A cell-based screen for resistance of Bcr-Abl-positive leukemia identifies the mutation pattern for PD166326, an alternative Abl kinase inhibitor.
In Philadelphia-positive (Ph(+)) leukemia, point mutations within the Bcr-Abl kinase domain emerged as a major mechanism of resistance to imatinib mesylate. We established a cell-based screening strategy for detection of clinically relevant point mutations using Bcr-Abl-transformed Ba/F3 cells. We identified 32 different single-point mutations within the kinase domain of Bcr-Abl. The pattern and frequency of mutations in this cell culture-based screen resembled the pattern and frequency observed in resistant patients. We then applied this screen to an alternative Abl kinase inhibitor. Using PD166326, the frequency of resistant colonies emerging at 5 to 10 times the median growth inhibition (IC50) of PD166326 was significantly lower than with imatinib. In addition, PD166326 produced a distinct pattern of Bcr-Abl mutations. The majority of mutations that came up with both imatinib and PD166326 could effectively be suppressed by increasing the dose of PD166326 to 50 to 500 nM. In contrast, only a few mutations could be suppressed by increasing the imatinib dose to 5 to 10 microM. However, 3 mutations affecting F317 displayed complete resistance to PD166326, but could be effectively inhibited by standard concentrations of imatinib. Thus, this robust and simple screening system provides a rational basis for combinatorial and sequential treatment strategies in targeted cancer therapy.
Department of Internal Medicine III, Technical University of Munich, D-81675 Munich, Germany.
Nikolas
N
von Bubnoff
Darren R
DR
Veach
Heiko
H
van der Kuip
Walter E
WE
Aulitzky
Jana
J
Sänger
Petra
P
Seipel
William G
WG
Bornmann
Christian
C
Peschel
Bayard
B
Clarkson
Justus
J
Duyster
D000818:Animals; D001549:Benzamides; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D016313:Genes, abl / Q000187:drug effects*; D006801:Humans; D000068877:Imatinib Mesylate; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics; D051379:Mice; D010879:Piperazines / Q000494:pharmacology; D017354:Point Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D014162:Transfection
D001549:Benzamides; C473352:PD 166326; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl