Wright Gabriela, Golubeva Volha, Remsing Rix Lily L, Berndt Norbert, Luo Yunting, Ward Grace A, Gray Jhanelle E, Schonbrunn Ernst, Lawrence Harshani R, Monteiro Alvaro N A, Rix Uwe
Drug Discovery Department, H. Lee Moffitt Cancer Center & Research Institute , Tampa, Florida 33612, United States.
Cancer Epidemiology, H. Lee Moffitt Cancer Center & Research Institute , Tampa, Florida 33612, United States.
ACS Chem Biol. 2017 Jul 21;12(7):1883-1892. doi: 10.1021/acschembio.7b00147. Epub 2017 Jun 7.
Inhibition of the WEE1 tyrosine kinase enhances anticancer chemotherapy efficacy. Accordingly, the WEE1 inhibitor AZD1775 (previously MK-1775) is currently under evaluation in clinical trials for cancer in combination with chemotherapy. AZD1775 has been reported to display high selectivity and is therefore used in many studies as a probe to interrogate WEE1 biology. However, AZD1775 also exhibits anticancer activity as a single agent although the underlying mechanism is not fully understood. Using a chemical proteomics approach, we here describe a proteome-wide survey of AZD1775 targets in lung cancer cells and identify several previously unknown targets in addition to WEE1. In particular, we observed polo-like kinase 1 (PLK1) as a new target of AZD1775. Importantly, in vitro kinase assays showed PLK1 and WEE1 to be inhibited by AZD1775 with similar potency. Subsequent loss-of-function experiments using RNAi for WEE1 and PLK1 suggested that targeting PLK1 enhances the pro-apoptotic and antiproliferative effects observed with WEE1 knockdown. Combination of RNAi with AZD1775 treatment suggested WEE1 and PLK1 to be the most relevant targets for mediating AZD1775's anticancer effects. Furthermore, disruption of WEE1 by CRISPR-Cas9 sensitized H322 lung cancer cells to AZD1775 to a similar extent as the potent PLK1 inhibitor BI-2536 suggesting a complex crosstalk between PLK1 and WEE1. In summary, we show that AZD1775 is a potent dual WEE1 and PLK1 inhibitor, which limits its use as a specific molecular probe for WEE1. However, PLK1 inhibition makes important contributions to the single agent mechanism of action of AZD1775 and enhances its anticancer effects.
抑制WEE1酪氨酸激酶可增强抗癌化疗疗效。因此，WEE1抑制剂AZD1775（以前称为MK-1775）目前正在与化疗联合用于癌症的临床试验中进行评估。据报道，AZD1775具有高选择性，因此在许多研究中用作探究WEE1生物学特性的探针。然而，AZD1775作为单一药物也表现出抗癌活性，尽管其潜在机制尚未完全了解。我们采用化学蛋白质组学方法，对肺癌细胞中AZD1775的靶点进行了全蛋白质组范围的研究，并鉴定出除WEE1之外的几个先前未知的靶点。特别是，我们观察到polo样激酶1（PLK1）是AZD1775的新靶点。重要的是，体外激酶分析表明，AZD1775对PLK1和WEE1的抑制效力相似。随后使用针对WEE1和PLK1的RNAi进行的功能丧失实验表明，靶向PLK1可增强WEE1基因敲低所观察到的促凋亡和抗增殖作用。RNAi与AZD1775处理的联合应用表明，WEE1和PLK1是介导AZD1775抗癌作用的最相关靶点。此外，CRISPR-Cas9介导的WEE1基因敲除使H322肺癌细胞对AZD1775的敏感性与强效PLK1抑制剂BI-2536相似，这表明PLK1和WEE1之间存在复杂的相互作用。总之，我们表明AZD1775是一种有效的WEE1和PLK1双重抑制剂，这限制了它作为WEE1特异性分子探针的用途。然而，抑制PLK1对AZD1775的单一药物作用机制有重要贡献，并增强了其抗癌效果。
