. Stromal fibroblasts in fact secreted various humoral factors, including interleukin-8, C-C motif chemokine 2 and macrophage migration inhibitory factor. The stromal support of B-cell lymphoma cells was attributed to the PI3K pathway, which was associated with upregulation of prosurvival MCL1 and downregulation of apoptotic BIM. Furthermore, fibroblast-conditioned media promoted the growth of B-cell lymphoma cell lines, which was suppressed by pharmacological inhibition of the PI3K pathway. Collectively, LN-associated stromal fibroblasts might supply various humoral factors to support B-cell lymphoma cells via the PI3K pathway.
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3170 - A ROLE OF THE TRANSCRIPTION FACTOR IRF8 IN BASOPHIL AND MAST CELL DEVELOPMENT Haruka Sasaki1, Daisuke Kurotaki1, Naoki Osato2, Hideaki Sato1, Izumi Sasaki3, Shin-ichi Koizumi1, Hongsheng Wang4, Chika Kaneda1, Akira Nishiyama1, Tsuneyasu Kaisho3, Hiroyuki Aburatani2, Herbert C. Morse III 4, Keiko Ozato5, and Tomohiko Tamura1 1 Dept. of Immunol., Yokohama City Univ. Grad. Sch. of Med., Yokohama, Japan; 2 Div. of Genome Sci., Res. Ctr. for Adv. Sci. and Technol., Univ. of Tokyo, Tokyo, Japan; 3Lab. for Immune Reg., IFReC, Osaka Univ., Osaka, Japan; 4NIAID, NIH, Rockville, Maryland, USA; 5NICHD, NIH, Bethesda, Maryland, USA Basophils and mast cells are key effector cells in host defense against infection and allergic responses. It has been shown that hematopoietic stem cells differentiate into basophil progenitors (BaPs) and mast cell progenitors (MCPs) via several progenitors such as granulocyte progenitors (GPs), pre-basophil and mast cell progenitors (preBMPs), and basophil/mast cell progenitors (BMCPs). BaPs and MCPs differentiate into mature basophils and mast cells, respectively. Several transcription factors, such as C/EBPa and GATA2, are important for this developmental pathway. Interferon regulatory factor-8 (IRF8) is a transcription factor known to regulate the development of several myeloid lineages. In this study, we found that Irf8–/– mice retained GPs, BMCPs, and mast cells, but lack pre-BMPs, BaPs, and basophils. MCPs were severely reduced in the bone marrow but were normally distributed in peripheral tissues in Irf8–/– mice. Using IRF8-GFP chimera knock-in mice, we show that GPs but not downstream populations expressed IRF8. Interestingly, in vivo transfer and in vitro culture experiments revealed the impaired intrinsic differentiation potential of Irf8–/– GPs towards not only basophils but also mast cells. Transcriptome analysis in Irf8–/– GPs and computational DNA motif analysis predicted that GATA2 may critically act downstream of IRF8. Indeed, retroviral transduction of GATA2 into Irf8–/– hematopoietic progenitor cells rescued basophil and mast cell differentiation in vitro. These results suggest that IRF8 acts at the GP stage to induce Gata2 expression, thereby promoting the development of basophils and mast cells. We also provide a possible explanation for the seemingly discrepant data described above regarding the effect of IRF8 on mast cell development.
3171 - M-CSF INSTRUCTS BOTH CELL DIVISION AND CELL IDENTITY IN HSC THROUGH INDEPENDENT TRANSCRIPTION FACTOR CIRCUITS Sandrine Sarrazin1, Prashanth K. Kandalla1, Noushine Mossadegh-Keller1, Leon Espinosa2, and Michael Sieweke1,3 1 Centre d’Immunologie de Marseille-Luminy (CIML), Marseille, France; 2 Laboratoire de Chimie Bacterienne UPR 9043, Marseille, France; 3Max-Delbr€uckCentrum f€ur Molekulare Medizin (MDC), Berlin, Germany We showed recently that M-CSF could instruct myeloid gene expression and differentiation preference of HSC by activation of the myeloid master regulator PU.1 without changing proliferation rate demonstrating that stem cells are direct targets of lineage instruction by cytokines. Interestingly, we observed previously that in absence of the transcription factor MafB, M-CSF does induce both myeloid lineage commitment and proliferation of HSC. To decipher the molecular mechanisms underlying this, we monitored cell division of wt and MafB deficient HSC in response to M-CSF by video-microscopy and analyzed mRNA expression of cell cycle regulators by nanofluidic real time PCR of individual HSC. In absence of MafB, HSC entered the cycle at a higher rate in response to M-CSF and showed a gene expression signature typical of cycling cells. Among the upstream cell cycle regulators, we saw a specific increase of Myc expression selectively upon M-CSF stimulation in absence of MafB, suggesting that MCSF-induced cell division in HSC is controlled by Myc under the repressive effect of MafB. On the other hand, MafB deficient HSC are still sensitive to M-CSF-induced myeloid commitment in vivo as individual transplanted highly purified MafB deficient HSC showed rapid activation of PU.1 in response to M-CSF. As in wt HSC, PU.1 activation in MafB deficient HSC was dependent on M-CSF signaling and gene expression profiling of single MafB deficient HSC demonstrated that M-CSF induced a myeloid signature as in wt HSC. Taken together our data indicate that external cytokine cues such as M-CSF can instruct both cell identity change and cell division in HSC acting on PU.1 and c-Myc controlled pathways respectively. We also showed that MafB restricts the sensitivity threshold for the activation of myeloid specific commitment divisons induced by M-CSF, leading to an integrated control of cell cycle and lineage choice in HSC.
3168 - OVEREXPRESSION OF RUNX1 SHORT ISOFORM PLAYS A PIVOTAL ROLE IN THE DEVELOPMENT OF MYELODYSPLASTIC SYNDROMES/MYELOPROLIFERATIVE NEOPLASMS Hiroko Sakurai1, Yuka Harada1,5, Hirotaka Matsui2, Hideaki Nakajima3, Toshio Kitamura4, Norio Komatsu1, and Hironori Harada1 1 Juntendo University School of Medicine, Tokyo, Japan; 2Kumamoto University, Kumamoto, Japan; 3Keio University School of Medicine, Tokyo, Japan; 4Institute of Medical Science, The University of Tokyo, Tokyo, Japan; 5Bunkyo Gakuin University, Tokyo, Japan RUNX1 mutations have been shown to contribute to the development of myeloid neoplasms. Although the mutations have been analyzed, its expression level has not been investigated. Therefore, we attempt to clarify the expression of RUNX1 in the pathogenesis of myeloid neoplasms. Expression levels of full length isoform (RUNX1b) and short isoform (RUNX1a which has a dominant negative effect on RUNX1b) in CD34+ cells from patients with myeloid neoplasms were examined. A part of patients with myelodysplasitc syndromes (MDS) or myelodysplastic syndrome / myeloproliferative neoplasms (MDS/MPN) showed RUNX1a overexpression. During the disease progression, the expression of RUNX1a became higher. We next analyzed the mechanism of RUNX1a overexpression. Splicing factor mutations, mainly SRSF2 and U2AF1, were detected frequently in MDS and MDS/MPN. Patients with splicing factor mutations showed higher RUNX1a expression than patients without the mutations. To confirm that the splicing factor mutations affect the expression of RUNX1a, we performed enforced expression of SRSF2 p.P95H mutant in a MDS-derived cell line, TF-1. After a single cell sorting, expanding clones demonstrated higher expression of RUNX1a than mock cells. Moreover, higher SRSF2 p.P95H intensity among the clones seemed to induce higher expression of RUNX1a. On the other hand, RUNX1b expression was reduced in all clones. Furthermore, intronic mutations including splice sites were detected, which may be another mechanism of RUNX1a overexpression. These results suggest that overexpression of RUNX1a may play a pivotal role in the development of MDS and MDS/MPN, in addition to RUNX1 mutations.
3169 - LYMPH NODE-ASSOCIATED STROMAL FIBROBLASTS AS A POTENTIAL SOURCE OF HUMORAL FACTORS TO SUPPORT B-CELL LYMPHOMA CELLS VIA THE PHOSPHATIDYLINOSITOL 3-KINASE PATHWAY Akihiko Sakamoto1, Kazuyuki Shimada1,2, Miyuki Katayama3, Fumihiko Hayakawa1, Akihiro Tomita1, and Hitoshi Kiyoi1 1 Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Institute for Advanced Research, Nagoya University, Nagoya, Japan; 3Division of Molecular Medicine, Aichi Cancer Center Research Institute, Nagoya, Japan Although various malignant lymphomas arise from lymph nodes (LNs), microenvironmental support of lymphoma cells remains to be understood. Here we demonstrate LN-associated stromal fibroblasts as a potential source of various humoral factors to support B-cell lymphoma cells via the phosphatidylinositol 3-kinase (PI3K) pathway. All procedures were approved by the institutional review board. We first successfully expanded stromal cells in vitro from primary LN samples with various hematological diseases, which were identified as fibroblasts by the marker expression. These fibroblasts supported the survival of LN samples with various hematological diseases (P 5 9.8 10 4; n 5 11). Furthermore, stromal fibroblasts supported the survival of primary B-cell lymphoma cells, which was seemingly correlated with their vulnerability. We then examined the underlying mechanism using patient-derived B-cell lymphoma cells, which were successfully expanded in vitro when cocultured with stromal fibroblasts. Fibroblast-conditioned media promoted the viability of B-cell lymphoma cells in a dose-dependent manner, suggesting a contribution of humoral factors. Stromal fibroblasts in fact secreted various humoral factors, including interleukin-8, C-C motif chemokine 2 and macrophage migration inhibitory factor. The stromal support of B-cell lymphoma cells was attributed to the PI3K pathway, which was associated with upregulation of prosurvival MCL1 and downregulation of apoptotic BIM. Furthermore, fibroblast-conditioned media promoted the growth of B-cell lymphoma cell lines, which was suppressed by pharmacological inhibition of the PI3K pathway. Collectively, LN-associated stromal fibroblasts might supply various humoral factors to support B-cell lymphoma cells via the PI3K pathway.
S93
3170 - A ROLE OF THE TRANSCRIPTION FACTOR IRF8 IN BASOPHIL AND MAST CELL DEVELOPMENT Haruka Sasaki1, Daisuke Kurotaki1, Naoki Osato2, Hideaki Sato1, Izumi Sasaki3, Shin-ichi Koizumi1, Hongsheng Wang4, Chika Kaneda1, Akira Nishiyama1, Tsuneyasu Kaisho3, Hiroyuki Aburatani2, Herbert C. Morse III 4, Keiko Ozato5, and Tomohiko Tamura1 1 Dept. of Immunol., Yokohama City Univ. Grad. Sch. of Med., Yokohama, Japan; 2 Div. of Genome Sci., Res. Ctr. for Adv. Sci. and Technol., Univ. of Tokyo, Tokyo, Japan; 3Lab. for Immune Reg., IFReC, Osaka Univ., Osaka, Japan; 4NIAID, NIH, Rockville, Maryland, USA; 5NICHD, NIH, Bethesda, Maryland, USA Basophils and mast cells are key effector cells in host defense against infection and allergic responses. It has been shown that hematopoietic stem cells differentiate into basophil progenitors (BaPs) and mast cell progenitors (MCPs) via several progenitors such as granulocyte progenitors (GPs), pre-basophil and mast cell progenitors (preBMPs), and basophil/mast cell progenitors (BMCPs). BaPs and MCPs differentiate into mature basophils and mast cells, respectively. Several transcription factors, such as C/EBPa and GATA2, are important for this developmental pathway. Interferon regulatory factor-8 (IRF8) is a transcription factor known to regulate the development of several myeloid lineages. In this study, we found that Irf8–/– mice retained GPs, BMCPs, and mast cells, but lack pre-BMPs, BaPs, and basophils. MCPs were severely reduced in the bone marrow but were normally distributed in peripheral tissues in Irf8–/– mice. Using IRF8-GFP chimera knock-in mice, we show that GPs but not downstream populations expressed IRF8. Interestingly, in vivo transfer and in vitro culture experiments revealed the impaired intrinsic differentiation potential of Irf8–/– GPs towards not only basophils but also mast cells. Transcriptome analysis in Irf8–/– GPs and computational DNA motif analysis predicted that GATA2 may critically act downstream of IRF8. Indeed, retroviral transduction of GATA2 into Irf8–/– hematopoietic progenitor cells rescued basophil and mast cell differentiation in vitro. These results suggest that IRF8 acts at the GP stage to induce Gata2 expression, thereby promoting the development of basophils and mast cells. We also provide a possible explanation for the seemingly discrepant data described above regarding the effect of IRF8 on mast cell development.
3171 - M-CSF INSTRUCTS BOTH CELL DIVISION AND CELL IDENTITY IN HSC THROUGH INDEPENDENT TRANSCRIPTION FACTOR CIRCUITS Sandrine Sarrazin1, Prashanth K. Kandalla1, Noushine Mossadegh-Keller1, Leon Espinosa2, and Michael Sieweke1,3 1 Centre d’Immunologie de Marseille-Luminy (CIML), Marseille, France; 2 Laboratoire de Chimie Bacterienne UPR 9043, Marseille, France; 3Max-Delbr€uckCentrum f€ur Molekulare Medizin (MDC), Berlin, Germany We showed recently that M-CSF could instruct myeloid gene expression and differentiation preference of HSC by activation of the myeloid master regulator PU.1 without changing proliferation rate demonstrating that stem cells are direct targets of lineage instruction by cytokines. Interestingly, we observed previously that in absence of the transcription factor MafB, M-CSF does induce both myeloid lineage commitment and proliferation of HSC. To decipher the molecular mechanisms underlying this, we monitored cell division of wt and MafB deficient HSC in response to M-CSF by video-microscopy and analyzed mRNA expression of cell cycle regulators by nanofluidic real time PCR of individual HSC. In absence of MafB, HSC entered the cycle at a higher rate in response to M-CSF and showed a gene expression signature typical of cycling cells. Among the upstream cell cycle regulators, we saw a specific increase of Myc expression selectively upon M-CSF stimulation in absence of MafB, suggesting that MCSF-induced cell division in HSC is controlled by Myc under the repressive effect of MafB. On the other hand, MafB deficient HSC are still sensitive to M-CSF-induced myeloid commitment in vivo as individual transplanted highly purified MafB deficient HSC showed rapid activation of PU.1 in response to M-CSF. As in wt HSC, PU.1 activation in MafB deficient HSC was dependent on M-CSF signaling and gene expression profiling of single MafB deficient HSC demonstrated that M-CSF induced a myeloid signature as in wt HSC. Taken together our data indicate that external cytokine cues such as M-CSF can instruct both cell identity change and cell division in HSC acting on PU.1 and c-Myc controlled pathways respectively. We also showed that MafB restricts the sensitivity threshold for the activation of myeloid specific commitment divisons induced by M-CSF, leading to an integrated control of cell cycle and lineage choice in HSC.