INHIBITION OF THE ABL KINASE-ACTIVITY BLOCKS THE PROLIFERATION OF BCRABL(+) LEUKEMIC-CELLS AND INDUCES APOPTOSIS/

The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leuke mias (ALL) are associated with the expression of BCR/ABL proteins. Thi s knowledge has nor yet been translated into any specific tool to cont rol ABL driven neoplastic cells growth, CGP57148B is an ATP-competitiv e inhibitor of the ABL protein kinase; it has been shown to inhibit th e kinase activity of ABL both in vitro and in vivo and to inhibit the growth of v-abl and bcr/abl transfectants, as well as the in vitro for mation of bone marrow (BM)-derived colonies in the presence of growth factors in some CML patients. These studies were performed to investig ate the activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal, BCR/ABL positive and negativ e cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thir teen BCR/ABL negative lines, both neoplastic(KG1, SU-DHL-1, U937, Daud i, NB4, NB4.306) and derived from normal cells (PHA blasts, LAK, fibro blasts, LCL, renal epithelial cells, endothelial cells, CD34(+) cells) , and 14 fresh leukemic samples were tested using a tritiated thymidin e uptake assay. The in vivo phosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annexi n V/propidium binding test. The induction of differentiation was assay ed by immunofluorescence using multiple antibodies. All six BCR/ABL(+) lines showed a dose dependent inhibition of their spontaneous prolife rative rate, which was not accompanied by differentiation. The treatme nt caused, within minutes, dephosphorylation of the BCR/ABL protein, f ollowed in 16-24 hours by a decrease in cycling cells and induction of apoptosis, No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations less than or equal to 3 mu M, with the exception of fibroblasts and CD34 c ells, Proliferation inhibition was observed also when using fresh samp les obtained from two Ph+ ALL and 12 consecutive CML patients. Inducti on of apoptosis was observed in these samples too. The activity of CGP 57148B can be monitored in ex vivo isolated or cultured cells using a simple and reproducible assay, without the need for exogenously added growth factors. This molecule possibly exerts its effects through the inhibition of the kinase activity of BCR/ABL and the subsequent initia tion of apoptosis, without inducing cell differentiation. Some normal cells are also affected.