- Email: [email protected]
P57KIP2 in developmental haematopoiesis
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3080 - THE SIGNALING BALANCE BETWEEN LYSOPHOSPHATIDIC ACID RECEPTOR 1 AND 3 IN BONE MARROW-DERIVED MESENCHYMAL STEM CELLS DETERMINES PROGRESSION OF MULTIPLE MYELOMA Masahiko Kanehira, Tohru Fujiwara, Shinji Nakajima, Yoko Okitsu, Yasushi Onishi, Noriko Fukuhara, Ryo Ichinohasama, and Hideo Harigae Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan Multiple myeloma (MM) is one of the hematologic malignancies characterized by accumulation of neoplastic plasma cells. Although novel therapeutic agents have significantly improved the survival of MM patients, MM is still a mostly incurable disease. The most common reason of relapsed and refractory MM is that myeloma cells intimately communicate with bone marrow stroma, which is known to secrete soluble factors advantageous to proliferation and drug resistance of myeloma cells. Therefore, novel approaches for understanding the crosstalk between myeloma cells and stroma are urgently needed for MM therapy. Recently, there are accumulating evidences that bone marrow-derived mesenchymal stem cells (MSC) act as stroma and support growth and drug resistance of myeloma cells. Interestingly, we observed that lysophosphatidic acid (LPA), a bioactive lipid mediator, stimulates MSC to produce IL-6, known as an important cytokine for MM progression. Thus, we investigated whether inhibition of LPA signaling in MSC interferes with communication between myeloma cells and MSC. MSC up-regulated production of autotoxin, one of enzymes for LPA biosynthesis, under conditions cultured with myeloma cells-derived conditioned medium. To date, six receptors have been reported to recognize LPA (LPA1-6). Inhibition of LPA1 signaling promotes proliferation of MSC, whereas inhibition of LPA3 signaling induces senescence of MSC followed by transdifferentiation toward cancer-associated fibroblasts. In murine xenograft model, myeloma cells coinjected with LPA1-silenced MSC had less tumorigenicity compared to myeloma cells coinjected with control MSC. Whereas, myeloma cells coinjected with LPA3-silenced MSC formed larger subcutaneous tumor masses with an increase of microvessels compared to myeloma cells coinjected with control MSC. Taken together, these results identified the balance between LPA1- and LPA3signaling in MSC as an important determinant in MM progression, and implied the LPA and LPA receptor axis as a crucial regulator of MM-stroma interaction. 3081 - THE ROLE OF CDKN1C/P57KIP2 IN DEVELOPMENTAL HAEMATOPOIESIS Chrysa Kapeni1,2 and Katrin Ottersbach1 1 MRC Centre for Regenerative Medicine, Edinburgh, United Kingdom; 2CIMR, Cambridge, United Kingdom The first adult-repopulating haematopoietic stem cells (HSCs) emerge at day (E) 10.5 of gestation in the aorta-gonads-mesonephros (AGM) region of the mouse embryo. From E11, HSCs are also found in the foetal liver, the yolk sac and placenta. However, the HSC migration route between those tissues and the regulatory mechanisms that control HSC emergence and differentiation are still obscure. We have identified a cell cycle inhibitor, p57Kip2, to be upregulated in the AGM at the time of HSC emergence and have confirmed strong expression in the AGM, as well as the foetal liver and the placenta. To evaluate the effect of p57Kip2 on embryonic HSCs, we isolated embryonic haematopoietic tissues at E11 and E12, and assessed their reconstituting capacity in vivo by transplantations. Among the tissues analysed so far, p57Kip2 KO embryos showed increased HSC activity in the E12 AGM but not in the placenta, foetal liver or yolk sac. Interestingly, although no effect of p57Kip2 deletion was observed in the primary recipients, serial transplants revealed a self-renewal advantage for p57Kip2-deficient HSCs from all the tissues tested as far, confirming a functional role for p57Kip2 in developing blood cells. We are also trying to understand the mechanism by which p57Kip2 exerts its effect on HSCs. Thus far, we have detected p57Kip2 expression in sympathoadrenal progenitors surrounding the dorsal aorta at the time of HSC emergence with an increase in these cells in the absence of p57Kip2, indicating a link between p57Kip2 expression and catecholamines. Previous work from our lab has established that catecholamine secretion promotes HSC production, which suggests a possible indirect pathway for p57Kip2 to affect HSCs. We are currently performing molecular profiling assays to further characterise the cell populations, including sympathoadrenal cells that comprise the AGM microenvironment. Moreover, we are continuing to produce a detailed description of p57Kip2 expression in HSCs and their supportive cells throughout key developmental points for haematopoiesis.
S71
3082 - AUTOLOGOUS HAEMATOPOIETIC STEM CELL TRANSPLANTATION REQUIRES RECIPIENT BM MACROPHAGES Simranpreet Kaur1, Liza J. Raggatt1, Rebecca Jacobsen1, Susan Millard1, Valerie Barbier1, Bianca Nowlan1, Ingrid G. Winkler1, Kelli P. MacDonald2, Andrew C. Perkins1, David A. Hume3, Jean P. Levesque1, and Allison R. Pettit1 1 Mater Research Institute-Univeristy of Queensland, Brisbane, Queensland, Australia; 2Queensland Institute of Medical Research, Brisbane, Queensland, Australia; 3Roslin Institute-University of Edinburgh, Scotland, United Kingdom Bone marrow (BM) macrophages (Macs) are pivotal in maintaining haematopoietic stem cell (HSC) niches. To examine Mac contributions to BM recovery post-autologous transplantation (Tx), we mapped BM-Mac persistence and origin. Recipient Macgreen mice, that express GFP in myeloid cells, were myeloablated using lethal irradiation (11.5 Gy) and transplanted with B6.SJL BM. Flow cytometry analyses of BM 2-16 weeks (wk) post-Tx revealed that a small portion of GFP+F4/ 80+CD11b+Ly6Gneg recipient BM-Macs were present post-Tx and persisted throughout the time course. Monocytes and granulocytes were 100% donor-derived supporting efficient ablation of recipient HSC. These persisting BM-Macs co-expressed HSC-niche Mac markers CD169, VCAM-1 and ER-HR3. A 5.6 fold expansion of recipient BM-Macs occurred between wk 2 and 5 post-Tx and coincided with increased BM residence of long-term (LT) donor HSC. In situ, GFP+F480+ recipient BM-Macs localized to HSC niche-enriched perivascular microenvironments within both central and endosteal regions. These data suggest that persistence of recipient BM-Macs post-myeloablation is required to reform BM HSC niches and promote successful HSC engraftment after autologous Tx. We selectively depleted recipient BM-Macs using CD169-DTR mice transplanted with syngeneic ubiquitous GFP BM. Depletion of CD169+ recipient BM-Macs abated donor LT-HSC BM engraftment by 80% at 5 wk post-Tx. Additionally the haematopoietic repopulation potential of donor HSC from CD169 depleted recipients was compromised in competitive Tx assays. Overall, BM contains a myeloablation-resistant self-repopulating Mac subset whose 1) expansion in the early post-Tx recovery period correlates with increased BM occupancy of HSC and 2) presence is necessary for efficient and/or sustained HSC engraftment. Therapeutic targeting of these resilient self-repopulating BMMacs may accelerate stem cell engraftment and haematopoietic recovery post autologous BM Tx. 3083 - GATA2 REPORTER EXPRESSION REVEALS STAGES OF ESC HEMATOPOIETIC PROGENITOR GENERATION AND FACILITATES STUDY OF GPR56 FUNCTION M.-L. Kauts1,2, P. Kaimakis2, P. Solaimani2, R. Van der Linden2, and E. Dzierzak1,2 1 U of Edinburgh, Edinburgh, United Kingdom; 2Erasmus MC, Rotterdam, Netherlands In the mouse embryo the production of the hematopoietic system occurs in spatio-temporally distinct stages. Hematopoietic stem cells (HSCs) are generated during the final definitive stage. HSCs arise from hemogenic endothelium in a transdifferentiation process (endothelial-to-hematopoietic transition; EHT) which is tightly controlled by a combination of transcription and extrinsic factors. The Gata2 transcription factor has a pivotal role in this process. By differentiating novel Gata2-Venus embryonic stem cells (G2V ESC), which facilitate the prospective isolation of Gata2+ cells, we demonstrate here that Gata2 is a relevant reporter for the generation of hematopoietic progenitors in vitro. Our analyses of Gata2 temporal expression show that it correlates with the temporal onset of primitive and definitive hematopoietic stages and to the stages observed in vivo. Hence, the G2V ESC differentiation system serves as an excellent model for recapitulating hematopoietic development in vitro and with improvements may allow for generation of HSCs. A recent whole-transcriptome study of EHT revealed a novel HSC molecule: G-protein coupled receptor 56 (Gpr56) (Solaimani, 2014). Whether signaling via Gpr56 is required for HSC generation, and how it functions in EHT is unknown. To elucidate this we are using G2V reporter ESCs expressing constitutively active Gpr56. Progenitor and transplantation assays with these ESC-derived Gata2+ hematopoietic cells are in progress so as to determine the potential of Gpr56 to enhance hematopoiesis. In addition, hematopoietic differentiation using Gpr56 knock-down G2V ESCs will allow further investigation of a requirement for Gpr56 in HSC development. Our preliminary data reveal that Gpr56 is enriched in Gata2+ cells during ESC hematopoietic differentiation and that primitive and definitive programs are inhibited when the Rho-associated protein kinase pathway (activated by GPRs) is blocked. Our ongoing experiments with our unique G2V ESCs will elucidate whether induction of Gpr56 is necessary and sufficient for de novo generation of long-term repopulating HSCs.
3080 - THE SIGNALING BALANCE BETWEEN LYSOPHOSPHATIDIC ACID RECEPTOR 1 AND 3 IN BONE MARROW-DERIVED MESENCHYMAL STEM CELLS DETERMINES PROGRESSION OF MULTIPLE MYELOMA Masahiko Kanehira, Tohru Fujiwara, Shinji Nakajima, Yoko Okitsu, Yasushi Onishi, Noriko Fukuhara, Ryo Ichinohasama, and Hideo Harigae Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan Multiple myeloma (MM) is one of the hematologic malignancies characterized by accumulation of neoplastic plasma cells. Although novel therapeutic agents have significantly improved the survival of MM patients, MM is still a mostly incurable disease. The most common reason of relapsed and refractory MM is that myeloma cells intimately communicate with bone marrow stroma, which is known to secrete soluble factors advantageous to proliferation and drug resistance of myeloma cells. Therefore, novel approaches for understanding the crosstalk between myeloma cells and stroma are urgently needed for MM therapy. Recently, there are accumulating evidences that bone marrow-derived mesenchymal stem cells (MSC) act as stroma and support growth and drug resistance of myeloma cells. Interestingly, we observed that lysophosphatidic acid (LPA), a bioactive lipid mediator, stimulates MSC to produce IL-6, known as an important cytokine for MM progression. Thus, we investigated whether inhibition of LPA signaling in MSC interferes with communication between myeloma cells and MSC. MSC up-regulated production of autotoxin, one of enzymes for LPA biosynthesis, under conditions cultured with myeloma cells-derived conditioned medium. To date, six receptors have been reported to recognize LPA (LPA1-6). Inhibition of LPA1 signaling promotes proliferation of MSC, whereas inhibition of LPA3 signaling induces senescence of MSC followed by transdifferentiation toward cancer-associated fibroblasts. In murine xenograft model, myeloma cells coinjected with LPA1-silenced MSC had less tumorigenicity compared to myeloma cells coinjected with control MSC. Whereas, myeloma cells coinjected with LPA3-silenced MSC formed larger subcutaneous tumor masses with an increase of microvessels compared to myeloma cells coinjected with control MSC. Taken together, these results identified the balance between LPA1- and LPA3signaling in MSC as an important determinant in MM progression, and implied the LPA and LPA receptor axis as a crucial regulator of MM-stroma interaction. 3081 - THE ROLE OF CDKN1C/P57KIP2 IN DEVELOPMENTAL HAEMATOPOIESIS Chrysa Kapeni1,2 and Katrin Ottersbach1 1 MRC Centre for Regenerative Medicine, Edinburgh, United Kingdom; 2CIMR, Cambridge, United Kingdom The first adult-repopulating haematopoietic stem cells (HSCs) emerge at day (E) 10.5 of gestation in the aorta-gonads-mesonephros (AGM) region of the mouse embryo. From E11, HSCs are also found in the foetal liver, the yolk sac and placenta. However, the HSC migration route between those tissues and the regulatory mechanisms that control HSC emergence and differentiation are still obscure. We have identified a cell cycle inhibitor, p57Kip2, to be upregulated in the AGM at the time of HSC emergence and have confirmed strong expression in the AGM, as well as the foetal liver and the placenta. To evaluate the effect of p57Kip2 on embryonic HSCs, we isolated embryonic haematopoietic tissues at E11 and E12, and assessed their reconstituting capacity in vivo by transplantations. Among the tissues analysed so far, p57Kip2 KO embryos showed increased HSC activity in the E12 AGM but not in the placenta, foetal liver or yolk sac. Interestingly, although no effect of p57Kip2 deletion was observed in the primary recipients, serial transplants revealed a self-renewal advantage for p57Kip2-deficient HSCs from all the tissues tested as far, confirming a functional role for p57Kip2 in developing blood cells. We are also trying to understand the mechanism by which p57Kip2 exerts its effect on HSCs. Thus far, we have detected p57Kip2 expression in sympathoadrenal progenitors surrounding the dorsal aorta at the time of HSC emergence with an increase in these cells in the absence of p57Kip2, indicating a link between p57Kip2 expression and catecholamines. Previous work from our lab has established that catecholamine secretion promotes HSC production, which suggests a possible indirect pathway for p57Kip2 to affect HSCs. We are currently performing molecular profiling assays to further characterise the cell populations, including sympathoadrenal cells that comprise the AGM microenvironment. Moreover, we are continuing to produce a detailed description of p57Kip2 expression in HSCs and their supportive cells throughout key developmental points for haematopoiesis.
S71
3082 - AUTOLOGOUS HAEMATOPOIETIC STEM CELL TRANSPLANTATION REQUIRES RECIPIENT BM MACROPHAGES Simranpreet Kaur1, Liza J. Raggatt1, Rebecca Jacobsen1, Susan Millard1, Valerie Barbier1, Bianca Nowlan1, Ingrid G. Winkler1, Kelli P. MacDonald2, Andrew C. Perkins1, David A. Hume3, Jean P. Levesque1, and Allison R. Pettit1 1 Mater Research Institute-Univeristy of Queensland, Brisbane, Queensland, Australia; 2Queensland Institute of Medical Research, Brisbane, Queensland, Australia; 3Roslin Institute-University of Edinburgh, Scotland, United Kingdom Bone marrow (BM) macrophages (Macs) are pivotal in maintaining haematopoietic stem cell (HSC) niches. To examine Mac contributions to BM recovery post-autologous transplantation (Tx), we mapped BM-Mac persistence and origin. Recipient Macgreen mice, that express GFP in myeloid cells, were myeloablated using lethal irradiation (11.5 Gy) and transplanted with B6.SJL BM. Flow cytometry analyses of BM 2-16 weeks (wk) post-Tx revealed that a small portion of GFP+F4/ 80+CD11b+Ly6Gneg recipient BM-Macs were present post-Tx and persisted throughout the time course. Monocytes and granulocytes were 100% donor-derived supporting efficient ablation of recipient HSC. These persisting BM-Macs co-expressed HSC-niche Mac markers CD169, VCAM-1 and ER-HR3. A 5.6 fold expansion of recipient BM-Macs occurred between wk 2 and 5 post-Tx and coincided with increased BM residence of long-term (LT) donor HSC. In situ, GFP+F480+ recipient BM-Macs localized to HSC niche-enriched perivascular microenvironments within both central and endosteal regions. These data suggest that persistence of recipient BM-Macs post-myeloablation is required to reform BM HSC niches and promote successful HSC engraftment after autologous Tx. We selectively depleted recipient BM-Macs using CD169-DTR mice transplanted with syngeneic ubiquitous GFP BM. Depletion of CD169+ recipient BM-Macs abated donor LT-HSC BM engraftment by 80% at 5 wk post-Tx. Additionally the haematopoietic repopulation potential of donor HSC from CD169 depleted recipients was compromised in competitive Tx assays. Overall, BM contains a myeloablation-resistant self-repopulating Mac subset whose 1) expansion in the early post-Tx recovery period correlates with increased BM occupancy of HSC and 2) presence is necessary for efficient and/or sustained HSC engraftment. Therapeutic targeting of these resilient self-repopulating BMMacs may accelerate stem cell engraftment and haematopoietic recovery post autologous BM Tx. 3083 - GATA2 REPORTER EXPRESSION REVEALS STAGES OF ESC HEMATOPOIETIC PROGENITOR GENERATION AND FACILITATES STUDY OF GPR56 FUNCTION M.-L. Kauts1,2, P. Kaimakis2, P. Solaimani2, R. Van der Linden2, and E. Dzierzak1,2 1 U of Edinburgh, Edinburgh, United Kingdom; 2Erasmus MC, Rotterdam, Netherlands In the mouse embryo the production of the hematopoietic system occurs in spatio-temporally distinct stages. Hematopoietic stem cells (HSCs) are generated during the final definitive stage. HSCs arise from hemogenic endothelium in a transdifferentiation process (endothelial-to-hematopoietic transition; EHT) which is tightly controlled by a combination of transcription and extrinsic factors. The Gata2 transcription factor has a pivotal role in this process. By differentiating novel Gata2-Venus embryonic stem cells (G2V ESC), which facilitate the prospective isolation of Gata2+ cells, we demonstrate here that Gata2 is a relevant reporter for the generation of hematopoietic progenitors in vitro. Our analyses of Gata2 temporal expression show that it correlates with the temporal onset of primitive and definitive hematopoietic stages and to the stages observed in vivo. Hence, the G2V ESC differentiation system serves as an excellent model for recapitulating hematopoietic development in vitro and with improvements may allow for generation of HSCs. A recent whole-transcriptome study of EHT revealed a novel HSC molecule: G-protein coupled receptor 56 (Gpr56) (Solaimani, 2014). Whether signaling via Gpr56 is required for HSC generation, and how it functions in EHT is unknown. To elucidate this we are using G2V reporter ESCs expressing constitutively active Gpr56. Progenitor and transplantation assays with these ESC-derived Gata2+ hematopoietic cells are in progress so as to determine the potential of Gpr56 to enhance hematopoiesis. In addition, hematopoietic differentiation using Gpr56 knock-down G2V ESCs will allow further investigation of a requirement for Gpr56 in HSC development. Our preliminary data reveal that Gpr56 is enriched in Gata2+ cells during ESC hematopoietic differentiation and that primitive and definitive programs are inhibited when the Rho-associated protein kinase pathway (activated by GPRs) is blocked. Our ongoing experiments with our unique G2V ESCs will elucidate whether induction of Gpr56 is necessary and sufficient for de novo generation of long-term repopulating HSCs.