Abstract B027: CRISPR screens identify thioredoxin reductase 1 as a target for inducing disulfidptosis in IDH1 -mutant glioma models
Astrocytoma and oligodendroglioma are initiated by missense mutations in isocitrate dehydrogenase 1 and 2 genes (IDH1/2). The mutant IDH1 protein acquires neomorphic activity, producing (R)-2-hydroxyglutarate, which interferes with α-ketoglutarate-dependent epigenetic processes. These effects result in metabolic and epigenetic changes that impair cellular differentiation and promotes tumorigenesis Whole-genome CRISPR/Cas9 knockout screens were conducted using isogenic IDH1-mutant and wild-type (WT) U-87 MG cells and identified the thioredoxin reductase 1 gene (TXNRD1) as a selective dependency in IDH1-mutants. This work sought to validate this dependency and detail the underlying mechanism using thioredoxin reductase 1 (TrxR1) inhibitors and TXNRD1 knockout (KO) models. Antiproliferative potency of TrxR1 inhibitors (auranofin, TRi-1) were similar in IDH1 WT and mutant cells when assessed in both 2D monolayer and 3D soft agar colony assays. Glutathione levels and reduced:oxidized glutathione ratios were decreased similarly in IDH1-mutant and WT cells following TrxR1 inhibition, consistent with increased oxidative stress. Reactive oxygen species (ROS) measured using ROS-Glo™ or CM-H2DCFDA dye were increased following acute treatment with TRi-1 or auranofin. In the ROS-Glo™ studies, ROS levels were modestly higher in IDH1-mutant compared with WT cells. Loss of membrane integrity prevented CM-H2DCFDA dye retention and decreased the ability to accurately assess intracellular ROS. This observed change was consistent with induction of disulfidptosis, which was supported by rapid morphological rounding of cells upon TrxR1 inhibitor treatment, suggestive of actin cytoskeletal collapse. Rescue of TrxR1 inhibitor-treated cultures was achieved using co-treatment with reducing agents (NAC, DTT or TCEP), supporting disulfidptosis as the cell death mechanism. Overall, both IDH1-mutant and WT cells displayed similar sensitivity to TrxR1 inhibition. To confirm the effect of TXNRD1 KO observed in the screen, multiguide RNA-Cas9 ribonucleoproteins were used to generate TXNRD1 knockouts. Neither IDH1-mutant nor WT cells tolerated acute TXNRD1 KO, although some surviving clonal KO lines were developed after several weeks. These KO clones displayed slower growth kinetics and impaired growth in soft agar. In contrast to TrxR1 inhibition, TXNRD1 KO clones displayed decreased CM-H2DCFDA signal compared with unedited cells, suggesting lower basal ROS levels. Quantitative proteomics implicated changes in several metabolic pathways, including amino acid, fatty acid and NRF2 activity, as compensatory mechanisms of TXNRD1 loss in both IDH1-mutant and WT cells. Despite these changes, glutathione levels and reduced:oxidized glutathione ratios were similar in TXNRD1 KO and unedited cell lines. These findings demonstrate that glioma models are dependent on TXNRD1 for survival, but acquire adaptive antioxidant changes to survive TXNRD1 loss. TrxR1 inhibition is acutely cytotoxic, via induction of disulfidptosis, with similar effects in IDH1-mutant and WT models. Sophia F. O'Brien-Gortner, Dinar Rani K, Daniel Conole, Tet-Woo Lee, Stephen MF. Jamieson, Dean C. Singleton. CRISPR screens identify thioredoxin reductase 1 as a target for inducing disulfidptosis in IDH1-mutant glioma models [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr B027.