Contributions
Abstract: S123
Type: Oral Presentation
Presentation during EHA23: On Friday, June 15, 2018 from 12:30 - 12:45
Location: Room K1
Background
Cohesin is a multimeric protein complex, which was originally implicated in sister chromatid cohesion and more recently, in long-range regulation of gene expression by stabilizing 3-dememsional structure of the genome. Recently, multiple components of the cohesin complex are identified as recurrent targets of somatic mutations in various myeloid malignancies, of which STAG2 is most frequently mutated and inactivated; however, the leukemogenic mechanism of mutated-STAG2 remain largely unknown.
Aims
The aim of the study is to investigate the functional role of STAG2 in leukemogenesis as well as normal hematopoiesis.
Methods
We generated Stag2 conditional knockout (cKO) mice having an Mx1-cre allele, in which Stag2 deletion was induced by polyIC injection.
Results
Stag2 cKO mice showed a mild leukocytopenia. Flow cytometry of bone marrow cells revealed an increase in the frequency of hematopoietic stem and progenitor cells (HSPCs) defined as Lin−Sca-1+Kit+(LSK) cells in Stag2 cKO mice. In the cell cycle kinetics analysis, Stag2 cKO HSPCs were characterized by an increase in the proportion of cells in S/G2/M phases. Within the myeloid progenitor (MP) compartment, we observed increased common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs), and decreased megakaryocyte/erythroid (MEPs) and common lymphoid progenitors (CLPs). Moreover, CD11b+Gr-1+ myeloid cells were increased in the bone marrow of Stag2 cKO mice. These results suggest that Stag2 deficiency causes skewing toward myeloid lineage.
In repeated methylcellulose cultures, Stag2 deficient BM cells showed an enhanced serial replating capacity, suggesting an increased self-renewal potential of Stag2 deficient hematopoietic stem cells (HSCs). In competitive repopulation assays, Stag2 cKO-derived cells showed reduced chimerism in the peripheral blood compared to wild-type mice-derived cells. In the bone marrow, by contrast, the chimerism of Stag2 cKO cells was not significantly changed, but tended to increase in the LSK, CMP and GMP fractions and decrease in the MEP and CLP fractions. These results suggest that Stag2 deficiency could enhance the self-renewal capacity of HSCs, while differentially impacting on their contribution to different hematopoietic cell fractions due most likely to the deregulated differentiation of HSCs.
We next investigated differential expression profiles between Stag2 WT and cKO cells using RNA sequencing of HSPC fractions. We observed significant up-regulation of key hematopoietic regulators, including Gata1, Gata2 and Runx1, which are implicated in the differentiation of HSCs. Of particular interest in this regard, assays for transposase accessible chromatin with sequencing (ATAC-seq) revealed a significant enrichment of the binding sites for Gata1, Gata2, and Runx1 in the open chromatin regions in Stag2-deleted HSPCs. We also observed up-regulated expression of genes related to myeloid programs, such as Mpo and Cebpa, while expression of several genes involved in the lymphoid development such as Cd9 and Irf4 were down regulated. These results indicate that Stag2-deficient HSCs are primed to differentiate toward myeloid lineage, which is in agreement with in vivo hematopoietic phenotypes.
Conclusion
Our results demonstrate that Stag2 loss leads to the impaired hematopoietic differentiation and enhances the self-renewal potential of HSCs, likely through the modulation of gene expression and chromatin accessibility, which may contribute to leukemogenesis in STAG2-mutated cells.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Abstract: S123
Type: Oral Presentation
Presentation during EHA23: On Friday, June 15, 2018 from 12:30 - 12:45
Location: Room K1
Background
Cohesin is a multimeric protein complex, which was originally implicated in sister chromatid cohesion and more recently, in long-range regulation of gene expression by stabilizing 3-dememsional structure of the genome. Recently, multiple components of the cohesin complex are identified as recurrent targets of somatic mutations in various myeloid malignancies, of which STAG2 is most frequently mutated and inactivated; however, the leukemogenic mechanism of mutated-STAG2 remain largely unknown.
Aims
The aim of the study is to investigate the functional role of STAG2 in leukemogenesis as well as normal hematopoiesis.
Methods
We generated Stag2 conditional knockout (cKO) mice having an Mx1-cre allele, in which Stag2 deletion was induced by polyIC injection.
Results
Stag2 cKO mice showed a mild leukocytopenia. Flow cytometry of bone marrow cells revealed an increase in the frequency of hematopoietic stem and progenitor cells (HSPCs) defined as Lin−Sca-1+Kit+(LSK) cells in Stag2 cKO mice. In the cell cycle kinetics analysis, Stag2 cKO HSPCs were characterized by an increase in the proportion of cells in S/G2/M phases. Within the myeloid progenitor (MP) compartment, we observed increased common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs), and decreased megakaryocyte/erythroid (MEPs) and common lymphoid progenitors (CLPs). Moreover, CD11b+Gr-1+ myeloid cells were increased in the bone marrow of Stag2 cKO mice. These results suggest that Stag2 deficiency causes skewing toward myeloid lineage.
In repeated methylcellulose cultures, Stag2 deficient BM cells showed an enhanced serial replating capacity, suggesting an increased self-renewal potential of Stag2 deficient hematopoietic stem cells (HSCs). In competitive repopulation assays, Stag2 cKO-derived cells showed reduced chimerism in the peripheral blood compared to wild-type mice-derived cells. In the bone marrow, by contrast, the chimerism of Stag2 cKO cells was not significantly changed, but tended to increase in the LSK, CMP and GMP fractions and decrease in the MEP and CLP fractions. These results suggest that Stag2 deficiency could enhance the self-renewal capacity of HSCs, while differentially impacting on their contribution to different hematopoietic cell fractions due most likely to the deregulated differentiation of HSCs.
We next investigated differential expression profiles between Stag2 WT and cKO cells using RNA sequencing of HSPC fractions. We observed significant up-regulation of key hematopoietic regulators, including Gata1, Gata2 and Runx1, which are implicated in the differentiation of HSCs. Of particular interest in this regard, assays for transposase accessible chromatin with sequencing (ATAC-seq) revealed a significant enrichment of the binding sites for Gata1, Gata2, and Runx1 in the open chromatin regions in Stag2-deleted HSPCs. We also observed up-regulated expression of genes related to myeloid programs, such as Mpo and Cebpa, while expression of several genes involved in the lymphoid development such as Cd9 and Irf4 were down regulated. These results indicate that Stag2-deficient HSCs are primed to differentiate toward myeloid lineage, which is in agreement with in vivo hematopoietic phenotypes.
Conclusion
Our results demonstrate that Stag2 loss leads to the impaired hematopoietic differentiation and enhances the self-renewal potential of HSCs, likely through the modulation of gene expression and chromatin accessibility, which may contribute to leukemogenesis in STAG2-mutated cells.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research