Expression profiling of HeLa cell line treated with CDK9 inhibitor. Homo sapiens

CDK9 is the kinase subunit of P-TEFb that enables RNA polymerase (Pol) II to transit from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to the lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9’s activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb’s loss of activity, only the simultaneous inhibition of CDK9 and MYC can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy. We used microarrays to examine the global impact on gene expression by imhibiting CDK9 at different time durations. Overall design: HeLa cell lines treated with CDK9 inhibitor at different time points

CDK9（Cyclin-dependent kinase 9，细胞周期蛋白依赖性激酶9）是P-TEFb（Positive Transcription Elongation Factor b，正性转录延伸因子b）的激酶亚基，可介导RNA聚合酶II（RNA polymerase II）从启动子近端暂停状态向有效转录延伸阶段转换。尽管CDK9作为治疗靶点受到广泛关注，但由于缺乏高选择性抑制剂，相关研究进展十分有限。本研究报道了i-CDK9的开发：该抑制剂可强效抑制CDK9对底物的磷酸化作用，并引发全基因组范围内的RNA聚合酶II暂停。尽管多数基因的表达水平出现下调，但经持续i-CDK9处理后，MYC及其他初级应答基因的表达反而上调。溴结构域蛋白BRD4（Bromodomain-containing protein 4）对这一表达上调过程至关重要：它可从7SK snRNP（7SK small nuclear ribonucleoprotein complex，7SK小细胞核核糖核蛋白复合物）中捕获P-TEFb并递送至靶基因，同时还能增强CDK9的活性及其对抑制剂的抵抗性。由于i-CDK9诱导的MYC表达及其与P-TEFb的结合可弥补P-TEFb的活性损失，仅同时抑制CDK9与MYC才能有效诱导癌细胞的生长停滞与凋亡，这提示联合治疗策略具备潜在应用价值。本研究通过基因芯片（microarray）检测了不同时长的CDK9抑制对基因表达的全局影响。实验整体设计：将海拉细胞系用CDK9抑制剂处理，并设置不同时间梯度。