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Erythropoiesis in DMT1 mutant mice
Poster Presentations/ Experimental Hematology 42 (2014) S23–S68
P1176 - DECLINED PRESENTATION ACTIVATING FLT3-ITD RECEPTOR MUTATIONS IN AML ARE ASSOCIATED WITH A SPECIFIC EPIGENETIC SIGNATURE COMPOSED OF A DISCRETE SUBSET RUNX1-BOUND DNASEI HYPERSENSITIVE SITES ENRICHED FOR AP-1 AND C/EBP MOTIFS Joaquin Zacarias Cabeza1, Pierre Cauchy1, Sally James2, Jason Piper3, Anetta Ptasinska1, Maria-Rosaria Imperato1, Martina Canestraro1, Salam Assi2, Maarten Hoogenkamp1, David Westhead2, Sascha Ott3, Constanze Bonifer1, and Peter Cockerill1 1 Institute of Biomedical Research, University of Birmingham, Birmingham, United Kingdom; 2University of Leeds, Leeds, United Kingdom; 3University of Warwick, Coventry, United Kingdom Acute myeloid leukemia (AML) with a FLT3 internal tandem duplication (FLT3-ITD) mutation is characterised by constitutive activation of MAPK and JAK/STAT signaling pathways. To identify FLT-ITD target genes and enhancers, we performed mRNA array analysis in parallel with genome-wide mapping of DNaseI Hypersensitive Sites (DHSs) in AML samples. This data was also used together with our recently developed genomewide DHS footprinting methodology to predict occupancy of transcription factor binding sites. We compared karyotypically normal (KN) AMLs bearing FLT3-ITD mutations to KN AMLs lacking this mutation, and to normal CD34+ peripheral blood stem cells (PBSCs). We found specific DHSs that were consistently enriched in the FLT3-ITD AML samples compared to normal CD34+ cells and other KN AMLs. These potential enhancers were enriched for DNA motifs for the transcription factors RUNX1, AP-1, and C/ EBP. Many of these motifs were predicted by DHS footprinting to be preferentially occupied in FLT3-ITD+ AML and not in PBSCs. We found that both Erk1/2 and AP-1 were constitutively active in FLT3-ITD+ cell lines, suggesting that the MAPK dependent activation of AP-1 is a major contributor to the reprogramming of the genome in FLT3-ITD+ AML. This is also probably the main mechanism leading to the AML-specific binding of RUNX1 to many additional sites that are not occupied in normal cells. The FLT3ITD target DHSs were often linked to genes that were upregulated. RUNX1 apparently contributed to this because many of these target genes were instead down-regulated in a FLT3-ITD+ patient also carrying a RUNX1 mutation. Interestingly, the FLT3-ITD-specific DHSs were not enriched for motifs for STAT or IRF family proteins that may also respond to FLT3 signaling. In summary, our data suggests that RUNX1 together with MAPK-inducible factors plays a major role in reprogramming gene expression in FLT3-ITD AML.
S67
P1178 - ERYTHROPOIESIS IN DMT1 MUTANT MICE Zuzana Zidova1, Pavla Koralkova1, Katarina Kapralova1, Renata Mojzikova1, Dalibor Dolezal1, Daniel Garcia-Santos2, Vladimir Divoky1, Prem Ponka2, and Monika Horvathova1 1 Department of Biology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic; 2Lady Davis Institute for Medical Research, Jewish General Hospital and the Department of Physiology, McGill University, Montreal, Quebec, Canada Deficiency of the divalent metal transporter 1 (DMT1) leads to hypochromic microcytic anemia. We have previously shown that DMT1 deficiency impairs erythroid differentiation and induces apoptosis of erythroid cells. The potential involvement of heme oxygenase 1, which co-regulates erythroid differentiation by controlling the heme regulatory pool in erythroid cells, is under investigation. In the presented study, we analyzed metabolic processes and survival of mature erythrocytes in order to address potential involvement of erythrocyte defect in the pathophysiology of the disease. We observed an accelerated clearance of CFSE-labeled DMT1-mutant erythrocytes from circulating blood as compared to wild-type erythrocytes. In vitro, DMT1-mutant erythrocytes showed significantly increased Annexin V binding after exposure to hyper-osmotic shock and glucose depletion, respectively. Exaggeration of an anti-oxidative defense in DMT1-mutant mice was confirmed by increased activity of oxidative stress-related enzymes. Accelerated anaerobic glycolysis and reduced ATP/ADP ratio detected in DMT1-mutant erythrocytes indicate enhanced demand for ATP to maintain erythrocyte membrane stability. We propose that DMT1 deficiency negatively affects metabolism and life span of mature erythrocytes; two other aspects of defective erythropoiesis which contribute to the pathophysiology of the disease. Grant support: Czech Grant Agency Grant No. P305/11/ 1745; Ministry of Health Czech Republic Grant No. NT13587; The European Structural Funds Project CZ.1.07/2.3.00/20.0164 and Internal Grant of Palacky University Olomouc LF_2014_011.
P1177 - DECLINED PRESENTATION EPIGENETIC COMPARISON OF THE NEURAL DIFFERENTIATION POTENTIAL OF MESENCHYMAL STEM CELLS DERIVED FROM HUMAN CHORION AND BONE MARROW Reihane Ziadlou Royan Institute, Tehran, Islamic Republic of Iran
P1179 - ENDOTHELIAL CELL MUTAGENESIS DURING DEVELOPMENT YIELDS HEMATOPOIETIC ABNORMALITIES Safiyyah Ziyad1, Jesse Riordan2, Kristine Huynh1, Benjamin Brett2, Todd Scheetz2, Adam Dupuy2, and Maria Iruela-Arispe1 1 MCDB, UCLA, Los Angeles, California, USA; 2University of Iowa, Iowa City, Iowa, USA
Finding a reliable and abundant source of stem cells for the replacement of missing neurons in nervous system diseases requires extensive characterization of neural differentiation associated markers in different sources of mesenchymal stem cells (MSCs). In neural differentiation, modification of histones plays an important role in regulation of gene expression. More recently, chorion derived stem cells of human placenta have been described as abundant, ethically acceptable, and easily accessible source of cells which have not been affected by the limitations of using bone marrow (BM) MSCs. In this study, neural differentiation potential, expression level and epigenetic profile of NESTIN gene as a neural differentiation marker were compared in mesenchymal stem cells derived from human chorion and bone marrow. For this respect, chorion and bone marrow MSCs have been isolated and cultured. The capacity of these sources of MSCs to differentiate into neural like cells were compared by immunocytochemistry analysis. The expression level of NESTIN, as a marker for neural progenitor cells, in MSCs derived from BM and chorion were evaluated quantitatively by real-time PCR technique. In addition, histone 3 modifications on the regulatory region of NESTIN were evaluated by ChIP real time-PCR technique. Immunocytochemical data showed that chorion derived MSCs expressed all analysed neural markers, including NESTIN, higher than BM MSCs. According to quantitative real time PCR analysis, chorion derived MSCs demonstrate significantly higher expression level of NESTIN in comparsion to BM. Furthermore, epigenetic profile was in agreement with the remarkable higher expression level of NESTIN gene in chorion derived MSCs in comparison to BM-MSCs. Finally, it is concluded that chorion derived MSCs have more potential for differentiation to neural cell lineages; so, they could be regarded as a promising source of stem cells for cell therapy of neurological disorders.
Lineage tracing analysis has revealed that hematopoietic stem cells originate from an endothelial intermediate with hemogenic capacity at mid gestation. Here we explored the biological impact of endothelial mutations in the development of hematopoietic diseases. We demonstrate that mutagenesis initiated in the hemogenic endothelium yields hematopoietic abnormalities in a mouse model. Specifically, we employed constitutively active VE-Cadherin-Cre to induce transposon mutagenesis in endothelial cells starting at E8.5. The majority (69%) of mutant mice developed an enlarged spleen and/or thymus and abnormal blood profile. A subset of the mutant animals exclusively presented with an enlarged spleen showing expansion of the red pulp zone. Blood leukocyte cytospins revealed blast-like cell predominance. The mutated genes associated with the enlarged spleen phenotype were indicative of a myeloid/ erythroid malignancy. Another subset of mutant animals exhibited an enlarged thymus. The mutated genes associated with the enlarged thymus suggest a T-lymphocyte basis. A large number of mutations correlate well with those previously identified in association with hematopoietic malignancies generated from global transposon mutagenesis screens and those driven by Vav, CD4, or Lck-Cre. However, a subset of mutations was unique to this screen, and they are likely to represent genes active during the hemogenic endothelial or early HSC stage. In summary, these findings not only underscore the endothelial origin of hematopoietic cells, but also suggest an endothelial origin of a subset of hematopoietic diseases.
P1176 - DECLINED PRESENTATION ACTIVATING FLT3-ITD RECEPTOR MUTATIONS IN AML ARE ASSOCIATED WITH A SPECIFIC EPIGENETIC SIGNATURE COMPOSED OF A DISCRETE SUBSET RUNX1-BOUND DNASEI HYPERSENSITIVE SITES ENRICHED FOR AP-1 AND C/EBP MOTIFS Joaquin Zacarias Cabeza1, Pierre Cauchy1, Sally James2, Jason Piper3, Anetta Ptasinska1, Maria-Rosaria Imperato1, Martina Canestraro1, Salam Assi2, Maarten Hoogenkamp1, David Westhead2, Sascha Ott3, Constanze Bonifer1, and Peter Cockerill1 1 Institute of Biomedical Research, University of Birmingham, Birmingham, United Kingdom; 2University of Leeds, Leeds, United Kingdom; 3University of Warwick, Coventry, United Kingdom Acute myeloid leukemia (AML) with a FLT3 internal tandem duplication (FLT3-ITD) mutation is characterised by constitutive activation of MAPK and JAK/STAT signaling pathways. To identify FLT-ITD target genes and enhancers, we performed mRNA array analysis in parallel with genome-wide mapping of DNaseI Hypersensitive Sites (DHSs) in AML samples. This data was also used together with our recently developed genomewide DHS footprinting methodology to predict occupancy of transcription factor binding sites. We compared karyotypically normal (KN) AMLs bearing FLT3-ITD mutations to KN AMLs lacking this mutation, and to normal CD34+ peripheral blood stem cells (PBSCs). We found specific DHSs that were consistently enriched in the FLT3-ITD AML samples compared to normal CD34+ cells and other KN AMLs. These potential enhancers were enriched for DNA motifs for the transcription factors RUNX1, AP-1, and C/ EBP. Many of these motifs were predicted by DHS footprinting to be preferentially occupied in FLT3-ITD+ AML and not in PBSCs. We found that both Erk1/2 and AP-1 were constitutively active in FLT3-ITD+ cell lines, suggesting that the MAPK dependent activation of AP-1 is a major contributor to the reprogramming of the genome in FLT3-ITD+ AML. This is also probably the main mechanism leading to the AML-specific binding of RUNX1 to many additional sites that are not occupied in normal cells. The FLT3ITD target DHSs were often linked to genes that were upregulated. RUNX1 apparently contributed to this because many of these target genes were instead down-regulated in a FLT3-ITD+ patient also carrying a RUNX1 mutation. Interestingly, the FLT3-ITD-specific DHSs were not enriched for motifs for STAT or IRF family proteins that may also respond to FLT3 signaling. In summary, our data suggests that RUNX1 together with MAPK-inducible factors plays a major role in reprogramming gene expression in FLT3-ITD AML.
S67
P1178 - ERYTHROPOIESIS IN DMT1 MUTANT MICE Zuzana Zidova1, Pavla Koralkova1, Katarina Kapralova1, Renata Mojzikova1, Dalibor Dolezal1, Daniel Garcia-Santos2, Vladimir Divoky1, Prem Ponka2, and Monika Horvathova1 1 Department of Biology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic; 2Lady Davis Institute for Medical Research, Jewish General Hospital and the Department of Physiology, McGill University, Montreal, Quebec, Canada Deficiency of the divalent metal transporter 1 (DMT1) leads to hypochromic microcytic anemia. We have previously shown that DMT1 deficiency impairs erythroid differentiation and induces apoptosis of erythroid cells. The potential involvement of heme oxygenase 1, which co-regulates erythroid differentiation by controlling the heme regulatory pool in erythroid cells, is under investigation. In the presented study, we analyzed metabolic processes and survival of mature erythrocytes in order to address potential involvement of erythrocyte defect in the pathophysiology of the disease. We observed an accelerated clearance of CFSE-labeled DMT1-mutant erythrocytes from circulating blood as compared to wild-type erythrocytes. In vitro, DMT1-mutant erythrocytes showed significantly increased Annexin V binding after exposure to hyper-osmotic shock and glucose depletion, respectively. Exaggeration of an anti-oxidative defense in DMT1-mutant mice was confirmed by increased activity of oxidative stress-related enzymes. Accelerated anaerobic glycolysis and reduced ATP/ADP ratio detected in DMT1-mutant erythrocytes indicate enhanced demand for ATP to maintain erythrocyte membrane stability. We propose that DMT1 deficiency negatively affects metabolism and life span of mature erythrocytes; two other aspects of defective erythropoiesis which contribute to the pathophysiology of the disease. Grant support: Czech Grant Agency Grant No. P305/11/ 1745; Ministry of Health Czech Republic Grant No. NT13587; The European Structural Funds Project CZ.1.07/2.3.00/20.0164 and Internal Grant of Palacky University Olomouc LF_2014_011.
P1177 - DECLINED PRESENTATION EPIGENETIC COMPARISON OF THE NEURAL DIFFERENTIATION POTENTIAL OF MESENCHYMAL STEM CELLS DERIVED FROM HUMAN CHORION AND BONE MARROW Reihane Ziadlou Royan Institute, Tehran, Islamic Republic of Iran
P1179 - ENDOTHELIAL CELL MUTAGENESIS DURING DEVELOPMENT YIELDS HEMATOPOIETIC ABNORMALITIES Safiyyah Ziyad1, Jesse Riordan2, Kristine Huynh1, Benjamin Brett2, Todd Scheetz2, Adam Dupuy2, and Maria Iruela-Arispe1 1 MCDB, UCLA, Los Angeles, California, USA; 2University of Iowa, Iowa City, Iowa, USA
Finding a reliable and abundant source of stem cells for the replacement of missing neurons in nervous system diseases requires extensive characterization of neural differentiation associated markers in different sources of mesenchymal stem cells (MSCs). In neural differentiation, modification of histones plays an important role in regulation of gene expression. More recently, chorion derived stem cells of human placenta have been described as abundant, ethically acceptable, and easily accessible source of cells which have not been affected by the limitations of using bone marrow (BM) MSCs. In this study, neural differentiation potential, expression level and epigenetic profile of NESTIN gene as a neural differentiation marker were compared in mesenchymal stem cells derived from human chorion and bone marrow. For this respect, chorion and bone marrow MSCs have been isolated and cultured. The capacity of these sources of MSCs to differentiate into neural like cells were compared by immunocytochemistry analysis. The expression level of NESTIN, as a marker for neural progenitor cells, in MSCs derived from BM and chorion were evaluated quantitatively by real-time PCR technique. In addition, histone 3 modifications on the regulatory region of NESTIN were evaluated by ChIP real time-PCR technique. Immunocytochemical data showed that chorion derived MSCs expressed all analysed neural markers, including NESTIN, higher than BM MSCs. According to quantitative real time PCR analysis, chorion derived MSCs demonstrate significantly higher expression level of NESTIN in comparsion to BM. Furthermore, epigenetic profile was in agreement with the remarkable higher expression level of NESTIN gene in chorion derived MSCs in comparison to BM-MSCs. Finally, it is concluded that chorion derived MSCs have more potential for differentiation to neural cell lineages; so, they could be regarded as a promising source of stem cells for cell therapy of neurological disorders.
Lineage tracing analysis has revealed that hematopoietic stem cells originate from an endothelial intermediate with hemogenic capacity at mid gestation. Here we explored the biological impact of endothelial mutations in the development of hematopoietic diseases. We demonstrate that mutagenesis initiated in the hemogenic endothelium yields hematopoietic abnormalities in a mouse model. Specifically, we employed constitutively active VE-Cadherin-Cre to induce transposon mutagenesis in endothelial cells starting at E8.5. The majority (69%) of mutant mice developed an enlarged spleen and/or thymus and abnormal blood profile. A subset of the mutant animals exclusively presented with an enlarged spleen showing expansion of the red pulp zone. Blood leukocyte cytospins revealed blast-like cell predominance. The mutated genes associated with the enlarged spleen phenotype were indicative of a myeloid/ erythroid malignancy. Another subset of mutant animals exhibited an enlarged thymus. The mutated genes associated with the enlarged thymus suggest a T-lymphocyte basis. A large number of mutations correlate well with those previously identified in association with hematopoietic malignancies generated from global transposon mutagenesis screens and those driven by Vav, CD4, or Lck-Cre. However, a subset of mutations was unique to this screen, and they are likely to represent genes active during the hemogenic endothelial or early HSC stage. In summary, these findings not only underscore the endothelial origin of hematopoietic cells, but also suggest an endothelial origin of a subset of hematopoietic diseases.