Blood Cells, Molecules, and Diseases (1997) 23(19) Oct 15: 380-394 C. Gambacorti-Passerini, et al. Article No. MD970155 Division of Experimental Oncology D and Medical Oncology C, Istituto Nazionale Tumori, Milan, Italy; 1 Sect. of Hematology and Division of Pediatric Hematology, University of Milan, S.Gerardo Hospital, Monza, Italy; 2 Division of Experimental Oncology, European Oncology Institute, Milan, Italy; 3 Oncology Research Department, Novartis International Inc., K125.4.20, Basel, Switzerland. 4 Reprint request to: Carlo Gambacorti, M.D., Division of Experimental Oncology D, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milano, Italy, phone 392239-0818, fax 392239-0764, email: gambacorti@istitutotumori.mi.it 1079-9796/97 Copyright (c) by The Blood Cells Foundation, La Jolla, California USA All rights of reproduction in any form reserved Published by Academic Press Established by Springer-Verlag, Inc. in 1975 380 Inhibition of the ABL Kinase Activity Blocks the Proliferation of BCR/ABL Leukemic Cells and Induces Apoptosis + Submitted 09/29/97 (communicated by Ernest Beutler, M.D., 09/30/97) Carlo Gambacorti-Passerini , Philipp le Coutre , Luca Mologni , Mirco Fanelli , 1, 2 1 1 1, 3 Carla Bertazzoli , Edoardo Marchesi , Massimo Di Nicola , Andrea Biondi , Gian Marco Corneo , 1 1 1 2 2 Daniela Belotti , Enrico Pogliani , Nicholas B. Lydon 2 2 4 ABSTRACT: The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression of BCR/ABL proteins. This knowledge has not yet been translated into any specific tool to control ABL driven neoplastic cells growth. CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the 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 formation of bone marrow (BM)-derived colonies in the presence of growth factors in some CML patients. These studies were performed to investigate the activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal, BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic (KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal cells (PHA blasts, LAK, fibroblasts, LCL, renal epithelial cells, endothelial cells, CD34 cells), and 14 fresh leukemic samples were tested using a tritiated + thymidine uptake assay. The in vivo phosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annexin V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multiple antibodies. All six BCR/ABL lines showed a dose dependent inhibition of their spontaneous proliferative rate, which + was not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed 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 3 μM, with the exception of fibroblasts and CD34 cells. Proliferation inhibition was observed also when using fresh samples obtained from two Ph+ ALL and 12 consecutive CML patients. Induction of apoptosis was observed in these samples too. The activity of CGP57148B 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 initiation of apoptosis, without inducing cell differentiation. Some normal cells are also affected.