CDK4/6 Inhibition Induces Senescence and Enhances Radiation Response by Disabling DNA Damage Repair in Oral Cavity Squamous Cell Carcinoma

SIMPLE SUMMARY: Human papilloma virus–negative (HPV(−)) oral cavity squamous cell carcinoma (OCSCC) is the leading cause of mortality amongst head and neck cancers. Radiation resistance remains a prime cause of treatment failure in OCSCC. Overall, failure to cure locally advanced OCSCC remains a formidable challenge. The aim of our study was to exploit the hyperactive CDK4/6 axis in HPV(−) OCSCC by targeting it with the p16 mimetic palbociclib and to assess the resulting effect on susceptibility to radiation. Our study demonstrates that both homologous recombination (HR) and Non-homologous end joining pathway (NHEJ), two critical DNA damage repair pathways, are compromised after palbociclib-induced senescence in OCSCC cells, leading to enhance radiation sensitivity. Our findings provide important insight towards a promising treatment paradigm in OCSCC. ABSTRACT: Purpose: HPV(−) OCSCC resists radiation treatment. The CDKN2A gene, encoding p16INK4A, is commonly disrupted in OCSCC. p16 inhibits CDK4/CDK6, leading to cell cycle arrest, but the biological sequelae of CDK4/6 inhibition in OCSCC remains understudied. This study examines whether inhibition of CDK4/6 enhances radiation response in OCSCC. Methods: MTT assays were performed in OCSCC cell lines HN5 and CAL27 following treatment with palbociclib. Clonogenic survival and synergy were analyzed after radiation (RT-2 or 4Gy), palbociclib (P) (0.5 µM or 1 µM), or concurrent combination treatment (P+RT). DNA damage/repair and senescence were examined. CDK4/6 were targeted via siRNA to corroborate P+RT effects. Three-dimensional immortalized spheroids and organoids derived from patient tumors (conditionally reprogrammed OCSCC CR-06 and CR-18) were established to further examine and validate responses to P+RT. Results: P+RT demonstrated reduced viability and synergy, increased β-gal expression (~95%), and ~two-fold higher γH2AX. Rad51 and Ku80 were reduced after P+RT, indicating impairment of both HR and NHEJ. siCDK4/6 increased senescence with radiation. Spheroids showed reduced proliferation and size with P+RT. CR-06 and CR-18 further demonstrated three-fold reduced proliferation and organoids size with P+RT. Conclusion: Targeting CDK4/6 can lead to improved efficacy when combined with radiation in OCSCC by inducing senescence and inhibiting DNA damage repair.