2. Academic Validation
  3. Deciphering acquired resistance mechanisms to sustained auxin-inducible protein degradation in cells and mice

Deciphering acquired resistance mechanisms to sustained auxin-inducible protein degradation in cells and mice

  • bioRxiv. 2025 Sep 23:2025.09.21.677607. doi: 10.1101/2025.09.21.677607.
Judith Hyle 1,  Zhenling Liu 1,  Shaela Fields 1,  Jifeng Yang 1,  Xinyan Chen 1,  Wenjie Qi 2,  Qiong Zhang 2,  Byoung-Kyu Cho 3,  Young Ah Goo 3,  Xiuling Li 4,  Jack Sublett 4,  Qianqian Li 1,  Liusheng He 5,  Jonathon Klein 6,  Peng Xu 7,  Shondra M Pruett-Miller 6,  Beisi Xu 2,  Chunliang Li 1
Affiliations

Affiliations

  • 1 Department of Tumor Cell Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
  • 2 Center for Applied Bioinformatics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
  • 3 Mass Spectrometry Technology Access Center at the McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA.
  • 4 Genetically Engineered Mouse Models Shared Resource, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
  • 5 Flow Cytometry and Cell Sorting Shared Resource, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
  • 6 Center for Advanced Genome Engineering, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
  • 7 Cyrus Tang Medical Institute, National Clinical Research Center for Hematologic Diseases, State Key Laboratory of Radiation Medicine and Protection, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, Jiangsu 215123, PR China.
PMID: 41040178 DOI: 10.1101/2025.09.21.677607
Abstract

Targeted protein degradation is a favorable strategy for studying the immediate downstream effects of protein loss-of-function. An appealing platform among these technologies is 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 due to the high concentrations of Auxin required in the original AID1 system is no longer a concern, making it possible to study 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 silencing of the OsTIR1 adaptor protein. Resistance to Auxin degradation was also acquired in mouse primary CtcfminiAID/miniAID knock-in B-ALL cells through missense mutations of the OsTIR1(F74G) protein in vivo and ex vivo. In summary, our innovative study expands our understanding of the AID system and cautions careful consideration of design for future applications in mammalian system.

Keywords

CTCF; auxin-inducible degron; genome editing; leukemia; resistance.

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