Recharacterization of the Tumor Suppressive Mechanism of RSL3 Identifies the Selenoproteome as a Druggable Pathway in Colorectal Cancer

Abstract Ferroptosis is a non-apoptotic form of cell death driven by iron-dependent lipid peroxide accumulation. Colorectal cancer (CRC) cells feature elevated intracellular iron and reactive oxygen species (ROS) that heighten ferroptosis sensitivity. The ferroptosis inducer (S)-RSL3 ([1S,3R]-RSL3) is widely described as a selective inhibitor of the selenocysteine-containing enzyme (selenoprotein) glutathione peroxidase 4 (GPX4), which detoxifies lipid peroxides utilizing glutathione. However, through chemical controls utilizing the (R) stereoisomer of RSL3 ([1R,3R]-RSL3) that does not bind GPX4, combined with inducible genetic knockdowns of GPX4 in CRC cell lines, we revealed here that GPX4 dependency does not always align with (S)-RSL3 sensitivity, questioning the current characterization of GPX4 as the primary target of (S)-RSL3. Affinity pull-down mass spectrometry with modified (S)-RSL3 probes identified multiple selenoprotein targets, indicating broad selenoprotein inhibition. Further investigation of the therapeutic potential of broadly disrupting the selenoproteome as a therapeutic strategy in CRC showed that the selenoprotein inhibitor auranofin, an FDA-approved gold-salt, chemically induced oxidative cell death and ferroptosis in CRC models in vitro and in vivo. Similarly, genetic perturbation of ALKBH8, a tRNA-selenocysteine methyltransferase required for selenoprotein translation, suppressed CRC growth. In summary, these findings recharacterize the mechanism of (S)-RSL3 beyond GPX4 inhibition and establish selenoproteome disruption as a CRC therapeutic strategy.