Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
PLoS One. 2012;7(8):e43437. doi: 10.1371/journal.pone.0043437. Epub 2012 Aug 16.
The 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.
In 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.
Mutations 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.
非受体酪氨酸激酶 JAK2 参与一组骨髓增殖性肿瘤，包括真性红细胞增多症、原发性血小板增多症和原发性骨髓纤维化。JAK2 选择性抑制剂目前正在临床试验中进行评估。耐药性慢性髓性白血病患者的数据表明，小分子抑制剂的治疗会通过 BCR-ABL 的突变或扩增产生耐药性。我们假设，小分子 JAK2 抑制剂的治疗也会在 JAK2 中产生抑制剂耐药突变体。
为了预先确定对 JAK2 抑制剂具有抗性的 JAK2 突变，我们利用 TEL-JAK2 进行了体外随机诱变筛选，以寻找对 JAK 抑制剂-I 具有抗性的 JAK2 等位基因。对分离出的突变进行了评估，以确定它们在存在抑制剂的情况下维持细胞生长、刺激下游信号通路和磷酸化新型 JAK2 底物的能力。
突变仅发生在 JAK2 的激酶结构域中。该突变体panel 赋予了对高浓度抑制剂的抗性，同时伴有 Stat5、Erk1/2 和 Akt 通路的持续激活。使用 JAK2 底物，在比野生型蛋白抑制作用高 200 倍的抑制剂浓度下，观察到突变型 JAK2 激酶的催化活性增强。当在 Jak2 V617F 等位基因的背景下测试突变体 panel 时，我们观察到一些突变赋予了对抑制剂的抗性，验证了 TEL-JAK2 在初始筛选中的应用。这些结果表明，小分子抑制剂选择了 JAK2 抑制剂耐药等位基因，下一代 JAK2 抑制剂的设计应考虑到在抑制剂耐药筛选中出现的突变位置。
