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

Ferroptosis is a nonapoptotic form of cell death driven by iron-dependent lipid peroxide accumulation. Colorectal cancer cells feature elevated intracellular iron and reactive oxygen species 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 using glutathione. However, through chemical controls using the (R) stereoisomer of RSL3 (1R,3R)-RSL3 that does not bind GPX4, combined with inducible genetic knockdowns of GPX4 in colorectal cancer cell lines, we revealed in this study that GPX4 dependency does not always align with (S)-RSL3 sensitivity, thereby 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 colorectal cancer showed that the selenoprotein inhibitor auranofin, an FDA-approved gold salt, chemically induced oxidative cell death and ferroptosis in colorectal cancer models in vitro and in vivo. Similarly, genetic perturbation of ALKBH8, a tRNA-selenocysteine methyltransferase required for selenoprotein translation, suppressed colorectal cancer growth. In summary, these findings recharacterize the mechanism of (S)-RSL3 beyond GPX4 inhibition and establish selenoproteome disruption as a colorectal cancer therapeutic strategy. SIGNIFICANCE: Chemoproteomic profiling reveals that RSL3 functions through pan-selenoprotein inhibition beyond GPX4 and identifies ALKBH8, a tRNA-selenocysteine methyltransferase essential for selenoprotein translation, as a therapeutic target to disrupt redox balance in colorectal cancer. See related commentary by Short, p. 2775.