Inhibition of CDK12 induces cancer cell dependence on activated P-TEFb via the ATM-stimulated p53 and NF-kB transcriptional programs

P-TEFb and CDK12 facilitate transcription elongation by RNA polymerase II and play prominent roles in cancer. Understanding their functional interplay could inform novel anti-cancer strategies. While inhibition of CDK12 downregulates unique sets of genes, eliciting genomic instability that is being exploited for novel therapies, little is known about the significance of transcriptional induction in CDK12-targeted cells. We show that inhibition of CDK12 in colon cancer-derived cells activates P-TEFb and induces genes of key cancer signaling pathways, including p53 and NF-kB. Mechanistically, cancer cells become exquisitely dependent on P-TEFb through activation of p53-dependent apoptosis and attenuation of NF-kB-dependent proliferation. Furthermore, we show that the DNA damage-responsive ATM kinase mediates these effects. While ATM is required for the synthetic lethality of CDK12 and P-TEFb co-targeting in p53-proficient cells, co-inhibition of ATM and CDK12 synergizes in decreasing viability of p53-deficient cells. Finally, pairwise targeting of CDK12, P-TEFb and transcription initiation kinase CDK7 stimulates p53-dependent apoptosis of cancer cell spheroids. We propose that the stimulation of Pol II pause release by P-TEFb at the signal-responsive genes underlies the dependence of CDK12-targeted cancer cells on P-TEFb. Together, our work provides a rationale for combinatorial targeting of CDK12 and P-TEFb or the induced oncogenic pathways in cancer. Overall design: Precision run-on sequencing (PRO-seq) analysis of nascent transcription in human colorectal carcinoma cell line, HCT116, during the inhibition of CDK12 and CDK9.

P-TEFb与细胞周期蛋白依赖性激酶12（Cyclin-Dependent Kinase 12，CDK12）可通过RNA聚合酶II（RNA polymerase II）促进转录延伸，并在癌症发生发展中发挥关键调控作用。阐明二者的功能互作关系可为新型抗癌策略的开发提供理论支撑。尽管抑制CDK12可下调特定基因集，诱发基因组不稳定性且该特性已被应用于新型疗法的研发，但目前对于CDK12靶向细胞中转录诱导事件的生物学意义仍知之甚少。

本研究发现，在结直肠癌细胞中抑制CDK12可激活P-TEFb，并诱导p53与核因子κB（NF-κB）等关键癌症信号通路相关基因的表达。机制层面分析显示，癌细胞可通过激活p53依赖的细胞凋亡通路、削弱NF-κB依赖的增殖信号，变得对P-TEFb产生极度依赖。进一步研究表明，DNA损伤应答通路中的ATM激酶（ATM kinase）可介导上述效应。在p53功能完整的细胞中，ATM是CDK12与P-TEFb联合靶向产生合成致死效应的必要条件；而联合抑制ATM与CDK12则可协同降低p53缺陷细胞的存活率。

最后，同时靶向CDK12、P-TEFb以及转录起始激酶细胞周期蛋白依赖性激酶7（Cyclin-Dependent Kinase 7，CDK7），可诱导癌细胞球状体发生p53依赖的细胞凋亡。本研究提出，在信号应答基因处，P-TEFb对RNA聚合酶II暂停释放的促进作用，是CDK12靶向癌细胞依赖P-TEFb的分子基础。综上，本研究为联合靶向CDK12与P-TEFb，或是靶向本研究中发现的诱导型致癌通路提供了理论依据。

总体实验设计：对人类结直肠癌细胞系HCT116在CDK12与细胞周期蛋白依赖性激酶9（Cyclin-Dependent Kinase 9，CDK9）抑制过程中的新生转录，开展精准run-on测序（Precision run-on sequencing，PRO-seq）分析。