- Email: [email protected]
Mitochondria are implicated in the regulation of terminal erythropoiesis
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
treatment option, but still, 5-year event-free survival reaches only about 50%. Hyperactive RAS signaling caused by genetic alterations in CBL, KRAS, NF1, NRAS or PTPN11 is the main driving event in JMML. So far, there is no clear understanding of how RAS pathway mutations relate to the heterogeneous disease biology and variable clinical outcome observed. We hypothesized that DNA methylation profiling, alone or in combination with genetic alterations, might provide a molecular basis for disease classification. DNA methylome analysis of 167 JMML samples (plus exome sequencing and expression profiling in 50 and 15 cases, respectively) identified three subgroups with low, intermediate and high methylation levels (LM, IM and HM). The HM group was enriched for patients with somatic PTPN11 mutations and includes poor outcome cases while the LM group was enriched for somatic NRAS & CBL mutations as well as for Noonan-patients and represents the good prognosis group. The IM group was enriched for cases with monosomy 7 and somatic KRAS mutations. Differentially methylated sites were enriched for regions decorated with H3K27me3 or PRC2 components and for genes associated with oncogenic RASsignaling. Integrative analysis revealed frequent co-occurrence of $2 mutations activating the RAS-RAF-MEK-ERK pathway in the HM & IM groups. This finding was paralleled by a significant up-regulation of DNMT1 & DNMT3B suggesting aberrant activation of the DNA methylation machinery in this context. Together, we identified three JMML subgroups characterized by distinct clinical and biological features. We further provide evidence for a molecular mechanism by which additional genetic events, presumably further activating the RAS-RAF-MEK-ERK pathway, mediate DNA hypermethylation via up-regulation of DNMTs in aggressive JMML cases.
3103 - MITOCHONDRIA ARE IMPLICATED IN THE REGULATION OF TERMINAL ERYTHROPOIESIS Raymond Liang1 and Saghi Ghaffari2 1 Icahn School of Medicine at Mount Sinai, Astoria, United States; 2Icahn School of Medicine at Mount Sinai, New York, United States
Erythroblast enucleation, the process of nucleus removal, is a critical step for red blood cell (RBC) formation and function. Our data suggests a novel mechanism required for enucleation, which involves mitochondrial activity. Outside of their role in generating heme, mitochondrial function during other erythroid specific processes is unknown. We observed that prior to murine erythroblast enucleation, mitochondria migrate distal to the extruding nucleus. Immunofluorescence images of FACS sorted erythroblasts at distinct stages of maturation show increasing mitochondrial polarization with maturation. This observation was further confirmed by ImageStream analysis that combines microfluidics with imaging to allow high-throughput imaging of single cells. With ImageStream we could distinguish cells in the process of enucleating dependent on the degree of nucleus extrusion. We found a significant positive correlation between erythroblast enucleation and mitochondrial polarization and predicted that mitochondria function might relate to the energetic demands of the enucleation process. To test this, we tracked total mitochondria abundance and mitochondrial membrane potential (MMP), a measure of mitochondrial activity, using fluorescent probes. Although mitochondrial abundance and MMP decrease as erythroblasts mature, a transient increase in MMP is associated with enucleating cells. We also sorted primary late stage erythroblasts and allowed them to mature towards enucleated RBCs in the presence of inhibitors of the electron transport chain (ETC), which lead to decreased ATP production. Treatment with these drugs led to a significant decrease in erythroblast enucleation rates compared to DMSO control treated erythroblasts. The treatment was specific to enucleation and not other erythroblast maturation processes. Notably, this effect was not due to increased cell death and was reversible through a wash step. Overall our data support a novel role for mitochondria in promoting the last stages of erythroblast enucleation, potentially through ATP generation and/or other co-factors produced through the ETC.
3102 - GATA-3 SUPPRESSES IRF8 TO PROMOTE HUMAN T-CELL LINEAGE COMMITMENT Kai Ling Liang, Inge Van de Walle, Laurentijn Tilleman, Filip Van Nieuwerburgh, and Tom Taghon
3104 - A ROLE FOR MICROGLIA IN HEMATOPOIETIC STEM/PROGENITOR CELL EMERGENCE IN THE EMBRYONIC BRAIN Zhuan Li1, Samanta Mariani2, Chris Vink2, and Elaine Dzierzak2
Ghent University, Ghent, Belgium
1
The thymus is an organ where T-cell development takes place. However, human thymic progenitors have the capacity to differentiate into cells of other haematopoietic lineages. This multi-lineage differentiation potential is lost progressively as thymic progenitors undergo sequential T-cell lineage specification and commitment. The former is dependent on Notch1 signalling and results in loss of myeloid and Bcell potential. At the commitment stage, T-cell specific transcription factors, rather than Notch signalling, are essential to suppress residual non-T potential. Recently, we reported that GATA-3 is a key driver in human T-lineage commitment by repressing natural killer (NK) cell fate through down-modulation of Notch signalling. Now, transcriptomic analyses of GATA-3 transduced human haematopoietic progenitors allowed us to identify IRF8 as a novel target gene. In comparison to the hypomorphic KRR-mutant of GATA-3, enforced expression of GATA-3 wild type significantly downregulated endogenous IRF8 RNA and protein expression. The effect was reversed in a GATA-3 knockdown setting. IRF8 is a member of interferon regulatory factor family that plays an important role in human haematopoiesis. In human, IRF8 is highly expressed in all haematopoietic lineages except T-cells. Dysregulation of IRF8 has been implicated in human leukaemia and mutations in IRF8 are clinically associated with human dendritic cell (DC) and NK cell immunodeficiency. As such, we hypothesize that negative regulation of IRF8 by GATA-3 is important to promote human T-lineage commitment. Using multi-coloured flow cytometry, we showed that IRF8 expression is restricted in ex vivo uncommitted (CD34+CD1a-) human thymic progenitors. Developmental trajectory of human thymic progenitors, defined by early T-cell markers, revealed dynamic and sequential expressions of IRF8 and GATA-3. This enabled us to identify, within the uncommitted pool of thymic progenitors, new subpopulations that reflect successive extinction of DC and NK lineage choices. Using in vitro co-culture assay, we recapitulated this previously uncharacterized transitional cell populations during human T-lineage commitment.
S83
QMRI, Edinburgh, United Kingdom; 2The University of Edinburgh, Edinburgh, United Kingdom
The mouse embryonic head is one of the sites for haematopoietic stem and progenitor cell (HSPC) development. Transplantation of subdissected head region cells has shown HSC localization in the hindbrain and branchial arches (HBA). Recent studies reveal a dynamic interaction of primitive macrophages with aortic HSPCs in zebrafish embryos. It is becoming clear by fate mapping that under physiological conditions, microglia cells (macrophages in the head, MV) are derived from the yolk sac. However, it is unknown what role(s) MV play in HSPC emergence in the head. In our study using MacGreen (Csf1r-GFP) embryos, we found in the HBA (E9.5-11) that more than 95% of MV (CD45+F4/80+CD11b+Gr1-) are GFP+. Flow cytometry showed that most MV in the E10 HBA express CX3CR1 and CXCR4. qRTPCR analysis showed that E10 HBA endothelial cells (CD31+CD45-CD41-, EC) express CX3CL1, which encodes the ligand of CX3CR1. In CX3CR1-/- embryos, the frequency of MV in the HBA is reduced. Interestingly, at E10 the percentage of haematopoietic progenitors (cKit+CD41lo) is decreased in the CX3CR1 deficient HBA, but it is not decreased in the E11 HBA. To test if MV play a role in endothelial cell-to-haematopoietic cell-transition (EHT), HBA EC were cultured with/without microglia in an OP9-DL1 coculture system. The presence of MV boosted the HPC output from EC more than 2-fold compared to the control group (EC only). qRTPCR experiments showed MV (MacGreen+) express significantly higher levels of pro-inflammatory factor genes (IL-1b, IL-1c, TNFb) compared to MacGreen- cells. Interestingly, EC expressed higher levels of receptor genes (IL1R1, TNFbR1) than CD41+/CD45+ or CD31-CD41-CD45- cells, suggesting that MV are involved in hematopoietic cell emergence in the HBA through the IL-1 and/or TNFa pathway. To examine if MV function in vivo, MV were specifically ablated in Csf1r-Cre;RosaDTA mice. In the HBA (E9.5-11), all the MV were depleted. Methylcellulose culture data showed the numbers of hematopoietic progenitors were reduced significantly after MV ablation in the HBA. In summary, these data suggest that microglia cells play important roles in hematopoietic cell emergence in HBA.
treatment option, but still, 5-year event-free survival reaches only about 50%. Hyperactive RAS signaling caused by genetic alterations in CBL, KRAS, NF1, NRAS or PTPN11 is the main driving event in JMML. So far, there is no clear understanding of how RAS pathway mutations relate to the heterogeneous disease biology and variable clinical outcome observed. We hypothesized that DNA methylation profiling, alone or in combination with genetic alterations, might provide a molecular basis for disease classification. DNA methylome analysis of 167 JMML samples (plus exome sequencing and expression profiling in 50 and 15 cases, respectively) identified three subgroups with low, intermediate and high methylation levels (LM, IM and HM). The HM group was enriched for patients with somatic PTPN11 mutations and includes poor outcome cases while the LM group was enriched for somatic NRAS & CBL mutations as well as for Noonan-patients and represents the good prognosis group. The IM group was enriched for cases with monosomy 7 and somatic KRAS mutations. Differentially methylated sites were enriched for regions decorated with H3K27me3 or PRC2 components and for genes associated with oncogenic RASsignaling. Integrative analysis revealed frequent co-occurrence of $2 mutations activating the RAS-RAF-MEK-ERK pathway in the HM & IM groups. This finding was paralleled by a significant up-regulation of DNMT1 & DNMT3B suggesting aberrant activation of the DNA methylation machinery in this context. Together, we identified three JMML subgroups characterized by distinct clinical and biological features. We further provide evidence for a molecular mechanism by which additional genetic events, presumably further activating the RAS-RAF-MEK-ERK pathway, mediate DNA hypermethylation via up-regulation of DNMTs in aggressive JMML cases.
3103 - MITOCHONDRIA ARE IMPLICATED IN THE REGULATION OF TERMINAL ERYTHROPOIESIS Raymond Liang1 and Saghi Ghaffari2 1 Icahn School of Medicine at Mount Sinai, Astoria, United States; 2Icahn School of Medicine at Mount Sinai, New York, United States
Erythroblast enucleation, the process of nucleus removal, is a critical step for red blood cell (RBC) formation and function. Our data suggests a novel mechanism required for enucleation, which involves mitochondrial activity. Outside of their role in generating heme, mitochondrial function during other erythroid specific processes is unknown. We observed that prior to murine erythroblast enucleation, mitochondria migrate distal to the extruding nucleus. Immunofluorescence images of FACS sorted erythroblasts at distinct stages of maturation show increasing mitochondrial polarization with maturation. This observation was further confirmed by ImageStream analysis that combines microfluidics with imaging to allow high-throughput imaging of single cells. With ImageStream we could distinguish cells in the process of enucleating dependent on the degree of nucleus extrusion. We found a significant positive correlation between erythroblast enucleation and mitochondrial polarization and predicted that mitochondria function might relate to the energetic demands of the enucleation process. To test this, we tracked total mitochondria abundance and mitochondrial membrane potential (MMP), a measure of mitochondrial activity, using fluorescent probes. Although mitochondrial abundance and MMP decrease as erythroblasts mature, a transient increase in MMP is associated with enucleating cells. We also sorted primary late stage erythroblasts and allowed them to mature towards enucleated RBCs in the presence of inhibitors of the electron transport chain (ETC), which lead to decreased ATP production. Treatment with these drugs led to a significant decrease in erythroblast enucleation rates compared to DMSO control treated erythroblasts. The treatment was specific to enucleation and not other erythroblast maturation processes. Notably, this effect was not due to increased cell death and was reversible through a wash step. Overall our data support a novel role for mitochondria in promoting the last stages of erythroblast enucleation, potentially through ATP generation and/or other co-factors produced through the ETC.
3102 - GATA-3 SUPPRESSES IRF8 TO PROMOTE HUMAN T-CELL LINEAGE COMMITMENT Kai Ling Liang, Inge Van de Walle, Laurentijn Tilleman, Filip Van Nieuwerburgh, and Tom Taghon
3104 - A ROLE FOR MICROGLIA IN HEMATOPOIETIC STEM/PROGENITOR CELL EMERGENCE IN THE EMBRYONIC BRAIN Zhuan Li1, Samanta Mariani2, Chris Vink2, and Elaine Dzierzak2
Ghent University, Ghent, Belgium
1
The thymus is an organ where T-cell development takes place. However, human thymic progenitors have the capacity to differentiate into cells of other haematopoietic lineages. This multi-lineage differentiation potential is lost progressively as thymic progenitors undergo sequential T-cell lineage specification and commitment. The former is dependent on Notch1 signalling and results in loss of myeloid and Bcell potential. At the commitment stage, T-cell specific transcription factors, rather than Notch signalling, are essential to suppress residual non-T potential. Recently, we reported that GATA-3 is a key driver in human T-lineage commitment by repressing natural killer (NK) cell fate through down-modulation of Notch signalling. Now, transcriptomic analyses of GATA-3 transduced human haematopoietic progenitors allowed us to identify IRF8 as a novel target gene. In comparison to the hypomorphic KRR-mutant of GATA-3, enforced expression of GATA-3 wild type significantly downregulated endogenous IRF8 RNA and protein expression. The effect was reversed in a GATA-3 knockdown setting. IRF8 is a member of interferon regulatory factor family that plays an important role in human haematopoiesis. In human, IRF8 is highly expressed in all haematopoietic lineages except T-cells. Dysregulation of IRF8 has been implicated in human leukaemia and mutations in IRF8 are clinically associated with human dendritic cell (DC) and NK cell immunodeficiency. As such, we hypothesize that negative regulation of IRF8 by GATA-3 is important to promote human T-lineage commitment. Using multi-coloured flow cytometry, we showed that IRF8 expression is restricted in ex vivo uncommitted (CD34+CD1a-) human thymic progenitors. Developmental trajectory of human thymic progenitors, defined by early T-cell markers, revealed dynamic and sequential expressions of IRF8 and GATA-3. This enabled us to identify, within the uncommitted pool of thymic progenitors, new subpopulations that reflect successive extinction of DC and NK lineage choices. Using in vitro co-culture assay, we recapitulated this previously uncharacterized transitional cell populations during human T-lineage commitment.
S83
QMRI, Edinburgh, United Kingdom; 2The University of Edinburgh, Edinburgh, United Kingdom
The mouse embryonic head is one of the sites for haematopoietic stem and progenitor cell (HSPC) development. Transplantation of subdissected head region cells has shown HSC localization in the hindbrain and branchial arches (HBA). Recent studies reveal a dynamic interaction of primitive macrophages with aortic HSPCs in zebrafish embryos. It is becoming clear by fate mapping that under physiological conditions, microglia cells (macrophages in the head, MV) are derived from the yolk sac. However, it is unknown what role(s) MV play in HSPC emergence in the head. In our study using MacGreen (Csf1r-GFP) embryos, we found in the HBA (E9.5-11) that more than 95% of MV (CD45+F4/80+CD11b+Gr1-) are GFP+. Flow cytometry showed that most MV in the E10 HBA express CX3CR1 and CXCR4. qRTPCR analysis showed that E10 HBA endothelial cells (CD31+CD45-CD41-, EC) express CX3CL1, which encodes the ligand of CX3CR1. In CX3CR1-/- embryos, the frequency of MV in the HBA is reduced. Interestingly, at E10 the percentage of haematopoietic progenitors (cKit+CD41lo) is decreased in the CX3CR1 deficient HBA, but it is not decreased in the E11 HBA. To test if MV play a role in endothelial cell-to-haematopoietic cell-transition (EHT), HBA EC were cultured with/without microglia in an OP9-DL1 coculture system. The presence of MV boosted the HPC output from EC more than 2-fold compared to the control group (EC only). qRTPCR experiments showed MV (MacGreen+) express significantly higher levels of pro-inflammatory factor genes (IL-1b, IL-1c, TNFb) compared to MacGreen- cells. Interestingly, EC expressed higher levels of receptor genes (IL1R1, TNFbR1) than CD41+/CD45+ or CD31-CD41-CD45- cells, suggesting that MV are involved in hematopoietic cell emergence in the HBA through the IL-1 and/or TNFa pathway. To examine if MV function in vivo, MV were specifically ablated in Csf1r-Cre;RosaDTA mice. In the HBA (E9.5-11), all the MV were depleted. Methylcellulose culture data showed the numbers of hematopoietic progenitors were reduced significantly after MV ablation in the HBA. In summary, these data suggest that microglia cells play important roles in hematopoietic cell emergence in HBA.