Contributions
Abstract: PB1922
Type: Publication Only
Background
BCR-ABL tyrosine kinase inhibitors (TKIs) are the mainstay of the treatment from chronic myeloid leukemia (CML). However, new generation TKIs (dasatinib, nilotinib, bosutinib and ponatinib) increase the risk of arterial occlusive events compared with the first generation (imatinib). To date, the mechanism(s) of these side effects is poorly understood but clinical data suggest that new generation TKIs might accelerate atherogenesis, in which endothelial cells play major function (e.g. lipid deposition, leukocyte recruitment).
Aims
This research aims to determine the effect of BCR-ABL TKIs on endothelial cell survival and major functions (endothelial cell migration and expression of adhesion molecules) using an in vitro model.
Methods
All experiments were performed on endothelial cells derived from human umbilical vein (HUVEC). Viability after 24h of TKI treatment was assessed using MTS and LDH assays following standard protocols. Reactive oxygen species (ROS) generation was quantified using a ROS detection reagent (CM-H2DCFDA) following the manufacturer instructions. The cell-surface expression of 3 adhesion molecules (ICAM-1, VCAM-1 and E-selectin) after 4-hour TNF-α activation (10ng/mL) was measured by on-cell ELISA and flow cytometry. Migration of endothelial cells was evaluated by a scratch assay (i.e. scratch of a confluent monolayer with a pipette tip and monitoring of the wound closure by inverted microscopy).
Results
Dasatinib, nilotinib and ponatinib at high concentration reduce cell metabolism indicating an inhibition of proliferation or induction of apoptosis by these 3 TKIs. Additionally, high-dose ponatinib (0.5µM) induces necrosis as demonstrated by increased LDH release. The analysis of ROS generation demonstrates that the 5 TKIs do not induce oxidative stress and do not alter cell viability by this way. Leukocyte recruitment is also an important process in atherogenesis and requires the expression of adhesion molecules by activated endothelial cells. The expression of ICAM-1, VCAM-1 and E-selectin was quantified by on-cell ELISA in HUVEC which demonstrated a decreased of ICAM-1, VCAM-1 and E-selectin expression with dasatinib, nilotinib and ponatinib at high concentration (0.5µM, 2µM and 0.5µM respectively) and no or little impact of imatinib and bosutinib. This reduction correlates with the decreased viability of endothelial cells with these 3 treatments, an hypothesis that has been confirmed by flow cytometry. Finally, we evaluate the migration ability of endothelial cell after treatment by scratch assay. High dose dasatinib inhibits endothelial cell migration whereas other TKIs do not have any impact. Therefore, dasatinib might induce arterial occlusive events through impaired vascular wound healing.
Conclusion
Over the 5 commercialized BCR-ABL TKIs, dasatinib, nilotinib and ponatinib possess the most impact on endothelial cells. They reduce viability by inducing apoptosis, necrosis or inhibiting cell proliferation, which possibly help to the development of atherosclerosis through impaired endothelium permeability, enabling migration and trapping of lipoprotein into the intima. Additionally, dasatinib reduces endothelial cell migration, which might contribute to formation of arterial thrombosis. Determination of mechanism(s) by which TKIs promote cardiovascular events is required to implement appropriate risk minimization measures and select patients to whom the prescription of these drugs should be avoided.
Session topic: 8. Chronic myeloid leukemia - Clinical
Keyword(s): Arterial Thrombosis, Chronic myeloid leukemia, Endothelial cell, Tyrosine kinase inhibitor
Abstract: PB1922
Type: Publication Only
Background
BCR-ABL tyrosine kinase inhibitors (TKIs) are the mainstay of the treatment from chronic myeloid leukemia (CML). However, new generation TKIs (dasatinib, nilotinib, bosutinib and ponatinib) increase the risk of arterial occlusive events compared with the first generation (imatinib). To date, the mechanism(s) of these side effects is poorly understood but clinical data suggest that new generation TKIs might accelerate atherogenesis, in which endothelial cells play major function (e.g. lipid deposition, leukocyte recruitment).
Aims
This research aims to determine the effect of BCR-ABL TKIs on endothelial cell survival and major functions (endothelial cell migration and expression of adhesion molecules) using an in vitro model.
Methods
All experiments were performed on endothelial cells derived from human umbilical vein (HUVEC). Viability after 24h of TKI treatment was assessed using MTS and LDH assays following standard protocols. Reactive oxygen species (ROS) generation was quantified using a ROS detection reagent (CM-H2DCFDA) following the manufacturer instructions. The cell-surface expression of 3 adhesion molecules (ICAM-1, VCAM-1 and E-selectin) after 4-hour TNF-α activation (10ng/mL) was measured by on-cell ELISA and flow cytometry. Migration of endothelial cells was evaluated by a scratch assay (i.e. scratch of a confluent monolayer with a pipette tip and monitoring of the wound closure by inverted microscopy).
Results
Dasatinib, nilotinib and ponatinib at high concentration reduce cell metabolism indicating an inhibition of proliferation or induction of apoptosis by these 3 TKIs. Additionally, high-dose ponatinib (0.5µM) induces necrosis as demonstrated by increased LDH release. The analysis of ROS generation demonstrates that the 5 TKIs do not induce oxidative stress and do not alter cell viability by this way. Leukocyte recruitment is also an important process in atherogenesis and requires the expression of adhesion molecules by activated endothelial cells. The expression of ICAM-1, VCAM-1 and E-selectin was quantified by on-cell ELISA in HUVEC which demonstrated a decreased of ICAM-1, VCAM-1 and E-selectin expression with dasatinib, nilotinib and ponatinib at high concentration (0.5µM, 2µM and 0.5µM respectively) and no or little impact of imatinib and bosutinib. This reduction correlates with the decreased viability of endothelial cells with these 3 treatments, an hypothesis that has been confirmed by flow cytometry. Finally, we evaluate the migration ability of endothelial cell after treatment by scratch assay. High dose dasatinib inhibits endothelial cell migration whereas other TKIs do not have any impact. Therefore, dasatinib might induce arterial occlusive events through impaired vascular wound healing.
Conclusion
Over the 5 commercialized BCR-ABL TKIs, dasatinib, nilotinib and ponatinib possess the most impact on endothelial cells. They reduce viability by inducing apoptosis, necrosis or inhibiting cell proliferation, which possibly help to the development of atherosclerosis through impaired endothelium permeability, enabling migration and trapping of lipoprotein into the intima. Additionally, dasatinib reduces endothelial cell migration, which might contribute to formation of arterial thrombosis. Determination of mechanism(s) by which TKIs promote cardiovascular events is required to implement appropriate risk minimization measures and select patients to whom the prescription of these drugs should be avoided.
Session topic: 8. Chronic myeloid leukemia - Clinical
Keyword(s): Arterial Thrombosis, Chronic myeloid leukemia, Endothelial cell, Tyrosine kinase inhibitor