Time-resolved proteomic and phosphoproteomic analysis reveals convergent and divergent biological perturbations induced by FDA-approved CDK4/6 inhibitors in hormone receptor-positive breast cancers

Palbociclib, ribociclib, and abemaciclib, which are FDA-approved CDK4/6 inhibitors, constitute the standard first-line treatment for advanced hormone receptor-positive (HR⁺) breast cancers. Although these drugs share nominal targets and demonstrate comparable first-line efficacy, significant divergences exist in their cross-line therapeutic responses upon first-line progression, indicating distinct mechanisms of action. To systematically elucidate their similarities and differences, we employed quantitative proteomics and phosphoproteomics to investigate and compare the molecular expression characteristics induced by these drugs. As a result, the three CDK4/6 inhibitors exhibited distinct anti-proliferative potencies in HR⁺ breast cancer cells. Dynamic proteomic and phosphoproteomic profiling demonstrated cell cycle arrest and mTORC1 pathway downregulation by all inhibitors, with abemaciclib exerting the most potent suppression. Subsequent kinase activity analysis indicated an upregulation of AKT1 kinase activity following CDK4/6 inhibitor stimulation. Furthermore, acridine orange staining and flow cytometry indicated that abemaciclib elevated lysosomal proteins and acidification while increasing reactive oxygen species (ROS). Collectively, this study deepens the understanding of shared and drug-specific molecular characteristics and mechanisms of CDK4/6 inhibitors at multi-omic levels. Moreover, it provides an experimental basis and potential directions for customizing combination therapies based on pathway vulnerabilities, as well as exploration of novel therapeutic modes and drugs.