Abstract
BackgroundThe non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic myeloid leukemia patients demonstrate that treatment with a small-molecule inhibitor generates resistance via mutation or amplification of BCR-ABL. We hypothesize that treatment with small molecule inhibitors of JAK2 will similarly generate inhibitor-resistant mutants in JAK2.MethodologyIn order to identify inhibitor-resistant JAK2 mutations a priori, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signaling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor.ConclusionsMutations were found exclusively in the kinase domain of JAK2. The panel of mutations conferred resistance to high concentrations of inhibitor accompanied by sustained activation of the Stat5, Erk1/2, and Akt pathways. Using a JAK2 substrate, enhanced catalytic activity of the mutant JAK2 kinase was observed in inhibitor concentrations 200-fold higher than is inhibitory to the wild-type protein. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles, and the design of next-generation JAK2 inhibitors should consider the location of mutations arising in inhibitor-resistant screens.
Highlights
When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen
Myeloproliferative neoplasms (MPNs) are diseases characterized by an excess production of one or more fully differentiated blood cell types, and can be precursors to more severe disorders including myelodysplastic syndrome and acute leukemia [1,2,3]
XL1-Red competent E. coli were transformed with pMPG2TEL-JAK2(5-12), producing a mutagenized library
Summary
Myeloproliferative neoplasms (MPNs) are diseases characterized by an excess production of one or more fully differentiated blood cell types, and can be precursors to more severe disorders including myelodysplastic syndrome and acute leukemia [1,2,3]. Ligation of the receptor results in JAK2 cross-phosphorylation and activation of downstream pathways including the STAT family of transcription factors, the PI3kinase/Akt survival pathway, and the ERK kinase pathway. Induction of these pathways results in transcription of genes required for survival and differentiation. The JAK2 V617F mutation is thought to relieve the negative regulatory role of the dualspecificity kinase domain and is is weakly oncogenic, able to transform specific cell lines to cytokine independence [9]. The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We hypothesize that treatment with small molecule inhibitors of JAK2 will generate inhibitor-resistant mutants in JAK2
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