CDK4/6 inhibition radiosensitizes p16-negative squamous cell carcinomas of the aerodigestive tract via cell cycle arrest

INTRODUCTION: CDK4/6 inhibitors have quickly entered clinical guidelines for luminal breast cancer, but their role in other cancer types remains to be defined. Squamous cell carcinomas not driven by human papillomavirus (HPV-negative SCCs) are frequently treated with radiotherapy, and concurrent treatment with CDK4/6 inhibitors has been proposed. We sought to investigate the efficacy of this combination across a range of HPV-negative SCCs and define molecular biomarkers of response that could be used for treatment selection. MATERIALS AND METHODS: We employed clonogenic assays and a previously described long-term viability assay for measuring radiation response to identify radioresistant cells within HPV-negative SCC cell lines. Among consistently radioresistant lines, we conducted drug/radiation combinations to measure additive effects of the CDK4/6 inhibitors, abemaciclib and palbociclib. Mechanistic effects of drug/radiation combinations were further investigated by flow cytometry, immunocytochemistry, and western blot. Efficacy was validated in vivo using an HPV-negative SCC cell line xenograft. RESULTS: Among the 19 HPV-negative SCC cell lines, 6 were consistently radioresistant using both clonogenic and viability assays. We validated the use of the viability assay for drug/radiation combinations using known radiosensitizing drug, cisplatin, before applying it to the CDK4/6 inhibitors. Five of the 6 radioresistant cell lines displayed a favorable combination effect with both CDK4/6 inhibitors. Sensitivity to the combination was reliant on drug-induced cell cycle arrest and correlated with lack of p16 activity in the presence of Rb. Combining abemaciclib with radiation reduced tumor growth in xenografted mice. CONCLUSIONS: CDK4/6 inhibitors are good candidates for further preclinical and clinical evaluation in combination with radiation in HPV-negative SCCs due to their effects on cell cycle phase in cells with Rb protein activity and low p16 levels. The long-term viability assay allows for efficient testing of drug/radiation combinations and can be considered for future experiments as part of a bench-to-bedside translational pipeline.