Src tyrosine kinase regulates adhesion and chemotaxis in Waldenstrom macroglobulinemia

Abstract

Purpose: Waldenstrom macroglobulinemia is a lymphoplasmacytic lymphoma characterized by widespread involvement of the bone marrow. Despite different options of therapy, Waldenstrom macroglobulinemia is still incurable. Src tyrosine kinase has been shown to play a central role in the regulation of a variety of biological processes, such as cell proliferation, migration, adhesion, and survival in solid tumors. We sought to determine whether the protein tyrosine kinase Src regulates adhesion, migration, and survival in Waldenstrom macroglobulinemia.

Experimental design: We tested the expression of Src tyrosine kinase in Waldenstrom macroglobulinemia and normal cells, and the effect of the specific Src inhibitor AZD0530 on the adhesion, migration, cell cycle, and survival of a Waldenstrom macroglobulinemia cell line and patient samples. Moreover, we tested the effect of AZD0530 on cytoskeletal and cell cycle signaling in Waldenstrom macroglobulinemia.

Results: We show that Src is overexpressed in Waldenstrom macroglobulinemia cells compared with control B cells, and that the use of the Src inhibitor AZD0530 led to significant inhibition of adhesion, migration, and cytoskeletal signaling induced by SDF1. Moreover, inhibition of Src activity induced G(1) cell cycle arrest; however, it had minimal effect on survival of Waldenstrom macroglobulinemia cells, and no significant effect on survival of normal cells.

Conclusions: Taken together, these results delineate the role of Src kinase activity in Waldenstrom macroglobulinemia and provide the framework for future clinical trials using Src inhibitors in combination with other drugs to improve the outcome of patients with Waldenstrom macroglobulinemia.

Figure 1. Expressed of Src tyrosine kinase in WM and the effect of SDF1 and AZD0530 on its phosphorylation

(A) Immunoblotting for Src and pSrc expression in cell from four WM patients, BCWM1 cell line and CD19+ cells from healthy donors. (B) The effect of AZD0530 on Basal activation of Src in BCWM1. (C) Time dependent activation of Src by SDF1 in BCWM1. (D) The effect of AZD0530 on the SDF1-induced cytoskeletal-signaling. β-actin and tubulin served as loading control.

Figure 2. The role of Src in adhesion of WM to BM components

The effect of AZD0530 on (A) SDF1-induced adhesion of BCWM1 to fibronectin, (B) SDF1-induced adhesion of cells from three WM patients to fibronectin, and (C) adhesion of BCWM1 to BM stromal cells and endothelial cells. (A) *p<0.030, **p<0.013 (B) Patient-1 *p<0.010, **p<0.018; Patient-2 *p<0.029, **p<0.050), Patient-3 *p< 0.046, **p< 0.0160 (C) *p<0.026, **p<0.021.

Figure 3. The role of Src in SDF1-induced chemotaxis of WM to BM components

The effect of AZD0530 on SDF1-induced chemotaxis of (A) BCWM1 and (B) cells from three WM patients. (C) The effect of AZD0530 on SDF1-induced chemotaxis of BCWM1 in the presence and absence of the CXCR4 inhibitor AMD3100 (A) *p<0.035, **p<0.013 (B) Patient-1 *p<0.002, **p<0.003; Patient-2 *p<0.002, **p<0.008; Patient-3 *p< 0.015, **p< 0.050 (C) *p<0.007, **p<0.043, ***p<0.009, ****p<0.006.

Figure 4. The role of Src in SDF1-induced actin polymerization

Representative immune-fluorescence images showing that AZD0530 inhibited the SDF1-induced polymerization of actin in BCWM1 cells. Left column: polymerized actin detected by phalloidin (red), middle column: DAPI staining of cell nuclei (blue), and right column: the merge of the actin polymerization and nuclei images.

Figure 5. The role of Src in cell cycle regulation

(A) The effect of increasing concentrations of AZD0530 on the cell cycle of BCWM1 cells, showing dose-dependent G1-arrest. (B) The effect of AZD0530 on protein related to cell proliferation and cell cycle in BCWM1 cells detected by immune-blotting, showing a decrease of the activation proliferative and cell cycle promoting of proteins, and an increase of cell cycle inhibitory proteins.