2563 - Co-Inhibition of BUB1 and CDK4/6 Augments Radiation Response in TNBC, Glioblastoma, and Lung Cancer

Purpose/Objective(s): Solid tumors including glioblastoma (GBM), triple negative breast cancer (TNBC), and lung cancer (LC) remain difficult to treat due to their rapid proliferation, genomic instability, and resistance to standard therapies. Aberrant activation of cell-cycle regulators such as CDK4/6 drives unchecked tumor growth and contributes to therapeutic failure. BUB1, a serine/threonine kinase essential for the spindle assembly checkpoint, is frequently dysregulated in aggressive cancers and has recently emerged as a candidate therapeutic target.

Materials/Methods: Cell proliferation, clonogenic survival, and immunoblot analyses were conducted in GBM, TNBC, and LC cell lines treated with the BUB1 inhibitor BAY1816032, CDK4/6 inhibitors (ribociclib, abemaciclib), and radiation. Drug cytotoxicity and synergy were quantified across multiple treatment schedules using combination index (CI) analysis. CDK4/6 inhibitor resistant derivatives were generated to evaluate BUB1 mediated mechanisms of resistance. DNA damage and homologous recombination (HR) repair were assessed by gH2AX, RAD51, and RPA-BrdU colocalization studies. In vivo efficacy was tested using SUM159 mammary fat-pad xenografts treated with BUB1i, CDK4/6i, and radiation.

Results: BUB1 inhibition significantly reduced viability across all models tested. Combined BUB1i + CDK4/6i therapy produced robust synergy (CI<1) in both RB-wild-type and RB-null cell lines. The addition of BUB1i markedly enhanced CDK4/6i-mediated radiosensitization in the majority of cell lines. In vivo, BUB1i further sensitized SUM159 tumors to CDK4/6 inhibition and radiation. Newly established CDK4/6i-resistant lines displayed BUB1 overexpression, and BUB1i partially restored CDK4/6i sensitivity. Mechanistically, BUB1i amplified CDK4/6i-induced impairment of HR repair, as evidenced by increased gH2AX accumulation and altered RAD51, RPA and BrdU foci dynamics. At later time points, combination treated cells exhibited larger RPA and BrdU foci with increased RPA-BrdU colocalization, consistent with persistent ssDNA, stalled replication forks, and impaired HR-mediated repair.

Conclusion: Dual inhibition of BUB1 and CDK4/6 represents a promising therapeutic strategy for enhancing radiosensitivity and overcoming intrinsic or acquired resistance in aggressive solid tumors. These results support the development of combination approaches targeting complementary G1/S and G2/M cell-cycle nodes to improve radiotherapy efficacy and limit tumor adaptation.