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Redefining the human blood progenitor hierarchy across development
S104
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3213 - IFNa PROMOTES DIFFERENTIATION OF BCR-ABL+ LEUKEMIC CELLS THROUGH UPREGULATING C/EBPb Asumi Yokota1, Hideyo Hirai1, Yoshihiro Hayashi2,3, Akihiro Tamura1, Atsushi Sato1, Masaki Iwasa1,3, Aya Fujishiro1,3, Tsukimi Shoji1, Yasuo Miura1, and Taira Maekawa1 1 Dept. of Transfusion Medicine/Cell Therapy, Kyoto University Hospital, Kyoto City, Kyoto, Japan; 2Div. of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA; 3Div. of Gastroenterology and Hematology, Shiga University of Medical Science, Otsu, Japan A leucine zipper transcription factor, C/EBPb is essential for emergency granulopoiesis under stress conditions including infection and cytokine stimulation (Hirai H et al, Nat Immunol 2006; Satake et al. J Immunol 2012). We have previously shown that C/EBPb is upregulated by BCR-ABL-STAT5 signaling and promotes both myeloid expansion and exhaustion of CML stem cells (Hayashi Y et al, Leukemia 2013). We hypothesized that CML stem cells are maintained in BM microenvironment by suppressing expression or function of C/EBPb and that CML stem cells could be eradicated by C/EBPb upregulation mediated through signaling independent of BCR/ABL. Although IFNa, a cytokine that induces normal HSC exhaustion, has been used for therapy of CML, the molecular mechanisms underlying its effect on CML stem cells remain elusive. Here we investigated the significance of C/EBPb in the effect of IFNa on BCR-ABL-expressing leukemic cells. When BCR-ABL-transduced EML cells, a mouse HSC-like cell line, were treated with IFNa, STAT1, STAT3 and STAT5 were promptly phosphorylated, and C/EBPb was upregulated even in the presence of imatinib, suggesting that IFNa was able to upregulate C/ EBPb through activation of STATs independently of BCR-ABL. To examine further the effect of IFNa-C/EBPb axis on CML stem cells, BM cells from WTor C/EBPb KO mice were retrovirally transduced with BCR-ABL and subjected to colony assay with or without IFNa. In the presence of IFNa, myeloid differentiation of WT leukemic cells was accelerated and their replating ability diminished rapidly, while such IFNa-mediated changes were cancelled in C/EBPb KO leukemic cells. Collectively, these results suggest that IFNa promotes myeloid differentiation and exhaustion of leukemic cells through STAT-C/EBPb pathway. Further investigation is underway to elucidate the involvement of C/EBPb in the in vivo effects of IFNa on exhaustion of CML stem cells.
3214 - GENOME-WIDE RNA TOMOGRAPHY ANALYSIS OF THE MICROENVIRONMENT PROMOTING HEMATOPOIETIC STEM CELL EMERGENCE IN THE EMBRYO AORTA Laurent Yvernogeau, Jan Philipp Junker, Alexander van Oudenaarden, and Catherine Robin Hubrecht Institute-KNAW & University Medical Center Utrecht, Utrecht, Netherlands The first hematopoietic stem cells (HSCs) emerge during embryonic development and are part of cell aggregates attached to the endothelial layer of the aorta (referred to as Intra-Aortic Hematopoietic Clusters or IAHCs). All HSCs originate from specialized endothelial cells named hemogenic endothelial cells via an endothelialto-hematopoietic transition (EHT) process. IAHC and HSC production is tightly controlled in time and space by regulatory signals emitted by the surrounding microenvironment. In mouse embryo, the number and location of IAHCs along the aorta is well known. IAHCs start to appear at embryonic day (E)9.5. Their number reaches a peak at E11.5, before to progressively decrease. HSCs are first detected at E10.5 in IAHCs located to the central part of the aorta. We established the precise cartography of IAHCs in the chicken embryo and found that IAHCs are restricted to the floor (similarly to human embryo) and to the upper part of the aorta (anterior to the vitelline artery-aorta connection). This precise and specific distribution of IAHCs in the chicken and mouse aortas makes both models powerful tools to trigger the molecular signals coming from the surrounding tissues and restricting hematopoietic activity to specific areas. A genome-wide RNA tomography technique (tomo-seq) was recently developed to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo. By applying the tomo-seq technique on whole aortas (subdissected with the surrounding mesenchyme) and on thick transversal embryo sections (collected from the anterior and posterior parts of the aorta), we aim to decipher genes that are (differentially) expressed in specific regions during IAHC (and HSC) generation. The comparison of both species should unravel conserved molecular mechanisms (possibly also conserved in human embryo). Our original approach should help identifying potent candidate genes and pathways involved in the regulation of the first HSCs generated during embryonic development.
3215 - THE CLINICAL IMPACT OF BOTH POINT MUTATED AND ALTERNATIVELY SPLICED BCR-ABL IN CML PATIENTS: RESULT OF HIGHLY-SENSITIVE, DEEP SEQUENCING STUDY Junichiro Yuda1, Toshihiro Miyamoto1, Jun Odawara1, Yasuyuki Ohkawa2, and Koichi Akashi1 1 Department of Medicine and Biosystemic Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Department of Advanced Medical Initiatives, Kyushu University, Fukuoka, Japan Background: Alternatively-spliced (AS) BCR-ABL, such as retention of 35bp intronic nucleotides at Exon 8/9 splice junction (Ins35) is important obstacles to achieve optimal response to TKI. This variant possesses a stop codon in kinase domain(KD), resulting in generation of ‘‘function dead’’ BCR-ABL, whereas it shows structural changes and failure to TKI binding. It is still controversial whether AS BCR-ABL has clinical impact on resistance for TKIs. Methods: We analyzed 394 samples from 72 CML-CP pts. cDNA was synthesized from extracted RNA samples, and BCR-ABL was amplified by long-range nested PCR method. These amplicons were deeply sequenced to detect BCR-ABL variants by using HiSeq 1500 (illumina). Result: In all 7 pts analyzed at diagnosis, CML cells have wild-type (WT) BCR-ABL but also have small amount of AS BCR-ABL. After TKI treatment, two types of splicing variants became evident: the Ins35 and the Intron 8-retained BCR-ABL. In newly diagnosed CML-CP, total amount of BCR-ABL quickly decreased, whereas the relative frequency of AS BCR-ABL increased, suggesting that clones possessing AS BCR-ABL might become dominant through TKI therapy. In 70 pts with deep molecular response under TKI treatment for O1 year, AS BCR-ABL was detected in 28 (72%) out of 39 pts who could not achieved MR 4.5, however it was detected in only 4 (13%) out of 31 pts who could achieved MR 4.5 during any points of treatment. Thus, emergence of AS BCR-ABL was significantly more frequent in pts retaining only MR3.0 (p!0.05). In these pts, total amount of BCR-ABL gradually decreased, while AS BCR-ABL did not, resulting in the poor response to TKI. There was no mutation of splicing machinery and splice site in pts with AS BCR-ABL. Conclusion: Our highly-sensitive quantification system revealed that persistence of AS BCR-ABL correlates with failure to achieve MR4.5 rather than point mutations during TKI therapy. Quantification of AS BCR-ABL variants is a beneficial method to predict clinical outcomes in CML pts. 3216 - REDEFINING THE HUMAN BLOOD PROGENITOR HIERARCHY ACROSS DEVELOPMENT Sasan Zandi1,2, Faiyaz Notta1, Naoya Takayama1, Stephanie Dobson1, Olga Gan1, Kerstin B. Kaufmann1, Jessica McLeod1, and John E. Dick1,2 1 Princess Margaret Cancer Centre, UHN, Toronto, Ontario, Canada; 2Dep of Molecular Genetics, University of Toronto, Canada Blood is one of the most regenerative organs with more than 1011 newly produced cells every day for the lifetime of the organism. Regulation of this massive output is predominantly achieved through the hierarchical organization of stem cells and a series of oligopotent progenitors that become increasingly restricted to unipotent progenitors. The standard model is based on the presence of oligopotent cells as they define the path from stem cells to unipotent progenitors. Although the standard model is still used extensively as an operational paradigm, further cell purification and functional clonal assays have led to key revisions of the model. Despite these advances there are still considerable uncertainty remains concerning the myelo-erythroid branch of human hematopoiesis and weather the hierarchical model is the same across development. We developed a novel cell-sorting scheme by including markers associated with erythroid (Er) and megakaryocytic (Mk) fates and resolve the fates of thousands of single human CD34+ cells. We then mapped the progenitor hierarchy across human developmental stages. More than 85% of clones in CMP fraction from human fetal liver (FL) and bone marrow (BM) were either unipotent or bipotent without concurrent mix (Mk-Er-My) potential. Only in FL and cord blood (CB) less than 5% of CMP clones were mixed, suggesting that unlike FL in BM multipotent cells are restricted to stem cell compartment. In FL, a striking 60% of clones from a sub-fraction of MPP were of Mk/Er type and the remainder of this subset was composed of Mk-only and Er-only clones. A substantial number of Mks emerged as part of mixed clones from HSCs and MPP in CB, and more evident in BM, supporting the hypothesis that Mk branching occurred directly from a multipotent cell. These unexpected data suggest that multipotency is restricted to the stem cell compartment by adulthood, whereas unilineage fates predominate the progenitor compartment forming a simple ‘two-tier’ hierarchy, with few intervening oligopotent intermediates.
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3213 - IFNa PROMOTES DIFFERENTIATION OF BCR-ABL+ LEUKEMIC CELLS THROUGH UPREGULATING C/EBPb Asumi Yokota1, Hideyo Hirai1, Yoshihiro Hayashi2,3, Akihiro Tamura1, Atsushi Sato1, Masaki Iwasa1,3, Aya Fujishiro1,3, Tsukimi Shoji1, Yasuo Miura1, and Taira Maekawa1 1 Dept. of Transfusion Medicine/Cell Therapy, Kyoto University Hospital, Kyoto City, Kyoto, Japan; 2Div. of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA; 3Div. of Gastroenterology and Hematology, Shiga University of Medical Science, Otsu, Japan A leucine zipper transcription factor, C/EBPb is essential for emergency granulopoiesis under stress conditions including infection and cytokine stimulation (Hirai H et al, Nat Immunol 2006; Satake et al. J Immunol 2012). We have previously shown that C/EBPb is upregulated by BCR-ABL-STAT5 signaling and promotes both myeloid expansion and exhaustion of CML stem cells (Hayashi Y et al, Leukemia 2013). We hypothesized that CML stem cells are maintained in BM microenvironment by suppressing expression or function of C/EBPb and that CML stem cells could be eradicated by C/EBPb upregulation mediated through signaling independent of BCR/ABL. Although IFNa, a cytokine that induces normal HSC exhaustion, has been used for therapy of CML, the molecular mechanisms underlying its effect on CML stem cells remain elusive. Here we investigated the significance of C/EBPb in the effect of IFNa on BCR-ABL-expressing leukemic cells. When BCR-ABL-transduced EML cells, a mouse HSC-like cell line, were treated with IFNa, STAT1, STAT3 and STAT5 were promptly phosphorylated, and C/EBPb was upregulated even in the presence of imatinib, suggesting that IFNa was able to upregulate C/ EBPb through activation of STATs independently of BCR-ABL. To examine further the effect of IFNa-C/EBPb axis on CML stem cells, BM cells from WTor C/EBPb KO mice were retrovirally transduced with BCR-ABL and subjected to colony assay with or without IFNa. In the presence of IFNa, myeloid differentiation of WT leukemic cells was accelerated and their replating ability diminished rapidly, while such IFNa-mediated changes were cancelled in C/EBPb KO leukemic cells. Collectively, these results suggest that IFNa promotes myeloid differentiation and exhaustion of leukemic cells through STAT-C/EBPb pathway. Further investigation is underway to elucidate the involvement of C/EBPb in the in vivo effects of IFNa on exhaustion of CML stem cells.
3214 - GENOME-WIDE RNA TOMOGRAPHY ANALYSIS OF THE MICROENVIRONMENT PROMOTING HEMATOPOIETIC STEM CELL EMERGENCE IN THE EMBRYO AORTA Laurent Yvernogeau, Jan Philipp Junker, Alexander van Oudenaarden, and Catherine Robin Hubrecht Institute-KNAW & University Medical Center Utrecht, Utrecht, Netherlands The first hematopoietic stem cells (HSCs) emerge during embryonic development and are part of cell aggregates attached to the endothelial layer of the aorta (referred to as Intra-Aortic Hematopoietic Clusters or IAHCs). All HSCs originate from specialized endothelial cells named hemogenic endothelial cells via an endothelialto-hematopoietic transition (EHT) process. IAHC and HSC production is tightly controlled in time and space by regulatory signals emitted by the surrounding microenvironment. In mouse embryo, the number and location of IAHCs along the aorta is well known. IAHCs start to appear at embryonic day (E)9.5. Their number reaches a peak at E11.5, before to progressively decrease. HSCs are first detected at E10.5 in IAHCs located to the central part of the aorta. We established the precise cartography of IAHCs in the chicken embryo and found that IAHCs are restricted to the floor (similarly to human embryo) and to the upper part of the aorta (anterior to the vitelline artery-aorta connection). This precise and specific distribution of IAHCs in the chicken and mouse aortas makes both models powerful tools to trigger the molecular signals coming from the surrounding tissues and restricting hematopoietic activity to specific areas. A genome-wide RNA tomography technique (tomo-seq) was recently developed to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo. By applying the tomo-seq technique on whole aortas (subdissected with the surrounding mesenchyme) and on thick transversal embryo sections (collected from the anterior and posterior parts of the aorta), we aim to decipher genes that are (differentially) expressed in specific regions during IAHC (and HSC) generation. The comparison of both species should unravel conserved molecular mechanisms (possibly also conserved in human embryo). Our original approach should help identifying potent candidate genes and pathways involved in the regulation of the first HSCs generated during embryonic development.
3215 - THE CLINICAL IMPACT OF BOTH POINT MUTATED AND ALTERNATIVELY SPLICED BCR-ABL IN CML PATIENTS: RESULT OF HIGHLY-SENSITIVE, DEEP SEQUENCING STUDY Junichiro Yuda1, Toshihiro Miyamoto1, Jun Odawara1, Yasuyuki Ohkawa2, and Koichi Akashi1 1 Department of Medicine and Biosystemic Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Department of Advanced Medical Initiatives, Kyushu University, Fukuoka, Japan Background: Alternatively-spliced (AS) BCR-ABL, such as retention of 35bp intronic nucleotides at Exon 8/9 splice junction (Ins35) is important obstacles to achieve optimal response to TKI. This variant possesses a stop codon in kinase domain(KD), resulting in generation of ‘‘function dead’’ BCR-ABL, whereas it shows structural changes and failure to TKI binding. It is still controversial whether AS BCR-ABL has clinical impact on resistance for TKIs. Methods: We analyzed 394 samples from 72 CML-CP pts. cDNA was synthesized from extracted RNA samples, and BCR-ABL was amplified by long-range nested PCR method. These amplicons were deeply sequenced to detect BCR-ABL variants by using HiSeq 1500 (illumina). Result: In all 7 pts analyzed at diagnosis, CML cells have wild-type (WT) BCR-ABL but also have small amount of AS BCR-ABL. After TKI treatment, two types of splicing variants became evident: the Ins35 and the Intron 8-retained BCR-ABL. In newly diagnosed CML-CP, total amount of BCR-ABL quickly decreased, whereas the relative frequency of AS BCR-ABL increased, suggesting that clones possessing AS BCR-ABL might become dominant through TKI therapy. In 70 pts with deep molecular response under TKI treatment for O1 year, AS BCR-ABL was detected in 28 (72%) out of 39 pts who could not achieved MR 4.5, however it was detected in only 4 (13%) out of 31 pts who could achieved MR 4.5 during any points of treatment. Thus, emergence of AS BCR-ABL was significantly more frequent in pts retaining only MR3.0 (p!0.05). In these pts, total amount of BCR-ABL gradually decreased, while AS BCR-ABL did not, resulting in the poor response to TKI. There was no mutation of splicing machinery and splice site in pts with AS BCR-ABL. Conclusion: Our highly-sensitive quantification system revealed that persistence of AS BCR-ABL correlates with failure to achieve MR4.5 rather than point mutations during TKI therapy. Quantification of AS BCR-ABL variants is a beneficial method to predict clinical outcomes in CML pts. 3216 - REDEFINING THE HUMAN BLOOD PROGENITOR HIERARCHY ACROSS DEVELOPMENT Sasan Zandi1,2, Faiyaz Notta1, Naoya Takayama1, Stephanie Dobson1, Olga Gan1, Kerstin B. Kaufmann1, Jessica McLeod1, and John E. Dick1,2 1 Princess Margaret Cancer Centre, UHN, Toronto, Ontario, Canada; 2Dep of Molecular Genetics, University of Toronto, Canada Blood is one of the most regenerative organs with more than 1011 newly produced cells every day for the lifetime of the organism. Regulation of this massive output is predominantly achieved through the hierarchical organization of stem cells and a series of oligopotent progenitors that become increasingly restricted to unipotent progenitors. The standard model is based on the presence of oligopotent cells as they define the path from stem cells to unipotent progenitors. Although the standard model is still used extensively as an operational paradigm, further cell purification and functional clonal assays have led to key revisions of the model. Despite these advances there are still considerable uncertainty remains concerning the myelo-erythroid branch of human hematopoiesis and weather the hierarchical model is the same across development. We developed a novel cell-sorting scheme by including markers associated with erythroid (Er) and megakaryocytic (Mk) fates and resolve the fates of thousands of single human CD34+ cells. We then mapped the progenitor hierarchy across human developmental stages. More than 85% of clones in CMP fraction from human fetal liver (FL) and bone marrow (BM) were either unipotent or bipotent without concurrent mix (Mk-Er-My) potential. Only in FL and cord blood (CB) less than 5% of CMP clones were mixed, suggesting that unlike FL in BM multipotent cells are restricted to stem cell compartment. In FL, a striking 60% of clones from a sub-fraction of MPP were of Mk/Er type and the remainder of this subset was composed of Mk-only and Er-only clones. A substantial number of Mks emerged as part of mixed clones from HSCs and MPP in CB, and more evident in BM, supporting the hypothesis that Mk branching occurred directly from a multipotent cell. These unexpected data suggest that multipotency is restricted to the stem cell compartment by adulthood, whereas unilineage fates predominate the progenitor compartment forming a simple ‘two-tier’ hierarchy, with few intervening oligopotent intermediates.