Jia Xinglong, Zhang Jingdan, Pan Lulu, He Jingliang, Zhu Mingrui, Zhao Lei, Zhang Xingyu, Zhao Wensi, Xie Dong, Shen Xiaoyan, Liu Bin, Tan Minjia
School of Pharmacy, Fudan University, Shanghai, China.
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
Cell Death Discov. 2025 Aug 25;11(1):404. doi: 10.1038/s41420-025-02677-5.
PF-3758309, a pyrrolopyrimidine-based inhibitor of p21-activated kinase 4 (PAK4), has demonstrated preclinical anti-tumor activity. However, due to poor pharmacokinetics and off-target effects, it has not advanced to clinical use. In this study, we conducted a comprehensive multi-omics analysis, including proteomics, transcriptomics, and ubiquitinomics, to investigate the mechanism of PF-3758309 in HCT116 cells. Our results revealed that PF-3758309 promotes the degradation of RNA polymerase II subunit proteins (POLR2A/B/E) via the cullin-RING ligase pathway. This process is mediated by the E3 ubiquitin ligase DNA damage-binding protein 2 (DDB2), and is independent of PAK4. Furthermore, the small-molecule inhibitor MLN4924, which blocks NEDD8-activating enzyme, reversed the degradation of POLR2A/B/E, supporting the role of ubiquitin-proteasome pathways in this process. Functional assays confirmed that PF-3758309 inhibits tumor cell growth and migration by promoting ubiquitination-dependent degradation of POLR2A/B/E. These findings uncover a previously unrecognized mechanism of PF-3758309's anti-tumor activity and provide a basis for further investigation into its therapeutic potential.
PF-3758309是一种基于吡咯并嘧啶的p21激活激酶4（PAK4）抑制剂，已显示出临床前抗肿瘤活性。然而，由于其药代动力学不佳和脱靶效应，它尚未进入临床应用。在本研究中，我们进行了全面的多组学分析，包括蛋白质组学、转录组学和泛素组学，以研究PF-3758309在HCT116细胞中的作用机制。我们的结果表明，PF-3758309通过cullin-RING连接酶途径促进RNA聚合酶II亚基蛋白（POLR2A/B/E）的降解。这一过程由E3泛素连接酶DNA损伤结合蛋白2（DDB2）介导，且独立于PAK4。此外，阻断NEDD8激活酶的小分子抑制剂MLN4924逆转了POLR2A/B/E的降解，支持了泛素-蛋白酶体途径在这一过程中的作用。功能试验证实，PF-3758309通过促进POLR2A/B/E的泛素化依赖性降解来抑制肿瘤细胞的生长和迁移。这些发现揭示了PF-3758309抗肿瘤活性的一种先前未被认识的机制，并为进一步研究其治疗潜力提供了基础。
