BUB1 and CDK4/6 Dual Inhibition Increases Radiation Sensitivity in Glioblastoma, Lung Cancer, and Triple-Negative Breast Cancer.

Background: Solid tumors including glioblastoma (GBM), lung cancer (LC), and triple-negative breast cancer (TNBC) exhibit marked radioresistance driven by dysregulated cell-cycle control and genomic instability. Although CDK4/6 inhibitors suppress tumor proliferation, their radiosensitizing capacity is limited by persistent DNA repair. BUB1, a mitotic checkpoint kinase overexpressed in aggressive cancers, has emerged as a regulator of DNA damage signaling. We tested whether co-targeting BUB1 and CDK4/6 enhances radiosensitivity across solid tumors. Methods: GBM, LC, and TNBC cell lines were treated with BUB1 inhibitor BAY1816032, CDK4/6 inhibitors ribociclib and abemaciclib, and radiation. Proliferation, clonogenic survival, immunoblotting, and combination index analyses assessed cytotoxicity and synergy. CDK4/6-resistant models were generated to examine resistance. In vivo efficacy was evaluated using SUM159 xenografts. DNA damage and homologous recombination repair were measured by gH2AX, RAD51, RPA, BrdU foci and comet assay. The resection branchpoint was assessed by phospho-RPA, with ATR inhibition and BLM or EXO1 depletion testing resection dependence. Results: BUB1 inhibition increased cytotoxicity in vitro and improved therapeutic response in vivo. Combined BUB1 and CDK4/6 inhibition showed strong synergy (C.I. < 1) and enhanced radiosensitization in RB+ models. CDK4/6-resistant cells displayed increased BUB1 expression, and BUB1 inhibition partially restored sensitivity. Mechanistically, dual inhibition intensified homologous recombination defects, marked by persistent gH2AX and altered RAD51, RPA, and BrdU dynamics, consistent with sustained single stranded DNA and impaired HR repair. Persistent RPA32 Ser33 phosphorylation reflects ATR-dependent resection that requires BLM and EXO1 at later stages, supporting sustained resection and unresolved repair leading to increased cell death. Conclusions: Dual inhibition of BUB1 and CDK4/6 represents a promising therapeutic strategy for enhancing radiosensitivity in GBM, lung cancer, and TNBC, particularly in Rb-intact settings.