ARD1 facilitates ferroptosis evasion via the induction of glutathione synthesis through catalysis of PABPC1 acetylation in HCC [CRISPR screen]

Aberrant upregulation of intracellular antioxidant glutathione (GSH) is implicated in promoting tumor proliferation, inducing drug resistance, and inhibiting ferroptosis across various malignancies, including hepatocellular carcinoma (HCC). However, the mechanism underlying the GSH metabolism reprogramming in HCC remains poorly understood. In this study, we employed a genome-wide CRISPR-Cas9 screen and RNA-seq to identify ARD1 as a pivotal facilitator of de novo GSH synthesis in HCC. Notably, ARD1 upregulation is positively correlated with elevated GSH levels and a poor prognosis in HCC patients. In vivo and in vitro functional assays revealed that ARD1 promotes HCC cell proliferation and inhibits ferroptosis in a GSH-dependent manner. LC-MS/MS-based stable isotope labeling revealed that ARD1 increases GSH levels by stabilizing the ?-glutamylcysteine ligase catalytic subunit (GCLC) transcript. Overall, this research underscores the crucial role of ARD1 in GSH metabolic reprogramming and ferroptosis regulation in HCC and reveals a novel strategy for ferroptosis-based targeted therapy for HCC. Overall design: The CRISPER-PoolTM KOUT human library containing 123,441 sgRNAs targeting 19,050 genes and 1,864 miRNAs was purchased from Genechem Co., Ltd. (Shanghai, China). After Huh-7 cells were transduced with various concentrations of the lentiviral library (2.5%-60%) for 72 hours, 2.5 µg/ml puromycin (Biosharp, China) was added to the medium, and the functional multiplicities of infection (MOIs) were assessed via a CCK-8 assay. A total of 2 × 108 Huh-7 cells were transduced with the corresponding amounts of lentiviral particles at functional MOIs to ensure 500-fold coverage and subjected to selection for cells stably infected for 72 hours with 2.5 µg/ml puromycin. The transduced cells were then divided into two groups and treated with or without the glutathione synthesis inhibitor buthionine sulfoximine (BSO; MCE, USA) at the IC20. Following 24 days of selection, at least 5 × 107 cells were collected and subjecred to DNA extraction, PCR amplification and sgRNA sequencing.