Contributions
Abstract: S122
Type: Oral Presentation
Presentation during EHA23: On Friday, June 15, 2018 from 12:15 - 12:30
Location: Room K1
Background
Myeloid malignancies are a heterogeneous continuum of neoplasia that share many common characteristics, such as altered transcriptional regulation caused by recurrent mutations of signalling, transcription and epigenetic factors. With a frequency of 15% across all myeloid malignancies, several of these mutations occur in members of the cohesin complex (STAG2, RAD21, SMC1a or SMC3, less frequent STAG1). The mutations are mutually exclusive, are predicted to cause loss of function alleles and a decrease of complex activity.
Aims
To determine a mechanism for the establishment of cohesin-deficient myeloid neoplasia.
Methods
Utilizing a model of haematopoietic stem and progenitor differentiation (HPC-7), we performed integrative analysis of cohesin binding, gene expression, chromatin state and 3D-DNA topology at genome scale, assessing different cellular states - haematopoietic progenitors, erythroid and myeloid cells - followed by matched analysis after inducible shRNA-mediated knockdown (KD) of multiple cohesin members. We finally validated the observed patterns in vitro and in vivo using a murine model of Flt3-ITD/Npm1c AML after inducible KD of Stag2 or Smc1a.
Results
We demonstrated alterations of cohesin member expression upon commitment down the erythroid but not myeloid lineage. This variable expression of cohesin translated into differing dosages at chromatin during differentiation; cohesin binding increased in the erythroid lineage at active cis-regulatory elements (enhancers/promoters), while the global cohesin dosage remained low in the myeloid lineage. Binding dynamics correlated to the H3K27 acetylation (H3K27ac), but not to gene expression, Ctcf binding or promoter interaction frequency.
Suprisingly, KD of cohesin members only minimally affected binding of the residual cohesin complex to active promoters/enhancers in immature cells, resulting in discrete changes of H3K27ac and gene expression. However, during differentiation of Stag2-perturbed haematopoiesis, significant biases of cohesin binding to active cis-regulatory elements were seen, with impaired dosage of cohesin demonstrated at many promoters. In erythroid progenitors, these promoters consistently specified for key erythroid genes, such as Klf1 or Gata1, which we had shown to require increased cohesin binding during erythropoiesis. The lack of cohesin complex binding led to impairment of H3K27ac, promoter interactions, gene expression and, functionally, a block of differentiation. Conversely, in the myeloid lineage, cohesin redistribution following Stag2-KD led to a relative increase at promoters of genes that are crucial for proliferation, including Myc, Id1 and Foxo1, inducing myeloid expansion.
We confirmed our findings in a murine model of Flt3-ITD/Npm1c AML where limiting concentrations of Stag2, by shRNA-KD, caused genome-wide cohesin and H3K27ac redistribution. This generated significant alterations of transcription, a more primitive and aggressive phenotype and, in limiting dilution transplants, a marked increase in leukemia stem cell frequency (1/2812 for WT-AML to 1/25 for shS2-AML).
Conclusion
Cohesin member perturbation redistributes the binding of the remaining cohesin proteins to active promoters/enhancers, altering H3K27ac, 3D-DNA interactions and transcription. This leads to distinct and opposite patterns in the erythroid and myeloid lineages and explain the loss of differentiation, increased self-renewal and proliferation phenotypes, thereby providing a mechanistic model for (pre)leukaemic development of cohesin-impaired haematopoiesis.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Myeloid malignancies
Abstract: S122
Type: Oral Presentation
Presentation during EHA23: On Friday, June 15, 2018 from 12:15 - 12:30
Location: Room K1
Background
Myeloid malignancies are a heterogeneous continuum of neoplasia that share many common characteristics, such as altered transcriptional regulation caused by recurrent mutations of signalling, transcription and epigenetic factors. With a frequency of 15% across all myeloid malignancies, several of these mutations occur in members of the cohesin complex (STAG2, RAD21, SMC1a or SMC3, less frequent STAG1). The mutations are mutually exclusive, are predicted to cause loss of function alleles and a decrease of complex activity.
Aims
To determine a mechanism for the establishment of cohesin-deficient myeloid neoplasia.
Methods
Utilizing a model of haematopoietic stem and progenitor differentiation (HPC-7), we performed integrative analysis of cohesin binding, gene expression, chromatin state and 3D-DNA topology at genome scale, assessing different cellular states - haematopoietic progenitors, erythroid and myeloid cells - followed by matched analysis after inducible shRNA-mediated knockdown (KD) of multiple cohesin members. We finally validated the observed patterns in vitro and in vivo using a murine model of Flt3-ITD/Npm1c AML after inducible KD of Stag2 or Smc1a.
Results
We demonstrated alterations of cohesin member expression upon commitment down the erythroid but not myeloid lineage. This variable expression of cohesin translated into differing dosages at chromatin during differentiation; cohesin binding increased in the erythroid lineage at active cis-regulatory elements (enhancers/promoters), while the global cohesin dosage remained low in the myeloid lineage. Binding dynamics correlated to the H3K27 acetylation (H3K27ac), but not to gene expression, Ctcf binding or promoter interaction frequency.
Suprisingly, KD of cohesin members only minimally affected binding of the residual cohesin complex to active promoters/enhancers in immature cells, resulting in discrete changes of H3K27ac and gene expression. However, during differentiation of Stag2-perturbed haematopoiesis, significant biases of cohesin binding to active cis-regulatory elements were seen, with impaired dosage of cohesin demonstrated at many promoters. In erythroid progenitors, these promoters consistently specified for key erythroid genes, such as Klf1 or Gata1, which we had shown to require increased cohesin binding during erythropoiesis. The lack of cohesin complex binding led to impairment of H3K27ac, promoter interactions, gene expression and, functionally, a block of differentiation. Conversely, in the myeloid lineage, cohesin redistribution following Stag2-KD led to a relative increase at promoters of genes that are crucial for proliferation, including Myc, Id1 and Foxo1, inducing myeloid expansion.
We confirmed our findings in a murine model of Flt3-ITD/Npm1c AML where limiting concentrations of Stag2, by shRNA-KD, caused genome-wide cohesin and H3K27ac redistribution. This generated significant alterations of transcription, a more primitive and aggressive phenotype and, in limiting dilution transplants, a marked increase in leukemia stem cell frequency (1/2812 for WT-AML to 1/25 for shS2-AML).
Conclusion
Cohesin member perturbation redistributes the binding of the remaining cohesin proteins to active promoters/enhancers, altering H3K27ac, 3D-DNA interactions and transcription. This leads to distinct and opposite patterns in the erythroid and myeloid lineages and explain the loss of differentiation, increased self-renewal and proliferation phenotypes, thereby providing a mechanistic model for (pre)leukaemic development of cohesin-impaired haematopoiesis.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Myeloid malignancies