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Originally published as JCO Early Release 10.1200/JCO.2008.19.8853 on December 15 2008 © 2009 American Society of Clinical Oncology.
Activity of Bosutinib, Dasatinib, and Nilotinib Against 18 Imatinib-Resistant BCR/ABL Mutants
Department of Clinical Medicine and Prevention, University of Milano-Bicocca, S Gerardo Hospital, Monza, Milan, Italy
Department of Oncology, Wyeth Pharmaceuticals, Pearl River, NY To the Editor: The treatment of chronic myeloid leukemia (CML) has been radically modified by the discovery of imatinib, a selective inhibitor of the chimeric protein BCR/ABL that is the cause of the disease.1-3 Patients with Philadelphia-positive (Ph+) acute lymphoblastic leukemia also benefited from imatinib availability, although to a lesser extent. Imatinib is able to bind the inactive conformation of BCR/ABL, preventing adenosine triphosphate from entering its binding pocket.4 A variable portion of patients experience resistance to imatinib therapy, depending on the phase and type of disease.5 Resistance can arise from different mechanisms, such as BCR/ABL amplification6 and low imatinib bioavailability,7 but in the vast majority of patients the decreased efficacy of imatinib therapy is due to point mutations into the protein sequence.8 Mutation sites can be schematically clustered in four regions: the phosphate-binding loop (P loop), the imatinib binding site, the catalytic domain, and the activation loop. Mutations can affect the drug-protein interaction directly as well as indirectly if their presence shifts the thermodynamic equilibrium from the inactive toward the active conformation of the enzyme.8 At present, more than 50 mutation sites and more than 70 individual mutations conferring different levels of resistance have been found in CML patients.8 Recently, several new inhibitors have been developed to obtain an increased potency and a broad range of activity against known imatinib-resistant mutants. Nilotinib is an imatinib derivative approximately 30-fold more potent than imatinib.9 Dasatinib is a dual-specific SRC and ABL inhibitor, structurally unrelated to imatinib, that is able to bind and inhibit both the active and inactive conformations of ABL, resulting in 100- to 300-fold higher activity than imatinib.10,11 Bosutinib is a dual SRC/ABL inhibitor that is active in the low nanomolar range against BCR/ABL.12,13 The lack of activity against KIT and platelet-derived growth factor receptor (PDGFR)13 (two common off-targets of other tyrosine kinase inhibitors [TKIs] such as imatinib, nilotinib, and dasatinib), the in vitro synergism with imatinib,14 and the benign toxicity profile of this drug15-17 render it an attractive candidate for single or combination treatments for Ph+ leukemias. Bosutinib is now in phase III clinical trials, and phase II studies have shown good activity in patients resistant to imatinib or other TKIs.15-17 Despite this increasing interest, limited data are available on the activity of bosutinib against known imatinib-resistant BCR/ABL mutations.12,13 Although second-generation TKIs are more potent than imatinib, their activity against leukemic stem cells seems limited.18 It is known that resistance to second-generation drugs can arise, and the analysis of mutation profiles reveals substantial differences among different TKIs.19,20 The availability of at least three TKIs to treat a patient resistant to imatinib can basically follow two different approaches. The choice of a certain TKI can be made on an empirical basis; for example, because a certain patient has not been previously exposed to that particular TKI. This strategy is widely used but is not the most rational one because of the different profiles of each TKI. Therefore, the possibility to compare directly the different activities of TKIs against a given mutation would be of remarkable importance in clinical practice. Such a tool could be used, in a manner similar to that of an antibiogram for bacterial diseases, guiding the choice of the most appropriate inhibitor for each patient. In our study, we investigated the activity of bosutinib, dasatinib, imatinib, and nilotinib against a panel of 18 mutated forms of BCR/ABL associated with imatinib resistance in CML and Ph+ acute lymphoblastic leukemia patients. The bosutinib was provided by Wyeth Pharmaceuticals (Pearl River, NY); the other three TKIs were synthesized by Enrico Rosso, MD, University of Venice, Italy. Inhibitors were tested simultaneously in the same laboratory and using the same reagents. The residues involved in the mutations were chosen to cover all the different domains of the protein and to include the eight most common forms of BCR/ABL mutations found in patients; in fact, the panel of mutants used here would cover more than 85% of patients with mutations.8 The Ba/F3 murine cell line was stably transfected with pcDNA3 vector containing either the wild-type or selected mutated forms of BCR/ABL. Mutants obtained by transfection were preferred over those obtained by infection because the BCR/ABL expression levels of the former ones are more comparable to those observed in fresh leukemic cells (data not shown), whereas clones obtained by infection usually present much higher expression levels.
The antiproliferative activity for bosutinib and the other three TKIs was assessed by tritiated thymidine incorporation assay, and the relative concentration that inhibits 50% (IC50) increase over wild-type BCR/ABL was calculated. We classified the relative resistance (RR) values in four categories: sensitive (RR
To our knowledge, we present here the first comprehensive characterization of bosutinib, tested against 18 imatinib-resistant BCR/ABL mutants and compared with dasatinib and nilotinib. The data presented here will need to be verified using correlates obtained from clinical studies. However, some positive findings can already be observed. For example, bosutinib clinical activity was already noted in patients presenting the F359V or P-loop mutations,15-17 which are notoriously insensitive to nilotinib, or F317L, which is known to be poorly sensitive to dasatinib.23 The different activity profiles of the TKIs tested and the presence of multiple mutants in the same patient suggest that frequent screening for mutations should be performed, and that a combined treatment with simultaneous/sequential administration of different TKIs could be tested in future clinical studies. Our study points out the differences in the activity spectra of the four TKIs against the 18 BCR/ABL mutations considered. The activity pattern presented in this study will help physicians develop rational, patient-tailored therapy. These results offer physicians a tool to use new TKIs in the most efficient way for their patients. AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a "U" are those for which no compensation was received; those relationships marked with a "C" were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment or Leadership Position: Frank Boschelli, Wyeth Research (C) Consultant or Advisory Role: None Stock Ownership: Frank Boschelli, Wyeth Research Honoraria: None Research Funding: Carlo Gambacorti-Passerini, Wyeth Research Expert Testimony: None Other Remuneration: None
NOTES published online ahead of print at www.jco.org on December 15, 2008 REFERENCES
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Copyright © 2009 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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2), moderately resistant (RR 2.01 to 4), resistant (RR 4.01 to 10), or highly resistant (RR > 10). The results are listed in 
