Mouse double minute-2 (MDM2)-p53 inhibitor alrizomadlin (APG-115) is an investigational agent known to induce apoptosis of TP53-wild type cancer cells (Aguilar et al, J Med Chem 2017). Emerging evidence suggests that activation of p53 by alrizomadlin also promotes antitumor immunity in the tumor microenvironment (Fang et al, JITC 2019; Zhou et al, Nat Immunol 2021), but the links between these processes are incompletely understood. Pyroptosis refers to inflammatory programmed cell death. Central to this process is the family of gasdermins, which can form pores in cell plasma membranes, resulting in lysis and release of immune stimulants. In cells expressing these proteins, GSDME can be cleaved by caspase-3, which converts noninflammatory apoptosis to pyroptosis (Zhang et al, Nature 2020). In this context, caspase-3/GSDME appears to represent a switch between apoptosis and pyroptosis. Given that alrizomadlin elicits its apoptogenic activity primarily by activating caspase-3, we hypothesized that the MDM2-p53 inhibitor might also induce pyroptosis in GSDME-expressing cells by cleaving caspases. Our study demonstrated that alrizomadlin uniformly elicited robust pyroptosis in GSDME-expressing cancer cells. After treatment with alrizomadlin, the dying cells showed swelling with plasma membrane blebbing characteristic of pyroptosis. Alrizomadlin treatment upregulated biomarkers of pyroptosis, inducing activation of caspase-3, promoting cleavage of GSDME, and increasing release of lactate dehydrogenase (LDH). After a short drug exposure of 24 hours, alrizomadlin elicited concurrent apoptotic and pyroptotic tumor cell death, as evidenced by coexistence of the hallmarks of apoptosis (PARP-1 cleavage) and pyroptosis (caspase-3 cleavage and activated GSDME) markers. When the treatment duration was extended to 48 hours, cleavage of GSDME continued to increase whereas PARP-1 cleavage became nearly undetectable, suggesting a conversion from apoptosis to pyroptosis. Inhibition of caspases by pan-caspase inhibitor Q-VD-OPh (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone) abolished not only alrizomadlin-induced caspase-3 and GSDME cleavage but also reduced LDH release and cell death without inducing morphologic changes associated with pyroptosis. Taken together, these results suggest that, in addition to apoptosis, MDM2-p53 inhibitor alrizomadlin induces caspase-mediated pyroptosis in GSDME-expressing cancer cells. In this study, we reveal for the first time that apoptosis-inducing, alrizomadlin induces both apoptosis and pyroptosis in GSDME-expressing cancer cells. GSDME-dependent pyroptosis may be a previously unrecognized mechanism of action for alrizomadlin to exert antitumor immunity, with potentially important implications for clinical development of therapy involving MDM2-p53 inhibition.

Citation Format: Qiuqiong Tang, Douglas D. Fang, Huidan Yu, Bingxing Wu, Yan Yin, Dajun Yang, Yifan Zhai. Inhibition of MDM2-p53 interaction by alrizomadlin (APG-115) induces pyroptotic cell death in gasdermin E (GSDME)-expressing cancer cells [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 2998.