Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells

Mei Huang; Jay F. Dorsey; P. K. Epling-Burnette; Ramadevi Nimmanapalli; Terry H. Landowski; Linda B. Mora; Guilian Niu; Dominic Sinibaldi; Fanqi Bai; Alan Kraker; Hua Yu; Lynn Moscinski; Sheng Wei; Julie Djeu; William S. Dalton; Kapil Bhalla; Thomas P. Loughran; Jie Wu; Richard Jove

doi:10.1038/sj.onc.1206028

Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells

Mei Huang, Jay F. Dorsey, P. K. Epling-Burnette, Ramadevi Nimmanapalli, Terry H. Landowski, Linda B. Mora, Guilian Niu, Dominic Sinibaldi, Fanqi Bai, Alan Kraker, Hua Yu, Lynn Moscinski, Sheng Wei, Julie Djeu, William S. Dalton, Kapil Bhalla, Thomas P. Loughran, Jie Wu, Richard Jove

Cancer Center

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Abstract

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr-Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr-Abl kinase. In this study, we show that blocking Bcr-Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC₅₀ of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr-Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr-Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr-Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr-Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.

Original language	English (US)
Pages (from-to)	8804-8816
Number of pages	13
Journal	Oncogene
Volume	21
Issue number	57
DOIs	https://doi.org/10.1038/sj.onc.1206028
State	Published - Dec 12 2002

Keywords

Apoptosis
Bcr-Abl
CML
Cell cycle
PD180970
Stat5

ASJC Scopus subject areas

Molecular Biology
Genetics
Cancer Research

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10.1038/sj.onc.1206028

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Huang, M., Dorsey, J. F., Epling-Burnette, P. K., Nimmanapalli, R., Landowski, T. H., Mora, L. B., Niu, G., Sinibaldi, D., Bai, F., Kraker, A., Yu, H., Moscinski, L., Wei, S., Djeu, J., Dalton, W. S., Bhalla, K., Loughran, T. P., Wu, J., & Jove, R. (2002). Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells. Oncogene, 21(57), 8804-8816. https://doi.org/10.1038/sj.onc.1206028

Huang, M, Dorsey, JF, Epling-Burnette, PK, Nimmanapalli, R, Landowski, TH, Mora, LB, Niu, G, Sinibaldi, D, Bai, F, Kraker, A, Yu, H, Moscinski, L, Wei, S, Djeu, J, Dalton, WS, Bhalla, K, Loughran, TP, Wu, J & Jove, R 2002, 'Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells', Oncogene, vol. 21, no. 57, pp. 8804-8816. https://doi.org/10.1038/sj.onc.1206028

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title = "Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells",

abstract = "Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr-Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr-Abl kinase. In this study, we show that blocking Bcr-Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC50 of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr-Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr-Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr-Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr-Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.",

keywords = "Apoptosis, Bcr-Abl, CML, Cell cycle, PD180970, Stat5",

author = "Mei Huang and Dorsey, {Jay F.} and Epling-Burnette, {P. K.} and Ramadevi Nimmanapalli and Landowski, {Terry H.} and Mora, {Linda B.} and Guilian Niu and Dominic Sinibaldi and Fanqi Bai and Alan Kraker and Hua Yu and Lynn Moscinski and Sheng Wei and Julie Djeu and Dalton, {William S.} and Kapil Bhalla and Loughran, {Thomas P.} and Jie Wu and Richard Jove",

note = "Funding Information: This work was supported by NCI grants CA55652 and CA82533 (to R Jove). We thank members of the laboratory for valuable discussions and the Moffitt Cancer Center Tumor Bank, Flow Cytometry and Molecular Imaging Core Facilities.",

year = "2002",

month = dec,

day = "12",

doi = "10.1038/sj.onc.1206028",

language = "English (US)",

volume = "21",

pages = "8804--8816",

journal = "Oncogene",

issn = "0950-9232",

publisher = "Springer Nature",

number = "57",

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T1 - Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells

AU - Huang, Mei

AU - Dorsey, Jay F.

AU - Epling-Burnette, P. K.

AU - Nimmanapalli, Ramadevi

AU - Landowski, Terry H.

AU - Mora, Linda B.

AU - Niu, Guilian

AU - Sinibaldi, Dominic

AU - Bai, Fanqi

AU - Kraker, Alan

AU - Yu, Hua

AU - Moscinski, Lynn

AU - Wei, Sheng

AU - Djeu, Julie

AU - Dalton, William S.

AU - Bhalla, Kapil

AU - Loughran, Thomas P.

AU - Wu, Jie

AU - Jove, Richard

N1 - Funding Information: This work was supported by NCI grants CA55652 and CA82533 (to R Jove). We thank members of the laboratory for valuable discussions and the Moffitt Cancer Center Tumor Bank, Flow Cytometry and Molecular Imaging Core Facilities.

PY - 2002/12/12

Y1 - 2002/12/12

N2 - Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr-Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr-Abl kinase. In this study, we show that blocking Bcr-Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC50 of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr-Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr-Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr-Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr-Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.

AB - Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr-Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr-Abl kinase. In this study, we show that blocking Bcr-Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC50 of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr-Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr-Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr-Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr-Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.

KW - Apoptosis

KW - Bcr-Abl

KW - CML

KW - Cell cycle

KW - PD180970

KW - Stat5

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Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells

Abstract

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