Association between imatinib-resistant BCR-ABL mutation-negative leukemia and persistent activation of LYN kinase.
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.
Wu
Ji
J
Departments of Experimental Therapeutics, The M. D. Anderson Cancer Center, Houston, TX, USA.
Meng
Feng
F
Kong
Ling-Yuan
LY
Peng
Zhenghong
Z
Ying
Yunming
Y
Bornmann
William G
WG
Darnay
Bryant G
BG
Lamothe
Betty
B
Sun
Hanshi
H
Talpaz
Moshe
M
Donato
Nicholas J
NJ
D000328:Adult; D000368:Aged; D000818:Animals; D000970:Antineoplastic Agents / Q000009:adverse effects / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D001549:Benzamides; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D016044:Fusion Proteins, bcr-abl; D006801:Humans; D000068877:Imatinib Mesylate; D015151:Immunoblotting; D047468:Immunoprecipitation; D054438:Leukemia, Myeloid, Chronic, Atypical, BCR-ABL Negative / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008819:Mice, Nude; D008875:Middle Aged; D010766:Phosphorylation; D010879:Piperazines / Q000009:adverse effects / Q000627:therapeutic use*; D047428:Protein Kinase Inhibitors / Q000009:adverse effects / Q000627:therapeutic use*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011743:Pyrimidines / Q000009:adverse effects / Q000627:therapeutic use*; D019061:src-Family Kinases / Q000378:metabolism*
D000970:Antineoplastic Agents; D001549:Benzamides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D020169:Caspases