FLT3 signals via the adapter protein Grb10 and overexpression of Grb10 leads to aberrant cell proliferation in acute myeloid leukemia

Abstract

The adaptor protein Grb10 plays important roles in mitogenic signaling. However, its roles in acute myeloid leukemia (AML) are predominantly unknown. Here we describe the role of Grb10 in FLT3-ITD-mediated AML. We observed that Grb10 physically associates with FLT3 in response to FLT3-ligand (FL) stimulation through FLT3 phospho-tyrosine 572 and 793 residues and constitutively associates with oncogenic FLT3-ITD. Furthermore endogenous Grb10-FLT3 association was observed in OCI-AML-5 cells. Grb10 expression did not alter FLT3 receptor activation or stability in Ba/F3-FLT3 cells. However, expression of Grb10 enhanced FL-induced Akt phosphorylation without affecting Erk or p38 phosphorylation in Ba/F3-FLT3-WT and Ba/F3-FLT3-ITD. Selective Grb10 depletion reduced Akt phosphorylation in Ba/F3-FLT3-WT and OCI-AML-5 cells. Grb10 transduces signal from FLT3 by direct interaction with p85 and Ba/F3-FLT3-ITD cells expressing Grb10 exhibits higher STAT5 activation. Grb10 regulates the cell cycle by increasing cell population in S-phase. Expression of Grb10 furthermore resulted in an increased proliferation and survival of Ba/F3-FLT3-ITD cells as well as increased colony formation in semisolid culture. Grb10 expression was significantly increased in AML patients compared to healthy controls and was also elevated in patients carrying FLT3-ITD mutants. The elevated Grb10 expression partially correlated to relapse as well as to poor prognosis. These results suggest that Grb10 binds to both normal and oncogenic FLT3 and induces PI3K-Akt and STAT5 signaling pathways resulting in an enhanced proliferation, survival and colony formation of hematopoietic cells.

Figure 1

Grb10 interacts with FLT3 in response to FL‐stimulation. (A) COS‐1 cells were co‐transfected with pcDNA3‐FLAG‐Grb10 and pcDNA3‐FLT3‐WT expression plasmids. Six hours after transfection, cells were serum‐starved overnight and then stimulated by FL or not for 5 min before lysis. Cell lysates were immunoprecipitated with an anti‐FLT3 or anti‐FLAG antibody followed by Western blotting analysis. (B) COS‐1 cells were co‐transfected with pcDNA3‐FLAG‐Grb10 and pcDNA3‐FLT3‐ITD expression plasmids. Six hours after transfection, cells were serum‐starved overnight and then stimulated by FL or not for 5 min before lysis. Cell lysates were immunoprecipitated with an anti‐FLT3 or anti‐FLAG antibody followed by Western blotting analysis. (C) OCI‐AML‐5 cells were lysed and subjected to the immunoprecipitation analysis with an anti‐FLT3 antibody. Then proteins were separated by SDS–PAGE followed by Western blotting analysis. (D) COS‐1 cells were transfected with pcDNA3‐FLAG‐Grb10 and pcDNA3‐FLT3‐K644A plasmids. Cells were then serum‐starved, stimulated with FL before lysis, immunoprecipitated and analyzed by Western blotting. (E) Grb10 and FLT3 transfected COS‐1 cells were stimulated for different time points after overnight starvation. Cells were then processed for immunoprecipitation and Western blotting analysis. (F) Amount of FLT3 from three individual experiments was quantified and normalized against Grb10 expression. (G) Levels of phospho‐Y‐FLT3 were quantified from the three individual experiments and plotted against relative binding. R2 values were calculated by GraphPad Prism 5.

Figure 2

Grb10–FLT3 interaction leads to Grb10 tyrosine phosphorylation. (A) COS‐1 cells were co‐transfected with pcDNA3‐FLAG‐Grb10 and/or pcDNA3‐FLT3‐WT expression plasmids. Six hours after transfection, cells were serum‐starved overnight and then stimulated by FL or not for 5 min before lysis. Cell lysates were used for Western blotting analysis. (B) Grb10 and FLT3 transfected COS‐1 cells were stimulated for different time points after overnight starvation. Cells were lysed and analyzed by Western blotting. (C) Levels of phospho‐Grb10 from three individual experiments were quantified and normalized against Grb10 expression. (D) Levels of phospho‐Y‐FLT3 and relative binding from three individual experiments were plotted against average phospho‐Grb10 values. (E) Grb10 and FLT3 transfected COS‐1 cells were treated with 10 μM PP1 or 15 μM PP2 for 60 min and stimulated for 5 min after overnight starvation. Cells were lysed and analyzed by Western blotting. R2 values were calculated by GraphPad Prism.

Figure 3

Grb10 interacts with FLT3 tyrosine 572 and 793 residues. (A) phospho‐peptides corresponding to 12 known tyrosine phosphorylation sites in FLT3 were immobilized on UltraLink. Peptide‐bound slurry was incubated with Grb10‐transfected COS‐1 cell lysates and after pulled‐down; proteins were processed for Western blotting using an anti‐Grb10 antibody. (B) COS‐1 cells were transfected with Grb10 and FLT3 wild‐type or mutant plasmids. Cells were then serum‐starved, stimulated with FL before lysis, immunoprecipitated and analyzed by Western blotting.

Figure 4

Expression of Grb10 did not alter receptor stability. (A) Ba/F3 cells were transfected with pcDNA3‐FLAG‐Grb10‐WT or empty vector by 4D‐nucleofector. One day after transfection cells were subjected to 0.8 mg/ml G‐418 selection. Expression levels were checked by Western blotting. (B) Grb10 or empty vector‐transfected Ba/F3 cells were further transfected with FLT3‐WT or FLT‐ITD retroviral plasmids (pMSCVpuro) and selected with 1.2 μg/ml of puromycin. Surface expression was assessed by flow cytometry with an anti‐FLT3 antibody. Dark area shows the FLT3 expression. (C) Cells were serum starved for 4 h and then stimulated with FL. Stimulation was stopped with cold PBS followed by lysis. Cell lysates were subjected to immunoprecipitation with an anti‐FLT3 antibody and then analyzed by Western blotting. Autoradiograms were quantified with Multi‐Gauge software. Statistical analysis was done using GraphPad Prism. (D) Cells were serum starved and treated with cycloheximide for 4 h. After FL‐stimulation cells were labeled with a PE‐conjugated anti‐FLT3 antibody and analyzed by flow cytometry. (E) Cells were serum starved and treated with cycloheximide for 4 h before FL‐stimulation. After stimulation cells were lysed and immunoprecipitated with an anti‐FLT3 antibody followed by Western blotting. Band intensity was measured by Multi‐Gauge software.

Figure 5

Grb10 increases Akt phosphorylation but not Erk phosphorylation. (A) Ba/F3‐FLT3‐WT cells transfected with Grb10 or empty vector were serum starved for 4 h and stimulated with FL for different time points. After lysis total cell lysate was analyzed by Western blotting. (B) Ba/F3‐FLT3‐WT cells were transfected with Grb10 shRNA or control shRNA by 4D‐nucleofector. One day after transfection cells were subjected to 0.8 mg/ml G‐418 selection. Expression levels were checked by Western blotting. (C) Ba/F3‐FLT3‐WT cells transfected with Grb10 shRNA or control shRNA were serum starved for 4 h and stimulated with FL for different time points. After lysis total cell lysate was analyzed by Western blotting. (D) Ba/F3‐FLT3‐ITD cells transfected with Grb10 or empty vector were serum starved for 4 h and stimulated with FL for different time points. After lysis total cell lysate was analyzed by Western blotting. (E) OCI‐AML‐5 cells were transfected with Grb10 siRNA or control siRNA by 4D‐nucleofector. Two days after transfection cells were lysed and Grb10 expression levels were checked by Western blotting. (F) OCI‐AML‐5 cells transfected with Grb10 siRNA or control siRNA were serum starved for overnight one day after transfection and stimulated with FL for different time points. After lysis total cell lysate was analyzed by Western blotting. (G) Autoradiograms from experiments (A, C, D, F) were quantified with Multi‐Gauge software. Statistical analysis was done using GraphPad Prism.

Figure 6

PI3K activity but not SFKs activity is required for Grb10‐mediated Akt activation. (A) Ba/F3‐FLT3‐WT/Grb10 cells were serum starved for 4 h and treated with 10 microM of either LY294002 (PI3K inhibitor), PP1, PP2 (Src inhibitor) or DMSO for 1 h followed by FL stimulation. Total cell lysates after lysis were analyzed by Western blotting. (B) Ba/F3‐FLT3‐WT/Grb10 cells were serum starved for 4 h and treated with Src inhibitor (PP1) or DMSO for 1 h followed by FL stimulation. Total cell lysates after lysis were analyzed by Western blotting. (C) Ba/F3‐FLT3‐WT/Grb10 cells were serum starved for 4 h and treated with Syk inhibitor II or DMSO for 1 h followed by FL stimulation. Total cell lysates after lysis were analyzed by Western blotting.

Figure 7

Grb10 transduces signal through interaction with p85. (A) Ba/F3‐FLT3‐WT cells transfected with Grb10 or empty vector were serum starved for 4 h and stimulated with FL for different time points. After lysis total cell lysate was analyzed by Western blotting. Autoradiograms were quantified with Multi‐Gauge software. Statistical analysis was done using GraphPad Prism. (B) Ba/F3‐FLT3‐WT cells transfected with Grb10 or empty vector were serum starved for 4 h and stimulated with FL for different time points. After lysis SHP2 protein was immunoprecipitated with an anti‐SHP2 antibody followed by Western blotting analysis. Autoradiograms were quantified with Multi‐Gauge software. Statistical analysis was done using GraphPad Prism. (C) COS‐1 cells transfected with FLT3 and Grb10 were serum starved for 4 h and stimulated with FL for 5 min. After lysis endogenous p85 was immunoprecipitated with an anti‐p85 antibody and analyzed by Western blotting. (A) OCI‐AML‐5 cells were serum starved for 4 h and stimulated with FL for 5 min. After lysis endogenous p85 was immunoprecipitated with an anti‐p85 antibody and analyzed by Western blotting.

Figure 8

Grb10 increases S‐phase cell population followed by an increased cell proliferation and survival. (A) Ba/F3‐FLT3‐ITD cells transfected with Grb10 or empty vector were cultured without any cytokine or 100 ng/ml FL for 46 h followed by 2 h EdU incubation. Cells were then fixed and labeled with Alexa Fluor 488 and 7‐AAD and then analyzed by flow cytometry. (B) Ba/F3‐FLT3‐WT cells transfected with Grb10 or empty vector were cultured without any cytokine or 100 ng/ml FL or IL‐3 for 48 h and then subjected to the trypan blue exclusion assay. (C) Ba/F3‐FLT3‐ITD cells transfected with Grb10 or empty vector were cultured without any cytokine or 100 ng/ml FL or IL‐3 for 48 h and then subjected to the trypan blue exclusion assay. (D) Cells were cultured without any cytokine or 100 ng/ml FL for 46 h followed by 2 h EdU incubation. Cells were then fixed and labeled with Alexa Fluor 647 and then analyzed by flow cytometry. (E) OCI‐AML‐5 cells were transfected with Grb10 siRNA or control siRNA were cultured without any cytokine or 100 ng/ml FL or IL‐3 for 72 h and then subjected to the trypan blue exclusion assay. (F) Ba/F3‐FLT3‐WT cells were cultured without any cytokine or 100 ng/ml FL for 48 h and then labeled with Annexin‐V‐PE and 7‐AAD followed by flow cytometry analysis. (G) Ba/F3‐FLT3‐ITD cells were cultured without any cytokine or 100 ng/ml FL for 48 h and then labeled with Annexin‐V‐PE and 7‐AAD followed by flow cytometry analysis.

Figure 9

Grb10 increases FLT3‐ITD‐induced colony formation as well as STAT5 activation. (A) Ba/F3‐FLT3‐ITD cells transfected with Grb10 or empty vector were serum starved for 4 h. After lysis STAT5 protein was immunoprecipitated with an anti‐STAT5 antibody followed by Western blotting analysis. (B) Ba/F3‐FLT3‐ITD cells transfected with Grb10 shRNA or control shRNA were serum starved for 4 h. After lysis STAT5 protein was immunoprecipitated with an anti‐STAT5 antibody followed by Western blotting analysis. (C) Autoradiograms from experiments (A, B) were quantified with Multi‐Gauge software. Statistical analysis was done using GraphPad Prism. (D) Ba/F3‐FLT3‐ITD cells were cultured without any cytokine or 100 ng/ml FL for 5 days in semisolid culture medium.

Figure 10

Grb10 expression leukemia patient samples. (A) Grb10 expression was analyzed from microarray data of 20 healthy donor and 26 AML patient samples. In CML dataset 17 patient samples were analyzed. Dataset from different experiments were normalized and scaled. (B) Grb10 expression was analyzed from the microarray dataset of acute promyelocytic leukemia patients carrying either wild‐type FLT3 or FLT3‐ITD mutation. (C) Microarray datasets of 54 pediatric AML patients having complete remission after treatment or relapsed, 21 patients having B‐cell chronic lymphocytic leukemia (B‐CLL) with indolent or progressive disease and 44 pediatric patients having acute lymphoblastic leukemia (ALL) with responsiveness or resistance to drug induced apoptosis were analyzed for Grb10 expression. (D) PTEN expression was analyzed from microarray data of 20 healthy donor and 26 AML patient samples. In CML dataset 17 patient samples were analyzed. Dataset from different experiments were normalized and scaled. Error bar shows SEM, and t‐test was performed to determine significance.