MLKL confers ferroptosis resistance in colorectal cancer cells via impaired ESCRT-III-mediated membrane repair.

Abstract

Background: Ferroptosis resistance is a major challenge in cancer therapy, contributing to poor prognosis and unsatisfactory treatment outcomes. Thus, identifying key regulators of ferroptosis is essential for developing strategies to overcome this limitation. Mixed lineage kinase domain-like protein (MLKL) is well established as the executioner of necroptosis, but emerging evidence suggests broader functions beyond necroptosis. A previous study demonstrated that MLKL regulates ferroptosis in an acute kidney injury model. However, its role in ferroptosis regulation in cancer remains largely unexplored.

Results: Bioinformatics analyses revealed that MLKL expression positively correlates with glutathione metabolism-related genes in colorectal cancer (CRC) patients and with reduced sensitivity to GPX4 inhibitors across pan-cancer cell lines. Functional studies demonstrated that MLKL depletion sensitizes not only CRC cells but also multiple cancer cell types to ferroptosis induced by GPX4 inhibitors. Mechanistically, MLKL-deficient CRC cells exhibited reduced intracellular glutathione levels and dysregulation of the NRF2/KEAP1/SLC7A11 antioxidant axis. However, KEAP1 knockdown in MLKL-depleted cells partially reduced ferroptosis sensitivity, indicating that additional mechanisms are involved. Proteomic analysis identified CHMP5, a component of the ESCRT-III membrane repair machinery, as a key downstream effector associated with MLKL. MLKL depletion suppressed CHMP5 expression and impaired its translocation to the plasma membrane during ferroptosis induction. Consistent with defective membrane repair, MLKL-knockdown CRC cells exhibited increased HMGB1 release following GPX4 inhibitor treatment, indicating enhanced membrane damage. Notably, CHMP5 overexpression reduced ferroptosis in MLKL-knockdown cells, highlighting that MLKL regulates ferroptosis susceptibility in part through CHMP5-dependent ESCRT-III signaling.

Conclusions: This study reveals a previously unrecognized role for MLKL in ferroptosis regulation through coordinating NRF2/KEAP1/SLC7A11-mediated glutathione metabolism and ESCRT-III-dependent membrane repair. These findings identify MLKL as a potential regulator of ferroptosis resistance and warrant further studies to evaluate the therapeutic relevance of MLKL-targeting strategies in colorectal cancer.