CD97 EXPRESSION MEDIATES MIGRATION AND ADHESION OF FLT3-ITD POSITIVE ACUTE MYELOID LEUKEMIA CELLS AND MODULATES THE BONE MARROW STROMAL MICROENVIRONMENT
(Abstract release date: 05/19/16)
EHA Library. Wobus M. 06/09/16; 132446; E897
Dr. Manja Wobus
Contributions
Contributions
Abstract
Abstract: E897
Type: Eposter Presentation
Background
Bone marrow niches are specialized microenvironments that facilitate homing and survival of normal hematopoietic stem and progenitor cells (HSPCs) as well as leukemic stem cells (LCSs). Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. However, defined mechanisms mediating interactions of acute myeloid leukemia (AML) cells with the microenvironment remain largely unexplored. We have recently demonstrated that expression of the adhesion GPCR CD97 is elevated in blasts of FLT3-ITD positive AML patients.
Aims
Here, we investigated the underlying mechanisms in more detail and the impact on mesenchymal stromal cells (MSCs) as a main cellular component of the bone marrow niche in vitro.
Methods
FLT3-ITD MV4-11 and FLT3 wildtype OCI-AML3 AML cells were used for the in vitro experiments. CD97 knock-down was achieved by lentiviral transduction of plko1.6/shRNACD97. Primary MSCs were isolated from bone marrow aspirates of healthy donors and co-cultured with AML cell lines in direct or indirect manner. Expression analyses were carried out by flow cytometry and Western blot. Trans-well migration was analysed in a Boyden chamber assay and the deformation capacity of the AML cells was investigated by real-time deformability cytometry (RT-DC).
Results
CD97 knock-down by lentiviral transduction of plko1.6/shRNACD97 in MV4-11 leukemic cells caused inhibited trans-well migration towards FCS and LPA which is at least in part Rho-ROCK pathway dependent. Adhesion to a stromal layer was significantly decreased within two days and immunoblotting revealed inhibited Akt phosphorylation in the CD97 knock-down cells. The expression of the lysophosphatidic acid (LPA) receptor correlated with CD97 levels in FLT3-ITD MV4-11 but not in FLT3 wildtype OCI-AML3 cells indicating a physical interaction of these receptors. Changes in size as well as the deformation capacity after retroviral transduction of leukemic cells suggested effects on actin-myosin cytoskeleton dynamics.In the second part of the study, we tested the impact of leukemic cells and their CD97 expression on the MSC phenotype. FACS analysis was performed after three days of MSC incubation with leukemic cell lines or their conditioned medium, respectively. Interestingly, CD90 and CD146 expression levels were increased by about 50% after coculture with MV4-11 wildtype cells but declined to the basic level after incubation with CD97 knock-down cells. In contrast, the expression levels of CD73 were increased by MV4-11 and even further elevated by CD97 knock-down cells. Comparable results were observed after MSC culture with conditioned medium of MV4-11 cells.
Conclusion
In summary, our data suggest a modulation of the bone marrow microenvironment by FLT3-ITD positive leukemic cells expressing CD97 in association with LPA receptor rendering it a potential new theragnostic target.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Bone marrow stroma, Flt3-ITD, Mesenchymal stem cell
Type: Eposter Presentation
Background
Bone marrow niches are specialized microenvironments that facilitate homing and survival of normal hematopoietic stem and progenitor cells (HSPCs) as well as leukemic stem cells (LCSs). Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. However, defined mechanisms mediating interactions of acute myeloid leukemia (AML) cells with the microenvironment remain largely unexplored. We have recently demonstrated that expression of the adhesion GPCR CD97 is elevated in blasts of FLT3-ITD positive AML patients.
Aims
Here, we investigated the underlying mechanisms in more detail and the impact on mesenchymal stromal cells (MSCs) as a main cellular component of the bone marrow niche in vitro.
Methods
FLT3-ITD MV4-11 and FLT3 wildtype OCI-AML3 AML cells were used for the in vitro experiments. CD97 knock-down was achieved by lentiviral transduction of plko1.6/shRNACD97. Primary MSCs were isolated from bone marrow aspirates of healthy donors and co-cultured with AML cell lines in direct or indirect manner. Expression analyses were carried out by flow cytometry and Western blot. Trans-well migration was analysed in a Boyden chamber assay and the deformation capacity of the AML cells was investigated by real-time deformability cytometry (RT-DC).
Results
CD97 knock-down by lentiviral transduction of plko1.6/shRNACD97 in MV4-11 leukemic cells caused inhibited trans-well migration towards FCS and LPA which is at least in part Rho-ROCK pathway dependent. Adhesion to a stromal layer was significantly decreased within two days and immunoblotting revealed inhibited Akt phosphorylation in the CD97 knock-down cells. The expression of the lysophosphatidic acid (LPA) receptor correlated with CD97 levels in FLT3-ITD MV4-11 but not in FLT3 wildtype OCI-AML3 cells indicating a physical interaction of these receptors. Changes in size as well as the deformation capacity after retroviral transduction of leukemic cells suggested effects on actin-myosin cytoskeleton dynamics.In the second part of the study, we tested the impact of leukemic cells and their CD97 expression on the MSC phenotype. FACS analysis was performed after three days of MSC incubation with leukemic cell lines or their conditioned medium, respectively. Interestingly, CD90 and CD146 expression levels were increased by about 50% after coculture with MV4-11 wildtype cells but declined to the basic level after incubation with CD97 knock-down cells. In contrast, the expression levels of CD73 were increased by MV4-11 and even further elevated by CD97 knock-down cells. Comparable results were observed after MSC culture with conditioned medium of MV4-11 cells.
Conclusion
In summary, our data suggest a modulation of the bone marrow microenvironment by FLT3-ITD positive leukemic cells expressing CD97 in association with LPA receptor rendering it a potential new theragnostic target.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Bone marrow stroma, Flt3-ITD, Mesenchymal stem cell
Abstract: E897
Type: Eposter Presentation
Background
Bone marrow niches are specialized microenvironments that facilitate homing and survival of normal hematopoietic stem and progenitor cells (HSPCs) as well as leukemic stem cells (LCSs). Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. However, defined mechanisms mediating interactions of acute myeloid leukemia (AML) cells with the microenvironment remain largely unexplored. We have recently demonstrated that expression of the adhesion GPCR CD97 is elevated in blasts of FLT3-ITD positive AML patients.
Aims
Here, we investigated the underlying mechanisms in more detail and the impact on mesenchymal stromal cells (MSCs) as a main cellular component of the bone marrow niche in vitro.
Methods
FLT3-ITD MV4-11 and FLT3 wildtype OCI-AML3 AML cells were used for the in vitro experiments. CD97 knock-down was achieved by lentiviral transduction of plko1.6/shRNACD97. Primary MSCs were isolated from bone marrow aspirates of healthy donors and co-cultured with AML cell lines in direct or indirect manner. Expression analyses were carried out by flow cytometry and Western blot. Trans-well migration was analysed in a Boyden chamber assay and the deformation capacity of the AML cells was investigated by real-time deformability cytometry (RT-DC).
Results
CD97 knock-down by lentiviral transduction of plko1.6/shRNACD97 in MV4-11 leukemic cells caused inhibited trans-well migration towards FCS and LPA which is at least in part Rho-ROCK pathway dependent. Adhesion to a stromal layer was significantly decreased within two days and immunoblotting revealed inhibited Akt phosphorylation in the CD97 knock-down cells. The expression of the lysophosphatidic acid (LPA) receptor correlated with CD97 levels in FLT3-ITD MV4-11 but not in FLT3 wildtype OCI-AML3 cells indicating a physical interaction of these receptors. Changes in size as well as the deformation capacity after retroviral transduction of leukemic cells suggested effects on actin-myosin cytoskeleton dynamics.In the second part of the study, we tested the impact of leukemic cells and their CD97 expression on the MSC phenotype. FACS analysis was performed after three days of MSC incubation with leukemic cell lines or their conditioned medium, respectively. Interestingly, CD90 and CD146 expression levels were increased by about 50% after coculture with MV4-11 wildtype cells but declined to the basic level after incubation with CD97 knock-down cells. In contrast, the expression levels of CD73 were increased by MV4-11 and even further elevated by CD97 knock-down cells. Comparable results were observed after MSC culture with conditioned medium of MV4-11 cells.
Conclusion
In summary, our data suggest a modulation of the bone marrow microenvironment by FLT3-ITD positive leukemic cells expressing CD97 in association with LPA receptor rendering it a potential new theragnostic target.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Bone marrow stroma, Flt3-ITD, Mesenchymal stem cell
Type: Eposter Presentation
Background
Bone marrow niches are specialized microenvironments that facilitate homing and survival of normal hematopoietic stem and progenitor cells (HSPCs) as well as leukemic stem cells (LCSs). Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. However, defined mechanisms mediating interactions of acute myeloid leukemia (AML) cells with the microenvironment remain largely unexplored. We have recently demonstrated that expression of the adhesion GPCR CD97 is elevated in blasts of FLT3-ITD positive AML patients.
Aims
Here, we investigated the underlying mechanisms in more detail and the impact on mesenchymal stromal cells (MSCs) as a main cellular component of the bone marrow niche in vitro.
Methods
FLT3-ITD MV4-11 and FLT3 wildtype OCI-AML3 AML cells were used for the in vitro experiments. CD97 knock-down was achieved by lentiviral transduction of plko1.6/shRNACD97. Primary MSCs were isolated from bone marrow aspirates of healthy donors and co-cultured with AML cell lines in direct or indirect manner. Expression analyses were carried out by flow cytometry and Western blot. Trans-well migration was analysed in a Boyden chamber assay and the deformation capacity of the AML cells was investigated by real-time deformability cytometry (RT-DC).
Results
CD97 knock-down by lentiviral transduction of plko1.6/shRNACD97 in MV4-11 leukemic cells caused inhibited trans-well migration towards FCS and LPA which is at least in part Rho-ROCK pathway dependent. Adhesion to a stromal layer was significantly decreased within two days and immunoblotting revealed inhibited Akt phosphorylation in the CD97 knock-down cells. The expression of the lysophosphatidic acid (LPA) receptor correlated with CD97 levels in FLT3-ITD MV4-11 but not in FLT3 wildtype OCI-AML3 cells indicating a physical interaction of these receptors. Changes in size as well as the deformation capacity after retroviral transduction of leukemic cells suggested effects on actin-myosin cytoskeleton dynamics.In the second part of the study, we tested the impact of leukemic cells and their CD97 expression on the MSC phenotype. FACS analysis was performed after three days of MSC incubation with leukemic cell lines or their conditioned medium, respectively. Interestingly, CD90 and CD146 expression levels were increased by about 50% after coculture with MV4-11 wildtype cells but declined to the basic level after incubation with CD97 knock-down cells. In contrast, the expression levels of CD73 were increased by MV4-11 and even further elevated by CD97 knock-down cells. Comparable results were observed after MSC culture with conditioned medium of MV4-11 cells.
Conclusion
In summary, our data suggest a modulation of the bone marrow microenvironment by FLT3-ITD positive leukemic cells expressing CD97 in association with LPA receptor rendering it a potential new theragnostic target.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Bone marrow stroma, Flt3-ITD, Mesenchymal stem cell
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