Bcr–Abl as target for therapeutic kinase inhibition

Protein tyrosine kinases are enzymes that transfer phosphate groups from ATP to substrate proteins, thereby governing cellular processes such as growth and differentiation. Tight regulation of tyrosine kinases is indispensable and, if not maintained, deregulated kinase activity can lead to transformation and initiation of malignancy. The Ph chromosome, first described as a shortened chromosome 22,¹ results from a reciprocal translocation between the long arms of chromosomes 9 and 22,² and is present in approximately 95% of chronic myeloid leukemia (CML) patients and up to 20% of adult acute lymphoblastic leukemia (ALL).^3,4 The Ph translocation gives rise to the oncogenic Bcr–Abl fusion protein that is characterized by a constitutively active tyrosine kinase. Bcr–Abl is sufficient to cause CML in mice,⁵ and its transforming capacity strictly depends on tyrosine kinase activity.⁶ This makes Bcr–Abl an attractive target for therapeutic intervention in CML and Ph+ ALL.

The 2-phenylaminopyrimidine class of small-molecule kinase inhibitors was identified using a high throughput screen of compound libraries at Ciba-Geigy (now Novartis).⁷ Imatinib mesylate, a derivative of the initial lead compound, was found to inhibit autophosphorylation of four kinases: the receptor tyrosine kinase platelet-derived growth factor receptor (PDGF-R) and , the receptor tyrosine kinase cKit and the protein tyrosine kinase Abl, including its close homolog Arg.^8,9,10,11,12 Imatinib has been demonstrated to specifically inhibit oncogenic fusion proteins including Bcr–Abl p210,¹³ p185,¹⁴ TEL (ETV6)-Abl,¹⁵ and TEL-PDGF-R.¹⁵ Moreover, the compound inhibits cKit harboring oncogenic mutations.¹⁶ In addition, imatinib proved to be active in a murine model of BCR–ABL-induced leukemia.^17,18

Imatinib is active in CML and Ph+ ALL

Phase I clinical studies showed impressive activity of the drug especially in late chronic phase CML (patients who failed interferon-based therapy) with 98% of the patients achieving complete hematologic responses (CHR).¹⁹ Also in blast crisis CML and relapsed or refractory Ph+ ALL, imatinib induced encouraging remissions.²⁰ Overall response rates (CHR or marrow response or return to chronic phase) were 21/38 patients (55%) in myeloid blast crisis CML including four patients (11%) with CHR and 14/20 patients (70%) in lymphoid blast crisis or Ph+ ALL with four patients (20%) achieving a CHR.²⁰ In accordance with the dose–response relation in chronic phase CML and comparison of pharmacokinetic with in vitro data, doses of 400 mg for chronic phase CML to 600 mg for advanced phase CML were recommended for subsequent protocols.²¹

Phase II clinical studies in late chronic phase (interferon-refractory and interferon-intolerant),²² accelerated phase,²³ and myeloid blast crisis CML²⁴ confirmed phase I data and established imatinib as an effective and well-tolerated therapy. In late chronic phase CML, imatinib induced a CHR in 95% of 454 patients. A major cytogenetic response (0–35% Ph+ cells in metaphase in bone marrow) was observed in 60%.²² In myeloid blast crisis CML, the administration of imatinib at 400 or 600 mg once daily led to sustained hematologic responses that lasted at least 4 weeks in 31% out of 229 patients, including 8% with a CHR.²⁴ In patients with relapsed or refractory Ph+ ALL or lymphoid blast crisis CML, imatinib (400 or 600 mg once daily) induced sustained responses in 27% out of 48 patients with a CHR in 6% of Ph+ ALL cases, and in 2/8 patients with lymphoid blast crisis CML including one patient with a sustained CHR.²⁵

Clinical resistance to imatinib

Whereas almost all chronic phase CML patients durably respond, remissions in CML blast crisis and Ph+ ALL are only of short duration. In the phase I study, virtually all patients (19/20) with lymphoid blast crisis CML and Ph+ ALL, and 31/38 (82%) patients with myeloid blast crisis CML relapsed within several months (median 58 days for lymphoid blast crisis and 84 days for myeloid blast crisis), despite continued therapy with imatinib.²⁰ In the phase II studies, estimated overall survival was 4.9/6.6 months in patients with Ph+ ALL/lymphoid blast crisis CML,²⁵ and 6.9 months in myeloid blast crisis CML,²⁴ with an estimated overall survival rate of 40% at 6 months in Ph+ ALL and 43% at 9 months in myeloid blast crisis CML, respectively.^24,25

Mechanisms of resistance to imatinib at the molecular level

The high activity of imatinib in chronic phase CML is evidently because of absolute dependence of the malignant clone on Bcr–Abl and its selective inhibition by imatinib. In advanced phase CML or Ph+ ALL, the combination of a high number of proliferating tumor cells and genomic instability^26,27,28 may lead to a particular high chance that secondary genetic alterations occur.²⁹ Clones that have acquired a genetic alteration that confers resistance to imatinib are selected by the drug. Intense research over the past 2 years elucidated mechanisms of resistance towards imatinib at the molecular level. Figure 1 illustrates molecular mechanisms of resistance towards imatinib that became evident so far. When a resistant clone still depends on Bcr–Abl, the oncoprotein must be active in the presence of imatinib (see Figure 1), either by an increase of Bcr–Abl protein (Figure 1b) or by mutation of sites that are critical for binding the drug (Figure 1c). If those secondary hits do not affect BCR–ABL, and independently mediate oncogenicity, these secondary genetic alterations may predominate in 'driving' the malignant clone (Figure 1d) despite effective Bcr–Abl kinase inhibition by imatinib. In either case, clinical resistance to imatinib will be the consequence.

Figure 1.

Mechanisms of resistance towards imatinib. (a) Imatinib is available within the cell at a sufficient quantity for inhibition of all Bcr–Abl-molecules. (b) Overexpression of Bcr–Abl allows the leukemic cell to maintain a baseline level of signaling that is sufficient for cell survival even in the presence of imatinib. (c) Specific mutations within the Abl kinase domain prevent binding of imatinib but still allow binding of ATP thus retaining Bcr–Abl kinase active. (d) Genetic instability may induce secondary genetic alterations that contribute to the Bcr–Abl-independent growth and/or survival of the malignant clone. (e) Increased levels of 1 acid glycoprotein might reduce the amount of unbound imatinib available for inhibition of Bcr–Abl within the cell.

Full figure and legend (161K)

In vitro models of imatinib resistance

Bcr–Abl-transformed cell lines, become – at least to some extent – resistant to imatinib after prolonged culture in the presence of imatinib at suboptimal concentrations. Such resistant sublines of LAMA84,^30,31 Ba/F3.p210,^31,32 K562,³² and AR230³¹ display amplification of the BCR–ABL gene and overexpression of the Bcr–Abl protein. Amplification of target genes that are part of critical biosynthetic pathways has been observed in neoplastic cells resistant to chemotherapeutic drugs, eg, amplification of the dihydrofolate reductase gene in methotrexate resistance.³³ Resistance to imatinib in Bcr–Abl-transformed cell lines is incomplete, occurring at concentrations where the residual activity of overexpressed Bcr–Abl may be sufficient for cell survival. High amounts of Bcr–Abl may thus allow the cell to maintain a 'baseline' level of signaling sufficient for cell survival even in the presence of imatinib.¹⁸ Interestingly, Bcr–Abl overexpression and amplification of BCR–ABL in resistant sublines of LAMA84-r were reversible after the withdrawal of imatinib, and LAMA84-r cells at the same time regained sensitivity to imatinib.³⁴

Three imatinib-resistant human cell lines (K562-r, LAMA84-r, and AR230-r) were examined for overexpression of the MDR-1 gene, coding for the P-glycoprotein (Pgp), which functions as a multidrug resistance-associated drug transporter in the plasma membrane. In comparison with its sensitive counterparts, only LAMA84-r overexpressed Pgp, and sensitivity to imatinib was partially restored when Pgp was inhibited with verapamil.³¹

There was no evidence for BCR–ABL gene mutations in resistant sublines of LAMA84, Ba/F3.p210, K562, and AR230,^30,31,32 consistent with the hypothesis that BCR–ABL point mutations may emerge from pre-existing clones in vivo that are selected for resistance in the presence of imatinib. The generation and selection of mutant forms of BCR–ABL may require in vivo conditions and therefore may not occur in vitro in a cell line derived from a single leukemic clone. However, the acquisition of a resistant phenotype in a human myeloid blast crisis KBM5 cell line by continuous exposure to imatinib was associated with only a marginal increase in the number of BCR–ABL gene copies and the level of Bcr–Abl protein.³⁵ Over and above, a single BCR–ABL point mutation (T315I) was detected in a fraction of the BCR–ABL gene copies in each of the clonally derived KBM5-STI571^R1.0 sublines, but not in parental KBM5 cells, suggesting that the mutation arose during drug exposure in vitro.³⁵ However, it cannot be ruled out that BCR–ABL point mutations emerge from clones that pre-exist in vivo and are selected for resistance in the presence of imatinib in vivo or in vitro. Thus, T315I mutations in KBM5 cells may be detected not until the presence of imatinib allows selection of single mutated cells. On the other hand, resistant sublines of LAMA84, Ba/F3.p210, K562, and AR230 may as well contain mutated BCR–ABL that has not been detected because the mutation affects only a portion of BCR–ABL transcripts, as it is the case for KBM5 cells.

Molecular mechanisms of imatinib resistance detected in patients

In an attempt to clarify the mechanism of resistance in imatinib-treated patients, an analysis performed by Gorre et al³⁶ resolved several issues. In cases of relapsed blast crisis CML and Ph+ ALL, resistance was associated with active Bcr–Abl kinase despite continued imatinib treatment, indicating that the disease still was driven by Bcr–Abl.³⁶ Patient cells displayed an impaired sensitivity to imatinib ex vivo, demonstrating that resistance represented a cell-intrinsic phenomenon. In three out of 11 cases, amplification of the BCR–ABL gene was found.³⁶ In one of those patients, BCR–ABL amplification disappeared after imatinib was discontinued,³⁶ indicating that amplification indeed was a consequence of selection in the presence of imatinib and confirming in vitro observations in resistant cell lines.^31,34 In addition, nine patients were analyzed for the presence of point mutations in BCR–ABL that might confer resistance by interfering with binding of imatinib at the kinase domain. Strikingly, a change of C to T at nucleotide 1091 leading to an exchange of a threonine at position 315 to isoleucine (T315I) was found in six out of nine cases. One patient had both, BCR–ABL amplification and mutation. A crystal structure analysis of the catalytic domain of Abl in complex to a variant form of imatinib predicted the presence of a threonine at position 315 to be a key requirement for binding imatinib.³⁷ An exchange to isoleucine at this position prevents the formation of a critical hydrogen bond and adds an extra hydrocarbon group in its side chain, thereby leading to a steric clash with the drug (see Figure 2).³⁷ Confirming these considerations, Bcr–Abl T315I remained phosphorylated in vitro in the presence of imatinib at concentrations sufficient to inhibit autophosphorylation of wild-type Bcr–Abl.³⁶

Figure 2.

Positions of BCR–ABL gene mutations. Ribbon representation of the c-Abl kinase domain in complex with imatinib. The polypeptide backbone is shown in white, helices, sheets, and loops in pale pink, imatinib in green; side chains of mutated residues (M244, G250, Q252, Y253, E255, F311, T315, F317, M351, E355, F359, V379, L387, and H396) in red. Generated with PDB 1IEP available from http://www.rcsb.org/pdb/.

Full figure and legend (271K)

Soon after T315I had been described, a number of additional point mutations in BCR–ABL were identified in cases of refractory or relapsed CML or Ph+ ALL (see Table 1^{38,39,40,41,42,43,44,45,46,47,48} ). Importantly, it became evident that hematopoietic cell lines transduced with different mutant forms of BCR–ABL indeed display resistance towards imatinib.⁴² Interestingly, there were differences in the extent of in vitro resistance: some mutant forms of Bcr–Abl (Y253H, E255K, E255V, and T315I; see Table 1) displayed IC₅₀ values (concentrations where 50% of inhibition of biologic activities exhibited by Bcr–Abl occurs) that by far exceed concentrations measured in patients⁴⁹ treated with imatinib.^42,43 In contrast, the BCR–ABL mutant H396P had IC50 values that were nearby concentrations measured in treated patients.^42,43 This indicates that increasing the dosage of imatinib may be beneficial in some cases of imatinib resistance due to mutated BCR–ABL.

Table 1 - BCR–ABL gene mutations detected in cases of CML and/or Ph+ ALL resistant to treatment with imatinib.

Full table

Although all mutants examined so far demonstrated to be kinase-active, a recent report added two important issues to the matter: the kinase activity and ATP affinity in relation to wild-type Abl.⁵⁰ In this study, T315I retained only 61% of its kinase activity, suggesting that a possibly pre-existing clone harboring T315I may confer a growth advantage over wild-type Bcr–Abl only in the presence of imatinib, and, vice versa, stopping imatinib may allow the wild-type clone to come up as the predominant clone again. Moreover, T315I displayed a two-fold increase in its ATP affinity relative to wild-type Abl.⁵⁰ This may contribute to resistance in this and other mutations affecting the ATP binding site.

Genomic instability of Ph+ cells has been demonstrated at the cytogenetic and nucleotide level^26,27,28 and can be regarded as a prerequisite for the formation of BCR–ABL gene amplification (Figure 1b), mutation (Figure 1c), and additional genetic changes (Figure 1d) that contribute to the evolution of leukemia. Clonal evolution was demonstrated in 19/36 patients with imatinib resistance,⁴⁸ indicating selection of a leukemic clone occurred that has acquired genetic alterations in addition to BCR–ABL. The nature and frequency of those genetic abnormalities remain to be elucidated. Also in murine models of Ph+ leukemia, clonal selection has been observed in imatinib resistance (see 'Imatinib resistance in murine models of Bcr–Abl-induced leukemia' section).

Frequency of point mutations in relapsed vs primary resistant patients

Initially, BCR–ABL gene mutations were reported predominantly in cases of relapsing blast crisis CML or Ph+ ALL.^{36,42,44,46,51} The discrepant frequency of BCR–ABL gene mutations that was initially reported by different authors^38,39,40 may be explained in part by differences in the technology that has been used for detection. In addition, patient selection may have a major impact on the frequency reported by different authors. It is obvious that mutations represent the major cause of resistance in relapsed patients who initially experienced a hematologic response receiving imatinib (see Table 2^{41,42,47,48,51}). Relapsed patients who have been reported so far mainly suffered from advanced phase CML and Ph+ ALL. In contrast, in hematologic^45,48 or cytogenetic^41,45,47 nonresponders, mutations in BCR–ABL have been detected less frequently, indicating that other mechanisms, such as amplification of the BCR–ABL gene,^36,48 or additional genetic alterations⁴⁸ may be important there. Those reports predominantly contained chronic and accelerated phase CML patients.

Table 2 - Frequency of mutated BCR–ABL in CML and Ph+ ALL in all resistant patients vs cases with initial response only.

Full table

Taken together, BCR–ABL mutations seem to occur frequently in relapsing CML and Ph+ ALL after an initial response to imatinib, whereas in primary resistance, other mechanisms may predominate.

Important lessons learned from crystal structure and computer modeling

Three years ago, Schindler et al³⁷ examined the structural mechanism for inhibition of the Abl kinase by imatinib. A crystal structure analysis of the murine Abl kinase domain in complex with a variant of imatinib³⁷ and two recent publications reporting the crystal structure of imatinib in complex with the kinase domain of human Abl^52,53 clarified that this drug achieves high specificity by recognizing the kinase domain of Abl when the activation loop (residues 381–402) adopts a closed, inactive conformation. In this conformation, the binding site of the substrate tyrosine is occupied by the region surrounding Y393, the major autophosphorylation site of Abl, and Y393 is not phosphorylated (see Figure 3). Imatinib, exclusively binding to the closed, inactive conformation, might therefore be regarded as an inhibitor of Abl kinase activation.^52,53 Moreover, a number of residues within the ATP binding site were predicted to be crucial for specific binding due to direct interaction with the inhibitor. Sequence comparison of the inhibitor binding region of different protein kinases revealed two positions (T315 and A380) with bulkier side chains in imatinib-insensitive, compared to imatinib-sensitive, kinases.⁵⁴ Thus, a complex mechanism of interaction seems to be the key for the selectivity of this compound.

Figure 3.

Proposed mechanism for impaired binding of imatinib to H396P/R. The activation loop of c-Abl in the closed, inactive conformation and the catalytic site of the kinase are shown. Imatinib (green) can only bind to this closed conformation. In this condition, the activation loop (residues 381–402) is folded into the active site of the kinase, mimicking bound substrate. Y393, the major autophosphorylation site in Abl, is not phosphorylated, and is presented by a -sheet (blue) to point into the active site, forming a hydrogen bond with D363 and R367. This -sheet, a part of the activation loop, contains H396 (red). An exchange of this histidine to proline or arginine may lead to an extended conformation of the activation loop, thereby impairing the binding of imatinib. Generated with PDB 1IEP available from http://www.rcsb.org/pdb/.

Full figure and legend (226K)

Strikingly, the results of crystallographic and computational studies perfectly matched the results of mutational analysis performed from patients: some of the residues, which have been identified to be critical, were actually found to be mutated in resistant patients. And, moreover, it is possible now to explain the insensitivity of mutated BCR–ABL at the molecular level. It is evident that BCR–ABL point mutants detected in patients with imatinib resistance can be divided into different categories: some are located at residues that are in direct contact with imatinib (see Figure 2). Mutations affecting such positions (eg T315, F317, and F359, see Table 1, Figure 2) may lead to a steric clash with the drug. The remaining mutations affect regions that have to adopt a specific conformation in order to allow binding of imatinib. Mutations at positions G250, Q252, and E255 are thought to destabilize the nucleotide-binding loop (P loop) that forms a part of the hydrophobic binding pocket for the drug (see Table 1, Figure 2). Y253 stabilizes a region that forms a part of the binding pocket via a hydrogen bond with an asparagine at position 322 (see Table 1). Furthermore, Y272F in c-Abl, corresponding to Y253F in Bcr–Abl, activates transformation by c-Abl,⁵⁵ and was the first residue in the c-Abl catalytic domain demonstrated to induce resistance to imatinib.⁵⁶ There is evidence that Y272F in c-Abl and Y253F in Bcr–Abl confer a gain of function by interference with the binding of a putative cellular protein inhibitor, leading to an increase of catalytic activity in vivo, but not in vitro.^46,50,55 Positions V379, L387, and H396 are located within the activation loop. Their mutation is assumed to destabilize the autoinhibitory conformation of the activation loop, which is required for imatinib to bind (Table 1, Figure 2).

Role of 1 acid glycoprotein

At clinically relevant concentrations, imatinib is thought to be bound to plasma proteins, such as albumin and 1 acid glycoprotein (AGP).⁵⁷ When the amount of drug that is available within the cell is diminished because of extrinsic or host factors such as AGP plasma levels, selection of a clone that is partially resistant to imatinib because of mutation or amplification of BCR–ABL may be possible when the intracellular inhibitor concentration drops below values necessary for inhibition of that particular clone. Indeed, it has been proposed that increased levels of AGP might reduce the amount of unbound imatinib available for inhibition of Bcr–Abl (see Figure 1e).⁵⁸ Both, the tumor burden in a mouse model⁵⁸ and the CML disease stage in humans,⁵⁹ correlated with plasma AGP levels, and a priori elevated plasma AGP levels led to a less rapid response to imatinib in human CML.⁵⁹ However, initially elevated plasma AGP levels were reversible in the course of treatment and did not affect the efficacy of the drug.⁵⁹ In addition, changes of imatinib plasma levels, indicating an effect of increased AGP levels on the distribution of imatinib, have not been found in relapsing patients in a phase I clinical trial.¹⁹Moreover, AGP purified from both normal and CML patients plasma failed to bind imatinib and did not abrogate inhibition of Ph+cells by imatinib in vitro.⁶⁰ In summary, the ability of AGP to bind and inhibit imatinib is a matter of debate, and the proof for AGP being a relevant mechanism of resistance in CML is still to be furnished.

Imatinib resistance in murine models of Bcr–Abl-induced leukemia

The establishment of a murine CML bone marrow retroviral transduction and transplantation model made it possible to investigate resistance to imatinib in mice. The CML-like myeloproliferative disorder in mice responds to treatment with imatinib with respect to hematologic response and prolongation of survival.^61,62 Treatment with imatinib was in some cases associated with enhanced expression of BCR–ABL at the RNA and/or protein level, but there was no correlation with response.⁶¹ In one study, seven out of nine mice relapsed with ALL after they initially responded to imatinib.⁶² Secondary transplantation of resistant mice suggested that resistance to imatinib arose because of cell-intrinsic factors.⁶² Sequencing of the BCR–ABL kinase domain revealed no mutations.⁶² In vitro, imatinib still inhibited Bcr–Abl in resistant cells. Southern blot analysis of imatinib-treated mice showed a reduction in the number of viral integration sites,^61,62 indicating clonal selection. Altogether, murine CML-like disease responds well to imatinib treatment but is not curable. The selection of clones that express Bcr–Abl at high levels may lead to resistant disease in some cases. However, clonal evolution seems to be the main mechanism of resistance towards imatinib in mice when a retroviral transduction and transplantation model is used.

Strategies to overcome or prevent resistance

The clinical activity of imatinib confirms the importance of the elevated Bcr-Abl tyrosine-kinase activity as the driving force in CML and Ph+ ALL. Resistance to the drug develops within short periods of time in advanced phase CML and Ph+ ALL. Recent observations also show mutations in relapsed accelerated and even in chronic phase CML.^41,45,47,48 Moreover, mutated BCR–ABL has recently been tracked back to pre-treatment samples in chronic phase⁴⁵ and myeloid blast crisis CML,⁴⁷ indicating that imatinib selects for a pre-existing mutated clone that allows Bcr–Abl to be kinase-active in the presence of the drug. However, Bcr–Abl can still be considered as target for therapeutic intervention even in resistant disease. To prevent or overcome resistance, it will be crucial to use combinations of different inhibitors or molecular-based therapies together with conventional cytostatic agents. Principles learned from antibiotic resistance in infectious diseases may be applied to mutations conferring resistance to targeted therapy leading to a combination treatment as the standard of care.⁶³ In cases of mutations that confer partial resistance (Q252H, E355G, or H396P, see Table 1), increasing the imatinib dosage may result in a response even in the presence of the mutant clone.

Combination treatments with conventional antileukemic agents

In vitro data suggest additive and synergistic effects of imatinib in combination with antileukemic agents.^64,65,66,67 A promising observation is an increase of the synergistic activity with chemotherapeutic agents such as cytarabine or etoposide at higher levels of growth inhibition by imatinib.^65,67 Based on these findings, several clinical trials are investigating simultaneous or sequential regimens using imatinib together with established substances such as interferon and cytarabine (eg the German CML-study IV in chronic phase CML, CSTI571A-DE-01 in myeloid blast crisis CML, and GMALL-STI571-IFN in Ph+ ALL or lymphoid blast crisis CML). For a more comprehensive review of preclinical investigation of imatinib in conjunction with established and novel antileukemic agents, preliminary results of phase I/II combination studies and ongoing clinical trials, see LaRosée et al.⁶⁷

New molecular targeted strategies

Second generation inhibitors of Bcr–Abl may vary in potency and with respect to positions within the ATP-binding site critical for binding the drug. In addition, unlike imatinib, different compounds may bind Bcr–Abl regardless of the conformation of the activation loop. Thus, new inhibitors could maintain activity against mutated BCR–ABL. Moreover, combinations of different kinase inhibitors may prevent the selection of imatinib-resistant clones. PD173955 (Parke-Davis, now Pfizer global research & development, Ann Arbor, MI, USA), also a small molecule inhibitor of ATP- binding, has been found to be a more potent inhibitor of Bcr–Abl than imatinib.⁵³ Importantly and in contrast to imatinib, this substance demonstrated to bind Abl with the activation loop being in a conformation that resembles an activated kinase, and independent of its phosphorylation state.⁵³ Therefore, second-generation kinase inhibitors may be superior to imatinib with respect to the potency of Bcr–Abl inhibition. Apart from compounds that bind to the ATP-binding site, the transformation potential of Bcr–Abl may be specifically disrupted using compounds that bind to different domains in Bcr–Abl. Tyrphostins alter the binding of Bcr–Abl to substrates rather than blocking access to the ATP-binding site. Inhibition by tyrphostins lead to the degradation of Bcr–Abl and apoptosis of Bcr–Abl-positive cells.⁶⁸

Compounds acting on biologically relevant proteins other than Bcr–Abl are capable of specifically suppressing a Bcr–Abl-dependent phenotype. Inhibition of the molecular chaperone HSP90 with geldanamycin and its derivative 17-AAG induced Bcr–Abl degradation and apoptosis in cells expressing Bcr–Abl wild type,^69,70,71 and was active in cells expressing BCR–ABL mutants T315I and E255K.⁷² Farnesyl protein transferase inhibitors (FTI) were specifically designed to block oncogenic Ras signaling through disruption of Ras prenylation, thereby preventing its translocation to the membrane. Inhibition of other farnesylated targets, including centromere-associated proteins, may be important for the activity of this class of compounds.^73,74 In addition, the FTI SCH66336 was shown to inhibit the MDR-1 product Pgp,⁷⁵ which was overexpressed in the imatinib-resistant cell line LAMA84-r.³¹ SCH66336, apart from its activity in solid tumors, selectively inhibited Bcr–Abl expressing cell lines and primary human CML cells,⁷³ and was active in a murine model of Bcr–Abl-induced leukemia.^73,76 In addition, SCH66336 inhibited the proliferation of imatinib-resistant cell lines and the colony formation of mononuclear cells derived from imatinib-resistant patients with amplification of BCR–ABL.⁷⁷ The coadministration of both drugs, imatinib and SCH66336, resulted in a synergistic increase in apoptosis in imatinib-sensitive cells. Furthermore, only the combination induced apoptosis in cells resistant to imatinib because of BCR–ABL amplification.⁷⁷ In contrast, there was no synergistic increase of apoptotic cells when Ba/F3 cells expressing the T315I mutant were exposed to both substances.⁷⁷ The authors concluded that a combination of both substances may be active in patients in whom imatinib resistance results from BCR–ABL amplification. A distinct FTI, R115777, proved to be active in patients with chronic phase CML.⁷⁸

Targeting BCR–ABL messenger RNA using antisense oligodeoxynucleotides (ODN) inhibited leukemia blast cells in vitro,⁷⁹ was effective in mice,⁸⁰ and has been utilized for in vitro purging.⁸¹ However, problems of uptake, specificity, and toxicity limit the therapeutic use in patients. Small interfering RNAs (siRNA) may more specifically and efficiently interfere with the expression of BCR–ABL mRNA.⁸² Moreover, since the structure of the Bcr–Abl oligomerization domain has been resolved, it may be possible to design inhibitors that specifically disrupt homodimerization and thus activation of Bcr–Abl.⁸³

Finally, an elegant approach has been generated recently by Vigneri and Wang:⁸⁴ entrapment of Bcr–Abl in the nucleus was achieved by treatment of cells with a combination of imatinib, promoting the nuclear import of cells, and leptomycin B (LMB), blocking its nuclear export. Bcr–Abl, trapped in the nucleus, recovered its activity after removal of imatinib. In the nucleus, active Bcr–Abl selectively induced apoptosis of CML cells. Importantly, as long as Bcr–Abl is expressed, this approach would work even in cases where the leukemic clone does not depend on Bcr–Abl any more because of secondary alterations that bypass its dependence on the oncogene. However, LMB displayed an unfavorable toxic profile in a phase I clinical trial in solid tumors. Profound anorexia and malaise were identified as dose-limiting toxicity.⁸⁵ Thus, the use of LMB may be limited to ex vivo applications such as purging bone marrow of CML cells.⁸⁴

In conclusion, combinations of imatinib and second-generation kinase inhibitors with established or novel antileukemic agents are about to be translated into clinical trials, and are expected not only to further improve the efficacy of current treatment options but also to prevent resistance by early eradication of all Ph-+ cells.

Discontinuation of imatinib

When mutant BCR–ABL is monitored, it must be kept in mind that wild-type disease may outgrow the mutated clone after cessation of imatinib therapy, as it has been described for Y253H.⁴⁸ Moreover, stopping imatinib may revert a BCR–ABL-amplified phenotype, as demonstrated in a patient with blast crisis CML³⁶ and in vitro.^31,34 In the imatinib-resistant cell line LAMA84-r, a decrease of Bcr–Abl protein and BCR–ABL gene copies, coinciding with restoration of sensitivity to imatinib, was observed upon removal of imatinib.³⁴ In addition, resistant cell lines that overexpressed Bcr–Abl showed a transient loss of viability and reduction of proliferation after withdrawal of imatinib, possibly caused by a sudden increase of kinase-active Bcr–Abl.³⁴ Thus, patients with imatinib resistance may respond again after an interval without imatinib.

Conclusions

Imatinib, a specific inhibitor targeting the deregulated tyrosine kinase Bcr–Abl, is capable of inducing impressive response rates as single agent in chronic phase CML, and also demonstrated significant activity in advanced phase CML and Ph+ ALL. However, there are doubts whether imatinib given as single agent can eradicate the malignant clone, since resistant disease clones frequently emerge in Ph+ ALL and blast crisis CML, and were detected even in single cases of chronic phase CML. The drug itself selects for clones that express Bcr–Abl at high levels or harbor secondary genetic alterations such as gene mutations within the kinase domain of BCR–ABL that prevent the drug from binding its target and result in a kinase-active oncoprotein in the presence of its inhibitor. A number of mutations that occur at positions predicted to be important for binding imatinib and that confer resistance to imatinib in vitro have now been identified. Good news, that mechanisms underlying resistance have been elucidated within a short period of time and that active Bcr–Abl in imatinib-resistant Ph+ leukemia is still the target of choice in a substantial proportion of cases. Future efforts should therefore focus on preventing resistant clones to emerge. Strategies that are currently tested in clinical trials include combination therapies of imatinib with synergistically acting conventional chemotherapeutic agents. Since imatinib selects for kinase domain mutations that specifically in- capacitate imatinib but may not at the same time abrogate inhibition by alternate compounds, new small molecule inhibitors will be used to prevent and to overcome resistance. At the end, combination strategies will be used first line in order to completely eliminate leukemic cells as fast as possible, including putative pre-existing cells that contain mutated BCR–ABL.

References

1. Nowell PC, Hungerford DA. A minute chromosome in human granulocytic leukaemia. Science 1960; 132: 1497–1501. | ISI |
2. Rowley JD. A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine flourescence and Giemsa staining. Nature 1973; 243: 290–293. | Article | PubMed | ISI | ChemPort |
3. Faderl S, Talpaz M, Estrov Z, O'Brien S, Kurzrock R, Kantarjian HM. The biology of chronic myeloid leukaemia. N Engl J Med 1999; 341: 164–172. | Article | PubMed | ISI | ChemPort |
4. Sawyers CL. Chronic myeloid leukaemia. N Engl J Med 1999; 340: 1330–1340. | Article | PubMed | ISI | ChemPort |
5. Daley GQ, Van Etten RA, Baltimore D. Induction of chronic myelogenous leukaemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science 1990; 247: 824–830. | Article | PubMed | ISI | ChemPort |
6. Lugo TG, Pendergast AM, Muller AJ, Witte ON. Tyrosine kinase activity and transformation potency of bcr–abl oncogene products. Science 1990; 247: 1079–1082. | Article | PubMed | ISI | ChemPort |
7. Zimmermann J, Buchdunger E, Mett H, Meyer T, Lydon NB, Traxler P. (Phenylamino)pyrimidine (PAP) derivatives: a new class of potent and highly selective PDGF-receptor autophosphorylation inhibitors. Bioorg Med Chem Lett 1996; 6: 1221–1226. | Article | ISI | ChemPort |
8. Zimmermann J, Buchdunger E, Mett H, Meyer T, Lydon NB. Potent and selective inhibitors of the ABL-kinase: phenylaminopyrimidine (PAP) derivatives. Bioorg Med Chem Lett 1997; 7: 187–192. | Article | ISI | ChemPort |
9. Druker BJ, Lydon NB. Lessons learned from the development of an abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest 2000; 105: 3–7. | PubMed | ISI | ChemPort |
10. Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther 2000; 295: 139–145. | PubMed | ISI | ChemPort |
11. Heinrich MC, Griffith DJ, Druker BJ, Wait CL, Ott KA, Zigler AJ. Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor. Blood 2000; 96: 925–932. | PubMed | ISI | ChemPort |
12. Okuda K, Weisberg E, Gilliland DG, Griffin JD. ARG tyrosine kinase activity is inhibited by STI571. Blood 2001; 97: 2440–2448. | Article | PubMed | ISI | ChemPort |
13. Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr–Abl positive cells. Nat Med 1996; 2: 561–566. | Article | PubMed | ISI | ChemPort |
14. Beran M, Cao X, Estrov Z, Jeha S, Jin G, O'Brien S et al. Selective inhibition of cell proliferation and BCR–ABL phosphorylation in acute lymphoblastic leukemia cells expressing Mr 190,000 BCR–ABL protein by a tyrosine kinase inhibitor (CGP 57148). Clin Cancer Res 1998; 4: 1661–1672. | PubMed | ISI | ChemPort |
15. Carroll M, Ohno-Jones S, Tamura S, Buchdunger E, Zimmermann J, Lydon NB et al. CGP 57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCR–ABL, TEL–ABL, and TEL–PDGFR fusion proteins. Blood 1997; 90: 4947–4952. | PubMed | ISI | ChemPort |
16. Heinrich MC, Wait CL, Yee KW, Griffith DJ. STI571 inhibits the kinase activity of wild type and juxtamembrane c-Kit mutants but not the exon 17 D816V mutation associated with mastocytosis. Blood 2000; 96: 173b (Abstr.).
17. Buchdunger E, Zimmermann J, Mett H, Meyer T, Muller M, Druker BJ, Lydon NB. Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylamin pyrimidine derivative. Cancer Res 1996; 56: 100–104. | PubMed | ISI | ChemPort |
18. le Coutre P, Mologni L, Cleris L, Marchesi E, Buchdunger E, Giardini R et al. In vivo eradication of human BCR/ABL-positive leukemia cells with an ABL kinase inhibitor. J Natl Cancer Inst 1999; 91: 163–168. | Article | PubMed | ChemPort |
19. Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM et al. 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. | Article | PubMed | ISI | ChemPort |
20. Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Fernandes Reese S, Ford JM et al. 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. | Article | PubMed | ISI | ChemPort |
21. Peng B, Hayes M, Racine-Poon A, Druker BJ, Talpaz M, Sawyers CL et al. Clinical investigation of the PK/PD relationship for Glivec (STI571): a novel inhibitor of signal transduction. Proc Am Soc Clin Oncol 2001; 20: 280 (Abstr.).
22. Kantarjian HM, Sawyers CL, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-Passerini C et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002; 346: 645–652. | Article | PubMed | ISI | ChemPort |
23. Talpaz M, Silver RT, Druker BJ, Goldmann JM, Gambacorti-Passerini C, Guilhot F et al. 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 | PubMed | ISI | ChemPort |
24. Sawyers CL, Hochhaus A, Feldman E, Goldman JM, Miller CB, Ottmann OG et al. Imatinib induces hematologic and cytogenetic responses in patients with chronic myeloid leukemia in myeloid blast crisis: results of a Phase II study. Blood 2002; 99: 3530–3539. | Article | PubMed | ISI | ChemPort |
25. Ottmann OG, Druker BJ, Sawyers CL, Goldman JM, Reiffers J, Silver RT et al. A phase 2 study of imatinib in patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoid leukemias. Blood 2002; 100: 1965–1971. | Article | PubMed | ISI | ChemPort |
26. Canitrot Y, Lautier D, Laurent G, Frechet M, Ahmet A, Turhan AG et al. Mutator phenotype of BCR–ABL transfected cell lines and its association with enhanced expression of DNA polymerase beta. Oncogene 1999; 18: 2676–2680. | Article | PubMed | ISI | ChemPort |
27. Salloukh HF, Launeville P. Increase in mutant frequencies in mice expressing the BCR–ABL activated tyrosine kinase. Leukemia 2000; 14: 1401–1404. | Article | PubMed | ISI | ChemPort |
28. Ohyashiki K, Iwama H, Tauchi T, Shimamoto T, Hayashi S, Ando K et al. Telomere dynamics and genetic instability in disease progression of chronic myeloid leukemia. Leukemia Lymphoma 2000; 40: 49–56.
29. Blagosklonny MV. STI-571 must select for drug-resistant cells but 'no cell breathes fire out of its nostrils like a dragon. Leukemia 2002; 16: 570–572. | Article | PubMed | ISI | ChemPort |
30. le Coutre P, Tassi E, Varella-Garcia M, Barni R, Mologni L, Cabrita G et al. Induction of resistance to the Abelson inhibitor STI571 in human leukemia cells through gene amplification. Blood 2000; 95: 1758–1766. | PubMed | ISI | ChemPort |
31. Mahon FX, Deininger MW, Schultheis B, Chabrol J, Reiffers J, Goldman JM et al. Selection and characterization of BCR–ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. Blood 2000; 96: 1070–1079. | PubMed | ISI | ChemPort |
32. Weisberg E, Griffin JD. Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines. Blood 2000; 95: 3498–3505. | PubMed | ISI | ChemPort |
33. Kuo MT, Sen S, Hittelman WN, Hsu TC. Chromosomal fragile sites and DNA amplification in drug resistant cells. Biochem Pharmacol 1998; 56: 7–13.
34. Tipping AJ, Mahon FX, Lagarde V, Goldman JM, Melo JV. Restoration of sensitivity to STI571-resistant chronic myeloid leukemia cells. Blood 2001; 98: 3864–3867. | Article | PubMed | ChemPort |
35. Ricci C, Scappini B, Divoky V, Gatto S, Onida F, Verstovsek S et al. Mutation in the ATP-binding pocket of the ABL kinase domain in an STI571-resistant BCR/ABL-positive cell line. Cancer Res 2002; 62: 5995–5998. | PubMed | ChemPort |
36. 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: 876–880. | Article | PubMed | ISI | ChemPort |
37. Schindler T, Bornmann W, Pellicena P, Miller WT, Clarkson B, Kuriyan J. Structural mechanism for STI-571 inhibition of Abelson tyrosine kinase. Science 2000; 289:1938–1942. | Article | PubMed | ISI | ChemPort |
38. Barthe C, Cony-Makhoul P, Melo JV, Reiffers J, Mahon FX. Roots of clinical resistance to STI-571 cancer therapy. Science 2001; 293: 2163a. | Article |
39. Hochhaus A, Kreil S, Corbin A, La-Rosée P, Lahaye T, Berger U et al. Roots of clinical resistance to STI-571 cancer therapy. Science 2001; 293: 2163a. | Article |
40. Gorre M, Shah N, Ellwood K, Nicoll J, Sawyers CL. Roots of clinical resistance to STI-571 cancer therapy. Science 2001; 293: 2163a. | Article |
41. Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K et al. High frequency of point mutations clustered within the adenosine 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. | Article | PubMed | ISI | ChemPort |
42. von Bubnoff N, Schneller F, Peschel C, Duyster J. BCR–ABL gene mutations in relation to clinical resistance of Philadelphia-chromosome-positive leukaemia to STI571: a prospective study. Lancet 2002; 359: 487–491. | Article | PubMed | ISI | ChemPort |
43. von Bubnoff N, Schneller F, Peschel C, Duyster J. Different Bcr–Abl gene mutations can cause clinical resistance of Philadelphia-positive leukemia towards STI571. Blood 2001; 98: 770a (Abstr.).
44. Hofmann WK, Jones LC, Lemp NA, de Vos S, Gschaidmeier H, Hoelzer D et al. Ph(+) acute lymphoblastic leukemia resistant to the tyrosine kinase inhibitor STI571 has a unique BCR–ABL gene mutation. Blood 2002; 99: 1860–1862. | Article | PubMed | ISI |
45. Roche-Lestienne C, Soenen-Cornu V, Grardel-Duflos N, Lai JL, Philippe N, Facon T et al. Several types of mutations of the Abl gene can be found in chronic myeloid leukemia patients resistant to STI571, and they can pre-exist to the onset of treatment. Blood 2002; 100: 1014–1018. | Article | PubMed | ISI | ChemPort |
46. Roumiantsev S, Shah NP, Gorre ME, Nicoll J, Brasher BB, Sawyers CL et al. Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop. Proc Natl Acad Sci USA 2002; 99: 10700–10705.  | Article | PubMed | ChemPort |
47. Shah NP, Nicoll JM, Nagar B, Gorre ME, Paquette RL, Kuriyan J et al. Multiple BCR–ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2002; 2: 117–125. | Article | PubMed | ISI | ChemPort |
48. Hochhaus A, Kreil S, Corbin AS, La Rosée P, Müller MC, Lahaye T et al. Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. Leukemia 2002; 16: 2190–2196. | Article | PubMed | ISI | ChemPort |
49. Druker BJ, Mauro MJ. STI571: targeting BCR–ABL as therapy for CML. Oncologist 2001; 6: 233–238. | Article | PubMed | ISI | ChemPort |
50. Corbin AS, Buchdunger E, Furet P, Druker BJ. Analysis of the structural basis of specifity of inhibition of the Abl kinase by STI571. J Biol Chem 2002; 277: 32214–32219, Manuscript M111525200, published online June 20. | Article | PubMed | ISI | ChemPort |
51. von Bubnoff N, Gschaidmeier H, Schneller F, Peschel C, Duyster J. Differential sensitivity of BCR–ABL gene mutations responsible for clinical resistance towards glivec (STI571) and alternate kinase inhibitors. Onkologie 2002; 25: 6 (Abstr.).
52. Manley PW, Cowan-Jacob SW, Fabbro D, Fendrich G, Furet P, Guez V et al. Molecular interactions between Glivec and isoforms of the c-Abl kinase. Proc Am Assoc Cancer Res 2002; 4196 (Abstr.).
53. Nagar B, Bornmann WG, Pellicena P, Schindler T, Veach DR, Miller WT et al. Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and Imatinib (STI-571). Cancer Res 2002; 62: 4236–4243. | PubMed | ISI | ChemPort |
54. Warmuth M, Simon N, Mathes R, Mitina O, Forster K, Moarefi I et al. Point mutations at molecular gatekeeper positions of ABL confer STI571- but not PP1-resistance to Bcr–Abl-expressing cell lines. Blood 2001; 98: 770a.
55. Allen PB, Wiedemann M. An activating mutation in the ATP binding site of the ABL kinase domain. J Biol Chem 1996; 271: 19585–19591. | Article | PubMed | ISI | ChemPort |
56. Roumiantsev S, Brasher BB, Van Etten RA. A point mutation in the ABL catalytic domain induces resistance to the tyrosine kinase inhibitor STI571. Blood 2000; 16: 470a.
57. Capdeville R, Buchdunger E, Zimmermann J, Matter A. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat rev 2002; 1: 493–502. | ChemPort |
58. Gambacorti-Passerini C, Barni R, le Coutre P, Zucchetti M, Cabrita G, Cleris L et al. Role of alpha1 acid glycoprotein in the in vivo resistance of human BCR–ABL (+) leukemia cells to the abl inhibitor STI571. J Natl Cancer Inst 2000; 92: 1641–1650. | Article | PubMed | ChemPort |
59. le Coutre P, Kreuzer KA, Na I-K, Lupberger J, Holdhoff M, Appelt C et al. Determination of -1 acid glycoprotein in patients with Ph+ chronic myeloid leukemia during the first 13 weeks of therapy with STI571. Blood Cells Mol Dis 2002; 28: 75–85. | Article | PubMed |
60. Jorgensen HG, Elliott MA, Allan EK, Carr CE, Holyoake TL, Smith KD. -1 acid glycoprotein expressed in the plasma of chronic myeloid leukemia patients does not mediate significant in vitro resistance to STI571. Blood 2002; 99: 713–715. | Article | PubMed | ISI | ChemPort |
61. Wolff NC, Ilaria RL. Establishment of a murine model for therapy-treated chronic myelogenous leukemia using the tyrosine kinase inhibitor STI571. Blood 2001; 98: 2808–2816. | Article | PubMed | ISI | ChemPort |
62. Miething C, Grundler R, Hoepfl J, Mugler C, Gschaidmeier H. Mice with a CML-like disease relapse with ALL under STI571 treatment. Exp Hematol 2002; 30: 96a.
63. Luzzatto L, Melo JV. Acquired resistance to imatinib mesylate: selection for pre-existing mutant cells. Blood 2002; 100: 1105.
64. Thiesing JT, Ohno-Jones S, Kolibaba KS, Druker BJ. Efficacy of STI571, an Abl tyrosine kinase inhibitor, in conjunction with other antileukemic agents against Bcr–Abl-positive cells. Blood 2000; 96: 3195–3199. | PubMed | ISI | ChemPort |
65. Topaly J, Zeller WJ, Fruehauf S. Synergistic activity of the new Abl-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR–ABL-positive chronic myelogenous leukemia cells. Leukemia 2001; 15: 342–347. | Article | PubMed | ISI | ChemPort |
66. Kano Y, Akutsu M, Tsunoda S, Mano H, Sato Y, Honma Y et al. In vitro cytotoxic effects of a tyrosine kinase inhibitor STI571 in combination with commonly used antileukemic agents. Blood 2001; 97: 1999–2007. | Article | PubMed | ISI | ChemPort |
67. La Rosée P, O'Dwyer ME, Druker BJ. Insights from pre-clinical studies for new combination treatment regimens with the Bcr–Abl kinase inhibitor imatinib mesylate (Gleevec/Glivec) in chronic myelogenous leukemia: a translational perspective. Leukemia 2002; 16: 1213–1219. | Article | PubMed | ISI | ChemPort |
68. Mow BM, Chandra J, Svingen PA, Hallgren CG, Weisberg E, Kottke TJ et al. Effects of the Bcr/abl kinase inhibitors STI571 and adaphostin (NSC 680410) on chronic myelogenous leukemia cells in vitro. Blood 2002; 99: 664–671. | Article | PubMed | ISI | ChemPort |
69. An WG, Schulte TW, Neckers LM. The heat shock protein 90 antagonist geldanamycin alters chaperone association with p210bcr–abl and v-src proteins before their degradation by the proteasome. Cell Growth Differ 2000; 11: 355–360. | PubMed | ISI | ChemPort |
70. Blagosklonny MV, Fojo T, Bhalla KN, Kim JS, Trepel JB, Figg WD et al. The Hsp90 inhibitor geldanamycin selectively sensitizes Bcr–Abl- expressing leukemia cells to cytotoxic chemotherapy. Leukemia 2001; 15: 1537–1543. | Article | PubMed | ISI | ChemPort |
71. Nimmanapalli R, O'Bryan E, Bhalla K. Geldanamycin and its analogue 17-allylamino-17-demethoxygeldanamycin lowers Bcr–Abl levels and induces apoptosis and differentiation of Bcr- Abl-positive human leukemic blasts. Cancer Res 2001; 61: 1799–1804. | PubMed | ISI | ChemPort |
72. Gorre ME, Ellwood-Yen K, Chiosis G, Rosen N, Sawyers CL. BCR–ABL point mutants isolated from patients with imatinib mesylate-resistant chronic myeloid leukemia remain sensitive to inhibitors of the BCR–ABL chaperone heat shock protein 90. Blood 2002; 100: 3041–3044. | Article | PubMed | ISI | ChemPort |
73. Peters DG, Hoover RR, Gerlach MJ, Koh EY, Zhang H, Choe K et al. Activity of the farnesyl protein transferase inhibitor SCH66336 against BCR/ABL-induced murine leukemia and primary cells from patients with chronic myeloid leukemia. Blood 2001; 97: 1404–1412. | Article | PubMed | ChemPort |
74. Ashar HR, James L, Gray K, Carr D, Black S, Armstrong L et al. Farnesyl transferase inhibitors block the farnesylation of CENP-E and CENP-F and alter the association of CENP-E with the microtubules. J Biol Chem 2000; 275: 30451–30457. | Article | PubMed | ISI | ChemPort |
75. Wang E, Casciano CN, Clement RP, Johnson WW. The farnesyl protein transferase inhibitor SCH66336 is a potent inhibitor of MDR1 product P-glycoprotein. Cancer Res 2001; 61: 7325–7329. | PubMed | ISI | ChemPort |
76. Reichert A, Heisterkamp N, Daley GQ, Groffen J. Treatment of Bcr/Abl-positive acute lymphoblastic leukemia in P190 transgenic mice with the farnesyl transferase inhibitor SCH66336. Blood 2001; 97: 1399–1403. | Article | PubMed | ISI | ChemPort |
77. Hoover RR, Mahon FX, Melo JV, Daley GQ. Overcoming STI571 resistance with the farnesyl transferase inhibitor SCH66336. Blood 2002; 100: 1068–1071. | Article | PubMed | ISI | ChemPort |
78. Thomas D, Cortes J, ÓBrien SM, Manero GG, Kurzrock R, Giles FJ et al. R115777, a farnesyl transferase inhibitor (FTI), has significant anti-leukemia activity in patients with chronic myeloid leukemia (CML). Blood 2001; 98: 727a.
79. Szczylik C, Skorski T, Nicolaides NC, Manzella L, Malaguarnera L, Venturelli D et al. Selective inhibition of leukemia cell proliferation by BCR–ABL antisense oligodeoxynucleotides. Science 1991; 253: 562–565. | Article | PubMed | ChemPort |
80. Skorski T, Nieborowska-Skorska M, Nicolaides NC, Szczylik C, Iversen P, Iozzo RV et al. Suppression of Philadelphia¹ Leukemia cell growth in mice by BCR–ABL antisense oligodeoxynucleotide. Proc Natl Acad Sci USA 1994; 91: 4504–4508. | Article | PubMed | ChemPort |
81. De Fabritiis P, Petti MC, Montefusco E, De Propris MS, Sala R, Bellucci R et al. BCR–ABL antisense oligodeoxynucleotide in vitro purging and autologous bone marrow transplantation for patients with chronic myelogenous leukemia in advanced phase. Blood 1998; 91: 3156–3162. | PubMed | ISI | ChemPort |
82. Scherr M, Battmer K, Winkler T, Heidenreich O, Ganser A, Eder M. Specific inhibition of bcr–abl gene expression by small interfering RNA. Blood 2003; 101: 1566–1569, DOI 10.1182/blood-2002-06-1685, prepublished online september 26, 2002. | Article | PubMed | ISI | ChemPort |
83. Zhao X, Ghaffari S, Lodish H, Malashkevich VN, Kim PS. Structure of the Bcr–Abl oncoprotein oligomerization domain. Nat Struct Biol 2002; 9:117–120. | Article | PubMed | ISI | ChemPort |
84. Vigneri P, Wang JY. Induction of apoptosis in chronic myelogenous leukemia cells through nuclear entrapment of BCR–ABL tyrosine kinase. Nat Med 2001; 7: 228–234. | Article | PubMed | ISI | ChemPort |
85. Newlands ES, Rustin GJ, Brampton MH. Phase I trial of elactocin. Br J Cancer 1996; 74: 648–649. | PubMed | ISI | ChemPort |