Kitasamycin overcomes ferroptosis and immunotherapy resistance by targeting the HUWE1-NCOA4-FTH1 Axis

Clinicians typically avoid antibiotics use during immunotherapy due to concerns about reduced efficacy. However, cancer patients requiring antibiotics postoperatively or for infections urgently need options that provide antimicrobial coverage while potentially enhancing, rather than impairing, immunotherapy. Restoring ferroptosis susceptibility represents a promising strategy to overcome immunotherapy resistance, yet the role of antibiotics in modulating ferroptosis and interacting with immunotherapy remains unexplored. In this study, we screened 96 FDA-approved antibiotics across seven pharmacological classes and identified the macrolide kitasamycin as a specific and potent ferroptosis sensitizer in vitro and in vivo. Mechanistically, kitasamycin competitively bound to HUWE1, inhibiting its E3 ubiquitin ligase activity, which stabilized NCOA4 and activated the NCOA4-FTH1 ferritinophagy axis. Single-cell transcriptomics, flow cytometry, and multiplex immunohistochemistry revealed that kitasamycin induced immunogenic ferroptosis and reshaped anti-tumor T-cell immunity. Critically, kitasamycin potentiated immune checkpoint blockade (ICB)-mediated ferroptosis and overcame ICB resistance across multiple preclinical melanoma models, including B16F10 subcutaneous tumors, BRAF-PTEN-driven spontaneous tumors, and human sourced peripheral blood mononuclear cells (HsPBMCs)-humanized mouse models. Clinically, a high NCOA4, low HUWE1 signature correlated with ferroptosis activation, increased T-cell infiltration, and improved survival in ICB-treated patients, suggesting its potential as a predictive biomarker. Our findings positioned kitasamycin as a promising adjunct to immunotherapy for cancer patients requiring concurrent antibiotic therapy. Abbreviations: FTH1: ferritin heavy chain 1; ICB: immune checkpoint blockade; IFNG: interferon gamma; mIHC: multiplex immunohistochemistry; scRNA-seq: single-cell RNA sequencing.