Diving into AA9-mediated transglutaminase 2 inhibition reveals ferroptosis as driver of anticancer effects.

Ferroptosis recently gained considerable attention in breast cancer research, particularly in triple-negative subtypes. In this context, emerging evidence links transglutaminase 2 (TG2) to aggressive tumour phenotypes, highlighting pharmacological TG2 inhibition as a promising therapeutic approach in aggressive breast cancer settings. Comparative analysis of independent RNA-sequencing datasets from distinct cellular models treated with irreversible TG2 inhibitors revealed ferroptosis as the only significantly enriched and commonly deregulated process. In triple-negative breast cancer cells, treatment with the cell-permeable AA9 inhibitor triggered a coherent ferroptotic transcriptional program, characterized by downregulation of glutathione peroxidase 4 and the glutathione biosynthesis genes and by upregulation of stress-responsive and iron-handling factors. A covalent pull-down magnetic system combined with proteomics and bioinformatic target prediction identified TG2 as the primary intracellular target of AA9 and revealed a TG2-centered interactome involving, among the pathways, hypoxia signalling, cytoskeletal regulation, and apoptosis. Moreover, molecular docking and immunoprecipitation experiments demonstrated that TG2 is physically associated with BH3-interacting domain death agonist and Bcl-2-associated X protein, key mediators of ferroptosis and apoptosis. Transmission electron microscopy further confirmed that AA9 induced ferroptosis-associated mitochondrial alterations, prevented by ferrostatin-1 co-treatment. Overall, these findings confirm TG2 pharmacological targeting as a strategy to reprogram metabolic adaptation and cell death responses in aggressive tumour subtypes.