Greenall S A, Lim Y C, Mitchell C B, Ensbey K S, Stringer B W, Wilding A L, O'Neill G M, McDonald K L, Gough D J, Day B W, Johns T G
Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Victoria, Australia.
Brain Cancer Discovery Collaborative, Australia.
Oncogenesis. 2017 May 15;6(5):e336. doi: 10.1038/oncsis.2017.33.
High-grade glioma (HGG) is an incurable brain cancer. The transcriptomes of cells within HGG tumors are highly heterogeneous. This renders the tumors unresponsive or able to adapt to therapeutics targeted at single pathways, thereby causing treatment failure. To overcome this, we focused on cyclin-dependent kinase 7 (CDK7), a ubiquitously expressed molecule involved in two major drivers of HGG pathogenesis: cell cycle progression and RNA polymerase-II-based transcription. We tested the activity of THZ1, an irreversible CDK7 inhibitor, on patient-derived primary HGG cell lines and ex vivo HGG patient tissue slices, using proliferation assays, microarray analysis, high-resolution respirometry, cell cycle analysis and in vivo tumor orthografts. The cellular processes affected by CDK7 inhibition were analyzed by reverse transcriptase-quantitative PCR, western blot, flow cytometry and immunofluorescence. THZ1 perturbed the transcriptome and disabled CDK activation, leading to cell cycle arrest at G2 and DNA damage. THZ1 halted transcription of the nuclear-encoded mitochondrial ribosomal genes, reducing mitochondrial translation and oxidative respiration. It also inhibited the expression of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFR-α), reducing signaling flux through the AKT, extracellular-signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) downstream pathways. Finally, THZ1 disrupted nucleolar, Cajal body and nuclear speckle formation, resulting in reduced cytosolic translation and malfunction of the spliceosome and thus leading to aberrant mRNA processing. These findings indicate that CDK7 is crucial for gliomagenesis, validate CDK7 as a therapeutic target and provide new insight into the cellular processes that are affected by THZ1 and induce antitumor activity.
高级别胶质瘤(HGG)是一种无法治愈的脑癌。HGG肿瘤内细胞的转录组具有高度异质性。这使得肿瘤对针对单一信号通路的治疗无反应或能够适应这些治疗,从而导致治疗失败。为了克服这一问题,我们聚焦于细胞周期蛋白依赖性激酶7(CDK7),这是一种普遍表达的分子,参与HGG发病机制的两个主要驱动因素:细胞周期进程和基于RNA聚合酶II的转录。我们使用增殖试验、微阵列分析、高分辨率呼吸测定法、细胞周期分析和体内肿瘤原位移植模型,测试了不可逆CDK7抑制剂THZ1对患者来源的原发性HGG细胞系和离体HGG患者组织切片的活性。通过逆转录定量PCR、蛋白质免疫印迹、流式细胞术和免疫荧光分析受CDK7抑制影响的细胞过程。THZ1扰乱转录组并使CDK激活失能,导致细胞周期在G2期停滞和DNA损伤。THZ1停止核编码的线粒体核糖体基因的转录,减少线粒体翻译和氧化呼吸。它还抑制表皮生长因子受体(EGFR)和血小板衍生生长因子受体-α(PDGFR-α)等受体酪氨酸激酶的表达,减少通过下游AKT、细胞外信号调节激酶1/2(ERK1/2)和信号转导及转录激活因子3(STAT3)信号通路的信号通量。最后,THZ1破坏核仁、卡哈尔体和核斑点的形成,导致胞质翻译减少和剪接体功能异常,从而导致异常的mRNA加工。这些发现表明CDK7对胶质瘤发生至关重要,验证了CDK7作为治疗靶点的有效性,并为受THZ1影响并诱导抗肿瘤活性的细胞过程提供了新的见解。