- Email: [email protected]
Clonal analysis of hematopoietic stem cell precursors reveals developmental evolution of phenotypic and functional properties
S78
Poster Presentations/ Experimental Hematology 44 (2016) S56–S110
3070 - CLONAL ANALYSIS OF HEMATOPOIETIC STEM CELL PRECURSORS REVEALS DEVELOPMENTAL EVOLUTION OF PHENOTYPIC AND FUNCTIONAL PROPERTIES Brandon Hadland1,2,3, Barbara Varnum-Finney1, and Irwin Bernstein1 1 Fred Hutchinson Cancer Research Center, Seattle, USA; 2Seattle Childrens Hospital, Seattle, USA; 3University of Washington School of Medicine, Seattle, US During embryogenesis, HSC develop from specialized hemogenic endothelium (HE) in a process referred to as the endothelial to hematopoietic transition (EHT). Critical properties that define the HSC, such as the ability to self-renew, home, and provide multilineage hematopoiesis, must be acquired during the EHT to generate an HSC that is capable of long-term, multilineage reconstitution in transplanted recipients. Recent studies, however, suggest that the vast majority of hematopoietic progenitors that arise from HE prior to or simultaneously with HSC are already committed to lineage-restricted fates, suggesting unique cell intrinsic and/or niche-provided conditions are necessary to support the rare HSC fate during EHT. Thus, a greater understanding is needed of the ontogeny of HSC precursors, their phenotypic and functional properties, and the niche signals that are necessary for their maturation to bone fide HSC. Such knowledge will be critical to the in vitro engineering of HSC de novo such as from pluripotent stem cells for potential therapeutic and disease modeling applications. To this end, we previously showed that niche endothelial cells from the initial site of HSC emergence in the aorta-gonad-mesonephros region (AGM-EC) can promote the maturation of murine embryo-derived precursors to engrafting HSC during in vitro co-culture. By combining single cell index sorting with functional analysis by AGM-EC co-culture and transplantation in vivo, we have identified clonal HSC precursors as early as embryonic day 9.5 in the para-aortic splanchnopleura (precursor to the AGM region). At E9.5, we find that HSC precursors are rare, comprising at most a few per embryo. By E11.5, HSC precursors increase in number in the AGM and exhibit higher proliferative self-renewal capacity and reduced propensity to differentiation during AGM-EC co-culture. Importantly, index sorting has permitted identification of cell surface marker phenotypes, such as higher expression of EPCR, which can partially distinguish HSC precursors from other hematopoietic progenitors lacking HSC potential. Our findings offer novel insight into the unique properties of developing HSC, and provide a platform for further interrogation of their molecular and functional properties at the single cell level.
3071 - ROLE OF AMINO ACID METABOLISM IN CANCER PROGRESSION Ayuna Hattori and Takahiro Ito University of Georgia, Athens, USA Changes in cellular metabolism are common properties observed in various cancers. However it remains poorly understood whether metabolic changes directly affect cancer development and progression. In chronic myelogenous leukemia (CML), disease can progress from an indolent chronic phase to an aggressive blast crisis phase in which cellular differentiation is arrested and extensive self-renewal of cancer cells occur. By studying mouse CML models, we identified an amino transferase that mediates CML disease progression. Its gene expression is up-regulated during disease progression in mouse models of CML as well as in the human CML. Functional inhibition by shRNA or small molecule inhibition promotes cellular differentiation and significantly impairs the propagation of fatal blast crisis CML (BC-CML). We also identified upstream regulator of the aminotransferase expression by bioinformatics gene analysis of human leukemia and other cancers. Our data demonstrate that altered amino acid metabolism inhibits cellular differentiation and regulates leukemia propagation.
3072 - NUCLEAR FACTOR I/X PROTECTS MURINE HEMATOPOEITIC STEM AND PROGENITOR CELLS FROM STRESS-INDUCED APOPTOSIS IN EX VIVO CULTURE Trent Hall1,2, Megan Walker3, Miguel Ganuza3, Marie Bordas4, Per Holmfeldt5, and Shannon McKinney-Freeman3 1 St. Jude Children’s Research Hospital; 2University of Tennessee Health Science Center; 3St. Jude Children’s Research Hospital, Arlington, USA; 4 Paris 7 University, Paris, France; 5Uppsala University, Uppsala, Sweden Hematopoietic stem cell (HSC) transplantation is the primary therapy of many hematologic ailments and understanding the molecular mechanisms surrounding stable HSC engraftment is crucial to improving patient outcomes. We recently identified the transcription factor nuclear factor I/X (Nfix) as a critical regulator of hematopoietic stem and progenitor cell (HSPC) survival post-transplantation. Nfix-depleted HSPC are lost from the marrow between 7-10 days post-transplant and residual Nfix-depleted HSPC display elevated levels of apoptosis at these time-points. To further characterize the anti-apoptotic role of Nfix in HSPC biology, we overexpressed (OE) Nfix in Lineage- Sca-1+ c-Kit+ (LSK) cells that were then maintained ex vivo under serum-free conditions with supportive cytokines. These cultures were monitored for cell surface phenotype, growth, vector selection, and apoptosis. 7 days later, the frequency of phenotypic LSK cells was reduced by half (p 5 0.017) in Nfix OE cultures compared to controls. Consequently, LSK cells OE Nfix failed to robustly repopulate the hematopoietic system of lethally irradiated mice relative to controls. Interestingly, both control and Nfix OE cell cultures were predominately c-Kit+ CD71+, suggesting our cultures support the expansion of an erythroblast-like population. Strikingly, cells OE Nfix persisted in culture about two weeks longer than controls (p 5 0.0044). These cultures displayed a dramatic selection for vector+ cells around day 20 that coincided with the loss of control cultures, suggesting that this extended culture depended on Nfix OE. Nfix OE cultures also displayed 40% less apoptosis than control cultures (p 5 0.00026). Reduced apoptosis correlated with upregulation of Bcl-xL in cells OE Nfix. To further test the ability of Nfix to protect cells from conditions of stress, we repeated these experiments under conditions of reduced supportive cytokines. Reducing cytokine levels by 75% had no effect on the persistence of Nfix OE cells in culture, apoptosis, or cellular phenotype. Vector selection was also initiated earlier in these cultures. In contrast, control cultures displayed a significant decrease in growth rate by day 9 (p 5 0.038) and a 2.9-fold increase in apoptosis relative to Nfix OE cells (p5 0.037). Thus, Nfix OE protected these cultures from the stress of reduced cytokines. These data provide further evidence that Nfix promotes HSPC survival, particularly during stress.
3073 - LOSS OF HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN L (HNRNP L) LEADS TO MITOCHONDRIAL DYSFUNCTION, DNA DAMAGE RESPONSE AND CASPASE-DEPENDENT CELL DEATH IN HEMATOPOIETIC STEM CELLS Anne Helness1,2, Marie-Claude Gaudreau3, Damien Grapton4, Charles Vadnais1, Jennifer Fraszczak1, Brian Wilhelm5, Franc¸ois Robert1, Florian Heyd6, and Tarik Moroy1 1 Institut de recherches cliniques de Montreal (IRCM), Montreal, Canada; 2 McGill University, Montreal, Canada; 3MedImmune, LLC, Gaithersburg, USA, Gaithersburgh, USA; 4The Lady Davis Institute, Montreal, Canada, Montreal, Canada; 5Institute for Research in Immunology and Cancer (IRIC), Montreal, Canada, Montreal, Canada; 6Freie Universit€at Berlin, Berlin, Germany The proliferation and survival of hematopoietic stem cells (HSCs) has to be strictly coordinated to ensure the timely production of all blood cells. Here we report that the splice factor and RNA binding protein hnRNP L (heterogeneous nuclear ribonucleoprotein L) is required for hematopoiesis, since its genetic ablation in mice reduces almost all blood cell lineages and causes premature death of the animals. In agreement with this, we observed that hnRNP L deficient HSCs lack both the ability to self-renew and foster hematopoietic differentiation in transplanted hosts and show high levels of AnnexinV suggesting that they undergo apoptosis. HSCs lacking hnRNP L also display mitochondrial dysfunction and elevated levels of reactive oxygen species (ROS). Moreover, lin-c-Kit+ fetal liver cells from hnRNP L deficient mice show high p53 protein levels and up-regulation of p53 target genes indicating an ongoing DNA damage response possibly caused by elevated ROS levels. Finally, cells lacking hnRNP L also up-regulated the expression of the death receptors
Poster Presentations/ Experimental Hematology 44 (2016) S56–S110
3070 - CLONAL ANALYSIS OF HEMATOPOIETIC STEM CELL PRECURSORS REVEALS DEVELOPMENTAL EVOLUTION OF PHENOTYPIC AND FUNCTIONAL PROPERTIES Brandon Hadland1,2,3, Barbara Varnum-Finney1, and Irwin Bernstein1 1 Fred Hutchinson Cancer Research Center, Seattle, USA; 2Seattle Childrens Hospital, Seattle, USA; 3University of Washington School of Medicine, Seattle, US During embryogenesis, HSC develop from specialized hemogenic endothelium (HE) in a process referred to as the endothelial to hematopoietic transition (EHT). Critical properties that define the HSC, such as the ability to self-renew, home, and provide multilineage hematopoiesis, must be acquired during the EHT to generate an HSC that is capable of long-term, multilineage reconstitution in transplanted recipients. Recent studies, however, suggest that the vast majority of hematopoietic progenitors that arise from HE prior to or simultaneously with HSC are already committed to lineage-restricted fates, suggesting unique cell intrinsic and/or niche-provided conditions are necessary to support the rare HSC fate during EHT. Thus, a greater understanding is needed of the ontogeny of HSC precursors, their phenotypic and functional properties, and the niche signals that are necessary for their maturation to bone fide HSC. Such knowledge will be critical to the in vitro engineering of HSC de novo such as from pluripotent stem cells for potential therapeutic and disease modeling applications. To this end, we previously showed that niche endothelial cells from the initial site of HSC emergence in the aorta-gonad-mesonephros region (AGM-EC) can promote the maturation of murine embryo-derived precursors to engrafting HSC during in vitro co-culture. By combining single cell index sorting with functional analysis by AGM-EC co-culture and transplantation in vivo, we have identified clonal HSC precursors as early as embryonic day 9.5 in the para-aortic splanchnopleura (precursor to the AGM region). At E9.5, we find that HSC precursors are rare, comprising at most a few per embryo. By E11.5, HSC precursors increase in number in the AGM and exhibit higher proliferative self-renewal capacity and reduced propensity to differentiation during AGM-EC co-culture. Importantly, index sorting has permitted identification of cell surface marker phenotypes, such as higher expression of EPCR, which can partially distinguish HSC precursors from other hematopoietic progenitors lacking HSC potential. Our findings offer novel insight into the unique properties of developing HSC, and provide a platform for further interrogation of their molecular and functional properties at the single cell level.
3071 - ROLE OF AMINO ACID METABOLISM IN CANCER PROGRESSION Ayuna Hattori and Takahiro Ito University of Georgia, Athens, USA Changes in cellular metabolism are common properties observed in various cancers. However it remains poorly understood whether metabolic changes directly affect cancer development and progression. In chronic myelogenous leukemia (CML), disease can progress from an indolent chronic phase to an aggressive blast crisis phase in which cellular differentiation is arrested and extensive self-renewal of cancer cells occur. By studying mouse CML models, we identified an amino transferase that mediates CML disease progression. Its gene expression is up-regulated during disease progression in mouse models of CML as well as in the human CML. Functional inhibition by shRNA or small molecule inhibition promotes cellular differentiation and significantly impairs the propagation of fatal blast crisis CML (BC-CML). We also identified upstream regulator of the aminotransferase expression by bioinformatics gene analysis of human leukemia and other cancers. Our data demonstrate that altered amino acid metabolism inhibits cellular differentiation and regulates leukemia propagation.
3072 - NUCLEAR FACTOR I/X PROTECTS MURINE HEMATOPOEITIC STEM AND PROGENITOR CELLS FROM STRESS-INDUCED APOPTOSIS IN EX VIVO CULTURE Trent Hall1,2, Megan Walker3, Miguel Ganuza3, Marie Bordas4, Per Holmfeldt5, and Shannon McKinney-Freeman3 1 St. Jude Children’s Research Hospital; 2University of Tennessee Health Science Center; 3St. Jude Children’s Research Hospital, Arlington, USA; 4 Paris 7 University, Paris, France; 5Uppsala University, Uppsala, Sweden Hematopoietic stem cell (HSC) transplantation is the primary therapy of many hematologic ailments and understanding the molecular mechanisms surrounding stable HSC engraftment is crucial to improving patient outcomes. We recently identified the transcription factor nuclear factor I/X (Nfix) as a critical regulator of hematopoietic stem and progenitor cell (HSPC) survival post-transplantation. Nfix-depleted HSPC are lost from the marrow between 7-10 days post-transplant and residual Nfix-depleted HSPC display elevated levels of apoptosis at these time-points. To further characterize the anti-apoptotic role of Nfix in HSPC biology, we overexpressed (OE) Nfix in Lineage- Sca-1+ c-Kit+ (LSK) cells that were then maintained ex vivo under serum-free conditions with supportive cytokines. These cultures were monitored for cell surface phenotype, growth, vector selection, and apoptosis. 7 days later, the frequency of phenotypic LSK cells was reduced by half (p 5 0.017) in Nfix OE cultures compared to controls. Consequently, LSK cells OE Nfix failed to robustly repopulate the hematopoietic system of lethally irradiated mice relative to controls. Interestingly, both control and Nfix OE cell cultures were predominately c-Kit+ CD71+, suggesting our cultures support the expansion of an erythroblast-like population. Strikingly, cells OE Nfix persisted in culture about two weeks longer than controls (p 5 0.0044). These cultures displayed a dramatic selection for vector+ cells around day 20 that coincided with the loss of control cultures, suggesting that this extended culture depended on Nfix OE. Nfix OE cultures also displayed 40% less apoptosis than control cultures (p 5 0.00026). Reduced apoptosis correlated with upregulation of Bcl-xL in cells OE Nfix. To further test the ability of Nfix to protect cells from conditions of stress, we repeated these experiments under conditions of reduced supportive cytokines. Reducing cytokine levels by 75% had no effect on the persistence of Nfix OE cells in culture, apoptosis, or cellular phenotype. Vector selection was also initiated earlier in these cultures. In contrast, control cultures displayed a significant decrease in growth rate by day 9 (p 5 0.038) and a 2.9-fold increase in apoptosis relative to Nfix OE cells (p5 0.037). Thus, Nfix OE protected these cultures from the stress of reduced cytokines. These data provide further evidence that Nfix promotes HSPC survival, particularly during stress.
3073 - LOSS OF HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN L (HNRNP L) LEADS TO MITOCHONDRIAL DYSFUNCTION, DNA DAMAGE RESPONSE AND CASPASE-DEPENDENT CELL DEATH IN HEMATOPOIETIC STEM CELLS Anne Helness1,2, Marie-Claude Gaudreau3, Damien Grapton4, Charles Vadnais1, Jennifer Fraszczak1, Brian Wilhelm5, Franc¸ois Robert1, Florian Heyd6, and Tarik Moroy1 1 Institut de recherches cliniques de Montreal (IRCM), Montreal, Canada; 2 McGill University, Montreal, Canada; 3MedImmune, LLC, Gaithersburg, USA, Gaithersburgh, USA; 4The Lady Davis Institute, Montreal, Canada, Montreal, Canada; 5Institute for Research in Immunology and Cancer (IRIC), Montreal, Canada, Montreal, Canada; 6Freie Universit€at Berlin, Berlin, Germany The proliferation and survival of hematopoietic stem cells (HSCs) has to be strictly coordinated to ensure the timely production of all blood cells. Here we report that the splice factor and RNA binding protein hnRNP L (heterogeneous nuclear ribonucleoprotein L) is required for hematopoiesis, since its genetic ablation in mice reduces almost all blood cell lineages and causes premature death of the animals. In agreement with this, we observed that hnRNP L deficient HSCs lack both the ability to self-renew and foster hematopoietic differentiation in transplanted hosts and show high levels of AnnexinV suggesting that they undergo apoptosis. HSCs lacking hnRNP L also display mitochondrial dysfunction and elevated levels of reactive oxygen species (ROS). Moreover, lin-c-Kit+ fetal liver cells from hnRNP L deficient mice show high p53 protein levels and up-regulation of p53 target genes indicating an ongoing DNA damage response possibly caused by elevated ROS levels. Finally, cells lacking hnRNP L also up-regulated the expression of the death receptors