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Molecular Targets for Therapy

Oligomerization inhibition, combined with allosteric inhibition, abrogates the transformation potential of T315I-positive BCR/ABL

Abstract

The t(9;22) translocation leads to the formation of the chimeric bcr/abl fusion gene, which encodes the BCR/ABL fusion protein. In contrast to its physiological counterpart c-ABL, the BCR/ABL kinase is constitutively activated, inducing the leukemic phenotype. The N-terminus of c-ABL (Cap region) contributes to the regulation of its kinase function. It is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain known as the myristoyl-binding pocket in a process called ‘capping’, which results in an auto-inhibited conformation. Because the cap region is replaced by the N-terminus of BCR, the BCR/ABL ‘escapes’ this auto-inhibition. Allosteric inhibition by myristate ‘mimics’, such as GNF-2, is able to inhibit unmutated BCR/ABL, but not the BCR/ABL that harbors the ‘gatekeeper’ mutation T315I. In this study, we analyzed the possibility of increasing the efficacy of allosteric inhibition by blocking BCR/ABL oligomerization. We showed that inhibition of oligomerization was able to not only increase the efficacy of GNF-2 on unmutated BCR/ABL, but also overcome the resistance of BCR/ABL-T315I to allosteric inhibition. These results strongly suggest that the response to allosteric inhibition by GNF-2 is inversely related to the degree of oligomerization of BCR/ABL. In summary, our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants represented by the combination of oligomerization and allosteric inhibitors.

Introduction

Chronic myeloid leukemia is a clonal myeloproliferative disorder and accounts for 15% of leukemias in adults. Blasts of chronic myeloid leukemia patients present the Philadelphia chromosome (Ph+), which is the cytogenetic correlate of the t(9;22) translocation. Ph+ cases of acute lymphatic leukemia (ALL), which represent 25–30% of cases, define a subgroup of the high-risk ALL.1, 2

The t(9;22)-related translocation products are the BCR/ABL fusion proteins. The fusion of BCR to ABL leads to a constitutive activation of ABL tyrosine kinase activity. c-ABL is finely regulated by a variety of stimuli, whereas constitutively activated ABL induces aberrant proliferation and neoplastic transformation by the constitutive activation of RAS, PI3 kinase and Janus kinase/signal transducer and activator of transcription (STAT).2 Constitutively activated ABL tyrosine kinase is indispensable for the transformation potential of BCR/ABL. The cellular transformation and leukemogenesis are strictly dependent on the ABL tyrosine kinase activity of BCR/ABL.3, 4, 5, 6

Inhibition of BCR/ABL kinase activity using either selective ABL-kinase inhibitors, such as imatinib-mesylate (Glivec or Gleevec) or nilotinib (Tasigna), or the multitarget inhibitor dasatinib (sprycel), which competes with adenosine-5’-triphosphate for its binding site, is a valid concept for the causal therapy of Ph+ leukemia. Unfortunately, in advanced Ph+ leukemia, chronic myeloid leukemia-blast crisis, and Ph+ ALL exposure to these ABL kinase inhibitors, selects for resistant clones. Secondary resistance is mostly because of the acquisition of point mutations in BCR/ABL that change the affinity of the protein for these adenosine-5’-triphosphate competitors.7 These clones are selected by continuous exposure to ABL kinase inhibitors.4, 8 With the exception of the ‘gatekeeper’ mutation, T315I, the most clinically relevant mutations confer resistance against the first-generation ABL kinase inhibitor, imatinib, but can still be targeted using one of the second-generation ABL kinase inhibitors, dasatinib or nilotinib.7

Tetramerization of ABL through the N-terminal coiled-coil region (CC) of BCR is essential for aberrant ABL-kinase activation.9 The CC contains the α-1 (a.a. 5–15) and α-2 (a.a. 28–67) helices, which are separated by a flexible loop. The dimer interface is formed through direct interaction of each monomer’s helix-2 with each other, whereas helix-1 from one monomer swings back and packs against the ‘outside’ of the helix-2 dimer and interacts with helix-2 of the other monomer.10 The deletion of helix-2 impairs oligomerization and reduces the kinase activity.11 We recently showed that targeting the CC domain forces BCR/ABL into a monomeric conformation that abolishes its transformation potential by interfering with its kinase activity. This also increases the sensitivity of the leukemic cells to imatinib and overcomes the imatinib resistance of BCR/ABL harboring the 253 and E255K mutations.9, 12

The activation status of wild-type c-ABL by autophosphorylation is finely regulated by several regulation signals. Myristoylation of the N-terminus of c-ABL is involved in the regulation of the ABL kinase activity. The N-terminus of ABL is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain—the myristoyl-binding pocket—a process called ‘capping’.13 The ‘capping’ leads to conformational changes that allow the intramolecular docking of the Src homology 2 domain to the kinase domain. Hence, c-ABL takes on an auto-inhibited conformation. The N-terminal auto-inhibitory ‘Cap’ region is absent in BCR/ABL, and the absence of this ‘Cap’ might allow the fusion protein to ‘escape’ auto-inhibition.14 This lack of auto-inhibition mainly contributes to the constitutive activation of BCR/ABL.14 The absence of an N-terminal myristoylated domain activates c-ABL, consistent with its auto-regulatory role.15

To discover agents capable of targeting the BCR/ABL kinase through a mechanism distinct from adenosine-5’-triphosphate competition, an unbiased differential cytotoxicity screen of a combinatorial kinase-directed heterocycle library led to the identification of a new class of BCR/ABL inhibitors. One of these compounds, GNF-2, is a cell-permeable pyrimidine compound that binds to myristoyl-binding pocket and functions as an allosteric, non-adenosine-5’-triphosphate-competitive inhibitor of BCR/ABL. It possesses exclusive anti-proliferative activity against BCR/ABL-expressing factor-independent cells.16 Unfortunately, GNF-2 is unable to overcome the resistance of BCR/ABL harboring the T315I mutation.16

In this study, we analyzed whether it was possible to overcome the resistance of the BCR/ABL-T315I mutant against molecular therapeutic approaches by combining the allosteric inhibition of GNF-2 with the competitive peptide helix-2 to inhibit BCR/ABL oligomerization. This is of particular importance when the high frequency of resistance to ABL kinase inhibitor is considered, especially in patients with Ph+ ALL. Therefore, we focused our study on the Ph+ ALL-associated p185BCR/ABL because there is no difference in the response to oligomerization inhibition between chronic myeloid leukemia-associated p210BCR/ABL and p185BCR/ABL or with regard to the response to GNF-2.17

Materials and methods

Plasmids

The complementary DNAs encoding p185BCR/ABL, p185-T315, their respective mutants lacking the N-terminal CC domain (ΔCC-p185 and ΔCC-p185-T315I), green fluorescent protein (GFP) or helix-2 fused to GFP have been previously described.12 All retroviral expression vectors used in this study were based on the bi-cistronic vectors PINCO or PAULO, which were converted into Gateway-destination vectors, through introduction of a Gateway cassette, according to the manufacturer's instructions (Invitrogen, Karlsruhe, Germany). All related inserts were available in the Gateway entry-vector (pENTR1A) for recombination into the destination vectors using the ‘LR-clonase’ enzyme kit (Invitrogen). All other constructs have been previously described.12

Cell lines

The Ba/F3 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (Invitrogen) containing 10 ng/ml of interleukin-3 (Cell Concepts, Umkirch, Germany). Ecotropic Phoenix cells and Rat-1 cells were cultured in Dulbecco's modied Eagle's medium supplemented with 10% fetal calf serum. GNF-2 (Sigma-Adrich, Steinheim, Germany) was dissolved in dimethylsulfoxyde and added at a final concentration of 2 μM. Cell growth was assessed by dye exclusion using Trypan blue. Proliferation was assessed using the XTT proliferation kit (Roche, Mannheim, Germany), according to the manufacturer’s instructions.

Western blotting

Western blot analyses were performed according to the widely established protocols. The following antibodies were used: anti-ABL (α-ABL) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phosphorylated ABL specific for the phosphorylated tyrosine residue 245 (α-p-ABL-Y245) (Upstate-Biotechnology, Lake Placid, NY, USA), anti-BCR (α-BCR) (Santa Cruz Biotechnology), anti-phosphorylated BCR specific for the phosphorylated tyrosine residue 177 (α-p-BCR-Y177), anti-STAT5, and anti-phosphorylated STAT5 (Cell Signaling, Boston, MA, USA). Blocking and antibody incubation were performed in 5% low-fat dry milk, followed by washing in Tris-buffered saline (10 mM Tris-HCl pH 8, 150 mM NaCl) containing 0.1% Tween-20.

Retroviral infection and transfection

Ecotropic retroviral supernatants were obtained as previously described.9 For infection of the target cells, retronectin (Takara Bio Inc., Otsu, Japan) was used to enhance infection efficiency, according to the manufacturer's instructions. Target cells/well were seeded at a concentration of 2 × 105. Infection efficiency was measured after 48 h by determining the percentage of GFP-positive or ΔNGFR (nerve growth factor receptor)-positive cells using fluorescence-activated cell sorting analysis.

Proliferation-competition assay (PCA)

PCA was performed as previously described.12 In brief, Ba/F3 cells were infected with PAULO vectors harboring unmutated or mutant p185BCR/ABL and the low-affinity nerve growth factor receptor lacking the cytoplasmic activation domain (ΔNGFR). Interleukin-3 was removed from the media of infected Ba/F3 cells by washing the cells twice with phosphate buffered saline. The cells were continuously cultivated in the absence of interleukin-3 and super-infected with GFP and helix-2-GFP. GFP expression levels of day 2 were used to normalize the expression levels of the different experiments. Proliferation-competition between single- and double-infected cell fractions was monitored by fluorescence-activated cell sorting analysis of GFP expression.

Transformation—soft agar assay

Soft agar assays were performed using Rat-1 fibroblasts that were retrovirally transduced with PINCO vectors harboring unmutated or mutant p185BCR/ABL. Six-well plates were filled with Dulbecco's modied Eagle's medium supplemented with 10% fetal calf serum and 0.5% bacto-agar (DIFCO Laboratories, Detroit, MI, USA) (2 ml/well). Transduced Rat-1 cells (5 × 103) were suspended in ‘top-agar’, which consisted of Dulbecco's modied Eagle's medium supplemented with 10% fetal calf serum and 0.25% bacto-agar (1 ml/well), and then stacked in the wells. Colonies were counted after incubation for 15 days at 37 °C and 5% CO2.

Results

Targeting the oligomerization of BCR/ABL increases the efficacy of GNF-2 against unmutated p185BCR/ABL

To determine whether or not the inhibition of oligomerization improved the response to allosteric inhibition of cells that depend on the functionality of BCR/ABL for their survival, we exposed p185BCR/ABL-expressing Ba/F3 in the presence/absence of helix-2 to GNF-2 at a concentration of 2 μM, which was the half-maximal inhibitory concentration for growth inhibition in our system (data not shown), upon factor withdrawal and performed a PCA (Figure 1a). The combination with helix-2 accelerated and intensified the effects of GNF-2, suggesting a reciprocal interaction between the two compounds in blocking the BCR/ABL-dependent survival signaling upon factor withdrawal in these cells (Figure 1a).

Figure 1
figure1

Effects of the oligomerization inhibitor helix-2 on the response of p185BCR/ABL toward allosteric inhibition by GNF-2. (a) Schematic representation of the proliferation-competition assay. Factor-independent cells (that is, p185BCR/ABL-expressing Ba/F3 cells) were retrovirally infected with inert control peptides (green fluorescent protein (GFP)) or helix-2-GFP. In the case of inert GFP control, the percentage of GFP-positive cells remained constant from day 2 to day 10. In contrast, the expression of inhibitory peptides resulted in an outgrowth of peptide-expressing cells, which was revealed by progressive loss of helix-2-GFP expression. For proliferation-competition experiments p185BCR/ABL-transduced Ba/F3 cells, adapted to factor independence, were infected with either GFP or helix-2-GFP expressing retroviruses and plated 2 days after infection in the absence/presence of 2 μM GNF-2. (b) XTT assay for Ba/F3 cells expressing p185BCR/ABL upon exposure to 0.5 μM imatinib and/or 2 μM GNF-2 in the absence/presence of helix-2, as indicated. Proliferation status was revealed by determination of the metabolic activity of cells given by the reduction rate of XTT to formazan. The means ± s.d. of triplicates from one representative experiment out of three performed are given. (c) The apoptosis rate of Ba/F3 cells expressing p185BCR/ABL upon exposure to 2 μM GNF-2 in the absence/presence of helix-2, as indicated. The apoptosis rate was determined by staining with 7-aminoactinomycin D (7-AAD). The data represent the mean and s.d. of three independent experiments. (d) Transformation assay by colony formation in soft agar. Rat-1 cells were retrovirally transduced with the indicated constructs, and seeded at 5 × 103 cells/well in soft agar in six-well-plates. After 15 days, the colonies were counted, and the means ± s.d. of triplicates obtained from two representative experiments are presented.

Given the fact that PCA cannot reveal the effects of GNF-2 alone, we analyzed the effects of GNF-2 alone or in combination with helix-2 in comparison with the effects of helix-2 on the proliferation of p185BCR/ABL-expressing Ba/F3 cells by measuring the reduction of the tetrazolium salt XTT to formazan (XTT-assay). As a control, the cells were exposed to 0.5 μM imatinib. Imatinib and GNF-2 reduced the metabolic activity of the p185BCR/ABL-expressing Ba/F3 cells to the same extent, independently of the presence of helix-2 (Figure 1b).

To analyze whether the observed effects on proliferation were related to the induction of apoptosis, factor-independent p185BCR/ABL-expressing Ba/F3 cells in the presence/absence of helix-2 were exposed to 2 μM GNF-2 and stained with 7-aminoactinomycin D, which determines the apoptosis rate. As expected, GNF-2 increased the apoptosis rate as compared with the controls, whereas helix-2 did not increase the apoptosis rate (Figure 1c). Interestingly, the combination of GNF-2 and helix-2 induced an apoptosis rate close to 100% (Figure 1c).

BCR/ABL-dependent signaling ‘substitutes’ for interleukin-3 signaling in factor-dependent hematopoietic cell lines, such as Ba/F3. To further study the effects of GNF-2 in combination with the oligomerization inhibition, a soft agar assay was performed as a classic transformation assay in untransformed Rat-1 fibroblasts. Rat-1 cells were retrovirally transduced with p185BCR/ABL along with either GFP as a control or helix-2, and were then exposed to 2 μM of GNF-2. Empty-vector-transduced Rat-1 cells (mock) were used as controls. GNF-2 alone was able to reduce the transformation potential of BCR/ABL to the same extent as helix-2, but the combination of both nearly completely abolished the BCR/ABL-induced transformation (Figure 1d).

Taken together, these data suggest that the effect of GNF-2 may be enhanced by the monomerization of BCR/ABL.

Targeting the oligomerization of BCR/ABL increases the efficacy of GNF-2 against p185BCR/ABL harboring the T315I mutation

Both GNF-2 and helix-2 are known to have no effect on cells that express p185-T315I.12, 16 To analyze whether the combination of these two approaches are able to inhibit p185-T315I, the experiments described above were repeated using Ba/F3 cells that express p185-T315I. The known resistance of these cells against GNF-2 and helix-2 alone was confirmed in the PCA, the XTT and the apoptosis assays, as well as in the soft agar assay (Figures 2a–d). Interestingly, in combination, the two compounds were nearly able to completely abolish the capacity of the p185-T315I mutant to mediate factor-independent growth, as well as its transformation potential (Figures 2a–d).

Figure 2
figure2

Effects of the oligomerization inhibitor helix-2 on the response of p185-T315I toward allosteric inhibition by GNF-2. (a) Proliferation-competition assay. The factor-independent cells p185-T315I expressing Ba/F3 were retrovirally infected with inert control peptides (green fluorescent protein (GFP)) or helix-2-GFP upon exposure or not to 2 μM GNF-2. (b) XTT-assay for Ba/F3 cells expressing p185BCR/ABL upon exposure to 0.5 μM imatinib and/or 2 μM GNF-2 in the absence/presence of helix-2, as indicated. Proliferation status was revealed by determination of the metabolic activity of cells given by the reduction rate of XTT to formazan. The means ± s.d. of triplicates of one representative experiment out of three performed are given. (c) The apoptosis rate of Ba/F3 cells expressing p185-T315I upon exposure to 2 μM GNF-2 in the absence/presence of helix-2, as indicated. The apoptosis rate was determined by staining with 7-aminoactinomycin D (7-AAD). Data represent the mean and s.d. of three independent experiments. (d) Transformation assay using colony formation in soft agar. Rat-1 cells were retrovirally transduced with the indicated constructs and seeded at 5 × 103 cells/well in soft-agar in six-well-plates. After 15 days, the colonies were counted, and the means±s.d. of triplicates of two representative experiments are presented.

Taken together, these data strongly suggest that the resistance of T315I is because of a conformational change that renders the myristoyl-binding pocket inaccessible to GNF-2 and that results in kinase activity independent of oligomerization.

To confirm these data, we analyzed the effects of GNF-2 on a BCR/ABL lacking the N-terminal coiled-coil oligomerization interface in which the presence of T315I restores the transformation potential as well as the capacity to mediate factor-independent growth.17 As expected, GNF-2 completely reverted the effect of T315I on this loss-of-function mutant (Figure 3).

Figure 3
figure3

Role of oligomerization in the sensitivity of unmutated p185BCR/ABL and p185-T315I toward allosteric inhibition by GNF-2. To determine the factor-independent growth, Ba/F3 cells were retrovirally transduced with the indicated constructs and grown upon factor withdrawal with exposure to 2 μM GNF-2. For factor-independent growth, the number of viable cells was determined daily by Trypan blue dye exclusion. Data represent the mean and s.d. of three independent experiments.

In summary, these data show that the combination of the two molecular approaches overcomes the resistance of BCR/ABL-T315I against the oligomerization inhibition of helix-2 and the allosteric inhibition of GNF-2.

Effects of the combination of GNF-2 and helix-2 on the autophosphorylation of BCR/ABL and its downstream signaling

Our data suggest that GNF-2 and helix-2 are able to overcome the T315I-induced stabilization of kinase activity only in a combination. Therefore, we studied the effects of this combination on the autophosphorylation and downstream signaling of p185BCR/ABL and p185-T315I in Ba/F3 cells in comparison with the effects of the ‘single drug’ approaches. We have recently shown that the capacity of T315I to restore leukemogenic potential to the loss of function of BCR/ABL is closely related to its capacity to trans-phosphorylate endogenous BCR at position Y177.17

GNF-2 had a stronger effect on the autophosphorylation of unmutated p185BCR/ABL than did helix-2, and both interfered efficiently with the downstream signaling through STAT5 and inhibited the Y177 phosphorylation of endogenous BCR (Figure 4). These effects were strongly increased by the combination of both GNF-2 and helix-2. On p185-T315I, neither GNF-2 nor helix-2 alone was able to inhibit autophosphorylation, STAT5 activation and trans-phosphorylation of BCR. In contrast, the two agents in combination completely abolished the autophosphorylation, STAT5 activation and BCR trans-phosphorylation, confirming the observations described above.

Figure 4
figure4

The combination of helix-2 and GNF-2 inhibits the autophosphorylation of p185-T315I and dependent signaling pathways. Western blot analysis of lysates of Ba/F3 cells expressing the indicated transgenes using antibodies directed against: c-ABL, ABL Y245 (anti-phospho-ABL), BCR (anti-BCR, which did not detect BCR/ABL), BCR phosphorylated at Y177 (anti-phospho-BCR), signal transducer and activator of transcription 5 (STAT5), phosphorylated STAT5 (anti-phospho-STAT5) and β-tubulin (anti-β-tubulin). Molecular mass references (kDa) are presented, and c-ABL and β-tubulin were used as a loading control.

Taken together, these data show that GNF-2, in combination with helix-2, was able to block the aberrant kinase activity of BCR/ABL harboring the T315I mutation, which is oligomerization independent and is resistant to allosteric inhibition.

Discussion

The major therapeutic challenge is to efficiently treat patients with BCR/ABL harboring the T315I mutation. Unfortunately, T315I not only confers resistance against ABL kinase inhibitor, but also against the inhibition of oligomerization by helix-212 and the allosteric inhibition by GNF-2.16

The T315I mutation is most recalcitrant to inhibition because of a combination of several factors, including steric hindrance of drug binding, loss of a key hydrogen-bonding interaction with the T315 side-chain hydroxyl group that is exploited by imatinib, nilotinib and dasatinib, and potentially through increasing aberrant intrinsic kinase activity accompanied with aberrant substrate phosphorylation.17, 18

Our data strongly suggest that both the sensitivity of unmutated p185BCR/ABL and the resistance of p185-T315I toward the allosteric inhibition by GNF-2 is dependent on oligomerization. In fact, the inhibition of oligomerization by the competitive helix-2 peptide not only increased the response of unmutated p185BCR/ABL, but also restored the response of p185-T315I to the level of unmutated p185BCR/ABL. This was also confirmed by the high sensitivity of the oligomerization-deficient p185-T315I mutant toward GNF-2. The relationship between the resistance against GNF-2 and oligomerization was already anticipated by the work of Adrian et al.,16 who showed that activation of the kinase activity of ABL by the oligomerization interfaces of nucleophosmin (NPM) or of Translocation-ETS-Like (TEL) was not reverted by GNF-2. We previously showed that the helix-loop-helix oligomerization interface of TEL, directly fused to the ABL portion of the fusion protein helix-loop-helix/ABL, showed enhanced ABL kinase activity along with a decreased response to imatinib, as compared with unmutated p185BCR/ABL or other ABL chimeras.9 Furthermore, the helix-loop-helix/ABL chimera formed complexes of higher molecular weight than even the unmutated p185BCR/ABL.9 This indicated that the level of resistance against GNF-2 might increase in parallel with the grade of oligomerization or high-molecular-weight formation by the ABL fusion proteins, which seems to parallel the sensitivity toward imatinib, which also decreases with the oligomerization status of ABL fusion proteins.9 Thus, one can speculate that GNF-2 shows a reduced capacity to bind myristoyl-binding pocket when BCR/ABL is present as an oligomer as compared with a monomer. Given that the oligomerization of BCR/ABL is a dynamic process, an equilibrium between monomers and oligomers must exist. It is possible to increase the sensitivity of unmutated p185BCR/ABL as well as that of p185-T315I, for allosteric inhibition, through a switch of this balance in favor of monomers by competitive peptides such as helix-2.

Given its increasing significance as a molecular therapy approach for Ph+ leukemia, the oligomerization inhibition should be further developed. There are different strategies to render the helix-2 ‘drugable’. Direct chemical modifications or packaging into liposomes increase its stability in the serum, together with specific tags for the membrane permeability. Furthermore, it could serve as a lead structure for computer-based design of small molecules.

Taken together, our results establish a new approach for the molecular targeting of unmutated BCR/ABL, as well as BCR/ABL harboring the multiresistance mutation T315I, which is the combination of oligomerization inhibitors and allosteric inhibitors.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

This study was supported by a grant from the Deutsche Forschungsgemeinschaft to MR (DFG-RU 728/3-1). MR is further funded by grants from Deutsche Krebshilfe e.V. (DKH-107063 and DKH-107741), Deutsche José Carreras Leukämie-Stiftung e.V. (DJCLS–R 07/27f) and the Alfred und Angelika Gutermuth Foundation.

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Mian, A., Oancea, C., Zhao, Z. et al. Oligomerization inhibition, combined with allosteric inhibition, abrogates the transformation potential of T315I-positive BCR/ABL. Leukemia 23, 2242–2247 (2009). https://doi.org/10.1038/leu.2009.194

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Keywords

  • Philadelphia chromosome-positive leukemia
  • BCR/ABL
  • imatinib-resistance
  • ‘gatekeeper’ mutation T315I
  • inhibition of oligomerization
  • allosteric inhibition

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