su-5614 and Leukemia--Myeloid

Autotaxin (ATX) has been reported to act as a motility and growth factor in a variety of cancer cells. The ATX protein acts as a secreted lysophospholipase D by converting lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which signals via G-protein-coupled receptors and has important functions in cell migration and proliferation. This study demonstrates that ATX expression is specifically upregulated and functionally active in acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) mutation of the FLT3 receptor gene. Moreover, ATX expression was also found in normal human CD34+ progenitor cells and selected myeloid and lymphoid subpopulations. Enforced expression of mutant FLT3-ITD by retroviral vector transduction increased ATX mRNA in selected cell lines, whereas inhibition of FLT3-ITD signaling by sublethal doses of PKC412 or SU5614 led to a significant downregulation of ATX mRNA and protein levels. In the presence of LPC, ATX expression significantly increased proliferation. LPA induced proliferation, regardless of ATX expression, and induced chemotaxis in all tested human leukemic cell lines and human CD34(+) progenitors. LPC increased chemotaxis only in cells with high expression of endogenous ATX by at least 80%, demonstrating the autocrine action of ATX. Inhibition of ATX using a small molecule inhibitor selectively induced killing of ATX-expressing cell lines and reduced motility in these cells. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration during hematopoiesis and that deregulation of ATX contributes to the pathogenesis of AML.

Topics: Acute Disease; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; fms-Like Tyrosine Kinase 3; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Hematopoietic Stem Cells; Humans; Indoles; K562 Cells; Leukemia, Myeloid; Lysophosphatidylcholines; Lysophospholipids; Mutation; Oligonucleotide Array Sequence Analysis; Phosphoric Diester Hydrolases; Protein Kinase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Staurosporine; Tandem Repeat Sequences

The correlation between drug sensitivity in vitro and the mutation status of the FLT3 receptor gene was evaluated in tumour cells from 17 previously untreated AML patients. Tumour cells with internal tandem duplication (ITD) in the FLT3 receptor gene were significantly more sensitive to the FLT3 inhibitor SU5614 than tumour cells with wild type FLT3. Combinations of SU5614 with etoposide and amsacrine showed better effect (p<0.05) compared with the respective single drugs. Our results suggest that the FLT3 inhibitor SU5614 may have a therapeutic potential, especially in combination with other cytotoxic agents, in patients with FLT3-ITD positive AML.

Topics: Acute Disease; Antineoplastic Agents; fms-Like Tyrosine Kinase 3; Humans; Indoles; Leukemia, Myeloid; Point Mutation; Polymerase Chain Reaction; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Tumor Cells, Cultured

Activating mutations in the juxtamembrane domain (FLT3-length mutations, FLT3-LM) and in the protein tyrosine kinase domain (TKD) of FLT3 (FLT3-TKD) represent the most frequent genetic alterations in acute myeloid leukemia (AML) and define a molecular target for therapeutic interventions by protein tyrosine kinase (PTK) inhibitors. We could show that distinct activating FLT3-TKD mutations at position D835 mediate primary resistance to FLT3 PTK inhibitors in FLT3-transformed cell lines. In the presence of increasing concentrations of the FLT3 PTK inhibitor SU5614, we generated inhibitor resistant Ba/F3 FLT3-internal tandem duplication (ITD) cell lines (Ba/F3 FLT3-ITD-R1-R4) that were characterized by a 7- to 26-fold higher IC50 (concentration that inhibits 50%) to SU5614 compared with the parental ITD cells. The molecular characterization of ITD-R1-4 cells demonstrated that specific TKD mutations (D835N and Y842H) on the ITD background were acquired during selection with SU5614. Introduction of these dual ITD-TKD, but not single D835N or Y842H FLT3 mutants, in Ba/F3 cells restored the FLT3 inhibitor resistant phenotype. Our data show that preexisting or acquired mutations in the PTK domain of FLT3 can induce drug resistance to FLT3 PTK inhibitors in vitro. These findings provide a molecular basis for the evaluation of clinical resistance to FLT3 PTK inhibitors in patients with AML.

Topics: Acute Disease; Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Apoptosis; Cell Division; Cell Line, Transformed; Cytarabine; DNA-Binding Proteins; Drug Resistance, Neoplasm; Enzyme Inhibitors; fms-Like Tyrosine Kinase 3; Genistein; Humans; Indoles; Leukemia, Myeloid; MAP Kinase Signaling System; Milk Proteins; Mutagenesis; Phenotype; Phosphorylation; Protein Structure, Tertiary; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; STAT5 Transcription Factor; Staurosporine; Trans-Activators; Tyrphostins

Activating mutations of FLT3 have been detected in patients with acute myeloid leukemia (AML). Two distinct types of FLT3 mutations are most common: internal tandem duplication (ITD) of sequences coding for the juxtamembrane domain and point mutations at codon 835 (Asp835) within the kinase domain. Both types of mutations constitutively activate the tyrosine kinase activity of FLT3 in experimental systems and result in factor-independent proliferation of Ba/F3 and 32D cells. Recently, novel mutations within the activation loop were identified in patients with AML: deletion of isoleucine 836 (Ile836del) and an exchange of isoleucine 836 to methionine plus an arginine insertion (Ile836Met+Arg). To examine whether the Ile836 mutations result in constitutive activation of the FLT3 receptor, we introduced both mutant FLT3 cDNAs transiently into HEK 293 cells. Both mutant FLT3 receptors were constitutively autophosphorylated in the absence of ligand and kinase activity led to constitutive activation of downstream signaling cascades as determined by activation of the STAT5 (signal transducer and activator of transcription 5) pathway. When stably expressed in the growth factor-dependent cell lines Ba/F3 and 32D, both deletion and insertion mutants led to factor-independent proliferation, indicating that both mutants have transforming capabilities. We then examined the sensitivity of the FLT3 ITD, FLT3 Asp835Tyr, and the novel FLT3 receptor mutants toward the kinase inhibitors AG1296, PKC412, and SU5614. We show that these FLT3 kinase inhibitors have distinct inhibitory potencies against different activating FLT3 receptor mutants. These results suggest that it may be useful to determine the exact kind of FLT3 mutation when applying receptor kinase inhibitors in clinical trials.

Topics: Acute Disease; Amino Acid Substitution; Animals; Cell Division; Cell Line; Codon; DNA-Binding Proteins; Drug Resistance; Enzyme Activation; Enzyme Inhibitors; fms-Like Tyrosine Kinase 3; Hematopoietic Stem Cells; Humans; Indoles; Leukemia, Myeloid; Membrane Proteins; Mice; Milk Proteins; Mutagenesis, Insertional; Mutation, Missense; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Recombinant Proteins; Sequence Deletion; STAT5 Transcription Factor; Staurosporine; Structure-Activity Relationship; Trans-Activators; Transfection; Tyrphostins

Angiogenesis, the process of new blood vessel formation, is a critical process during growth and metastasis of solid tumors and might also represent a promising therapeutical target in patients with acute myeloid leukemia (AML).. In this study, we analyzed the expression of vascular endothelial growth factor receptors (VEGFR)-1/2 and its ligand VEGF in AML cell lines and characterized the inhibitory activity of the protein tyrosine kinase (PTK) inhibitor SU5614 on human endothelial and leukemic cells.. Intracellular VEGF expression was detected in 9 of 10 leukemic cell lines. In contrast, VEGFR-1 and VEGFR-2 expression was restricted to 6 and 2 out of 10 cell lines, respectively. Although SU5614 was a potent inhibitor of the VEGF-induced endothelial cell sprouting in vitro, the sensitivity of leukemic cells toward the growth inhibitory activity of the compound was determined by the c-kit, but not by the VEGFR-1/2 expression. SU5614 induced growth arrest and apoptosis in c-kit-expressing Kasumi-1, UT-7, and M-07e cells and inhibited the stem cell factor (SCF)-induced tyrosine phosphorylation of c-kit. The sensitivity of Kasumi-1 cells towards the growth inhibitory activity of SU5614 was caused by an autocrine production of SCF, but not by transforming mutations of c-kit.. Our data provide strong evidence that SU5614 has a dual mode of action, and by direct inhibition of c-kit in AML cells and by inhibition of VEGFR-2 in endothelial cells, it might represent a novel treatment option for patients with c-kit+ AML.

Topics: Acute Disease; Animals; Apoptosis; Autocrine Communication; Cell Division; Culture Media, Serum-Free; Drug Resistance; Endothelial Growth Factors; Endothelium, Vascular; Enzyme Inhibitors; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Growth Inhibitors; Hematopoietic Stem Cells; Humans; Indoles; Leukemia, Myeloid; Lymphokines; Mice; Neoplasm Proteins; Neovascularization, Pathologic; Oncogene Proteins, Fusion; Phosphorylation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-kit; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factors