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Mitochondrial dynamics regulate lineage potential of hematopoietic stem cells
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3104 - DYNAMIC IN VIVO MEASUREMENT OF ACUTE LYMPHOBLASTIC LEUKAEMIA INTERACTIONS WITHIN BONE MARROW MICROENVIRONMENTS Cristina Lo Celso Imperial College London, London, United Kingdom The bone marrow (BM) contains multiple, distinct microenvironments, or niches, that regulate the function of haematopoietic stem and progenitors cells to ensure a balanced production of all mature blood cells. It has been suggested that blood cancers might be dependent on signals from these niches during development of disease and evasion from chemotherapy. Leukaemia is known to remodel the BM microenvironment, and therefore its interaction with it must be dynamic. However, our current knowledge of leukaemia biology is derived ex vivo from flow cytometric analysis, which therefore lacks information about cell localization within its tissue of origin, and static images, which cannot describe the dynamics of leukaemia interaction with the BM microenvironment. We have developed an intra-vital microscopy system to visualize the interaction of Notch-driven T cell acute lymphoblastic leukaemia (T-ALL) with known components of the BM niche. We were able to collect 3 dimensional data on the distribution of T-ALL cells across entire BM cavities, to time-lapse record the behaviour of identified leukaemia cells and colonies, and to re-image individual mice prior to, and following chemotherapy. Our results reveal that T-ALL cells are not dependent on specific BM microenvironments for propagation of disease or selection of a subpopulation of chemo-resistant clones, instead suggesting that a stochastic mechanism underlies both processes. In contrast, T-ALL leads to rapid remodelling of the endosteal niche in late stages of disease, including a complete loss of mature osteoblastic cells whilst perivascular niches are maintained. These findings provide the first dynamic analysis of leukaemia cell interactions with the bone marrow microenvironment in vivo and imply that cell intrinsic mechanisms, rather than signals from specific niches, are responsible for BM invasion and survival of chemo-resistant T-ALL. These same mechanisms are likely to lead to the extensive microenvironment remodelling observed in late stages of disease.
3105 - INVESTIGATION OF THE ROLE OF TRIB2 IN NORMAL MURINE HEMATOPOIESIS Kai Ling Liang1,2, Caitriona O’Connor2, Tommie V. McCarthy1, and Karen Keeshan2 1 University College Cork, Cork, Ireland; 2Paul O’Gorman Leukemia Research Centre, Institute of Cancer Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom TRIB2 is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1, 2, 3). Pathologically, TRIB2 induces potent murine acute myeloid leukemia and is associated with acute lymphoblastic leukemia. However, the normal hematopoietic role of TRIB2 remains elusive. Here, we studied murine hematopoiesis after Trib2 ablation. At the steady state, Trib2 loss did not adversely affect peripheral blood cell counts and populations. Trib2-/- mice had similar bone marrow cellularity compared to wild type (WT) mice and no significant differences in the populations of hematopoietic stem and progenitor cells. However, Trib2-/- mice had significantly higher thymic cellularity. Both Trib2-/- and WT mice had similar numbers of immature double negative (DN)1-4 thymic subsets. However, Trib2-/DN cells proliferated faster as indicated by the higher expression of Ki-67, which contributed to the increased number of double and single positive (DP and SP) mature thymic subsets. To evaluate the impact of Trib2-/- on stress hematopoiesis, we treated both groups of mice with 5-fluorouracil (5-FU) in vivo and compared their hematopoietic recovery at different time points. Trib2 loss did not impair the bone marrow multilineage hematopoietic recovery but increased SP T cells detected in the peripheral blood. At 16 and 24 hours post treatment, Trib2-/- mice had significantly reduced numbers of DN1 c-Kithi precursors, DN3L and DPbl cycling subsets, and increased apoptosis, respectively. At day 4 and 14 post treatment, Trib2-/- mice had significantly higher thymic cellularity, frequency and number of thymic subsets compared to WT mice. DN1 c-Kit- precursors expanded significantly more in Trib2-/compared to WT mice. Our results demonstrate that, in the absence of TRIB2, thymocytes are more sensitive to 5-FU induced cell death and thymopoietic recovery is accelerated. These data suggest TRIB2 regulates the differentiation and survival of intrathymic precursors and potentially has a role in the checkpoints of thymopoiesis.
S77
3106 - MITOCHONDRIAL DYNAMICS REGULATE LINEAGE POTENTIAL OF HEMATOPOIETIC STEM CELLS Larry Luchsinger, Mariana Justino Almeida, and Hans Snoeck Columbia Univeristy, New York, New York, USA Hematopoiesis is sustained over an entire lifetime through continuous maintenance and self-renewal of hematopoietic stem cells (HSCs). However, a coherent picture of how these processes are achieved by the HSC to regulate homeostatic responses of the hematopoietic system in vivo has not yet emerged. Moreover, the molecular mechanisms underlying the recently identified clonal heterogeneity among HSCs in lineage potential and self-renewal capacity are unknown. The transcription factor Prdm16 is required for HSC maintenance and self-renewal. We show that regulation of mitochondrial dynamics is a target of the Prdm16 transcriptional program. Our data show that Prdm16 maintains mitochondrial hyperfusion in HSC through transcriptional activation of Mitofusin 2 (Mfn2). HSCs retrovirally transduced with Mfn2 showed enhanced function in competitive and serial repopulation assays compared to control vector. Furthermore, our studies reveal that Mfn2 and mitochondrial hyperfusion facilitates increased steady-state intracellular [Ca2+] buffering in HSCs, leading to decreased baseline [Cai2+] and consequently, lower calcineurin phosphatase activity thereby inhibiting nuclear translocation of the transcription factor, NFAT. Pharmacological inhibition of NFAT increased the mRNA expression of lymphoid-biased HSC markers IL7Ra and Sox4 in vitro and NFAT inhibition increased the lymphoid reconstitution potential of HSCs in vivo. Furthermore, a significant decrease in Mfn2 expression and mitochondrial fusion was observed in 2yr old HSCs compared to young controls which may correlate with lymphoid deficiencies and a myeloid-biased HSC phenotype observed with age. Despite the prevalent use of glycolysis in HSCs for energetic metabolism, we show mitochondria play a critical role in conferring lymphoid lineage potential and may underlie the conspicuous heterogeneity of HSC differentiation potential. Insights from these findings will broadly impact our understanding of HSC biology and advance the field towards improving targeted therapies to treat diseases of HSC origin.
3107 - ASYMMETRIC CELL DIVISION OF HEMATOPOIETIC STEM CELLS Dirk Loeffler and Timm Schroeder Cell Systems Dynamics, Department of Biosystems Science and Engineering (D-BSSE), ETH Z€urich, Basel, Switzerland Hematopoietic stem cells (HSC) maintain their numbers while replenishing all mature blood cells. Asymmetric cell division is often discussed as the underlying mechanism balancing HSC self-renewal and differentiation. In this model future daughter cell fates are determined by the unequal inheritance of cell fate determinants. This subsequently leads to the differentiation of one daughter while the other retains its stem cell capacity. However, this mechanism could never been shown directly and it cannot be excluded that HSCs divide symmetrically and later commitment to differentiation is determined by mechanisms not related to division. Previous studies have identified asymmetrically inherited proteins during invertebrate and vertebrate progenitor divisions. However, the low frequency of HSCs and technical challenges hampered the study of events during their mitosis. The evidence for asymmetric inheritance of proteins during hematopoietic stem and progenitor cell division relies on a limited number of studies, none of which was so far able to demonstrate that the asymmetric inheritance of proteins determines future daughter cell fates. We develop bioimaging systems for continuous quantification of single hematopoietic stem and progenitor cells to link asymmetric inheritance of cell fate determinants to future cell fates. Our results question current dogmas about the mechanism of asymmetric HSC division.
3104 - DYNAMIC IN VIVO MEASUREMENT OF ACUTE LYMPHOBLASTIC LEUKAEMIA INTERACTIONS WITHIN BONE MARROW MICROENVIRONMENTS Cristina Lo Celso Imperial College London, London, United Kingdom The bone marrow (BM) contains multiple, distinct microenvironments, or niches, that regulate the function of haematopoietic stem and progenitors cells to ensure a balanced production of all mature blood cells. It has been suggested that blood cancers might be dependent on signals from these niches during development of disease and evasion from chemotherapy. Leukaemia is known to remodel the BM microenvironment, and therefore its interaction with it must be dynamic. However, our current knowledge of leukaemia biology is derived ex vivo from flow cytometric analysis, which therefore lacks information about cell localization within its tissue of origin, and static images, which cannot describe the dynamics of leukaemia interaction with the BM microenvironment. We have developed an intra-vital microscopy system to visualize the interaction of Notch-driven T cell acute lymphoblastic leukaemia (T-ALL) with known components of the BM niche. We were able to collect 3 dimensional data on the distribution of T-ALL cells across entire BM cavities, to time-lapse record the behaviour of identified leukaemia cells and colonies, and to re-image individual mice prior to, and following chemotherapy. Our results reveal that T-ALL cells are not dependent on specific BM microenvironments for propagation of disease or selection of a subpopulation of chemo-resistant clones, instead suggesting that a stochastic mechanism underlies both processes. In contrast, T-ALL leads to rapid remodelling of the endosteal niche in late stages of disease, including a complete loss of mature osteoblastic cells whilst perivascular niches are maintained. These findings provide the first dynamic analysis of leukaemia cell interactions with the bone marrow microenvironment in vivo and imply that cell intrinsic mechanisms, rather than signals from specific niches, are responsible for BM invasion and survival of chemo-resistant T-ALL. These same mechanisms are likely to lead to the extensive microenvironment remodelling observed in late stages of disease.
3105 - INVESTIGATION OF THE ROLE OF TRIB2 IN NORMAL MURINE HEMATOPOIESIS Kai Ling Liang1,2, Caitriona O’Connor2, Tommie V. McCarthy1, and Karen Keeshan2 1 University College Cork, Cork, Ireland; 2Paul O’Gorman Leukemia Research Centre, Institute of Cancer Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom TRIB2 is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1, 2, 3). Pathologically, TRIB2 induces potent murine acute myeloid leukemia and is associated with acute lymphoblastic leukemia. However, the normal hematopoietic role of TRIB2 remains elusive. Here, we studied murine hematopoiesis after Trib2 ablation. At the steady state, Trib2 loss did not adversely affect peripheral blood cell counts and populations. Trib2-/- mice had similar bone marrow cellularity compared to wild type (WT) mice and no significant differences in the populations of hematopoietic stem and progenitor cells. However, Trib2-/- mice had significantly higher thymic cellularity. Both Trib2-/- and WT mice had similar numbers of immature double negative (DN)1-4 thymic subsets. However, Trib2-/DN cells proliferated faster as indicated by the higher expression of Ki-67, which contributed to the increased number of double and single positive (DP and SP) mature thymic subsets. To evaluate the impact of Trib2-/- on stress hematopoiesis, we treated both groups of mice with 5-fluorouracil (5-FU) in vivo and compared their hematopoietic recovery at different time points. Trib2 loss did not impair the bone marrow multilineage hematopoietic recovery but increased SP T cells detected in the peripheral blood. At 16 and 24 hours post treatment, Trib2-/- mice had significantly reduced numbers of DN1 c-Kithi precursors, DN3L and DPbl cycling subsets, and increased apoptosis, respectively. At day 4 and 14 post treatment, Trib2-/- mice had significantly higher thymic cellularity, frequency and number of thymic subsets compared to WT mice. DN1 c-Kit- precursors expanded significantly more in Trib2-/compared to WT mice. Our results demonstrate that, in the absence of TRIB2, thymocytes are more sensitive to 5-FU induced cell death and thymopoietic recovery is accelerated. These data suggest TRIB2 regulates the differentiation and survival of intrathymic precursors and potentially has a role in the checkpoints of thymopoiesis.
S77
3106 - MITOCHONDRIAL DYNAMICS REGULATE LINEAGE POTENTIAL OF HEMATOPOIETIC STEM CELLS Larry Luchsinger, Mariana Justino Almeida, and Hans Snoeck Columbia Univeristy, New York, New York, USA Hematopoiesis is sustained over an entire lifetime through continuous maintenance and self-renewal of hematopoietic stem cells (HSCs). However, a coherent picture of how these processes are achieved by the HSC to regulate homeostatic responses of the hematopoietic system in vivo has not yet emerged. Moreover, the molecular mechanisms underlying the recently identified clonal heterogeneity among HSCs in lineage potential and self-renewal capacity are unknown. The transcription factor Prdm16 is required for HSC maintenance and self-renewal. We show that regulation of mitochondrial dynamics is a target of the Prdm16 transcriptional program. Our data show that Prdm16 maintains mitochondrial hyperfusion in HSC through transcriptional activation of Mitofusin 2 (Mfn2). HSCs retrovirally transduced with Mfn2 showed enhanced function in competitive and serial repopulation assays compared to control vector. Furthermore, our studies reveal that Mfn2 and mitochondrial hyperfusion facilitates increased steady-state intracellular [Ca2+] buffering in HSCs, leading to decreased baseline [Cai2+] and consequently, lower calcineurin phosphatase activity thereby inhibiting nuclear translocation of the transcription factor, NFAT. Pharmacological inhibition of NFAT increased the mRNA expression of lymphoid-biased HSC markers IL7Ra and Sox4 in vitro and NFAT inhibition increased the lymphoid reconstitution potential of HSCs in vivo. Furthermore, a significant decrease in Mfn2 expression and mitochondrial fusion was observed in 2yr old HSCs compared to young controls which may correlate with lymphoid deficiencies and a myeloid-biased HSC phenotype observed with age. Despite the prevalent use of glycolysis in HSCs for energetic metabolism, we show mitochondria play a critical role in conferring lymphoid lineage potential and may underlie the conspicuous heterogeneity of HSC differentiation potential. Insights from these findings will broadly impact our understanding of HSC biology and advance the field towards improving targeted therapies to treat diseases of HSC origin.
3107 - ASYMMETRIC CELL DIVISION OF HEMATOPOIETIC STEM CELLS Dirk Loeffler and Timm Schroeder Cell Systems Dynamics, Department of Biosystems Science and Engineering (D-BSSE), ETH Z€urich, Basel, Switzerland Hematopoietic stem cells (HSC) maintain their numbers while replenishing all mature blood cells. Asymmetric cell division is often discussed as the underlying mechanism balancing HSC self-renewal and differentiation. In this model future daughter cell fates are determined by the unequal inheritance of cell fate determinants. This subsequently leads to the differentiation of one daughter while the other retains its stem cell capacity. However, this mechanism could never been shown directly and it cannot be excluded that HSCs divide symmetrically and later commitment to differentiation is determined by mechanisms not related to division. Previous studies have identified asymmetrically inherited proteins during invertebrate and vertebrate progenitor divisions. However, the low frequency of HSCs and technical challenges hampered the study of events during their mitosis. The evidence for asymmetric inheritance of proteins during hematopoietic stem and progenitor cell division relies on a limited number of studies, none of which was so far able to demonstrate that the asymmetric inheritance of proteins determines future daughter cell fates. We develop bioimaging systems for continuous quantification of single hematopoietic stem and progenitor cells to link asymmetric inheritance of cell fate determinants to future cell fates. Our results question current dogmas about the mechanism of asymmetric HSC division.