- Email: [email protected]
The dual function of LMO2 in driving erythroid cell fate
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Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
upregulated in CD9+ HSCs upon Thpo signaling. Thpo stimulated CD9+ HSCs exhibited increase of phosphorylated-STAT3 (S727) which is known to translocate to and activate mitochondria metabolism. Our data indicate that Thpo-mediated exit from cell cycle quiescence is coupled with emergence of Mk-biased HSCs. While facilitating cell cycle proliferation, Thpo signaling metabolically fortifies Mk-biased HSCs through mitochondrial activation, enabling them fit for Mk production. This suggests clonal expansion of HSCs which are metabolically adjusted for Mk-differentiation during stress hematopoietic states.
3150 - FUBP1 PROMOTES LEUKEMIA PROGRESSION BY REGULATION OF CELL CYCLE AND APOPTOSIS Van T. Hoang1, Katharina Gerlach1, Uta Kuller-M€uller2, Eva Weissenberger1, Daniela Krause1, and Martin Z€ornig1 1 Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt, Germany; 2German Red Cross Blood Donor Service Baden-Wuerttemberg – Hessen, Frankfurt, Germany
We have shown the important role of FUBP1 (Far Upstream Element Binding Protein 1) in the maintenance of hematopoietic stem cells (HSCs) (Rabenhorst, Thalheimer et al. 2015). Fubp1-/- gene trap mice died in utero at day E15.5 with reduced numbers of total fetal liver cells, and their HSCs failed to engraft long-term. In adult mice, FUBP1 is also required for HSC self-renewal, and it promotes cell proliferation and inhibits apoptosis through regulation of target gene expression (e.g. c-myc, cyclin D2, p21, p27). We then decided to investigate a potential function of the molecule in chronic (CML) and acute myeloid leukemia (AML). Eppert et al. demonstrated an increased expression of FUBP1 in CD34+ CD38- cells compared to total bone marrow (BM) derived from human primary AML in their microarray analysis (Eppert, Takenaka et al. 2011). To study its role in leukemia development and leukemia initiating cells, and to test a potential treatment of leukemia with FUBP1 inhibitors, we employed the retroviral transduction/transplantation mouse model of CML and AML induced by BCR-ABL1 and MLL-AF9, respectively. Mice that received BM transduced with the oncogene plus Fubp1 shRNA survived longer than the control mice. BCR-ABL+ Fubp1 shRNA+ CML cell populations including total BM, Lin- and LSK cells showed more apoptosis and less proliferation. Furthermore, pharmacological inhibition of FUBP1 improved the survival of AML mice significantly, even to a larger extent than Ara-C treatment. Next, we analyzed FUBP1 expression in biopsies derived from CML (n511) and AML patients (n572) by immunohistochemistry and compared with healthy BM (n58). Of note, no enhanced FUBP1 expression was observed in leukemic samples. However, we noticed a shorter overall survival in AML patients with strong FUBP1 expression compared to those with no or moderate expression, suggesting that FUBP1 levels might correlate with the aggressiveness of leukemia. In summary, our data suggest that FUBP1 acts as a cell cycle regulator and anti-apoptotic factor in leukemia, similar to its oncogenic role in solid tumors, and the protein can be a potential target for therapeutic purpose.
3149 - FOXP2 IS ESSENTIAL FOR THE QUIESCENT STATE AND SELF-RENEWAL CAPACITY OF HEMATOPOIETIC STEM CELLS Kentaro Hosokawa, Saki Morimoto, Yuya Kunisaki, Tomoko Hyoda, Yasufumi Uehara, Haruka Imanishi, and Fumio Arai
3151 - THE DUAL FUNCTION OF LMO2 IN DRIVING ERYTHROID CELL FATE Trang Hoang1,2, Marie-Claude Sincennes3, Veronique Lisi4, Diogo Veiga5, Magali Humbert6, Francois Major7, Bachir Affar8, and Alain Verreault7
Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
1 Institute for Research in Immunology and Cancer, Montreal, Canada; 2Montreal, Canada; 3University of Ottawa, Ottawa, Canada; 4University of California, Santa Barbara, Santa Barbara, United States; 5Jackson Genomics Institute, Farmington, United States; 6Bern University, Bern, Switzerland; 7IRIC, University of Montreal, Montreal, Canada; 8University of Montreal, Montreal, Canada
Cell cycle quiescence is crucial for maintenance of the hematopoietic stem cells (HSCs) activity. Activation of the cell cycle and continued proliferation reduced the self-renewal capacity and eventually resulted in the reduction of HSC pool. In this study, we found that Foxp2 – a forkhead transcription factor which tightly associated with the speech and language development – specifically expressed in the quiescent long-term (LT)-HSCs. It is reported that Foxp2 is the responsible gene of the inherited speech and language disorder while the role of Foxp2 in the regulation of bone marrow (BM) HSCs is poorly understood. We then examined the function of Foxp2 in the maintenance of HSCs. First, we analyzed the function of Foxp2 in the maintenance of colony formation capacity of HSCs. Knockdown of Foxp2 in LT-HSCs decreased the number of colony forming unit in culture (CFU-C) while overexpression of Foxp2 increased CFU-C formation. Notably, overexpression of Foxp2 in HSCs increased the number of immature colonies (CFU-GEMM) compared with the control. We next examined the effect of the overexpression and knockdown of Foxp2 in LT-HSCs in the long-term reconstitution (LTR) ability of HSCs. We found that the knockdown of Foxp2 reduced the engraftment of donor HSCs in peripheral blood and reduced the number of quiescent HSCs. Conversely, the overexpression of Foxp2 maintained LTR ability of donor HSCs. To clarify the molecular mechanism of Foxp2 in the maintenance of HSC function, we analyzed the effect of exogenous Foxp2 on the expression of cell cycle-related genes and found that Foxp2 overexpression upregulated the expression of Cdkn1a (p21) in HSCs. These data suggest that Foxp2 contributes the maintenance of the quiescent state of HSCs through the induction of p21.
Erythroid precursors and pro-erythroblasts are among the highest proliferative cells in the bone marrow. In the erythroid lineage, LMO2 protein levels are highest in pro-erythroblasts and progressively decrease with maturation through four stages of differentiation to orthochromatic erythroid cells. Interestingly, this correlates with decreased proportion of cells in S/G2/M from 52% to 25%. Moreover, decreasing LMO2 levels in pro-erythroblasts was sufficient to lower the proportion of cycling cells to 25%, indicating that LMO2 levels control their high cycling status. LMO2, a LIM only domain protein, does not bind DNA but interacts with the SCL and GATA1 transcription factors to drive erythroid gene expression program. The proteomics of the SCL-LMO2 pentameric complex formed on DNA indicate that LMO2 is stoichiometric with SCL tethered on erythroid gene promoters. Nonetheless, 25% of the nuclear pool of LMO2 was not associated with SCL, indicating that LMO2 has SCL-independent functions. Through a proteome-wide screen for novel LMO2 interaction partners in Kit+Lin- hematopoietic progenitors, we uncovered un unexpected role for LMO2 in directly controlling DNA replication, via interaction with three essential replication enzymes: POLD1, PRIM1 and MCM6. In sharp contrast, SCL does not interact with these enzymes. Moreover, tethering LMO2 to DNA is sufficient to recruit these replication enzymes to DNA and converts LMO2-bound sequences into origins of replication. Interestingly, ectopic LMO2 expression interfered with the production of TER119+ cells in the bone marrow. In contrast, decreasing LMO2 protein levels, either via short hairpin RNA or ectopic expression of miR-223, was sufficient to decrease the proliferative status of proerythroblasts and favor their differentiation. We conclude that LMO2 controls cell fate in the erythroid
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
lineage via two mechanisms. First, LMO2 controls the expression of erythroid genes through a well characterized interaction with the SCL complex. Second, LMO2 interacts with the DNA replication complex that drives entry into S phase in erythroid precursors and prevents commitment to terminal erythroid differentiation.
S99
3153 - METABOLIC PROFILE OF LEUKEMIC CELLS INFLUENCES RESPONSE TO CURRENT THERAPY Katerina Hlozkova1, Alena Pecinova2, David Pajuelo Reguera2, Marketa Simcikova1, Marketa Zaliova1, Natividad Alquezar Artieda1, Alena Musilova1, Tomas Mracek2, Jan Trka1, and Julia Starkova1 1 CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, Second Faculty of Medicine, Charles University, Prague, Czech Republic; 2 Institute of Physiology, the Czech Academy of Sciences, Prague, Czech Republic
L-asparaginase (ASNase) is one of the crucial components of acute lymphoblastic leukemia (ALL) therapy. We have previously shown that ASNase triggers metabolic reprogramming of leukemic cells. Nevertheless, this relation is reciprocal since basal cell metabolic state can dramatically influence response to ASNase treatment. In this context we decided to pursue relation between sensitivity to ASNase and metabolic profile of leukemic cells. Glycolysis and oxidative phosphorylation (OXPHOS) activity were measured on Seahorse Analyser XF24. Metabolic rates and sensitivity to drugs (ASNase, vincristine (VCR) and daunorubicin (DNR)) of 19 leukemic cell lines (B-ALL, T-ALL, AML and CML) were determined. Hierarchical cluster analysis revealed that glycolysis and OXPHOS clustered leukemic cells separately according to their lineage origin. Myeloid leukemia and T-ALL cell lines clustered together, whilst B-ALL cell lines gathered into the second cluster. Next, we identified metabolic processes which correlated with sensitivity to tested cytostatics. The results showed that higher sensitivity to ASNase present in ALL correlated the most significantly with lower basal respiration, higher oxidative metabolism and higher ATP synthase activity (p50.008). To functionally test the selected results, we treated leukemic cell lines with Oligomycin A (Oligo), the inhibitor of ATP synthase. Oligo treatment decreased sensitivity to ASNase compared to cells treated with ASNase alone. On the other hand, Oligo treatment did not disturb cytostatic effect of VCR and DNR. We discovered that leukemia cells present very distinct metabolic profile depending on their lineage origin which can be reflected in their response to the therapy. We assume that different sensitivity to ASNase of B-ALL and T-ALL often observed in patients could be explained by different metabolic predisposition. Moreover, data also indicate that higher ATP synthase activity correlated with higher sensitivity to ASNase administration. Applicability of VCR and DNR in both lymphoid and myeloid leukemias is possible due to the mechanism of action independent of bioenergetic processes. Supported by 15-28848A.
3152 - DECLINED PRESENTATION AND PUBLICATION
3154 - CHARACTERIZATION OF HSC IN BMI1-OVEREXPRESSING MICE Isabel Hidalgo-Gavilan1 and David Bryder2 1
Lund University, Lund, Sweden; 2Stem Cell Center, Lund University, Lund, Sweden
Bmi1 has been reported as a master regulator of several types of stem cells, including hematopoietic stem cells (HSCs). In addition, Bmi1 has been found overexpressed in several hematologic tumours such as lymphomas, leukaemia or myelodysplastic syndromes, where its high expression also confers worse prognosis, although the causality of these observations is to a large extent lacking. To date, most studies exploring the normal role of Bmi-1 have utilized loss-of-function models. Here, we developed a novel and inducible gain-of-function Bmi1 model. While Bmi1 overexpression alone does not appear to cause any hematopoietic abnormalities, long-term analysis of serially transplanted HSCs suggests intrinsic effects of prolonged Bmi-1 expression on hematopoietic output. Continuous work aims to unravel the effect of Bmi1 overexpressing HSC in the settings of additional/secondary stressors.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
upregulated in CD9+ HSCs upon Thpo signaling. Thpo stimulated CD9+ HSCs exhibited increase of phosphorylated-STAT3 (S727) which is known to translocate to and activate mitochondria metabolism. Our data indicate that Thpo-mediated exit from cell cycle quiescence is coupled with emergence of Mk-biased HSCs. While facilitating cell cycle proliferation, Thpo signaling metabolically fortifies Mk-biased HSCs through mitochondrial activation, enabling them fit for Mk production. This suggests clonal expansion of HSCs which are metabolically adjusted for Mk-differentiation during stress hematopoietic states.
3150 - FUBP1 PROMOTES LEUKEMIA PROGRESSION BY REGULATION OF CELL CYCLE AND APOPTOSIS Van T. Hoang1, Katharina Gerlach1, Uta Kuller-M€uller2, Eva Weissenberger1, Daniela Krause1, and Martin Z€ornig1 1 Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt, Germany; 2German Red Cross Blood Donor Service Baden-Wuerttemberg – Hessen, Frankfurt, Germany
We have shown the important role of FUBP1 (Far Upstream Element Binding Protein 1) in the maintenance of hematopoietic stem cells (HSCs) (Rabenhorst, Thalheimer et al. 2015). Fubp1-/- gene trap mice died in utero at day E15.5 with reduced numbers of total fetal liver cells, and their HSCs failed to engraft long-term. In adult mice, FUBP1 is also required for HSC self-renewal, and it promotes cell proliferation and inhibits apoptosis through regulation of target gene expression (e.g. c-myc, cyclin D2, p21, p27). We then decided to investigate a potential function of the molecule in chronic (CML) and acute myeloid leukemia (AML). Eppert et al. demonstrated an increased expression of FUBP1 in CD34+ CD38- cells compared to total bone marrow (BM) derived from human primary AML in their microarray analysis (Eppert, Takenaka et al. 2011). To study its role in leukemia development and leukemia initiating cells, and to test a potential treatment of leukemia with FUBP1 inhibitors, we employed the retroviral transduction/transplantation mouse model of CML and AML induced by BCR-ABL1 and MLL-AF9, respectively. Mice that received BM transduced with the oncogene plus Fubp1 shRNA survived longer than the control mice. BCR-ABL+ Fubp1 shRNA+ CML cell populations including total BM, Lin- and LSK cells showed more apoptosis and less proliferation. Furthermore, pharmacological inhibition of FUBP1 improved the survival of AML mice significantly, even to a larger extent than Ara-C treatment. Next, we analyzed FUBP1 expression in biopsies derived from CML (n511) and AML patients (n572) by immunohistochemistry and compared with healthy BM (n58). Of note, no enhanced FUBP1 expression was observed in leukemic samples. However, we noticed a shorter overall survival in AML patients with strong FUBP1 expression compared to those with no or moderate expression, suggesting that FUBP1 levels might correlate with the aggressiveness of leukemia. In summary, our data suggest that FUBP1 acts as a cell cycle regulator and anti-apoptotic factor in leukemia, similar to its oncogenic role in solid tumors, and the protein can be a potential target for therapeutic purpose.
3149 - FOXP2 IS ESSENTIAL FOR THE QUIESCENT STATE AND SELF-RENEWAL CAPACITY OF HEMATOPOIETIC STEM CELLS Kentaro Hosokawa, Saki Morimoto, Yuya Kunisaki, Tomoko Hyoda, Yasufumi Uehara, Haruka Imanishi, and Fumio Arai
3151 - THE DUAL FUNCTION OF LMO2 IN DRIVING ERYTHROID CELL FATE Trang Hoang1,2, Marie-Claude Sincennes3, Veronique Lisi4, Diogo Veiga5, Magali Humbert6, Francois Major7, Bachir Affar8, and Alain Verreault7
Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
1 Institute for Research in Immunology and Cancer, Montreal, Canada; 2Montreal, Canada; 3University of Ottawa, Ottawa, Canada; 4University of California, Santa Barbara, Santa Barbara, United States; 5Jackson Genomics Institute, Farmington, United States; 6Bern University, Bern, Switzerland; 7IRIC, University of Montreal, Montreal, Canada; 8University of Montreal, Montreal, Canada
Cell cycle quiescence is crucial for maintenance of the hematopoietic stem cells (HSCs) activity. Activation of the cell cycle and continued proliferation reduced the self-renewal capacity and eventually resulted in the reduction of HSC pool. In this study, we found that Foxp2 – a forkhead transcription factor which tightly associated with the speech and language development – specifically expressed in the quiescent long-term (LT)-HSCs. It is reported that Foxp2 is the responsible gene of the inherited speech and language disorder while the role of Foxp2 in the regulation of bone marrow (BM) HSCs is poorly understood. We then examined the function of Foxp2 in the maintenance of HSCs. First, we analyzed the function of Foxp2 in the maintenance of colony formation capacity of HSCs. Knockdown of Foxp2 in LT-HSCs decreased the number of colony forming unit in culture (CFU-C) while overexpression of Foxp2 increased CFU-C formation. Notably, overexpression of Foxp2 in HSCs increased the number of immature colonies (CFU-GEMM) compared with the control. We next examined the effect of the overexpression and knockdown of Foxp2 in LT-HSCs in the long-term reconstitution (LTR) ability of HSCs. We found that the knockdown of Foxp2 reduced the engraftment of donor HSCs in peripheral blood and reduced the number of quiescent HSCs. Conversely, the overexpression of Foxp2 maintained LTR ability of donor HSCs. To clarify the molecular mechanism of Foxp2 in the maintenance of HSC function, we analyzed the effect of exogenous Foxp2 on the expression of cell cycle-related genes and found that Foxp2 overexpression upregulated the expression of Cdkn1a (p21) in HSCs. These data suggest that Foxp2 contributes the maintenance of the quiescent state of HSCs through the induction of p21.
Erythroid precursors and pro-erythroblasts are among the highest proliferative cells in the bone marrow. In the erythroid lineage, LMO2 protein levels are highest in pro-erythroblasts and progressively decrease with maturation through four stages of differentiation to orthochromatic erythroid cells. Interestingly, this correlates with decreased proportion of cells in S/G2/M from 52% to 25%. Moreover, decreasing LMO2 levels in pro-erythroblasts was sufficient to lower the proportion of cycling cells to 25%, indicating that LMO2 levels control their high cycling status. LMO2, a LIM only domain protein, does not bind DNA but interacts with the SCL and GATA1 transcription factors to drive erythroid gene expression program. The proteomics of the SCL-LMO2 pentameric complex formed on DNA indicate that LMO2 is stoichiometric with SCL tethered on erythroid gene promoters. Nonetheless, 25% of the nuclear pool of LMO2 was not associated with SCL, indicating that LMO2 has SCL-independent functions. Through a proteome-wide screen for novel LMO2 interaction partners in Kit+Lin- hematopoietic progenitors, we uncovered un unexpected role for LMO2 in directly controlling DNA replication, via interaction with three essential replication enzymes: POLD1, PRIM1 and MCM6. In sharp contrast, SCL does not interact with these enzymes. Moreover, tethering LMO2 to DNA is sufficient to recruit these replication enzymes to DNA and converts LMO2-bound sequences into origins of replication. Interestingly, ectopic LMO2 expression interfered with the production of TER119+ cells in the bone marrow. In contrast, decreasing LMO2 protein levels, either via short hairpin RNA or ectopic expression of miR-223, was sufficient to decrease the proliferative status of proerythroblasts and favor their differentiation. We conclude that LMO2 controls cell fate in the erythroid
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
lineage via two mechanisms. First, LMO2 controls the expression of erythroid genes through a well characterized interaction with the SCL complex. Second, LMO2 interacts with the DNA replication complex that drives entry into S phase in erythroid precursors and prevents commitment to terminal erythroid differentiation.
S99
3153 - METABOLIC PROFILE OF LEUKEMIC CELLS INFLUENCES RESPONSE TO CURRENT THERAPY Katerina Hlozkova1, Alena Pecinova2, David Pajuelo Reguera2, Marketa Simcikova1, Marketa Zaliova1, Natividad Alquezar Artieda1, Alena Musilova1, Tomas Mracek2, Jan Trka1, and Julia Starkova1 1 CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, Second Faculty of Medicine, Charles University, Prague, Czech Republic; 2 Institute of Physiology, the Czech Academy of Sciences, Prague, Czech Republic
L-asparaginase (ASNase) is one of the crucial components of acute lymphoblastic leukemia (ALL) therapy. We have previously shown that ASNase triggers metabolic reprogramming of leukemic cells. Nevertheless, this relation is reciprocal since basal cell metabolic state can dramatically influence response to ASNase treatment. In this context we decided to pursue relation between sensitivity to ASNase and metabolic profile of leukemic cells. Glycolysis and oxidative phosphorylation (OXPHOS) activity were measured on Seahorse Analyser XF24. Metabolic rates and sensitivity to drugs (ASNase, vincristine (VCR) and daunorubicin (DNR)) of 19 leukemic cell lines (B-ALL, T-ALL, AML and CML) were determined. Hierarchical cluster analysis revealed that glycolysis and OXPHOS clustered leukemic cells separately according to their lineage origin. Myeloid leukemia and T-ALL cell lines clustered together, whilst B-ALL cell lines gathered into the second cluster. Next, we identified metabolic processes which correlated with sensitivity to tested cytostatics. The results showed that higher sensitivity to ASNase present in ALL correlated the most significantly with lower basal respiration, higher oxidative metabolism and higher ATP synthase activity (p50.008). To functionally test the selected results, we treated leukemic cell lines with Oligomycin A (Oligo), the inhibitor of ATP synthase. Oligo treatment decreased sensitivity to ASNase compared to cells treated with ASNase alone. On the other hand, Oligo treatment did not disturb cytostatic effect of VCR and DNR. We discovered that leukemia cells present very distinct metabolic profile depending on their lineage origin which can be reflected in their response to the therapy. We assume that different sensitivity to ASNase of B-ALL and T-ALL often observed in patients could be explained by different metabolic predisposition. Moreover, data also indicate that higher ATP synthase activity correlated with higher sensitivity to ASNase administration. Applicability of VCR and DNR in both lymphoid and myeloid leukemias is possible due to the mechanism of action independent of bioenergetic processes. Supported by 15-28848A.
3152 - DECLINED PRESENTATION AND PUBLICATION
3154 - CHARACTERIZATION OF HSC IN BMI1-OVEREXPRESSING MICE Isabel Hidalgo-Gavilan1 and David Bryder2 1
Lund University, Lund, Sweden; 2Stem Cell Center, Lund University, Lund, Sweden
Bmi1 has been reported as a master regulator of several types of stem cells, including hematopoietic stem cells (HSCs). In addition, Bmi1 has been found overexpressed in several hematologic tumours such as lymphomas, leukaemia or myelodysplastic syndromes, where its high expression also confers worse prognosis, although the causality of these observations is to a large extent lacking. To date, most studies exploring the normal role of Bmi-1 have utilized loss-of-function models. Here, we developed a novel and inducible gain-of-function Bmi1 model. While Bmi1 overexpression alone does not appear to cause any hematopoietic abnormalities, long-term analysis of serially transplanted HSCs suggests intrinsic effects of prolonged Bmi-1 expression on hematopoietic output. Continuous work aims to unravel the effect of Bmi1 overexpressing HSC in the settings of additional/secondary stressors.