Effect of the bcr/abl kinase inhibitors AG957 and NSC 680410 on chronic myelogenous leukemia cells in vitro

The tyrphostin AG957 (NSC 654705) inhibits p210(bcr/abl), the transforming kinase responsible for most eases of chronic myelogenous leukemia (CML), Th e present studies were performed to determine the fate of AG957-treated cel ls and assess the selectivity of AG957 for CML myeloid progenitors, When K5 62 cells (derived from a patient with blast crisis CML) were treated with A G957, dose- and time-dependent p210(ber/abl) down-regulation was followed b y mitochondrial release of cytochrome c, activation of caspase-9 and caspas e-3, and apoptotic morphological changes. These apoptotic changes were inhi bited by transfection with cDNA encoding dominant negative caspase-9 but no t dominant-negative FADD or blocking anti-Fas antibodies, In additional exp eriments, a 24-h AG957 exposure caused dose-dependent inhibition of K562 co lony formation in soft agar. To extend these studies to clinical samples of CML, peripheral blood mononuclear cells from 10 chronic phase CML patients and normal controls were assayed for the growth of hematopoietic colonies in vitro in the presence of increasing concentrations of AG957. These assay s demonstrated selectivity of AG957 for CML progenitors, with median IC(50) s (CML versus normal) of 7.3 versus >20 mu M AG957 in granulocyte colony-fo rming cells (P < 0.001), 5.3 versus >20 mu M in granulocyte/macrophage colo ny-forming cells (P < 0.05), and 15.5 versus >20 mu M in erythroid colony-f orming cells (P > 0.05). The adamantyl ester of AG957 (NSC 680410) down-reg ulated p210(bcr/abl) in K562 cells and inhibited granulocyte colony formati on in CML specimens at lower concentrations; without enhanced toxicity in n ormal progenitors. These observations not only demonstrate that AG957-induc ed p210(bcr/abl) down-regulation is followed by activation of the cytochrom e c/Apaf-1/caspase-9 pathway but also indicate that this class of kinase in hibitor exhibits selectivity worthy of further evaluation.