Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a promising therapeutic strategy for cervical cancer. However, the mitochondrial factors governing ferroptosis sensitivity in this malignancy remain incompletely understood. PET117, a conserved mitochondrial protein, has been implicated in mitochondrial homeostasis, yet its role in ferroptosis regulation and cervical cancer pathophysiology is unknown. Here, we report a novel role of PET117 in regulating ferroptosis. PET117 expression was significantly elevated in cervical cancer tissues and loss of PET117 in HeLa cells markedly suppressed erastin- and RSL3-induced ferroptosis. Mechanistically, PET117 deficiency attenuated intracellular reactive oxygen species (ROS) accumulation, lipid peroxidation, and iron overload. Mitochondrial proteomics and RNA-seq revealed extensive remodeling of the mitochondrial proteome and ferroptosis-related transcriptional networks upon PET117 depletion. Notably, integrative analysis of mitochondrial and nascent proteomes identified acyl-CoA synthetase family member 2 (ACSF2) as a downstream target of PET117. These findings establish PET117 as a novel regulator of ferroptosis in cervical cancer, thereby linking mitochondrial function to ferroptosis regulation.

Kinase inhibitors have achieved great success in targeted cancer therapy, yet the evident limitations in their effectiveness persist due to therapeutic resistance. To gain insight into the molecular mechanisms and thwart resistance, we profiled the time‐resolved nascent protein perturbations in response to drug therapy using metabolic labeling and facilitated the identification of 2238 proteins via liquid chromatography tandem mass spectrometry (LC–MS/MS). Among these, 51 proteins exhibited upregulation, whereas 105 proteins showed downregulation following a 24‐h drug treatment. Clustering analysis revealed that the differential proteins were mainly enriched in metabolic‐related pathways. Combined with changes in whole‐protein levels, we noticed significant fluctuations in the metabolism‐related protein nicotinamide N ‐methyltransferase (NNMT). Additionally, NNMT overexpression diminished drug effectiveness, whereas its inhibition enhanced therapeutic efficacy. An increase in NNMT was also found in drug‐resistant cells, and the NNMT inhibitor JBSNF‐000088 inhibited the proliferation of resistant cells. Subsequent phosphoproteomic analysis indicated that the effects of NNMT overexpression on transcription factors, proteins involved in the Rho GTPases cycle, and cell‐cycle‐related proteins may be related to tumor resistance. In summary, our study provides unique insights into nascent protein perturbations during the initial stages of drug therapy and identified NNMT as a promising target for delaying and overcoming therapeutic resistance.