Y654 of ß-catenin is essential for FLT3/ITD-related tyrosine phosphorylation and nuclear localization of ß-catenin.

ß-Catenin plays a dual role as a key effecter in the regulation of adherens junctions as well as a transcriptional co-activator. Tyrosine phosphorylation of ß-catenin affects the cell adhesion, migration, and gene transcription in many types of human cancer cells, including acute myeloid leukemia cells with FLT3 internal tandem duplication (FLT3/ITD-AML). Here, we investigated the relationship between three tyrosine residues (Y86, Y142, and Y654) in ß-catenin and oncogenic FLT3/ITD kinase. In the experiments using COS-7 cells expressing FLT3/ITD and Wt or mutant ß-catenin, FLT3/ITD phosphorylated Y654, and this residue was essential for ß-catenin's nuclear localization by FLT3/ITD. Promoter-reporter assays demonstrated that Y654 phosphorylation of ß-catenin was closely related to TCF transcriptional activity. In vitro kinase assays, using recombinant FLT3 and biotinylated ß-catenin peptide including Y654 showed that FLT3 directly phosphorylated Y654 of ß-catenin. These results explain how FLT3/ITD affects the tyrosine phosphorylation, nuclear localization, and transcriptional activity of ß-catenin. Targeting Y654 phosphorylation may lead to the development of novel approaches to therapy for FLT3/ITD-AML.

KW-2449, a novel multikinase inhibitor, suppresses the growth of leukemia cells with FLT3 mutations or T315I-mutated BCR/ABL translocation.

KW-2449, a multikinase inhibitor of FLT3, ABL, ABL-T315I, and Aurora kinase, is under investigation to treat leukemia patients. In this study, we examined its possible modes of action for antileukemic effects on FLT3-activated, FLT3 wild-type, or imatinib-resistant leukemia cells. KW-2449 showed the potent growth inhibitory effects on leukemia cells with FLT3 mutations by inhibition of the FLT3 kinase, resulting in the down-regulation of phosphorylated-FLT3/STAT5, G(1) arrest, and apoptosis. Oral administration of KW-2449 showed dose-dependent and significant tumor growth inhibition in FLT3-mutated xenograft model with minimum bone marrow suppression. In FLT3 wild-type human leukemia, it induced the reduction of phosphorylated histone H3, G(2)/M arrest, and apoptosis. In imatinib-resistant leukemia, KW-2449 contributed to release of the resistance by the simultaneous down-regulation of BCR/ABL and Aurora kinases. Furthermore, the antiproliferative activity of KW-2449 was confirmed in primary samples from AML and imatinib-resistant patients. The inhibitory activity of KW-2449 is not affected by the presence of human plasma protein, such as alpha1-acid glycoprotein. These results indicate KW-2449 has potent growth inhibitory activity against various types of leukemia by several mechanisms of action. Our studies indicate KW-2449 has significant activity and warrants clinical study in leukemia patients with FLT3 mutations as well as imatinib-resistant mutations.

FLT3/ ITD regulates leukaemia cell adhesion through α4ß1 integrin and Pyk2 signalling.

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. ß1 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 α4ß1 integrin to soluble VCAM-1. α4ß1 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 ß1 integrin and Pyk2 indicating the signal crosstalk between FLT3, ß1 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.

Irrespective of CD34 expression, lineage-committed cell fraction reconstitutes and re-establishes transformed Philadelphia chromosome-positive leukemia in NOD/SCID/IL-2Rgammac-/- mice.

Stem cells of acute myeloid leukemia (AML) have been identified as immunodeficient mouse-repopulating cells with a Lin(-)CD34(+)38(-) phenotype similar to normal hematopoietic stem cells. To identify the leukemia-propagating stem cell fraction of Philadelphia chromosome-positive (Ph(+)) leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n = 3) or Ph(+) acute lymphoblastic leukemia (n = 3) into NOD/SCID/IL-2Rgammac(-/-) mice. Engrafted cells were almost identical to the original leukemia cells as to phenotypes, IGH rearrangements, and karyotypes. CD34(+)CD38(-)CD19(+), CD34(+)38(+)CD19(+), and CD34(-)CD38(+)CD19(+) fractions could self-renew and transfer the leukemia, whereas the CD34(-)CD38(+)CD19(+) fraction did not stably propagate in NOD/SCID mice. These findings suggest that leukemia-repopulating cells in transformed Ph(+) leukemia are included in a lineage-committed but multilayered fraction, and that CD34(+) leukemia cells potentially emerge from CD34(-) populations.

BCR-ABL-independent and RAS / MAPK pathway-dependent form of imatinib resistance in Ph-positive acute lymphoblastic leukemia cell line with activation of EphB4.

OBJECTIVE: We investigated the mechanism responsible for imatinib (IM) resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) cell lines. METHODS: We established cell lines from a patient with Ph(+) ALL at the time of first diagnosis and relapsed phase and designated as NPhA1 and NPhA2, respectively. We also derived IM-resistant cells, NPhA2/STIR, from NPhA2 under gradually increasing IM concentrations. RESULTS: NPhA1 was sensitive to IM (IC(50) 0.05 microm) and NPhA2 showed mild IM resistance (IC(50) 0.3 microm). NPhA2/STIR could be maintained in the presence of 10 microm IM. Phosphorylation of MEK and ERK was slightly elevated in NPhA2 and significantly elevated in NPhA2/STIR compared to NPhA1 cells. After treatment with IM, phosphorylation of MEK and ERK was not suppressed but rather increased in NPhA2 and NPhA2/STIR. Active RAS was also increased markedly in NPhA2/STIR after IM treatment. The expression of BCL-2 was increased in NPhA2 compared to NPhA1, but no further increase in NPhA2/STIR. Proliferation of NPhA2/STIR was significantly inhibited by a combination of MEK inhibitor and IM. Analysis of tyrosine phosphorylation status with a protein tyrosine kinase array showed increased phosphorylation of EphB4 in NPhA2/STIR after IM treatment. Although transcription of EphB4 was suppressed in NPhA1 and NPhA2 after IM treatment, it was not suppressed and its ligand, ephrinB2, was increased in NPhA2/STIR. Suppression of EphB4 transcripts by introducing short hairpin RNA into NPhA2/STIR partially restored their sensitivity to IM. CONCLUSIONS: These results suggest a new mechanism of IM resistance mediated by the activation of RAS/MAPK pathway and EphB4.

Prognostic implication and biological roles of RhoH in acute myeloid leukaemia.

The Rho family of small GTPases, including Rho, Rac and Cdc42, has been well characterised as a molecular switch that transduces signals from plasma membrane to the downstream effectors. RhoH gene, a member of the Rho family, is specifically expressed in haematopoietic cells. The known function of RhoH is antagonising Rac and mediating activation of ZAP-70 in T lymphocytes; however, biological roles of RhoH in myeloid cells remain unknown. Here, we analysed the prognostic implication of the expression level of the RhoH gene transcript in bone marrow samples from 90 newly diagnosed acute myeloid leukaemia (AML) patients using a real-time fluorescence detection method. Kaplan-Meier analysis demonstrated that low expression of the RhoH transcript was a predictor of worse prognosis in both overall and disease-free survival. Multivariate analysis demonstrated that low expression of RhoH was an independent unfavourable prognostic factor for both overall and disease-free survival of AML patients. Overexpression of RhoH leads to dephosphorylation of Bad at Serine 75 residue possibly through deactivation of Rac. It is possible that low expression of RhoH (i.e. high GTP-Rac) contributes to chemotherapy resistance in leukaemia cells. Our result suggests that inhibition of Rac and its signalling components might provide a useful anti-leukaemic strategy.