Deciphering resistance mechanisms to auxin-inducible protein degradation in mammalian cells

Targeted protein degradation is a favorable strategy for studying the immediate downstream effects of protein loss-of-function, such as the auxin-inducible degron (AID) system. Although this system has been applied extensively to cell and animal models, degradation resistance to long-term auxin treatment has not been studied. With the advent of the new AID2 system, cellular toxicity caused by the high auxin concentrations required in the original AID1 system is no longer a concern, enabling the study of protein degradation over extended periods. In this study, we derived multiple miniAID-tagged knock-in human cell lines and a Ctcf-miniAID knock-in mouse strain to investigate mechanisms of degradation resistance. We revealed four independent resistance mechanisms, including a nonsense mutation in the CTCF coding sequence that removed the miniAID peptide, a missense point mutation in the miniAID coding region that disrupted ubiquitin complex targeting, and reduced expression of OsTIR1 adaptor protein. Resistance to auxin degradation was also observed in mouse primary Ctcf^{miniAID/miniAID} knock-in B-cell acute lymphoblastic leukemia cells through missense mutations of the OsTIR1^(F74G) protein ex vivo and in vivo. By systematically characterizing degron resistance mechanisms in mammalian cells, we identified potential limitations of the AID system for long-term protein degradation studies.