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Crosstalk between BCR/ABL oncoprotein and CXCR4 signaling through a Src family kinase in human leukemia cells.

Ptasznik A, Urbanowska E, Chinta S, Costa MA, Katz BA, Stanislaus MA, Demir G, Linnekin D, Pan ZK, Gewirtz AM - J. Exp. Med. (2002)

Bottom Line: Thus, BCR/ABL perturbs Lyn function through a tyrosine kinase-dependent mechanism.Accordingly, the blockade of Lyn tyrosine kinase inhibits both BCR/ABL-dependent and CXCR4-dependent cell movements.These results define a Src tyrosine kinases-dependent mechanism whereby BCR/ABL (and potentially other oncoproteins) dysregulates G protein-coupled receptor signaling and function of mammalian precursors.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6100, USA. andrzejp2@cs.com

ABSTRACT
Stromal-derived factor (SDF)-1 and its G protein-coupled receptor, CXCR4, regulate stem/progenitor cell migration and retention in the marrow and are required for hematopoiesis. We show here an interaction between CXCR4 and the Src-related kinase, Lyn, in normal progenitors. We demonstrate that CXCR4-dependent stimulation of Lyn is associated with the activation of phosphatidylinositol 3-kinase (PI3-kinase). This chemokine signaling, which involves a Src-related kinase and PI3-kinase, appears to be a target for BCR/ABL, a fusion oncoprotein expressed only in leukemia cells. We show that the binding of phosphorylated BCR/ABL to Lyn results in the constitutive activation of Lyn and PI3-kinase, along with a total loss of responsiveness of these kinases to SDF-1 stimulation. Inhibition of BCR/ABL tyrosine kinase with STI571 restores Lyn responsiveness to SDF-1 signaling. Thus, BCR/ABL perturbs Lyn function through a tyrosine kinase-dependent mechanism. Accordingly, the blockade of Lyn tyrosine kinase inhibits both BCR/ABL-dependent and CXCR4-dependent cell movements. Our results demonstrate, for the first time, that Lyn-mediated pathological crosstalk exists between BCR/ABL and the CXCR4 pathway in leukemia cells, which disrupts chemokine signaling and chemotaxis, and increases the ability of immature cells to escape from the marrow. These results define a Src tyrosine kinases-dependent mechanism whereby BCR/ABL (and potentially other oncoproteins) dysregulates G protein-coupled receptor signaling and function of mammalian precursors.

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Effect of PP2 on CXCR4-dependent and BCR/ABL-dependent cell movements. Inhibition of CXCR4-dependent cell movements in Lyn-deficient primary cells. (A) The inhibition of SDF-1–regulated chemotaxis by PP2 was determined as described under Materials and Methods and Results in BCR/ABL-negative HL-60 cells (MigR1) and BCR/ABL-positive HL-60 cells (Mig210). The initial number of BCR/ABL-negative MigR1 cells that migrated to medium alone was set to 100%. The results shown (mean ± SD) are averages of six experiments. (B) The inhibition of SDF-1–regulated chemotaxis by PP2 was determined in hematopoietic precursor cells Mo7e as described under Materials and Methods and Results. The results shown are representative of six experiments. (C) The suppression of CXCR4-dependent chemotaxis by PP2, but not by its inactive analogue PP3, in CD34+ human primary myeloid cells. PMA-induced migration is normal in PP2-pretreated CD34+ cells. Treatment and migration were performed as described in Materials and Methods. The results shown represent the average ± range of four separate determinations with different normal cell donors. (D) The reduction of SDF-1-induced migration in Lyn-deficient mononuclear bone marrow cells from knock-out mice. PMA-induced migration appears to be normal in these cells. Data represents the mean and standard deviation of eight samples with eight different animals. Treatment with factors and migration assays were performed as described in Materials and Methods.
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fig5: Effect of PP2 on CXCR4-dependent and BCR/ABL-dependent cell movements. Inhibition of CXCR4-dependent cell movements in Lyn-deficient primary cells. (A) The inhibition of SDF-1–regulated chemotaxis by PP2 was determined as described under Materials and Methods and Results in BCR/ABL-negative HL-60 cells (MigR1) and BCR/ABL-positive HL-60 cells (Mig210). The initial number of BCR/ABL-negative MigR1 cells that migrated to medium alone was set to 100%. The results shown (mean ± SD) are averages of six experiments. (B) The inhibition of SDF-1–regulated chemotaxis by PP2 was determined in hematopoietic precursor cells Mo7e as described under Materials and Methods and Results. The results shown are representative of six experiments. (C) The suppression of CXCR4-dependent chemotaxis by PP2, but not by its inactive analogue PP3, in CD34+ human primary myeloid cells. PMA-induced migration is normal in PP2-pretreated CD34+ cells. Treatment and migration were performed as described in Materials and Methods. The results shown represent the average ± range of four separate determinations with different normal cell donors. (D) The reduction of SDF-1-induced migration in Lyn-deficient mononuclear bone marrow cells from knock-out mice. PMA-induced migration appears to be normal in these cells. Data represents the mean and standard deviation of eight samples with eight different animals. Treatment with factors and migration assays were performed as described in Materials and Methods.

Mentions: As shown in Fig. 5 A the initial number of BCR/ABL-negative MigR1 cells that migrated to medium alone was arbitrarily set to 100%. The number of MigR1 cells that migrated to medium plus SDF-1 was approximately fivefold higher. Similar results were obtained using another Lyn-expressing (unpublished data), BCR/ABL-negative hematopoietic cell line, M07e (Fig. 5 B). In contrast, BCR/ABL-positive Mig210 cells had a strong chemotactic response to both medium alone or medium plus SDF-1. The presence of SDF-1 in the medium had no significant effect on Mig210 cell migration. This result indicates that BCR/ABL oncoprotein blocks CXCR4-regulated chemotaxis, but increases spontaneous cell motility. Chemotaxis, both CXCR4-dependent (in MigR1 or M07e cells) and BCR/ABL-dependent (in Mig210 cells), was effectively blocked by PP2 pretreatment. It indicates that a Src family kinase is required for chemotaxis and is shared between CXCR4 signaling and BCR/ABL signaling in hematopoietic cells. These results are highly consistent with participation of Lyn in BCR/ABL and CXCR4 signal transduction as demonstrated in Figs. 1–4.


Crosstalk between BCR/ABL oncoprotein and CXCR4 signaling through a Src family kinase in human leukemia cells.

Ptasznik A, Urbanowska E, Chinta S, Costa MA, Katz BA, Stanislaus MA, Demir G, Linnekin D, Pan ZK, Gewirtz AM - J. Exp. Med. (2002)

Effect of PP2 on CXCR4-dependent and BCR/ABL-dependent cell movements. Inhibition of CXCR4-dependent cell movements in Lyn-deficient primary cells. (A) The inhibition of SDF-1–regulated chemotaxis by PP2 was determined as described under Materials and Methods and Results in BCR/ABL-negative HL-60 cells (MigR1) and BCR/ABL-positive HL-60 cells (Mig210). The initial number of BCR/ABL-negative MigR1 cells that migrated to medium alone was set to 100%. The results shown (mean ± SD) are averages of six experiments. (B) The inhibition of SDF-1–regulated chemotaxis by PP2 was determined in hematopoietic precursor cells Mo7e as described under Materials and Methods and Results. The results shown are representative of six experiments. (C) The suppression of CXCR4-dependent chemotaxis by PP2, but not by its inactive analogue PP3, in CD34+ human primary myeloid cells. PMA-induced migration is normal in PP2-pretreated CD34+ cells. Treatment and migration were performed as described in Materials and Methods. The results shown represent the average ± range of four separate determinations with different normal cell donors. (D) The reduction of SDF-1-induced migration in Lyn-deficient mononuclear bone marrow cells from knock-out mice. PMA-induced migration appears to be normal in these cells. Data represents the mean and standard deviation of eight samples with eight different animals. Treatment with factors and migration assays were performed as described in Materials and Methods.
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fig5: Effect of PP2 on CXCR4-dependent and BCR/ABL-dependent cell movements. Inhibition of CXCR4-dependent cell movements in Lyn-deficient primary cells. (A) The inhibition of SDF-1–regulated chemotaxis by PP2 was determined as described under Materials and Methods and Results in BCR/ABL-negative HL-60 cells (MigR1) and BCR/ABL-positive HL-60 cells (Mig210). The initial number of BCR/ABL-negative MigR1 cells that migrated to medium alone was set to 100%. The results shown (mean ± SD) are averages of six experiments. (B) The inhibition of SDF-1–regulated chemotaxis by PP2 was determined in hematopoietic precursor cells Mo7e as described under Materials and Methods and Results. The results shown are representative of six experiments. (C) The suppression of CXCR4-dependent chemotaxis by PP2, but not by its inactive analogue PP3, in CD34+ human primary myeloid cells. PMA-induced migration is normal in PP2-pretreated CD34+ cells. Treatment and migration were performed as described in Materials and Methods. The results shown represent the average ± range of four separate determinations with different normal cell donors. (D) The reduction of SDF-1-induced migration in Lyn-deficient mononuclear bone marrow cells from knock-out mice. PMA-induced migration appears to be normal in these cells. Data represents the mean and standard deviation of eight samples with eight different animals. Treatment with factors and migration assays were performed as described in Materials and Methods.
Mentions: As shown in Fig. 5 A the initial number of BCR/ABL-negative MigR1 cells that migrated to medium alone was arbitrarily set to 100%. The number of MigR1 cells that migrated to medium plus SDF-1 was approximately fivefold higher. Similar results were obtained using another Lyn-expressing (unpublished data), BCR/ABL-negative hematopoietic cell line, M07e (Fig. 5 B). In contrast, BCR/ABL-positive Mig210 cells had a strong chemotactic response to both medium alone or medium plus SDF-1. The presence of SDF-1 in the medium had no significant effect on Mig210 cell migration. This result indicates that BCR/ABL oncoprotein blocks CXCR4-regulated chemotaxis, but increases spontaneous cell motility. Chemotaxis, both CXCR4-dependent (in MigR1 or M07e cells) and BCR/ABL-dependent (in Mig210 cells), was effectively blocked by PP2 pretreatment. It indicates that a Src family kinase is required for chemotaxis and is shared between CXCR4 signaling and BCR/ABL signaling in hematopoietic cells. These results are highly consistent with participation of Lyn in BCR/ABL and CXCR4 signal transduction as demonstrated in Figs. 1–4.

Bottom Line: Thus, BCR/ABL perturbs Lyn function through a tyrosine kinase-dependent mechanism.Accordingly, the blockade of Lyn tyrosine kinase inhibits both BCR/ABL-dependent and CXCR4-dependent cell movements.These results define a Src tyrosine kinases-dependent mechanism whereby BCR/ABL (and potentially other oncoproteins) dysregulates G protein-coupled receptor signaling and function of mammalian precursors.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6100, USA. andrzejp2@cs.com

ABSTRACT
Stromal-derived factor (SDF)-1 and its G protein-coupled receptor, CXCR4, regulate stem/progenitor cell migration and retention in the marrow and are required for hematopoiesis. We show here an interaction between CXCR4 and the Src-related kinase, Lyn, in normal progenitors. We demonstrate that CXCR4-dependent stimulation of Lyn is associated with the activation of phosphatidylinositol 3-kinase (PI3-kinase). This chemokine signaling, which involves a Src-related kinase and PI3-kinase, appears to be a target for BCR/ABL, a fusion oncoprotein expressed only in leukemia cells. We show that the binding of phosphorylated BCR/ABL to Lyn results in the constitutive activation of Lyn and PI3-kinase, along with a total loss of responsiveness of these kinases to SDF-1 stimulation. Inhibition of BCR/ABL tyrosine kinase with STI571 restores Lyn responsiveness to SDF-1 signaling. Thus, BCR/ABL perturbs Lyn function through a tyrosine kinase-dependent mechanism. Accordingly, the blockade of Lyn tyrosine kinase inhibits both BCR/ABL-dependent and CXCR4-dependent cell movements. Our results demonstrate, for the first time, that Lyn-mediated pathological crosstalk exists between BCR/ABL and the CXCR4 pathway in leukemia cells, which disrupts chemokine signaling and chemotaxis, and increases the ability of immature cells to escape from the marrow. These results define a Src tyrosine kinases-dependent mechanism whereby BCR/ABL (and potentially other oncoproteins) dysregulates G protein-coupled receptor signaling and function of mammalian precursors.

Show MeSH
Related in: MedlinePlus