Pyrrolo[1,2-B][1,2,5] benzodiazepines (PBTDs) Compounds Induce Apoptosis in Chronic Myeloid Leukaemia Cells from Patients at Onset, Imatinib and Second Generation TK Inhibitors (Dasatinib, Nilotinib) Resistant.

CML is a clonal myeloproliferative disease in which Bcr-Abl deregulated tyrosine kinase (TK) activity impairs blood cells homeostasis leading to altered growth, homing and apoptosis. Imatinib mesylate (Glivec, Novartis), an ATP competitor molecule, is the gold standard therapy for Bcr-Abl positive diseases, but resistance is increasingly encountered, mainly due to point mutations in the abl kinase portion of the molecule. Second generation TK inhibitors (Dasatinib, Brystol-Myers Squibb and Nilotinib, Novartis) can bind mutated forms of the protein, but T315I mutation appear unresponsive to TK treatments. Apoptosis is a cell suicide mechanism activated to remove redundant, damaged, or infected cells. An essential component of the apoptotic machinery, the caspase family, consists of a group of intracellular cysteine proteases that can be divided into initiator/upstream caspases (2, 8, 9, and 10 caspases), which activate the downstream/effector caspases (3, 6 and 7 caspases). Cleavage of a selected group of substrates by effector caspases is responsible for dismantling of essential cell components, which results in morphological and biochemical changes that characterise apoptotic cell death: cytoskeletal rearrangement, cell membrane blebbing, nuclear condensation and DNA fragmentation. Based on cell line experiments we focused our attention on some members of the pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs) family to test their potential apoptotic activity in CML. Important apoptotic activity was demonstrated on K562, K562 R-IM, HL -60 ,U937 and Jurkatt cell lines, as evidenced with the concentration and the percentage of the cell death quantified by measuring PI-uptake by flow cytometry, DNA fragmentation, and analyzed by agarose gel electrophoresis generating a characteristic ladder pattern of discontinuous DNA fragments. Three PBTDs coumpounds (RS 678, RS779, RS 2630 RS) were then incubated with peripheral blood and/or bone marrow cells isolated from 30 CML untreated, 25 imatinib-resistant, 10 Dasatinib resistant, and 1 Nilotinib resistant patients. Cells were incubated at the concentration 10μM for,16,24, and 48 hours. Apoptotic DNA fragmentation was tested by agarose gels electrophoresis and was present in all but one patients in a percentage varying from 60 to 85%, compared to 0 to 5% of internal controls. Among the 25 imatinib-resistant patient 23 were tested for abl mutation using D-HPLC. Five patients showed T315I, one F317L, one each M351V, D276G, Y253H, and 2 patients showed 2 mutations (F317l and M315T; and G250G, and F359V). Only one patient Dasatinib reistant carrying M351V mutation showed very low apoptosis (5–10%), while the 5 patients with T315I mutation underwent high apoptotic DNA fragmentation. Apoptosis seem to be triggered by PBTDs through activation of initiator caspases 8 and 9 and effector caspases 3 as eveidenced by western blotting. Although more experiments are needed, these compounds appear promising for a preclinical approach in CML.