CELL-EXTRINSIC HAEMATOPOIETIC IMPACT OF EZH2 INACTIVATION IN FOETAL LIVER ENDOTHELIAL CELLS
(Abstract release date: 05/19/16)
EHA Library. Neo W. 06/10/16; 135177; S144
Dr. Wen Hao Neo
Contributions
Contributions
Abstract
Abstract: S144
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:30 - 12:45
Location: Room H5
Background
The cell-intrinsic role of many different epigenetic factors in the regulation of normal haematopoiesis and development of blood cancers is well established. However, the possible role and function of epigenetic regulators in components of the haematopoietic niche remains largely unexplored. Ezh2, is a histone methyltransferase and key component of the polycomb repressive complex 2 (PRC2). Loss of function of PRC2 causes a cell-intrinsic defect in adult haematopoietic stem cells (HSCs) and block in T- and B-lymphopoiesis. Further, both increased and reduced PRC2 activity has been implicated in a range of blood cancers. However, the haematopoietic impact of Ezh2 inactivation in components of the niche has not been explored.
Aims
Recent evidence supports that vascular endothelial cells (VECs) are key components of the haematopoietic niche during foetal development and we therefore aimed to study the haematopoietic impact of Ezh2 depletion in foetal liver (FL) VECs.
Methods
Ezh2 was depleted in haematopoietic cells with or without deletion in VECs using Tie2-Cre (Tie2-KO) or Vav-iCre (Vav-KO) respectively, and the impact on foetal haematopoiesis was explored through phenotypic and functional assays.
Results
At E12.5, both Vav-KO and Tie2-KO embryos showed close to 100% recombination efficiency in haematopoietic cells, whereas only Tie2-KO showed high levels of recombination in VECs (CD45-Ter119-CD31+). There was a striking difference in survival, with embryonic lethality at E13.5 in Tie2-KO embryos in contrast to Vav-KO which survived beyond E18.5. Numbers of phenotypic HSCs (LSKCD48-CD150+) were normal in Tie2-KO at E12.5 and expanded through to E18.5 in Vav-KO embryos, confirmed in functional (in vivo reconstitution) assays, excluding an impact of Ezh2 loss on FL HSCs. In contrast, there was a Tie2-KO specific loss of E12.5 FL cellularity (fc=0.44; p=0.0006) and reduction in progenitor cells (LSKCD48+CD150-; fc=0.33; p<0.0001) that was not present in Vav-KO embryos. Furthermore, Tie2-KO but not Vav-KO E12.5 FLs were severely anaemic, with a marked loss of CD71+Ter119+ erythroid precursors (fc=0.07; p=0.0010). We reasoned that depletion of Ezh2 in VECs was causing a cell-extrinsic suppression of FL progenitors, particularly those of erythropoietic lineage. This Tie2-KO phenotype was highly reminiscent of that seen in Steel mice, and although KitL mRNA expression was increased (fc=3.62; p=0.0291), immunofluorescence staining demonstrated an almost complete loss of the membrane bound form of KitL (mKL) in Tie2-KO FL. The loss of mKL was not VEC-specific, but also affected Tie2-KO hepatoblasts (CD45-Ter119-CD31-Alb+), which express KitL but are not targeted by Tie2-Cre. As mKL is a known target of Mmp9, and Mmp9 is a known target of Ezh2, we next examined Mmp9 mRNA expression and showed this was upregulated in Tie2-KO FLs (fc=12.41; p=0.0236). In order to confirm the mechanistic role of Mmp9, we demonstrated that Ezh2-depleted VECs from Tie2-KO FL had reduced capability to support erythropoietic colony formation in vitro in co-culture assays (fc=0.22; p=0.0036), which could be fully rescued with Mmp9 inhibitor treatment.
Conclusion
These data demonstrate a marked cell-extrinsic haematopoietic impact of PRC2 inactivation in VECs through loss of mKL expression caused by a marked upregulation of Mmp9 expression. This provides evidence that modulation of epigenetic regulators can have a major cell-extrinsic impact on haematopoiesis, of relevance for the development of epigenetic therapies, including Ezh2 inhibitors.
Session topic: Stem cells and the microenvironment
Keyword(s): Erythropoieisis, EZH2, Kit, MMP
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:30 - 12:45
Location: Room H5
Background
The cell-intrinsic role of many different epigenetic factors in the regulation of normal haematopoiesis and development of blood cancers is well established. However, the possible role and function of epigenetic regulators in components of the haematopoietic niche remains largely unexplored. Ezh2, is a histone methyltransferase and key component of the polycomb repressive complex 2 (PRC2). Loss of function of PRC2 causes a cell-intrinsic defect in adult haematopoietic stem cells (HSCs) and block in T- and B-lymphopoiesis. Further, both increased and reduced PRC2 activity has been implicated in a range of blood cancers. However, the haematopoietic impact of Ezh2 inactivation in components of the niche has not been explored.
Aims
Recent evidence supports that vascular endothelial cells (VECs) are key components of the haematopoietic niche during foetal development and we therefore aimed to study the haematopoietic impact of Ezh2 depletion in foetal liver (FL) VECs.
Methods
Ezh2 was depleted in haematopoietic cells with or without deletion in VECs using Tie2-Cre (Tie2-KO) or Vav-iCre (Vav-KO) respectively, and the impact on foetal haematopoiesis was explored through phenotypic and functional assays.
Results
At E12.5, both Vav-KO and Tie2-KO embryos showed close to 100% recombination efficiency in haematopoietic cells, whereas only Tie2-KO showed high levels of recombination in VECs (CD45-Ter119-CD31+). There was a striking difference in survival, with embryonic lethality at E13.5 in Tie2-KO embryos in contrast to Vav-KO which survived beyond E18.5. Numbers of phenotypic HSCs (LSKCD48-CD150+) were normal in Tie2-KO at E12.5 and expanded through to E18.5 in Vav-KO embryos, confirmed in functional (in vivo reconstitution) assays, excluding an impact of Ezh2 loss on FL HSCs. In contrast, there was a Tie2-KO specific loss of E12.5 FL cellularity (fc=0.44; p=0.0006) and reduction in progenitor cells (LSKCD48+CD150-; fc=0.33; p<0.0001) that was not present in Vav-KO embryos. Furthermore, Tie2-KO but not Vav-KO E12.5 FLs were severely anaemic, with a marked loss of CD71+Ter119+ erythroid precursors (fc=0.07; p=0.0010). We reasoned that depletion of Ezh2 in VECs was causing a cell-extrinsic suppression of FL progenitors, particularly those of erythropoietic lineage. This Tie2-KO phenotype was highly reminiscent of that seen in Steel mice, and although KitL mRNA expression was increased (fc=3.62; p=0.0291), immunofluorescence staining demonstrated an almost complete loss of the membrane bound form of KitL (mKL) in Tie2-KO FL. The loss of mKL was not VEC-specific, but also affected Tie2-KO hepatoblasts (CD45-Ter119-CD31-Alb+), which express KitL but are not targeted by Tie2-Cre. As mKL is a known target of Mmp9, and Mmp9 is a known target of Ezh2, we next examined Mmp9 mRNA expression and showed this was upregulated in Tie2-KO FLs (fc=12.41; p=0.0236). In order to confirm the mechanistic role of Mmp9, we demonstrated that Ezh2-depleted VECs from Tie2-KO FL had reduced capability to support erythropoietic colony formation in vitro in co-culture assays (fc=0.22; p=0.0036), which could be fully rescued with Mmp9 inhibitor treatment.
Conclusion
These data demonstrate a marked cell-extrinsic haematopoietic impact of PRC2 inactivation in VECs through loss of mKL expression caused by a marked upregulation of Mmp9 expression. This provides evidence that modulation of epigenetic regulators can have a major cell-extrinsic impact on haematopoiesis, of relevance for the development of epigenetic therapies, including Ezh2 inhibitors.
Session topic: Stem cells and the microenvironment
Keyword(s): Erythropoieisis, EZH2, Kit, MMP
Abstract: S144
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:30 - 12:45
Location: Room H5
Background
The cell-intrinsic role of many different epigenetic factors in the regulation of normal haematopoiesis and development of blood cancers is well established. However, the possible role and function of epigenetic regulators in components of the haematopoietic niche remains largely unexplored. Ezh2, is a histone methyltransferase and key component of the polycomb repressive complex 2 (PRC2). Loss of function of PRC2 causes a cell-intrinsic defect in adult haematopoietic stem cells (HSCs) and block in T- and B-lymphopoiesis. Further, both increased and reduced PRC2 activity has been implicated in a range of blood cancers. However, the haematopoietic impact of Ezh2 inactivation in components of the niche has not been explored.
Aims
Recent evidence supports that vascular endothelial cells (VECs) are key components of the haematopoietic niche during foetal development and we therefore aimed to study the haematopoietic impact of Ezh2 depletion in foetal liver (FL) VECs.
Methods
Ezh2 was depleted in haematopoietic cells with or without deletion in VECs using Tie2-Cre (Tie2-KO) or Vav-iCre (Vav-KO) respectively, and the impact on foetal haematopoiesis was explored through phenotypic and functional assays.
Results
At E12.5, both Vav-KO and Tie2-KO embryos showed close to 100% recombination efficiency in haematopoietic cells, whereas only Tie2-KO showed high levels of recombination in VECs (CD45-Ter119-CD31+). There was a striking difference in survival, with embryonic lethality at E13.5 in Tie2-KO embryos in contrast to Vav-KO which survived beyond E18.5. Numbers of phenotypic HSCs (LSKCD48-CD150+) were normal in Tie2-KO at E12.5 and expanded through to E18.5 in Vav-KO embryos, confirmed in functional (in vivo reconstitution) assays, excluding an impact of Ezh2 loss on FL HSCs. In contrast, there was a Tie2-KO specific loss of E12.5 FL cellularity (fc=0.44; p=0.0006) and reduction in progenitor cells (LSKCD48+CD150-; fc=0.33; p<0.0001) that was not present in Vav-KO embryos. Furthermore, Tie2-KO but not Vav-KO E12.5 FLs were severely anaemic, with a marked loss of CD71+Ter119+ erythroid precursors (fc=0.07; p=0.0010). We reasoned that depletion of Ezh2 in VECs was causing a cell-extrinsic suppression of FL progenitors, particularly those of erythropoietic lineage. This Tie2-KO phenotype was highly reminiscent of that seen in Steel mice, and although KitL mRNA expression was increased (fc=3.62; p=0.0291), immunofluorescence staining demonstrated an almost complete loss of the membrane bound form of KitL (mKL) in Tie2-KO FL. The loss of mKL was not VEC-specific, but also affected Tie2-KO hepatoblasts (CD45-Ter119-CD31-Alb+), which express KitL but are not targeted by Tie2-Cre. As mKL is a known target of Mmp9, and Mmp9 is a known target of Ezh2, we next examined Mmp9 mRNA expression and showed this was upregulated in Tie2-KO FLs (fc=12.41; p=0.0236). In order to confirm the mechanistic role of Mmp9, we demonstrated that Ezh2-depleted VECs from Tie2-KO FL had reduced capability to support erythropoietic colony formation in vitro in co-culture assays (fc=0.22; p=0.0036), which could be fully rescued with Mmp9 inhibitor treatment.
Conclusion
These data demonstrate a marked cell-extrinsic haematopoietic impact of PRC2 inactivation in VECs through loss of mKL expression caused by a marked upregulation of Mmp9 expression. This provides evidence that modulation of epigenetic regulators can have a major cell-extrinsic impact on haematopoiesis, of relevance for the development of epigenetic therapies, including Ezh2 inhibitors.
Session topic: Stem cells and the microenvironment
Keyword(s): Erythropoieisis, EZH2, Kit, MMP
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 12:30 - 12:45
Location: Room H5
Background
The cell-intrinsic role of many different epigenetic factors in the regulation of normal haematopoiesis and development of blood cancers is well established. However, the possible role and function of epigenetic regulators in components of the haematopoietic niche remains largely unexplored. Ezh2, is a histone methyltransferase and key component of the polycomb repressive complex 2 (PRC2). Loss of function of PRC2 causes a cell-intrinsic defect in adult haematopoietic stem cells (HSCs) and block in T- and B-lymphopoiesis. Further, both increased and reduced PRC2 activity has been implicated in a range of blood cancers. However, the haematopoietic impact of Ezh2 inactivation in components of the niche has not been explored.
Aims
Recent evidence supports that vascular endothelial cells (VECs) are key components of the haematopoietic niche during foetal development and we therefore aimed to study the haematopoietic impact of Ezh2 depletion in foetal liver (FL) VECs.
Methods
Ezh2 was depleted in haematopoietic cells with or without deletion in VECs using Tie2-Cre (Tie2-KO) or Vav-iCre (Vav-KO) respectively, and the impact on foetal haematopoiesis was explored through phenotypic and functional assays.
Results
At E12.5, both Vav-KO and Tie2-KO embryos showed close to 100% recombination efficiency in haematopoietic cells, whereas only Tie2-KO showed high levels of recombination in VECs (CD45-Ter119-CD31+). There was a striking difference in survival, with embryonic lethality at E13.5 in Tie2-KO embryos in contrast to Vav-KO which survived beyond E18.5. Numbers of phenotypic HSCs (LSKCD48-CD150+) were normal in Tie2-KO at E12.5 and expanded through to E18.5 in Vav-KO embryos, confirmed in functional (in vivo reconstitution) assays, excluding an impact of Ezh2 loss on FL HSCs. In contrast, there was a Tie2-KO specific loss of E12.5 FL cellularity (fc=0.44; p=0.0006) and reduction in progenitor cells (LSKCD48+CD150-; fc=0.33; p<0.0001) that was not present in Vav-KO embryos. Furthermore, Tie2-KO but not Vav-KO E12.5 FLs were severely anaemic, with a marked loss of CD71+Ter119+ erythroid precursors (fc=0.07; p=0.0010). We reasoned that depletion of Ezh2 in VECs was causing a cell-extrinsic suppression of FL progenitors, particularly those of erythropoietic lineage. This Tie2-KO phenotype was highly reminiscent of that seen in Steel mice, and although KitL mRNA expression was increased (fc=3.62; p=0.0291), immunofluorescence staining demonstrated an almost complete loss of the membrane bound form of KitL (mKL) in Tie2-KO FL. The loss of mKL was not VEC-specific, but also affected Tie2-KO hepatoblasts (CD45-Ter119-CD31-Alb+), which express KitL but are not targeted by Tie2-Cre. As mKL is a known target of Mmp9, and Mmp9 is a known target of Ezh2, we next examined Mmp9 mRNA expression and showed this was upregulated in Tie2-KO FLs (fc=12.41; p=0.0236). In order to confirm the mechanistic role of Mmp9, we demonstrated that Ezh2-depleted VECs from Tie2-KO FL had reduced capability to support erythropoietic colony formation in vitro in co-culture assays (fc=0.22; p=0.0036), which could be fully rescued with Mmp9 inhibitor treatment.
Conclusion
These data demonstrate a marked cell-extrinsic haematopoietic impact of PRC2 inactivation in VECs through loss of mKL expression caused by a marked upregulation of Mmp9 expression. This provides evidence that modulation of epigenetic regulators can have a major cell-extrinsic impact on haematopoiesis, of relevance for the development of epigenetic therapies, including Ezh2 inhibitors.
Session topic: Stem cells and the microenvironment
Keyword(s): Erythropoieisis, EZH2, Kit, MMP
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