L-2-hydroxyglutarate modulates ferroptosis through Nrf2/Slc7a11 and Atf4/Chac1 axes in hepatocellular carcinoma

Liver tumors undergo profound metabolic reprogramming to support survival, proliferation, and metastasis. Through a metabolic library screen combined with network-level integration of transcriptomic and metabolomic data, we identified a regulatory role of L-2-hydroxyglutarate dehydrogenase (L2HGDH) and its substrate, L-2-hydroxyglutarate (L2HG), on ferroptosis in hepatocellular carcinoma (HCC), which exhibits a metabolic environment prone to L2HG accumulation. Mechanistically, L2HG was found to promote chromatin accessibility at ferroptosis-related gene loci. Specifically, L2HG conferred protection against ferroptosis by enhancing cystine uptake via NRF2/SLC7A11 activation, while concurrently sensitizing cells to ferroptosis through glutathione degradation via ATF4/CHAC1 induction. The ferroptosis-protective effect of L2HG was dependent on NRF2, the ablation of which restored near-normal metabolic profiles in a spontaneous liver tumor mouse model. These findings suggest that the metabolically accumulated L2HG plays a dual role in modulating both ferroptosis resistance and sensitivity, thereby influencing liver tumorigenesis. Targeting these metabolic axes may provide novel strategies for ferroptosis-based antitumor therapies. Overall design: RNA-seq were conducted for the HCC cell lines Hep3B and SNU398, treated with 1 mM TFMB-L2HG, cultured with HPLM medium for 48 hours before RNA purification with the PureLink RNA Mini Kit (Invitrogen). ATAC-seq was conducted for Hep3B cells with or without TFMB-L2HG treatment using the ATAC-seq kit (Active Motif) according to the manufacturer's instructions. Metabolic scale CRISPRa screen was conducted in HuH7 cells expressing dCas9-VPR and transfected with lentiviral packaged to target metabolic genes using the library (Addgene, #187080). The virus infected HuH7 cells were treated with 5 µM Imidazole ketone erastin to induce ferroptosis for 14 days. The genmic DNA from initial and final populations were subjected to PCR amplification and deep-sequenced using an Illumina HiSeq 2500 platform. Sequencing reads were aligned to the reference sgRNA library, and sgRNA abundance was quantified. Results were reported as percent total sgRNA reads.