Radiotherapy and DNA damage response inhibitors modestly sensitize HNSCC to NK cell killing, with ATM inhibition more effective than ATR inhibition

PURPOSE: Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC) remain challenging due to their reduced radiosensitivity. Targeting the DNA damage response (DDR) with kinase inhibitors may enhance tumor susceptibility to radiotherapy (RT). While RT is known to alter the immune phenotype of HNSCC cells, its impact on innate immune responses, particularly on natural killer (NK) cells, remains unclear. We therefore investigated how RT combined with ATM or ATR inhibition-key DDR kinases-affects NK cell-mediated killing and activation. METHODS: The tumor cell lines HSC4 and Cal33 (both HPV-negative) and UM-SCC-47 and UD-SCC‑2 (both HPV-positive) were treated with the DDR inhibitors VE-822 (ATRi) and AZD0156 (ATMi) and hypofractionated RT (2 × 5 Gy). After co-cultivation with primary human NK cells, tumor cell death was measured using flow cytometry after 24 h. Further, NK cell activation markers and the supernatant of tumor cell-NK cell co-cultures were measured and the effect of immune checkpoint inhibitors (durvalumab, monalizumab) was additionally investigated. RESULTS: Particularly the HPV-negative HSC4 cells pretreated with either ATMi or RT + ATMi showed significantly increased killing by NK cells. Reduced secretion of granulysin by NK cells was mainly found in the co-cultures of HPV-negative HNSCC treated with ATRi. Treatment with the immune checkpoint inhibitor durvalumab (anti-PD-L1) did not result in significant augmentation of NK cell-induced tumor cell killing in this setting. Conversely, treatment with monalizumab (anti-NKG2A) resulted in a modest increase. CONCLUSION: Natural killer cells showed only a limited contribution to the killing of HNSCC cells pretreated with RT or RT + DDRi. However, a subset of patients with head and neck tumors-such as those represented by the HSC4 model-might still benefit from combining RT with ATMi rather than ATRi to enhance NK cell-mediated tumor killing.