Introduction: Radiochemotherapy with the apoptosis stimulator Xevinapant was the first arm to ever demonstrate superiority over radiochemotherapy in a randomized (phase 2) trial in HNSCC. However, the following phase 3 Trilynx-study recently failed to achieve its primary endpoint. In this project we aim at identifying effective combinations for Xevinapant to optimize curative treatment for HPV-negative HNSCC patients by testing an ATR (Tuvusertib) and a PARP inhibitor (Olaparib) in comparison to cisplatin.

Material and methods: Effectiveness of ATR-inhibition was tested through the potential to inhibit radiation-induced G2 cell-cycle arrest. Cytostatic effects were assessed by proliferation assay, radiosensitization by colony formation assay. Apoptosis induction was assessed by Annexin V staining.

Results: Effective inhibition of radiation-induced G2-arrest was observed at only 30nm Tuvusertib in all 4 cell lines tested. Proliferation analyses showed varying sensitivities of the cell lines towards the different inhibitors. With the exception of HSC4, growth inhibition under combined treatment did not suggest more than additive effects. When combined with radiation in colony formation assays, our data suggest the most effective radiosensitization through ATR-inhibition and the least through Xevinapant. Xevinapant combinations mostly suggested additive rather than synergistic effects. Xevinapant and irradiation resulted in increased rates of apoptosis, moderately increased under combined inhibition.

Conclusions: ATR-inhibition resulted in the most distinct radiosensitization. Our current data suggest a mostly moderate benefit for combinations with Xevinapant. The contribution of the programmed cell death pathways apoptosis and necroptosis will further be investigated.

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