Abstract A070: Time-Dependent Oxidation and Aggregation of Vimentin upon Binding by β-lap Induced Immunogenic Cell Death

Abstract β-lapachone (β-lap) has been demonstrated as an NAD(P)H:quinone oxidoreductase1 (NQO1) bioactivatable compound. It initiated a futile redox cycle catalyzed by NQO1, resulting in reactive oxygen species (ROS) generation, with consumption of the reductive NAD(P)H. After a 4-h pulse of sufficiently lethal dose of β-lap, high levels of ROS diffused into nuclei to induce tumor-selective DNA damage and programmed necrosis in cancers overexpressing NQO1. Recently, our experiments showed that prolonged low dose β-lap administration exerted significant and NQO1 independent antitumor efficacy in mouse models with no overt signs of toxicity. Thus, we synthesized a photoactive β-lap probe to identify the molecular target of β-lap. Vimentin, a major constituent of the intermediate filament family of proteins, was identified as the direct binding target of β-lap. Vimentin was highly upregulated in multiple cancers including PDAC, and was a binding partner of β-lap with a dissociation constant at micromolar levels. Ligand binding assays in Panc1 cells employing β-lap as a specific competitor showed that vimentin was bound by β-lap-probe in a β-lap-competitive manner, and further confirmed that the in vivobinding target of β-lap was vimentin. Moreover, it was manifested that endogenous vimentin mRNA levels directly correlated with sensitivity to prolonged exposure of β-lap in a panel of pancreatic cancer cell lines, and knockout of vimentin in Panc1 cells markedly decreased lethality after prolonged exposure of β-lap. Mechanistically, we revealed that β-lap bound to vimentin and induced disulfide bonds that participated in disulfide cross-linking between a pair of vimentin dimers in a concentration and time dependent manner, forming vimentin oligomers of approximately 150 kDa. Prolonged exposure of β-lap promoted the disruption of filament architecture and vimentin degradation, ultimately resulting in cell death. In addition, the cell death by targeting vimentin exhibited a more inflammatory condition and enhanced DC activation. Together, our findings unveiled an unknown molecular target for β-lap and provided valuable insights into its mechanism of action. Targeting vimentin also presented a new strategy that inhibited the tumor growth and synergized with immunotherapy to disrupt immune evasion and reduce immune suppression against pancreatic cancers. Citation Format: Feng Qian, Lei Dou, Kaixin Wang, Yi Xia, Zhidong Chen, Yumeng Zu, Shuang Xi. Time-Dependent Oxidation and Aggregation of Vimentin upon Binding by β-lap Induced Immunogenic Cell Death [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A070.