Targeted Protein Degradation of NUDT5 Dissociates Catalytic Inhibition from Protein Loss in 6-Thioguanine Response

6-Thioguanine (6-TG) is an FDA-approved antimetabolite drug that is widely used clinically, including for 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 develop and validate a selective, cell-active NUDT5 degrader toolkit and orthogonally characterize target engagement, ternary complex formation, degradation kinetics, and proteome-wide selectivity. These degraders, in conjunction with orthogonal CRISPR knock-out and reconstitution experiments, support a 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.