A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF−/−, or p53−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.

多形性胶质母细胞瘤（Glioblastoma multiforme, GBM）是成人最常见的原发性脑恶性肿瘤，目前有效治疗手段十分有限。GBM肿瘤组织中存在具备神经干细胞分子与细胞学特征的细胞亚群，此类细胞是驱动肿瘤生长的核心群体。本研究对比了人源胶质母细胞瘤源性神经干细胞（glioblastoma-derived neural stem cells, GNS cells）与遗传背景正常的神经干细胞（neural stem cells, NS cells）对160种小分子激酶抑制剂的应答差异。研究采用活细胞成像（live-cell imaging）与高内涵图像分析（high content image analysis）工具，筛选得到化合物JNJ-10198409（简称J101），其可诱导GNS细胞阻滞于有丝分裂前中期，而对NS细胞无类似效应。抗体芯片（antibody microarrays）与激酶谱分析（kinase profiling）结果显示，J101的作用机制为抑制polo样激酶1（polo-like kinase 1, Plk1）的活性磷酸化形式（磷酸化T210位点, phospho T210），进而引发纺锤体结构异常并使细胞停滞在有丝分裂前中期。我们进一步发现，已进入临床开发阶段的强效特异性Plk1抑制剂（BI 2536、BI 6727与GSK 461364）可复刻J101的生物学表型，且对GNS细胞具有显著选择性杀伤作用。通过猪脑内皮细胞血脑屏障（blood-brain barrier, BBB）体外模型实验，我们还观察到上述临床候选化合物的体外血脑屏障通透性优于J101。通过对小鼠突变型NS细胞（INK4a/ARF基因敲除型，INK4a/ARF−/−；或p53基因敲除型，p53−/−）以及条件性p53 floxed NS细胞系的急性p53基因缺失实验进行分析，我们推测GNS细胞对BI 2536或J101的敏感性，可能源于其缺乏p53介导的代偿通路。综上，本研究数据表明GBM干细胞对Plk1抑制剂介导的增殖破坏极为敏感，此类抑制剂或具备快速转化为临床治疗药物的应用价值。