LPS STIMULATION INDUCES ROS FORMATION, APOPTOSIS, DNA DAMAGE AND OXIDATION OF CYSTEINE RESIDUES IN FLT3-ITD POSITIVE CELL LINES RESULTING IN FLT3-TYROSINE KINASE INHIBITOR RESISTANCE
(Abstract release date: 05/19/16)
EHA Library. Mueller C. 06/09/16; 132426; E877
Dr. Christian Mueller
Contributions
Contributions
Abstract
Abstract: E877
Type: Eposter Presentation
Background
Recurrent infections are associated with an increased incidence in the development of malignancies, including acute myeloid leukemia (AML). Inflammatory events directly induce formation of reactive oxygen species (ROS) and cause DNA damage, protein and lipid oxidation. In case of recurrent infections, bacterial peptides, like LPS, can lead to ROS induction. In particular, cysteine residues of proteins can directly be oxidized by ROS causing either loss or gain of function. However, it is still unclear how inflammatory events can induce these protein oxidation and to what extent the oxidation of cysteine residues in a subset of AML, the FLT3-ITD positive AML, causes a change of biological behavior.
Aims
The goal of our study was to characterize the role of LPS-induced oxidative stress in murine and human FLT3-ITD positive cell lines to identify responsible FLT3-ITD- and TLR related signaling pathways. To adress stress-induced cysteine oxidation of the FLT3-kinase, we generated cysteine-to-alanine mutants in murine myeloid 32D cells (FLT3-WT and FLT3-ITD) and investigated the induction of ROS formation, proliferation, cell cycle distribution and the response to TKI treatment (AC220, PKC412).
Methods
32D cells stably transduced with either FLT3-WT or FLT3-ITD and human FLT3-ITD positive MV4,11 were stimulated with LPS (5-50 ng/ml) for 2h and with 10 ng/ml LPS for different time-points (0-90 min), respectively. ROS formation (H2DCFDA method) and activation of FLT3-ITD and TLR-related signaling pathways were analyzed by immunoblotting. To analyze effects of chronic inflammation, FLT3-WT and FLT3-ITD positive cells were kept in culture for 12 days with repeated LPS stimulation (10 ng/ml every 48h). To address the role of cysteine oxidation within the FLT3-kinase, we mutated highly conserved cysteine residues C474 (extracellular domain) and C925 (intracellular domain) to alanine by site-directed mutagenesis and performed a retroviral transduction of 32D cells with the respective constructs. Subsequently, cysteine mutants were characterized for proliferation (MTT), ROS formation and their sensitivity to TKI treatment (AC220, PKC412; 5 to 50 nM, respectively) in comparison to standard FLT3-ITD mutant.
Results
LPS stimulation resulted in an increased time- and dose-dependent ROS induction in FLT3-ITD positive cells compared to WT cells (100ng/ml LPS for 2h: 6-fold). In addition, LPS stimulation resulted in a stronger activation of FLT3-ITD- and TLR-related signaling pathways in ITD-positive cells (Stat5, Akt, Erk, NFB). Long-term LPS stimulation over 12 days induced an enhanced ROS formation, apoptosis and accumulation of H2AX in both FLT3-WT, as well as in FLT3-ITD positive cells. To address the role of cysteine oxidation, we generated stable cysteine-to-alanine-mutants. However, the FLT3-ITD C925A mutation (intracelullar domain) resulted in an increase of ROS formation compared to the FLT3-ITD C474A mutation (extracellular domain; 25% increase). Furthermore, the C925A mutation resulted in a significant decrease in apoptosis after TKI treatment compared to non-mutated FLT3-ITD and the FLT3-ITD C474A mutant (AC220 50nM: 25%; PKC412 50nM: 40%). In addition, FLT3-ITD C925A mutant showed a reduced decrease in phosphorylation of Stat5 and Akt after short-term (1h) TKI treatment (AC220: 50, 100 nM; PKC412 10, 100nM) compared to non-mutated and FLT3-ITD C474A mutants.
Conclusion
Our study demonstrates that inflammatory events, like LPS stimulation, induce ROS formation, apoptosis and DNA damage in FLT3-WT and FLT3-ITD positive cells. This might lead to genetic instability and induction of critical mutations, e.g. within FLT3 kinase. Additionally, we could show that cysteine oxidation within the intracellular domain of FLT3-ITD resulted in a resistance to TKI treatment.
Session topic: E-poster
Keyword(s): AML, Flt3-ITD, Kinase inhibitor, Resistance
Type: Eposter Presentation
Background
Recurrent infections are associated with an increased incidence in the development of malignancies, including acute myeloid leukemia (AML). Inflammatory events directly induce formation of reactive oxygen species (ROS) and cause DNA damage, protein and lipid oxidation. In case of recurrent infections, bacterial peptides, like LPS, can lead to ROS induction. In particular, cysteine residues of proteins can directly be oxidized by ROS causing either loss or gain of function. However, it is still unclear how inflammatory events can induce these protein oxidation and to what extent the oxidation of cysteine residues in a subset of AML, the FLT3-ITD positive AML, causes a change of biological behavior.
Aims
The goal of our study was to characterize the role of LPS-induced oxidative stress in murine and human FLT3-ITD positive cell lines to identify responsible FLT3-ITD- and TLR related signaling pathways. To adress stress-induced cysteine oxidation of the FLT3-kinase, we generated cysteine-to-alanine mutants in murine myeloid 32D cells (FLT3-WT and FLT3-ITD) and investigated the induction of ROS formation, proliferation, cell cycle distribution and the response to TKI treatment (AC220, PKC412).
Methods
32D cells stably transduced with either FLT3-WT or FLT3-ITD and human FLT3-ITD positive MV4,11 were stimulated with LPS (5-50 ng/ml) for 2h and with 10 ng/ml LPS for different time-points (0-90 min), respectively. ROS formation (H2DCFDA method) and activation of FLT3-ITD and TLR-related signaling pathways were analyzed by immunoblotting. To analyze effects of chronic inflammation, FLT3-WT and FLT3-ITD positive cells were kept in culture for 12 days with repeated LPS stimulation (10 ng/ml every 48h). To address the role of cysteine oxidation within the FLT3-kinase, we mutated highly conserved cysteine residues C474 (extracellular domain) and C925 (intracellular domain) to alanine by site-directed mutagenesis and performed a retroviral transduction of 32D cells with the respective constructs. Subsequently, cysteine mutants were characterized for proliferation (MTT), ROS formation and their sensitivity to TKI treatment (AC220, PKC412; 5 to 50 nM, respectively) in comparison to standard FLT3-ITD mutant.
Results
LPS stimulation resulted in an increased time- and dose-dependent ROS induction in FLT3-ITD positive cells compared to WT cells (100ng/ml LPS for 2h: 6-fold). In addition, LPS stimulation resulted in a stronger activation of FLT3-ITD- and TLR-related signaling pathways in ITD-positive cells (Stat5, Akt, Erk, NFB). Long-term LPS stimulation over 12 days induced an enhanced ROS formation, apoptosis and accumulation of H2AX in both FLT3-WT, as well as in FLT3-ITD positive cells. To address the role of cysteine oxidation, we generated stable cysteine-to-alanine-mutants. However, the FLT3-ITD C925A mutation (intracelullar domain) resulted in an increase of ROS formation compared to the FLT3-ITD C474A mutation (extracellular domain; 25% increase). Furthermore, the C925A mutation resulted in a significant decrease in apoptosis after TKI treatment compared to non-mutated FLT3-ITD and the FLT3-ITD C474A mutant (AC220 50nM: 25%; PKC412 50nM: 40%). In addition, FLT3-ITD C925A mutant showed a reduced decrease in phosphorylation of Stat5 and Akt after short-term (1h) TKI treatment (AC220: 50, 100 nM; PKC412 10, 100nM) compared to non-mutated and FLT3-ITD C474A mutants.
Conclusion
Our study demonstrates that inflammatory events, like LPS stimulation, induce ROS formation, apoptosis and DNA damage in FLT3-WT and FLT3-ITD positive cells. This might lead to genetic instability and induction of critical mutations, e.g. within FLT3 kinase. Additionally, we could show that cysteine oxidation within the intracellular domain of FLT3-ITD resulted in a resistance to TKI treatment.
Session topic: E-poster
Keyword(s): AML, Flt3-ITD, Kinase inhibitor, Resistance
Abstract: E877
Type: Eposter Presentation
Background
Recurrent infections are associated with an increased incidence in the development of malignancies, including acute myeloid leukemia (AML). Inflammatory events directly induce formation of reactive oxygen species (ROS) and cause DNA damage, protein and lipid oxidation. In case of recurrent infections, bacterial peptides, like LPS, can lead to ROS induction. In particular, cysteine residues of proteins can directly be oxidized by ROS causing either loss or gain of function. However, it is still unclear how inflammatory events can induce these protein oxidation and to what extent the oxidation of cysteine residues in a subset of AML, the FLT3-ITD positive AML, causes a change of biological behavior.
Aims
The goal of our study was to characterize the role of LPS-induced oxidative stress in murine and human FLT3-ITD positive cell lines to identify responsible FLT3-ITD- and TLR related signaling pathways. To adress stress-induced cysteine oxidation of the FLT3-kinase, we generated cysteine-to-alanine mutants in murine myeloid 32D cells (FLT3-WT and FLT3-ITD) and investigated the induction of ROS formation, proliferation, cell cycle distribution and the response to TKI treatment (AC220, PKC412).
Methods
32D cells stably transduced with either FLT3-WT or FLT3-ITD and human FLT3-ITD positive MV4,11 were stimulated with LPS (5-50 ng/ml) for 2h and with 10 ng/ml LPS for different time-points (0-90 min), respectively. ROS formation (H2DCFDA method) and activation of FLT3-ITD and TLR-related signaling pathways were analyzed by immunoblotting. To analyze effects of chronic inflammation, FLT3-WT and FLT3-ITD positive cells were kept in culture for 12 days with repeated LPS stimulation (10 ng/ml every 48h). To address the role of cysteine oxidation within the FLT3-kinase, we mutated highly conserved cysteine residues C474 (extracellular domain) and C925 (intracellular domain) to alanine by site-directed mutagenesis and performed a retroviral transduction of 32D cells with the respective constructs. Subsequently, cysteine mutants were characterized for proliferation (MTT), ROS formation and their sensitivity to TKI treatment (AC220, PKC412; 5 to 50 nM, respectively) in comparison to standard FLT3-ITD mutant.
Results
LPS stimulation resulted in an increased time- and dose-dependent ROS induction in FLT3-ITD positive cells compared to WT cells (100ng/ml LPS for 2h: 6-fold). In addition, LPS stimulation resulted in a stronger activation of FLT3-ITD- and TLR-related signaling pathways in ITD-positive cells (Stat5, Akt, Erk, NFB). Long-term LPS stimulation over 12 days induced an enhanced ROS formation, apoptosis and accumulation of H2AX in both FLT3-WT, as well as in FLT3-ITD positive cells. To address the role of cysteine oxidation, we generated stable cysteine-to-alanine-mutants. However, the FLT3-ITD C925A mutation (intracelullar domain) resulted in an increase of ROS formation compared to the FLT3-ITD C474A mutation (extracellular domain; 25% increase). Furthermore, the C925A mutation resulted in a significant decrease in apoptosis after TKI treatment compared to non-mutated FLT3-ITD and the FLT3-ITD C474A mutant (AC220 50nM: 25%; PKC412 50nM: 40%). In addition, FLT3-ITD C925A mutant showed a reduced decrease in phosphorylation of Stat5 and Akt after short-term (1h) TKI treatment (AC220: 50, 100 nM; PKC412 10, 100nM) compared to non-mutated and FLT3-ITD C474A mutants.
Conclusion
Our study demonstrates that inflammatory events, like LPS stimulation, induce ROS formation, apoptosis and DNA damage in FLT3-WT and FLT3-ITD positive cells. This might lead to genetic instability and induction of critical mutations, e.g. within FLT3 kinase. Additionally, we could show that cysteine oxidation within the intracellular domain of FLT3-ITD resulted in a resistance to TKI treatment.
Session topic: E-poster
Keyword(s): AML, Flt3-ITD, Kinase inhibitor, Resistance
Type: Eposter Presentation
Background
Recurrent infections are associated with an increased incidence in the development of malignancies, including acute myeloid leukemia (AML). Inflammatory events directly induce formation of reactive oxygen species (ROS) and cause DNA damage, protein and lipid oxidation. In case of recurrent infections, bacterial peptides, like LPS, can lead to ROS induction. In particular, cysteine residues of proteins can directly be oxidized by ROS causing either loss or gain of function. However, it is still unclear how inflammatory events can induce these protein oxidation and to what extent the oxidation of cysteine residues in a subset of AML, the FLT3-ITD positive AML, causes a change of biological behavior.
Aims
The goal of our study was to characterize the role of LPS-induced oxidative stress in murine and human FLT3-ITD positive cell lines to identify responsible FLT3-ITD- and TLR related signaling pathways. To adress stress-induced cysteine oxidation of the FLT3-kinase, we generated cysteine-to-alanine mutants in murine myeloid 32D cells (FLT3-WT and FLT3-ITD) and investigated the induction of ROS formation, proliferation, cell cycle distribution and the response to TKI treatment (AC220, PKC412).
Methods
32D cells stably transduced with either FLT3-WT or FLT3-ITD and human FLT3-ITD positive MV4,11 were stimulated with LPS (5-50 ng/ml) for 2h and with 10 ng/ml LPS for different time-points (0-90 min), respectively. ROS formation (H2DCFDA method) and activation of FLT3-ITD and TLR-related signaling pathways were analyzed by immunoblotting. To analyze effects of chronic inflammation, FLT3-WT and FLT3-ITD positive cells were kept in culture for 12 days with repeated LPS stimulation (10 ng/ml every 48h). To address the role of cysteine oxidation within the FLT3-kinase, we mutated highly conserved cysteine residues C474 (extracellular domain) and C925 (intracellular domain) to alanine by site-directed mutagenesis and performed a retroviral transduction of 32D cells with the respective constructs. Subsequently, cysteine mutants were characterized for proliferation (MTT), ROS formation and their sensitivity to TKI treatment (AC220, PKC412; 5 to 50 nM, respectively) in comparison to standard FLT3-ITD mutant.
Results
LPS stimulation resulted in an increased time- and dose-dependent ROS induction in FLT3-ITD positive cells compared to WT cells (100ng/ml LPS for 2h: 6-fold). In addition, LPS stimulation resulted in a stronger activation of FLT3-ITD- and TLR-related signaling pathways in ITD-positive cells (Stat5, Akt, Erk, NFB). Long-term LPS stimulation over 12 days induced an enhanced ROS formation, apoptosis and accumulation of H2AX in both FLT3-WT, as well as in FLT3-ITD positive cells. To address the role of cysteine oxidation, we generated stable cysteine-to-alanine-mutants. However, the FLT3-ITD C925A mutation (intracelullar domain) resulted in an increase of ROS formation compared to the FLT3-ITD C474A mutation (extracellular domain; 25% increase). Furthermore, the C925A mutation resulted in a significant decrease in apoptosis after TKI treatment compared to non-mutated FLT3-ITD and the FLT3-ITD C474A mutant (AC220 50nM: 25%; PKC412 50nM: 40%). In addition, FLT3-ITD C925A mutant showed a reduced decrease in phosphorylation of Stat5 and Akt after short-term (1h) TKI treatment (AC220: 50, 100 nM; PKC412 10, 100nM) compared to non-mutated and FLT3-ITD C474A mutants.
Conclusion
Our study demonstrates that inflammatory events, like LPS stimulation, induce ROS formation, apoptosis and DNA damage in FLT3-WT and FLT3-ITD positive cells. This might lead to genetic instability and induction of critical mutations, e.g. within FLT3 kinase. Additionally, we could show that cysteine oxidation within the intracellular domain of FLT3-ITD resulted in a resistance to TKI treatment.
Session topic: E-poster
Keyword(s): AML, Flt3-ITD, Kinase inhibitor, Resistance
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