CDK9异常活化参与了超增强子介导的短寿命蛋白质转录，这些蛋白质对癌细胞的生存至关重要。靶向CDK9已在临床试验中对不同类型的癌症显示出强效的抗肿瘤活性。然而，有关CDK9抑制剂耐药性的研究和知识非常有限。在本研究中，我们建立了一种获得对高选择性CDK9抑制剂BAY1251152耐药性的AML细胞系。通过基因组测序，我们在CDK9的激酶结构域中鉴定到一个L156F突变，该突变也是CDK9基因中的编码SNP。通过使用CRISPR/Cas9将L156F插入癌细胞中，我们发现单个CDK9 L156F突变可以驱动对CDK9抑制剂的耐药性，无论是ATP竞争性抑制剂还是PROTAC降解剂。机制上，由于位阻作用，CDK9 L156F破坏了与抑制剂的结合，进一步，该突变影响了CDK9蛋白的热稳定性和催化活性。为了克服由CDK9-L156F突变介导的耐药性，我们发现了一种化合物IHMT-CDK9-36，该化合物对CDK9 WT和L156F突变体均表现出强效的抑制活性。总之，我们报道了CDK9抑制剂的一种新的耐药机制，并为未来CDK9抑制剂的开发提供了一种新的化学结构。© 2023 中国药学会和中国医学科学院药物研究所。Elsevier B.V.负责出版和主持。

Abnormally activated CDK9 participates in the super-enhancer mediated transcription of short-lived proteins required for cancer cell survival. Targeting CDK9 has shown potent anti-tumor activity in clinical trials among different cancers. However, the study and knowledge on drug resistance to CDK9 inhibitors are very limited. In this study, we established an AML cell line with acquired resistance to a highly selective CDK9 inhibitor BAY1251152. Through genomic sequencing, we identified in the kinase domain of CDK9 a mutation L156F, which is also a coding SNP in the CDK9 gene. By knocking in L156F into cancer cells using CRISPR/Cas9, we found that single CDK9 L156F could drive the resistance to CDK9 inhibitors, not only ATP competitive inhibitor but also PROTAC degrader. Mechanistically, CDK9 L156F disrupts the binding with inhibitors due to steric hindrance, further, the mutation affects the thermal stability and catalytic activity of CDK9 protein. To overcome the drug resistance mediated by the CDK9-L156F mutation, we discovered a compound, IHMT-CDK9-36 which showed potent inhibition activity both for CDK9 WT and L156F mutant. Together, we report a novel resistance mechanism for CDK9 inhibitors and provide a novel chemical scaffold for the future development of CDK9 inhibitors.© 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.