su-5614 and Leukemia--Myeloid--Acute

Flt3 is a tyrosine kinase receptor expressed on hematopoietic cells. Activating mutations of this receptor are encountered in over one-fourth of acute myelogenous leukemia (AML) cases and activate multiple intracellular pathways leading to cell proliferation, inhibition of apoptosis, and blockage of differentiation in leukemic blasts. AML with flt3 mutations has a worse prognosis than AML with normal flt3, at least in younger patients. Several flt3 inhibitors are in various stages of preclinical and clinical development and it is hoped that specific therapies against AML with flt3 mutations will soon be available to the clinician.

Topics: Adult; Animals; Enzyme Inhibitors; fms-Like Tyrosine Kinase 3; Humans; Indoles; Leukemia, Myeloid, Acute; Mice; Middle Aged; Prognosis; Proto-Oncogene Proteins; Pyrroles; Randomized Controlled Trials as Topic; Receptor Protein-Tyrosine Kinases; Treatment Outcome

FLT3 activating mutations are the most frequent single genetic abnormality in patients with acute myeloid leukemia. Thus targeting the FLT3 activated kinase is a promising treatment approach. We wanted to test whether the protein tyrosine kinase inhibitor SU5614 selectively eliminates leukemic stem cells while sparing their normal counterparts.

Topics: Adult; Aged; Cells, Cultured; fms-Like Tyrosine Kinase 3; Hematopoietic Stem Cells; Humans; Indoles; Leukemia, Myeloid, Acute; Middle Aged; Protein Kinase Inhibitors; Protein-Tyrosine Kinases

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.

Topics: Animals; Apoptosis; Blotting, Western; Bone Marrow Transplantation; Cell Division; DNA-Binding Proteins; Enzyme Inhibitors; Flow Cytometry; fms-Like Tyrosine Kinase 3; Gene Expression; Green Fluorescent Proteins; Humans; Indoles; Leukemia, Myeloid, Acute; Luminescent Proteins; Mice; Milk Proteins; Mitogen-Activated Protein Kinases; Mutagenesis; Mutation; Myeloproliferative Disorders; Polymerase Chain Reaction; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; STAT5 Transcription Factor; Trans-Activators; Transfection; Tumor Cells, Cultured

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.. We analyzed the transforming potential and the signaling of FLT3-ITD mutants in Ba/F3 cells and in primary AML blasts.. 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.. 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.

Topics: Amino Acid Sequence; Cell Transformation, Neoplastic; DNA-Binding Proteins; Enzyme Activation; fms-Like Tyrosine Kinase 3; Humans; Indoles; Leukemia, Myeloid, Acute; Milk Proteins; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Phosphorylation; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; STAT3 Transcription Factor; STAT5 Transcription Factor; Trans-Activators

Activating length mutations in the juxtamembrane (JM) domain of the FLT3 gene (FLT3-LM) and mutations in the catalytic domain (FLT3D835/836) of this receptor tyrosine kinase represent the most frequent genetic alterations in acute myeloid leukemia (AML). Here, we describe a 6-bp insertion in the activation loop of FLT3 between codons 840 and 841 of FLT3 (FLT3-840GS) in 2 unrelated patients with AML. Screening for other activating mutations of FLT3, KIT, and NRAS showed no further genetic alterations in patients carrying the FLT3-840GS. In functional analyses we could show that this mutant is hyperphosphorylated on tyrosine and confers interleukin 3-independent growth to Ba/F3 cells, which can be inhibited by a specific FLT3 protein tyrosine kinase (PTK) inhibitor. Our results show for the first time that in addition to known mutations in the JM and the catalytic domain, further activating length mutations exist in the FLT3 gene.

Topics: Aged; Animals; Cell Division; Codon; DNA Mutational Analysis; Enzyme Inhibitors; Exons; Fatal Outcome; Female; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Leukemic; Humans; Indoles; Interleukin-3; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid, Acute; Male; Mice; Mutagenesis, Insertional; Neoplasm Proteins; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured