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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 human K562 cells(A, E) K562 cells and NB4 cells were cultured at the indicated concentration of rapamycin overnight. The rapamycin activity was determined by phosphorylation of the p70S6K protein. Total p70S6K was used a control. (B, F) K562 and NB4 cells were cultured in the indicated concentration of rapamycin for 48 hours and the fold proliferation of the cells was determined by manual cell count. (C, G) K562 cells and NB4 cells were cultured in the presence of ABT-737 for 48 hours. The cell viability were determined by both PI staining and trypan blue exclusion. Both methods gave similar results. (D, H) K562 and NB4 cells were cultured in either rapamycin alone (10 nM), ABT-737 alone (5µM), or the combination of both for 48 hours. Cell viability was determined as described in Methods.
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Figure 6: Rapamycin combined with ABT-737 synergistically kills human K562 cells(A, E) K562 cells and NB4 cells were cultured at the indicated concentration of rapamycin overnight. The rapamycin activity was determined by phosphorylation of the p70S6K protein. Total p70S6K was used a control. (B, F) K562 and NB4 cells were cultured in the indicated concentration of rapamycin for 48 hours and the fold proliferation of the cells was determined by manual cell count. (C, G) K562 cells and NB4 cells were cultured in the presence of ABT-737 for 48 hours. The cell viability were determined by both PI staining and trypan blue exclusion. Both methods gave similar results. (D, H) K562 and NB4 cells were cultured in either rapamycin alone (10 nM), ABT-737 alone (5µM), or the combination of both for 48 hours. Cell viability was determined as described in Methods.

Mentions: Since rapamycin alone was not effective at killing the BaF3 cells, rapamycin was combined with ABT-737, an inhibitor of bcl-2/bcl-XL. ABT-737 was toxic in a dose-dependent manner up to 10 µM to all BaF3 cell lines (Fig. 5C). However, when 5 µM ABT-737 was combined with a concentration of 1 nM rapamycin, a striking synergy was observed in cell lines expressing TEL-JAK2, BCR-ABL, and STAT5aS711F increasing from ~20% to >80% killing (Fig. 5D). To extend this observation further, the effects of rapamycin or ABT-737 alone were assayed in human BCR-ABL positive K562 cells. Human myeloproliferative neoplasms are more complex genetically than the primary BM cell or BaF3 model cells. Despite inhibition of phosphorylation of p70S6K at concentrations above 10 nM (Fig. 6A), rapamycin alone had minimal effects on these cells (Fig. 6B). K562 cells were then exposed to ABT-737 which displayed very low toxicity at concentrations ≤ 5 µM and up to 30% death at 10 µM (Fig. 6C). However, the combination of 10 µM ABT-737 and a non-toxic concentration of 1 nM rapamycin gave a synergistic increase in the percentage of cell killing (Fig. 6D). In contrast, NB4 cells were more sensitive to rapamycin alone but showed no synergy when combined with ABT-737 (Fig. 6E–H) indicating that the effect is not generalizable to all types of leukemia cells. Various doses and timing were tested for NB4 cells and no evidence of synergy was observed (data not shown). Similar results were also obtained in HL-60 cells (data not shown).


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 human K562 cells(A, E) K562 cells and NB4 cells were cultured at the indicated concentration of rapamycin overnight. The rapamycin activity was determined by phosphorylation of the p70S6K protein. Total p70S6K was used a control. (B, F) K562 and NB4 cells were cultured in the indicated concentration of rapamycin for 48 hours and the fold proliferation of the cells was determined by manual cell count. (C, G) K562 cells and NB4 cells were cultured in the presence of ABT-737 for 48 hours. The cell viability were determined by both PI staining and trypan blue exclusion. Both methods gave similar results. (D, H) K562 and NB4 cells were cultured in either rapamycin alone (10 nM), ABT-737 alone (5µM), or the combination of both for 48 hours. Cell viability was determined as described in Methods.
© Copyright Policy
Related In: Results  -  Collection

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Figure 6: Rapamycin combined with ABT-737 synergistically kills human K562 cells(A, E) K562 cells and NB4 cells were cultured at the indicated concentration of rapamycin overnight. The rapamycin activity was determined by phosphorylation of the p70S6K protein. Total p70S6K was used a control. (B, F) K562 and NB4 cells were cultured in the indicated concentration of rapamycin for 48 hours and the fold proliferation of the cells was determined by manual cell count. (C, G) K562 cells and NB4 cells were cultured in the presence of ABT-737 for 48 hours. The cell viability were determined by both PI staining and trypan blue exclusion. Both methods gave similar results. (D, H) K562 and NB4 cells were cultured in either rapamycin alone (10 nM), ABT-737 alone (5µM), or the combination of both for 48 hours. Cell viability was determined as described in Methods.
Mentions: Since rapamycin alone was not effective at killing the BaF3 cells, rapamycin was combined with ABT-737, an inhibitor of bcl-2/bcl-XL. ABT-737 was toxic in a dose-dependent manner up to 10 µM to all BaF3 cell lines (Fig. 5C). However, when 5 µM ABT-737 was combined with a concentration of 1 nM rapamycin, a striking synergy was observed in cell lines expressing TEL-JAK2, BCR-ABL, and STAT5aS711F increasing from ~20% to >80% killing (Fig. 5D). To extend this observation further, the effects of rapamycin or ABT-737 alone were assayed in human BCR-ABL positive K562 cells. Human myeloproliferative neoplasms are more complex genetically than the primary BM cell or BaF3 model cells. Despite inhibition of phosphorylation of p70S6K at concentrations above 10 nM (Fig. 6A), rapamycin alone had minimal effects on these cells (Fig. 6B). K562 cells were then exposed to ABT-737 which displayed very low toxicity at concentrations ≤ 5 µM and up to 30% death at 10 µM (Fig. 6C). However, the combination of 10 µM ABT-737 and a non-toxic concentration of 1 nM rapamycin gave a synergistic increase in the percentage of cell killing (Fig. 6D). In contrast, NB4 cells were more sensitive to rapamycin alone but showed no synergy when combined with ABT-737 (Fig. 6E–H) indicating that the effect is not generalizable to all types of leukemia cells. Various doses and timing were tested for NB4 cells and no evidence of synergy was observed (data not shown). Similar results were also obtained in HL-60 cells (data not shown).

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