- Email: [email protected]
HLX regulates hematopoiesis by modulating cell metabolism
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
KLH and KLH arms, respectively (p50.42). Immune response analysis revealed a greater change in immune response genes in the Id-KLH arm after transplant, and a significant decrease in mRNA expression of immune inhibitory genes by NanoString nCounter in the Id-KLH group who had achieved near complete remission or better. Sixteen (100%) and 19 (95%) patients went on to receive maintenance therapy in the Id-KLH and KLH arms, respectively (p50.42). After a median follow up of 27.4 months, 2-year PFS was 64% and 84% in Id-KLH and KLH arms, respectively (p50.10). Conclusion: Id-KLH vaccine and vaccine-primed costimulated T cells can be safely administered in the setting of auto-HCT. Gene expression profile showed a correlation between the activity of immune response genes and the depth of clinical response.
3060 - ERNAS: A POTENTIAL PROXY FOR ENHANCER STATE DURING BLOOD DIFFERENTIATION AT THE SINGLE-CELL LEVEL Blanca Pijuan-Sala, Fernando Calero-Nieto, Nicola Wilson, and Berthold G€ottgens
3059 - HLX REGULATES HEMATOPOIESIS BY MODULATING CELL METABOLISM Indre Piragyte1, Thomas Clapes1, Ramon Klein Geltink1, Na Yin1, Aikaterini Polyzou1, Javier Langa Oliva2, Cora Beckmann3, Erika Pearce1, Angelika Rambold1, Freidrich Kapp3, Marina Mione4, Alexander Polyzos5, and Eirini Trompouki1
3061 - ALTERED HSC FATE UNDERLIES ABERRANT BLOOD PHENOTYPES IN RHEUMATOID ARTHRITIS Eric Pietras, Giovanny Hernandez, Susan Kuldanek, Rabe Jennifer, Gregory Kirkpatrick, Leila Noetzli, Sumitra Acharya, Biniam Adane, Craig Jordan, Jorge DiPaola, Michael Holers, and Nirmal Banda
MPI of Immunobiology and Epigenetics, Freiburg, Germany; 2University of Bern, Bern, Switzerland; 3University of Freiburg, Freiburg, Germany; 4University of Trento, Trento, Italy; 5Biomedical Research Foundation Academy of Athens, Athens, Greece
S71
University of Cambridge, Cambridge, United Kingdom Blood differentiation is a dynamic process governed by a series of regulatory events leading to continuous changes in gene expression. It is widely accepted that this process is mainly orchestrated by enhancers, inter- or intra-genic DNA regions where transcription factors bind to promote the activation of gene expression. Recently, it has been shown that enhancers can also function as transcriptional units, which produce short and unstable transcripts, called enhancer RNAs or eRNAs. While their functionality is still controversial, some studies have shown that enhancer expression correlates with enhancer activity as well as with its target gene expression. These findings open exciting avenues in the field of gene regulation: can we use eRNAs as a surrogate for enhancer state? If so, could we capture cells before they start expressing key blood-related genes and study the differentiation pathway in more detail? Unfortunately, all the aforementioned results have been inferred from bulk experiments, which average gene expression profiles from multiple cells, thus obscuring the heterogeneity that can exist within cell populations. Therefore, a single-cell approach to assess the feasibility of eRNAs as predictors of enhancer state at the single-cell level is needed. The goal of this project is to assess the enhancer RNA expression for a few blood-related genes, such as Tal1, Gata1 and Gata2, and to evaluate how well eRNAs correlate with gene expression and whether they may be used as a predictor to infer novel progenitor cell states. Preliminary results suggest that eRNA expression can be captured at the single-cell level with single-cell RNA-seq and that, due to their low abundance, increasing the sequencing depth could potentially boost their detection. With my poster presentation, I will provide an update on the questions highlighted above, with a particular focus on potential insights into eRNA biology that may be obtained through the use of single-cell techniques.
University of Colorado Anschutz Medical Campus, Aurora, United States
1
Upregulation of transcription factor HLX (H2.0-like Homeobox) is frequently observed in patients with AML. Since developmental pathways are often reactivated in cancer, we asked whether hlx1 plays a role during hematopoietic development in zebrafish. Endothelial cell (EC) specific overexpression of human HLX led to aberrant production of hematopoietic stem and progenitor cells (HSPCs) and increased number of immature myeloid cells at 48 hpf mimicking a preleukemic phenotype. On the other hand, hlx1 morphants had diminished numbers of HSPCs, a defect that could be rescued by overexpressing human HLX from either an EC or HSPCs specific promoter. Edu-labelling of EC, the precursors of HSPCs, from overexpression or morphant fish showed hyper-or-hypo-proliferation of these cells respectively. RNA-seq analysis of ECs revealed that overexpression of HLX led to downregulation of mitochondrial electron chain enzymes (e.g., NDUFS4 and SDHD) and upregulation of the peroxisome proliferator activated receptor family members (PPARs) together with enzymes important for fatty acid oxidation (FAO). Downregulation of hlx1 caused upregulation of the mitochondrial chain enzymes and downregulation of PPARs. These results suggested that HLX regulates the proliferative potential of hematopoietic cells by fine tuning mitochondrial potential and FAO. Indeed PPAR agonists could rescue HSPC generation in zebrafish morphants, while PPAR antagonists reversed the differentiation block of myeloid cells in zebrafish. To test whether role of HLX is conserved, we used human progenitor CD34+ cells. In accordance with our results in zebrafish, overexpression of HLX led to increased number of CFU-C, decreased oxidative phosphorylation capacity and mitochondrial potential, whereas downregulation had the opposite effect. ChIP-seq experiments revealed that HLX binds to mitochondrial genes (e.g., NDUFS4 and OPA1), and also genes involved in lipid metabolism (e.g., PPARD, and CPT2). In summary we establish HLX as a direct regulator of the proliferative potential of HSPCs by modulating their metabolic status.
Rheumatoid arthritis (RA) is a debilitating chronic autoimmune disorder characterized by joint inflammation and progressive degradation, particularly in the hands and feet. Notably, RA is also characterized by a systemic inflammatory phenotype that includes increased production of pro-inflammatory cytokines such as interleukin (IL)-1 and TNF, and blood system dysfunction typified by chronic anemia of disease, impaired lymphopoiesis and production of tissue-damaging myeloid cells. However, the direct impact and mechanism of RA-driven inflammation on hematopoietic stem cell fate and function has not been well characterized. In the present study, we used a mouse collagen-induced arthritis (CIA) model of human RA, which faithfully recapitulates many features of human RA. Strikingly, hematopoietic output was profoundly altered in CIA mice, with concurrent expansion of myeloid cells in the bone marrow at the expense of lymphoid and erythroid lineages. Consistent with these observations, we observed a significant decrease in the number of erythroid and lymphoid progenitors (CLP) in the bone marrow and thymus. On the other hand, we observed a significant and specific increase in the number of myeloidbiased MPP3. Notably, these changes occur in concert with a significant remodeling of the bone marrow stroma, resulting in reduced numbers of endothelial, mesenchymal and osteoblast-lineage cells in the bone. Consistent with these observations, molecular analyses of HSCs from CIA mice indicates the activation of key myeloid lineage determinants that may drive myeloid overproduction. Current investigations are now addressing the mechanism of myeloid expansion, the contribution of key pro-inflammatory cytokines to this phenotype, and the impact of aberrant autoimmune processes in initiating inflammatory hematopoiesis prior to the onset of clinically apparent arthritis. Altogether, our findings demonstrate a profound hematopoietic remodeling in the context of RA that likely fuels the disease process, and identifies potential mechanisms by which current and future therapeutic approaches can modulate blood production to prevent/delay disease or improve patient outcomes.
KLH and KLH arms, respectively (p50.42). Immune response analysis revealed a greater change in immune response genes in the Id-KLH arm after transplant, and a significant decrease in mRNA expression of immune inhibitory genes by NanoString nCounter in the Id-KLH group who had achieved near complete remission or better. Sixteen (100%) and 19 (95%) patients went on to receive maintenance therapy in the Id-KLH and KLH arms, respectively (p50.42). After a median follow up of 27.4 months, 2-year PFS was 64% and 84% in Id-KLH and KLH arms, respectively (p50.10). Conclusion: Id-KLH vaccine and vaccine-primed costimulated T cells can be safely administered in the setting of auto-HCT. Gene expression profile showed a correlation between the activity of immune response genes and the depth of clinical response.
3060 - ERNAS: A POTENTIAL PROXY FOR ENHANCER STATE DURING BLOOD DIFFERENTIATION AT THE SINGLE-CELL LEVEL Blanca Pijuan-Sala, Fernando Calero-Nieto, Nicola Wilson, and Berthold G€ottgens
3059 - HLX REGULATES HEMATOPOIESIS BY MODULATING CELL METABOLISM Indre Piragyte1, Thomas Clapes1, Ramon Klein Geltink1, Na Yin1, Aikaterini Polyzou1, Javier Langa Oliva2, Cora Beckmann3, Erika Pearce1, Angelika Rambold1, Freidrich Kapp3, Marina Mione4, Alexander Polyzos5, and Eirini Trompouki1
3061 - ALTERED HSC FATE UNDERLIES ABERRANT BLOOD PHENOTYPES IN RHEUMATOID ARTHRITIS Eric Pietras, Giovanny Hernandez, Susan Kuldanek, Rabe Jennifer, Gregory Kirkpatrick, Leila Noetzli, Sumitra Acharya, Biniam Adane, Craig Jordan, Jorge DiPaola, Michael Holers, and Nirmal Banda
MPI of Immunobiology and Epigenetics, Freiburg, Germany; 2University of Bern, Bern, Switzerland; 3University of Freiburg, Freiburg, Germany; 4University of Trento, Trento, Italy; 5Biomedical Research Foundation Academy of Athens, Athens, Greece
S71
University of Cambridge, Cambridge, United Kingdom Blood differentiation is a dynamic process governed by a series of regulatory events leading to continuous changes in gene expression. It is widely accepted that this process is mainly orchestrated by enhancers, inter- or intra-genic DNA regions where transcription factors bind to promote the activation of gene expression. Recently, it has been shown that enhancers can also function as transcriptional units, which produce short and unstable transcripts, called enhancer RNAs or eRNAs. While their functionality is still controversial, some studies have shown that enhancer expression correlates with enhancer activity as well as with its target gene expression. These findings open exciting avenues in the field of gene regulation: can we use eRNAs as a surrogate for enhancer state? If so, could we capture cells before they start expressing key blood-related genes and study the differentiation pathway in more detail? Unfortunately, all the aforementioned results have been inferred from bulk experiments, which average gene expression profiles from multiple cells, thus obscuring the heterogeneity that can exist within cell populations. Therefore, a single-cell approach to assess the feasibility of eRNAs as predictors of enhancer state at the single-cell level is needed. The goal of this project is to assess the enhancer RNA expression for a few blood-related genes, such as Tal1, Gata1 and Gata2, and to evaluate how well eRNAs correlate with gene expression and whether they may be used as a predictor to infer novel progenitor cell states. Preliminary results suggest that eRNA expression can be captured at the single-cell level with single-cell RNA-seq and that, due to their low abundance, increasing the sequencing depth could potentially boost their detection. With my poster presentation, I will provide an update on the questions highlighted above, with a particular focus on potential insights into eRNA biology that may be obtained through the use of single-cell techniques.
University of Colorado Anschutz Medical Campus, Aurora, United States
1
Upregulation of transcription factor HLX (H2.0-like Homeobox) is frequently observed in patients with AML. Since developmental pathways are often reactivated in cancer, we asked whether hlx1 plays a role during hematopoietic development in zebrafish. Endothelial cell (EC) specific overexpression of human HLX led to aberrant production of hematopoietic stem and progenitor cells (HSPCs) and increased number of immature myeloid cells at 48 hpf mimicking a preleukemic phenotype. On the other hand, hlx1 morphants had diminished numbers of HSPCs, a defect that could be rescued by overexpressing human HLX from either an EC or HSPCs specific promoter. Edu-labelling of EC, the precursors of HSPCs, from overexpression or morphant fish showed hyper-or-hypo-proliferation of these cells respectively. RNA-seq analysis of ECs revealed that overexpression of HLX led to downregulation of mitochondrial electron chain enzymes (e.g., NDUFS4 and SDHD) and upregulation of the peroxisome proliferator activated receptor family members (PPARs) together with enzymes important for fatty acid oxidation (FAO). Downregulation of hlx1 caused upregulation of the mitochondrial chain enzymes and downregulation of PPARs. These results suggested that HLX regulates the proliferative potential of hematopoietic cells by fine tuning mitochondrial potential and FAO. Indeed PPAR agonists could rescue HSPC generation in zebrafish morphants, while PPAR antagonists reversed the differentiation block of myeloid cells in zebrafish. To test whether role of HLX is conserved, we used human progenitor CD34+ cells. In accordance with our results in zebrafish, overexpression of HLX led to increased number of CFU-C, decreased oxidative phosphorylation capacity and mitochondrial potential, whereas downregulation had the opposite effect. ChIP-seq experiments revealed that HLX binds to mitochondrial genes (e.g., NDUFS4 and OPA1), and also genes involved in lipid metabolism (e.g., PPARD, and CPT2). In summary we establish HLX as a direct regulator of the proliferative potential of HSPCs by modulating their metabolic status.
Rheumatoid arthritis (RA) is a debilitating chronic autoimmune disorder characterized by joint inflammation and progressive degradation, particularly in the hands and feet. Notably, RA is also characterized by a systemic inflammatory phenotype that includes increased production of pro-inflammatory cytokines such as interleukin (IL)-1 and TNF, and blood system dysfunction typified by chronic anemia of disease, impaired lymphopoiesis and production of tissue-damaging myeloid cells. However, the direct impact and mechanism of RA-driven inflammation on hematopoietic stem cell fate and function has not been well characterized. In the present study, we used a mouse collagen-induced arthritis (CIA) model of human RA, which faithfully recapitulates many features of human RA. Strikingly, hematopoietic output was profoundly altered in CIA mice, with concurrent expansion of myeloid cells in the bone marrow at the expense of lymphoid and erythroid lineages. Consistent with these observations, we observed a significant decrease in the number of erythroid and lymphoid progenitors (CLP) in the bone marrow and thymus. On the other hand, we observed a significant and specific increase in the number of myeloidbiased MPP3. Notably, these changes occur in concert with a significant remodeling of the bone marrow stroma, resulting in reduced numbers of endothelial, mesenchymal and osteoblast-lineage cells in the bone. Consistent with these observations, molecular analyses of HSCs from CIA mice indicates the activation of key myeloid lineage determinants that may drive myeloid overproduction. Current investigations are now addressing the mechanism of myeloid expansion, the contribution of key pro-inflammatory cytokines to this phenotype, and the impact of aberrant autoimmune processes in initiating inflammatory hematopoiesis prior to the onset of clinically apparent arthritis. Altogether, our findings demonstrate a profound hematopoietic remodeling in the context of RA that likely fuels the disease process, and identifies potential mechanisms by which current and future therapeutic approaches can modulate blood production to prevent/delay disease or improve patient outcomes.