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Depletion of STAT5 blocks TEL-SYK-induced APMF-type leukemia with myelofibrosis and myelodysplasia in mice.

Sprissler C, Belenki D, Maurer H, Aumann K, Pfeifer D, Klein C, Müller TA, Kissel S, Hülsdünker J, Alexandrovski J, Brummer T, Jumaa H, Duyster J, Dierks C - Blood Cancer J (2014)

Bottom Line: LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates.SYK inhibitor treatment (R788) of diseased TEL-SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis.Stat5 was identified as a major downstream mediator of TEL-SYK in vitro as well as in vivo.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Hematology/Oncology, University Medical Center Freiburg, Freiburg, Germany [2] University of Freiburg, Schaenzlestrasse 1, Freiburg, Germany.

ABSTRACT
The spleen tyrosine kinase (SYK) was identified as an oncogenic driver in a broad spectrum of hematologic malignancies. The in vivo comparison of three SYK containing oncogenes, SYK(wt), TEL-SYK and IL-2-inducible T-cell kinase (ITK)-SYK revealed a general myeloexpansion and the establishment of three different hematologic (pre)diseases. SYK(wt) enhanced the myeloid and T-cell compartment, without leukemia/lymphoma development. ITK-SYK caused lethal T-cell lymphomas and the cytoplasmic TEL-SYK fusion induced an acute panmyelosis with myelofibrosis-type acute myeloid leukemia (AML) with up to 50% immature megakaryoblasts infiltrating bone marrow, spleen and liver, additional MPN features (myelofibrosis and granulocyte expansion) and MDS stigmata with megakaryocytic and erythroid dysplasia. LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates. SYK inhibitor treatment (R788) of diseased TEL-SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis. Stat5 was identified as a major downstream mediator of TEL-SYK in vitro as well as in vivo. Consequently, targeted deletion of Stat5 in vivo completely abrogated TEL-SYK-induced AML and myelofibrosis development, proving Stat5 as a major driver of SYK-induced transformation. Our experiments highlight the important role of SYK in AML and myelofibrosis and prove SYK and STAT5 inhibitors as potent treatment options for those diseases.

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TEL–SYK induces activation of STAT5 in vivo. (a) Intracellular phospho flow cytometry for p-STAT5/6, p-PLCγ2 and p-SLP76 of GFP+ myeloid spleen cells coming from control, SYKwt, ITK–SYK and TEL–SYK-transplanted Balb/c mice. Graphs show the mean fluorescence intensity (MFI) per mouse. TEL–SYK shows highest activation of STAT5 and STAT6 in vivo, whereas SYK and ITK–SYK show a strong activation of PLCγ2 and SLP76. *P<0.05; **P<0.01; ***P<0.001, unpaired t-test. (b) Intracellular phospho-flow cytometry for phosphorylated STAT5/6, PLCγ2 and SLP76 of 32D TEL–SYK cells, KG-1 cells (AML cell line) and SET-2 cells (megakaryocytic leukemia cell line) treated with the SYK inhibitor R406 for 5 min, 30 min or 2 h. For p-STAT6, p-PLCγ2 and p-SLP76 only the 30 min time point is shown. (c) 32D cells with IL-3, 32D TEL–SYK cells without IL-3, KG-1 cells and SET-2 cells were all treated with R406 for 48 h and apoptosis was assessed by AnnexinV/7-AAD staining. Graph shows the mean relative percentage of viable cells (AnnexinV-/7-AAD-) compared with the dimethylsulfoxide (DMSO) control+s.e.m from three independent experiments (n=3). (d) Total viable cell numbers were assessed by counting the cells with a Neubauer counting chamber after treatment with SYK inhibitor over 48–72 h. Graphs are shown for the DMSO control, the 2 μM and the 4 μM R406 concentration for 32D cells+IL-3, 32D TEL–SYK cells without IL-3, KG-1 cells and SET-2 cells. (e) 32D BCR–ABL and 32D TEL–SYK cells grown without IL-3 were treated with 5 and 10 μM pimozide for 48 h and apoptosis was assessed by AnnexinV/7-AAD staining. Graph shows the mean relative percentage of viable cells (AnnexinV-/7-AAD-) compared with the DMSO control+s.e.m from three independent experiments (n=3). *P<0.05; **P<0.01; ***P<0.001, paired t-test. (f) Growth curves of 32D BCR–ABL and 32D TEL–SYK cells grown without IL-3 and treated with DMSO, 5 and 10 μM pimozide were performed over 48 h in three independent experiments (n=3) by counting Trypan blue negative cells in a Neubauer chamber.
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fig7: TEL–SYK induces activation of STAT5 in vivo. (a) Intracellular phospho flow cytometry for p-STAT5/6, p-PLCγ2 and p-SLP76 of GFP+ myeloid spleen cells coming from control, SYKwt, ITK–SYK and TEL–SYK-transplanted Balb/c mice. Graphs show the mean fluorescence intensity (MFI) per mouse. TEL–SYK shows highest activation of STAT5 and STAT6 in vivo, whereas SYK and ITK–SYK show a strong activation of PLCγ2 and SLP76. *P<0.05; **P<0.01; ***P<0.001, unpaired t-test. (b) Intracellular phospho-flow cytometry for phosphorylated STAT5/6, PLCγ2 and SLP76 of 32D TEL–SYK cells, KG-1 cells (AML cell line) and SET-2 cells (megakaryocytic leukemia cell line) treated with the SYK inhibitor R406 for 5 min, 30 min or 2 h. For p-STAT6, p-PLCγ2 and p-SLP76 only the 30 min time point is shown. (c) 32D cells with IL-3, 32D TEL–SYK cells without IL-3, KG-1 cells and SET-2 cells were all treated with R406 for 48 h and apoptosis was assessed by AnnexinV/7-AAD staining. Graph shows the mean relative percentage of viable cells (AnnexinV-/7-AAD-) compared with the dimethylsulfoxide (DMSO) control+s.e.m from three independent experiments (n=3). (d) Total viable cell numbers were assessed by counting the cells with a Neubauer counting chamber after treatment with SYK inhibitor over 48–72 h. Graphs are shown for the DMSO control, the 2 μM and the 4 μM R406 concentration for 32D cells+IL-3, 32D TEL–SYK cells without IL-3, KG-1 cells and SET-2 cells. (e) 32D BCR–ABL and 32D TEL–SYK cells grown without IL-3 were treated with 5 and 10 μM pimozide for 48 h and apoptosis was assessed by AnnexinV/7-AAD staining. Graph shows the mean relative percentage of viable cells (AnnexinV-/7-AAD-) compared with the DMSO control+s.e.m from three independent experiments (n=3). *P<0.05; **P<0.01; ***P<0.001, paired t-test. (f) Growth curves of 32D BCR–ABL and 32D TEL–SYK cells grown without IL-3 and treated with DMSO, 5 and 10 μM pimozide were performed over 48 h in three independent experiments (n=3) by counting Trypan blue negative cells in a Neubauer chamber.

Mentions: To verify if any of those pathways activated by the SYK oncogenes in vitro is also relevant in vivo, we analyzed BM and spleen cells of SYK oncogene carrying mice by using phospho-flow cytometry of GFP+ cells. TEL–SYK positive murine myeloid cells showed a strongly increased phosphorylation of STAT5 and STAT6 in vivo, supporting the in vitro results (Figure 7a). In contrast, other phosphorylation events like PLCγ2 activation could not be confirmed in vivo and SLP76 phosphorylation was only moderately increased. The phosphorylation pattern of SYKwt cells was completely different, with a strongly increased phosphorylation of PLCγ2 and SLP76, but not STAT5 and STAT6, which is in line with the in vitro results. According to those findings, we hypothesized that STAT5 and STAT6 phosphorylation, but not SLP76 and PLCγ2, make the difference in between malignant transformation caused by TEL–SYK or only myeloid expansion as caused by SYKwt. STAT5 is known to be a major player in oncogenic transformation of myeloid cells and is activated in myeloproliferative diseases and different types of AML. To verify that STAT5 is directly regulated by the oncogenic TEL–SYK kinase fusion in myeloid cells, we treated TEL–SYK 32D cells with the SYK inhibitor R406 for up to 2 h. SYK inhibition in TEL–SYK 32D cells resulted in a fast dephosphorylation of STAT5 within 10 min and induction of apoptosis and reduced proliferation in the absence of IL-3 (Figures 7b–d). SYK inhibition did not reduce STAT6 phosphorylation nor phosphorylation of SLP76 or PLCγ2 within these 2 h, pointing towards those genes as secondary phosphorylation events.


Depletion of STAT5 blocks TEL-SYK-induced APMF-type leukemia with myelofibrosis and myelodysplasia in mice.

Sprissler C, Belenki D, Maurer H, Aumann K, Pfeifer D, Klein C, Müller TA, Kissel S, Hülsdünker J, Alexandrovski J, Brummer T, Jumaa H, Duyster J, Dierks C - Blood Cancer J (2014)

TEL–SYK induces activation of STAT5 in vivo. (a) Intracellular phospho flow cytometry for p-STAT5/6, p-PLCγ2 and p-SLP76 of GFP+ myeloid spleen cells coming from control, SYKwt, ITK–SYK and TEL–SYK-transplanted Balb/c mice. Graphs show the mean fluorescence intensity (MFI) per mouse. TEL–SYK shows highest activation of STAT5 and STAT6 in vivo, whereas SYK and ITK–SYK show a strong activation of PLCγ2 and SLP76. *P<0.05; **P<0.01; ***P<0.001, unpaired t-test. (b) Intracellular phospho-flow cytometry for phosphorylated STAT5/6, PLCγ2 and SLP76 of 32D TEL–SYK cells, KG-1 cells (AML cell line) and SET-2 cells (megakaryocytic leukemia cell line) treated with the SYK inhibitor R406 for 5 min, 30 min or 2 h. For p-STAT6, p-PLCγ2 and p-SLP76 only the 30 min time point is shown. (c) 32D cells with IL-3, 32D TEL–SYK cells without IL-3, KG-1 cells and SET-2 cells were all treated with R406 for 48 h and apoptosis was assessed by AnnexinV/7-AAD staining. Graph shows the mean relative percentage of viable cells (AnnexinV-/7-AAD-) compared with the dimethylsulfoxide (DMSO) control+s.e.m from three independent experiments (n=3). (d) Total viable cell numbers were assessed by counting the cells with a Neubauer counting chamber after treatment with SYK inhibitor over 48–72 h. Graphs are shown for the DMSO control, the 2 μM and the 4 μM R406 concentration for 32D cells+IL-3, 32D TEL–SYK cells without IL-3, KG-1 cells and SET-2 cells. (e) 32D BCR–ABL and 32D TEL–SYK cells grown without IL-3 were treated with 5 and 10 μM pimozide for 48 h and apoptosis was assessed by AnnexinV/7-AAD staining. Graph shows the mean relative percentage of viable cells (AnnexinV-/7-AAD-) compared with the DMSO control+s.e.m from three independent experiments (n=3). *P<0.05; **P<0.01; ***P<0.001, paired t-test. (f) Growth curves of 32D BCR–ABL and 32D TEL–SYK cells grown without IL-3 and treated with DMSO, 5 and 10 μM pimozide were performed over 48 h in three independent experiments (n=3) by counting Trypan blue negative cells in a Neubauer chamber.
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Related In: Results  -  Collection

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Show All Figures
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fig7: TEL–SYK induces activation of STAT5 in vivo. (a) Intracellular phospho flow cytometry for p-STAT5/6, p-PLCγ2 and p-SLP76 of GFP+ myeloid spleen cells coming from control, SYKwt, ITK–SYK and TEL–SYK-transplanted Balb/c mice. Graphs show the mean fluorescence intensity (MFI) per mouse. TEL–SYK shows highest activation of STAT5 and STAT6 in vivo, whereas SYK and ITK–SYK show a strong activation of PLCγ2 and SLP76. *P<0.05; **P<0.01; ***P<0.001, unpaired t-test. (b) Intracellular phospho-flow cytometry for phosphorylated STAT5/6, PLCγ2 and SLP76 of 32D TEL–SYK cells, KG-1 cells (AML cell line) and SET-2 cells (megakaryocytic leukemia cell line) treated with the SYK inhibitor R406 for 5 min, 30 min or 2 h. For p-STAT6, p-PLCγ2 and p-SLP76 only the 30 min time point is shown. (c) 32D cells with IL-3, 32D TEL–SYK cells without IL-3, KG-1 cells and SET-2 cells were all treated with R406 for 48 h and apoptosis was assessed by AnnexinV/7-AAD staining. Graph shows the mean relative percentage of viable cells (AnnexinV-/7-AAD-) compared with the dimethylsulfoxide (DMSO) control+s.e.m from three independent experiments (n=3). (d) Total viable cell numbers were assessed by counting the cells with a Neubauer counting chamber after treatment with SYK inhibitor over 48–72 h. Graphs are shown for the DMSO control, the 2 μM and the 4 μM R406 concentration for 32D cells+IL-3, 32D TEL–SYK cells without IL-3, KG-1 cells and SET-2 cells. (e) 32D BCR–ABL and 32D TEL–SYK cells grown without IL-3 were treated with 5 and 10 μM pimozide for 48 h and apoptosis was assessed by AnnexinV/7-AAD staining. Graph shows the mean relative percentage of viable cells (AnnexinV-/7-AAD-) compared with the DMSO control+s.e.m from three independent experiments (n=3). *P<0.05; **P<0.01; ***P<0.001, paired t-test. (f) Growth curves of 32D BCR–ABL and 32D TEL–SYK cells grown without IL-3 and treated with DMSO, 5 and 10 μM pimozide were performed over 48 h in three independent experiments (n=3) by counting Trypan blue negative cells in a Neubauer chamber.
Mentions: To verify if any of those pathways activated by the SYK oncogenes in vitro is also relevant in vivo, we analyzed BM and spleen cells of SYK oncogene carrying mice by using phospho-flow cytometry of GFP+ cells. TEL–SYK positive murine myeloid cells showed a strongly increased phosphorylation of STAT5 and STAT6 in vivo, supporting the in vitro results (Figure 7a). In contrast, other phosphorylation events like PLCγ2 activation could not be confirmed in vivo and SLP76 phosphorylation was only moderately increased. The phosphorylation pattern of SYKwt cells was completely different, with a strongly increased phosphorylation of PLCγ2 and SLP76, but not STAT5 and STAT6, which is in line with the in vitro results. According to those findings, we hypothesized that STAT5 and STAT6 phosphorylation, but not SLP76 and PLCγ2, make the difference in between malignant transformation caused by TEL–SYK or only myeloid expansion as caused by SYKwt. STAT5 is known to be a major player in oncogenic transformation of myeloid cells and is activated in myeloproliferative diseases and different types of AML. To verify that STAT5 is directly regulated by the oncogenic TEL–SYK kinase fusion in myeloid cells, we treated TEL–SYK 32D cells with the SYK inhibitor R406 for up to 2 h. SYK inhibition in TEL–SYK 32D cells resulted in a fast dephosphorylation of STAT5 within 10 min and induction of apoptosis and reduced proliferation in the absence of IL-3 (Figures 7b–d). SYK inhibition did not reduce STAT6 phosphorylation nor phosphorylation of SLP76 or PLCγ2 within these 2 h, pointing towards those genes as secondary phosphorylation events.

Bottom Line: LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates.SYK inhibitor treatment (R788) of diseased TEL-SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis.Stat5 was identified as a major downstream mediator of TEL-SYK in vitro as well as in vivo.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Hematology/Oncology, University Medical Center Freiburg, Freiburg, Germany [2] University of Freiburg, Schaenzlestrasse 1, Freiburg, Germany.

ABSTRACT
The spleen tyrosine kinase (SYK) was identified as an oncogenic driver in a broad spectrum of hematologic malignancies. The in vivo comparison of three SYK containing oncogenes, SYK(wt), TEL-SYK and IL-2-inducible T-cell kinase (ITK)-SYK revealed a general myeloexpansion and the establishment of three different hematologic (pre)diseases. SYK(wt) enhanced the myeloid and T-cell compartment, without leukemia/lymphoma development. ITK-SYK caused lethal T-cell lymphomas and the cytoplasmic TEL-SYK fusion induced an acute panmyelosis with myelofibrosis-type acute myeloid leukemia (AML) with up to 50% immature megakaryoblasts infiltrating bone marrow, spleen and liver, additional MPN features (myelofibrosis and granulocyte expansion) and MDS stigmata with megakaryocytic and erythroid dysplasia. LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates. SYK inhibitor treatment (R788) of diseased TEL-SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis. Stat5 was identified as a major downstream mediator of TEL-SYK in vitro as well as in vivo. Consequently, targeted deletion of Stat5 in vivo completely abrogated TEL-SYK-induced AML and myelofibrosis development, proving Stat5 as a major driver of SYK-induced transformation. Our experiments highlight the important role of SYK in AML and myelofibrosis and prove SYK and STAT5 inhibitors as potent treatment options for those diseases.

Show MeSH
Related in: MedlinePlus