Abstract
Abstract Peposertib is a small-molecule inhibitor of DNA-PK, a key regulator of nonhomologous end joining (NHEJ). NHEJ inhibition along with irradiation (IR)-induced DNA double-strand breaks increases antitumor treatment efficacy. Here, we investigate responses of HNSCC models with distinct HPV and p53 status to the treatment with IR, DNA-PK inhibition, and their combination. Three groups of cell lines with various HPV/p53 genotypes (p53-wt/HPV-; p53-mutated/HPV-, and p53-wt/HPV+) were treated by peposertib, 4Gy IR, or a combination of both. In addition to viability and cell cycle assays, caspase 3 activity and senescence-associated β-galactosidase (β-gal) assays were used to evaluate cell fates such as apoptosis and senescence. yH2AX and RAD51 foci immunostainings were implemented to assess the levels of DNA damage and repair. NMRI-nu mice with subcutaneous xenografts of p53-wt/HPV+ and p53-wt/HPV- cell lines were treated with either fractionated 10Gy IR (via the small animal radiation therapy system SmART) alone or in combination with orally distributed peposertib for five consecutive days. Tumors were collected at the end of experiment and the immunostaining targeting Ki67, nuclear DNA fragmentation (TUNEL staining of apoptosis) and β-gal (senescence) was performed. Decreased number of viable cells after IR alone and particularly after combined treatment was observed in most cell lines. Inhibition of NHEJ combined with IR induces an abrogation of proliferation with different cell fates. Whereas HPV+ and p53-mutated cells undergo apoptosis due to a common alteration in the p53 pathways, p53-wt cells are preferentially eliminated through senescence. Elevated yH2AX foci formation after 24h and 48h in the combination treatment group indicates unresolved persistent DNA damage. In vivo, significant effects of IR and of the combination treatment on tumor growth control was observed in p53-wt/HPV+ but not in p53-wt/HPV- xenografts where the tumors relapsed towards end of follow-up. High level of proliferative and apoptotic cells and low level of senescent cells detected in HPV+ compared to HPV- may potentially determine a better treatment response in HPV+ tumors. Diverse pattern of viability and cell cycle distribution upon treatment with IR or combination in cells with dysfunctional p53 compared to cells with p53-wt confirmed the outcome dependence on p53 and HPV status. Peposertib radiosensitizes HNSCC tumors and leads to a better treatment response in dysfunctional p53 cells both in-vitro and in-vivo. Determination of the HPV and p53 status in a particular tumor might be necessary to effectively shape the intervention outcome when combining NHEJ targeting with radiation therapy. The work was supported by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945), who provided peposertib free of charge. Citation Format: Liana Hayrapetyan, Selina M. Roth, Lusine Hovhannisyan, Matúš Medo, Daniel M. Aebersold, Yitzhak Zimmer, Michaela Medová. p53-dependent treatment response to DNA-PK inhibition in combination with irradiation in head and neck squamous cell carcinoma (HNSCC) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 498.
Published Version
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