Selective cytotoxic mechanism of GTP-14564, a novel tyrosine kinase inhibitor in leukemia cells expressing a constitutively active Fms-like tyrosine kinase 3 (FLT3).
The receptor tyrosine kinase FLT3 is constitutively activated by an internal tandem duplication (ITD) mutation within the juxtamembrane domain in 20-30% of patients with acute myeloid leukemia. In this study, we identified GTP-14564 as a specific kinase inhibitor for ITD-FLT3 and investigated the molecular basis of its specificity. GTP-14564 inhibited the growth of interleukin-3-independent Ba/F3 expressing ITD-FLT3 at 1 microM, whereas a 30-fold higher concentration of GTP-14564 was required to inhibit FLT3 ligand-dependent growth of Ba/F3 expressing wild type FLT3 (wt-FLT3). However, this inhibitor suppressed the kinase activities of wt-FLT3 and ITD-FLT3 equally, suggesting that the signaling pathways for proliferation differ between wt-FLT3 and ITD-FLT3. Analysis of downstream targets of FLT3 using GTP-14564 revealed STAT5 activation to be essential for growth signaling of ITD-FLT3. In contrast, wt-FLT3 appeared to mainly use the MAPK pathway rather than the STAT5 pathway to transmit a proliferative signal. Further analysis demonstrated that the first two tyrosines in an ITD were critical for STAT5 activation and growth induction but that all of the tyrosines in the juxtamembrane region were dispensable in terms of the proliferation signals of wt-FLT3. These results indicate that an ITD mutation in FLT3 elicits an aberrant STAT5 activation that results in increased sensitivity to GTP-14564. Thus, FLT3-targeted inhibition is an attractive approach, with the potential for selective cytotoxicity, to the treatment of ITD-FLT3-positive acute myeloid leukemia.
Murata
Ken
K
Division of Hematopoietic Factors, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Kumagai
Hidetoshi
H
Kawashima
Toshiyuki
T
Tamitsu
Kaori
K
Irie
Mariko
M
Nakajima
Hideaki
H
Suzu
Shinya
S
Shibuya
Masabumi
M
Kamihira
Shimeru
S
Nosaka
Tetsuya
T
Asano
Shigetaka
S
Kitamura
Toshio
T
D000818:Animals; D001572:Benzofurans / Q000494:pharmacology*; D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005799:Genes, Dominant; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D020128:Inhibitory Concentration 50; D007377:Interleukin-3 / Q000378:metabolism; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D008894:Milk Proteins; D008954:Models, Biological; D008956:Models, Chemical; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000737:chemistry; D051941:fms-Like Tyrosine Kinase 3
D001572:Benzofurans; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C477524:GTP 14564; D007377:Interleukin-3; D008164:Luminescent Proteins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D011720:Pyrazoles; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D014443:Tyrosine; C497970:FLT3 protein, human; C497971:Flt3 protein, mouse; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D051941:fms-Like Tyrosine Kinase 3