ACTIVATION OF NRF2 IN BONE MARROW MESENCHYMAL STROMAL CELLS MEDIATES SURVIVAL AND CHEMOTHERAPY RESISTANCE IN ACUTE MYELOID LEUKAEMIA BLASTS
(Abstract release date: 05/19/16)
EHA Library. Loughran N. 06/09/16; 132438; E889
Ms. Niamh Loughran
Contributions
Contributions
Abstract
Abstract: E889
Type: Eposter Presentation
Background
The bone marrow microenvironment and the cells with in it play a critical role in acute myeloid leukaemia. NRF2 regulates a range of cytoprotective transcriptional responses and acts as a master regulator of oxidative and electrophilic stress. We have previously shown that NRF2 forms part of a major cell survival pathway in protecting the AML blasts from chemotherapy induced apoptosis. Here we hypothesise that NRF2 also functions to regulate pro-survival factors in Bone Marrow - Mesenchymal Stromal Cells (BM-MSC) from patients with AML and investigate whether targeting NRF2 in the BM-MSC could enhance the efficacy of cytotoxic chemotherapy in AML.
Aims
In this study we aim to characterise the NRF2 signalling pathway in the BM-MSC and investigate how NRF2 signalling regulates oxidative stress in BM-MSC-AML niche.
Methods
Primary AML blasts and BM-MSC were isolated from AML patients following informed consent and under approval from the UK National Research Ethics Service (LRCEref07/H0310/146). Cytarabine, daunorubicin and hydrogen peroxide (H2O2) were used to induce NRF2 activation in AML blasts and BM-MSC. Annexin V/PI staining was used to determine apoptosis of treated cells. N-acetyl cysteine (NAC) was used to determine the role of glutathione. Cell-Titer Glo assay was performed to determine cell viability. Dichlorofluorescein-H₂DCFDA (DCF) dye was used to determine levels of reactive oxygen species (ROS). Western blot analysis was used to determine NRF2 expression in drug treated samples.
Results
Results show that AML blasts have increased survival in vitro when co-cultured with BM-MSC. Co-culture with BM-MSC was also shown to confer protection to AML blasts in the presence of daunorubicin, cytarabine and H2O2. Lower levels of ROS were observed in the AML blasts after BM-MSC co-culture, as determined by DCF fluorescence. NRF2 in BM-MSC was found to be activated by daunorubicin and cytarabine, as depicted by an increased expression of NRF2 in the nucleus after drug treatment. Moreover depletion of NRF2 in the BM-MSC reduced the protection conferred to the AML cells by the BM-MSC in response to daunorubicin, cytarabine and H2O2. Furthermore, the addition of NAC was shown to decrease AML blast apoptosis in culture in these assays.
Conclusion
These results demonstrate that NRF2 is induced in the non-malignant BM-MSC in response to chemotherapy with the functional consequence of promoting survival in neighbouring AML blasts. We propose targeting the actions of NRF2 in the stromal cells as a novel strategy to increase the tumour cytotoxicity of current drugs used to treat patients with AML.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Bone marrow stroma, Chemoresistance, Transcription factor
Type: Eposter Presentation
Background
The bone marrow microenvironment and the cells with in it play a critical role in acute myeloid leukaemia. NRF2 regulates a range of cytoprotective transcriptional responses and acts as a master regulator of oxidative and electrophilic stress. We have previously shown that NRF2 forms part of a major cell survival pathway in protecting the AML blasts from chemotherapy induced apoptosis. Here we hypothesise that NRF2 also functions to regulate pro-survival factors in Bone Marrow - Mesenchymal Stromal Cells (BM-MSC) from patients with AML and investigate whether targeting NRF2 in the BM-MSC could enhance the efficacy of cytotoxic chemotherapy in AML.
Aims
In this study we aim to characterise the NRF2 signalling pathway in the BM-MSC and investigate how NRF2 signalling regulates oxidative stress in BM-MSC-AML niche.
Methods
Primary AML blasts and BM-MSC were isolated from AML patients following informed consent and under approval from the UK National Research Ethics Service (LRCEref07/H0310/146). Cytarabine, daunorubicin and hydrogen peroxide (H2O2) were used to induce NRF2 activation in AML blasts and BM-MSC. Annexin V/PI staining was used to determine apoptosis of treated cells. N-acetyl cysteine (NAC) was used to determine the role of glutathione. Cell-Titer Glo assay was performed to determine cell viability. Dichlorofluorescein-H₂DCFDA (DCF) dye was used to determine levels of reactive oxygen species (ROS). Western blot analysis was used to determine NRF2 expression in drug treated samples.
Results
Results show that AML blasts have increased survival in vitro when co-cultured with BM-MSC. Co-culture with BM-MSC was also shown to confer protection to AML blasts in the presence of daunorubicin, cytarabine and H2O2. Lower levels of ROS were observed in the AML blasts after BM-MSC co-culture, as determined by DCF fluorescence. NRF2 in BM-MSC was found to be activated by daunorubicin and cytarabine, as depicted by an increased expression of NRF2 in the nucleus after drug treatment. Moreover depletion of NRF2 in the BM-MSC reduced the protection conferred to the AML cells by the BM-MSC in response to daunorubicin, cytarabine and H2O2. Furthermore, the addition of NAC was shown to decrease AML blast apoptosis in culture in these assays.
Conclusion
These results demonstrate that NRF2 is induced in the non-malignant BM-MSC in response to chemotherapy with the functional consequence of promoting survival in neighbouring AML blasts. We propose targeting the actions of NRF2 in the stromal cells as a novel strategy to increase the tumour cytotoxicity of current drugs used to treat patients with AML.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Bone marrow stroma, Chemoresistance, Transcription factor
Abstract: E889
Type: Eposter Presentation
Background
The bone marrow microenvironment and the cells with in it play a critical role in acute myeloid leukaemia. NRF2 regulates a range of cytoprotective transcriptional responses and acts as a master regulator of oxidative and electrophilic stress. We have previously shown that NRF2 forms part of a major cell survival pathway in protecting the AML blasts from chemotherapy induced apoptosis. Here we hypothesise that NRF2 also functions to regulate pro-survival factors in Bone Marrow - Mesenchymal Stromal Cells (BM-MSC) from patients with AML and investigate whether targeting NRF2 in the BM-MSC could enhance the efficacy of cytotoxic chemotherapy in AML.
Aims
In this study we aim to characterise the NRF2 signalling pathway in the BM-MSC and investigate how NRF2 signalling regulates oxidative stress in BM-MSC-AML niche.
Methods
Primary AML blasts and BM-MSC were isolated from AML patients following informed consent and under approval from the UK National Research Ethics Service (LRCEref07/H0310/146). Cytarabine, daunorubicin and hydrogen peroxide (H2O2) were used to induce NRF2 activation in AML blasts and BM-MSC. Annexin V/PI staining was used to determine apoptosis of treated cells. N-acetyl cysteine (NAC) was used to determine the role of glutathione. Cell-Titer Glo assay was performed to determine cell viability. Dichlorofluorescein-H₂DCFDA (DCF) dye was used to determine levels of reactive oxygen species (ROS). Western blot analysis was used to determine NRF2 expression in drug treated samples.
Results
Results show that AML blasts have increased survival in vitro when co-cultured with BM-MSC. Co-culture with BM-MSC was also shown to confer protection to AML blasts in the presence of daunorubicin, cytarabine and H2O2. Lower levels of ROS were observed in the AML blasts after BM-MSC co-culture, as determined by DCF fluorescence. NRF2 in BM-MSC was found to be activated by daunorubicin and cytarabine, as depicted by an increased expression of NRF2 in the nucleus after drug treatment. Moreover depletion of NRF2 in the BM-MSC reduced the protection conferred to the AML cells by the BM-MSC in response to daunorubicin, cytarabine and H2O2. Furthermore, the addition of NAC was shown to decrease AML blast apoptosis in culture in these assays.
Conclusion
These results demonstrate that NRF2 is induced in the non-malignant BM-MSC in response to chemotherapy with the functional consequence of promoting survival in neighbouring AML blasts. We propose targeting the actions of NRF2 in the stromal cells as a novel strategy to increase the tumour cytotoxicity of current drugs used to treat patients with AML.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Bone marrow stroma, Chemoresistance, Transcription factor
Type: Eposter Presentation
Background
The bone marrow microenvironment and the cells with in it play a critical role in acute myeloid leukaemia. NRF2 regulates a range of cytoprotective transcriptional responses and acts as a master regulator of oxidative and electrophilic stress. We have previously shown that NRF2 forms part of a major cell survival pathway in protecting the AML blasts from chemotherapy induced apoptosis. Here we hypothesise that NRF2 also functions to regulate pro-survival factors in Bone Marrow - Mesenchymal Stromal Cells (BM-MSC) from patients with AML and investigate whether targeting NRF2 in the BM-MSC could enhance the efficacy of cytotoxic chemotherapy in AML.
Aims
In this study we aim to characterise the NRF2 signalling pathway in the BM-MSC and investigate how NRF2 signalling regulates oxidative stress in BM-MSC-AML niche.
Methods
Primary AML blasts and BM-MSC were isolated from AML patients following informed consent and under approval from the UK National Research Ethics Service (LRCEref07/H0310/146). Cytarabine, daunorubicin and hydrogen peroxide (H2O2) were used to induce NRF2 activation in AML blasts and BM-MSC. Annexin V/PI staining was used to determine apoptosis of treated cells. N-acetyl cysteine (NAC) was used to determine the role of glutathione. Cell-Titer Glo assay was performed to determine cell viability. Dichlorofluorescein-H₂DCFDA (DCF) dye was used to determine levels of reactive oxygen species (ROS). Western blot analysis was used to determine NRF2 expression in drug treated samples.
Results
Results show that AML blasts have increased survival in vitro when co-cultured with BM-MSC. Co-culture with BM-MSC was also shown to confer protection to AML blasts in the presence of daunorubicin, cytarabine and H2O2. Lower levels of ROS were observed in the AML blasts after BM-MSC co-culture, as determined by DCF fluorescence. NRF2 in BM-MSC was found to be activated by daunorubicin and cytarabine, as depicted by an increased expression of NRF2 in the nucleus after drug treatment. Moreover depletion of NRF2 in the BM-MSC reduced the protection conferred to the AML cells by the BM-MSC in response to daunorubicin, cytarabine and H2O2. Furthermore, the addition of NAC was shown to decrease AML blast apoptosis in culture in these assays.
Conclusion
These results demonstrate that NRF2 is induced in the non-malignant BM-MSC in response to chemotherapy with the functional consequence of promoting survival in neighbouring AML blasts. We propose targeting the actions of NRF2 in the stromal cells as a novel strategy to increase the tumour cytotoxicity of current drugs used to treat patients with AML.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Bone marrow stroma, Chemoresistance, Transcription factor
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