FLT3/ITD Regulates Leukemia Cell Adhesion through α4β1 Integrin and Pyk2 Signaling.

FLT3 (FMS-like receptor tyrosine kinase 3) is a member of the class III receptor tyrosine kinases, and is preferentially expressed in hematopoietic stem cells as well as in the brain, placenta and liver. The FLT3 ligand (FL) is expressed as a membrane-bound or soluble form by bone marrow stromal cells, stimulates the stem cells by itself or in cooperation with other cytokines. The FL-FLT3 signal transduction plays an important role in survival, proliferation, and differentiation of hematopoietic stem cells. Internal tandem duplication of FLT3 (FLT3/ITD) within its juxtamembrane domain occurs in 15 to 35% of adults in acute myeloid leukemia (AML). This mutation causes constitutive activation of FLT3 and is associated with a poor prognosis. The high relapse rate is in part due to the insufficient eradication of slow-cycling leukemic stem cells in the bone marrow niche. Adhesion molecules such as αa4β1 integrin and CD44 are crucial for the persistence of leukemic cells in the bone marrow microenvironment. β1 integrins mediate hematopoietic stem and progenitor cell homing and retention in the bone marrow and inhibit hematopoietic proliferation and differentiation. Having no intrinsic kinase activity, integrins recruit intracellular kinases, such as the focal adhesion kinase (FAK) or the related proline-rich tyrosine kinase 2 (Pyk2), to initiate signal transduction.

To investigate whether inhibition of FLT3/ITD kinase activity affects the leukemic cell adhesion through integrin and related kinase signaling.

NAMO-2, a cell line of acute myelomonocytic leukemia with FLT3/ITD was treated with FLT3 specific inhibitor FI-700. The addition of FI-700 inhibited adhesion of NAMO-2 cells on feeder cells within 1 hour. FI-700 did not induce apoptosis of NAMO-2 cells under this concentration, suggesting that FI-700 downregulates adhesion molecules. A flow cytometry assay was used to quantify the kinetics of sVCAM-1/Fc binding to NAMO-2 and MOLM-13, both bearing FLT3/ITD. Preincubation of both cells with FLT3 specific inhibitor FI-700 significantly inhibited the basal binding of αa4 integrin to VCAM-1. We then investigated whether FLT3 inhibitor inactivates Rap1, which is a critical mediator of inside-out signaling of αa4 integrin in Jurkat cells and eosinophil. FI-700 treatment of both NAMO-2 and MOLM-13 cells did not alter the GTP loading of Rap1, demonstrating that deactivation of αa4β1 integrin by FLT3 inhibitor is not mediated by Rap1. Previous study demonstrated that FLT3/ITD expression confers autonomous proliferation capability, and induces constitutive activation of downstream signaling molecules such as STAT5, MAP kinase and PI3K/Akt pathway. Consisting with that, FI-700 treatment dephosphorylates Serine 473 of Akt in NAMO-2 cell. Pyk2 is a FAK family of nonreceptor tyrosine kinase, which is abundantly expressed in hematopoietic cells. The stimulation of cytokine, chemokine, and adhesion receptors such as integrin results in Pyk2 phosphorylation, potential recruitment of Src-family kinases, and activation of other signaling pathways. Addition of FI-700 caused marked dephosphorylation of Pyk2 in NAMO-2 cells. Notably, both wtFLT3 and FLT3/ITD were co-immunoprecipitated with β1 integrin and Pyk2. These results demonstrated that FLT3 and β1 integrin form macromolecular complex with Pyk2.

The basal activity of αa4β1 integrins on FLT3/ITD positive cells is downregulated by FLT3 specific inhibitor FI-700. On the other hand, the inhibition of FLT3/ITD leads to the dephosphorylation of Tyr402 of Pyk2, which is crucial to relay multiple signal transduction pathway. These findings suggest that anti-FLT3 therapy might work as anti-adhesion therapy of AML.

Kiyoi:Kyowa Hakko Kirin Co., Ltd. : Consultancy. Naoe:Kyowa Hakko Kirin Co., Ltd. : Research Funding; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Wyeth K.K.: Research Funding.