背景:针对主轴组装检查点(SAC)的药物,如Aurora激酶B(AURKB)和双特异性蛋白激酶TTK的抑制剂,处于临床发展的不同阶段。然而,对SAC废除的细胞反应了解甚少,也没有用于患者选择的标志物.
方法:使用一组53种不同来源的肿瘤细胞系。通过MTT和流式细胞术分析药物的作用。通过FISH和Q-PCR确定拷贝数状态;通过nCounter和RT-Q-PCR确定mRNA表达,通过蛋白质印迹确定蛋白质表达。CRISPR-Cas9技术用于基因敲除(KO)和多西环素诱导的pTRIPZ载体用于异位表达。最后,通过将培养的细胞或肿瘤片段植入免疫缺陷小鼠进行体内实验。
结果:对AURKB和TTK抑制剂敏感的肿瘤细胞和患者来源的异种移植物(PDX)始终显示出BH3相互作用域死亡激动剂(BID)的高表达水平,而具有低BID的细胞系和PDX具有一致的抗性。基因沉默使BID过表达的细胞对SAC废除不敏感,而低BID细胞中的异位BID表达显着增加了敏感性。SAC废除诱导CASP-2的活化,导致CASP-3的裂解和仅在高水平的BID存在下广泛的细胞死亡。最后,一项患病率研究显示,6%的人实体瘤中存在高BIDmRNA.
结论:SAC废除后肿瘤细胞的命运由AURKB/CASP-2信号机制驱动,受投标级别监管。我们的结果为临床探索具有高BID表达的肿瘤中的SAC靶向药物铺平了道路。
Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection.
A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice.
Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors.
The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.