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1.
Blood ; 103(6): 2266-75, 2004 Mar 15.
Article in English | MEDLINE | ID: mdl-14604974

ABSTRACT

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.


Subject(s)
Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Leukemia, Myeloid , Milk Proteins , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Staurosporine/analogs & derivatives , Acute Disease , Animals , Antimetabolites, Antineoplastic/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Cell Division/drug effects , Cell Division/physiology , Cell Line, Transformed/drug effects , Cell Line, Transformed/physiology , Cytarabine/pharmacology , DNA-Binding Proteins/metabolism , Drug Resistance, Neoplasm , Genistein/pharmacology , Humans , MAP Kinase Signaling System/physiology , Mutagenesis , Phenotype , Phosphorylation/drug effects , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Receptor Protein-Tyrosine Kinases/chemistry , STAT5 Transcription Factor , Staurosporine/pharmacology , Trans-Activators/metabolism , Tyrphostins/pharmacology , fms-Like Tyrosine Kinase 3
2.
Clin Cancer Res ; 9(6): 2140-50, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12796379

ABSTRACT

PURPOSE: Activating length mutations in the juxtamembrane domain (FLT3-LM) and mutations in the tyrosine kinase domain (FLT3-TKD) of FLT3 represent the most frequent genetic alterations in acute myeloid leukemia (AML). However, the functional role of active FLT3 mutants in primary AML blast cells is not well characterized. EXPERIMENTAL DESIGN: We analyzed the transforming potential and the signaling of FLT3-ITD mutants in Ba/F3 cells and in primary AML blasts. RESULTS: FLT3-ITD mutants induce an autophosphorylation of the receptor, interleukin 3-independent growth in Ba/F3 cells, and a strong STAT5 and mitogen-activated protein kinase (MAPK) activation. In contrast to the FLT3-ITD mutants, the ligand-stimulated FLT3-WT receptor was unable to transduce a fully proliferative response in Ba/F3 and monocytic OCI-AML5 cells. The ligand-stimulated FLT3-WT receptor activated AKT and MAPK, but not STAT5. In primary blast cells from 60 patients with AML, FLT3 was expressed in 91.9% of patients carrying a FLT3-LM/TKD mutation compared with 77.8% in FLT3-LM/TKD-negative patients. STAT3 and STAT5 were constitutively activated in 76 and 63% of patients, respectively. In accordance with the results in Ba/F3 cells, a high FLT3 expression and the presence of a FLT3-LM was strongly associated with the STAT5 but not with the STAT3 activation in primary AML blast cells. Moreover, the constitutive tyrosine phosphorylation of STAT5 was efficiently down-regulated by a FLT3 protein tyrosine kinase inhibitor in AML cells expressing an active FLT3 mutant. CONCLUSIONS: Active FLT3 receptor mutants have transforming potential in hematopoietic cells and induce a strong activation of STAT5 in primary AML cells. The FLT3-STAT5 pathway contributes to the malignant phenotype and represents a promising molecular therapeutic target structure in AML.


Subject(s)
DNA-Binding Proteins/physiology , Leukemia, Myeloid, Acute/pathology , Milk Proteins , Proto-Oncogene Proteins/physiology , Receptor Protein-Tyrosine Kinases/physiology , Trans-Activators/physiology , Amino Acid Sequence , Cell Transformation, Neoplastic , Enzyme Activation , Humans , Indoles/pharmacology , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/therapy , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , Phosphorylation , STAT3 Transcription Factor , STAT5 Transcription Factor , fms-Like Tyrosine Kinase 3
3.
Blood ; 101(4): 1494-504, 2003 Feb 15.
Article in English | MEDLINE | ID: mdl-12406902

ABSTRACT

Activating mutations of the protein tyrosine kinase (PTK) FLT3 can be found in approximately 30% of patients with acute myeloid leukemia (AML), thereby representing the most frequent single genetic alteration in AML. These mutations occur in the juxtamembrane (FLT3 length mutations; FLT3-LMs) and the second tyrosine kinase domain of FLT3-TKD and confer interleukin 3 (IL-3)-independent growth to Ba/F3 cells. In the mouse bone marrow transplantation model, FLT3-LMs induce a myeloproliferative syndrome stressing their transforming activity in vivo. In this study, we analyzed the pro-proliferative and antiapoptotic potential of FLT3 in FLT3-LM/TKD-mutation-transformed Ba/F3 cells and AML-derived cell lines. The PTK inhibitor SU5614 has inhibitory activity for FLT3 and selectively induces growth arrest, apoptosis, and cell cycle arrest in Ba/F3 and AML cell lines expressing a constitutively activated FLT3. In addition, the compound reverts the antiapoptotic and pro-proliferative activity of FLT3 ligand (FL) in FL-dependent cells. No cytotoxic activity of SU5614 was found in leukemic cell lines that express a nonactivated FLT3 or no FLT3 protein. At the biochemical level, SU5614 down-regulated the activity of the hyperphosphorylated FLT3 receptor and its downstream targets, signal transducer and activator of (STAT) 3, STAT5, and mitogen-activated protein kinase (MAPK), and the STAT5 target genes BCL-X(L) and p21. Our results show that SU5614 is a PTK inhibitor of FLT3 and has antiproliferative and proapoptotic activity in AML-derived cell lines that endogenously express an activated FLT3 receptor. The selective and potent cytotoxicity of FLT3 PTK inhibitors support a clinical strategy of targeting FLT3 as a new molecular treatment option for patients with FLT3-LM/TKD-mutation(+) AML.


Subject(s)
Apoptosis/drug effects , Cell Division/drug effects , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Leukemia, Myeloid, Acute/pathology , Milk Proteins , Proto-Oncogene Proteins/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Blotting, Western , Bone Marrow Transplantation , DNA-Binding Proteins/antagonists & inhibitors , Flow Cytometry , Gene Expression , Green Fluorescent Proteins , Humans , Leukemia, Myeloid, Acute/genetics , Luminescent Proteins/genetics , Mice , Mitogen-Activated Protein Kinases , Mutagenesis , Mutation , Myeloproliferative Disorders/genetics , Polymerase Chain Reaction , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , STAT5 Transcription Factor , Trans-Activators/antagonists & inhibitors , Transfection , Tumor Cells, Cultured , fms-Like Tyrosine Kinase 3
4.
Exp Hematol ; 30(3): 262-71, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11882364

ABSTRACT

OBJECTIVE: Signal transducers and activators of transcription (STAT) factors are critical mediators in the signal transduction of cytokine receptors. In hematopoietic and epithelial cells, activation of STAT 1 induces apoptosis and growth arrest, whereas activation of STAT3 and STAT5 transduces growth-promoting signals. We and others have previously described a high expression and constitutive activation of STAT1, 3, and 5 in AML blasts. In this report we focused on the mechanisms and also the biologic relevance of STAT activation in AML.Results. RESULTS: We report here that a constitutive STAT activation can be detected in up to 95% of primary AML blasts. In vitro, neither induction of the leukemic fusion protein PML-RAR alpha in U937 cells nor expression of transforming ras-mutants, but several leukemic protein tyrosine kinase (PTK), strongly induced activation of STAT3 and 5. Stable transfection of BA/F3 cells with TEL-JAK2, TEL-ABL, and BCR-ABL resulted in IL-3 independent growth and strong activation of STAT3 and STAT5 by TEL-JAK2 and TEL-ABL, but not by BCR-ABL. In addition, expression of constitutive active mutants of STAT3 and STAT5 alone were sufficient to transform BA/F3 cells. CONCLUSIONS: These results show that STAT3 and STAT5 are activated in the majority of primary AML blasts and are major targets of leukemic fusion proteins with PTK activity. However, the STAT activation pattern by leukemic PTKs differed significantly and might reflect their transforming potential in acute (TEL-JAK2 and TEL-ABL) and chronic leukemias (p210BCR-ABL). The transforming capacity of constitutively activated STAT3 and STAT5 mutants strongly supports their fundamental role in the process of malignant transformation in hematopoietic cells.


Subject(s)
Cell Transformation, Neoplastic , DNA-Binding Proteins/metabolism , Hematopoietic Stem Cells/pathology , Milk Proteins , Oncogene Proteins, Fusion/physiology , Protein-Tyrosine Kinases/metabolism , Trans-Activators/metabolism , Animals , Cell Line , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/physiology , Gene Expression , Hematopoietic Stem Cells/metabolism , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Oncogene Proteins, Fusion/genetics , STAT1 Transcription Factor , STAT3 Transcription Factor , STAT5 Transcription Factor , Transfection
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