Cellular LanthaScreen and beta-lactamase reporter assays for high-throughput screening of JAK2 inhibitors.

The Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 5 pathway is responsible for regulation of cellular responses to a number of cytokines and growth factors. In hematopoietic cells, growth factors such as granulocyte macrophage-colony stimulating factor, interleukin-3, and erythropoietin induce the activation of JAK2, which leads to the phosphorylation, dimerization, and transactivation of STAT5 proteins. Dysregulation of JAK2 by activating mutations such as JAK2V617F results in constitutive phosphorylation of STAT5 and has been linked to numerous myeloproliferative disorders such as polycythemia vera. A cellular LanthaScreen (Invitrogen Corp., Carlsbad, CA) time-resolved Förster resonance energy transfer assay for wild-type JAK2 activity was developed. This assay utilized the growth factor-dependent human erythroleukemia TF1 cell line engineered to express a green fluorescent protein-STAT5 fusion protein. Furthermore, a complementary beta-lactamase reporter gene assay was developed to analyze the transcriptional activity of STAT5 downstream of JAK2 in TF1 cells. The same technologies were applied to the development of cellular assays for the interrogation of the disease-relevant JAK2V617F activating mutant. A small molecule inhibitor and Stealth (Invitrogen Corp.) RNA interference oligonucleotides were used to confirm the involvement of JAK2. Our results suggest that these cellular assays and validation tools represent powerful integrated methods for the analysis of physiological and disease-relevant JAK/STAT pathways within the physiological cellular context.