REACTIVE OXYGEN SPECIES (ROS) MODULATES SENSITIVITY TO FLT3-TYROSINE KINASE INHIBITORS IN MURINE AND HUMAN FLT3-ITD POSITIVE AML CELL LINES.
(Abstract release date: 05/19/16)
EHA Library. Mueller C. 06/09/16; 132434; E885
Dr. Christian Mueller
Contributions
Contributions
Abstract
Abstract: E885
Type: Eposter Presentation
Background
FLT3-ITD positive acute myeloid leukemia (AML) is characterized by an increased concentration of intracellular ROS and high extracellular concentrations of hydrogen peroxide (H2O2). Increased ROS levels regulate numerous cellular processes, such as cell cycle, apoptosis, metabolism and drug resistance. Interestingly, in leukemia driven by other oncogenes (e.g. Bcr-Abl) it has been described that modulation of ROS may improve the response to selective tyrosine kinase inhibitor (TKI) treatment. Some pathways, including STAT5/NOX4, have been reported to induce high ROS levels in FLT3-ITD-positive AML. However, it is unclear how FLT3-ITD positive AML cells modulate their cellular functions and/or drug resistance by auto-/paracrine ROS species. In addition, in leukemia driven by other oncogenes (e.g. Bcr-Abl) it has been described that modulation of ROS may improve the response to selective tyrosine kinase inhibitor (TKI) treatment.
Aims
Our goal was to identify the auto-/paracrine effects of hydrogen peroxide on proliferation and cell cycle progression in FLT3-ITD positive murine and human cell lines. In addition, we aimed to characterize the effects of H2O2 modulation with respect to the sensitivity to TKI treatement as a possible resistance-inducing factor.
Methods
Murine (32D, BaF3) and human (MOLM-13, MV4-11) FLT3-ITD positive cell lines were analyzed for intracellular ROS levels using H2DCFD, extracellular H2O2 concentration and activities of the scavenging enzymes SOD1, SOD2, catalase, and glutathione peroxidase (GPx). For the analysis of the role of extracellular H2O2, cells were incubated directly with H2O2 (10 µM) for 2h and assayed for the activation of FLT3-ITD-dependent signaling pathways (STAT5, Akt and Erk) using immunoblotting. For the analysis of the effects of H2O2-generating enzyme glucose oxidase and of the H2O2-degrading enzyme catalase, cells were cultured for 48h and assayed for proliferation (MTT) and cell cycle progression (PI). Furthermore, the effect of extracellular H2O2 concentration on TKI treatment (AC220, PKC412; each 5 to 50 nM) was analyzed by FACS.
Results
Stimulation of FLT3-ITD positive cells using H2O2 resulted in a strongly increased time-dependent activation of FLT3-ITD-associated signaling pathways (STAT5, Akt, Erk) as compared to untreated cells. This difference was more pronounced in ITD-positive cells in comparison to WT cells particularly for AKT activation. In addition, stimulation with H2O2 also increased the activities of SOD1/2 and catalase.The long-term increase in extracellular H2O2 concentration was mimicked experimentally by addition of glucose oxidase to the culture medium (0.75 U/ml). Interestingly, this resulted in enhanced proliferation and cell cycle progression, whereas cultivation with catalase led to decreased proliferation and cell cycle arrest.Combined treatment with catalase and TKIs (AC220, PKC412; 5 and 10 nM, respectively) resulted in a significantly higher rate of apoptosis. Of note, the response to both TKIs was significantly reduced when glucose oxidase (0.75 U/ml) was added to the medium. The decrease in apoptosis was associated with stabilization of the mitochondrial potential (TMRE assay), stable expression of the anti-apoptotic proteins Mcl-1 and Bcl-XL, and a reduced cleavage of caspase 3 and caspase 8.
Conclusion
Our results strongly suggest that H2O2 acts as an auto-/paracrine stimulator of proliferation and cell cycle progression in FLT3-ITD positive cells.. Furthermore, we could characterize H2O2 as a regulatory element with respect to TKI sensitivity. Our results indicate that combined inhibition of FLT3-ITD and H2O2 reducing agents/enzymes, like catalase, may have potential clinical application in treatment of FLT3-ITD positive AML patients.
Session topic: E-poster
Keyword(s): AML, FLT3, Kinase inhibitor, Reactive oxygen species
Type: Eposter Presentation
Background
FLT3-ITD positive acute myeloid leukemia (AML) is characterized by an increased concentration of intracellular ROS and high extracellular concentrations of hydrogen peroxide (H2O2). Increased ROS levels regulate numerous cellular processes, such as cell cycle, apoptosis, metabolism and drug resistance. Interestingly, in leukemia driven by other oncogenes (e.g. Bcr-Abl) it has been described that modulation of ROS may improve the response to selective tyrosine kinase inhibitor (TKI) treatment. Some pathways, including STAT5/NOX4, have been reported to induce high ROS levels in FLT3-ITD-positive AML. However, it is unclear how FLT3-ITD positive AML cells modulate their cellular functions and/or drug resistance by auto-/paracrine ROS species. In addition, in leukemia driven by other oncogenes (e.g. Bcr-Abl) it has been described that modulation of ROS may improve the response to selective tyrosine kinase inhibitor (TKI) treatment.
Aims
Our goal was to identify the auto-/paracrine effects of hydrogen peroxide on proliferation and cell cycle progression in FLT3-ITD positive murine and human cell lines. In addition, we aimed to characterize the effects of H2O2 modulation with respect to the sensitivity to TKI treatement as a possible resistance-inducing factor.
Methods
Murine (32D, BaF3) and human (MOLM-13, MV4-11) FLT3-ITD positive cell lines were analyzed for intracellular ROS levels using H2DCFD, extracellular H2O2 concentration and activities of the scavenging enzymes SOD1, SOD2, catalase, and glutathione peroxidase (GPx). For the analysis of the role of extracellular H2O2, cells were incubated directly with H2O2 (10 µM) for 2h and assayed for the activation of FLT3-ITD-dependent signaling pathways (STAT5, Akt and Erk) using immunoblotting. For the analysis of the effects of H2O2-generating enzyme glucose oxidase and of the H2O2-degrading enzyme catalase, cells were cultured for 48h and assayed for proliferation (MTT) and cell cycle progression (PI). Furthermore, the effect of extracellular H2O2 concentration on TKI treatment (AC220, PKC412; each 5 to 50 nM) was analyzed by FACS.
Results
Stimulation of FLT3-ITD positive cells using H2O2 resulted in a strongly increased time-dependent activation of FLT3-ITD-associated signaling pathways (STAT5, Akt, Erk) as compared to untreated cells. This difference was more pronounced in ITD-positive cells in comparison to WT cells particularly for AKT activation. In addition, stimulation with H2O2 also increased the activities of SOD1/2 and catalase.The long-term increase in extracellular H2O2 concentration was mimicked experimentally by addition of glucose oxidase to the culture medium (0.75 U/ml). Interestingly, this resulted in enhanced proliferation and cell cycle progression, whereas cultivation with catalase led to decreased proliferation and cell cycle arrest.Combined treatment with catalase and TKIs (AC220, PKC412; 5 and 10 nM, respectively) resulted in a significantly higher rate of apoptosis. Of note, the response to both TKIs was significantly reduced when glucose oxidase (0.75 U/ml) was added to the medium. The decrease in apoptosis was associated with stabilization of the mitochondrial potential (TMRE assay), stable expression of the anti-apoptotic proteins Mcl-1 and Bcl-XL, and a reduced cleavage of caspase 3 and caspase 8.
Conclusion
Our results strongly suggest that H2O2 acts as an auto-/paracrine stimulator of proliferation and cell cycle progression in FLT3-ITD positive cells.. Furthermore, we could characterize H2O2 as a regulatory element with respect to TKI sensitivity. Our results indicate that combined inhibition of FLT3-ITD and H2O2 reducing agents/enzymes, like catalase, may have potential clinical application in treatment of FLT3-ITD positive AML patients.
Session topic: E-poster
Keyword(s): AML, FLT3, Kinase inhibitor, Reactive oxygen species
Abstract: E885
Type: Eposter Presentation
Background
FLT3-ITD positive acute myeloid leukemia (AML) is characterized by an increased concentration of intracellular ROS and high extracellular concentrations of hydrogen peroxide (H2O2). Increased ROS levels regulate numerous cellular processes, such as cell cycle, apoptosis, metabolism and drug resistance. Interestingly, in leukemia driven by other oncogenes (e.g. Bcr-Abl) it has been described that modulation of ROS may improve the response to selective tyrosine kinase inhibitor (TKI) treatment. Some pathways, including STAT5/NOX4, have been reported to induce high ROS levels in FLT3-ITD-positive AML. However, it is unclear how FLT3-ITD positive AML cells modulate their cellular functions and/or drug resistance by auto-/paracrine ROS species. In addition, in leukemia driven by other oncogenes (e.g. Bcr-Abl) it has been described that modulation of ROS may improve the response to selective tyrosine kinase inhibitor (TKI) treatment.
Aims
Our goal was to identify the auto-/paracrine effects of hydrogen peroxide on proliferation and cell cycle progression in FLT3-ITD positive murine and human cell lines. In addition, we aimed to characterize the effects of H2O2 modulation with respect to the sensitivity to TKI treatement as a possible resistance-inducing factor.
Methods
Murine (32D, BaF3) and human (MOLM-13, MV4-11) FLT3-ITD positive cell lines were analyzed for intracellular ROS levels using H2DCFD, extracellular H2O2 concentration and activities of the scavenging enzymes SOD1, SOD2, catalase, and glutathione peroxidase (GPx). For the analysis of the role of extracellular H2O2, cells were incubated directly with H2O2 (10 µM) for 2h and assayed for the activation of FLT3-ITD-dependent signaling pathways (STAT5, Akt and Erk) using immunoblotting. For the analysis of the effects of H2O2-generating enzyme glucose oxidase and of the H2O2-degrading enzyme catalase, cells were cultured for 48h and assayed for proliferation (MTT) and cell cycle progression (PI). Furthermore, the effect of extracellular H2O2 concentration on TKI treatment (AC220, PKC412; each 5 to 50 nM) was analyzed by FACS.
Results
Stimulation of FLT3-ITD positive cells using H2O2 resulted in a strongly increased time-dependent activation of FLT3-ITD-associated signaling pathways (STAT5, Akt, Erk) as compared to untreated cells. This difference was more pronounced in ITD-positive cells in comparison to WT cells particularly for AKT activation. In addition, stimulation with H2O2 also increased the activities of SOD1/2 and catalase.The long-term increase in extracellular H2O2 concentration was mimicked experimentally by addition of glucose oxidase to the culture medium (0.75 U/ml). Interestingly, this resulted in enhanced proliferation and cell cycle progression, whereas cultivation with catalase led to decreased proliferation and cell cycle arrest.Combined treatment with catalase and TKIs (AC220, PKC412; 5 and 10 nM, respectively) resulted in a significantly higher rate of apoptosis. Of note, the response to both TKIs was significantly reduced when glucose oxidase (0.75 U/ml) was added to the medium. The decrease in apoptosis was associated with stabilization of the mitochondrial potential (TMRE assay), stable expression of the anti-apoptotic proteins Mcl-1 and Bcl-XL, and a reduced cleavage of caspase 3 and caspase 8.
Conclusion
Our results strongly suggest that H2O2 acts as an auto-/paracrine stimulator of proliferation and cell cycle progression in FLT3-ITD positive cells.. Furthermore, we could characterize H2O2 as a regulatory element with respect to TKI sensitivity. Our results indicate that combined inhibition of FLT3-ITD and H2O2 reducing agents/enzymes, like catalase, may have potential clinical application in treatment of FLT3-ITD positive AML patients.
Session topic: E-poster
Keyword(s): AML, FLT3, Kinase inhibitor, Reactive oxygen species
Type: Eposter Presentation
Background
FLT3-ITD positive acute myeloid leukemia (AML) is characterized by an increased concentration of intracellular ROS and high extracellular concentrations of hydrogen peroxide (H2O2). Increased ROS levels regulate numerous cellular processes, such as cell cycle, apoptosis, metabolism and drug resistance. Interestingly, in leukemia driven by other oncogenes (e.g. Bcr-Abl) it has been described that modulation of ROS may improve the response to selective tyrosine kinase inhibitor (TKI) treatment. Some pathways, including STAT5/NOX4, have been reported to induce high ROS levels in FLT3-ITD-positive AML. However, it is unclear how FLT3-ITD positive AML cells modulate their cellular functions and/or drug resistance by auto-/paracrine ROS species. In addition, in leukemia driven by other oncogenes (e.g. Bcr-Abl) it has been described that modulation of ROS may improve the response to selective tyrosine kinase inhibitor (TKI) treatment.
Aims
Our goal was to identify the auto-/paracrine effects of hydrogen peroxide on proliferation and cell cycle progression in FLT3-ITD positive murine and human cell lines. In addition, we aimed to characterize the effects of H2O2 modulation with respect to the sensitivity to TKI treatement as a possible resistance-inducing factor.
Methods
Murine (32D, BaF3) and human (MOLM-13, MV4-11) FLT3-ITD positive cell lines were analyzed for intracellular ROS levels using H2DCFD, extracellular H2O2 concentration and activities of the scavenging enzymes SOD1, SOD2, catalase, and glutathione peroxidase (GPx). For the analysis of the role of extracellular H2O2, cells were incubated directly with H2O2 (10 µM) for 2h and assayed for the activation of FLT3-ITD-dependent signaling pathways (STAT5, Akt and Erk) using immunoblotting. For the analysis of the effects of H2O2-generating enzyme glucose oxidase and of the H2O2-degrading enzyme catalase, cells were cultured for 48h and assayed for proliferation (MTT) and cell cycle progression (PI). Furthermore, the effect of extracellular H2O2 concentration on TKI treatment (AC220, PKC412; each 5 to 50 nM) was analyzed by FACS.
Results
Stimulation of FLT3-ITD positive cells using H2O2 resulted in a strongly increased time-dependent activation of FLT3-ITD-associated signaling pathways (STAT5, Akt, Erk) as compared to untreated cells. This difference was more pronounced in ITD-positive cells in comparison to WT cells particularly for AKT activation. In addition, stimulation with H2O2 also increased the activities of SOD1/2 and catalase.The long-term increase in extracellular H2O2 concentration was mimicked experimentally by addition of glucose oxidase to the culture medium (0.75 U/ml). Interestingly, this resulted in enhanced proliferation and cell cycle progression, whereas cultivation with catalase led to decreased proliferation and cell cycle arrest.Combined treatment with catalase and TKIs (AC220, PKC412; 5 and 10 nM, respectively) resulted in a significantly higher rate of apoptosis. Of note, the response to both TKIs was significantly reduced when glucose oxidase (0.75 U/ml) was added to the medium. The decrease in apoptosis was associated with stabilization of the mitochondrial potential (TMRE assay), stable expression of the anti-apoptotic proteins Mcl-1 and Bcl-XL, and a reduced cleavage of caspase 3 and caspase 8.
Conclusion
Our results strongly suggest that H2O2 acts as an auto-/paracrine stimulator of proliferation and cell cycle progression in FLT3-ITD positive cells.. Furthermore, we could characterize H2O2 as a regulatory element with respect to TKI sensitivity. Our results indicate that combined inhibition of FLT3-ITD and H2O2 reducing agents/enzymes, like catalase, may have potential clinical application in treatment of FLT3-ITD positive AML patients.
Session topic: E-poster
Keyword(s): AML, FLT3, Kinase inhibitor, Reactive oxygen species
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