Multi-omics analysis reveals RNA polymerase II degradation as a novel mechanism of PF-3758309's anti-tumor activity.

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.