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Chronic Myeloid Leukemia, BCR/ABL Studies and Myeloproliferative Disorders

The Bcr-Abl mutations T315I and Y253H do not confer a growth advantage in the absence of imatinib

Abstract

Mutations in the Bcr-Abl kinase domain are a frequent cause of imatinib resistance in patients with advanced CML or Ph+ ALL. The impact of these mutations on the overall oncogenic potential of Bcr-Abl and on the clinical course of the disease in the absence of imatinib is presently unclear. In this study, we analyzed the effects of the Bcr-Abl P-loop mutation Y253H and the highly imatinib resistant T315I mutation on kinase activity in vitro and transforming efficiency of Bcr-Abl in vitro and in vivo. Immunoprecipitated Bcr-AblY253H and Bcr-AblT315I proteins displayed similar kinase activities and substrate phosphorylation patterns as Bcr-Abl wildtype. We directly compared the proliferative capacity of mutant to wildtype Bcr-Abl in primary BM cells in vitro and in a murine transplantation model of CML by using a competitive repopulation assay. The results implicate that in the absence of imatinib, there is no growth advantage for cells carrying Bcr-AblT315I or Bcr-AblY253H compared to Bcr-Ablwt, whereas imatinib treatment clearly selects for leukemic cells expressing mutant Bcr-Abl both in vitro and in vivo. Thus, the analysed Bcr-Abl mutants confer imatinib resistance, but do not induce a growth advantage in the absence of imatinib.

Introduction

The constitutively activated tyrosine kinase Bcr-Abl is the product of the reciprocal chromosomal translocation t(9;22) and plays a central role in the pathogenesis of chronic myeloid leukemia (CML) and approximately 20% of adult acute lymphoblastic leukemia (ALL).1, 2 The development of imatinib, a specific inhibitor of the Bcr-Abl kinase, has had a significant impact on the therapy of CML and Ph+ ALL.3, 4 But whereas most patients with chronic phase CML show durable responses, nearly all patients suffering from advanced-phase CML and Ph+ ALL relapse despite continued therapy.5 Bcr-Abl amplification, clonal evolution and Bcr-Abl point mutations have been described as the most important mechanisms of drug resistance.6, 7, 8 Based on mutational and crystal structure analysis, Bcr-Abl point mutations impair imatinib binding by different mechanisms.9, 10 One group of amino acid exchanges located at the drug contact side (e.g. F311, T315 and F317) directly prevents imatinib binding, with the amino acid exchange at position 315 being the mutation most frequently found in patients.11 A second group of common mutations such as E255K/V and Y253H/F occurring at the nucleotide-binding (P) loop impairs the conformation of the kinase domain required for imatinib binding. A third group of mutations such as H396P/R is located in the activation loop.12 Although investigations in cell lines, crystallographic studies and clinical data have clearly established an important role for point mutations in the development of imatinib resistance, the impact of the mutations on the oncogenic potential of Bcr-Abl in vivo has not been thoroughly investigated. Furthermore, there are divergent data on the impact of these point mutations on the kinase activity and the transformation capacity of Bcr-Abl in vitro (Gorre et al. Blood 2004; 104:161a (abstract) and Grisworld et al. Blood 2004; 104:161a (abstract)).12, 13 The fact that some mutations have been detected in patients prior to imatinib therapy and reports that patients with P-loop mutations have a worse prognosis may indicate a pathophysiologic role for the point mutations in addition to mediating imatinib resistance.14, 15, 16, 17 This might be clinically relevant, since an increased kinase activity could allow selection of the mutant clone in the absence of imatinib and induce a more aggressive disease with a worse outcome. In contrast, a diminished kinase activity might lead to the replacement of mutant clones by Bcr-Ablwt expressing cells and thereby allow repeated responses after pausing kinase targeted treatment in analogy to antiretroviral therapy in HIV-disease.18 To delineate the effects of Bcr-Abl point mutations on kinase activity and transformation, we analysed cell lines and primary murine BM transformed by mutant or wild-type Bcr-Abl in vitro and in a syngeneic murine transplant model of CML.

Materials and methods

Imatinib

The tyrosine kinase inhibitor imatinib (STI-571) was kindly provided by Novartis Pharma AG, Basel, Switzerland.

DNA constructs

The retroviral MSCV-p210 Bcr-Abl wt and MIGRI vector were kind gifts from W Pear (Philadelphia, PA, USA).19, 20 The two mutated Bcr-Abl transcripts T315I and Y253H were cloned into the EcoRI site of the MIGRI retroviral vector coexpressing the enhanced green fluorescent protein (EGFP).

Retrovirus preparation

Phoenix™ ecotropic helper-free retroviral producer cells (G Nolan, Stanford, CA, USA) were grown in DMEM (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS). Retroviruses were generated by transient transfection of the different retroviral constructs using Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) according to the manufacturer's recommendations. Transfection efficiency of Phoenix™ cells was tested by flow cytometry analysis for EGFP-expression and Western blot for Bcr-Abl expression. Supernatants were titered by flow cytometry on 3T3 fibroblasts for the EGFP-expressing constructs, titers of EGFP negative constructs were adjusted according Bcr-Abl expression in infected NIH3T3 cells. Viral supernatants were tested to be negative for replication competent viral particles by serial passage on NIH3T3 cells.

Bone marrow infection, transplantation and animal studies

All animals were kept in a special caging system (Thoren, PA, USA) and received acidified water and food ad lib. All procedures were reviewed and approved by the universities supervisory animal care committee. Bone marrow cells from male Balb/c donor mice were collected 4 days after treatment with 150 mg/kg 5-FU (Ribosepharm, Munich, Germany). Cells were suspended in BBMM (IMDM, 20% FCS, 1% BSA) containing growth factors (mIL-3 10 ng/ml, IL-6 10 ng/ml, SCF 50 ng/ml (R&D Systems, Wiesbaden, Germany)) and prestimulated for 24 h. BM cells were transduced with viral supernatants by spin infection four times every 12 h as described previously.21 The cells were harvested 12 h after the last spin infection, washed with PBS, resuspended in Hanks balanced salt solution (Sigma-Aldrich, Irvine, UK) and MIG-Bcr-Abl mutant infected cells were tested for EGFP expression by FACS analysis. Lethally irradiated (800 rad) female Balb/c recipient mice were transplanted with 2.5 × 106 cells of a 3:1 mixture of Bcr-Ablwt and either Bcr-AblT315I or Bcr-AblY253H mutant infected BM cells via tail vein injection. This ratio was chosen since we aimed at a significant and easily identifiable population of Bcr-Abl mutant/EGFP+ cells initially, but also a not too high fraction of these cells in order to show selection upon addition of imatinib.

For serial transplantation, spleen cells from primary transplanted diseased mice were injected into sublethally irradiated (450 rad) female Balb/c mice. To monitor disease development and the relative growth of Bcr-Abl mutant infected cells compared to Bcr-Ablwt cells peripheral blood obtained from the tail vein of transplanted mice was analysed by PBC count (Vet abc blood counter, Scil, Viernheim, Germany) and flow cytometric analysis. Imatinib (STI-571) treatment was initiated two days after BMT or with rising leukocyte counts with 100 mg/kg twice a day via gavage.

Western blot

Western blots were performed as described before.22 For Bcr-Ablwt and mutant expression analysis, NIH3T3 murine fibroblast cells or Ba/F3 murine pre-B lymphocyte cells were infected with the different retroviral constructs. The infected cells were lysed, run on a polyacrylamide gel, blotted onto nylon membrane and probed with anti-Abl (8E9), antiphosphotyrosine (4G10) or anti-β-Actin antibodies (BD Pharmingen, Heidelberg, Germany and Biomol, Hamburg, Germany).

Kinase assays

Kinase assays were performed as described previously.22 Briefly, 293 cells were transfected with p210wt p210T315I or p210Y253H constructs in the MIG-vector and empty MIG-vector as control. After 48 h, the cells were lysed and the Bcr-Abl protein was precipitated with an anti-Abl antibody (K-12, Santa Cruz, Heidelberg, Germany) from the lysates of p210wt and p210mut transfected cells. Two control immunoprecipitations (IP) were included. Either an IP with an isotype control antibody from cells transfected with p210wt or an IP with the K-12 antibody from empty vector transfected cells was conducted. The kinase reactions were performed employing an artificial Abl-substrate.23 Subsequently, substrates were run on a 8% polyacrylamide gel, blotted onto nylon membrane, probed with antiphosphotyrosine antibody (4G10) and visualized by enhanced chemoluminescence (Pierce, Rockford, IL, USA). Equal substrate loading was controlled by amido-black staining of the membrane according to standard laboratory methods. The blots were scanned and quantitated with Image Quant software (Molecular Dynamics, Krefeld, Germany). Relative substrate phosphorylation was determined by calculating the ratio between the intensity of substrate phosphorylation and corresponding amido-black staining intensity. The ratio of the 20 min kinase reaction was defined as 100%.

Flow cytometry

Flow cytometry analysis of BM, spleen and peripheral blood cells from diseased mice was performed as previously described.21 Briefly, after staining with Fc-block and CD 45-Cy5 cells were incubated with PE-conjugated antibodies against CD11b, CD45R/B220 and CD90.2(Thy1.2) (BD Pharmingen, Heidelberg, Germany). Propidium iodide was used to exclude dead cells and samples were analysed on a Coulter XL2 cytometer.

In vitro liquid BM transformation and methylcellulose assay

To analyse the effect of the mutants on cell growth and resistance towards Imatinib, spleen cells from mice transplanted with a mixture of Bcr-Ablwt and either Bcr-AblT315I or Bcr-AblY253H infected BM cells were plated in liquid BM and methylcellulose assays. All experiments were performed with spleen cells plated in triplicate from at least two independently transplanted animals. Two different in vitro assays were performed with increasing imatinib concentrations (0, 0.25, 0.5 and 1 μ M imatinib). For the liquid BM transformation assay, the cells were suspended in BM medium (IMDM, 20% FCS, 1% BSA) without growth factors and plated in triplicate into 12-well tissue culture dishes with 1 × 105 cells per plate. Cells were regularly analysed by flow cytometry to determine the fraction of EGFP positive cells.

The methylcellulose assay was performed by plating 4 × 104 spleen cells/ml in triplicate wells in methylcellulose without growth factors (Methocult TM H4230, Stem Cell Technologies, Vancouver, Canada) in 12-well tissue culture dishes. After 14 days of incubation at 37°C, colony numbers from wells were counted, single colonies picked and the fraction of EGFP positive colonies determined by flow cytometry.

Results

Bcr-Ablwt Bcr-AblT315I and Bcr-AblY253H phosphorylate an artificial substrate with similar kinetics

Single amino acid changes may have a strong impact on the enzymatic activity of tyrosine kinases. Several reports suggested that Bcr-Abl mutations arising in imatinib resistant patients may influence the kinase activity of Bcr-Abl. To investigate potential differences induced by the T315I and Y253H mutations in Bcr-Abl, we examined the kinase activity of wildtype and mutant Bcr-Abl proteins by analyzing the substrate phosphorylation kinetics of Bcr-Abl immunoprecipitated from transiently transfected 293 cells (Figure 1a). We employed a peptide consisting of an optimized phosphorylation site for the Abl kinase of only seven amino acids (AIYAAPF) fused to the 3′ end of the EGFP sequence.23 In the kinase assay, all three Bcr-Abl proteins phosphorylated the substrate with similar efficiency and kinetics (Figure 1a), implicating that the steady-state kinase activity of the two mutants does not differ significantly from Bcr-Ablwt.

Figure 1
figure1

Kinase assay analyzing phosphorylation kinetics of a synthetic Abl-substrate by immunoprecipitated Bcr-Abl (BA) wildtype (wt) or mutant (T315I/Y253H). The kinase reaction was allowed to proceed for the indicated time. After blotting, the membranes were probed with anti-phosphotyrosine antibody and stained with amidoblack to control substrate loading. (a) Input control of 293T cells transiently transfected with MIG-BAwt, MIG-BAT315I or MIG-BAY253H constructs by anti-Abl Western blot. (b) Kinase assay with BAwt, BAT315I (c) or BAY253H (d). (e) Anti-phosphotyrosine, Abl- and β-actin-blot of Ba/F3 cells transformed by MIG-Bcr-Ablwt, MIG-Bcr-AblT315I or MIG-Bcr-AblY253H after 6 h incubation with 0, 1 and 5 μ M imatinib.

Next, we examined the phosphorylation patterns of Bcr-Ablwt and the two mutants in the murine pre-B cell line Ba/F3. A Western blot probed for total protein phosphorylation did not show salient differences in the overall phosphorylation pattern of Ba/F3 cells transformed by either wt or mutant Bcr-Abl (Figure 1b). As expected, the phosphotyrosine signals strongly decreased upon addition of imatinib in the Bcr-Ablwt cells, whereas the response to imatinib was diminished to some extent in the Y253H mutant and there was no response to imatinib in the T315I mutant (Figure 1b).

Establishment of a competitive repopulation assay

Our next aim was to directly compare the oncogenic potential of the Bcr-Abl mutants T315I and Y253H to Bcr-Ablwt in primary murine BM cells. For this purpose, we established a competitive repopulation assay by cloning the Bcr-AblT315I and the Bcr-AblY253H mutant in the bicistronic EGFP+ retroviral expression vector MIGRI, whereas the Bcr-Abl wt cDNA was expressed from the EGFP MSCV-vector, enabling us to measure the relative contribution of Bcr-Abl mutant infected cells to the overall leukemic cell population (Figure 2a). It has previously been shown that the bicistronic MIGRI vector which contains the MSCV retroviral LTR promoter followed by an IRES-EGFP cassette allows a slightly higher expression than the MSCV vector carrying a phosphoglycerate promoter driving a neomycin resistance gene.22 The small difference in activity of the two MSCV-based vectors is thought to be caused by promoter competition in the MSCV-pgk-neo vector. For this reason, we expressed the Bcr-Abl mutants in the MIG-constructs to ensure expression of the Bcr-Abl mutants is at least as high as expression of the Bcr-Ablwt construct. All three Bcr-Abl constructs were equally well expressed after infection of NIH3T3 cells (Figure 2b).

Figure 2
figure2

(a) Structure of the retroviral vectors MIG-Bcr-AblT315I/MIG-Bcr-AblY253H and MSCV-Bcr-Ablwt used in the bone marrow transplant assay. The Bcr-Ablwt cDNA was cloned into the EcoRI site of the MSCV vector. The T315I and Y253H mutants (Bcr-Ablmut) were cloned into the EcoRI site of the MIGRI retroviral vector coexpressing the enhanced green fluorescent protein (EGFP) via an internal ribosomal entry site (IRES). LTR, long terminal repeat. PPKG, murine phosphoglycerate kinase promoter controls expression of the neomycin resistance gene (Neor). (b) Western blot analysis of infected NIH3T3 cells for MSCV-BAwt and MIG-BAmutant protein expression. The blot was probed with anti-Abl and anti-β-actin antibodies. (c) Schematic protocol of retroviral transduction and transplantation procedure. A competitive BM repopulation assay was established by cotransplantation of EGFP Bcr-Ablwt and EGFP+ Bcr-Ablmut infected cells into lethally irradiated mice. Spleen cells from transplanted mice were subsequently employed for in vitro assays and serial transplantation.

To delineate the effects of imatinib treatment on Bcr-Ablwt and Bcr-AblT315I/Y253H cell populations in vivo, we employed a murine retroviral infection/transplantation model of CML. Since we reasoned that the very aggressive and rapidly fatal CML-like disease in primary transplanted mice precludes the analysis of more subtle differences in the overall growth kinetics of the Bcr-Ablwt- and mutant-infected leukemic cell populations in the absence of imatinib, we used leukemic cells from primary transplanted animals for the subsequent in vitro and in vivo analysis of the proliferative capacity of the two mutants as depicted in Figure 2c.

A mixture of 2.5 × 106 Bcr-Ablwt and Bcr-AblT315I or Bcr-AblY253H infected bone marrow cells were transplanted into three lethally irradiated female recipient mice per mutant in a ratio of 3:1 (Figure 2c). All transplanted mice rapidly developed a myeloproliferative disease characterized by massive expansion of myeloid cells in the peripheral blood, spleen and bone marrow and had to be sacrificed around day 14 after transplantation due to progressive illness (data not shown). In this first group of experiments with three animals per mutation, the fraction of leukemic cells expressing mutant Bcr-Abl was detectable at the initially transplanted ratio of about 20–26% (data not shown). The ratio of cells infected with the MIG and MSCV retroviral vectors was verified by real time PCR (Supplementary Figure 1). To investigate also subtle differences between the biologic activity of wt and mutant Bcr-Abl, we then proceeded to analyze competitive growth of leukemic cells from these mice by in vitro assays and serially transplantation into additional sublethally irradiated mice.

Primary murine bone marrow cells expressing Bcr-AblT315I and Bcr-AblY253H have no proliferative advantage in cell culture compared to Bcr-Ablwt expressing cells

Spleen-derived leukemic cells from the mice, which had developed CML-like disease were plated in liquid BM assays or methylcellulose. Two independently transplanted mice were analysed for each mutant. The mean initial percentage of EGFP+ cells was 15 and 18% for the Bcr-AblT315I mutant and 17 and 26% for the Bcr-AblY253H mutant.

In the absence of imatinib, the percentage of Bcr-AblT315I/EGFP+ cells initially decreased slightly but then stayed at the same level of approximately 20% over a week of culture in the liquid BM assay (Figure 3a). Similar growth characteristics held true for the Bcr-Ablwt/Bcr-AblY253H mixture of cells, where the mutant fraction also stabilized at around 20% EGFP+ cells (Figure 3b).

Figure 3
figure3

(a) Liquid transformation assay with spleen cells from mice transplanted with a mixture of EGFP Bcr-Ablwt and EGFP+ Bcr-AblT315I transformed cells. In three independent experiments, leukemic cells from two transplanted mice were plated in triplicate in IMDM/20% FCS without growth factors, and 0, 0.25, 0.5 and 1 μ M imatinib was added. The relative amount of EGFP+ Bcr-AblT315I-transformed cells was repeatedly determined by FACS analysis. (b) Liquid transformation assay performed as described in (a) with spleen cells from two mice independently transplanted with a mixture of EGFP Bcr-Ablwt and EGFP+ Bcr-AblY253H transformed cells and plated in triplicate. (c, d) Influence of imatinib on the relative contribution of Bcr-AblT315I and Bcr-AblY253H transformed cells to total colony number in a methylcellulose assay. Spleen cells from two independently transplanted mice were plated in triplicate in methylcellulose without growth factors in the presence of the indicated concentrations of imatinib. Colony numbers were counted after 14 days of culture and the percentage of EGFP+ colonies was determined by FACS analysis.

As a control for the transforming capacity of the Bcr-Abl mutants, we also cultured the cells in the presence of imatinib in liquid BM assays. The Bcr-AblT315I/EGFP+ fraction reached more than 90% during treatment with 0.5 or 1 μ M imatinib in the liquid BM assay (Figure 3a), and also the fraction of Bcr-AblY253H/EGFP-expressing cells increased after addition of imatinib (Figure 3b). For the methylcellulose assays, colonies were picked after 14 days and analyzed by flow cytometry. On average, 20 colonies were analyzed from each plate (range 15–25). Generally, the colony number decreased with higher imatinib concentrations (data not shown) and revealed a strong selection of EGFP+/Bcr-Abl mutant expressing cells upon addition of imatinib, whereas without imatinib there was no significant difference in the percentage of EGFP+/Bcr-Abl mutant cells compared to the initially plated ratio (Figure 3c and d).

Taken together, the in vitro assays failed to show a growth advantage for one of the Bcr-Abl mutants compared to Bcr-Ablwt in the absence of imatinib, but clearly demonstrated an imatinib dependent selection of the Bcr-Abl mutants already at low concentrations of the inhibitor.

Bcr-AblT315I/Y253H transformed cells do not show a growth advantage in serially transplanted mice compared to Bcr-Ablwt cells

To analyze the proliferative capacity of the Bcr-AblT315I mutant in a competitive repopulation assay in vivo, 3 × 106 spleen cells with approimately 25% EGFP/Bcr-AblT315I positive cells were serially transplanted to three sublethally irradiated mice. The mice developed a myeloproliferative syndrome with a latency of 10–15 days. We measured the fraction of CD11b and EGFP-expressing cells in the PB by flow cytometry. There were almost no B220 or Th1.2-expressing cells in the PB of the mice with CML disease (data not shown). With rising leukocyte counts, the fraction of EGFP/Bcr-AblT315I expressing cells stabilized at a mean expression of 25.7% (Figure 4a and b). In none of the analyzed animals a growth advantage for the Bcr-AblT315I mutant compared to Bcr-Ablwt transformed leukemic cells was evident.

Figure 4
figure4

(a) Representative flow cytometric analysis of the PB from a mouse transplanted with a 3:1 mixture of EGFP Bcr-Ablwt and EGFP+ Bcr-AblT315I infected cells which did not receive imatinib treatment. The x axis represents EGFP-expression in the FL1 channel, CD11b-PE was measured in the FL2 channel. The timepoint of analysis is indicated above the FACS window in days after transplantation. (b) Serial analysis of the leukocyte curve and EGFP fractions of the mouse from (a). The left y axis represents leukocyte number, the right y axis stands for the relative EGFP expression. (c) Representative flow cytometric analysis of the PB from a mouse transplanted with a 3:1 mixture of EGFP Bcr-Ablwt and EGFP+ Bcr-AblY253H infected cells without imatinib treatment. (d) Follow-up analysis of leukocyte counts and EGFP expression of the mouse from (c). (e) Relative EGFP-expression of a group of three mice transplanted with either a mixture of Bcr-AblT315I or Bcr-AblY253H and Bcr-Ablwt in the initial transplanted cell population and at the end stage of CML disease.

We then compared the growth characteristics of Bcr-AblY253H and Bcr-Ablwt transformed cells in vivo. Again, we serially transplanted 3 × 106 spleen cells to three sublethally irradiated mice. The recipient mice developed a MPD with a latency of 18–21 days. Similar to mice transplanted with the Bcr-AblT315I mutant, the percentage of EGFP/Bcr-AblY253H positive cells in all analyzed mice did not increase significantly over the initially transplanted ratio (Figure 4c and d). The comparison of the percentage of EGFP/Bcr-AblT315I/Y253H expressing cells at the beginning and at the end stage of their disease revealed no significant change in the ratio of the EGFP/Bcr-Abl mutant positive cells with a mean expression of 19.6% (Figure 4e). Taken together, these data indicated that in our model the analyzed Bcr-Abl mutants do not confer a significant growth advantage over Bcr-Ablwt in the absence of imatinib.

Selection of Bcr-AblT315I in the presence of imatinib in vivo

In the cell culture assays, addition of imatinib induced strong positive selection of the resistant Bcr-Abl mutations. To demonstrate that the leukemic cell clones harbouring Bcr-Abl mutants were capable to overgrow the Bcr-Ablwt cells in the presence of imatinib in mice, we serially transferred 3 × 106 leukemic cells transduced with Bcr-Ablwt/Bcr-AblT315I from a primary transplanted mouse into four secondary sublethally irradiated mice. To monitor the development of a CML-like disease and the fraction of cells expressing mutant Bcr-Abl, PB was analyzed for EGFP expression. In the imatinib-treated mice (by gavage with 100 mg/kg twice daily), there was a significant increase of EGFP+ cells in the PB over the course of disease (Figure 5a and b), whereas there was again no apparent positive selection in the two nontreated mice (Figure 5c).

Figure 5
figure5

(a) Consecutive flow cytometric analysis of the PB from a mouse serially transplanted with a 3:1 mixture of cells infected with either EGFP MSCV-Bcr-Ablwt or an EGFP+ MIG-Bcr-AblT315I construct and treated with 100 mg/kg imatinib bid from day 1 on via gavage. The x axis represents EGFP expression, the y axis expression of CD11b-PE. The time of analysis (number of days after transplantation) is indicated above the FACS window. (b) Graph showing the leukocyte curve as well as columns representing the percentage of EGFP/Bcr-AblT315I+ cells of the mouse from (a) over the course of the disease. (c) Relative EGFP/Bcr-AblT315I expression during CML disease development in altogether four mice serially transplanted as in (a). Two mice were either treated with 100 mg/kg imatinib or left untreated.

Discussion

The mechanisms involved in imatinib-resistance development have been extensively studied and mutations in the Bcr-Abl kinase domain, Bcr-Abl amplification and clonal evolution have most frequently been ascribed to treatment failure.24 Point mutations in the Abl kinase region have been found in all stages of CML, but occur much more frequently in advanced disease.25 Some investigators have detected mutant Abl transcripts prior to imatinib treatment.14, 15, 16 Furthermore, there have been reports that patients with a P-loop mutation carry an especially poor prognosis.17 These findings may implicate a broader, imatinib-independent role at least for some Bcr-Abl mutations. From biochemical, structural and cell culture assays, the impact on imatinib response has been established for most of the Abl mutations found in clinically resistant patients,26, 27, 28 whereas the effects of these mutations on Bcr-Abl kinase activity and on general disease phenotype are still under investigation, and there is some debate concerning the biological activity of these mutants (12, 13, Gorre et al. Blood 2004; 104:161a (abstract) and Grisworld et al. Blood 2004; 104:161a (abstract)).

Therefore, we initially addressed the influence of the T315I and Y253H mutation on Bcr-Abl kinase activity. For the kinase assay, we used immunoprecipitated Bcr-Abl reflecting steady state Bcr-Abl kinase activity. Since other frequently used Bcr-Abl substrates like CRKL bind to Bcr-Abl, a mutation in the Bcr-Abl sequence might potentially affect this binding and therefore have an impact on target phosphorylation kinetics. By employing a synthetic protein containing an optimal Abl-phosphorylation site as the substrate, we sought to exclude a potential influence mediated by an altered substrate specificity of the mutants. We found similar phosphorylation kinetics of the Bcr-AblT315I and the Bcr-AblY253H mutant compared to Bcr-Ablwt in the kinase assay. It has been previously shown that the Y253F mutation has a positive effect on c-Abl transforming potency without increasing its in vitro kinase activity.12, 29 It was speculated that the Y253F mutation, besides impeding imatinib binding by destabilizing the Abl P-loop and thus compromising the induced-fit mechanism, may interfere with binding of a negative regulator of Abl transforming activity.12 In our experiments, we did not see a significant difference in the overall protein phosphorylation pattern in Ba/F3 cells transformed by Bcr-Ablwt or the Bcr-Abl mutants. Although these findings do not fully rule out that a differentially binding regulator may exist, they suggest that the substrate specificity of Bcr-Abl in contrast to c-Abl may not be significantly influenced by the mutations. In a work published by Corbin et al.,30 a slightly reduced kinase activity of the c-AblT315I mutant compared to c-Ablwt was described. The fact that the authors have analyzed c-Abl purified from bacteria, in contrast to our assay where we looked at Bcr-Abl immunoprecipitated from murine B-lymphoid cells may explain why we did not see a substantial effect of the T315I mutation on Bcr-Abl kinase activiy. In a more recent work, an increased kinase activity of Bcr-AblT315I was reported.13 However, the authors only analyzed a short, truncated form of Bcr-Abl consisting only of the SH2, SH3 and kinase domain, making the results from this study difficult to interpret.

So far, no experimental animal models enabling the analysis of Bcr-Abl point mutations in vivo have been described. We employed a competitive BM repopulation/transformation assay to analyze the effects of two Bcr-Abl mutants commonly found in imatinib-resistant patients on the transformation and growth properties of leukemic cells in vivo in comparison to Bcr-Ablwt. By mixing EGFP BM cells transduced with a Bcr-Ablwt vector and cells infected with an EGFP+ vector coexpressing a Bcr-Abl mutant, we were able to identify the fraction of Bcr-Abl mutant expressing cells by flow cytometric analysis.

Bcr-Abl transformed primary murine BM cells proliferate in FCS-supplemented IMDM and form colonies in media containing methylcellulose in the absence of growth factors.31 To analyze the relative growth kinetics of Bcr-Ablwt and Bcr-Ablmut transformed murine BM cells, we plated leukemic cells from the primary transplanted mice in a liquid BM assay and in methylcellulose. Interestingly, the cells transformed by the Bcr-Abl mutants did not display a differential growth in the absence of imatinib, implicating that the two analyzed mutations do not have a significant impact on Bcr-Abl transforming potential in these assays.

As expected, we found that addition of imatinib selected for cells expressing the mutant Bcr-Abl cDNAs in the methylcellulose assay as well as in the liquid transformation assay in a dose-dependent manner. These findings underline the role of these mutations in resistance development and at the same time demonstrate the ability of our assay to detect differences in the proliferative capacity of the two populations.

In a in vivo analysis of the biologic activity of the T315I and the Y253H mutant, the fraction of Bcr-AblT315I/EGFP+ or Bcr-AblY253H/EGFP+ cells remained stable in the absence of imatinib, arguing against a selective advantage mediated by an increased transforming potential of the two mutations. In contrast, imatinib treatment clearly selected for leukemic cells expressing the Bcr-AblT315I mutant in vivo.

Taken together, our data demonstrate that both the Bcr-AblT315I as well as the Bcr-AblY253H mutation are selected for by imatinib treatment in vitro and in vivo, supporting their role in resistance development. On the other hand, we did not see a selection of either the T315I or the Y253H mutant in vitro or in vivo in the absence of imatinib, suggesting the mutations do not have a strong impact on transformation by Bcr-Abl. In our view, these findings would account for the fact that neither in untreated Bcr-Abl expressing cell lines nor in untreated CML or Ph+ ALL patients a high fraction of leukemic cells containing these mutations has been found to date. Clonal positive or negative selection of leukemic clones expressing Bcr-Abl mutants in patients in the absence of imatinib, which has been described in previous reports,16, 32 may have been caused by secondary genetic events influencing the growth characteristics of the leukemic cell. Thus, in the absence of imatinib, cellular adaptive changes, such as additional mutations, Bcr-Abl amplifications, immunologic escape mechanisms or other aberrations may account for the proliferative advantage of the leukemic clone. This would implicate that the response of a mutant clone towards imatinib withdrawal will have to be determined individually in each CML patient.

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Acknowledgements

This work was supported by a grant from the BMBF (NGFN II) to JD. CM was supported by a fellowship from the Deutsche Jose Carreras Leukämie Stiftung (DJCLS2001/NAT-2).

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Correspondence to J Duyster.

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Supplementary Information accompanies the paper on Leukemia's website (http://www.nature.com/leu)

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Miething, C., Feihl, S., Mugler, C. et al. The Bcr-Abl mutations T315I and Y253H do not confer a growth advantage in the absence of imatinib. Leukemia 20, 650–657 (2006). https://doi.org/10.1038/sj.leu.2404151

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Keywords

  • Bcr-Abl
  • CML
  • imatinib
  • resistance
  • mouse model

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