Huang Mei, Dorsey Jay F, Epling-Burnette P K, Nimmanapalli Ramadevi, Landowski Terry H, Mora Linda B, Niu Guilian, Sinibaldi Dominic, Bai Fanqi, Kraker Alan, Yu Hua, Moscinski Lynn, Wei Sheng, Djeu Julie, Dalton William S, Bhalla Kapil, Loughran Thomas P, Wu Jie, Jove Richard
Molecular Oncology, H Lee Moffitt Cancer Center, Research Institute, Tampa, Florida, FL 33612, USA.
Oncogene. 2002 Dec 12;21(57):8804-16. doi: 10.1038/sj.onc.1206028.
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 IC(50) 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.
慢性粒细胞白血病（CML）是一种骨髓增殖性疾病，其特征为BCR-ABL基因易位以及Abl酪氨酸激酶的组成性激活。在转录因子信号转导子与转录激活子（STAT）家族成员中，Stat5被Bcr-Abl激酶激活，并与CML的发病机制有关。我们最近鉴定出PD180970是一种新型且高效的Bcr-Abl激酶抑制剂。在本研究中，我们发现，在人K562 CML细胞系中使用PD180970阻断Bcr-Abl激酶活性会导致Stat5 DNA结合活性受到抑制，IC（50）为5 nM。此外，Abl激酶介导的Stat5激活的消除会抑制K562细胞的增殖并诱导其凋亡，但在Bcr-Abl阴性的髓系细胞系HEL 92.1.7和HL-60中则不会。从痘苗病毒载体表达的显性负性Stat5蛋白也会诱导K562细胞凋亡，这与早期研究一致，这些研究表明Stat5信号在CML细胞的生长和存活中起重要作用。RNA和蛋白质分析揭示了Stat5的几个候选靶基因，包括Bcl-x、Mcl-1、c-Myc和细胞周期蛋白D2，在用PD180970处理后它们的表达下调。此外，PD180970在源自CML患者的培养原代白血病细胞中抑制Stat5 DNA结合活性。为了检测CML患者标本中激活的Stat5，我们开发了一种免疫细胞化学检测方法，该方法可作为分子终点检测方法来监测Bcr-Abl信号的抑制。此外，PD180970阻断Stat5信号并诱导对STI-571（格列卫，伊马替尼）耐药的Bcr-Abl阳性细胞凋亡。总之，这些结果表明PD180970的作用机制涉及抑制Bcr-Abl介导的Stat5信号，并进一步证明该结构类别的化合物可能代表CML的潜在治疗药物。
