¹Molecular Oncology and Experimental Therapeutics Programs, H Lee Moffitt Cancer Center and Research Institute, University of South Florida College of Medicine, Tampa, FL, USA

²Department of Interdisciplinary Oncology, University of South Florida College of Medicine, Tampa, FL, USA

³Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa, FL, USA

⁴Cancer Pharmacology, Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, MI, USA

⁵Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa, FL, USA

Correspondence to: J Wu, Molecular Oncology Program, MRC 3-East, H Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA; Fax: (813) 903-6817

Bcr-Abl tyrosine kinase has been validated as a molecular target for the treatment of chronic myelogenous leukemia (CML). More recently, it has been reported that CML patients could develop resistance to the Bcr-Abl tyrosine kinase inhibitor, imatinib (STI571, Gleevec), pointing to the need for development of additional Bcr-Abl tyrosine kinase inhibitors or other therapeutic strategies. It was also found that a significant proportion of patients who received the Bcr-Abl inhibitor did not achieve complete cytogenetic response. Mechanisms for incomplete cytogenetic response to Bcr-Abl inhibition are not entirely clear. We report here three new pyrido[2,3-d]pyrimidine Bcr-Abl tyrosine kinase inhibitors, PD164199, PD173952, PD173958, that induced apoptosis of Bcr-Abl-dependent hematopoietic cells. An interleukin-3 (IL-3) autocrine loop was observed previously in primitive CD34⁺/Bcr-Abl⁺ leukemic cells in CML patients. Using 32Dp210^Bcr-Abland Baf3p210^Bcr-Abl cells as models, we tested whether IL-3 might protect Bcr-Abltransformed, IL-3-responsive cells from apoptosis caused by Bcr-Abl tyrosine kinase inhibition. Results of trypan blue exclusion, fluoroisothiocyanate-valyl-alanyl-aspartyl-[O-methyl] -fluoromethylketone (FITC-VAD-FMK), and Annexin-V/7-amino-actinomycin D (7-AAD) binding assays indicate that IL-3 could protect Bcr-Abl-transformed, IL-3 responsive hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors. This finding raises the possibility that the IL-3 autocrine loop found in primitive CD34⁺/Bcr-Abl⁺ cells in CML patients could contribute to the incomplete eradication of Bcr-Abl⁺ cells by Bcr-Abl inhibition.

Leukemia (2002) 16, 1589-1595. doi:10.1038/sj.leu.2402678

Bcr-Abl; leukemia; tyrosine kinase inhibitor; interleukin-3; apoptosis

Introduction

Chronic myelogenous leukemia (CML) is a malignant disorder of hematopoietic stem cells.^1,2 A cytogenetic characteristic of CML is the presence of the Philadelphia chromosome, which arises from the reciprocal t(9;22) chromosomal translocation and contains the Bcr-Abl fusion gene. The Bcr-Abl oncogene encodes either the p210^Bcr-Abl tyrosine kinase that is found in most cases of CML or the p185^Bcr-Abl tyrosine kinase that is found mostly in acute lymphoblastic leukemia.¹ Bcr-Abl confers cytokine-independent proliferative and anti-apoptotic activities to hematopoietic cells.² Genetic and biochemical studies have demonstrated that the tyrosine kinase activity of Bcr-Abl is essential for its transforming activity.^3,4,5,6 Moreover, the wild-type c-Abl tyrosine kinase does not appear to be essential for normal cell survival and proliferation. Therefore, Bcr-Abl tyrosine kinase represents an excellent, rational molecular target for therapy of CML. This concept has been validated by demonstration of the clinical efficacy of the Bcr-Abl tyrosine kinase inhibitor, imatinib (formerly CGP57148, STI571, Gleevec), in CML.^7,8,9,10,11

While imatinib has remarkable clinical efficacy in CML, primary and acquired resistance in some patients have been observed as a result of Bcr-Abl gene amplification, Bcr-Abl tyrosine kinase mutation or other unknown changes.^{8,12,13,14,15,16,17,18} Several mutations in the tyrosine kinase domain of Bcr-Abl have been identified from patients with acquired resistance to imatinib.^{12,13,14,15,16,17} Laboratory experiments have confirmed that Bcr-Abl gene harboring these mutations become resistant to inhibition by the Bcr-Abl tyrosine kinase inhibitor.^12,16 Therefore, while imatinib represents a major therapeutic advance for CML, further development of new, more potent Bcr-Abl tyrosine kinase inhibitors or other therapeutic strategies are warranted.

Furthermore, while complete hematologic responses in peripheral blood was achieved in virtually all patients with CML in the chronic phase when they were given a sufficiently high dose of imatinib, complete cytogenetic remissions in bone marrow was observed in 41% of these patients.⁹ This observation implies that a majority of these patients have one or more sub-populations of Bcr-Abl⁺ hematopioetic cells in bone marrow that are resistant to the Bcr-Abl tyrosine kinase inhibitor. Interleukin-3 (IL-3) is a known growth and survival factor for hematopoietic stem/progenitor cells. Expression of IL-3 receptor is repressed when hematopoietic progenitor cells differentiate. Interestingly, it was observed that primitive (CD34⁺) leukemic cells isolated from Bcr-Abl⁺ CML patients have an autocrine loop of IL-3 production and response.¹⁹

In this study, we identified three new Bcr-Abl tyrosine kinase inhibitors, which are among the most potent Abl tyrosine kinase inhibitors in vitro. Using two p210^Bcr-Abl-transformed, IL-3 responsive hematopoietic progenitor cells as models, we investigated the possibility that IL-3 might protect Bcr-Abl-transformed, IL-3 responsive cells from apoptosis caused by Bcr-Abl tyrosine kinase inhibitors. We found that while Bcr-Abl tyrosine kinase inhibitors induced apoptosis of these cells in the absence of IL-3, they were ineffective in the presence of IL-3. This finding suggests that IL-3 could play a critical role in protecting Bcr-Abl⁺ hematopoietic cells from Bcr-Abl tyrosine kinase inhibitors and may contribute to the incomplete cytogenetic response to Bcr-Abl inhibition observed in CML patients.

Materials and methods

Reagents and cells

PD159373, PD164199, PD173952, PD173958, and PD180970 (Table 1) were synthesized by Pfizer Global Research and Development.^20,21 Imatinib (STI571) was from Novartis Pharmaceuticals (East Hanover, NJ, USA). Recombinant Abl and Lyn tyrosine kinases were obtained from New England Biolab (Beverly, MA, USA) and Upstate Biotechnology (Lake Placid, NY, USA), respectively. A plasmid for GST-Csk was kindly provided by Dr Torkel Vang (University of Oslo, Oslo, Norway).²² GST-Csk protein was expressed in E. coli DH5 and affinity purified by standard procedure. Baculovirus-expressed human insulin receptor tyrosine kinase was a gift of Coralia Rivas (Memorial Sloan-Kettering Cancer Center, New York, USA).²³ Murine IL-3 was obtained from PeproTech (Rocky Hill, NJ, USA). K562 is a human CML cell line that contains p210^Bcr-Abl. 32Dp210^Bcr-Abl is a p210^Bcr-Abl-transformed mouse promyeloid 32D cell line, while Baf3p210^Bcr-Abl is a p210^Bcr-Abl-transformed mouse Baf3 pre-B cell line.²⁴ All cell lines were grown in RPMI 1640 containing 10% fetal bovine serum and 100 g/ml penicillinstreptomycin.

In vitro tyrosine kinase assays

Abl tyrosine kinase activity was determined using a synthetic peptide (EAIYAAPFAKKK) as substrate. The activities of Lyn and Csk tyrosine kinases were assayed using a Src substrate peptide (KVEKIGEGTYGVVYK). The activity of a recombinant insulin receptor tyrosine kinase was measured using Raytide (Calbiochem, La Jolla, CA, USA) as substrate. The conditions for the in vitro kinase assays were as provided previously.²⁵ Kinase reactions were performed in the presence of various concentrations of test compound (1 l in DMSO, total reaction mixture volume was 40 l). IC₅₀ was obtained as the concentration of compound required to inhibit tyrosine phosphorylation of substrate by 50%.

Cell viability, poly-(ADP-ribose) polymerase (PARP) cleavage, fluoroisothiocyanate-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (FITC-VAD-FMK) and Annexin-V/7-amino-actinomycin D (7-AAD binding assays)

Cells (1 ´ 10⁵ cells/ml) were plated in 2 ml RPMI/10% FCS. Tyrosine kinase inhibitors, IL-3 (10 ng/ml), or DMSO (solvent control) were added at the time of cell plating. The volume of DMSO was kept at 0.1% of the medium volume. Cell viability was determined by the trypan blue exclusion assay.²⁵ PARP cleavage assay and flow cytometric analysis of apoptosis using the Annexin-V/7-AAD binding assay were performed as described²⁵ except that 7-AAD (Via-Probe, PharMingen, San Diego, CA, USA), instead of propidium iodide, was used.

For flow cytometric analysis of apoptosis using the CaspACE FITC-VAD-FMK In Situ Marker (Promega, Madison, WI, USA), cells (1.5 ´ 10⁵ in 0.5 ml) were incubated with 10 M FITC-VAD-FMK in the dark at 37°C, 5% CO₂ for 20 min. After incubation, cells were washed twice with phosphate-buffered saline, resuspended in 0.5 ml phosphate-buffered saline, and analyzed immediately. At least 1 ´ 10⁴ gated-cells in each sample were analyzed. Untreated cells stained with FITC-VAD-FMK were used to compensate for the flow cytometric analysis.

Other assays

Immunoprecipitation and immunoblotting for analysis of tyrosine phosphorylation of Bcr-Abl and CrkL were performed as described.^25,26

Results

Analysis of pyrido[2,3-d]pyrimidine derivatives for Bcr-Abl tyrosine kinase inhibitory activities in vitro

We previously found that a pyrido[2,3-d]pyrimidine derivative, PD180970, was a potent Abl tyrosine kinase inhibitor.²⁵ To test whether other pyrido[2,3-d]pyrimidine derivatives also inhibit the Abl tyrosine kinase, we analyzed the Abl tyrosine kinase inhibitor activity of four other derivatives available to us (Table 1). As shown in Table 1, PD164199, PD173952 and PD173958 have a similar potency as PD180970 in inhibiting the Abl tyrosine kinase activity in vitro. The IC₅₀ of these three compounds for Abl tyrosine kinase inhibition in vitro was in the 0.6-1.7 nM range. On the other hand, PD159373 had only a small effect on Abl tyrosine kinase activity in vitro at the concentrations that we have tested. PD164199, PD173952, PD173958 and PD180970 also showed similar inhibitory activities towards the Lyn tyrosine kinase (IC₅₀: 0.3-2.8 nM), which is a hematopoietic cell Src-family kinase, consistent with these compounds as Src tyrosine kinase inhibitors. The Csk tyrosine kinase, which is a negative regulator of the Src family kinases, was inhibited by PD164199, PD173952, PD173958 and PD180970 at higher concentrations (IC₅₀: 6.6-10.8 nM), (Table 1). All five compounds did not inhibit the insulin receptor tyrosine kinase (Table 1).

Inhibition of p210^Bcr-Abl and CrkL tyrosine phosphorylation in human K562 cells by the pyrido[2,3-d]pyrimidine derivatives

To determine whether PD159373, PD164199, PD173952 and PD173958 inhibit Bcr-Abl tyrosine kinase activity in human CML cells, we treated K562 cells with various concentrations of these compounds (Figure 1a and b). Cells were also treated with the previously characterized PD180970 as a positive control. p210^Bcr-Abl and its substrate CrkL were immunoprecipitated from cell lysates and analyzed by immunoblotting with an anti-phosphotyrosine antibody (PY20). As shown in Figure 1a and b, PD164199, PD173952, PD173958 and PD180970 inhibited tyrosine phosphorylation of p210^Bcr-Abl and CrkL in K562 cells in a concentration-dependent manner. Consistent with the in vitro kinase assay, PD159373 did not inhibit p210^Bcr-Abl and CrkL tyrosine phosphorylation in K562 cells (Figure 1a and b). In additional experiments, we found that inhibition of the p210^Bcr-Abl tyrosine kinase correlated well with loss of STAT5 DNA binding activity and down-regulation of Bcl-xL expression in K562 cells but not in the Bcr-Ablnegative HEL leukemic cells that also have constitutively active STAT5 (data not shown).

Induction of K562 and MEG-01 cell death by pyrido[2,3-d]pyrimidine derivatives

We next analyzed the effects of pyrido[2,3-d]pyrimidine derivatives on cell viability of K562 cells. K562 cells were incubated with pyrido[2,3-d]pyrimidine derivatives (0.5 M) or DMSO (solvent) for 1-4 days and cell viability was determined. Figure 1c shows that incubation of K562 cells with PD164199, PD173952, PD173958 and PD180970 caused cell death, whereas PD159373 and the solvent control had no effect. Incubation with PD164199, PD173952, PD173958 and PD180970, but not PD159373, also induced cell death of another human Bcr-Abl⁺ leukemic cell line, MEG-01 (data not shown). In comparison, PD173952 and PD173958 had no effect on cell viability of the Bcr-Abl-negative HL60 and HEL leukemic cells as reported previously for PD180970,²⁵ whereas PD164199 did reduce cell viability of HL60 and HEL cells (data not shown).

The 116-kD PARP is cleaved specifically into 85-kDa and 25-kDa fragments during apoptotic cell death.²⁵ Figure 1d shows that no 85-kDa PARP fragment was detected in K562 and MEG-01 cells treated with DMSO or PD159373, whereas the 85-kDa fragment was present in K562 and MEG-01 cells treated for 24 or 48 h with PD164199, PD173952, PD173958 and PD180970. These results suggest that PD164199, PD173952, PD173958 and PD180970, but not PD159373, induced apoptosis of K562 and MEG-01 cells.

IL-3 protects p210^Bcr-Abl-transformed 32D and Baf3 cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors

During our preliminary studies with PD180970, we found that the viability of 32D cells was not affected by PD180970. 32D cells are IL-3 dependent and were grown in medium supplemented with IL-3. This observation suggested that PD180970, or a Bcr-Abl tyrosine kinase inhibitor in general, does not inhibit the IL-3-induced cell survival pathway. It was also observed previously that IL-3 could increase viable cell numbers of Bcr-Abl-transformed 32D cells when treated with imatinib.^4,27 These observations raise the possibility that IL-3 could protect IL-3 responsive, Bcr-Abl-transformed hematopoietic cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors.

To test this possibility, we determined the effects of PD180970, PD164199 and imatinib on 32Dp210^Bcr-Abl and Baf3p210^Bcr-Abl cells. These two cell lines were derived from IL-3-dependent 32Dcl3 and Baf3 cells by p210^Bcr-Abl transformation.²⁴ They can grow in medium without IL-3, but remain IL-3 responsive. In the absence of IL-3, incubation of 32Dp210^Bcr-Abl cells (Figure 2a) with PD180970 (50 nM), PD164199 (50 nM) or imatinib (1 M) resulted in timedependent decreases in cell viability. Similar results were obtained using Baf3p210^Bcr-Abl cells (Figure 2b). Cells treated with solvent (DMSO) remained viable through the time course (Figure 2a and b). Remarkably, the effects of PD180970, PD164199 and imatinib on viability of 32Dp210^Bcr-Abl and Baf3p210^Bcr-Abl cells were completely blocked when these cells were cultured in the presence of IL-3 (10 ng/ml) (Figure 2a and b). Additional experiments performed with PD173952 and PD173958 gave the same results (data not shown).

To determine whether PD180970, PD164199 and imatinib induce apoptosis of 32Dp210^Bcr-Abl and Baf3p210^Bcr-Abl cells and to confirm that IL-3 protects these cells from Bcr-Abl tyrosine kinase inhibitor-induced apoptosis, we first performed flow cytometric analysis using the CaspACE FITC-VAD-FMK Marker (Promega). This cell permeable compound binds to activated caspases and fluorescently labels apoptotic cells. Figure 2c shows that about 5% of solvent-treated 32Dp210^Bcr-Abl cells were labeled positive with the apoptosis marker. In the absence of IL-3, 32Dp210^Bcr-Abl cells treated with PD180970 (100 nM), PD164199 (100 nM) or imatinib (1 M) for 18 h were 42.9%, 70.9% or 41.8% positive, respectively, for the apoptosis marker (Figure 2c). Strikingly, when IL-3 was included in the medium, these numbers returned to the basal 5% level. Experiments performed in Baf3p210^Bcr-Abl cells gave similar results (not shown).

To further verify the above results, an Annexin-V/7-AAD binding assay was performed (Figure 2d). In solvent-treated cells, 10.1% cells were stained Annexin-V positive (early apoptotic cells) and 3.9% cells were stained Annexin-V/7-AAD double-positive (late apoptotic and necrotic cells). In the absence of IL-3, incubation with PD180970 (100 nM), PD164199 (100 nM) or imatinib (1 M) for 24 h increased the Annexin-V-positive population to 37.6%, 38.8% or 34.2% and the double-positive population to 34.6%, 49.8% or 33.9%, respectively (Figure 2d, upper panels). Again, the effects of PD180970, PD164199 and imatinib on induction of apoptosis of these cells disappeared when IL-3 was present in the medium (Figure 2d, lower panels). Therefore, although the FITC-VAD-FMK and Annexin-V/7-AAD binding assays gave slightly different basal staining levels, both assays clearly show that PD180970, PD164199 and imatinib did not induce apoptosis of 32Dp210^Bcr-Abl cells when IL-3 was present.

IL-3 does not prevent inhibition of p210^Bcr-Abl tyrosine kinase by its inhibitors

To assess if Bcr-Abl tyrosine kinase inhibitors inhibited p210^Bcr-Abl in 32Dp210^Bcr-Abl and Baf3p210^Bcr-Abl cells when IL-3 was present, we treated these cells with Bcr-Abl inhibitors in the presence or absence of IL-3, and analyzed tyrosine phosphorylation of p210^Bcr-Abl. As shown in Figure 3, p210^Bcr-Abl was phosphorylated on tyrosine in solvent-treated cells. PD180970, PD164199 and imatinib inhibited tyrosine phosphorylation of p210^Bcr-Abl in 32Dp210^Bcr-Abl and Baf3p210^Bcr-Abl cells regardless of whether IL-3 was in the cell culture medium (Figure 3). These results rule out the possibility that p210^Bcr-Abl tyrosine kinase was not inhibited in 32Dp210^Bcr-Abl and Baf3p210^Bcr-Abl cells in the presence of IL-3, and suggest that the protective effect of IL-3 is mediated by an IL-3-induced signal transduction pathway that does not require Bcr-Abl tyrosine kinase activity.

Discussion

Our in vitro kinase assays indicate that PD164199, PD173952 and PD173958, like the previously described PD180970,²⁵ are among the most potent Abl tyrosine kinase inhibitors that have been reported. p210^Bcr-Abl and CrkL tyrosine phosphorylation assays in K562 cells demonstrate that these compounds are able to inhibit Bcr-Abl tyrosine kinase activity and cause apoptotic cell death of Bcr-Abl-dependent cells. However, it remains to be determined which of these Bcr-Abl inhibitors has better pharmacokinetic properties in vivo.

Although it inhibits Bcr-Abl kinase, PD164199 has an additional undefined activity that also affects viability of Bcr-Abl-independent HL60 and HEL leukemic cells (data not shown). Nevertheless, this undefined activity does not appear to interfere with the IL-3-induced cell survival signaling pathway because PD164199 was unable to cause 32Dp210^Bcr-Abl and Baf3p210^Bcr-Abl cell death in the presence of IL-3.

Several laboratories have identified point mutations in the tyrosine kinase domain of Bcr-Abl protein derived from CML patients with acquired resistance to imatinib.^{12,13,14,15,16,17} It was demonstrated recently that these Bcr-Abl mutants could confer resistance to imatinib in Baf3 cells harboring these mutants.¹⁶ It will be of significant interest to determine if one or more of these imatinib-resistant Bcr-Abl mutants are sensitive to inhibition by PD164199, PD173952, PD173958 or PD180970.

PD164199, PD173952, PD173958 and PD180970 were originally identified as Src tyrosine kinase inhibitors.^20,21 Not surprisingly, our data show that they inhibit Lyn kinase. Interestingly, Csk, a negative regulator of Src family kinases, was inhibited by PD164199, PD173952, PD173958 and PD180970 at slightly higher drug concentrations (Table 1). Because Src family kinases are downstream of Csk in cell signaling and since these compounds are more potent as Src inhibitors, it is predicted that inhibition of Csk will not diminish the effectiveness of these compounds on blocking Src signaling in the cells.

Clinical studies have shown that a high proportion of CML patients do not have complete cytogenetic responses when treated with imatinib.^7,9 Several possibilities may be envisioned to explain the incomplete cytogenetic response observed in clinical studies. For example, a small subpopulation of Bcr-Abl inhibitor-resistant leukemic cells may already be present in a CML patient prior to the drug treatment. Importantly, it was observed that primitive CD34⁺/Bcr-Abl⁺ cells from CML patients have an IL-3 autocrine loop.¹⁹ Experiments presented in this study illustrate that IL-3 can protect IL-3 responsive, Bcr-Abl-transformed hematopoietic progenitor cells from apoptosis caused by Bcr-Abl tyrosine kinase inhibitors, including imatinib. This finding raises the possibility that the IL-3 autocrine loop found in primitive CD34⁺/Bcr-Abl⁺ cells could be one of the mechanisms responsible for incomplete eradication of Bcr-Abl⁺ leukemic cells from CML patients by the Bcr-Abl tyrosine kinase inhibitor. If further studies with clinical samples support this notion, one could predict that stimulation of differentiation of Bcr-Abl⁺ hematopoietic progenitor cells will lead to a better cytogenetic response to Bcr-Abl inhibition in CML patients.

Further studies are needed to identify the IL-3-dependent survival pathway and to evaluate feasibility of simultaneous inhibition of both IL-3-dependent and Bcr-Abl-dependent cell survival pathways for complete eradication of Bcr-Abl⁺ cells from CML patients. In addition, since PD164199, PD173952, PD173958 and PD180970 are also potent Src inhibitors, our finding that IL-3 can protect 32Dp210^Bcr-Abl and Baf3p210^Bcr-Abl cells from apoptosis induced by these compounds suggests that the IL-3-induced cell survival pathway is Src-independent.

Acknowledgments

We thank Jennifer Berger and Neha Patel for technical assistance, and Jodi Kroeger of the Moffitt Flow Cytometry Core for flow cytometric analysis. This study was supported in part by American Cancer Society Grant RPG0028901TBE (to JW), NIH grants CA77467 (to JW) and CA82533 (to RJ), and the Moffitt Cancer Center Core Facilities.

1 Sawyers C. Chronic myeloid leukemia. N Engl J Med 1999; 340: 1330-1340. MEDLINE

2 Deininger MWN, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia. Blood 2000; 96: 3343-3356. MEDLINE

3 Daley G, ven Etten R, Baltimore D. Induction of chronic myelogenous leukemia in mice by the p210bcr/abl gene of the Philadelphia chromosome. Science 1990; 247: 824-830. MEDLINE

4 Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon NB. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996; 2: 561-566. MEDLINE

5 Kaur G, Gazit A, Levitzki A, Stowe E, Cooney DA, Sausville EA. Tyrphostin induced growth inhibition: correlation with effect on p210bcr-abl autokinase activity in K562 chronic myelogenous leukemia. Anticancer Drugs 1994; 5: 213-222. MEDLINE

6 Sillaber C, Gesbert F, Frank DA, Sattler M, Griffin JD. STAT5 activation contributes to growth and viability in Bcr/Abl-transformed cells. Blood 2000; 95: 2118-2125. MEDLINE

7 Druker BJ, Talpaz M, Resta D, Peng B, Buchdunger E, Ford JM, Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S, Sawyers CL. Efficacy and safety of a specific inhibitor of the Bcr-Abl tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344: 1031-1037. MEDLINE

8 Druker BJ, Sawyer CL, Kantarjian H, Resta D, Reese SF, Ford J, Capdeville R, Talpaz M. Activity of a specific inhibitor of the Bcr-Abl tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 2001; 344: 1038-1042. MEDLINE

9 Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-Passerini C, Niederwieseer D, Resta D, Capdeville R, Zoellner U, Talpaz M, Druker B. Hematologic and cytogenetic responses to Imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002; 346: 645-652. MEDLINE

10 Talpaz M, Silver R, Druker B, Goldman J, Gambacorti-Passerini C, Guilhot F, Schiffer C, Fischer T, Deininger MWN, Lennard AL, Hochhaus A, Ottmann OG, Gratwohl A, Baccarani M, Stone R, Tura S, Mahon FX, Fermandes-Reese S, Gathmann I, Capdeville R, Kantarjian HM, Sawyers CL. Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood 2002; 99: 1928-1937. Article MEDLINE

11 Savage DG, Antman KH. Imatinib mesylate-a new oral targeted therapy. N Engl J Med 2002; 346: 683-693. MEDLINE

12 Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN, Sawyers CL. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001; 293: 876-879. Article MEDLINE

13 Barthe C, Cony-Makhoul P, Melo JV, Reiffers J, Mahon F-X. Roots of clinical resistance to STI-571 cancer therapy. Science 2001; 293: 2163a.

14 Hochhaus A, Kreil S, Corbin A, Rosee PL, Lahaye T, Berger U, Cross NCP, Linkesch W, Druker BJ, Hehlmann R. Roots of clinical resistance to STI-571 cancer therapy. Science 2001; 293: 2163a.

15 Hofmann WK, Jones LC, Lemp NA, de Vos S, Gschaidmeier H, Hoelzer D, Ottmann OG, Koeffler HP. Ph⁺ acute lymphblastic leukemia resistant to the tyrosine kinase inhibitor STI571 has a unique BCR-ABL gene mutation. Blood 2002; 99: 1860-1862. Article MEDLINE

16 Von Bubnoff N, Schneller F, Peschel C, Duyster J. Bcr-Abl gene mutations in relation to clinical resistance of Philadelphia-chromosome-positive leukemia to STI571: a prospective study. Lancet 2002; 359: 487-491. MEDLINE

17 Branford S, Zbigniew R, Walsh S, Grigg A, Arthur C, Taylor K, Herrmann R, Lynch KP, Hughes TP. High frequency of point mutations clustered within the adnosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 2002; 99: 3472-3475. MEDLINE

18 Sirulink A, Silver RT, Najfeld V. Marked ploid and BCR-ABL gene amplification in vivo in a patient treated with STI571. Leukemia 2001; 15: 1795-1797. MEDLINE

19 Jiang X, Lopez A, Holyoake T, Eaves A, Eaves C. Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia. Proc Natl Acad Sci USA 1999; 96: 12804-12809. MEDLINE

20 Kraker AJ, Hartl BG, Amar AM, Barvian MR, Showalter HDH, Moore CW. Biochemical and cellular effects of c-src selective pyrido[2, 3-d]pyrimidine tyrosine kinase inhibitors. Biochem Pharm 2000; 60: 885-898. MEDLINE

21 Klutchko SR, Hamby JM, Boschelli DH, Wu Z, Kraker AJ, Amar AM, Hartl BG, Shen C, Klohs WD, Steinkampf RW, Driscoll DL, Nelson JM, Elliott WL, Roberts BJ, Stoner CL, Vincent PW, Dykes DJ, Panek RL, Lu GH, Major TC, Dahring TK, Hallak H, Bradford LA, Showalter HD, Doherty AM. 2-Substituted aminopyrido[2,3-d]pyrimidin-7(8H)-ones. Structure-activity relationships against selected tyrosine kinases and in vitro and in vivo anticancer activity. J Med Chem 1998; 41: 3276-3292. MEDLINE

22 Vang T, Tasken K, Skalhegg BS, Hansson V, Levy FO. Kinetic properties of the C-terminal Src kinase, p50^csk. Biochim Biophys Acta 1998; 1384: 285-293. MEDLINE

23 Villalba M, Wente SR, Russell DS, Ahn JC, Reichelderfer CF, Rosen OM. Another version of the human insulin receptor kinase domain: expression, purification, and characterization. Proc Natl Acad Sci USA 1989; 86: 7848-7852. MEDLINE

24 Gu H, Griffin JD, Neel BG. Characterization of two SHP-2-associated binding proteins and potential substrates in hematopoietic cells. J Biol Chem 1997; 272: 16421-16430. MEDLINE

25 Dorsey JF, Jove R, Kraker AJ, Wu J. The pyrido[2,3-d]pyrimidine derivative PD180970 inhibits p210^Bcr-Abl tyrosine kinase and induces apoptosis of K562 leukemic cells. Cancer Res 2000; 60: 3127-3131. MEDLINE

26 Cunnick JM, Dorsey JF, Munoz-Antonia T, Mei L, Wu J. Requirement of SHP2 binding to Grb2-associated binder-1 for mitogen-activated protein kinase activation in response to lysophosphatidic acid and epidermal growth factor. J Biol Chem 2000; 275: 13842-13848. MEDLINE

27 Carrol M, Ohno-Jones S, Tamura S, Buchdunger E, Zimmermann J, Lydon NB, Gilliland G, Druker BJ. CGP57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCR-ABL, TEL-ABL, and TEL-PDGFR fusion proteins. Blood 1997; 90: 4947-4951. MEDLINE

Figure 1 Inhibitory effects of pyrido[2,3-d]pyrimidine derivatives on Bcr-Abl and on viability of Bcr-Abl positive cells. (a) and (b) K562 cells were treated with DMSO (0) or the indicated concentrations of inhibitors for 12 h. p210^Bcr-Abl (a) or CrkL (b) were immunoprecipitated from cell lysates of 6 ´ 10⁶ cells/each. One half of each immunoprecipitate was analyzed by immunoblotting with an anti-phosphotyrosine antibody (upper panels), the other half of each immunoprecipitate was analyzed by immunoblotting with an anti-Abl antibody (a, lower panels) or anti-CrkL antibody (b, lower panels). Arrowheads indicate p210^Bcr-Abl. A repeated experiment gave the same results. (c) K562 cells (1 ´ 10⁵ cells/ml, 2 ml/each) were treated with the indicated compounds (0.5 M/each) and cells viability was determined every 24 h. The data were derived from two duplicate experiments. (d) K562 and MEG-01 cells were treated with the indicated compounds (0.5 M/each) for 24 and 48 h. Cell lysates (40 g protein/each lane for K562 cells, 30 g protein/each lane for MEG-01 cells) were analyzed by immunoblotting with an anti-PARP antibody.

Figure 2 IL-3 protects 32Dp210^Bcr-Abl and Baf3p210^Bcr-Abl cells from apoptosis induced by PD180970, PD164199 and imatinib. (a) and (b) 32Dp210^Bcr-Abl and Baf3p210^Bcr-Abl cells (7 ´ 10⁴ cells/ml, 2 ml/each) were plated in medium with or without IL-3 (10 ng/ml) and incubated with PD180970 (50 nM), PD164199 (50 nM), imatinib (1 M), or DMSO (0.1%) for up to 96 h. Cell viability was determined every 24 h. Data were from two (a) or three (b) duplicate experiments. (c) Flow cytometric analysis of 32Dp210^Bcr-Abl cells using the FITC-VAD-FMK marker. Each panel contains two representative flow cytometric graphs from cells cultured with (thick line) and without (thin line) IL-3. M1 indicates labeled cells. The percentage labeled cells in each sample from two duplicate experiments (n = 4) are given. (d) 32Dp210^Bcr-Abl cells were analyzed by the Annexin-V/7-AAD binding assay. The graphs are representatives of two duplicate experiments and the percentage of Annexin-V positive (lower-right quadrant) and Annexin-V/7-AAD double positive (upper-right quadrant) cells from these experiments are given.

Figure 3 IL-3 does not prevent inhibition of Bcr-Abl tyrosine kinase by Bcr-Abl tyrosine kinase inhibitors in 32Dp210^Bcr-Abl and Baf3p210^Bcr-Abl cells. 32Dp210^Bcr-Abl (a) or Baf3p210^Bcr-Abl cells (b) were plated in the presence or absence of IL-3 (10 ng/ml) and treated with PD180970 (100 nM), PD164199 (100 nM) or imatinib (1 M) for 10 h. p210^Bcr-Abl was immunoprecipitated with an anti-Abl antibody and analyzed by immunoblotting with an anti-phosphotyrosine antibody (upper panels) or an anti-Abl antibody (lower panels). Arrows indicate the p210^Bcr-Abl band. Ptyr, anti-phosphotyrosine antibody. Abl, anti-Abl antibody.

Table 1 Inhibitory activities of pyrido[2,3-d]pyrimidine derivatives on protein tyrosine kinases in vitro