Random mutagenesis reveals residues of JAK2 critical in evading inhibition by a tyrosine kinase inhibitor.
Random mutagenesis reveals residues of JAK2 critical in evading inhibition by a tyrosine kinase inhibitor.
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DOI：
10.1371/journal.pone.0043437
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Barber DL
中科院分区：
文献类型：
--
作者：
Marit MR;Chohan M;Matthew N;Huang K;Kuntz DA;Rose DR;Barber DL
非受体酪氨酸激酶JAK2与一组骨髓增生性肿瘤有关，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进行临床试验评估。来自耐药的慢性粒细胞白血病患者的数据表明，用小分子抑制剂治疗通过突变或扩增bcr-abl而产生耐药性。我们假设，用JAK2的小分子抑制剂处理JAK2，同样会在JAK2中产生耐药突变。为了先验鉴定JAK2耐药突变，我们利用TEL-JAK2对JAK2耐药等位基因进行了体外随机突变筛选。分离的突变被评估为它们维持细胞生长，刺激下游信号通路，并在抑制剂存在的情况下磷酸化一种新的JAK2底物。突变仅在JAK2的激活域中发现。这一组突变赋予了对高浓度抑制剂的抗性，同时伴随着Stat5、ERK1/2和Akt通路的持续激活。使用JAK2底物，当抑制剂浓度比抑制野生型蛋白的浓度高200倍时，突变的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组时，我们观察到突变的子集会对抑制剂产生抗性，从而验证了TEL-JAK2在初始筛查中的使用。这些结果表明，小分子抑制剂选择JAK2抑制剂抗性等位基因，下一代JAK2抑制剂的设计应该考虑到出现在抑制剂耐药筛查中的突变的位置。
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.
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影响因子：
20.3
作者：
Kantarjian, Hagop M;Giles, Francis J;le Coutre, Philipp D
通讯作者：
le Coutre, Philipp D
影响因子：
64.5
作者：
Azam, M;Latek, RR;Daley, GQ
通讯作者：
Daley, GQ
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2.6
作者：
Li, Zhe;Xing, Shu;Zhao, Zhizhuang Joe
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Zhao, Zhizhuang Joe
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50.3
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Chan WW;Wise SC;Kaufman MD;Ahn YM;Ensinger CL;Haack T;Hood MM;Jones J;Lord JW;Lu WP;Miller D;Patt WC;Smith BD;Petillo PA;Rutkoski TJ;Telikepalli H;Vogeti L;Yao T;Chun L;Clark R;Evangelista P;Gavrilescu LC;Lazarides K;Zaleskas VM;Stewart LJ;Van Etten RA;Flynn DL
通讯作者：
Flynn DL
影响因子：
20.3
作者：
le Coutre, P;Tassi, E;Gambacorti-Passerini, C
通讯作者：
Gambacorti-Passerini, C