Selective cytotoxic mechanism of GTP-14564, a novel tyrosine kinase inhibitor in leukemia cells expressing a constitutively active Fms-like tyrosine kinase 3 (FLT3).

Abstract	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.
Authors	Ken Murata, Hidetoshi Kumagai, Toshiyuki Kawashima, Kaori Tamitsu, Mariko Irie, Hideaki Nakajima, Shinya Suzu, Masabumi Shibuya, Shimeru Kamihira, Tetsuya Nosaka, Shigetaka Asano, Toshio Kitamura
Journal	The Journal of biological chemistry (J Biol Chem) Vol. 278 Issue 35 Pg. 32892-8 (Aug 29 2003) ISSN: 0021-9258 [Print] United States
PMID	12815052 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References	Benzofurans DNA, Complementary DNA-Binding Proteins Enzyme Inhibitors GTP 14564 Interleukin-3 Luminescent Proteins Milk Proteins Proto-Oncogene Proteins Pyrazoles Recombinant Proteins STAT5 Transcription Factor Trans-Activators Green Fluorescent Proteins Tyrosine FLT3 protein, human Flt3 protein, mouse Protein-Tyrosine Kinases Receptor Protein-Tyrosine Kinases fms-Like Tyrosine Kinase 3
Topics	Animals Benzofurans (pharmacology) Blotting, Western Cell Division Cell Line DNA, Complementary (metabolism) DNA-Binding Proteins (metabolism) Dose-Response Relationship, Drug Electrophoresis, Polyacrylamide Gel Enzyme Inhibitors (pharmacology) Genes, Dominant Genetic Vectors Green Fluorescent Proteins Humans Inhibitory Concentration 50 Interleukin-3 (metabolism) Leukemia, Myeloid, Acute (metabolism) Luminescent Proteins (metabolism) MAP Kinase Signaling System Mice Milk Proteins Models, Biological Models, Chemical Mutation Phosphorylation Precipitin Tests Protein Structure, Tertiary Protein-Tyrosine Kinases (antagonists & inhibitors) Proto-Oncogene Proteins (metabolism) Pyrazoles (pharmacology) Receptor Protein-Tyrosine Kinases (metabolism) Recombinant Proteins (metabolism) Retroviridae (genetics) STAT5 Transcription Factor Signal Transduction Time Factors Trans-Activators (metabolism) Transfection Tumor Cells, Cultured Tyrosine (chemistry) fms-Like Tyrosine Kinase 3

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