AMN107, a novel aminopyrimidine inhibitor of p190 Bcr-Abl activation and of in vitro proliferation of Philadelphia-positive acute lymphoblastic leukemia cells

Authors


Abstract

BACKGROUND

Previous studies have shown that patients with Bcr-Abl–positive acute lymphoblastic leukemia (ALL) either have primary disease that is refractory to imatinib mesylate or develop disease recurrence after an initial response.

METHODS

The authors investigated the effects of a newly designed Bcr-Abl inhibitor, AMN107, by comparing its in vitro inhibitory potency on p190 Bcr-Abl ALL cell lines with that of imatinib.

RESULTS

In two Philadelphia (Ph)-positive ALL cell lines, AMN107 was found to be 30–40 times more potent than imatinib in inhibiting cellular proliferation. AMN107 was also more effective than imatinib in inhibiting phosphorylation of p190 Bcr-Abl tyrosine kinase in cell lines and primary ALL cells. The inhibition of cellular proliferation was associated with the induction of apoptosis in only one of the cell lines. No activity was observed in cell lines lacking the BCR-ABL genotype.

CONCLUSIONS

The results of the current study suggest the superior potency of AMN107 compared with imatinib in Ph-positive ALL and support clinical trials of AMN107 in patients with Ph-positive ALL. Cancer 2005. © 2005 American Cancer Society.

As predicted from in vitro studies,1, 2 imatinib mesylate (imatinib, STI 571) has proven to be a highly effective inhibitor of p210 Bcr-Abl tyrosine kinase in vivo, inducing high rates of complete hematologic and complete cytogenetic disease remissions in patients with chronic myelogenous leukemia (CML).3, 4 The initial in vitro studies2 also suggested a therapeutic potential in patients with acute lymphoblastic leukemia (ALL) possessing the Philadelphia (Ph) chromosome, who express the p 190 Bcr-Abl protein. Subsequent clinical studies in patients with chemotherapy-refractory or recurrent ALL documented antileukemic activity, but in a majority of patients the responses were less dramatic and of shorter duration.5, 6 To our knowledge there has been no satisfactory explanation for this clinical resistance. The emergence of imatinib resistance in patients with CML led to studies that identified point mutations within the Bcr-Abl kinase domain7–11 or increased levels of Bcr-Abl tyrosine kinase with or without amplification of the BCR-ABL genomic locus7, 9, 12–15 as the main mechanisms leading to clinical resistance to imatinib. Although mutations have been reported in ALL-derived cells,16 the evidence for their association with clinical resistance to imatinib is less compelling. An understanding of the molecular action of imatinib and the mechanisms of resistance stimulated the search for more active agents.

Promising results have been reported with small molecules that inhibit the tyrosine kinase activity of Bcr-Abl and of down-stream kinases, such a c-Src, which are believed to be involved in the intracellular signaling in the disease process.17 In addition, Bcr-Abl inhibitors, having greater potency than imatinib, might also be able to overcome the drug resistance associated with increased levels of Bcr-Abl kinase.

AMN107 is a new drug that is rationally designed based on the crystallographic structure of the imatinib–Bcr-Abl complex.18 The crystallographic analysis of the AMN107–Bcr-Abl complex (Fig. 1) documented that AMN107 binds to inactive conformation of the Abl kinase, thus delivering the same specificity profile as imatinib. The greater affinity of AMN107 compared with imatinib is the consequence of a better topologic fit to the protein and, relative to imatinib, a smaller binding activity contributed by the pyridinyl and pyrimidinyl groups compared with the total energy of AMN107, in which the trifluoromethyl phenyl group contributes greatly to the potency. This results in a lesser effect of some mutations on the overall activity of AMN107.9, 18

Figure 1.

Schematic diagram, based on an X-ray crystal structure, of the Abl kinase domain (cyan) in complex with AMN107 (yellow). The hinge region is shown in green, the glycine-rich loop is shown in red, and the activation loop is shown in magenta. In this structure, the activation loop adopts an inactive conformation similar to that observed in the imatinib-Abl complex..

We have recently reported promising results documenting the activity of AMN107 in both imatinib-sensitive and imatinib-resistant p210 Bcr-Abl myeloid leukemic cell lines.19 In the current study, we report that AMN107 is also more potent than imatinib in inhibiting proliferation of human p190 Bcr-Abl–expressing ALL cell lines. AMN107 also inhibited p190 Bcr-Abl phosphorylation in these cell lines, as well as in primary leukemic cells derived from patients with Ph-positive ALL.

MATERIALS AND METHODS

Cell Lines

Lymphoblastic cell lines were established in the laboratory of Dr. Z. Estrov (The University of Texas M. D. Anderson Cancer Center, Houston, TX). Two cell lines designated Z-119 and Z-181 were derived from patients with Ph-positive ALL. They all retained typical B-cell characteristics and phenotypes of the original tumors. Karyotypic analysis revealed t (9;22), and kinase assay detected p190 Bcr-Abl in both lines.20 Z-138, a cell line lacking the Ph chromosome, was derived from a patient with chronic lymphocytic leukemia and supervening ALL.

The Z-119 and Z-138 cell lines were maintained in Iscove modified Dulbecco medium (IMDM; Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS; Invitrogen). Z181 was maintained in minimal essential medium-α (Invitrogen) supplemented with 10% FBS (Invitrogen).

The acute myelomonocytic leukemic cell line HL60 and the monocytic cell line U937 were maintained in RPMI medium (Invitrogen) supplemented with 10% FBS and 1% penicillin-streptomycin. The Bcr-Abl p 210-positive myeloid cell line KBM521 was maintained in IMDM, supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin.

Primary leukemic cells were obtained from three patients with pre-B ALL with t (9;22). The presence of transcript coding for p190 tyrosine kinase was documented by reverse transcriptase-polymerase chain reaction in all three specimens. Informed consent was obtained from all patients for the use of tissue specimens for investigational purposes and the protocol was approved by the nstitutional review board.

Drugs

Imatinib (STI571) and AMN107 (NVP-AMN107-NX; 4-methyl-N-[3-(4-methyl-1H-imidazol)-5-(trifluoro-methyl) phenyl]-3-(3-pyridinyl)-2pyridinyl]amino] benzamide) were kindly provided by Novartis Pharma AG. Stock dilutions were prepared in dimethylsulfoxide (DMSO) and stored in 10 mM solution at −20 °C. Drug dilutions in DMSO were prepared to obtain final dilutions for cellular assays immediately before use.

Growth Inhibition Assay

MTS assay (CellTiter 96 Aqueous One Solution Reagent; Promega Corporation, WI) was used to measure the in vitro effect of imatinib and AMN107 on the growth of human leukemic cells. The assay was performed according to the manufacturer's recommendations with cells seeded in triplicate in 96-well microtiter plates (Falcon, Lincoln Park, NJ) and incubated in the presence of different drug concentrations for 72 hours. After 72 hours of exposure, the level of inhibition was measured as a percentage of control growth (no drug in the sample). The drug concentration resulting in 50% inhibition of the growth (IC50) was determined.

Detection of Caspase 3 Activity

To monitor caspase 3 and caspase 3-like activity in living cells by flow cytometry, the fluorogenic substrate PhiPhiLux G1D2 (Oncoimmunin, Gaithersburg, MD) was used. After treatment with imatinib or AMN107, cells were washed in Ca2+-free phosphate-buffered saline (PBS), resuspended in 25 μL of substrate solution, and incubated for 1 hour in a humidified chamber at 37 °C in the dark. They were then washed and resuspended in Ca2+-free PBS. Propidium iodide (PI) was added and cell samples were run on a flow cytometer (FACScan; Becton Dickinson, San Jose, CA) and obtained data were analyzed using CellQuest software (Becton Dickinson).

Cell Cycle Analysis and Evaluation of Apoptotic (Sub-G1 cells) by Flow Cytometry.

Cells were incubated in the presence of AMN107 or imatinib for 3 days. To investigate the extent of cell cycle disturbance and apoptosis, we harvested cells and performed experiments after 24 hours, 48 hours, and 72 hours of exposure to drugs. The drug concentrations chosen for these experiments were selected according to the sensitivities of the cell lines determined by the MTS assay.

After treatment, cells were collected, washed in Ca2+-free PBS, and fixed overnight in 70% cold ethanol at −20 °C. The cells were then washed twice in cold PBS, resuspended in hypotonic PI solution (25 μg/mL PI, 0.1% Triton X-100, 30 mg/mL polyethylene glycol, and 3600 U/mL RNAse, dissolved in 4 mM of sodium citrate buffer [pH 7.8]; Sigma Chemical Company, St. Louis, MO) and incubated for ≥ 1 hour at 4 °C in the dark. Cell cycle contents and sub-G1 cells were determined by flow cytometry (FACScan). Cell cycle distribution was analyzed using ModFit LT software (Becton Dickinson).

Immunoprecipitation of Bcr-Abl

The immunoprecipitation of Bcr-Abl was done as previously reported.2 Cells (20 × 106 cells per sample) treated with different concentrations of imatinib or AMN107 for 3 hours were collected, washed 3 times with cold PBS, resuspended in 250 μL of lysis buffer (10 mM sodium phosphate [pH 7.2], containing 100 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate, 5 mM ethylenediaminetetraacetic acid [EDTA], 1 mM phenymethylesulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM 4-(2-aminoethyl)benzensulfonyl hydrochloride (AEBSF; Sigma-Aldrich, St. Louis, MO) and × 1 Roche (Roche Diagnostics, Alameda, CA) complete Mini protease inhibitor cocktail) and incubated on ice for 1 hour. After centrifuging the cell lysate at 14,000 revolutions per minute (rpm) at 4 °C for 40 minutes, the supernatant fluid was removed and mixed with 25 μL of anti–Bcr-Abl P6D monoclonal antibody for 1 hour on ice, followed by adding 50 μL of Protein-A/G agarose slurry (sc-2003, Santa Cruz Biotechnology, Santa Cruz, CA) and incubated overnight at 4 °C with constant rotation. The antibody-protein complex was washed 3 times: once with RIPA buffer (100 mM NaCl in 10 mM phosphate buffer [pH 7.2], containing 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.05% SDS, 5 mM EDTA, and a cocktail of protease inhibitors), once with washing buffer (100 mM NaCl in 10 mM sodium phosphate [pH 7.2] and 0.1% Triton X-100), and, finally, once with 50 mM Tris-HCl buffer (pH 7.5). The protein complex was subjected to Western blot analysis.

Western Blot Analysis

The immunoprecipitated complex was eluted from the agarose with 2 × loading buffer and run (20 × 106 cells per line) on a 9.5% SDS-polyacrylamide gel electrophoresis gel. Western blot analysis was performed overnight at 4 °C. After blocking the nitrocellulose (Schleicher & Schuller, Keene, NH) with 5% nonfat milk prepared in PBS-0.1% Tween 20 for 5 hours, the membranes were incubated with mouse antiphosphotyrosine (py99; Santa Cruz Biotechnology) diluted in 2.5% bovine serum albumin and 2.5 % nonfat milk (dilution 1:8000), overnight at 4 °C. The active band for phosphorylated Bcr-Abl was detected using conjugated horseradish peroxidase (HRP)-sheep anti-mouse antibody (NA931V; Amersham, Arlington Heights, IL). Detection was performed by enhanced chemiluminescence as specified by the manufacturer (ECL, Amersham).

The same membranes were stripped, and reprobed with mouse anti–Bcr-Abl antibody (8E9; dilution 1:4000) overnight at 4 °C. The active band for Bcr-Abl was detected with HRP-sheep-anti-mouse antibody.

RESULTS

Effect of AMN107 on Cell Growth of Ph-Positive ALL Cell Lines and Its Comparative Potency to Imatinib

Treatment with AMN107 or imatinib for 3 days inhibited the proliferation of Z-119 cells with IC50 values of 19.3 nM and 620 nM, respectively (Fig. 2A), indicating that in this cell line, AMN107 was 32 times more potent than imatinib. Because of the slower growth of Z-181 cells, the in vitro drug exposure was extended to 4 days. After the 96 hours of treatment, the estimated IC50 values for AMN107 and imatinib were 1.6 nM and 63.9 nM, respectively (Fig. 2B), showing that AMN107 was 40 times more potent than imatinib. The Z138 Ph'-negative cells, used as negative controls, showed no response to comparable treatments with AMN107 (drug concentrations ≤ to 5 μM) or imatinib (drug concentrations ≤ 0.5 μM) (Fig. 2C). A similar lack of effect was also observed in the monocytic cell line, U937. This confirmed the selectivity of AMN107 against Bcr-Abl–positive cells.

Figure 2.

(A–C) Comparison of sensitivity to AMN107 and imatinib of 2 Philadelphia chromosome-positive cell lines expressing p190 Bcr-Abl (Z-119 and Z-181) and a Bcr-Abl–negative lymphoid cell line, exposed in vitro to increasing concentrations of the respective drug. The MTS assay was used to evaluate toxicity. The potency of AMN107 is expressed as a ratio between inhibitory concentrations (IC values) of imatinib and AMN107.

Cell Cycle Analysis, Caspase 3 Activity, and Apoptosis.

Exposure of Z-119 cells to increasing concentrations of either AMN107 or imatinib resulted in an accumulation of the cells in G0/G1 phases of the cell cycle. The proportion of cells in G0/G1 increased from 24 hours to 72 hours, indicating a time dependency (Table 1). At equipotent concentrations of imatinib and AMN107 (≈ inhibitory concentrations [IC]70), the effects of both drugs on cell cycle distribution were comparable (Table 1). Similar effects on the cell cycle distribution, although to a much lesser degree, were also observed in Z-181 cells with the effect of imatinib only marginally discernible (Table 1).

Table 1. Exposure of p190 Bcr-Ab1 ALL cells to AMN107 or Imatinib at Equivalent Doses (∼7∼IC70) Results in the Accumulation of Cells in the G0/G1 Phase of the Cell Cycle
Drug exposure in hrsPercentage of cells in G0/G1 phase
Z-119 cellsZ-181 cells
0Imatinib (1000 nM)AMN107 (25 nM)0Imatinib (100 nM)AMN107 (3 nM)
  1. ALL: acute lymphoblastic leukemia.

2432.968.070.744.760.151.4
4827.872.276.345.556.342.9
7219.179.778.243.360.344.5

Within 48 hours of exposure, both AMN107 and imatinib induced detectable increases in the caspase 3 activity and increases in the number of apoptotic cells in Z-119 cells. At equitoxic doses of both drugs, the ability of AMN107 to induce caspase 3 and apoptosis (Table 2) was comparable to that of imatinib. Exposure of Z-181 cells to concentrations of AMN107 or imatinib resulting in a comparable cytotoxic effect, as measured by the IC values, failed to induce a detectable activation of caspase 3 or an increase in the number of apoptotic cells (Table 2).

Table 2. Induction of Caspase 3 Activity and Apoptosis by Imatinib and AMN107 at Equitoxic Doses of Drugs (∼IC70) in a Ph' Positive ALL Cell Line Expressing p190 Bcr-Abl
Drug exposure in hrsPercentage of caspase 3-positive and apoptotic cells
Z-119Z-181
0Imatinib (1000 nM)AMN107 (25 nM)0Imatinib (100 nM)AMN107 (3 nM)
  1. Ph: Philadelphia; ALL: acute lymphoblastic leukemia.

Caspase 3      
245.18.47.14.54.85.0
484.520.816.84.45.75.5
726.428.832.53.95.65.0
Apoptosis      
2417.526.126.56.37.67.8
4813.335.938.17.411.010.2
7210.843.141.86.39.37.3

Inhibition of p190 Bcr-Abl Phosphorylation in ALL Cell Lines

Exposure of cells for 3 hours to AMN107 at concentrations of ≥ 125 nM resulted in complete inhibition of p190 Bcr-Abl tyrosine kinase in both Z-119 and Z-181 cell lines without affecting the level of Bcr-Abl expression. The Z-119 cells appeared to be more sensitive with almost complete inhibition of phosphorylation already seen after exposure to 12.5 nM of AMN107. To achieve a complete suppression of phosphorylation of the kinase by imatinib, concentrations of ≥ 2.5 μM and higher were necessary in both p190-positive cell lines. These results suggest a > 20 times higher potency of AMN107 compared with imatinib. The concentration of imatinib required to completely inhibit phosphorylation of p190 tyrosine kinase was comparable to that required for inhibition of phosphorylation of p210 Bcr-Abl as verified by the concomitant experiment with p210 Bcr-Abl–positive KBM5 cells, untreated and treated with 2.5 μM of imatinib, which were used as positive controls (Fig. 3).

Figure 3.

Bcr-Abl phosphorylation and expression after exposure of leukemic cells to AMN107 or imatinib. Cells were cultured at a density of 4 million cells per milliliter and exposed to various concentrations of AMN107 or imatinib for 3 hours and Lysed. Whole cell lysates (20 × 107 per sample) were immunoprecipitated with Bcr-Abl antibody. Western blot analysis using antiphosphotyrosine antibody was performed first, after which the membranes were stripped and reprobed with Bcr-Abl antibody. HL60 acute myelogenous leukemia cells were used as a negative control and KBM5 cells were used as Bcr-Abl–positive controls. (A) Cell lines. (B) Primary acute lymphoblastic leukemia cells expressing p190 Bcr-Abl.

Inhibition of p190 Bcr-Abl Phosphorylation in Primary Leukemic Cells Derived from Patients with Ph-Positive ALL

To assess the effect of AMN107 on the activity of p190 tyrosine kinase in primary leukemic cells, freshly obtained cells from 3 patients with recurrent, p190 Bcr-Abl ALL were exposed in vitro to AMN107 or imatinib at concentrations expected to suppress phosphorylation of the kinase in leukemic cell lines (see above). As shown in Figure 3B, the inhibition of phosphorylation was evident in samples from Patients 1 and 3 after exposure to 125 nM concentration of AMN107, but no discernible suppression was observed after exposure to 2500 nM of imatinib. In Patient 2, a barely detectable inhibition with both drugs was noted, with imatinib appearing slightly more effective.

DISCUSSION

Herein, we report that AMN107, a new inhibitor of Bcr-Abl tyrosine kinase, effectively inhibited the proliferation of human lymphoid leukemia cell lines expressing p190 Bcr-Abl. Similar to imatinib, the effect of AMN107 is selective for Bcr-Abl–expressing cell lines with no effect being observed in Bcr-Abl–negative leukemic cells treated with comparable concentrations of AMN107. Although slight differences in the potency of AMN107 against the 2 p190 Bcr-Abl–expressing cell lines were observed, the results taken together confirmed that AMN107 was approximately 30–40 times more potent than imatinib in inhibiting cell proliferation. AMN107 was also consistently (> 20 times) more potent than imatinib in inhibiting phosphorylation of p190 Bcr-Abl kinase in these cell lines. Preliminary results indicated that AMN107 also inhibited Bcr-Abl phosphorylation in primary p190 Bcr-Abl–expressing ALL cells to an extent at least comparable to imatinib.

At equieffective doses of both drugs, the overall response patterns to AMN107 and imatinib were similar in terms of induction of apoptosis and the effect on cell cycle distribution. Both drugs induced caspase 3 and an increase in the apoptotic cells in the same cell line (Z-119), whereas no such effect could be observed in Z-181 cells with either drug. These observations suggest that not only apoptosis but also other pathways leading to inhibition of cell growth are affected by AMN107 and imatinib. Because a similar heterogeneity in apoptotic response to AMN107 and imatinib was observed in p210 Bcr-Abl–expressing myeloid leukemia cell lines sensitive or resistant to imatinib,19 it is likely that various pathways downstream of Bcr-Abl contribute to the ultimate demise of the cells after p190 Bcr-Abl tyrosine kinase inactivation, leading to interruption of Bcr-Abl signaling. The sensitivity of both Ph-positive cell lines to AMN107 was approximately of the same order of magnitude, and AMN107 was effective in inhibiting Bcr-Abl phosphorylation. Thus, the major differences between the two cell lines in terms of the observed degree of induction of apoptosis and perturbation of the cell cycle appear to have secondary importance in the action of AMN107 as well as imatinib.

Whether the increased potency of AMN107 observed in Ph-positive ALL cell lines translates into clinical benefit will be ultimately tested in clinical trials. In a previous study,19 AMN107 proved more potent than imatinib in p210 Bcr-Abl myeloid leukemia cell lines sensitive or resistant to imatinib. The activity of AMN107, albeit weaker, was also documented in cell lines rendered resistant to imatinib by culturing in the presence of low concentrations of drug and characterized by genomic amplification of BCR-ABL and overexpression of wild-type p210 Bcr-Abl protein in 1 cell line and by T315I mutation of the adenosine triphosphate (ATP) coding domain of the ABL in the other.11, 14 The potency of AMN107 was lower in imatinib-resistant than in imatinib-sensitive cell lines, suggesting a degree of crossresistance with imatinib. Although a moderate decrease in the efficacy of AMN107 was observed in cells with resistance associated with Bcr-Abl amplification, the presence of T315I mutation conferred a high degree of resistance.19

The results of the current study complement the elegant in vitro and in vivo studies of AMN107 by Weisberg et al.18 In their experimental systems, which employed cell lines transfected with wild-type BCR-ABL (32D Bcr-Abl or Ba/F3 Bcr-Abl), AMN107 was approximately 10-fold more active than imatinib against cellular p210 and p190 Bcr-Abl. The investigators further reported a higher potency of AMN107 than that of imatinib in in vitro and in animal models in vivo, in cells transduced by several imatinib-resistant mutants of Bcr-Abl.18

It may be speculated that in cases of imatinib resistance arising from increased Bcr-Abl expression, AMN107 may be more effective because of its higher binding affinity, whereas in cases of imatinib resistance caused by point mutation, destabilizing the imatinib-binding conformation, the binding requirements of AMN107 may represent a likely advantage, particularly in the case of point mutations affecting amino acid residues which are part of the protein surface in contact with the drug.18

The results of our studies indicated that AMN107 was highly effective in inhibiting Bcr-Abl phosphorylation and in vitro proliferation of p190 Bcr-Abl lymphoblastic leukemia cell lines. The higher potency of AMN107 compared with imatinib may translate into a therapeutic advantage in clinical trials, particularly in patients with Ph-positive ALL and CML that are poorly controlled by imatinib.

Clinical trials have recently been initiated in patients with advanced stages of CML and refractory/recurrent Ph-positive ALL. A preliminary report of the initial results documents a promising activity of AMN107 in CML and ALL, both of which are clinically resistant to imatinib mesylate.22

Acknowledgements

The authors acknowledge Novartis A.B. for the free supply of AMN107 and imatinib.

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