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 Mutants1

Vol. 9, 1267–1273, April 2003 Clinical Cancer Research 1267

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 Mutants1

David R. Huron, Mercedes E. Gorre, be overcome by TK inhibitors of other structural classes, Alan J. Kraker, Charles L. Sawyers, Neal Rosen,2 suggesting new approaches for future anticancer drug de- and Mark M. Moasser velopment. PD166326 is a prototype of a new generation of anti-Bcr-abl compounds with picomolar potency and sub- Departments of Medicine [N. R., M. M. M.] and Cell Biology [N. R.], stantial activity against STI571-resistant mutants. Memorial Sloan-Kettering Cancer Center and Program in Pharmacology, Weill Graduate School of Medical Sciences, Cornell University [D. R. H.], New York, New York 10021; Department of Introduction Medicine and Molecular Biology Institute, University of California, CML3 is a myeloproliferative disorder of hematopoietic Los Angeles, California 90095 [M. E. G., C. L. S.]; and Cancer Pharmacology, Pfizer Global Research and Development, Ann Arbor, stem cells. The pathologic hallmark of this disease is the Phil- Michigan 48105 [A. J. K.] adelphia chromosome, which is seen in Ͼ90% of CML patients. The Philadelphia chromosome is the result of a t(9;22) chromo- somal translocation that results in the juxtaposition of 3Ј se- Abstract quences of the Abl TK proto-oncogene located on chromosome Inhibition of the constitutively active Bcr-abl tyrosine 9 with 5Ј sequences of the Bcr gene on chromosome 22. The kinase (TK) by STI571 has proven to be a highly effective resulting Bcr-abl fusion gene encodes a novel cytoplasmic pro- treatment for chronic myelogenous leukemia (CML). How- tein with constitutive TK activity (1). Expression of this fusion ever, STI571 is only transiently effective in blast crisis, and gene is seen in 90% of patients with CML, and experimental drug resistance emerges by amplification of or development evidence confirms that Bcr-abl expression is sufficient to induce of mutational changes in Bcr-abl. We have screened a family CML-like disease in mice (2). The oncogenic potency of of TK inhibitors of the pyrido [2,3-d]pyrimidine class, un- the Bcr-abl oncoprotein is quantitatively related to its TK ac- related to STI571, and describe here a compound with sub- tivity (3). stantial activity against STI-resistant mutant Bcr-abl pro- Efforts to develop novel treatments for CML have focused teins. This compound, PD166326, is a dual specificity TK on targeting the TK activity of Bcr-abl and has led to the inhibitor and inhibits src and abl in vitro with IC50sof6and development of STI571, a 2-phenylaminopyrimidine compound 8nM respectively. PD166326 inhibits the growth of K562 that is potent, and selective inhibitor of Abl, c-kit, and platelet- cells with IC50 of 300 pM, leading to apoptotic G1 arrest, derived growth factor-receptor TKs (4). This compound selec- whereas non-Bcr-abl cell types require >1000 times higher tively inhibits the growth of Bcr-abl-positive but not -negative concentrations. We tested the effects of PD166326 on two of cell lines (5, 6). STI571 has potent antileukemic activity in the clinically observed STI571-resistant Bcr-abl mutants. clinical studies producing complete hematological responses in PD166326 potently inhibits the E255K mutant Bcr-abl pro- 98% of patients in chronic phase CML, including complete tein and the growth of Bcr-ablE255K-driven cells. The cytogenetic responses in Ͼ50% (7, 8). Clinical response is T315I mutant Bcr-abl protein, which is mutated within associated with inhibition of Bcr-abl TK activity in vivo (7). the ATP-binding pocket, is resistant to PD166326; how- STI571 also produces hematological remissions in 50–70% of ever, the growth of Bcr-ablT315I-driven cells is partially patients with blast crisis, although these responses are frequently sensitive to this compound, likely through the inhibition of not durable, and patients relapse within 2–6 months of therapy Bcr-abl effector pathways. These findings show that TK (9). Relapse during STI571 therapy is associated with drug- drug resistance is a structure-specific phenomenon and can resistant 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 Bcr- Received 8/7/02; accepted 10/25/02. The costs of publication of this article were defrayed in part by the abl signaling. payment of page charges. This article must therefore be hereby marked Although the etiologic role of Bcr-abl in the pathogenesis advertisement in accordance with 18 U.S.C. Section 1734 solely to of CML is now well established, the signaling pathways by indicate this fact. which it transforms myeloid cells is much less understood. 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 3 The abbreviations used are: CML, chronic myelogenous leukemia; Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY TK, tyrosine kinase; RIPA, radioimmunoprecipitation assay; Bcr, break- 10021. Phone: (212) 639-2370; Fax: (212) 717-3627; E-mail: rosenn@ point cluster region; Abl, Abelson; PK, protein kinase; MAPK, mitogen- mskcc.org. activated protein kinase. Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. 1268 An Inhibitor of STI571-resistant Bcr-abl Mutants

Numerous substrates are tyrosine phosphorylated by Bcr-abl. Table 1 In vitro activities of TK inhibitors (all values in nM) These include adapter molecules (Crk, Shc, and p62DOK), pro- c-src IC50 c-abl IC50 teins associated with cytoskeletal functions (Paxillin, Fak, and Ј PD166406 21 300 Talin), proteins with catalytic functions (phosphatidylinositol 3 PD173955 22 110 kinase, peritoneal lymphocyte-␥, Ras-GTPase-activating pro- PD179483 9 40 tein, and Syp), and other proteins, including Bap-1, Cbl, and PD166326 6 8 Vav (reviewed in Ref. 12). Bcr-abl also has autophosphorylation STI571 Inactive 50 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 prolifera- washed once in PBS, cell nuclei were prepared by the method of tion through activation of the Ras/MAPK pathway; recruitment Nusse (16), and cell cycle distribution was determined by flow of Crkl, which can lead to altered cell adhesion; recruitment of cytometric analysis of DNA content using red fluorescence of phosphatidylinositol 3Ј kinase, leading to activation of the Akt 488 nm excited ethidium bromide-stained nuclei. pathway and antiapoptotic signaling; and phosphorylation of Protein Extraction and Western Blotting. Cells were Stat1 and Stat5 with activation of the Stat pathway (reviewed in washed in PBS once and lysed in modified RIPA buffer [10 mM Ref. 12). Bcr-abl also activates other nonreceptor TKs, includ- sodium phosphate (pH 7.2), 150 mM NaCl, 0.1% sodium dode- ing the src family kinases Hck and Lyn. Bcr-abl directly cyl sulfate, 1% NP40, 1% Na deoxycholate, 1 mM Na Vanadate, associates with Hck and Lyn and results in their increased and protease inhibitors]. Total cellular protein (50 ␮g) was activities (13). separated by SDS-PAGE, transferred to membrane, and immu- Novel therapies for CML need to address the emerging noblotted using antibodies to phosphotyrosine (Santa Cruz Bio- problem of clinical resistance to STI571. Because tumor pro- technology), c-abl (8E9), phospho-Hck (Santa Cruz Biotechnol- gression in patients receiving STI571 seems to be mediated by ogy), MAPK (Santa Cruz Biotechnology), and phospho-MAPK amplification of or mutations in the Bcr-abl gene that cause the (Promega). TK to be less efficiently inhibited by the drug, newer TK In Vitro Kinase Assay. C-abl kinase assays were per- inhibitors may be susceptible to the same mechanisms of resist- formed using purified recombinant c-abl and peptide substrate ance. We report here that STI571 resistance can be overcome by (New England Biolabs). Kinase assays were performed in 50 a novel TK inhibitor of a different class. We have been studying mM Tris-Cl (pH 7.5), 10 mM MgCl ,1mM EGTA, 2 mM DTT, a family of structurally unrelated TK inhibitors selective for src 2 0.2% Triton-X, 100 ␮M ATP, and 40 ␮M peptide substrate, in kinases. A member of this family of TK inhibitors was recently 100-␮l reaction volumes containing 50 units of c-abl enzyme reported to have substantial activity against Bcr-abl and Bcr- and 10 ␮Ci [32P] ␥-ATP. Reactions were allowed to proceed for abl-driven cells (14). In this study, we have screened this family 10 min at 30°C and stopped by the addition of EDTA and of TK inhibitors, designated previously as src-selective inhibi- boiling. Reaction products were spotted on phosphocellulose tors, find varying degrees of anti-abl activities, and identified a paper, washed several times with phosphoric acid and then compound with the most potent anti-Bcr-abl activity to date. acetone, and counted in scintillation fluid. Pilot experiments This compound shows picomolar antileukemic activity specifi- were initially performed to establish that these reaction condi- cally in Bcr-abl-driven cell lines, has substantial activity against tions were in linear range. some STI571-resistant Bcr-abl mutants, and provides a prototype for the next generation of CML therapies. Results PD166326 Inhibits c-Abl in Vitro. In screening a com- Materials and Methods pound library for inhibitors of c-src TK activity, a number of Cell Culture and Growth Assays. Cells were cultured pyrido[2,3-d]pyrimidines were described previously that are Ͻ in RPMI medium supplemented with 100 units/ml penicillin, ATP-competitive inhibitors of c-src with IC50s 20 nM and 100 ␮g/ml streptomycin, 4 mM glutamine, and 10% heat-inac- varying degrees of selectivity for c-src (15). We screened this

tivated fetal bovine serum and incubated at 37°Cin5%CO2. group of compounds for activity against c-abl using purified For growth assays, cells were seeded in 12-well clusters at recombinant c-abl and peptide substrate in in vitro kinase as-

10,000–20,000 cells/well. Cells were placed in media contain- says. The most potent compound was PD166326 with an IC50 of ing various concentrations of the drugs with vehicle (DMSO) 8nM (against c-abl) and 6 nM (against src; Table 1). The src Ͼ Ͻ never contributing 0.1%. After 4–7 days, cells were counted family kinase Lck is inhibited with IC50 5nM. This compound using a Coulter counter. All experiments were performed in also has activity against basic fibroblast growth factor, platelet- duplicate, and results were averaged. PD166326 was stored in a derived growth factor, and epidermal growth factor receptor Ϫ 10 mg/ml DMSO solution and stored at 70°C. The derivation TKs in vitro with IC50s of 62, 139, and 80 nM, respectively (15). and chemical structure of PD166326 has been published previ- PD166326 shows no significant activity against c-Jun-NH2- ously (15). terminal kinases, cyclic AMP-dependent PKA, PKB-␤, PKC-␣, Cell Cycle Assays. Cells were treated with indicated rho-dependent PK, casein kinase-2, and phosphorylase kinase. concentrations of PD166326 or vehicle (DMSO) for the indi- In comparison with PD166326, STI571 is a weaker inhibitor of ϭ cated times. For synchronization, cells were incubated in media Abl in vitro with an IC50 50 nM (Table 1). containing 5 ␮g/ml aphidicolin for 24 h, washed twice in PBS, PD166326 Inhibits Bcr-abl in Vivo. PD166326 also in- and replaced in growth media. At the time of harvest, cells were hibits Bcr-abl activity in cells as determined by Western blot

by this compound as shown by experiments with synchronized

cells. K562 cells were synchronized at the G1-S boundary with aphidicolin and released into PD166326 or vehicle, and cell cycle progression was studied over the following 24 h. These data show that PD166326 treatment does not interfere with

progression through the S, G2, or mitotic phases of the cell cycle, but PD166326-treated cells are unable to exit the G1 phase (Fig. 3). Similar experiments with nocodazole-synchro-

nized cells also confirm that PD166326 blocks G1 progression (data not shown). The inhibition of G1 progression and induction of apoptosis in K562 cells are similar to the effects reported previously for STI571 (17). These data show that PD166326 is a potent inhibitor of Bcr-abl kinase activity and inhibits Bcr-

abl-driven cell growth through the inhibition of G1 progression, leading to apoptotic cell death. PD166326 Inhibits Cells Driven by STI571-resistant Fig. 1 Log phase K562 cells were treated with STI571 or PD166326 for 24 h at the indicated concentrations and harvested in RIPA buffer, Mutant Bcr-abl. Resistance to STI571 treatment is associated and total cellular lysates were separated by SDS-PAGE, transferred to with mutations in the Bcr-abl oncoprotein that render it refrac- membrane, and immunoblotted using antiphosphotyrosine, anti-abl, an- tory to STI571 inhibition (10). Because PD166326 inhibits both ti-MAPK, or antiphosphoMAPK antibodies. The concentrations of Src and Abl, whereas STI571 only inhibits Abl, it may bind PD166326 shown here are considerably lower than shown for STI571, because Bcr-abl autophosphorylation is much more sensitive to Bcr-abl differently than STI571. This difference raises the pos- PD166326. These concentrations are within dose ranges that produce sibility that it may be effective against some mutant Bcr-abl growth inhibition with these two drugs. proteins. We compared the activities of PD166326 and STI571 against two such mutant Bcr-abl proteins derived from patients who have relapsed on STI571 therapy. The T315I mutation is frequently seen in relapsed patients, eliminates a critical Thre- Table 2 Cell growth IC50 for PD166326 (all values in nM) onine residue within the ATP-binding pocket of Abl, and greatly Cell type Bcr-abl PD166326 IC 50 reduces the binding affinity of STI571. The E255K mutation ϩ K562 0.3 also lies within a region of Bcr-abl commonly mutated in BaF3 p210Bcr-abl ϩ 6.0 MCF-7 (breast cancer) Ϫ 1000.0 relapsed patients; however, the structural basis for STI571 re- MDA-MB-468 (breast cancer) Ϫ 2000.0 sistance conferred by mutations in this region is not currently 32D (immortalized myeloid) Ϫ 800.0 understood. BaF3 mouse hematopoietic cell lines were stably BaF3 (immortalized myeloid) Ϫ 1000.0 transfected with either the wild-type p210Bcr-abl cDNA or the T315I or E255K mutant versions as described previously (10). The expression of Bcr-abl renders BaF3 cells interleukin-3 independent, whereas control cells transfected with vector alone analysis of Bcr-abl autophosphorylation in K562 cells. In these require interleukin-3 for growth. Although STI571 inhibits the Bcr-abl ϭ cells, Bcr-abl autophosphorylation is inhibited with IC50 of1nM wild-type p210 cells with IC50 500 nM, the T315I and Bcr-abl compared with 100 nM for STI571 (Fig. 1). Bcr-abl autophos- E255K mutant p210 cells are highly resistant. However, phorylation correlates with Bcr-abl-signaling activity as shown resistance to STI571 does not appear to confer cross-resistance by the parallel decline of MAPK activity with inhibition of to PD166326. PD166326 inhibits the autophosphorylation Bcr-abl in these assays (Fig. 1, bottom panel). of p210Bcr-ablE255K in vivo as effectively as the autophosphor- PD166326 Inhibits Bcr-abl-driven Cell Growth. The ylation of the wild-type p210Bcr-abl, whereas this mutant is biological activity and potency of PD166326 was initially eval- highly resistant to inhibition by STI571 (Fig. 4). However, the uated in cell growth assays using K562 cells. This compound p210Bcr-ablT315I mutant is resistant to PD166326 as it is to ϭ inhibits K562 cell growth with IC50 0.3 nM (Table 2). Another STI571. This is not surprising, considering the critical role of Bcr-abl-driven cell line, BaF3-p210Bcr-abl, is also extremely Thr (315) within the ATP-binding pocket (see “Discussion”). sensitive to PD166326 with an IC50 of6nM. The potent bio- To determine whether cell growth sensitivity to PD166326 logical activity of PD166326 is highly specific for Bcr-abl- correlates with inhibition of the mutant Bcr-abl oncoproteins, driven cells as additional hematopoetic and epithelial cell lines we also determined the sensitivity of the BaF3 cells driven by are only inhibited at two to three log higher concentrations and the wild-type and mutant Bcr-abl proteins. BaF3p210Bcr-abl cells ␮ ϭ IC50s in the 0.8–2 M range (Table 2). are very sensitive to PD166326 (IC50 6nM), and the E255K Bcr-abl PD166326 Inhibits G1 Progression. Further analysis re- mutant p210 cells remain relatively sensitive to this com- ϭ veals that PD166326 inhibits cell proliferation specifically in the pound (IC50 15 nM; Table 3). The effective inhibition of E255KBcr-abl G1 phase of the cell cycle. At concentrations that fully inhibit p210 activity at dose ranges that inhibit the growth the growth of Bcr-abl-positive cells but not other cell types, of these cells is additional evidence that STI571-resistant leu-

PD166326 leads to accumulation of cells in the G1 phase ac- kemic cells are driven by persistent activity of the mutated companied by a significant increase in the number of apoptotic Bcr-abl oncoprotein. In comparison, the T315I mutant cells are cells (Fig. 2). Additional phases of the cell cycle are not affected partially resistant to PD166326, although not fully resistant.

Fig. 2 In A, K562 cells were seeded at 40,000 cells/well of six-well clusters in 5 or 50 nM PD166326 or vehicle (DMSO) and counted each day for 5 days. Results are the average of duplicates and confirmed by additional similar experiments. In B, K562 cells were seeded at 2 million/10-cm dish in 5 or 50 nM PD166326 or vehicle (DMSO). After 48 h, cells were harvested, and cell cycle distribution was determined by flow cytometric analysis of DNA content using red fluorescence of 488 nm excited ethidium bromide-stained nuclei. PD166326-treated cells

show accumulation in the G1 phase, as well as large increases in cells with sub-G1 DNA content characteristic of apoptotic cells.

Quantitative analysis of cells with sub-G1 DNA content reveals 8% (DMSO), 34% (5 nM PD166326), and 44% (50 nM PD166326) apoptotic cells in the experimental arms.

p210T315I PD166326 inhibits BaF3 cells with IC50 of 150 nM homologous, and in some cases identical, among most members (Table 3). Although this is 25-fold weaker than the inhibition of of the src family, and therefore, these immunoblots reflect the the wild-type BaF3p210 cells, it may still be of therapetic value activation of any or multiple members of the src family. because it is 8-fold more potent than the inhibition of the BaF3-vector controls and non-Bcr-abl-driven cells (Tables 2 and 3). Although PD166326 inhibits the growth of Discussion Bcr-ablT315I BaF3p210 cells with IC50 of 150 nM, it fails to inhibit Although STI571 has revolutionized the treatment of CML, the autophosphorylation of the T351I Bcr-abl mutant at doses the problem of TK drug resistance is now emerging as a clinical Յ1 ␮M (Fig. 4), suggesting that its antiproliferative effects are reality. Resistance to STI571 appears to have a structural basis, mediated in part through mechanisms other than the inhibition and newer TK inhibitors may also be susceptible to similar of Bcr-abl (see “Discussion”). mechanisms of resistance. However, TK inhibitors of a different PD166326 Inhibits the Bcr-abl-induced Activation of structural class may have more favorable binding characteris- src Kinases. PD166326 is also active against src kinases, and tics. Dorsey et al. (14) initially reported that a src-selective TK its antileukemic effects may be in part related to its inhibition of inhibitor of the pyrido [2,3-d]pyrimidine class has substantial the src kinases Hck and Lyn, which function downstream of activity against Bcr-abl kinase. We have extended this finding Bcr-abl. The src kinases Hck and Lyn are activated by Bcr-abl by screening a family of src-selective pyrido [2,3-d]pyrimidines and may mediate some of the transforming functions of Bcr-abl. and identified a compound with the most potent activity against Phosphorylation of tyr (416) in the catalytic domain is required abl kinase. Here, we report the characterization of this com- for activation of src kinases, although the mechanism by which pound, PD166326, a novel dual specificity TK inhibitor that is Bcr-abl activates Hck and Lyn is not understood (see “Discus- Ͼ100-fold more potent than STI571 in vivo and inhibits K562

sion”). Inhibition of Bcr-abl by STI571 results in a parallel cells with an IC50 of 300 pM. It is unlikely that the potent growth inhibition of Hck activation in K562 cells (Figs. 1 and 5). In inhibitory activities of PD166326 are related to nonspecific these cells, PD166326 also inhibits Bcr-abl and Hck activation, activities because the potency of this compound appears to be although at 100-fold lower doses than seen with STI571 (Figs. specific for cell types driven by Bcr-abl kinase. Although Bcr-

1 and 5). Hck is also activated by mutant forms of Bcr-abl, and abl-driven cells are inhibited with IC50s in the 0.3–6nM range, in the mutant BaF3p210Bcr-ablE255K cells, PD166326 inhibits other cell types, including the hematopoietic cells BaF3 and Hck activation; this correlates with the observed inhibition of 32D, as well as epithelial cancer cells, including MCF-7 and Bcr-ablE255K autophosphorylation (Figs. 4 and 6) and inhibition MDA-MB-468 cells, which are driven by epidermal growth

of cell growth (Table 3). In contrast, the activation of Hck by the factor receptor overactivity, are inhibited with IC50sinthe T315I Bcr-abl mutant is not inhibited by PD166326 (Fig. 6), and 0.8–2 ␮M range (Table 2). The micromolar activity of this correlates with the observed resistance of Bcr-ablT315I ac- PD166326 against the growth of non-Bcr-abl-driven cells is tivity to PD166326 (Fig. 4). However, despite failure to inhibit most likely mediated through inhibition of additional cellular Bcr-abl activity and the consequent activation of Hck, targets because unlike Bcr-abl-positive cells, the growth of PD166326 inhibits the growth of BaF3p210Bcr-ablT315I cells Bcr-abl-negative cells is inhibited during the S phase of the cell

with IC50 of 150 nM, likely through additional mechanisms (see cycle (data not shown). The picomolar potency and cellular “Discussion”). The antiphospho-Hck antibodies used in these selectivity of PD166326 are significantly superior to STI571 in experiments specifically recognize the phosphorylated tyrosine vitro. residue in the catalytic domain of Hck. This epitope is highly Because Bcr-abl signaling is known to involve the src

Fig. 4 BaF3 transfectants were treated with the indicated concentrations of STI571 and PD166326 for 1 h and harvested in RIPA buffer, and total cellular lysates were separated by SDS-PAGE, transferred to membrane, and immunoblotted using antiphosphotyrosine antibodies.

Table 3 Cell growth IC50s against Bcr-abl mutant cells (all values in nM) Cell type STI571 PD166326 BaF3-vector Ͼ1000.0 1200.0 BaF3-p210wt 500.0 6.0 BaF3-p210T315I Ͼ1000.0 150.0 BaF3-p210E255K Ͼ1000.0 15.0

Fig. 3 K562 cells were synchronized in 5 ␮g/ml aphidicolin for 20 h, washed twice in PBS, and incubated in media containing 50 nM PD166326 or DMSO. Cells were harvested at 6, 12, 18, and 24 h after aphidicolin release, and cell cycle distribution was determined by flow Fig. 5 K562 cells were treated with the indicated concentrations of cytometry as described. STI571 or PD166326 for 1 h and harvested in RIPA buffer, and total cellular lysates were separated by SDS-PAGE and immunoblotted using anti-Hck p-Y416 antibodies. This antibody recognizes the phosphorylated tyrosine residue within the catalytic domain of most members of the src family. family kinases Hck and Lyn, and because PD166326 is also a potent inhibitor of src family kinases, it is plausible that the biological potency of this compound is related to dual inhibition of these two functionally related TKs. Hck associates with and inhibitory effects of this compound in Bcr-abl-driven cells is phosphorylates Bcr-abl on Tyr 177, leading to recruitment of difficult to know until such candidate targets are identified and Grb2/Sos and activation of the Ras pathway (18). Kinase- studied. defective Hck mutants suppress Bcr-abl-induced transforma- Because relapse on STI571 is associated with mutations in tion, suggesting that Hck-mediated signaling is essential for the Bcr-abl that alter the binding of STI571, understanding the transforming activity of Bcr-abl (19). The role of Lyn in Bcr-abl nature of the STI571 interaction with Abl is of fundamental signaling is less well studied. However, Lyn activity is also importance to overcome drug resistance. The crystal structure of elevated in acute myeloid leukemia cell lines, and in these cells, a variant STI571 in complex with the catalytic domain of Abl inhibition of Lyn expression using antisense molecules leads to was recently solved by Schindler et al. (21). STI binds within decreased proliferative activity; inhibition of Lyn kinase activity the ATP-binding pocket of Abl in its inactive conformation. using src family-selective pharmacological inhibitors leads to This interaction is critically affected by the conformation of the potent inhibition of cell growth and colony formation (20). It is Abl activation loop. When phosphorylated, this activation loop also possible that the potency of PD166326 is mediated through favors an open and activating conformation, which, by virtue of the inhibition of other, yet undiscovered cellular proteins, and its NH2-terminal anchor, interferes with STI571 binding to the our data do not exclude this possibility. However, the role of ATP-binding pocket. Consistent with this model, the binding of currently unknown cellular targets in mediating the growth STI571 is selective for the inactive conformation of Abl, and

findings. A number of amino acid residues mediate the binding of STI571 within the ATP-binding pocket, and among these, Thr (315) is critical for hydrogen bond formation with the drug (21). The T315I mutation, seen in STI571-resistant CML, pre- cludes hydrogen bonding with STI571 and results in a steric clash attributable to the extra hydrocardon group in Ile (10). Likewise, PD166326 does not inhibit the activity of Bcr-ablT315I in vivo (Fig. 4), suggesting that Thr (315) is also important for its binding within the ATP pocket of abl. However, PD166326 has some activity against BaF3p210T315I cells and inhibits their

growth with IC50 of 150 nM (Tables 2 and 3). This activity is related to Bcr-abl-driven growth because growth inhibition of non-Bcr-abl-driven cell types requires 5–15-fold higher concen- Fig. 6 BaF3 cells transfected with the wild-type and mutant Bcr-abl trations. Because PD166326 is a potent inhibitor of src kinases, constructs were treated with the indicated concentrations of STI571 and and because the src kinases Hck and Lyn mediate some of the PD166326 for 1 h and harvested. Total cellular lysates were separated by SDS-PAGE and immunoblotted using anti-Hck p-Y416 antibodies. transforming activities of Bcr-abl, it is possible that PD166326 This antibody recognizes the phosphorylated tyrosine residue within the inhibits the growth of BaF3p210T315I cells through the inhibi- catalytic domain of most members of the src family. tion of Hck and Lyn. However, seemingly inconsistent with this hypothesis, we fail to see inhibition of Hck Y416 phosphorylation in these cells at growth inhibitory concentrations (Fig. 6). However, this does not disprove the hypothesis because of this compound is unable to inhibit the catalytic activity of active limitations in assaying Hck activity in vivo. If PD166326 binds phosphorylated Abl (21). The broader activity of PD166326, with and inhibits the active Y416 phosphorylated conformation including activity against src kinases, suggests that unlike of Hck, then this catalytically inactive drug-Hck complex may STI571, it may not bind selectively to the inactive conformation remain stably in this phosphorylated conformation, and phos- of Abl, because in its active conformation, Abl bears consider- pho-Y416 Hck antibodies will be unable to demonstrate the in able structural homology to the src kinases (21). Although vivo inhibition of Hck catalytic function. In vitro kinase assays selectivity for the inactive conformation is postulated to confer do not help in this regard either, because during the process of a high degree of molecular specificity to STI571, this may be at cell lysis and immunoprecipitation, the Hck-PD166326 interac- the price of potency. PD166326 may be binding to both inactive tion is lost (data not shown). Therefore, in BaF3 p210T315I cells, and active conformations of Abl, leading to the more effective where Bcr-abl activity is resistant to PD166326, inhibition of inhibition of overall enzyme activity that we see in vitro (Table Hck activity may be responsible for the observed growth inhib- ϭ 1). In addition, phosphorylation of the activation loop of Abl is itory effects at IC50 150 nM despite persistent phosphorylation catalyzed by the src family kinase Hck in Bcr-abl-transformed of Hck at these doses. In addition, although Y416 is a site of cells. Because PD166326 also inhibits Hck, this may prevent autophosphorylation in src kinases, it may also be a substrate for phosphorylation of the activation loop, destabilizing the Abl phosphorylation by other kinases. In fact, in our experiments, active conformation. This allosteric mechanism, in addition to Hck Y416 phosphorylation status parallels Bcr-abl activity (com- the direct binding of PD166326 to the ATP-binding pocket, pare Figs. 4 with 6), which suggests that Hck Y416 may also be could provide dual mechanisms for its inhibition of Abl activa- a substrate for Bcr-abl. Although the activity of PD166326 tion and provide the basis for its increased potency. Validation against src kinases would suggest that it inhibits BaF3 p210T315I of these hypotheses awaits crystallographic studies of cells through a src family member, these experiments do not rule PD166326 bound to Abl. out the possibility that this cellular sensitivity is mediated PD166326 is noncross-resistant with STI571 and has sub- through the inhibition of other, yet unknown, kinases. stantial activity against the T315I and E255K STI571-resistant The structural basis for the STI571 resistance of the Bcr-abl mutants. This finding has important implications for the E255K-mutated Bcr-abl is less clear because the functional future design and use of TK inhibitors of all kinds, because it is significance of this residue is currently unknown. Interestingly, the first report showing that TK inhibitor resistance can be this mutation confers little resistance to PD166326 (Table 2 and overcome by another TK inhibitor of a different structural class. Fig. 4). PD166326 shows no loss of activity against Bcr- It is difficult to speculate on whether the development of resist- ablE255K autophosphorylation in vivo (Fig. 4) and only 2.5-fold ance to PD166326 will be just as likely as with STI571, but less activity against the growth of BaF3Bcr-ablE255K cells (Ta- because these compounds are structurally unrelated, resistance ble 3) compared with wild-type Bcr-abl controls. The cellular E255K to PD166326 will likely involve a different structural basis than IC50 of PD166326 against BaF3Bcr-abl cells (15 nM)is resistance to STI571. This distinction creates the opportunity for much lower than its activity in non-Bcr-abl-driven cell types strategies to prevent or overcome resistance, such as sequential (0.8–2 ␮M) and much greater than the activity of STI571 against or combination therapies. However, understanding drug sensi- this mutant. If the basis for Bcr-abl E255K resistance to STI571 tivity and resistance is of fundamental importance in this regard. is destabilization of the inactive conformation, and if PD166326 Although more studies are necessary to understand the in fact binds to the active conformation, then this would explain structural and cellular basis underlying STI571 resistance and why PD166326 is effective in inhibiting Bcr-ablE255K. How- PD166326 sensitivity, existing data at least partly explain our ever, validation of these hypotheses requires crystal structure

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Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. 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

David R. Huron, Mercedes E. Gorre, Alan J. Kraker, et al.

Clin Cancer Res 2003;9:1267-1273.

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