Abstract Actinomycin D (ActD) was the first anti-cancer antibiotic approved for the management of human cancers. However, the notorious toxicity profile limits its widespread application in cancers including cancers of the aerodigestive tract. Recent studies show that combining low-dose ActD with existing chemotherapies could potentially reduce the toxicity of chemotherapy drugs through p53 mediated cyclotherapy (protection of normal cells while eliminating cancer cells), a recently coined concept. An understanding of ActD’s effect on p53 signaling is critical for meaningful application of ActD in cyclotherapy-based combinations. This study evaluated the antitumor efficacy and mechanism of action of ActD in aerodigestive tract cancers. We found that ActD strongly inhibited the growth of a panel of aerodigestive tract cancer cell lines and induced efficient apoptosis although the sensitivity varies among cell lines. The IC50 values of ActD spanned between 0.021-2.96nM. Mechanistic studies revealed that ActD time- and dose-dependently increased the expression of total and phosphorylated p53 (ser15). Furthermore, ActD-induced apoptosis is dependent on p53 in cells expressing wild-type p53. Further studies demonstrated that ActD induced context-dependent differential expression of downstream targets p21 and PUMA without significant effects on p27. Interestingly, our study revealed that p53-p21 is the predominant pathway activated by low-dose ActD in cells with wild type p53. Therefore, our findings further rationalize the premise for future studies to explore the application of low-dose ActD as a chemoprotectant for cyclotherapy combinations in aerodigestive tract cancers. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence) Citation Format: Adeoluwa A. Adeluola, A. R. M Ruhul Amin. Low dose actinomycin D preferentially activates p53-p21 pathway in aerodigestive tract cancers: Implication for cyclotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1837.