Contributions
Abstract: EP655
Type: E-Poster Presentation
Session title: Chronic myeloid leukemia - Biology & Translational Research
Background
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm (MPN) driven by the BCR-ABL1 fusion gene. Athero-thrombotic complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKI’s). The mechanism underlying these complications is not fully understood but may involve off-target effects that promote vascular toxicity. In response to various stimuli, neutrophils can expel extracellular strands of decondensed DNA in complex with histones and additional proteins called neutrophil extracellular traps (NETs). These structures have the ability to ensnare and kill microbes but are also implicated in the pathogenesis of thrombosis.
Aims
Study NET formation in CML and evaluate a possible contribution of BCR-ABL1 and of TKI’s to NET formation.
Methods
Neutrophils were isolated from CML patients (n=9) and from age and gender matched controls (n=9) by Percoll gradient. NETosis was evaluated by measuring the levels of accepted NETosis markers. NET formation was assessed by the NETosis Assay Kit (Cayman chemical) as well as by immunofluorescence (IF). Expression of citrullinated histon H3 (H3cit), an established marker of NETosis and of PAD4, an initiator of this process, was determined by Western blot and IF. CD11b expression and reactive oxygen species (ROS) production were analyzed by FACS and ELISA, respectively. Assessments were performed in resting and in PMA/ionomycin stimulated neutrophils and after ex vivo treatment with clinically relevant concentrations of imatinib (5.3 µM), ponatinib (0.17 µM) and dasatinib (0.22 µM). To validate our findings and further study the effect of BCR-ABL1 and TKIs on NETosis, we prepared BCR-ABL1 retroviral transduced HoxB8-immortalized mouse hematopoietic progenitors which differentiate into neutrophils in vitro.
Results
Neutrophils from patients with CML had a significant increase in NETs formation as assessed by morphology and by an increase in DNA-associated neutrophil elastase compared to normal neutrophils, at baseline (p≤0.05, p≤0.03, respectively) and after PMA stimulation (p≤0.005, p≤0.002, respectively). In accordance, H3cit and PAD4 expression (staining and WB) and ROS generation were also higher in CML samples compared to controls (p≤0.04). Ponatinib treatment of neutrophils from CML patients significantly augmented DNA-associated neutrophil elastase levels as compared to DMSO, imatinib and dasatinib treated neutrophils (p≤0.05 for all comparisons). Ponatinib treatment also led to increased ROS levels in CML neutrophils as compared to DMSO and imatinib (p≤0.05). Differentiated BCR-ABL1-transduced HoxB8 neutrophils stimulated with ionomycin demonstrated increased H3cit & PAD4 expression. These processes were significantly reduced by Cl-amidine, a PAD4 inhibitor. Ponatinib and nilotinib increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with ionomycin. However, no significant difference was found in ROS generation of ionomycin stimulated HoxB8 neutrophils transduced with empty vector or with BCR-ABL1. Also, no difference was observed in ROS levels of HoxB8-BCR-ABL1 cells treated with the different TKIs.
Conclusion
Our findings support the hypothesis that CML is associated with increased NET formation which is further augmented by ponatinib, suggesting a possible role for NETs in promoting vascular toxicity in CML. In the BCR-ABL1/HOXB8 model, NETs formation is PAD4 dependent. Further studies are needed to assess the possible role of increased NETs formation in CML on the CVS associated with BCR-ABL1 and TKIs exposure.
Keyword(s): Chronic myeloid leukemia, Neutrophil function, Thrombosis, Tyrosine kinase inhibitor
Abstract: EP655
Type: E-Poster Presentation
Session title: Chronic myeloid leukemia - Biology & Translational Research
Background
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm (MPN) driven by the BCR-ABL1 fusion gene. Athero-thrombotic complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKI’s). The mechanism underlying these complications is not fully understood but may involve off-target effects that promote vascular toxicity. In response to various stimuli, neutrophils can expel extracellular strands of decondensed DNA in complex with histones and additional proteins called neutrophil extracellular traps (NETs). These structures have the ability to ensnare and kill microbes but are also implicated in the pathogenesis of thrombosis.
Aims
Study NET formation in CML and evaluate a possible contribution of BCR-ABL1 and of TKI’s to NET formation.
Methods
Neutrophils were isolated from CML patients (n=9) and from age and gender matched controls (n=9) by Percoll gradient. NETosis was evaluated by measuring the levels of accepted NETosis markers. NET formation was assessed by the NETosis Assay Kit (Cayman chemical) as well as by immunofluorescence (IF). Expression of citrullinated histon H3 (H3cit), an established marker of NETosis and of PAD4, an initiator of this process, was determined by Western blot and IF. CD11b expression and reactive oxygen species (ROS) production were analyzed by FACS and ELISA, respectively. Assessments were performed in resting and in PMA/ionomycin stimulated neutrophils and after ex vivo treatment with clinically relevant concentrations of imatinib (5.3 µM), ponatinib (0.17 µM) and dasatinib (0.22 µM). To validate our findings and further study the effect of BCR-ABL1 and TKIs on NETosis, we prepared BCR-ABL1 retroviral transduced HoxB8-immortalized mouse hematopoietic progenitors which differentiate into neutrophils in vitro.
Results
Neutrophils from patients with CML had a significant increase in NETs formation as assessed by morphology and by an increase in DNA-associated neutrophil elastase compared to normal neutrophils, at baseline (p≤0.05, p≤0.03, respectively) and after PMA stimulation (p≤0.005, p≤0.002, respectively). In accordance, H3cit and PAD4 expression (staining and WB) and ROS generation were also higher in CML samples compared to controls (p≤0.04). Ponatinib treatment of neutrophils from CML patients significantly augmented DNA-associated neutrophil elastase levels as compared to DMSO, imatinib and dasatinib treated neutrophils (p≤0.05 for all comparisons). Ponatinib treatment also led to increased ROS levels in CML neutrophils as compared to DMSO and imatinib (p≤0.05). Differentiated BCR-ABL1-transduced HoxB8 neutrophils stimulated with ionomycin demonstrated increased H3cit & PAD4 expression. These processes were significantly reduced by Cl-amidine, a PAD4 inhibitor. Ponatinib and nilotinib increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with ionomycin. However, no significant difference was found in ROS generation of ionomycin stimulated HoxB8 neutrophils transduced with empty vector or with BCR-ABL1. Also, no difference was observed in ROS levels of HoxB8-BCR-ABL1 cells treated with the different TKIs.
Conclusion
Our findings support the hypothesis that CML is associated with increased NET formation which is further augmented by ponatinib, suggesting a possible role for NETs in promoting vascular toxicity in CML. In the BCR-ABL1/HOXB8 model, NETs formation is PAD4 dependent. Further studies are needed to assess the possible role of increased NETs formation in CML on the CVS associated with BCR-ABL1 and TKIs exposure.
Keyword(s): Chronic myeloid leukemia, Neutrophil function, Thrombosis, Tyrosine kinase inhibitor