Inhibition Of Cdk4/6 In Head And Neck Squamous Cell Carcinoma Dismantles Key Dna Repair Pathways In Response To Radiation Treatment.

Abstract Introduction: Conventional treatment for head and neck squamous cell carcinoma (HNSCC) relies on surgery and radiation for cure; locoregional failure is often related to recurrence after radiation. CDKN2A, coding for the CDK4/6 inhibitory protein, p16INK4a (p16), is among the most commonly lost tumor suppressors in HNSCC. Palbociclib, a chemotherapeutic agent that is FDA-approved for breast cancer, is a selective CDK4/6 inhibitor. FOXM1 is a key cell cycle regulatory protein whose activation is controlled by CDK4/6. FOXM1 controls cell proliferation, DNA damage repair and suppresses senescence. The current study examines the role of CDK4/6 and its targets in response to radiation in HNSCC. We demonstrate inhibiting CDK4/6 can synergize with radiation, partially via suppression of FOXM1 signaling and impact on senescence. Experimental Procedure: Our study focuses on utilizing palbociclib to increase radiation response in HPV(-) oral cavity HNSCC and understanding mechanisms of potential radiosensitization. We examined cell viability after concurrent palbociclib and radiation (P+RT) application using proliferation assays in 2-dimensional (2D) and 3D organoid models of immortalized cell lines (HN5 and Cal27), and calculated synergy by Compusyn analysis. After 3-days treatment, we performed western blot to evaluate DNA damage response (DDR) proteins and β-galactosidase (β -gal) staining to evaluate the senescent phenotype. Flow cytometry studies were used to measure cell cycle arrest and QPCR to measure expression of cell cycle regulators. Results: P+RT (1µM palbociclib + 2 or 4Gy RT) demonstrated consistent synergy in both 2D and 3D viability assays. Comparing P+RT to RT alone, flow cytometry data revealed a 2-fold increase in the G1 arrested population, and DNA damage (measured by γ-H2AX level) increased by 2.3 fold. Additionally, Rad51 and Ku80 showed a 2 and 10-fold decrease respectively, suggesting an impact on DDR repair via both the homologous recombination and non-homologous end joining pathways. On transient knockdown of CDK-4 or 6, or both, we observed that senescence was the prevailing response - concurrent inhibition of CDK4 and CDK6 was the most profound, and levels of senescence based on β -gal staining results matched that observed with palbociclib treatment. P+RT demonstrated a robust 3-fold decrease in FOXM1 mRNA level and a 10-fold decrease in protein expression. We observed a 5-fold decrease in FOXM1 levels with CDK6 inhibition compared to a 2-fold decrease with CDK4 inhibition alone. Independently, FOXM1 inhibition induced apoptosis in HNSCC to a level higher than staurosporine-control treated cells. Conclusions: Our results suggest that palbociclib may make HNSCC more susceptible to RT via the induction of senescence related to suppressed signaling through FOXM1.We propose P+RT as a rational and potentially effective treatment strategy in HNSCC Citation Format: Nitisha Shrivastava, Carlos Thomas, Daniel Li, Cory D. Fulcher, Michael B. Prystowsky, Indranil Basu, Chandan Guha, Thomas J. Ow. Inhibition of CDK4/6 in head and neck squamous cell carcinoma dismantles key DNA repair pathways in response to radiation treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1958.