- Email: [email protected]
Role of septins in HSC aging
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3176 - BILE ACIDS SUPPORT EXPANDING HEMATOPOIETIC STEM/ PROGENITOR CELLS IN THE FETAL LIVER Valgardur Sigurdsson1, Hajime Takei2, Svetlana Soboleva1, Kavitha Siva1, Roman Galeev1, Fredrik Leeb-Lundberg3, Takashi Iida4, Hiroshi Nittono2, and Kenichi Miharada1 1 Div. Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden; 2Junshin Clinic Bile Acid Institute, Tokyo, Japan; 3Dept. Drug Target Discovery, Lund University, Lund, Sweden; 4Dept. Chemistry, College of Humanities and Sciences, Nihon University, Tokyo, Japan Hematopoietic stem cells (HSCs) are actively expanding in the fetal liver (FL) but the mechanisms enabling this have not been completely uncovered. HSCs are vulnerable to and quickly activate endoplasmic reticulum (ER) stress responses believed to be fueled by accumulation of unfolded / misfolded proteins (Miharada et al., Cell Rep. 2014). This raises the question how HSCs deal with the higher protein-folding requirement during expansion in the FL. Of note, FL-HSCs have unchanged levels of ER stress related genes despite their high proliferation status without an increase in heat shock protein levels, strongly indicating that other factor(s) block ER stress elevation. Here we demonstrate that bile acids (BA) act as natural chaperones to eliminate ER stress in the fetal liver. Reduction of BA levels using the FxR agonist, GW4064, significantly decreased the number of hematopoietic stem/progenitor cells, due to increased apoptosis caused by elevated ER stress levels. Similarly, dual deletion of Cyp27a1, a key BA synthetic enzyme, in both mother and fetus severely decreased total cellularity and number of HSCs in FL due to increased ER stress. Interestingly, FL of homozygotes grown in heterozygote mothers did not show any significant difference compared to littermate heterozygotes, suggesting that the contribution of maternal BA may be in FL is critical for HSCs. In both models, injection of TCA (a major BA in FL) or Salubrinal, an ER stress inhibitor, rescued the effects of BA reduction. Mechanistically, we can show that TCA is readily taken up by FL HSPCs and part of their function is to suppress the production of cytosolic aggreosomes. This leads to reduction of ER stress and the maintenance of functional HSCs after ex vivo culture. These findings propose a novel role for BA as a critical part of fetal hematopoiesis supporting expansion of HSCs.
3177 - ROLE OF SEPTINS IN HSC AGING Katharina Senger1, M.Carolina Florian1, and Hartmut Geiger1,2 1 University of Ulm, Ulm, Germany; 2CCHMC, Cincinnati, Ohio, USA Aging functionally impairs hematopoietic stem cells (HSCs). The underlying molecular mechanisms are poorly understood. The small Rho GTPase Cdc42 has been shown to be involved in HSC aging. It switches between an active (GTP bound) and an inactive (GDP bound) state, thereby regulating actin and tubulin organization as well as cell polarity in distinct cell types. Cdc42 activity is increased in aged compared to young HSCs. This is associated with the apolar distribution of polarity proteins and correlates with a decrease in stem cell function. Polarity is organized and maintained by cytoskeletal proteins. Septins, a family of 13 GTP-binding proteins, form filaments and act as scaffolds or diffusion barriers that segregate membrane areas into discrete domains. In yeast, Cdc42 was shown to act upstream of septins via effector proteins called gics or borgs for mammals. Moreover, in cell lines, ectopic expression of active Cdc42 causes a loss of septin filament assembly, probably by inhibiting the interaction of borgs with septins. Based on this current scientific knowledge, we hypothesize that septins play a role downstream of Cdc42 in the establishment and/or maintenance of polarity in LTHSCs, which gets lost upon aging and that borgs might be the possible link between Cdc42 and septins in this polarization pathway. Our data show that distinct septins are expressed at different levels in aged compared to young long-term repopulating HSCs (LT-HSCs). Selected septins present themselves with a polar distribution in HSCs, which is LT-HSC specific and regulated by the activity of Cdc42. As for septins, gene expression of distinct borgs changes upon HSC aging. Co-stainings of individual septins and borgs show co-localization within HSCs, as it is has been described in yeast before. Additional experiments are underway to define septin/borg interactions in HSCs mechanistically. Knockdown experiments of different borg proteins and subsequent transplantation are performed to determine the functional role of borgs in HSCs as well as to elucidate the signaling pathway by interfering with the Cdc42-septin axis. Clarifying mechanisms of cytoskeletal remodeling upon aging will help to improve our understanding of aging-associated hematopoietic dysfunction and disease.
S95
3178 - COMPARATIVE FUNCTIONAL ANALYSIS OF THE MOLECULAR NETWORK OF 7 SELECTED MLL FUSION PROTEINS Anna Skucha1, Alexey Stukalov2, Matthias Muhar4, Jacques Colinge5, Keiryn Bennett1, Johannes Zuber4, Giulio Superti-Furga1, and Florian Grebien3 1 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; 2Max-Planck Institute of Biotechnology, Munich, Germany; 3Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria; 4IMP, Vienna, Austria; 5Institut de Recherche en Cancerologie, Montpellier, France The Mixed Lineage Leukemia gene (MLL) is a frequent target of chromosomal rearrangements in human malignancies. Balanced translocations result in the fusion of the MLL gene to over 65 partner genes. Even though critical effectors of distinct MLL fusion proteins have been identified, it is not clear if these effectors are conserved among all MLL fusion proteins or if different molecular mechanisms of transformation exist. We aim to delineate common critical effectors of MLL fusion proteins that are presumed to employ different mechanisms of oncogenic transformation. Stable cell lines allowing for inducible expression of 7 selected, affinity-tagged MLL fusion proteins were prepared and the expression of transgenes was verified by qRTPCR and Western Blotting. Affinity purification coupled to mass spectrometry (APMS) identified known and novel interaction partners of 7 MLL fusions. Advanced statistical filtering using a novel algorithm developed by us yielded a densely connected protein-protein interaction network of O950 proteins. 128 proteins were found to interact with $5 of the 7 MLL-fusions. This list of conserved MLL-interactors is enriched for proteins with functions in chromatin metabolism and transcriptional control. To functionally dissect the conserved MLL-interactome, we employed a pooled RNAi screening approach. MLL-AF9-positive AML cells were transduced with pools of viral vectors allowing for the expression of 6 shRNA targeting the same candidate gene. Through a screening methodology that allows for simultaneous positive and negative selection readouts, we identified novel interactors of MLL fusion proteins that were selected for further validation experiments. In conclusion, we developed an experimental pipeline for the functional characterization of cellular effects of MLL fusion proteins in a comprehensive manner, allowing further insight into the molecular mechanisms of MLL-fusion dependent leukemogenesis.
3179 - IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF EARLY STRESS ERYTHROID PROGENITORS USING NOVEL SURFACE MARKERS AND IN VIVO TRACING Sofie Singbrant Soderberg1,2, Alexander Mattebo1,2, and Johan Flygare1,2 1 Lund University, Lund, Sweden; 2Lund Stem Cell Center, Lund, Sweden Stress erythropoiesis is a physiological response to severe anemia, and involves rapid generation of erythrocytes from progenitors in the spleen and liver. Compared to steady state erythropoiesis, stress erythropoiesis remains poorly characterized and precise identification of stress erythroid progenitors is important to understand its kinetics and regulation. Early stress erythroid progenitors, so called stress-BFU-Es, have previously been described as c-Kit+CD71lowTer119low. However, only 0.2% of these cells give rise to BFU-E colonies. In this study we identify CD9 as a novel surface marker greatly enriching for stress-BFU-Es. By combining CD9 with CD150 and Sca1 we have fractionated multi-potent progenitors, BFU-Es, and more mature CFU-Es during stress erythropoiesis. Evaluation of their differentiation potential in vitro demonstrated that cells co-expressing Sca1, CD150, and CD9 were multipotent, whereas loss of Sca1 limited the potential to the megakaryocytic/erythroid lineages. BFU-Es were Sca1-CD150+CD9+, while CD150+CD9- cells contained more mature CFU-Es and erythroblasts. In agreement, Sca1-CD150+CD9+ cells contained the majority of radio-protective day 8 spleen colonies (CFU-S8) when transplanted into irradiated recipients. Mature erythrocytes enucleate and loose the expression of regularly used tracing markers, making them hard to trace in vivo. By transplanting wild-type recipients with stress erythroid progenitors isolated from transgenic mice expressing Kusabira Orange also in enucleated cells (kindly provided by Dr. Nakauchi), we have determined their kinetics and full in vivo potential. In conclusion, we have identified novel surface marker combinations providing a 100-fold enrichment of early stress erythroid progenitors compared to state-of-theart. Furthermore, we have for the first time traced the full in vivo potential of stress erythroid progenitors after transplantation. Our results provide a valuable tool to increase the understanding of in vivo regulation of stress erythropoiesis, which holds promise of discovering new strategies to increase red blood cell production.
3176 - BILE ACIDS SUPPORT EXPANDING HEMATOPOIETIC STEM/ PROGENITOR CELLS IN THE FETAL LIVER Valgardur Sigurdsson1, Hajime Takei2, Svetlana Soboleva1, Kavitha Siva1, Roman Galeev1, Fredrik Leeb-Lundberg3, Takashi Iida4, Hiroshi Nittono2, and Kenichi Miharada1 1 Div. Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden; 2Junshin Clinic Bile Acid Institute, Tokyo, Japan; 3Dept. Drug Target Discovery, Lund University, Lund, Sweden; 4Dept. Chemistry, College of Humanities and Sciences, Nihon University, Tokyo, Japan Hematopoietic stem cells (HSCs) are actively expanding in the fetal liver (FL) but the mechanisms enabling this have not been completely uncovered. HSCs are vulnerable to and quickly activate endoplasmic reticulum (ER) stress responses believed to be fueled by accumulation of unfolded / misfolded proteins (Miharada et al., Cell Rep. 2014). This raises the question how HSCs deal with the higher protein-folding requirement during expansion in the FL. Of note, FL-HSCs have unchanged levels of ER stress related genes despite their high proliferation status without an increase in heat shock protein levels, strongly indicating that other factor(s) block ER stress elevation. Here we demonstrate that bile acids (BA) act as natural chaperones to eliminate ER stress in the fetal liver. Reduction of BA levels using the FxR agonist, GW4064, significantly decreased the number of hematopoietic stem/progenitor cells, due to increased apoptosis caused by elevated ER stress levels. Similarly, dual deletion of Cyp27a1, a key BA synthetic enzyme, in both mother and fetus severely decreased total cellularity and number of HSCs in FL due to increased ER stress. Interestingly, FL of homozygotes grown in heterozygote mothers did not show any significant difference compared to littermate heterozygotes, suggesting that the contribution of maternal BA may be in FL is critical for HSCs. In both models, injection of TCA (a major BA in FL) or Salubrinal, an ER stress inhibitor, rescued the effects of BA reduction. Mechanistically, we can show that TCA is readily taken up by FL HSPCs and part of their function is to suppress the production of cytosolic aggreosomes. This leads to reduction of ER stress and the maintenance of functional HSCs after ex vivo culture. These findings propose a novel role for BA as a critical part of fetal hematopoiesis supporting expansion of HSCs.
3177 - ROLE OF SEPTINS IN HSC AGING Katharina Senger1, M.Carolina Florian1, and Hartmut Geiger1,2 1 University of Ulm, Ulm, Germany; 2CCHMC, Cincinnati, Ohio, USA Aging functionally impairs hematopoietic stem cells (HSCs). The underlying molecular mechanisms are poorly understood. The small Rho GTPase Cdc42 has been shown to be involved in HSC aging. It switches between an active (GTP bound) and an inactive (GDP bound) state, thereby regulating actin and tubulin organization as well as cell polarity in distinct cell types. Cdc42 activity is increased in aged compared to young HSCs. This is associated with the apolar distribution of polarity proteins and correlates with a decrease in stem cell function. Polarity is organized and maintained by cytoskeletal proteins. Septins, a family of 13 GTP-binding proteins, form filaments and act as scaffolds or diffusion barriers that segregate membrane areas into discrete domains. In yeast, Cdc42 was shown to act upstream of septins via effector proteins called gics or borgs for mammals. Moreover, in cell lines, ectopic expression of active Cdc42 causes a loss of septin filament assembly, probably by inhibiting the interaction of borgs with septins. Based on this current scientific knowledge, we hypothesize that septins play a role downstream of Cdc42 in the establishment and/or maintenance of polarity in LTHSCs, which gets lost upon aging and that borgs might be the possible link between Cdc42 and septins in this polarization pathway. Our data show that distinct septins are expressed at different levels in aged compared to young long-term repopulating HSCs (LT-HSCs). Selected septins present themselves with a polar distribution in HSCs, which is LT-HSC specific and regulated by the activity of Cdc42. As for septins, gene expression of distinct borgs changes upon HSC aging. Co-stainings of individual septins and borgs show co-localization within HSCs, as it is has been described in yeast before. Additional experiments are underway to define septin/borg interactions in HSCs mechanistically. Knockdown experiments of different borg proteins and subsequent transplantation are performed to determine the functional role of borgs in HSCs as well as to elucidate the signaling pathway by interfering with the Cdc42-septin axis. Clarifying mechanisms of cytoskeletal remodeling upon aging will help to improve our understanding of aging-associated hematopoietic dysfunction and disease.
S95
3178 - COMPARATIVE FUNCTIONAL ANALYSIS OF THE MOLECULAR NETWORK OF 7 SELECTED MLL FUSION PROTEINS Anna Skucha1, Alexey Stukalov2, Matthias Muhar4, Jacques Colinge5, Keiryn Bennett1, Johannes Zuber4, Giulio Superti-Furga1, and Florian Grebien3 1 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; 2Max-Planck Institute of Biotechnology, Munich, Germany; 3Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria; 4IMP, Vienna, Austria; 5Institut de Recherche en Cancerologie, Montpellier, France The Mixed Lineage Leukemia gene (MLL) is a frequent target of chromosomal rearrangements in human malignancies. Balanced translocations result in the fusion of the MLL gene to over 65 partner genes. Even though critical effectors of distinct MLL fusion proteins have been identified, it is not clear if these effectors are conserved among all MLL fusion proteins or if different molecular mechanisms of transformation exist. We aim to delineate common critical effectors of MLL fusion proteins that are presumed to employ different mechanisms of oncogenic transformation. Stable cell lines allowing for inducible expression of 7 selected, affinity-tagged MLL fusion proteins were prepared and the expression of transgenes was verified by qRTPCR and Western Blotting. Affinity purification coupled to mass spectrometry (APMS) identified known and novel interaction partners of 7 MLL fusions. Advanced statistical filtering using a novel algorithm developed by us yielded a densely connected protein-protein interaction network of O950 proteins. 128 proteins were found to interact with $5 of the 7 MLL-fusions. This list of conserved MLL-interactors is enriched for proteins with functions in chromatin metabolism and transcriptional control. To functionally dissect the conserved MLL-interactome, we employed a pooled RNAi screening approach. MLL-AF9-positive AML cells were transduced with pools of viral vectors allowing for the expression of 6 shRNA targeting the same candidate gene. Through a screening methodology that allows for simultaneous positive and negative selection readouts, we identified novel interactors of MLL fusion proteins that were selected for further validation experiments. In conclusion, we developed an experimental pipeline for the functional characterization of cellular effects of MLL fusion proteins in a comprehensive manner, allowing further insight into the molecular mechanisms of MLL-fusion dependent leukemogenesis.
3179 - IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF EARLY STRESS ERYTHROID PROGENITORS USING NOVEL SURFACE MARKERS AND IN VIVO TRACING Sofie Singbrant Soderberg1,2, Alexander Mattebo1,2, and Johan Flygare1,2 1 Lund University, Lund, Sweden; 2Lund Stem Cell Center, Lund, Sweden Stress erythropoiesis is a physiological response to severe anemia, and involves rapid generation of erythrocytes from progenitors in the spleen and liver. Compared to steady state erythropoiesis, stress erythropoiesis remains poorly characterized and precise identification of stress erythroid progenitors is important to understand its kinetics and regulation. Early stress erythroid progenitors, so called stress-BFU-Es, have previously been described as c-Kit+CD71lowTer119low. However, only 0.2% of these cells give rise to BFU-E colonies. In this study we identify CD9 as a novel surface marker greatly enriching for stress-BFU-Es. By combining CD9 with CD150 and Sca1 we have fractionated multi-potent progenitors, BFU-Es, and more mature CFU-Es during stress erythropoiesis. Evaluation of their differentiation potential in vitro demonstrated that cells co-expressing Sca1, CD150, and CD9 were multipotent, whereas loss of Sca1 limited the potential to the megakaryocytic/erythroid lineages. BFU-Es were Sca1-CD150+CD9+, while CD150+CD9- cells contained more mature CFU-Es and erythroblasts. In agreement, Sca1-CD150+CD9+ cells contained the majority of radio-protective day 8 spleen colonies (CFU-S8) when transplanted into irradiated recipients. Mature erythrocytes enucleate and loose the expression of regularly used tracing markers, making them hard to trace in vivo. By transplanting wild-type recipients with stress erythroid progenitors isolated from transgenic mice expressing Kusabira Orange also in enucleated cells (kindly provided by Dr. Nakauchi), we have determined their kinetics and full in vivo potential. In conclusion, we have identified novel surface marker combinations providing a 100-fold enrichment of early stress erythroid progenitors compared to state-of-theart. Furthermore, we have for the first time traced the full in vivo potential of stress erythroid progenitors after transplantation. Our results provide a valuable tool to increase the understanding of in vivo regulation of stress erythropoiesis, which holds promise of discovering new strategies to increase red blood cell production.