Triplet radio-chemo-immunotherapy reprograms the PD-L1-CDK6 axis to remodel the tumor microenvironment and overcome immune resistance in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) remains a highly aggressive malignancy with limited therapeutic success and frequent resistance to standard chemoradiotherapy. Beyond its cytotoxic effects, radiotherapy (RT) has emerged as a potent immune modulator capable of reshaping the tumor microenvironment (TME). However, the molecular mechanisms linking RT to immune reprogramming and adaptive therapeutic resistance remain incompletely defined. In this study, we identify a previously underexplored IFNγ-PD-L1-CDK6-STAT3 signaling axis as a central driver of immune evasion and oncogenic persistence in HNSCC. Transcriptomic analyses of TCGA-HNSC datasets revealed coordinated upregulation of IFNγ, CD274 (PD-L1), and CDK6 in tumor tissues, with elevated CDK6 expression associated with poorer overall survival. Using in vitro and in vivo models, we demonstrate that clinically relevant X-ray irradiation generated by a linear accelerator induces time-dependent upregulation of PD-L1, IFNγ, CDK6, and STAT3 within 12-48 h following irradiation, reflecting an adaptive immune response coupled with activation of pro-survival transcriptional programs. Pharmacologic inhibition of CDK6 with palbociclib attenuated radiation-induced PD-L1 expression, enhanced apoptosis, and potentiated T-cell- and natural killer (NK)-cell-mediated cytotoxicity. In an HNSCC xenograft model, combined RT + anti-PD-L1 + palbociclib therapy significantly suppressed tumor growth, increased infiltration of CD8⁺ and CD4⁺ T cells, promoted repolarization of tumor-associated macrophages (TAMs) toward an M1 phenotype, and reduced immunosuppressive signaling compared with monotherapy or dual-treatment arms. Mechanistically, RT acted as an immune primer, PD-L1 blockade relieved checkpoint-mediated immune suppression, and CDK6 inhibition disrupted a pro-survival feedback loop sustaining immune resistance. Collectively, this triplet strategy enhances tumor immunogenicity while counteracting RT-induced adaptive immune resistance, providing a mechanistically integrated therapeutic approach to overcome treatment resistance in HNSCC.