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Effective targeting of STAT5-mediated survival in myeloproliferative neoplasms using ABT-737 combined with rapamycin.

Li G, Miskimen KL, Wang Z, Xie XY, Tse W, Gouilleux F, Moriggl R, Bunting KD - Leukemia (2010)

Bottom Line: In either case, mice lacking Gab2 or treated with rapamycin showed attenuated myeloid hyperplasia and modestly improved survival, but the effects were not cytotoxic and were reversible.To improve on this approach, we combined in vitro targeting of STAT5-mediated AKT/mTOR using rapamycin with inhibition of the STAT5 direct target genes bcl-2 and bcl-X(L) using ABT-737.Therefore, targeting distinct STAT5-mediated survival signals, for example, bcl-2/bcl-X(L) and AKT/mTOR may be an effective therapeutic approach for human myeloproliferative neoplasms.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology-Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA.

ABSTRACT
Signal transducer and activator of transcription-5 (STAT5) is a critical transcription factor for normal hematopoiesis and its sustained activation is associated with hematologic malignancy. A persistently active mutant of STAT5 (STAT5a(S711F)) associates with Grb2-associated binding protein 2 (Gab2) in myeloid leukemias and promotes growth in vitro through AKT activation. Here we have retrovirally transduced wild-type or Gab2(-/-) mouse bone marrow cells expressing STAT5a(S711F) and transplanted into irradiated recipient mice to test an in vivo myeloproliferative disease model. To target Gab2-independent AKT/mTOR activation, we treated wild-type mice separately with rapamycin. In either case, mice lacking Gab2 or treated with rapamycin showed attenuated myeloid hyperplasia and modestly improved survival, but the effects were not cytotoxic and were reversible. To improve on this approach, we combined in vitro targeting of STAT5-mediated AKT/mTOR using rapamycin with inhibition of the STAT5 direct target genes bcl-2 and bcl-X(L) using ABT-737. Striking synergy with both drugs was observed in mouse BaF3 cells expressing STAT5a(S711F), TEL-JAK2 or BCR-ABL and in the relatively single agent-resistant human BCR-ABL-positive K562 cell line. Therefore, targeting distinct STAT5-mediated survival signals, for example, bcl-2/bcl-X(L) and AKT/mTOR may be an effective therapeutic approach for human myeloproliferative neoplasms.

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Rapamycin combined with ABT-737 synergistically kills transduced BaF3 cells expressing STAT5aS711F, BCR-ABL, or TEL-JAK2A. BaF3 cells were transduced with the IR-GFP, STAT5aS711F, BCR-ABL, or TEL-JAK2 vector. The cells were treated with 1 nM rapamycin overnight. Recombinant IL-3 at a minimal concentration of 0.01 ng/ml was added to IR-GFP cells to maintain their viability. Cell extracts were resolved by SDS-PAGE and immunoblot was performed with the indicated antibody. B. Cells were cultured in serial concentrations of rapamycin for 48 hours and cell expansion was measured by manual scoring using a hemacytometer. C. Cells were cultured with the indicated concentration of ABT-737 for 48 hours and cell viability was measured by trypan blue exclusion. D. Cells were cultured in 0.2 ng/ml IL3 or without IL3 as indicated. For each group, the cells were treated with either rapamycin (1nM) or ABT-737 (5µm) alone or combination. After 48 hours, cell death was determined by trypan blue exclusion assay. P values shown are for comparison of ABT-737 alone vs. ABT-737/Rapamycin combination groups.
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Figure 5: Rapamycin combined with ABT-737 synergistically kills transduced BaF3 cells expressing STAT5aS711F, BCR-ABL, or TEL-JAK2A. BaF3 cells were transduced with the IR-GFP, STAT5aS711F, BCR-ABL, or TEL-JAK2 vector. The cells were treated with 1 nM rapamycin overnight. Recombinant IL-3 at a minimal concentration of 0.01 ng/ml was added to IR-GFP cells to maintain their viability. Cell extracts were resolved by SDS-PAGE and immunoblot was performed with the indicated antibody. B. Cells were cultured in serial concentrations of rapamycin for 48 hours and cell expansion was measured by manual scoring using a hemacytometer. C. Cells were cultured with the indicated concentration of ABT-737 for 48 hours and cell viability was measured by trypan blue exclusion. D. Cells were cultured in 0.2 ng/ml IL3 or without IL3 as indicated. For each group, the cells were treated with either rapamycin (1nM) or ABT-737 (5µm) alone or combination. After 48 hours, cell death was determined by trypan blue exclusion assay. P values shown are for comparison of ABT-737 alone vs. ABT-737/Rapamycin combination groups.

Mentions: We next tested whether the mTOR inhibitor rapamycin would be effective at suppressing growth of a broader set of hematopoietic cells expressing human disease relevant mutations. BaF3 cells expressing STAT5aS711F were compared with BaF3 cells expressing BCR-ABL or TEL-JAK2. All 3 lines were cytokine-independent and all were sensitive to rapamycin-induced growth suppression. Cell lines expressing only the IR-GFP empty vector still required IL-3 for growth stimulation. The BaF3 cells were cultured in a low dose of 0.01 ng/ml IL-3 and this concentration was sufficient to keep the control cells alive during 48 hours without significant loss of viability. Overexpression of STAT5aS711F enhanced Akt activation and downstream phosphorylation of p70S6 kinase (p70S6K) and AKT relative to the IR-GFP control (Fig. 5A). Treatment with rapamycin for 24 hours at the concentration of 1 nM effectively blocked STAT5aS711F mediated growth and suppressed p70S6K without having any direct impact on STAT5 tyrosine or Akt serine phosphorylation. It is worthy to note that although rapamycin significantly inhibited proliferation (Fig. 5B), it didn’t induce significant loss of cell viability in any of the BaF3 cell lines.


Effective targeting of STAT5-mediated survival in myeloproliferative neoplasms using ABT-737 combined with rapamycin.

Li G, Miskimen KL, Wang Z, Xie XY, Tse W, Gouilleux F, Moriggl R, Bunting KD - Leukemia (2010)

Rapamycin combined with ABT-737 synergistically kills transduced BaF3 cells expressing STAT5aS711F, BCR-ABL, or TEL-JAK2A. BaF3 cells were transduced with the IR-GFP, STAT5aS711F, BCR-ABL, or TEL-JAK2 vector. The cells were treated with 1 nM rapamycin overnight. Recombinant IL-3 at a minimal concentration of 0.01 ng/ml was added to IR-GFP cells to maintain their viability. Cell extracts were resolved by SDS-PAGE and immunoblot was performed with the indicated antibody. B. Cells were cultured in serial concentrations of rapamycin for 48 hours and cell expansion was measured by manual scoring using a hemacytometer. C. Cells were cultured with the indicated concentration of ABT-737 for 48 hours and cell viability was measured by trypan blue exclusion. D. Cells were cultured in 0.2 ng/ml IL3 or without IL3 as indicated. For each group, the cells were treated with either rapamycin (1nM) or ABT-737 (5µm) alone or combination. After 48 hours, cell death was determined by trypan blue exclusion assay. P values shown are for comparison of ABT-737 alone vs. ABT-737/Rapamycin combination groups.
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Related In: Results  -  Collection

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Figure 5: Rapamycin combined with ABT-737 synergistically kills transduced BaF3 cells expressing STAT5aS711F, BCR-ABL, or TEL-JAK2A. BaF3 cells were transduced with the IR-GFP, STAT5aS711F, BCR-ABL, or TEL-JAK2 vector. The cells were treated with 1 nM rapamycin overnight. Recombinant IL-3 at a minimal concentration of 0.01 ng/ml was added to IR-GFP cells to maintain their viability. Cell extracts were resolved by SDS-PAGE and immunoblot was performed with the indicated antibody. B. Cells were cultured in serial concentrations of rapamycin for 48 hours and cell expansion was measured by manual scoring using a hemacytometer. C. Cells were cultured with the indicated concentration of ABT-737 for 48 hours and cell viability was measured by trypan blue exclusion. D. Cells were cultured in 0.2 ng/ml IL3 or without IL3 as indicated. For each group, the cells were treated with either rapamycin (1nM) or ABT-737 (5µm) alone or combination. After 48 hours, cell death was determined by trypan blue exclusion assay. P values shown are for comparison of ABT-737 alone vs. ABT-737/Rapamycin combination groups.
Mentions: We next tested whether the mTOR inhibitor rapamycin would be effective at suppressing growth of a broader set of hematopoietic cells expressing human disease relevant mutations. BaF3 cells expressing STAT5aS711F were compared with BaF3 cells expressing BCR-ABL or TEL-JAK2. All 3 lines were cytokine-independent and all were sensitive to rapamycin-induced growth suppression. Cell lines expressing only the IR-GFP empty vector still required IL-3 for growth stimulation. The BaF3 cells were cultured in a low dose of 0.01 ng/ml IL-3 and this concentration was sufficient to keep the control cells alive during 48 hours without significant loss of viability. Overexpression of STAT5aS711F enhanced Akt activation and downstream phosphorylation of p70S6 kinase (p70S6K) and AKT relative to the IR-GFP control (Fig. 5A). Treatment with rapamycin for 24 hours at the concentration of 1 nM effectively blocked STAT5aS711F mediated growth and suppressed p70S6K without having any direct impact on STAT5 tyrosine or Akt serine phosphorylation. It is worthy to note that although rapamycin significantly inhibited proliferation (Fig. 5B), it didn’t induce significant loss of cell viability in any of the BaF3 cell lines.

Bottom Line: In either case, mice lacking Gab2 or treated with rapamycin showed attenuated myeloid hyperplasia and modestly improved survival, but the effects were not cytotoxic and were reversible.To improve on this approach, we combined in vitro targeting of STAT5-mediated AKT/mTOR using rapamycin with inhibition of the STAT5 direct target genes bcl-2 and bcl-X(L) using ABT-737.Therefore, targeting distinct STAT5-mediated survival signals, for example, bcl-2/bcl-X(L) and AKT/mTOR may be an effective therapeutic approach for human myeloproliferative neoplasms.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology-Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA.

ABSTRACT
Signal transducer and activator of transcription-5 (STAT5) is a critical transcription factor for normal hematopoiesis and its sustained activation is associated with hematologic malignancy. A persistently active mutant of STAT5 (STAT5a(S711F)) associates with Grb2-associated binding protein 2 (Gab2) in myeloid leukemias and promotes growth in vitro through AKT activation. Here we have retrovirally transduced wild-type or Gab2(-/-) mouse bone marrow cells expressing STAT5a(S711F) and transplanted into irradiated recipient mice to test an in vivo myeloproliferative disease model. To target Gab2-independent AKT/mTOR activation, we treated wild-type mice separately with rapamycin. In either case, mice lacking Gab2 or treated with rapamycin showed attenuated myeloid hyperplasia and modestly improved survival, but the effects were not cytotoxic and were reversible. To improve on this approach, we combined in vitro targeting of STAT5-mediated AKT/mTOR using rapamycin with inhibition of the STAT5 direct target genes bcl-2 and bcl-X(L) using ABT-737. Striking synergy with both drugs was observed in mouse BaF3 cells expressing STAT5a(S711F), TEL-JAK2 or BCR-ABL and in the relatively single agent-resistant human BCR-ABL-positive K562 cell line. Therefore, targeting distinct STAT5-mediated survival signals, for example, bcl-2/bcl-X(L) and AKT/mTOR may be an effective therapeutic approach for human myeloproliferative neoplasms.

Show MeSH
Related in: MedlinePlus