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1.
Blood ; 131(4): 426-438, 2018 01 25.
Article in English | MEDLINE | ID: mdl-29187377

ABSTRACT

An activating mutation of Fms-like tyrosine kinase 3 (FLT3) is the most frequent genetic alteration associated with poor prognosis in acute myeloid leukemia (AML). Although many FLT3 inhibitors have been clinically developed, no first-generation inhibitors have demonstrated clinical efficacy by monotherapy, due to poor pharmacokinetics or unfavorable safety profiles possibly associated with low selectivity against FLT3 kinase. Recently, a selective FLT3 inhibitor, quizartinib, demonstrated favorable outcomes in clinical studies. However, several resistant mutations emerged during the disease progression. To overcome these problems, we developed a novel FLT3 inhibitor, FF-10101, designed to possess selective and irreversible FLT3 inhibition. The co-crystal structure of FLT3 protein bound to FF-10101 revealed the formation of a covalent bond between FF-10101 and the cysteine residue at 695 of FLT3. The unique binding brought high selectivity and inhibitory activity against FLT3 kinase. FF-10101 showed potent growth inhibitory effects on human AML cell lines harboring FLT3 internal tandem duplication (FLT3-ITD), MOLM-13, MOLM-14, and MV4-11, and all tested types of mutant FLT3-expressing 32D cells including quizartinib-resistant mutations at D835, Y842, and F691 residues in the FLT3 kinase domain. In mouse subcutaneous implantation models, orally administered FF-10101 showed significant growth inhibitory effect on FLT3-ITD-D835Y- and FLT3-ITD-F691L-expressing 32D cells. Furthermore, FF-10101 potently inhibited growth of primary AML cells harboring either FLT3-ITD or FLT3-D835 mutation in vitro and in vivo. These results indicate that FF-10101 is a promising agent for the treatment of patients with AML with FLT3 mutations, including the activation loop mutations clinically identified as quizartinib-resistant mutations.


Subject(s)
Amides/therapeutic use , Antineoplastic Agents/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Mutation , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/genetics , Amides/pharmacokinetics , Amides/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Molecular Docking Simulation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , fms-Like Tyrosine Kinase 3/chemistry
2.
Sci Rep ; 7(1): 5519, 2017 07 17.
Article in English | MEDLINE | ID: mdl-28717217

ABSTRACT

ROS1 rearrangement is observed in 1-2% of non-small cell lung cancers (NSCLC). The ROS1 tyrosine kinase inhibitor (TKI) crizotinib has induced marked tumour shrinkage in ROS1-rearranged cancers. However, emergence of acquired resistance to TKI is inevitable within a few years. Previous findings indicate that cabozantinib overcomes secondary mutation-mediated crizotinib-resistance in ROS1-fusion-positive cells. Here we attempted to establish cabozantinib-resistant cells by N-ethyl-N-nitrosourea mutagenesis screening using CD74-ROS1-expressing Ba/F3 cells. Two resistant cell lines with CD74-ROS1 F2004V or F2075C mutations, which are homologous to ALK F1174 or F1245 mutations, survived in the presence of a low dose of ROS1-TKI. Removal of ROS1-TKI from these TKI-addicted cells induced excessive activation of ROS1 tyrosine kinase followed by apoptosis. We succeeded in recapturing the TKI-addicted phenotype using doxycycline-inducible CD74-ROS1 mutant over-expression in Ba/F3 cells, suggesting that excessive ROS1 oncogenic signaling itself induced apoptosis instead of cell growth. Phosphoproteomic analysis and high-throughput inhibitor screening revealed that excessive ROS1 signaling in the TKI-addicted cells phosphorylated or activated apoptosis-related molecules such as FAF1 or p38. Collectively, our findings partly clarify molecular mechanisms of excessive ROS1 oncogenic signaling that mediates paradoxical induction of apoptosis.


Subject(s)
Apoptosis/drug effects , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Anilides/pharmacology , Antigens, Differentiation, B-Lymphocyte/genetics , Antigens, Differentiation, B-Lymphocyte/metabolism , Cell Line, Tumor , Doxycycline/pharmacology , Drug Resistance, Neoplasm/genetics , Ethylnitrosourea/pharmacology , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Humans , Imidazoles/pharmacology , Mutagenesis, Site-Directed , Phosphopeptides/analysis , Protein Structure, Tertiary , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Pyridines/pharmacology , Signal Transduction/drug effects , Tandem Mass Spectrometry , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolism
3.
Mol Cancer Ther ; 16(10): 2130-2143, 2017 10.
Article in English | MEDLINE | ID: mdl-28751539

ABSTRACT

Neurotrophic receptor tyrosine kinase 1 (NTRK1) gene rearrangement leads to constitutive activation of NTRK1, which induces high-transforming ability. NTRK-rearranged cancers have been identified in several cancer types, such as glioblastoma, non-small cell lung cancer, and colorectal cancer. Although there are currently no clinically approved inhibitors that target NTRK1, several tyrosine kinase inhibitors (TKI), such as entrectinib and LOXO-101, are in clinical trials. The purpose of this study was to identify potential mechanisms of resistance to NTRK inhibitors and find potential therapeutic strategies to overcome the resistance. We examined the sensitivity of TPM3-NTRK1-transformed Ba/F3 cells and TPM3-NTRK1-harboring KM12 cells to multiple NTRK inhibitors. Acquired NTRK inhibitor-resistant mutations were screened by N-ethyl-N-nitrosourea mutagenesis with Ba/F3-TPM3-NTRK1 cells or by the establishment of NTRK-TKI-resistant cells from KM12 cells continuously treated with NTRK-TKIs. We identified multiple novel NTRK-TKI resistance mutations in the NTRK1 kinase domain, including G595R, and insulin growth factor receptor type 1 (IGF1R) bypass pathway-mediated resistance. After identifying the resistance mechanisms, we performed drug screening with small-molecule inhibitors to overcome the resistance. As a result, we found that ponatinib and nintedanib effectively inhibited the survival of TPM3-NTRK1-G667C but not G595R mutants, both of which showed resistance to entrectinib or larotrectinib (LOXO-101). Furthermore, cabozantinib with an IGF1R inhibitor such as OSI-906 could overcome bypass pathway-mediated resistance. We developed a comprehensive model of acquired resistance to NTRK inhibitors in cancer with NTRK1 rearrangement and identified cabozantinib as a therapeutic strategy to overcome the resistance. Mol Cancer Ther; 16(10); 2130-43. ©2017 AACR.


Subject(s)
Drug Resistance, Neoplasm/genetics , Neoplasms/drug therapy , Protein Kinase Inhibitors/administration & dosage , Receptor, trkA/genetics , Anilides/administration & dosage , Benzamides/administration & dosage , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Gene Rearrangement/genetics , Humans , Indazoles/administration & dosage , Mutation , Neoplasms/genetics , Neoplasms/pathology , Pyrazoles/administration & dosage , Pyridines/administration & dosage , Pyrimidines/administration & dosage , Receptor, IGF Type 1 , Receptor, trkA/antagonists & inhibitors , Receptors, Somatomedin/antagonists & inhibitors
4.
Anal Chim Acta ; 635(2): 207-13, 2009 Mar 09.
Article in English | MEDLINE | ID: mdl-19216880

ABSTRACT

A novel chelating ligand, 2,4-[bis-(2,4-dihydroxybenzylidene)]-dihydrazinoquinazoline (DBHQ), was synthesized, and the fluorescence characteristics of its complex with metal ions were investigated. Thirty-five different metal ions were tested for the emission of fluorescence in the presence of DBHQ in aqueous solutions in a pH range of 3.0-10.5 (at a difference of 0.5 for each metal). It was observed that DBHQ fluoresces intensely at 470nm with an excitation wavelength of 405nm in the presence of Ga(3+) or Al(3+) in the pH range 3.0-4.0. The other metal ions did not show fluorescence with DBHQ. Although the presence of Cu(2+), Co(2+) and Fe(3+) decreased the fluorescence intensity of DBHQ-Ga(3+), the addition of a fluoride ion (NaF) recovered the fluorescence by masking the interfering ions. In addition, the fluoride ions were found to enhance the sensitive determination of Ga(3+) because the fluorescence intensity of DBHQ-Ga(3+) was further increased approximately 2.5-fold in the presence of F(-) (phi=0.658) from that in the absence of F(-) (phi=0.401). The fluoride ions also masked the Al(3+) ions, which emit fluorescence on chelation with DBHQ. Therefore, a selective and sensitive detection of Ga(3+) was achieved by using DBHQ in the presence of F(-). The detection limit of Ga(3+) was approximately 50nmolL(-1) (3.5ppb). The proposed method was applicable to determine Ga(3+) in river water.


Subject(s)
Benzylidene Compounds/chemistry , Chelating Agents/chemistry , Fluorescent Dyes/chemistry , Gallium/analysis , Quinazolines/chemistry , Spectrometry, Fluorescence/methods , Benzylidene Compounds/chemical synthesis , Chelating Agents/chemical synthesis , Fluorescent Dyes/chemical synthesis , Metals/chemistry , Quinazolines/chemical synthesis , Rivers , Schiff Bases/chemistry , Water Pollutants, Chemical/analysis
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