- Email: [email protected]
2012 - FIBRE PREVENTS HIGH SALT DIET-INDUCED GUT PERMEABILITY, HAEMATOPOIETIC STEM CELL MOBILISATION AND ATHEROSCLEROSIS
Short Talk Presentations / Experimental Hematology 2019;76 (Suppl): S42−S50 2010 - IDENTIFYING NECESSARY CHROMATIN STATES AND TARGETS FOR HEMOGENIC SPECIFICATION AND REPROGRAMMING Elizabeth Howell1,2, Bing He3, Melike Lakadamyali1, Nancy Speck1, Kai Tan3, Peng Gao3, Amanda Yzaguirre4 1 University of Pennsylvania, Phildelphia, United States; 2Department of Cell and Developmental Biology, Perelman School of Medicine, Phildelphia, United States; 3Children’s Hospital of Philadelphia, Philadelphia, United States; 4Salk Institute, San Diego, United States Hematopoietic stem cells (HSCs) originate from a subset of endothelium in the embryo known as hemogenic endothelium. Hemogenic endothelium must undergo an endothelial to hematopoietic transition (EHT) to form HSCs. EHT requires the activity of the transcription factor (TF) RUNX1. Ectopic pan-endothelial expression of RUNX1 in mouse embryos is able to specify embryonic endothelium as hemogenic. However, as compared to embryonic endothelium, RUNX1 is not as effective at specifying fetal endothelium as hemogenic. To understand the difference in the competency of embryonic versus fetal endothelium to be specified as hemogenic, we examined differences in chromatin at these two developmental stages. ATAC-seq revealed that RUNX1 appears to be specifying embryonic endothelium through a canonical EHT program. To orchestrate this EHT transcriptional program, RUNX1 participates with other hematopoietic TFs, such as FLI1, ETS1, SPI1, and GFI1. Following RUNX1 induction in embryonic endothelium, immune genes and motility related genes are upregulated. In fetal endothelium, however, RUNX1 induction results in a more complex picture. To determine if these differences were due to underlying chromatin differences between fetal and embryonic endothelium, we examined control embryonic and fetal endothelium using stochastic optical reconstruction microscopy (STORM). STORM imaging suggests that there are global differences in chromatin structure between fetal and embryonic endothelium. Fetal endothelium appears to have more densely packed chromatin that is located more peripherally than embryonic endothelium. Taken together, these data show that the reduced specification efficiency by RUNX1 observed between embryonic and fetal endothelium is due, at least in part, to chromatin structure.
2011 - ENGINEERING A HEMATOPOIETIC STEM CELL NICHE BY REVITALIZING MESENCHYMAL STEM CELLS Fumio Nakahara1, Daniel Borger2, Qiaozhi Wei2, Sandra Pinho2, Maria Maryanovich2 , Ali Zahalka2, Masako Suzuki3 , Cristian Cruz2, Zichen Wang4, Chunliang Xu2 , Philip Boulais2, Avi Ma’ayan4 , John Greally3, Paul Frenette2 1 Department of Hematology and Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan, Bunkyo-ku, Japan; 2Department of Cell Biology, Albert Einstein College of Medicine, Bronx, United States; 3Center for Epigenomics, Department of Genetics, Albert Einstein College of Medicine, Bronx, United States; 4 Department of Pharmacological Sciences, Mount Sinai Center for Bioinformatics, Icahn School of Medicine at Mount Sinai, New York, United States Hematopoietic stem cells (HSCs) are maintained by bone marrow (BM) niches in vivo, but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors by Nestin-GFP+ mesenchymal-derived stem cells (MSCs) is downregulated upon culture, suggesting that transcriptional rewiring may contribute to this reduced HSC maintenance potential. Using an RNA sequencing screen, we identified 5 transcription factors (Klf7, Ostf1, Xbp1, Irf3, Irf7) that restored HSC niche function in cultured BM-derived MSCs. These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs showed higher repopulation capacity and protected lethally irradiated recipient mice. Competitive reconstitution assays revealed 7-fold expansion of functional HSCs by rMSCs. rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress. We have also identified Mef2c as an important transcription factor downstream of KOXII. Our results suggest that all five KOXII genes are necessary to fully restore the niche activity in MSCs ex vivo. Some of these genes may act singly, perhaps by preventing MSC differentiation or driving expansion of hematopoietic growth factors. Our results thus establish a new platform that provides critical insight in the regulatory network of the HSC niche and forms the basis toward the engineering of supportive niches for curative cell therapies.
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2012 - FIBRE PREVENTS HIGH SALT DIET-INDUCED GUT PERMEABILITY, HAEMATOPOIETIC STEM CELL MOBILISATION AND ATHEROSCLEROSIS Man Lee, Olivia Cooney, Annas Al-sharea, Michelle Flynn, Gerard Pernes, Graeme Lancaster, Andrew Murphy Baker Heart & Diabetes Institute, Melbourne, Australia A high salt diet (HSD) is a key risk factor for atherosclerotic-cardiovascular disease (CVD). We show that a HSD (3.6% NaCl) promotes the maturation of pathogenic TH17 cells in the bone marrow (BM) and promotes the breakdown of the BM niche, by reducing leptin receptor mesenchymal cells (LepR +MSCs) and increasing the number of osteoclasts in atherosclerotic-prone Apoe-/- mice. This led to the egress of haematopoietic stem and progenitor cells (HSPCs) into the spleen, inducing monocytosis via extramedullary myelopoiesis which plays a causal role in atherosclerosis. Administering antiIL17A antibody and zoledronic acid restored LepR+MSCs and osteoclast levels in the BM back to control (NSD; 1% NaCl) while preventing HSPC egress and atherogenesis. Recently, a HSD has been found to alter the microbiome, promoting intestinal permeability and TH17 maturation. Thus, we supplemented HSD-mice with fibre (400mg/kg), to restore the microbiome and prevent gut permeability. Fibre prevented HSD-induced intestinal permeability and TH17 maturation. Fibre also reduced BM TH17 cells and restored the levels of HSD-induced LepR+MSCs and osteoclasts back to control levels, preventing HSPC egress to the spleen. This reduced the number of inflammatory monocytes entering into the atherosclerotic lesion, inhibiting atherogenesis. These findings demonstrate that fibre supplementation can prevent HSDdriven inflammatory effects in the gut and the breakdown of the BM niche, to prevent atherogenesis.
2014 - DYNAMIN-DEPENDENT ENDOCYTOSIS IS REQUIRED FOR MULTIPLE NICHE SIGNALS THAT ARE CRUCIAL FOR LEUKEMIA STEM CELL ACTIVITY AND CHEMORESISTANCE Cedric Tremblay1, sung chiu1, Jesslyn Saw1, Stefan Sonderegger1, Ngoc Chau2, Adam McCluskey3, Phillip Robinson4, Stephen Jane5, David Curtis1 1 Australian Centre for Blood Diseases (ACBD) - Monash University, Melbourne, Australia; 2Children’s Medical Reserch Institute, Sydney, Australia; 3University of Newcastle, Newcastle, Australia; 4 Children’s Medical Research Institute, Sydney, Australia; 5Clinical Haematology, Alfred Hospital, Prahran, Australia The hierarchical model posits that acute leukemias arise from leukemia stem cells (LSCs), which display stem cell-like properties like long-term self-renewal and differentiation to generate the heterogeneity observed in the tumor at diagnosis. Elimination of LSCs and their ancestral clones, pre-leukemic stem cells (pre-LSCs), is critical for longterm cure as they are the source of relapse. How these cells survive high-dose therapy remains poorly defined, but may include quiescence and pro-survival signals provided by the microenvironment. Strategies to interfere with specific signals from the niche have shown promising results, but may be subject to compensatory mechanisms due to the inherent plasticity of LSCs. The transactivation of signalling pathways downstream of growth factors from the niche is dependent on receptor-mediated endocytosis, a generic process dependent on Dynamins. Given the important role of the microenvironment for relapse-inducing cells, we postulated that inhibition of Dynamin might impair their properties and sensitize them to chemotherapy. Here, we show that the Dynamin small molecule inhibitor Dynole 34-2 blocks multiple signalling pathways in pre-LSCs and LSCs by preventing endocytosis. Using the Lmo2-transgenic mouse model of T-cell acute lymphoblastic leukemia (T-ALL), we found that Dynole 34-2 impairs self-renewal of pre-LSCs and sensitizes relapse-inducing cells to chemotherapy. We also found that Dynole 34-2 delayed leukemia onset in mice treated with high-dose therapy. Treatment of different patient-derived xenografts revealed that that inhibition of Dynamin activity with Dynole 34-2 represents an effective therapeutic strategy for different subtypes of human T-ALL. In summary, our finding that Dynamin activity is essential for LSC activity and chemoresistance in acute leukemia paves the way for novel therapeutic strategies.
2011 - ENGINEERING A HEMATOPOIETIC STEM CELL NICHE BY REVITALIZING MESENCHYMAL STEM CELLS Fumio Nakahara1, Daniel Borger2, Qiaozhi Wei2, Sandra Pinho2, Maria Maryanovich2 , Ali Zahalka2, Masako Suzuki3 , Cristian Cruz2, Zichen Wang4, Chunliang Xu2 , Philip Boulais2, Avi Ma’ayan4 , John Greally3, Paul Frenette2 1 Department of Hematology and Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan, Bunkyo-ku, Japan; 2Department of Cell Biology, Albert Einstein College of Medicine, Bronx, United States; 3Center for Epigenomics, Department of Genetics, Albert Einstein College of Medicine, Bronx, United States; 4 Department of Pharmacological Sciences, Mount Sinai Center for Bioinformatics, Icahn School of Medicine at Mount Sinai, New York, United States Hematopoietic stem cells (HSCs) are maintained by bone marrow (BM) niches in vivo, but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors by Nestin-GFP+ mesenchymal-derived stem cells (MSCs) is downregulated upon culture, suggesting that transcriptional rewiring may contribute to this reduced HSC maintenance potential. Using an RNA sequencing screen, we identified 5 transcription factors (Klf7, Ostf1, Xbp1, Irf3, Irf7) that restored HSC niche function in cultured BM-derived MSCs. These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs showed higher repopulation capacity and protected lethally irradiated recipient mice. Competitive reconstitution assays revealed 7-fold expansion of functional HSCs by rMSCs. rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress. We have also identified Mef2c as an important transcription factor downstream of KOXII. Our results suggest that all five KOXII genes are necessary to fully restore the niche activity in MSCs ex vivo. Some of these genes may act singly, perhaps by preventing MSC differentiation or driving expansion of hematopoietic growth factors. Our results thus establish a new platform that provides critical insight in the regulatory network of the HSC niche and forms the basis toward the engineering of supportive niches for curative cell therapies.
S45
2012 - FIBRE PREVENTS HIGH SALT DIET-INDUCED GUT PERMEABILITY, HAEMATOPOIETIC STEM CELL MOBILISATION AND ATHEROSCLEROSIS Man Lee, Olivia Cooney, Annas Al-sharea, Michelle Flynn, Gerard Pernes, Graeme Lancaster, Andrew Murphy Baker Heart & Diabetes Institute, Melbourne, Australia A high salt diet (HSD) is a key risk factor for atherosclerotic-cardiovascular disease (CVD). We show that a HSD (3.6% NaCl) promotes the maturation of pathogenic TH17 cells in the bone marrow (BM) and promotes the breakdown of the BM niche, by reducing leptin receptor mesenchymal cells (LepR +MSCs) and increasing the number of osteoclasts in atherosclerotic-prone Apoe-/- mice. This led to the egress of haematopoietic stem and progenitor cells (HSPCs) into the spleen, inducing monocytosis via extramedullary myelopoiesis which plays a causal role in atherosclerosis. Administering antiIL17A antibody and zoledronic acid restored LepR+MSCs and osteoclast levels in the BM back to control (NSD; 1% NaCl) while preventing HSPC egress and atherogenesis. Recently, a HSD has been found to alter the microbiome, promoting intestinal permeability and TH17 maturation. Thus, we supplemented HSD-mice with fibre (400mg/kg), to restore the microbiome and prevent gut permeability. Fibre prevented HSD-induced intestinal permeability and TH17 maturation. Fibre also reduced BM TH17 cells and restored the levels of HSD-induced LepR+MSCs and osteoclasts back to control levels, preventing HSPC egress to the spleen. This reduced the number of inflammatory monocytes entering into the atherosclerotic lesion, inhibiting atherogenesis. These findings demonstrate that fibre supplementation can prevent HSDdriven inflammatory effects in the gut and the breakdown of the BM niche, to prevent atherogenesis.
2014 - DYNAMIN-DEPENDENT ENDOCYTOSIS IS REQUIRED FOR MULTIPLE NICHE SIGNALS THAT ARE CRUCIAL FOR LEUKEMIA STEM CELL ACTIVITY AND CHEMORESISTANCE Cedric Tremblay1, sung chiu1, Jesslyn Saw1, Stefan Sonderegger1, Ngoc Chau2, Adam McCluskey3, Phillip Robinson4, Stephen Jane5, David Curtis1 1 Australian Centre for Blood Diseases (ACBD) - Monash University, Melbourne, Australia; 2Children’s Medical Reserch Institute, Sydney, Australia; 3University of Newcastle, Newcastle, Australia; 4 Children’s Medical Research Institute, Sydney, Australia; 5Clinical Haematology, Alfred Hospital, Prahran, Australia The hierarchical model posits that acute leukemias arise from leukemia stem cells (LSCs), which display stem cell-like properties like long-term self-renewal and differentiation to generate the heterogeneity observed in the tumor at diagnosis. Elimination of LSCs and their ancestral clones, pre-leukemic stem cells (pre-LSCs), is critical for longterm cure as they are the source of relapse. How these cells survive high-dose therapy remains poorly defined, but may include quiescence and pro-survival signals provided by the microenvironment. Strategies to interfere with specific signals from the niche have shown promising results, but may be subject to compensatory mechanisms due to the inherent plasticity of LSCs. The transactivation of signalling pathways downstream of growth factors from the niche is dependent on receptor-mediated endocytosis, a generic process dependent on Dynamins. Given the important role of the microenvironment for relapse-inducing cells, we postulated that inhibition of Dynamin might impair their properties and sensitize them to chemotherapy. Here, we show that the Dynamin small molecule inhibitor Dynole 34-2 blocks multiple signalling pathways in pre-LSCs and LSCs by preventing endocytosis. Using the Lmo2-transgenic mouse model of T-cell acute lymphoblastic leukemia (T-ALL), we found that Dynole 34-2 impairs self-renewal of pre-LSCs and sensitizes relapse-inducing cells to chemotherapy. We also found that Dynole 34-2 delayed leukemia onset in mice treated with high-dose therapy. Treatment of different patient-derived xenografts revealed that that inhibition of Dynamin activity with Dynole 34-2 represents an effective therapeutic strategy for different subtypes of human T-ALL. In summary, our finding that Dynamin activity is essential for LSC activity and chemoresistance in acute leukemia paves the way for novel therapeutic strategies.