Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.
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We thank N. Kwiatkowski for critical reading of the manuscript, W. Kaelin for sharing referenced cell lines and the dual luciferase plasmids (Dana-Farber Cancer Institute, pLL3.7-EF1a-IRES-Gateway-nluc-2xHA-IRES2-fluc-hCL1-P2A-Puro), R. Kunz and the Thermo Fisher Scientific Center for Multiplexed Proteomics at the Harvard Medical School for the quantitative proteomics and phosphoproteomics assessment, and S. Nabet, A. Aguirre, W. Hahn, and members of the Bradner and Gray laboratories for helpful discussions. This work was supported by an American Cancer Society Postdoctoral Fellowship PF-17-010-01-CDD (B.N.), the Claudia Adams Barr Program in Innovative Basic Cancer Research (D.L.B.), Damon Runyon Cancer Research Foundation DRG-2196-14 (D.L.B.), and generous philanthropic gifts from the Hale Center for Pancreatic Cancer Research and the Katherine L. and Steven C. Pinard Research Fund.
The authors claim the following competing financial interests: International Patent Application Nos. PCT/US2016/039048, PCT/US2016/046087, PCT/US2016/046088, PCT/US2016/046089, each filed in the name of Dana-Farber Cancer Institute, Inc. D.L.B., J.P., and A.S. are now employees of Novartis. G.E.W. is a consultant for C4 Therapeutics. N.S.G. is a Scientific Founder and member of the Scientific Advisory Board of Syros Pharmaceuticals, C4 Therapeutics, and Petra Pharmaceuticals and is the inventor on IP licensed to these entities. J.E.B. is a Scientific Founder of Syros Pharmaceuticals, SHAPE Pharmaceuticals, Acetylon Pharmaceuticals, Tensha Therapeutics (now Roche), and C4 Therapeutics and is the inventor on IP licensed to these entities. J.E.B. is now an executive and shareholder in Novartis AG.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Table 1–6, Supplementary Figures 1–15
Synthetic procedures and characterization of dFKBP-1, dTAG-7, dTAG-13, dTAG-48, dTAG-51, bio-SLF, bio-Thal
Entire list of normalized and scaled quantitative mass spectrometry-based proteomics data. Triplicate values from biologically independent samples of normalized percent relative abundance of quantified proteins are presented for NIH/3T3 cells expressing FKBP12F36V-KRASG12V treated with DMSO, 1 µM dTAG-13 for one hour, and 1 µM dTAG-13 for four hours.
Entire list of normalized and scaled quantitative mass spectrometry-based phosphoserine/threonine data. Triplicate values from biologically independent samples of normalized percent relative abundance of quantified phosphosites are presented for NIH/3T3 cells expressing FKBP12F36V-KRASG12V treated with DMSO, 1 µM dTAG-13 for one hour, and 1 µM dTAG-13 for four hours.
Entire list of normalized and scaled quantitative mass spectrometry-based phosphotyrosine data. Triplicate values from biologically independent samples of normalized percent relative abundance of quantified phosphosites are presented for NIH/3T3 cells expressing FKBP12F36V-KRASG12V treated with DMSO, 1 µM dTAG-13 for one hour, and 1 µM dTAG-13 for four hours.
ERCC spike-in normalized FPKM values of top 200 upregulated and 200 downregulated expressed transcripts upon comparison of mock transduced (control) NIH/3T3 cells or NIH/3T3 cells expressing FKBP12F36V-KRASG12V treated with DMSO. Triplicate FPKM values from biologically independent samples are presented for control NIH/3T3 cells treated with DMSO and NIH/3T3 cells expressing FKBP12F36V-KRASG12V treated with DMSO, 1 µM dTAG-13, or 10 nM trametinib. Dataset accompanies heatmap in Fig. 5c.
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Nabet, B., Roberts, J.M., Buckley, D.L. et al. The dTAG system for immediate and target-specific protein degradation. Nat Chem Biol 14, 431–441 (2018). https://doi.org/10.1038/s41589-018-0021-8
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