Abstract 5982: Mechanisms underlying the anti-cancer activity of new, non-covalent thioredoxin reductase inhibitors

Abstract Triple negative breast cancer (TNBC) makes up only 15-20% of all breast cancer cases, yet it contributes to 50% of all breast cancer related mortalities. This highlights the urgent need to develop novel therapeutics for the treatment of TNBC. The thioredoxin (Trx) system is an important cellular antioxidant pathway that plays crucial roles in redox homeostasis and supports proliferation, apoptosis, and gene transcription; Thioredoxin reductases (TXNRDs) are the central enzymes in this pathway. Of note, TXNRDs are elevated in TNBC to help tumors cope with excessive redox stress resulting from deregulated metabolism and replicative stress, thus presenting a potential vulnerability. A series of breakthrough studies by our collaborators identified a novel regulatory and druggable “doorstop” pocket in this family of enzymes to which they have developed inhibitors for. These novel TXNRD(i)s show anti-cancer activity in a panel of TNBC lines, by inhibiting proliferation, inducing cell death, inhibiting clonogenic survival, mammosphere forming efficiency and invasion. Importantly, these TXNRD(i)s also inhibit tumor growth in a xenograft model of TNBC. Our goal is to understand the mechanisms underlying the pleiotropic anti-cancer activities elicited by these TXNRD inhibitors. We hypothesize our novel TXNRD(i)s work, in part, by increasing and shifting the balance of redox stress to block proliferation and induce cell death in TNBC cells. To understand major pathways implicated, we conducted transcriptomics (RNAsequencing) in TNBC cells treated with two lead TXNRD(i)s, 8VP101 and 9VP19. Bioinformatic analysis (GSEA) indicates multiple proliferation and mRNA related pathways predominantly enriched by 8VP101. This is consistent with induction of redox stress. To corroborate this finding, cells were treated with 8VP and ROS levels were determined by incubating cells with DCFDA, a ROS sensitive probe, and analyzed by flow cytometry. We find a significant induction in ROS levels comparable to auranofin, a non-specific covalent inhibitor of TXNRDs. Another predominant pathway emerging from GSEA was regulation of cell cycle. Cell cycle analysis in cells treated with 8VP also shows an accumulation in G1. Based on our data, we conclude that our novel TXNRD(i)s elicit anti-cancer activity by elevating intracellular ROS leading to cell cycle arrest and induction of apoptosis and necroptosis. Our findings have important implications for advancing new therapies for TNBC. Citation Format: Abigail Rullo, Brenna Flowers, Keacha Chang, Pavel Petukhov, Irida Kastrati. Mechanisms underlying the anti-cancer activity of new, non-covalent thioredoxin reductase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5982.