INTEGRATED ANALYSIS OF THE HUMAN HEMATOPOIETIC NON-CODING RNA LANDSCAPE REVEALS LNC-RNA STEM CELL SIGNATURE IN AML
(Abstract release date: 05/19/16)
EHA Library. Klusman J. 06/10/16; 135154; S121
Dr. Jan-Henning Klusman
Contributions
Contributions
Abstract
Abstract: S121
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:15 - 12:30
Location: Hall A3
Background
Long non-coding RNAs (lncRNAs) and miRNAs have emerged as crucial regulators of gene expression, epigenetics and cell fate decisions.
Aims
We sought to establish a comprehensive resource for the exploration of non-coding RNAs across the human hematopoietic hierarchy, to understand their role in the pathogenesis of acute myeloid leukemia (AML).
Methods
Here we present an integrated quantitative and functional analysis of the miRNA-, lncRNA- and mRNA-transcriptome of purified human hematopoietic stem cells (HSCs) and their differentiated progenies, including granulocytes, monocytes, T-cells, NK-cells, B-cells, megakaryocytes and erythroid precursors, which we correlated with the ncRNA expression profile of 46 pediatric AML samples to establish a core lncRNA stem cell signature in AML.
Results
For each blood cell population, RNA from 5 healthy donors was hybridized onto three microarray platforms (Arraystar lncRNA V2.0, NCode™-miRNA/-ncRNA), yielding a coverage of more than 40,000 lncRNAs, 25,000 mRNAs and 900 miRNAs on 146 arrays. Compared to mRNAs, the mean expression level of lncRNAs was nearly 2-fold lower (p<2.2x10-16), highlighting the challenge for RNA-Seq to provide adequate coverage of these rare transcripts. T-distributed stochastic neighbor embedding (t-SNE) on lncRNA and miRNA genes robustly structured the dataset into groups of samples that matched the input populations, demonstrating their unique ncRNA expression profiles. Self-organizing maps revealed clusters of lncRNAs and mRNAs that were coordinately expressed in HSCs and during lineage commitment. To demonstrate their functionality, we knocked down LINC00173 from the granulocytic core signature using two independent shRNA constructs, which resulted in diminished granulocytic in vitro differentiation (2-fold reduction in percentage of CD66b+/CD13+ granulocytes, p≤0.05), myeloid colony-formation (1.5-2-fold, p≤0.05) and nuclear lobulation (MGG-staining). Accordingly, CRISPR-mediated transcriptional repression of nuclear localized LINC00173 (RNA-FISH validated by qRT-RNA of fractionated RNA) using dCas9-KRAB and three sgRNAs per locus reduced proliferation of myeloid NB4 cells (2-3-fold, p≤0.01).Having established a global human hematopoietic lncRNA expression resource, we extended our findings to malignant hematopoiesis. Linear (PCA) and nonlinear (t-SNE) dimensionality reduction of 46 pediatric AML samples including Down syndrome AMKL, core-binding factor AMLs (inv[16] or t[8;21]) and MLL-rearranged leukemias mapped most samples to a space between HSCs and differentiated cells together with the myeloid progenitors. A subset of AML-samples mapped closely to healthy HSCs, including most of the DS-AMKLs and MLL-AMLs. We identified a stem-cell associated lncRNA signature that was absent in healthy differentiated progenies, but upregulated in AML samples. A mesoscale CRISPRi screening in AML cell lines suggested the importance of the lncRNA stem cell core signature for the maintenance of leukemic growth.
Conclusion
The definition of a core lncRNA stem cell signature in normal HSCs and AML blasts will guide our way towards an improved understanding of self-renewal and the underlying transcriptional program, which is hijacked during malignant transformation.
Session topic: AML Biology - Novel mechanisms of leukemogenesis
Keyword(s): Acute myeloid leukemia, Expression profiling, Hematopoiesis
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:15 - 12:30
Location: Hall A3
Background
Long non-coding RNAs (lncRNAs) and miRNAs have emerged as crucial regulators of gene expression, epigenetics and cell fate decisions.
Aims
We sought to establish a comprehensive resource for the exploration of non-coding RNAs across the human hematopoietic hierarchy, to understand their role in the pathogenesis of acute myeloid leukemia (AML).
Methods
Here we present an integrated quantitative and functional analysis of the miRNA-, lncRNA- and mRNA-transcriptome of purified human hematopoietic stem cells (HSCs) and their differentiated progenies, including granulocytes, monocytes, T-cells, NK-cells, B-cells, megakaryocytes and erythroid precursors, which we correlated with the ncRNA expression profile of 46 pediatric AML samples to establish a core lncRNA stem cell signature in AML.
Results
For each blood cell population, RNA from 5 healthy donors was hybridized onto three microarray platforms (Arraystar lncRNA V2.0, NCode™-miRNA/-ncRNA), yielding a coverage of more than 40,000 lncRNAs, 25,000 mRNAs and 900 miRNAs on 146 arrays. Compared to mRNAs, the mean expression level of lncRNAs was nearly 2-fold lower (p<2.2x10-16), highlighting the challenge for RNA-Seq to provide adequate coverage of these rare transcripts. T-distributed stochastic neighbor embedding (t-SNE) on lncRNA and miRNA genes robustly structured the dataset into groups of samples that matched the input populations, demonstrating their unique ncRNA expression profiles. Self-organizing maps revealed clusters of lncRNAs and mRNAs that were coordinately expressed in HSCs and during lineage commitment. To demonstrate their functionality, we knocked down LINC00173 from the granulocytic core signature using two independent shRNA constructs, which resulted in diminished granulocytic in vitro differentiation (2-fold reduction in percentage of CD66b+/CD13+ granulocytes, p≤0.05), myeloid colony-formation (1.5-2-fold, p≤0.05) and nuclear lobulation (MGG-staining). Accordingly, CRISPR-mediated transcriptional repression of nuclear localized LINC00173 (RNA-FISH validated by qRT-RNA of fractionated RNA) using dCas9-KRAB and three sgRNAs per locus reduced proliferation of myeloid NB4 cells (2-3-fold, p≤0.01).Having established a global human hematopoietic lncRNA expression resource, we extended our findings to malignant hematopoiesis. Linear (PCA) and nonlinear (t-SNE) dimensionality reduction of 46 pediatric AML samples including Down syndrome AMKL, core-binding factor AMLs (inv[16] or t[8;21]) and MLL-rearranged leukemias mapped most samples to a space between HSCs and differentiated cells together with the myeloid progenitors. A subset of AML-samples mapped closely to healthy HSCs, including most of the DS-AMKLs and MLL-AMLs. We identified a stem-cell associated lncRNA signature that was absent in healthy differentiated progenies, but upregulated in AML samples. A mesoscale CRISPRi screening in AML cell lines suggested the importance of the lncRNA stem cell core signature for the maintenance of leukemic growth.
Conclusion
The definition of a core lncRNA stem cell signature in normal HSCs and AML blasts will guide our way towards an improved understanding of self-renewal and the underlying transcriptional program, which is hijacked during malignant transformation.
Session topic: AML Biology - Novel mechanisms of leukemogenesis
Keyword(s): Acute myeloid leukemia, Expression profiling, Hematopoiesis
Abstract: S121
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:15 - 12:30
Location: Hall A3
Background
Long non-coding RNAs (lncRNAs) and miRNAs have emerged as crucial regulators of gene expression, epigenetics and cell fate decisions.
Aims
We sought to establish a comprehensive resource for the exploration of non-coding RNAs across the human hematopoietic hierarchy, to understand their role in the pathogenesis of acute myeloid leukemia (AML).
Methods
Here we present an integrated quantitative and functional analysis of the miRNA-, lncRNA- and mRNA-transcriptome of purified human hematopoietic stem cells (HSCs) and their differentiated progenies, including granulocytes, monocytes, T-cells, NK-cells, B-cells, megakaryocytes and erythroid precursors, which we correlated with the ncRNA expression profile of 46 pediatric AML samples to establish a core lncRNA stem cell signature in AML.
Results
For each blood cell population, RNA from 5 healthy donors was hybridized onto three microarray platforms (Arraystar lncRNA V2.0, NCode™-miRNA/-ncRNA), yielding a coverage of more than 40,000 lncRNAs, 25,000 mRNAs and 900 miRNAs on 146 arrays. Compared to mRNAs, the mean expression level of lncRNAs was nearly 2-fold lower (p<2.2x10-16), highlighting the challenge for RNA-Seq to provide adequate coverage of these rare transcripts. T-distributed stochastic neighbor embedding (t-SNE) on lncRNA and miRNA genes robustly structured the dataset into groups of samples that matched the input populations, demonstrating their unique ncRNA expression profiles. Self-organizing maps revealed clusters of lncRNAs and mRNAs that were coordinately expressed in HSCs and during lineage commitment. To demonstrate their functionality, we knocked down LINC00173 from the granulocytic core signature using two independent shRNA constructs, which resulted in diminished granulocytic in vitro differentiation (2-fold reduction in percentage of CD66b+/CD13+ granulocytes, p≤0.05), myeloid colony-formation (1.5-2-fold, p≤0.05) and nuclear lobulation (MGG-staining). Accordingly, CRISPR-mediated transcriptional repression of nuclear localized LINC00173 (RNA-FISH validated by qRT-RNA of fractionated RNA) using dCas9-KRAB and three sgRNAs per locus reduced proliferation of myeloid NB4 cells (2-3-fold, p≤0.01).Having established a global human hematopoietic lncRNA expression resource, we extended our findings to malignant hematopoiesis. Linear (PCA) and nonlinear (t-SNE) dimensionality reduction of 46 pediatric AML samples including Down syndrome AMKL, core-binding factor AMLs (inv[16] or t[8;21]) and MLL-rearranged leukemias mapped most samples to a space between HSCs and differentiated cells together with the myeloid progenitors. A subset of AML-samples mapped closely to healthy HSCs, including most of the DS-AMKLs and MLL-AMLs. We identified a stem-cell associated lncRNA signature that was absent in healthy differentiated progenies, but upregulated in AML samples. A mesoscale CRISPRi screening in AML cell lines suggested the importance of the lncRNA stem cell core signature for the maintenance of leukemic growth.
Conclusion
The definition of a core lncRNA stem cell signature in normal HSCs and AML blasts will guide our way towards an improved understanding of self-renewal and the underlying transcriptional program, which is hijacked during malignant transformation.
Session topic: AML Biology - Novel mechanisms of leukemogenesis
Keyword(s): Acute myeloid leukemia, Expression profiling, Hematopoiesis
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:15 - 12:30
Location: Hall A3
Background
Long non-coding RNAs (lncRNAs) and miRNAs have emerged as crucial regulators of gene expression, epigenetics and cell fate decisions.
Aims
We sought to establish a comprehensive resource for the exploration of non-coding RNAs across the human hematopoietic hierarchy, to understand their role in the pathogenesis of acute myeloid leukemia (AML).
Methods
Here we present an integrated quantitative and functional analysis of the miRNA-, lncRNA- and mRNA-transcriptome of purified human hematopoietic stem cells (HSCs) and their differentiated progenies, including granulocytes, monocytes, T-cells, NK-cells, B-cells, megakaryocytes and erythroid precursors, which we correlated with the ncRNA expression profile of 46 pediatric AML samples to establish a core lncRNA stem cell signature in AML.
Results
For each blood cell population, RNA from 5 healthy donors was hybridized onto three microarray platforms (Arraystar lncRNA V2.0, NCode™-miRNA/-ncRNA), yielding a coverage of more than 40,000 lncRNAs, 25,000 mRNAs and 900 miRNAs on 146 arrays. Compared to mRNAs, the mean expression level of lncRNAs was nearly 2-fold lower (p<2.2x10-16), highlighting the challenge for RNA-Seq to provide adequate coverage of these rare transcripts. T-distributed stochastic neighbor embedding (t-SNE) on lncRNA and miRNA genes robustly structured the dataset into groups of samples that matched the input populations, demonstrating their unique ncRNA expression profiles. Self-organizing maps revealed clusters of lncRNAs and mRNAs that were coordinately expressed in HSCs and during lineage commitment. To demonstrate their functionality, we knocked down LINC00173 from the granulocytic core signature using two independent shRNA constructs, which resulted in diminished granulocytic in vitro differentiation (2-fold reduction in percentage of CD66b+/CD13+ granulocytes, p≤0.05), myeloid colony-formation (1.5-2-fold, p≤0.05) and nuclear lobulation (MGG-staining). Accordingly, CRISPR-mediated transcriptional repression of nuclear localized LINC00173 (RNA-FISH validated by qRT-RNA of fractionated RNA) using dCas9-KRAB and three sgRNAs per locus reduced proliferation of myeloid NB4 cells (2-3-fold, p≤0.01).Having established a global human hematopoietic lncRNA expression resource, we extended our findings to malignant hematopoiesis. Linear (PCA) and nonlinear (t-SNE) dimensionality reduction of 46 pediatric AML samples including Down syndrome AMKL, core-binding factor AMLs (inv[16] or t[8;21]) and MLL-rearranged leukemias mapped most samples to a space between HSCs and differentiated cells together with the myeloid progenitors. A subset of AML-samples mapped closely to healthy HSCs, including most of the DS-AMKLs and MLL-AMLs. We identified a stem-cell associated lncRNA signature that was absent in healthy differentiated progenies, but upregulated in AML samples. A mesoscale CRISPRi screening in AML cell lines suggested the importance of the lncRNA stem cell core signature for the maintenance of leukemic growth.
Conclusion
The definition of a core lncRNA stem cell signature in normal HSCs and AML blasts will guide our way towards an improved understanding of self-renewal and the underlying transcriptional program, which is hijacked during malignant transformation.
Session topic: AML Biology - Novel mechanisms of leukemogenesis
Keyword(s): Acute myeloid leukemia, Expression profiling, Hematopoiesis
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