Journal of the National Cancer Institute Advance Access originally published online on June 24, 2008
JNCI Journal of the National Cancer Institute 2008 100(13):926-939; doi:10.1093/jnci/djn188
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© The Author 2008. Published by Oxford University Press.
ARTICLES |
Association Between Imatinib-Resistant BCR-ABL Mutation-Negative Leukemia and Persistent Activation of LYN Kinase
Affiliations of authors: Departments of Experimental Therapeutics (JW, FM, LYK, BGD, BL, NJD) and Experimental Diagnostic Imaging (ZP, YY, WGB), The M. D. Anderson Cancer Center, Houston, TX; Division of Hematology/Oncology, Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI (HS, MT)
Correspondence to: Nicholas J. Donato, PhD, Division of Hematology/Oncology, Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, 1500 E. Medical Center Dr, Rm CCGC 4306, Ann Arbor, MI 48109 (e-mail: ndonato{at}med.umich.edu).
Background: Imatinib is a tyrosine kinase inhibitor that is used to treat chronic myelogenous leukemia (CML). BCR-ABL mutations are associated with failure of imatinib treatment in many CML patients. LYN kinase regulates survival and responsiveness of CML cells to inhibition of BCR-ABL kinase, and differences in LYN regulation have been found between imatinib-sensitive and -resistant CML cell lines.
Methods: We evaluated cells from 12 imatinib-resistant CML patients with mutation-negative BCR-ABL and from six imatinib-sensitive patients who discontinued therapy because of imatinib intolerance. Phosphorylation of BCR-ABL and LYN was assessed in patient cells and cell lines by immunoblotting with activation state–specific antibodies, co-immunoprecipitation studies, and mass spectroscopy analysis of phosphopeptides. Cell viability, caspase activation, and apoptosis were also measured. Mutations were analyzed by sequencing. The effect of silencing LYN with short interfering RNAs (siRNAs) or reducing activation by treatment with tyrosine kinase inhibitors was evaluated in cell lines and patient cells.
Results: Imatinib treatment suppressed LYN phosphorylation in cells from imatinib-sensitive CML patients and imatinib-sensitive cell lines. Imatinib treatment blocked BCR-ABL signaling but did not suppress LYN phosphorylation in cells from imatinib-resistant patients, and persistent activation of LYN kinase was not associated with mutations in LYN kinase or its carboxyl-terminal regulatory domains. Unique LYN phosphorylation sites (tyrosine-193 and tyrosine-459) and associated proteins (c-Cbl and p80) were identified in cells from imatinib-resistant patients. Reducing LYN expression (siRNA) or activation (dasatinib) was associated with loss of cell survival and cytogenetic or complete hematologic responses in imatinib-resistant disease.
Conclusions: LYN activation was independent of BCR-ABL in cells from imatinib-resistant patients. Thus, LYN kinase may be involved in imatinib resistance in CML patients with mutation-negative BCR-ABL and its direct inhibition is consistent with clinical responses in these patients.
| CONTEXT AND CAVEATS Prior knowledge The tyrosine kinase inhibitor imatinib is used to treat chronic myelogenous leukemia (CML). Failure of imatinib treatment in many but not all CML patients is associated with BCR-ABL mutations. LYN kinase regulates survival and responsiveness of CML cells to inhibition of BCR-ABL kinase, and differences in LYN regulation have been found between imatinib-sensitive and -resistant CML cell lines. Study design In vitro study of imatinib-sensitive and -resistant CML cell lines and of cells isolated from imatinib-sensitive CML patients and from imatinib-resistant patients without BCR-ABL mutations. Contribution Imatinib treatment suppressed LYN phosphorylation in cells from imatinib-sensitive CML patients and cell lines but not in cells from imatinib-resistant patients who were BCR-ABL mutation negative. Unique LYN phosphorylation sites and associated proteins were identified in cells from imatinib-resistant patients. Reducing LYN expression with short interfering RNAs or activation with tyrosine kinase inhibitors was associated with loss of cell survival and cytogenetic or complete hematologic responses in imatinib-resistant disease. Implication LYN kinase appears to be involved in imatinib-resistant CML. Limitations Sample availability and access to patient material were limited, and repetitive analyses were not usually possible. Although the concentration of tyrosine kinase inhibitors used was in line with pharmacologically achievable levels, the cellular concentration of each inhibitor may vary widely between patients and may only partially reflect the concentrations used. Therefore, the effects described for kinase inhibitor activities may only partially reflect their clinical activity.
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Manuscript received May 30, 2007; revised April 24, 2008; accepted May 8, 2008.
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J Natl Cancer Inst 2008 100: 907.
J Natl Cancer Inst 2008 100: 907.
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