fi-700 and Leukemia--Myeloid--Acute

Topics: Animals; Aurora Kinases; Drug Design; Drug Evaluation, Preclinical; Enzyme Inhibitors; fms-Like Tyrosine Kinase 3; Humans; Leukemia; Leukemia, Myeloid, Acute; Mutation; Protein Serine-Threonine Kinases; Pyridines; Pyrimidines

Internal tandem duplication of FMS-like receptor tyrosine kinase 3 (FLT3/ITD) within its juxtamembrane domain is a frequent mutation in adult acute myeloid leukaemia (AML). This mutation causes constitutive activation of FLT3 and is associated with poor prognosis. The high relapse rate of FLT3/ITD-positive AML might be partly because of insufficient eradication of slow-cycling leukaemic stem cells in the bone marrow microenvironment. β1 integrin mediates haematopoietic stem and progenitor cell homing along with their retention in the bone marrow and also inhibits haematopoietic proliferation and differentiation. Here, we demonstrate that inhibition of FLT3/ITD kinase activity by a FLT3 selective inhibitor named FI-700 decreases affinity of α4β1 integrin to soluble VCAM-1. α4β1 integrin deactivation by FI-700 is independent of Rap1, which is the critical regulator of integrin inside-out signalling. In addition, selective inhibition of FLT3/ITD induces Pyk2 dephosphorylation together with the inhibition of phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Both wild-type and ITD-FLT3 proteins co-immunoprecipitated with β1 integrin and Pyk2 indicating the signal crosstalk between FLT3, β1 integrin and Pyk2. These results collectively indicated that the inhibition of FLT3 kinase might contribute not only to the induction of apoptosis, but also to the leukaemia cell detachment from the bone marrow microenvironment in the treatment of AML.

Topics: Animals; Cell Adhesion; Cell Line, Tumor; Coculture Techniques; fms-Like Tyrosine Kinase 3; Focal Adhesion Kinase 2; Gene Duplication; Humans; Integrin alpha4beta1; Leukemia, Myeloid, Acute; Mice; Multiprotein Complexes; Mutation; Phosphorylation; Pyridines; Pyrimidines; Shelterin Complex; Signal Transduction; Tandem Repeat Sequences; Telomere-Binding Proteins; Vascular Cell Adhesion Molecule-1

Fms-like tyrosine kinase-3 (FLT3) inhibitors have been used to overcome the dismal prognosis of acute myeloid leukemia (AML) with FLT3 mutations. Clinical results with FLT3 inhibitor monotherapy have shown that bone marrow responses are commonly less pronounced than peripheral blood responses. We investigated the role of p53 in bone marrow stromal cells in stromal cell-mediated resistance to FLT3 inhibition in FLT3 mutant AML. While the FLT3 inhibitor FI-700 induced apoptosis in FLT3 mutant AML cells, apoptosis induction was diminished under stromal coculture conditions. Protection appeared to be mediated, in part, by CXCL12 (SDF-1)/CXCR4 signaling. The protective effect of stromal cells was significantly reduced by pre-exposure to the HDM2 inhibitor Nutlin-3a. p53 activation by Nutlin-3a was not cytotoxic to stromal cells, but reduced CXCL12 mRNA levels and secretion of CXCL12 partially through p53-mediated HIF-1α down-regulation. Results show that p53 activation in stroma cells blunts stroma cell-mediated resistance to FLT3 inhibition, in part through down-regulation of CXCL12. This is the first report of Nutlin effect on the bone marrow environment. We suggest that combinations of HDM2 antagonists and FLT3 inhibitors may be effective in clinical trials targeting mutant FLT3 leukemias.

Topics: Adult; Apoptosis; Bone Marrow Cells; Chemokine CXCL12; Down-Regulation; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Leukemic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; Leukemia, Myeloid, Acute; Mesenchymal Stem Cells; Mutation; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-mdm2; Pyridines; Pyrimidines; Tumor Cells, Cultured; Tumor Microenvironment; Tumor Suppressor Protein p53; Young Adult

Treatment using Fms-like tyrosine kinase-3 (FLT3) inhibitors is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML. We found that FI-700 immediately reduced antiapoptotic Mcl-1 levels and enhanced Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/internal tandem duplication cells through the Mcl-1/Noxa axis. FI-700 induced proteasome-mediated degradation of Mcl-1, resulting in the reduced ability of Mcl-1 to sequester proapoptotic Bim. Nutlin-3 induced Noxa, which displaced Bim from Mcl-1. The FI-700/Nutlin-3 combination profoundly activated Bax and induced apoptosis. Our findings suggest that FI-700 actively enhances p53 signaling toward mitochondrial apoptosis and that a combination strategy aimed at inhibiting FLT3 and activating p53 signaling could potentially be effective in AML.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Doxorubicin; fms-Like Tyrosine Kinase 3; G1 Phase; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Piperazines; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Pyridines; Pyrimidines; Tumor Suppressor Protein p53