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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 exclusively induces factor independency and STAT5/6 phosphorylation in myeloid and B-lymphoid cells. (a) Expression of SYKwt, ITK–SYK and TEL–SYK after retroviral infection and GFP-sorting of the IL-3 dependent cell lines BaF3 and 32D. For detection of SYKwt and ITK–SYK by western blot, a primary SYK antibody was used. For detection of TEL–SYK, we used a TEL antibody. (b) Growth curves for IL-3-depleted BaF3 and 32D cells expressing the different oncogenes over 5 days were performed by counting Trypan blue negative cells via Neubauer chamber. (c) Intracellular phospho flow cytometry for phosphorylated STAT5/6, PLCγ2 and SLP76 in BaF3 cells (upper panel) and 32D cells (lower panel) expressing the different constructs. (d) Western blot of BaF3 and 32D cells expressing either control vector or the different SYK oncogenes with antibodies against phosphorylated STATs, PLCγ, JAK2, SLP76, ERK and AKT. Left panel shows the phosphorylated protein, right panel the total protein.
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fig6: TEL–SYK exclusively induces factor independency and STAT5/6 phosphorylation in myeloid and B-lymphoid cells. (a) Expression of SYKwt, ITK–SYK and TEL–SYK after retroviral infection and GFP-sorting of the IL-3 dependent cell lines BaF3 and 32D. For detection of SYKwt and ITK–SYK by western blot, a primary SYK antibody was used. For detection of TEL–SYK, we used a TEL antibody. (b) Growth curves for IL-3-depleted BaF3 and 32D cells expressing the different oncogenes over 5 days were performed by counting Trypan blue negative cells via Neubauer chamber. (c) Intracellular phospho flow cytometry for phosphorylated STAT5/6, PLCγ2 and SLP76 in BaF3 cells (upper panel) and 32D cells (lower panel) expressing the different constructs. (d) Western blot of BaF3 and 32D cells expressing either control vector or the different SYK oncogenes with antibodies against phosphorylated STATs, PLCγ, JAK2, SLP76, ERK and AKT. Left panel shows the phosphorylated protein, right panel the total protein.

Mentions: To further examine the differences in signal transduction between TEL–SYK, SYKwt and ITK-SYK dependent on the background of the transformed cell, we overexpressed the oncogenes in myeloid (32D cells) and B-lymphoid (BaF3 cells) cell lines (Figure 6a).


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 exclusively induces factor independency and STAT5/6 phosphorylation in myeloid and B-lymphoid cells. (a) Expression of SYKwt, ITK–SYK and TEL–SYK after retroviral infection and GFP-sorting of the IL-3 dependent cell lines BaF3 and 32D. For detection of SYKwt and ITK–SYK by western blot, a primary SYK antibody was used. For detection of TEL–SYK, we used a TEL antibody. (b) Growth curves for IL-3-depleted BaF3 and 32D cells expressing the different oncogenes over 5 days were performed by counting Trypan blue negative cells via Neubauer chamber. (c) Intracellular phospho flow cytometry for phosphorylated STAT5/6, PLCγ2 and SLP76 in BaF3 cells (upper panel) and 32D cells (lower panel) expressing the different constructs. (d) Western blot of BaF3 and 32D cells expressing either control vector or the different SYK oncogenes with antibodies against phosphorylated STATs, PLCγ, JAK2, SLP76, ERK and AKT. Left panel shows the phosphorylated protein, right panel the total protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219468&req=5

fig6: TEL–SYK exclusively induces factor independency and STAT5/6 phosphorylation in myeloid and B-lymphoid cells. (a) Expression of SYKwt, ITK–SYK and TEL–SYK after retroviral infection and GFP-sorting of the IL-3 dependent cell lines BaF3 and 32D. For detection of SYKwt and ITK–SYK by western blot, a primary SYK antibody was used. For detection of TEL–SYK, we used a TEL antibody. (b) Growth curves for IL-3-depleted BaF3 and 32D cells expressing the different oncogenes over 5 days were performed by counting Trypan blue negative cells via Neubauer chamber. (c) Intracellular phospho flow cytometry for phosphorylated STAT5/6, PLCγ2 and SLP76 in BaF3 cells (upper panel) and 32D cells (lower panel) expressing the different constructs. (d) Western blot of BaF3 and 32D cells expressing either control vector or the different SYK oncogenes with antibodies against phosphorylated STATs, PLCγ, JAK2, SLP76, ERK and AKT. Left panel shows the phosphorylated protein, right panel the total protein.
Mentions: To further examine the differences in signal transduction between TEL–SYK, SYKwt and ITK-SYK dependent on the background of the transformed cell, we overexpressed the oncogenes in myeloid (32D cells) and B-lymphoid (BaF3 cells) cell lines (Figure 6a).

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