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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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Depletion of Stat5 blocks TEL–SYK-induced leukemia and myelofibrosis. (a) Western blot showing depletion of Stat5 in Stat5fl/flMx1Cre BMCs versus control Stat5fl/fl BMCs from mice treated with three times injection of Poly (I:C) and 35 days after transplantation. (b) Intracellular phospho-Stat5 staining was assessed by flow cytometry from BMCs from mice treated as in A (n=4 per group). (c) Photograps showing spleens of three TEL–SYK control and three TEL–SYK Stat5−/− mice in comparison after 35 days. (d) Spleen and liver weights from TEL–SYK control and TEL–SYK Stat5−/− mice. (e) Peripheral blood of TEL–SYK control and TEL–SYK Stat5−/− mice (n=4) was analyzed for red blood cell count, hematocrit, WBC count, thrombocytes and granulocytes 35 days post transplantation with the ADVIA120 blood analyzer. Graphs show results for total WBCs and the percentage of granulocytes. (f) Photographs from blood smears of control TEL–SYK and TEL–SYK Stat5−/− mice, which were stained with May-Grünwald/Giemsa. Bars, 50 μm. (g) GFP contents, CD11b+/GFP+ expression, CD90+3+/GFP+ expression and Ter119+/GFP+ expression within total live cells was assessed by flow cytometry analysis in BM and spleen cells from TEL–SYK control and TEL–SYK Stat5−/− mice 35 days after transplantation (n=4). *P<0.05; **P<0.01; ***P<0.001, unpaired t-test. (h) Representative images showing hematoxylin/eosin and reticulin (left image) stained BM, spleen and liver slides from TEL–SYK control and TEL–SYK Stat5−/− mice. Bars, 50 μm.
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fig8: Depletion of Stat5 blocks TEL–SYK-induced leukemia and myelofibrosis. (a) Western blot showing depletion of Stat5 in Stat5fl/flMx1Cre BMCs versus control Stat5fl/fl BMCs from mice treated with three times injection of Poly (I:C) and 35 days after transplantation. (b) Intracellular phospho-Stat5 staining was assessed by flow cytometry from BMCs from mice treated as in A (n=4 per group). (c) Photograps showing spleens of three TEL–SYK control and three TEL–SYK Stat5−/− mice in comparison after 35 days. (d) Spleen and liver weights from TEL–SYK control and TEL–SYK Stat5−/− mice. (e) Peripheral blood of TEL–SYK control and TEL–SYK Stat5−/− mice (n=4) was analyzed for red blood cell count, hematocrit, WBC count, thrombocytes and granulocytes 35 days post transplantation with the ADVIA120 blood analyzer. Graphs show results for total WBCs and the percentage of granulocytes. (f) Photographs from blood smears of control TEL–SYK and TEL–SYK Stat5−/− mice, which were stained with May-Grünwald/Giemsa. Bars, 50 μm. (g) GFP contents, CD11b+/GFP+ expression, CD90+3+/GFP+ expression and Ter119+/GFP+ expression within total live cells was assessed by flow cytometry analysis in BM and spleen cells from TEL–SYK control and TEL–SYK Stat5−/− mice 35 days after transplantation (n=4). *P<0.05; **P<0.01; ***P<0.001, unpaired t-test. (h) Representative images showing hematoxylin/eosin and reticulin (left image) stained BM, spleen and liver slides from TEL–SYK control and TEL–SYK Stat5−/− mice. Bars, 50 μm.

Mentions: To further validate the role of STAT5 downstream of oncogenic SYK, we overexpressed TEL–SYK in BMCs from conditional Stat5fl/flMx1Cre mice versus control Stat5fl/fl mice.40 This experimental system was previously used to demonstrate the addiction of BCR–ABL-transformed leukemic cells to Stat5 signaling, whereas normal adult hematopoiesis was rarely affected (mild reduction of hematocrit and thrombocytes within the first 8 weeks of Stat5 depletion).41 TEL–SYK/Stat5fl/flMx1Cre BMCs and TEL–SYK/ Stat5fl/fl control BMCs were transplanted into irradiated recipients and treated with Poly-I:C 7 days after transplantation to allow full engraftment of normal and leukemic cells before depletion of Stat5. Stat5 depletion could be detected by western blot analysis of total Stat5 protein levels and by intracellular phospho-Stat5 staining of hematopoietic cells derived from the BM at the end of the experiment (Figures 8a and b). Thirty-two days after transplantation, the control group developed a lethal disease with leukocytosis and increase in myeloid cells in the peripheral blood and the experiment was terminated. Comparison of the spleens showed a dramatic difference with TEL–SYK control spleens being enlarged to a medium weight of 630±79 mg, whereas TEL–SYK/Stat5−/− spleens were normal in size and weight (mean 121±9 mg; Figures 8c and d). Analysis of the peripheral blood at the time of death revealed a leukocytosis in the TEL–SYK control group with an average of 40 × 103 cells/μl mainly consisting of mature granulocytes, whereas TEL–SYK/Stat5−/− mice had normal WBC counts in between 1 and 3 × 103 cells/μl (Figures 8e and f) without enhancement of the myeloid population. Further analysis of BMCs and spleen cells showed, that the Stat5−/− did not only prevent the expansion of TEL–SYK+ myeloid cells, but blocked the expansion of the complete TEL–SYK+ population (GFP+ cells within total BMCs; Figure 8f). Histological evaluation revealed that not only the AML phenotype (liver and spleen infiltration with immature blasts, leukocytosis) was suppressed by the depletion of Stat5, but that also myelofibrosis was abrogated (Figure 8h), pointing toward Stat5 as the major mediator of TEL–SYK-induced disease development.


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)

Depletion of Stat5 blocks TEL–SYK-induced leukemia and myelofibrosis. (a) Western blot showing depletion of Stat5 in Stat5fl/flMx1Cre BMCs versus control Stat5fl/fl BMCs from mice treated with three times injection of Poly (I:C) and 35 days after transplantation. (b) Intracellular phospho-Stat5 staining was assessed by flow cytometry from BMCs from mice treated as in A (n=4 per group). (c) Photograps showing spleens of three TEL–SYK control and three TEL–SYK Stat5−/− mice in comparison after 35 days. (d) Spleen and liver weights from TEL–SYK control and TEL–SYK Stat5−/− mice. (e) Peripheral blood of TEL–SYK control and TEL–SYK Stat5−/− mice (n=4) was analyzed for red blood cell count, hematocrit, WBC count, thrombocytes and granulocytes 35 days post transplantation with the ADVIA120 blood analyzer. Graphs show results for total WBCs and the percentage of granulocytes. (f) Photographs from blood smears of control TEL–SYK and TEL–SYK Stat5−/− mice, which were stained with May-Grünwald/Giemsa. Bars, 50 μm. (g) GFP contents, CD11b+/GFP+ expression, CD90+3+/GFP+ expression and Ter119+/GFP+ expression within total live cells was assessed by flow cytometry analysis in BM and spleen cells from TEL–SYK control and TEL–SYK Stat5−/− mice 35 days after transplantation (n=4). *P<0.05; **P<0.01; ***P<0.001, unpaired t-test. (h) Representative images showing hematoxylin/eosin and reticulin (left image) stained BM, spleen and liver slides from TEL–SYK control and TEL–SYK Stat5−/− mice. Bars, 50 μm.
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fig8: Depletion of Stat5 blocks TEL–SYK-induced leukemia and myelofibrosis. (a) Western blot showing depletion of Stat5 in Stat5fl/flMx1Cre BMCs versus control Stat5fl/fl BMCs from mice treated with three times injection of Poly (I:C) and 35 days after transplantation. (b) Intracellular phospho-Stat5 staining was assessed by flow cytometry from BMCs from mice treated as in A (n=4 per group). (c) Photograps showing spleens of three TEL–SYK control and three TEL–SYK Stat5−/− mice in comparison after 35 days. (d) Spleen and liver weights from TEL–SYK control and TEL–SYK Stat5−/− mice. (e) Peripheral blood of TEL–SYK control and TEL–SYK Stat5−/− mice (n=4) was analyzed for red blood cell count, hematocrit, WBC count, thrombocytes and granulocytes 35 days post transplantation with the ADVIA120 blood analyzer. Graphs show results for total WBCs and the percentage of granulocytes. (f) Photographs from blood smears of control TEL–SYK and TEL–SYK Stat5−/− mice, which were stained with May-Grünwald/Giemsa. Bars, 50 μm. (g) GFP contents, CD11b+/GFP+ expression, CD90+3+/GFP+ expression and Ter119+/GFP+ expression within total live cells was assessed by flow cytometry analysis in BM and spleen cells from TEL–SYK control and TEL–SYK Stat5−/− mice 35 days after transplantation (n=4). *P<0.05; **P<0.01; ***P<0.001, unpaired t-test. (h) Representative images showing hematoxylin/eosin and reticulin (left image) stained BM, spleen and liver slides from TEL–SYK control and TEL–SYK Stat5−/− mice. Bars, 50 μm.
Mentions: To further validate the role of STAT5 downstream of oncogenic SYK, we overexpressed TEL–SYK in BMCs from conditional Stat5fl/flMx1Cre mice versus control Stat5fl/fl mice.40 This experimental system was previously used to demonstrate the addiction of BCR–ABL-transformed leukemic cells to Stat5 signaling, whereas normal adult hematopoiesis was rarely affected (mild reduction of hematocrit and thrombocytes within the first 8 weeks of Stat5 depletion).41 TEL–SYK/Stat5fl/flMx1Cre BMCs and TEL–SYK/ Stat5fl/fl control BMCs were transplanted into irradiated recipients and treated with Poly-I:C 7 days after transplantation to allow full engraftment of normal and leukemic cells before depletion of Stat5. Stat5 depletion could be detected by western blot analysis of total Stat5 protein levels and by intracellular phospho-Stat5 staining of hematopoietic cells derived from the BM at the end of the experiment (Figures 8a and b). Thirty-two days after transplantation, the control group developed a lethal disease with leukocytosis and increase in myeloid cells in the peripheral blood and the experiment was terminated. Comparison of the spleens showed a dramatic difference with TEL–SYK control spleens being enlarged to a medium weight of 630±79 mg, whereas TEL–SYK/Stat5−/− spleens were normal in size and weight (mean 121±9 mg; Figures 8c and d). Analysis of the peripheral blood at the time of death revealed a leukocytosis in the TEL–SYK control group with an average of 40 × 103 cells/μl mainly consisting of mature granulocytes, whereas TEL–SYK/Stat5−/− mice had normal WBC counts in between 1 and 3 × 103 cells/μl (Figures 8e and f) without enhancement of the myeloid population. Further analysis of BMCs and spleen cells showed, that the Stat5−/− did not only prevent the expansion of TEL–SYK+ myeloid cells, but blocked the expansion of the complete TEL–SYK+ population (GFP+ cells within total BMCs; Figure 8f). Histological evaluation revealed that not only the AML phenotype (liver and spleen infiltration with immature blasts, leukocytosis) was suppressed by the depletion of Stat5, but that also myelofibrosis was abrogated (Figure 8h), pointing toward Stat5 as the major mediator of TEL–SYK-induced disease development.

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