Targeting the FBXO5-DOK6 axis to overcome temozolomide resistance in glioblastoma via proteasome-cytomechanics regulation.

The development of resistance to temozolomide (TMZ) represents a critical challenge in the management of glioblastoma (GBM), yet the molecular basis for this resistance is still not fully elucidated. In this study, we utilized a custom-designed CRISPR/Cas9 sgRNA library that targets 1117 genes associated with ubiquitination, combined with a positive-negative dual screening strategy, to identify pivotal factors in TMZ resistance. This approach revealed F-Box Protein 5 (FBXO5), an E3 ligase subunit, as a major contributor to TMZ resistance. Correlation analyses indicated that high levels of FBXO5 are associated with poor treatment response and reduced survival in GBM patients. Further investigation showed that FBXO5 promotes K48-linked polyubiquitination and subsequent proteasomal breakdown of DOK6, a protein whose depletion enhances mechanical rigidity in tumor cells and initiates survival signaling. Additionally, through virtual screening of a natural product library containing 4500 compounds, we identified Theaflavin 3,3'-digallate (TF3) as a selective inhibitor of FBXO5. This compound was found to synergistically improve TMZ sensitivity both in vitro and in vivo. Our results highlight a critical proteasome-cytomechanics pathway in GBM chemoresistance and suggest that targeting FBXO5 could be an effective therapeutic strategy for treating patients with GBM.