RFC4 Drives Temozolomide Resistance in Glioblastoma by Activating STK38-BECN1-Dependent Autophagy

Glioblastoma (GBM) remains intractable owing to treatment resistance and near-inevitable relapse. While autophagy has been demonstrated to contribute to temozolomide (TMZ) resistance, its regulatory mechanisms remain unclear. RFC4, a replication factor implicated in tumor progression, has not yet been systematically investigated in glioma for its potential role in autophagy regulation. In this work, multi-omics analyses and immunohistochemistry were used to evaluate clinical relevance of RFC4. TMZ-resistant GBM cell lines and orthotopic mouse models were used for functional studies. Autophagy was assessed via LC3B-II quantification, immunofluorescence, and electron microscopy. Structural interactions were mapped using truncation mutants, GST pull-down assays, and molecular docking. We found that RFC4 played oncogenic roles in GBM tissues, correlating with poor prognosis and TMZ resistance. TMZ treatment increased RFC4 enhancer accessibility and stabilized STK38, a kinase that regulates autophagosome formation. RFC4-STK38 interaction promoted BECN1 recruitment, thereby driving autophagy. Critically, phosphorylation of STK38 at T444 was essential for stabilizing the RFC4-STK38-BECN1 complex, whereas the phospho-deficient STK38-T444A mutation impaired BECN1 binding and autophagosome maturation. In vivo, RFC4 overexpression conferred TMZ resistance; however, this resistance was reversed by chloroquine-mediated autophagy inhibition. Co-overexpression of RFC4 and STK38 predicted the worst patient survival, and STK38 knockdown induced synthetic lethality in this context. Together, this study identified the RFC4-STK38-BECN1 axis as a master regulator of TMZ resistance via autophagy. The T444 phosphorylation could be a druggable vulnerability and its disruption reversed pro-survival autophagy. Clinically, RFC4/STK38 co-expression stratified high-risk GBM patients. Preclinically, targeting RFC4-STK38 synergized with TMZ to overcome resistance. These findings elucidated a novel GBM resistance mechanism and provided a translational roadmap for precision therapy.