Advances in Brief A Novel Pyridopyrimidine Inhibitor of Abl Kinase Is a Picomolar Inhibitor of Bcr-abl-driven K562 Cells and Is Effective against STI571-resistant Bcr-abl Mutants

Inhibition of the constitutively active Bcr-abl tyrosine kinase (TK) by STI571 has proven to be a highly effective treatment for chronic myelogenous leukemia (CML). However, STI571 is only transiently effective in blast crisis, and drug resistance emerges by amplification of or development of mutational changes in Bcr-abl. We have screened a family of TK inhibitors of the pyrido [2,3-d]pyrimidine class, unrelated to STI571, and describe here a compound with substantial activity against STI-resistant mutant Bcr-abl proteins. This compound, PD166326, is a dual specificity TK inhibitor and inhibits src and abl in vitro with IC50s of 6 and 8 nM respectively. PD166326 inhibits the growth of K562 cells with IC50 of 300 pM, leading to apoptotic G1 arrest, whereas non-Bcr-abl cell types require >1000 times higher concentrations. We tested the effects of PD166326 on two of the clinically observed STI571-resistant Bcr-abl mutants. PD166326 potently inhibits the E255K mutant Bcr-abl protein and the growth of Bcr-ablE255K-driven cells. The T315I mutant Bcr-abl protein, which is mutated within the ATP-binding pocket, is resistant to PD166326; however, the growth of Bcr-ablT315I-driven cells is partially sensitive to this compound, likely through the inhibition of Bcr-abl effector pathways. These findings show that TK drug resistance is a structure-specific phenomenon and can be overcome by TK inhibitors of other structural classes, suggesting new approaches for future anticancer drug development. PD166326 is a prototype of a new generation of anti-Bcr-abl compounds with picomolar potency and substantial activity against STI571-resistant mutants. Introduction CML is a myeloproliferative disorder of hematopoietic stem cells. The pathologic hallmark of this disease is the Philadelphia chromosome, which is seen in 90% of CML patients. The Philadelphia chromosome is the result of a t(9;22) chromosomal translocation that results in the juxtaposition of 3 sequences of the Abl TK proto-oncogene located on chromosome 9 with 5 sequences of the Bcr gene on chromosome 22. The resulting Bcr-abl fusion gene encodes a novel cytoplasmic protein with constitutive TK activity (1). Expression of this fusion gene is seen in 90% of patients with CML, and experimental evidence confirms that Bcr-abl expression is sufficient to induce CML-like disease in mice (2). The oncogenic potency of the Bcr-abl oncoprotein is quantitatively related to its TK activity (3). Efforts to develop novel treatments for CML have focused on targeting the TK activity of Bcr-abl and has led to the development of STI571, a 2-phenylaminopyrimidine compound that is potent, and selective inhibitor of Abl, c-kit, and plateletderived growth factor-receptor TKs (4). This compound selectively inhibits the growth of Bcr-abl-positive but not -negative cell lines (5, 6). STI571 has potent antileukemic activity in clinical studies producing complete hematological responses in 98% of patients in chronic phase CML, including complete cytogenetic responses in 50% (7, 8). Clinical response is associated with inhibition of Bcr-abl TK activity in vivo (7). STI571 also produces hematological remissions in 50–70% of patients with blast crisis, although these responses are frequently not durable, and patients relapse within 2–6 months of therapy (9). Relapse during STI571 therapy is associated with drugresistant Bcr-abl TK activation. This is associated with amplification of the Bcr-abl gene, mutations within the ATP-binding pocket, and other regions of Bcr-abl (10, 11). Effective treatment of patients in initial blast crisis or relapse after STI571 therapy awaits novel strategies to more effectively inhibit Bcrabl signaling. Although the etiologic role of Bcr-abl in the pathogenesis of CML is now well established, the signaling pathways by which it transforms myeloid cells is much less understood. Received 8/7/02; accepted 10/25/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by The Belfer Foundation and Taub Foundation (N. R., M. M. M.), USPHS National Research Service Award GM07185 (M. E. G.), and Leukemia and Lymphoma Society (C. L. S.). C. L. S. is a Doris Duke Distinguished Clinical Scientist. 2 To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-2370; Fax: (212) 717-3627; E-mail: rosenn@ mskcc.org. 3 The abbreviations used are: CML, chronic myelogenous leukemia; TK, tyrosine kinase; RIPA, radioimmunoprecipitation assay; Bcr, breakpoint cluster region; Abl, Abelson; PK, protein kinase; MAPK, mitogenactivated protein kinase. 1267 Vol. 9, 1267–1273, April 2003 Clinical Cancer Research Research. on June 9, 2017. © 2003 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Numerous substrates are tyrosine phosphorylated by Bcr-abl. These include adapter molecules (Crk, Shc, and p62), proteins associated with cytoskeletal functions (Paxillin, Fak, and Talin), proteins with catalytic functions (phosphatidylinositol 3 kinase, peritoneal lymphocyte, Ras-GTPase-activating protein, and Syp), and other proteins, including Bap-1, Cbl, and Vav (reviewed in Ref. 12). Bcr-abl also has autophosphorylation activity. In fact, Bcr-abl may be signaling through several pathways. Autophosphorylation provides docking sites for adapter proteins, including Grb2 and Shc, which can lead to proliferation through activation of the Ras/MAPK pathway; recruitment of Crkl, which can lead to altered cell adhesion; recruitment of phosphatidylinositol 3 kinase, leading to activation of the Akt pathway and antiapoptotic signaling; and phosphorylation of Stat1 and Stat5 with activation of the Stat pathway (reviewed in Ref. 12). Bcr-abl also activates other nonreceptor TKs, including the src family kinases Hck and Lyn. Bcr-abl directly associates with Hck and Lyn and results in their increased activities (13). Novel therapies for CML need to address the emerging problem of clinical resistance to STI571. Because tumor progression in patients receiving STI571 seems to be mediated by amplification of or mutations in the Bcr-abl gene that cause the TK to be less efficiently inhibited by the drug, newer TK inhibitors may be susceptible to the same mechanisms of resistance. We report here that STI571 resistance can be overcome by a novel TK inhibitor of a different class. We have been studying a family of structurally unrelated TK inhibitors selective for src kinases. A member of this family of TK inhibitors was recently reported to have substantial activity against Bcr-abl and Bcrabl-driven cells (14). In this study, we have screened this family of TK inhibitors, designated previously as src-selective inhibitors, find varying degrees of anti-abl activities, and identified a compound with the most potent anti-Bcr-abl activity to date. This compound shows picomolar antileukemic activity specifically in Bcr-abl-driven cell lines, has substantial activity against some STI571-resistant Bcr-abl mutants, and provides a prototype for the next generation of CML therapies. Materials and Methods Cell Culture and Growth Assays. Cells were cultured in RPMI medium supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, 4 mM glutamine, and 10% heat-inactivated fetal bovine serum and incubated at 37°C in 5% CO2. For growth assays, cells were seeded in 12-well clusters at 10,000–20,000 cells/well. Cells were placed in media containing various concentrations of the drugs with vehicle (DMSO) never contributing 0.1%. After 4–7 days, cells were counted using a Coulter counter. All experiments were performed in duplicate, and results were averaged. PD166326 was stored in a 10 mg/ml DMSO solution and stored at 70°C. The derivation and chemical structure of PD166326 has been published previously (15). Cell Cycle Assays. Cells were treated with indicated concentrations of PD166326 or vehicle (DMSO) for the indicated times. For synchronization, cells were incubated in media containing 5 g/ml aphidicolin for 24 h, washed twice in PBS, and replaced in growth media. At the time of harvest, cells were washed once in PBS, cell nuclei were prepared by the method of Nusse (16), and cell cycle distribution was determined by flow cytometric analysis of DNA content using red fluorescence of 488 nm excited ethidium bromide-stained nuclei. Protein Extraction and Western Blotting. Cells were washed in PBS once and lysed in modified RIPA buffer [10 mM sodium phosphate (pH 7.2), 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% NP40, 1% Na deoxycholate, 1 mM Na Vanadate, and protease inhibitors]. Total cellular protein (50 g) was separated by SDS-PAGE, transferred to membrane, and immunoblotted using antibodies to phosphotyrosine (Santa Cruz Biotechnology), c-abl (8E9), phospho-Hck (Santa Cruz Biotechnology), MAPK (Santa Cruz Biotechnology), and phospho-MAPK (Promega). In Vitro Kinase Assay. C-abl kinase assays were performed using purified recombinant c-abl and peptide substrate (New England Biolabs). Kinase assays were performed in 50 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.2% Triton-X, 100 M ATP, and 40 M peptide substrate, in 100l reaction volumes containing 50 units of c-abl enzyme and 10 Ci [P] -ATP. Reactions were allowed to proceed for 10 min at 30°C and stopped by the addition of EDTA and boiling. Reaction products were spotted on phosphocellulose paper, washed several times with phosphoric acid and then acetone, and counted in scintillation fluid. Pilot experiments were initially performed to establish that these reaction conditions were in linear range.