Targeting Riboflavin Kinase Induces Ferroptosis and Apoptosis in Triple-Negative Breast Cancer

Background: Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous subtype of breast cancer lacking estrogen receptor, progesterone receptor, and HER2 expression. Due to the absence of actionable molecular targets, patients rely heavily on chemotherapy, often facingearly recurrence and poor prognosis. There is an urgent need for novel therapeutic strategies that utilize alternative cell death mechanisms beyond conventional apoptosis. Methods: To identify metabolic vulnerabilities specific to TNBC, we employed an integrative strategy combining genome-scale metabolic modeling based on patient transcriptomic data with CRISPR-Cas9 dependency datasets. Riboflavin kinase (RFK), an enzyme that converts riboflavin into FMN and FAD, was identified as a top-ranked candidate target. Functional validation was conducted via genetic knockdown and pharmacological inhibition using roseoflavin. Cellular proliferation was assessed by WST assay and crystal violet staining. Apoptosis and ferroptosis were evaluated by Annexin V/PI flow cytometry, western blotting, JC-1 and C11-BODIPY fluorescence, ROS and MDA assays, and glutathione quantification. In vivo efficacy was tested in orthotopic xenograft models using RFK-silenced TNBC cells or roseoflavin-treated mice. Immunohistochemical analyses (Ki67, 4-HNE, TUNEL) were used to assess tumor proliferation, ferroptosis, and apoptosis, respectively. Results: RFK suppression significantly inhibited TNBC cell proliferation in vitro and in vivo . Mechanistically, RFK loss reduced glutathione levels, increased intracellular ROS accumulation,and enhanced lipid peroxidation, resulting in mitochondrial dysfunction and concurrent induction of ferroptosis and apoptosis. In TNBC xenograft models, RFK knockdown or roseoflavin treatment markedly reduced tumor growth, enhanced lipid peroxidation, and increased cell death. Transcriptomic analyses suggest that TNBC tumors, exhibiting heightened ferroptosis susceptibility, may engage in metabolic reprogramming,characterized by upregulation of genes involved in riboflavin uptake, flavin cofactor biosynthesis, and glutathione synthesis, as a compensatory adaptation toenhance redox buffering capacity and resist ferroptotic stress. Conclusions: Our study identifiedRFK as a TNBC-specific metabolic vulnerability, regulatingredox homeostasis and cell death pathways. Targeting RFK represents a promising therapeutic strategy for TNBC, as it induced both ferroptosis and apoptosis. These findings underscore the potential of exploiting the riboflavin–FMN/FAD–glutathione axis as a redox metabolic checkpoint in ferroptosis-prone TNBC.