The Risk of Arterial Thrombosis in Patients With Chronic Myeloid Leukemia Treated With Second and Third Generation BCR-ABL Tyrosine Kinase Inhibitors May Be Explained by Their Impact on Endothelial Cells:An In-Vitro Study

BCR-ABL tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia, inducing deep molecular responses, largely improving patient survival and rendering treatment-free remission possible. However, three of the five BCR-ABL TKIs, dasatinib, nilotinib, and ponatinib, increase the risk of developing arterial thrombosis. Prior investigations reported that nilotinib and ponatinib affect the endothelium, but the mechanisms by which they exert their toxic effects are still unclear. The impact of dasatinib and bosutinib on endothelial cells has been poorly investigated. Here, we aimed to provide an in vitro homogenous evaluation of the effects of BCR-ABL TKIs on the endothelium, with a special focus on the type of cell death to elucidate the mechanisms responsible for the potential cytotoxic effects of BCR-ABL TKIs nilotinib and ponatinib on endothelial cells. We tested the five BCR-ABL TKIs at three concentrations on human umbilical venous endothelial cells (HUVECs). This study highlights the endothelial toxicity of ponatinib and provides insights about the mechanisms by which it affects endothelial cell viability. Ponatinib induced apoptosis and necrosis of HUVECs after 72 h. Dasatinib affected endothelial cells in vitro by inhibiting their proliferation and decreased wound closure as soon as 24 h of treatment and even at infra-therapeutic dose (0.005 µM). Comparatively, imatinib, nilotinib, and bosutinib had little impact on endothelial cells at therapeutic concentrations. They did not induce apoptosis nor necrosis, even after 72 h of treatment but they inhibited HUVEC proliferation. Overall, this study reports various effects of BCR-ABL TKIs on endothelial cells and suggests that ponatinib and dasatinib induce arterial thrombosis through endothelial dysfunction.</p

Study of the potential role of extracellular vesicles in thrombosis associated with paroxysmal nocturnal hemoglobinuria and red blood cell concentrates transfusions

The impact of extracellular vesicles (EVs) in the physiopathology of thrombosis has been deeply studied. In paroxysmal nocturnal hemoglobinuria (PNH) patients, thrombosis is the main complication, thereby, it takes a big place in the patient’s management. These patients may also receive red blood cell (RBC) transfusion, however, the procoagulant activity inside the concentrates may be increased due to EV release with storage time. The first aim of this thesis was the study of the EV responsibility in the pathogenesis of thrombosis in PNH. This purpose has been reached, first, by the analyze of the procoagulant activity in PNH patients at different time of treatment with eculizumab. Besides this study, a cellular model of the disease has been developed to assess the procoagulant activity of EVs released by PNH (-like) cells. The second aim of this thesis was the assessment of procoagulant activity EV-driven inside RBC concentrates through storage time. Two studies have been led in this purpose. For both, whole blood was collected in 12 healthy volunteers and RBC concentrates have been prepared. In the first study, EVs were sampled from packed-RBCs 13 times from day 0 to day 42. The amount and the procoagulant activity EV-driven were measured after 6 months of freezing. The second study was performed by implementing the limitation of the first one (i.e. the high number of samplings and the freezing of samples). In this purpose the samplings were decreased from 13 to 3 and the procoagulant activity has been measured both in fresh and frozen samples. This work allowed highlighting a decrease of EV amount and activity during eculizumab treatment in PNH patients. In PNH-context, a role of procoagulant leucocytes derived EVs has also been shown thanks to the cellular model. Finally, it also permitted to be reassuring about thrombotic risk after the transfusion of old RBC concentrates.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Transfer of multidrug resistance among acute myeloid leukemia cells via extracellular vesicles and their microRNA cargo.

The treatment of acute leukemia is still challenging due in part to the development of resistance and relapse. This chemotherapeutics resistance is established by clonal selection of resistant variants of the cancer cells. Recently, a horizontal transfer of chemo-resistance among cancer cells via extracellular vesicles (EVs) has been suggested. The aim of this research was to investigate the role of EVs in chemo-resistance in acute myeloid leukemia. For this purpose, the sensitive strain of the promyelocytic leukemia HL60 cell line was studied along with its multi-resistant strain, HL60/AR that overexpresses the multidrug resistance protein 1 (MRP-1). A chemo-resistance transfer between the two strains was established by treating HL60 cells with EVs generated by HL60/AR. This study reveals that EVs from HL60/AR can interact with HL60 cells and transfer at least partially, their chemo-resistance. EVs-treated cells begin to express MRP-1 probably due to a direct transfer of MRP-1 and nucleic acids transported by EVs. In this context, two microRNAs were highlighted for their high differential expression in EVs related to sensitive or chemo-resistant cells: miR-19b and miR-20a. Because circulating microRNAs are found in all biological fluids, these results bring out their potential clinical use as chemo-resistance biomarkers in acute myeloid leukemia