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2035 – AGE-RELATED DOWNREGULATION OF LMNA IMPACTS HUMAN HEMATOPOIETIC STEM CELL FUNCTION
Experimental Hematology 2019;76 (Suppl):e1−e6
LATE-BREAKING ORAL PRESENTATIONS 2034 − RAB5C-MEDIATED ENDOCYTIC TRAFFICKING REGULATES HEMATOPOIETIC STEM AND PROGENITOR CELL DEVELOPMENT THROUGH NOTCH AND AKT SIGNALING Feng Liu Institute of Zoology, Chinese Academy of Sciences, Beijing, China (People’s Republic) A better understanding of molecular mechanisms underlying hematopoietic stem and progenitor cell (HSPC) development may lead to improved protocols to generate functional and transplantable HSPCs ex vivo. Here we report that Rab5c is essential for HSPC specification by endocytic trafficking of Notch and AKT signaling in zebrafish embryos. Rab5c deficiency leads to defects in HSPC production. Mechanistically, Rab5c regulates hemogenic endothelium specification by endocytic trafficking of Notch ligand and receptor. We further show that the interaction between Rab5c and Appl1 in endosome is required for the survival of endothelial cells in the aorta-gonad-mesonephros region through AKT signaling. Interestingly, Rab5c over-activation can also lead to defects in HSPC production, which is attributed to excessive endolysosomal trafficking inducing Notch signaling defect. Taken together, our findings establish a previously unrecognized role of Rab5c-mediated endocytic trafficking in HSPC development, and provide new insights into how spatiotemporal signals are orchestrated to accurately execute cell fate transition. 2035 − AGE-RELATED DOWNREGULATION OF LMNA IMPACTS HUMAN HEMATOPOIETIC STEM CELL FUNCTION Hsuan-Ting Huang, Emmalee Adelman, Dean Wade, Gabriel Gaidosh, Ramin Shiekhattar, Maria Figueroa University of Miami, Miami, United States Hematopoietic stem cells, essential for the production of blood cells, decline in function with aging. Among these changes with aging are increased frequency of mutations in epigenetic modifiers. We analyzed the epigenetic landscape of Lineage-CD34+CD38- human HSC-enriched fraction (HSCe) from young (18-30 years), middleaged (45-55 years), and aged (65-75 years) individuals by comparing changes in gene expression by RNA sequencing and histone marks (H3K4me3, H3K4me1, H3K27ac, H3K27me3) by ChIP sequencing. Integrative analysis of the histone profiles by k-means clustering identified 12 clusters of genes defined by their specific pattern of age-associated epigenetic changes, which were accompanied by changes in gene expression affecting 1,133 genes between young and aged HSCe. The LMNA gene, which encodes the nuclear lamina protein Lamin A/C, was one of the most downregulated genes with aging (7.9-fold, p=1.9 £ 10 13). This downregulation was mediated by loss of active histone marks H3K4me3 at the LMNA gene promoter, as well as loss of H3K27ac at two putative enhancer regions. ShRNA-mediated knockdown of LMNA in young, CD34+ hematopoietic stem and progenitor cells (HSPCs) resulted in an increase in myeloid colony formation (n=7 biological replicates with an average of 33% in total colony increase), consistent with the aging phenotype. Moreover, knockdown of LMNA in CD34+ cells impaired myeloid differentiation in liquid culture as determined by persistence of CD34 expression (n=5, p≤0.0001) with a delay in CD11b expression (n=5, p≤0.05). Initial results by STORM super resolution imaging show loss of LMNA leads to enlarged nuclear size and changes in chromatin localization. In conclusion, loss of LMNA recapitulates features of HSC aging, and restoring LMNA levels may be important for restoring normal function in aged HSCs.
2036 − INFLAMMAGING AND T-ALL: ONCOGENECYTOKINE INTERACTIONS AND THEIR ROLE IN LEUKEMOGENESIS Gabriela Segat1,2 , Rachel Wong1 , Kateryna Tyshchenko1, Manabu Kusakabe3, Ann Sun1, Damoun Torabi1, Samuel Gusscott1, Andrew Weng1 1 Terry Fox Laboratory, BC Cancer Research Centre, Vancouver, Canada; 2 University of British Columbia, Vancouver, Canada; 3 University of Tsukuba, Tsukuba, Japan T-cell acute lymphoblastic leukemia (T-ALL) can affect any age group; however, disease outcome is worse in older patients. Aging has been described to be accompanied by an increase in the body’s proinflammatory status, a phenomenon referred as “inflammaging”. By analysing two T-ALL transcriptome datasets, we have found that inflammation pathways are upregulated in older patients, whereas younger patients present upregulation of cell cycle pathways, suggesting that inflammaging is also recapitulated in T-ALL. Here, we hypothesize that T-ALL oncogenes can cooperate with specific cytokines/stimuli and their signaling pathways to create distinct leukemic phenotypes. To test this, human cord blood CD34+ cells were transduced with different T-ALL oncogenes and grown in co-culture with stromal cells under different pro-inflammatory conditions. We observed that most of the transduced cells had their cell growth negatively affected by the proinflammatory conditions, although some oncogene combinations showed more tolerance to these conditions than others. Activated NOTCH1+TLX3 transduced cells represented the only gene combination that showed significantly higher cell growth in the presence of interferon-gamma (IFNg) when compared to non-transduced cells in the same culture. IL-15 and IFNg potentiated differentiation arrest observed with certain oncogene combinations. Normal uncommitted T-cell progenitors (CD7+CD5+CD44 +CD1a-) responded to IL-15 via STAT5 and AKT phosphorylation, and this response was enhanced by chronic treatment with IL-15. Our results suggest that proinflammatory conditions can alter the effect of certain oncogenes on T-cell progenitors and influence leukemogenesis, potentially leading to distinct biological behaviors of resulting leukemias. These findings may lead to identification of cytokine dependencies associated with particular T-ALL genetic subtypes. 2037 − DACH1 DOWNREGULATION MARKS A ‘LYMPHOIDPRIMED PROGENITOR’ IN EARLY HAEMATOPOIESIS Shalin Naik1,2, Daniela Zalcenstein3, Luyi Tian3 , Jaring Schreuder3 , Sara Tomei3, Dawn Lin3, Kirsten Fairfax3, Jess Bolden3, Andrew Jarrat3 , Mark McKenzie3 , Warren Alexander3 , Ladina di Rago3, Adrienne Hilton3, Doug Hilton3, Jacob Jackson4 , Matt McCormack4, Ashley Ng3, Matt Ritchie3 1 Immunology Division, Melbourne, Australia; 2Walter & Eliza Hall Institute of Medical Research, Melbourne, Australia; 3WEHI, Melbourne, Australia; 4Monash University, Melbourne, Australia A classical view of blood cell development is that multipotent haematopoietic stem and progenitor cells (HSPCs) become lineagerestricted at defined stages. The Lin−c-kit+Sca1+Flt3+ stage, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Through single cell RNA-sequencing, we identify heterogeneous expression of Dach1 and associated genes in this fraction where it co-expressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1−
LATE-BREAKING ORAL PRESENTATIONS 2034 − RAB5C-MEDIATED ENDOCYTIC TRAFFICKING REGULATES HEMATOPOIETIC STEM AND PROGENITOR CELL DEVELOPMENT THROUGH NOTCH AND AKT SIGNALING Feng Liu Institute of Zoology, Chinese Academy of Sciences, Beijing, China (People’s Republic) A better understanding of molecular mechanisms underlying hematopoietic stem and progenitor cell (HSPC) development may lead to improved protocols to generate functional and transplantable HSPCs ex vivo. Here we report that Rab5c is essential for HSPC specification by endocytic trafficking of Notch and AKT signaling in zebrafish embryos. Rab5c deficiency leads to defects in HSPC production. Mechanistically, Rab5c regulates hemogenic endothelium specification by endocytic trafficking of Notch ligand and receptor. We further show that the interaction between Rab5c and Appl1 in endosome is required for the survival of endothelial cells in the aorta-gonad-mesonephros region through AKT signaling. Interestingly, Rab5c over-activation can also lead to defects in HSPC production, which is attributed to excessive endolysosomal trafficking inducing Notch signaling defect. Taken together, our findings establish a previously unrecognized role of Rab5c-mediated endocytic trafficking in HSPC development, and provide new insights into how spatiotemporal signals are orchestrated to accurately execute cell fate transition. 2035 − AGE-RELATED DOWNREGULATION OF LMNA IMPACTS HUMAN HEMATOPOIETIC STEM CELL FUNCTION Hsuan-Ting Huang, Emmalee Adelman, Dean Wade, Gabriel Gaidosh, Ramin Shiekhattar, Maria Figueroa University of Miami, Miami, United States Hematopoietic stem cells, essential for the production of blood cells, decline in function with aging. Among these changes with aging are increased frequency of mutations in epigenetic modifiers. We analyzed the epigenetic landscape of Lineage-CD34+CD38- human HSC-enriched fraction (HSCe) from young (18-30 years), middleaged (45-55 years), and aged (65-75 years) individuals by comparing changes in gene expression by RNA sequencing and histone marks (H3K4me3, H3K4me1, H3K27ac, H3K27me3) by ChIP sequencing. Integrative analysis of the histone profiles by k-means clustering identified 12 clusters of genes defined by their specific pattern of age-associated epigenetic changes, which were accompanied by changes in gene expression affecting 1,133 genes between young and aged HSCe. The LMNA gene, which encodes the nuclear lamina protein Lamin A/C, was one of the most downregulated genes with aging (7.9-fold, p=1.9 £ 10 13). This downregulation was mediated by loss of active histone marks H3K4me3 at the LMNA gene promoter, as well as loss of H3K27ac at two putative enhancer regions. ShRNA-mediated knockdown of LMNA in young, CD34+ hematopoietic stem and progenitor cells (HSPCs) resulted in an increase in myeloid colony formation (n=7 biological replicates with an average of 33% in total colony increase), consistent with the aging phenotype. Moreover, knockdown of LMNA in CD34+ cells impaired myeloid differentiation in liquid culture as determined by persistence of CD34 expression (n=5, p≤0.0001) with a delay in CD11b expression (n=5, p≤0.05). Initial results by STORM super resolution imaging show loss of LMNA leads to enlarged nuclear size and changes in chromatin localization. In conclusion, loss of LMNA recapitulates features of HSC aging, and restoring LMNA levels may be important for restoring normal function in aged HSCs.
2036 − INFLAMMAGING AND T-ALL: ONCOGENECYTOKINE INTERACTIONS AND THEIR ROLE IN LEUKEMOGENESIS Gabriela Segat1,2 , Rachel Wong1 , Kateryna Tyshchenko1, Manabu Kusakabe3, Ann Sun1, Damoun Torabi1, Samuel Gusscott1, Andrew Weng1 1 Terry Fox Laboratory, BC Cancer Research Centre, Vancouver, Canada; 2 University of British Columbia, Vancouver, Canada; 3 University of Tsukuba, Tsukuba, Japan T-cell acute lymphoblastic leukemia (T-ALL) can affect any age group; however, disease outcome is worse in older patients. Aging has been described to be accompanied by an increase in the body’s proinflammatory status, a phenomenon referred as “inflammaging”. By analysing two T-ALL transcriptome datasets, we have found that inflammation pathways are upregulated in older patients, whereas younger patients present upregulation of cell cycle pathways, suggesting that inflammaging is also recapitulated in T-ALL. Here, we hypothesize that T-ALL oncogenes can cooperate with specific cytokines/stimuli and their signaling pathways to create distinct leukemic phenotypes. To test this, human cord blood CD34+ cells were transduced with different T-ALL oncogenes and grown in co-culture with stromal cells under different pro-inflammatory conditions. We observed that most of the transduced cells had their cell growth negatively affected by the proinflammatory conditions, although some oncogene combinations showed more tolerance to these conditions than others. Activated NOTCH1+TLX3 transduced cells represented the only gene combination that showed significantly higher cell growth in the presence of interferon-gamma (IFNg) when compared to non-transduced cells in the same culture. IL-15 and IFNg potentiated differentiation arrest observed with certain oncogene combinations. Normal uncommitted T-cell progenitors (CD7+CD5+CD44 +CD1a-) responded to IL-15 via STAT5 and AKT phosphorylation, and this response was enhanced by chronic treatment with IL-15. Our results suggest that proinflammatory conditions can alter the effect of certain oncogenes on T-cell progenitors and influence leukemogenesis, potentially leading to distinct biological behaviors of resulting leukemias. These findings may lead to identification of cytokine dependencies associated with particular T-ALL genetic subtypes. 2037 − DACH1 DOWNREGULATION MARKS A ‘LYMPHOIDPRIMED PROGENITOR’ IN EARLY HAEMATOPOIESIS Shalin Naik1,2, Daniela Zalcenstein3, Luyi Tian3 , Jaring Schreuder3 , Sara Tomei3, Dawn Lin3, Kirsten Fairfax3, Jess Bolden3, Andrew Jarrat3 , Mark McKenzie3 , Warren Alexander3 , Ladina di Rago3, Adrienne Hilton3, Doug Hilton3, Jacob Jackson4 , Matt McCormack4, Ashley Ng3, Matt Ritchie3 1 Immunology Division, Melbourne, Australia; 2Walter & Eliza Hall Institute of Medical Research, Melbourne, Australia; 3WEHI, Melbourne, Australia; 4Monash University, Melbourne, Australia A classical view of blood cell development is that multipotent haematopoietic stem and progenitor cells (HSPCs) become lineagerestricted at defined stages. The Lin−c-kit+Sca1+Flt3+ stage, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Through single cell RNA-sequencing, we identify heterogeneous expression of Dach1 and associated genes in this fraction where it co-expressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1−