- Email: [email protected]
erythrocyte progenitor
Poster Presentations/ Experimental Hematology 42 (2014) S23–S68
P1112 - DECLINED PRESENTATION NITROGEN MONOXIDE INHIBITS HAEM SYNTHESIS IN MOUSE RETICULOCYTES Marc Mikhael1,2, Shan Soe-Lin2,1, Sameer Apte1,2, and Prem Ponka1,2 1 Physiology, McGill University, Montreal, Quebec, Canada; 2Lady Davis Institute, Montreal, Quebec, Canada AI (anaemia of inflammation) often manifests in patients with chronic immune activation due to cancer, chronic infections, autoimmune disorders, rheumatoid arthritis and other diseases. The pathogenesis of AI is complex and involves cytokine-mediated inhibition of erythropoiesis, insufficient erythropoietin production and diminished sensitivity of erythroid progenitors to this hormone, and retention of iron in haemoglobin-processing macrophages. NO (nitric oxide) is a gaseous molecule produced by activated macrophages that has been identified as having numerous effects on iron metabolism. In the present study, we explore the possibility that NO affects iron metabolism in reticulocytes and our results suggest that NO may also contribute to AI. We treated reticulocytes with the NO donor SNP (sodium nitroprusside). The results indicate that NO inhibits haem synthesis dramatically and rapidly at the level of erythroid-specific 5-aminolaevulinic acid synthase 2, which catalyses the first step of haem synthesis in erythroid cells. We also show that NO leads to the inhibition of iron uptake via the Tf (transferrin)-Tf receptor pathway. In addition, NO also causes an increase in (eukaryotic initiation factor) phosphorylation levels and decreases globin translation. The profound impairment of haem synthesis, iron uptake and globin translation in reticulocytes by NO raises the possibility that this gas may also contribute to AI.
P1113 - THE TRANSCRIPTION ELONGATION CONTROL BY IKAROS AND NURD IS REQUIRED FOR NORMAL HEMATOPOIESIS Stefania Bottardi2,1, Lionel Mavoungou2,1, Helen Pak2,1, Yahia A. Lakehal2,1, and Eric Milot2,1 1 l’Universite de Montreal, Montreal, Quebec, Canada; 2H^opital MaisonneuveRosemont, Montreal, Quebec, Canada IKAROS is a critical regulator of hematopoietic cell fate, lineage commitment and differentiation. In collaboration with the Nucleosome Remodeling and Deacetylation (NuRD) complex, it promotes gene repression and activation. However, it is unclear how the IKAROS association with NuRD, a HDAC-containing complex, supports transcription activation. IKAROS also associates with the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. We recently found out that IKAROS assembles a novel complex containing NuRD, P-TEFb and is required for the simultaneous recruitment of P-TEFb components along with the P-TEFb activator Protein Phosphatase 1a (PP1) to specific genes. We showed that IKAROS acquires the capacity to bind and activate P-TEFb only when it is expressed at high levels. The knockdown of NuRD subunits indicated that relief of Pol II promoterproximal pausing and efficient elongation require NuRD occupancy at the genes transcribed regions. Finally, we demonstrated that an IKAROS mutant unable to bind the P-TEFb activator PP1 does not sustain gene expression and impedes normal differentiation of Ik null hematopoietic progenitor cells.
S51
P1114 - IDENTIFICATION AND CHARACTERIZATION OF THERAPY RESISTANCE DETERMINANTS IN LEUKEMIA Michael Milyavsky1, Olga Gan2, Mark van Delft2, Sean P. McDermott2, Adi Zipin-Roitman1, Alla Buzina3, Troy Ketela3, Liran Shlush2, Stephanie Xie2, Veronique Voisin2, Jason Moffat3, Mark Minden2, and John Dick2 1 Pathology, Tel Aviv University, Tel Aviv, Israel; 2Ontario Institute for Cancer Research, Toronto, Ontario, Canada; 3Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada Acute Myeloid Leukemia (AML) cure remains challenging due to resistance mechanisms operating in the minority of leukemia cells with high regenerative potential (Leukemia Stem cells, LSC). Currently, molecular determinants governing escape of LSC from DNA damaging therapy remain largely undefined. We, therefore, devised genome-wide shRNA screen using a library of 80,000 lentiviral shRNA vectors targeting 16,000 human genes to isolate genes responsible for inducing human leukemia cell survival and regeneration following multiple challenges with DNA damaging agent, such as irradiation. As the result, we isolated numerous shRNA clones that drastically increase leukemia cell regeneration after genotoxic injury. Some of the screen candidates are known DNA damage response players (e.g. p53 and CHEK2), but the majority of the high confidence hits remains uncharacterized in terms of their involvement in stress responses. Here, we report studies aimed to further characterize a small subset of hits implicated in the epigenetic regulation (e.g. SMYD2), provide their on-target validation and initial insights into protective mechanisms. Furthermore, using bioinformatics approaches, we attempted to integrate our functional genomics screen hits with the transcriptional signatures of human AML samples that differ in their resistance toward standard chemotherapy drug. The role of SMYD2 in mediating LSC chemotherapy resistance in xenotransplantation model of human AML is currently under investigation We believe that our integrated experimental platform will identify crucial determinants of LSC resistance that could be used as prognostic markers and, most importantly, will provide a foundation for their targeting.
P1115 - HUMAN MEGAKARYOCYTE PROGENITOR EMERGES INDEPENDENT OF MEGAKARYOCYTE/ERYTHROCYTE PROGENITOR Kohta Miyawaki, Hirotake Kusumoto, Hiromi Iwasaki, and Koichi Akashi Kyushu University, Fukuoka, Japan In human hematopoiesis, the megakaryocyte (Meg) lineage is supposed to diverge from bipotent MEP, which resides downstream of CMP. Here, we report prospective isolation of Meg progenitor (MegP), which emerges directly from CMP bypassing MEP. Because CD41 is known as a Meg lineage-specific marker, we first searched for the CD41-expressing cell population in the CD34 positive stem/progenitor fraction of healthy individuals. A fraction (w10%) of CMPs expressed CD41, but neither MEPs nor GMPs expressed detectable levels of CD41. Purified CD41+CMPs displayed myeloblast-like immature morphologies, and w1% of the cells possessed 4N nuclei, indicating that these cells are committed to Meg lineage. Although CD41-CMPs generated all types of myeloid colonies, CD41+CMPs exclusively gave rise to Meg colonies but completely lacked erythroid or granulocyte/monocyte (GM) lineage potentials. Moreover, CD41+CMPs possessed a distinct gene expression profile with up-regulation of Meg lineage-specific genes and down-regulation of erythroid and GM lineage-affiliated genes. Therefore, we defined the CD41+CMP as a human MegP. The Meg colony-producing potential of MegPs was almost thirty times as strong as that of MEPs, indicating that MEPs are largely committed to erythroid lineage. This MegP phenotype was detectable in CD41-CMP cultures but not in MEP cultures, suggesting that MegP develops downstream of CMP independent of MEP. Importantly, MegP pool significantly expanded in essential thrombocythemia (ET) patients. The newly-identified MegP robustly contributes to physiological megakaryopoiesis, thus is a therapeutic target for various thrombopoietic disorders.
P1112 - DECLINED PRESENTATION NITROGEN MONOXIDE INHIBITS HAEM SYNTHESIS IN MOUSE RETICULOCYTES Marc Mikhael1,2, Shan Soe-Lin2,1, Sameer Apte1,2, and Prem Ponka1,2 1 Physiology, McGill University, Montreal, Quebec, Canada; 2Lady Davis Institute, Montreal, Quebec, Canada AI (anaemia of inflammation) often manifests in patients with chronic immune activation due to cancer, chronic infections, autoimmune disorders, rheumatoid arthritis and other diseases. The pathogenesis of AI is complex and involves cytokine-mediated inhibition of erythropoiesis, insufficient erythropoietin production and diminished sensitivity of erythroid progenitors to this hormone, and retention of iron in haemoglobin-processing macrophages. NO (nitric oxide) is a gaseous molecule produced by activated macrophages that has been identified as having numerous effects on iron metabolism. In the present study, we explore the possibility that NO affects iron metabolism in reticulocytes and our results suggest that NO may also contribute to AI. We treated reticulocytes with the NO donor SNP (sodium nitroprusside). The results indicate that NO inhibits haem synthesis dramatically and rapidly at the level of erythroid-specific 5-aminolaevulinic acid synthase 2, which catalyses the first step of haem synthesis in erythroid cells. We also show that NO leads to the inhibition of iron uptake via the Tf (transferrin)-Tf receptor pathway. In addition, NO also causes an increase in (eukaryotic initiation factor) phosphorylation levels and decreases globin translation. The profound impairment of haem synthesis, iron uptake and globin translation in reticulocytes by NO raises the possibility that this gas may also contribute to AI.
P1113 - THE TRANSCRIPTION ELONGATION CONTROL BY IKAROS AND NURD IS REQUIRED FOR NORMAL HEMATOPOIESIS Stefania Bottardi2,1, Lionel Mavoungou2,1, Helen Pak2,1, Yahia A. Lakehal2,1, and Eric Milot2,1 1 l’Universite de Montreal, Montreal, Quebec, Canada; 2H^opital MaisonneuveRosemont, Montreal, Quebec, Canada IKAROS is a critical regulator of hematopoietic cell fate, lineage commitment and differentiation. In collaboration with the Nucleosome Remodeling and Deacetylation (NuRD) complex, it promotes gene repression and activation. However, it is unclear how the IKAROS association with NuRD, a HDAC-containing complex, supports transcription activation. IKAROS also associates with the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. We recently found out that IKAROS assembles a novel complex containing NuRD, P-TEFb and is required for the simultaneous recruitment of P-TEFb components along with the P-TEFb activator Protein Phosphatase 1a (PP1) to specific genes. We showed that IKAROS acquires the capacity to bind and activate P-TEFb only when it is expressed at high levels. The knockdown of NuRD subunits indicated that relief of Pol II promoterproximal pausing and efficient elongation require NuRD occupancy at the genes transcribed regions. Finally, we demonstrated that an IKAROS mutant unable to bind the P-TEFb activator PP1 does not sustain gene expression and impedes normal differentiation of Ik null hematopoietic progenitor cells.
S51
P1114 - IDENTIFICATION AND CHARACTERIZATION OF THERAPY RESISTANCE DETERMINANTS IN LEUKEMIA Michael Milyavsky1, Olga Gan2, Mark van Delft2, Sean P. McDermott2, Adi Zipin-Roitman1, Alla Buzina3, Troy Ketela3, Liran Shlush2, Stephanie Xie2, Veronique Voisin2, Jason Moffat3, Mark Minden2, and John Dick2 1 Pathology, Tel Aviv University, Tel Aviv, Israel; 2Ontario Institute for Cancer Research, Toronto, Ontario, Canada; 3Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada Acute Myeloid Leukemia (AML) cure remains challenging due to resistance mechanisms operating in the minority of leukemia cells with high regenerative potential (Leukemia Stem cells, LSC). Currently, molecular determinants governing escape of LSC from DNA damaging therapy remain largely undefined. We, therefore, devised genome-wide shRNA screen using a library of 80,000 lentiviral shRNA vectors targeting 16,000 human genes to isolate genes responsible for inducing human leukemia cell survival and regeneration following multiple challenges with DNA damaging agent, such as irradiation. As the result, we isolated numerous shRNA clones that drastically increase leukemia cell regeneration after genotoxic injury. Some of the screen candidates are known DNA damage response players (e.g. p53 and CHEK2), but the majority of the high confidence hits remains uncharacterized in terms of their involvement in stress responses. Here, we report studies aimed to further characterize a small subset of hits implicated in the epigenetic regulation (e.g. SMYD2), provide their on-target validation and initial insights into protective mechanisms. Furthermore, using bioinformatics approaches, we attempted to integrate our functional genomics screen hits with the transcriptional signatures of human AML samples that differ in their resistance toward standard chemotherapy drug. The role of SMYD2 in mediating LSC chemotherapy resistance in xenotransplantation model of human AML is currently under investigation We believe that our integrated experimental platform will identify crucial determinants of LSC resistance that could be used as prognostic markers and, most importantly, will provide a foundation for their targeting.
P1115 - HUMAN MEGAKARYOCYTE PROGENITOR EMERGES INDEPENDENT OF MEGAKARYOCYTE/ERYTHROCYTE PROGENITOR Kohta Miyawaki, Hirotake Kusumoto, Hiromi Iwasaki, and Koichi Akashi Kyushu University, Fukuoka, Japan In human hematopoiesis, the megakaryocyte (Meg) lineage is supposed to diverge from bipotent MEP, which resides downstream of CMP. Here, we report prospective isolation of Meg progenitor (MegP), which emerges directly from CMP bypassing MEP. Because CD41 is known as a Meg lineage-specific marker, we first searched for the CD41-expressing cell population in the CD34 positive stem/progenitor fraction of healthy individuals. A fraction (w10%) of CMPs expressed CD41, but neither MEPs nor GMPs expressed detectable levels of CD41. Purified CD41+CMPs displayed myeloblast-like immature morphologies, and w1% of the cells possessed 4N nuclei, indicating that these cells are committed to Meg lineage. Although CD41-CMPs generated all types of myeloid colonies, CD41+CMPs exclusively gave rise to Meg colonies but completely lacked erythroid or granulocyte/monocyte (GM) lineage potentials. Moreover, CD41+CMPs possessed a distinct gene expression profile with up-regulation of Meg lineage-specific genes and down-regulation of erythroid and GM lineage-affiliated genes. Therefore, we defined the CD41+CMP as a human MegP. The Meg colony-producing potential of MegPs was almost thirty times as strong as that of MEPs, indicating that MEPs are largely committed to erythroid lineage. This MegP phenotype was detectable in CD41-CMP cultures but not in MEP cultures, suggesting that MegP develops downstream of CMP independent of MEP. Importantly, MegP pool significantly expanded in essential thrombocythemia (ET) patients. The newly-identified MegP robustly contributes to physiological megakaryopoiesis, thus is a therapeutic target for various thrombopoietic disorders.