Targeted Protein Degradation of NUDT5 Reveals an Unexpected Non-Enzymatic Role in 6-Thioguanine-Mediated Toxicity
6-Thioguanine (6-TG), an FDA-approved antimetabolite drug, is widely used in the treatment of leukemia. Its cellular effects require metabolic activation and are regulated through interactions with various proteins such as NUDT15, which catalyzes the hydrolysis of the active 6-TG metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP. Recent genome-wide CRISPR loss-of-function studies have identified another NUDIX hydrolase, NUDT5, as a crucial mediator of 6-TG toxicity. Here, we present the development and characterization of potent and selective NUDT5 degraders, guided by a cell-based assay screening strategy. These degraders, in conjunction with orthogonal CRISPR knock-out and reconstitution experiments, reveal a novel and unexpected, non-enzymatic role for NUDT5 in modulating the cellular response to 6-TG. Depletion of NUDT5 protein is antagonistic to NUDT15 inhibition, suggesting a distinct mode-of-action with potential implications for patient therapy.