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.

Author information

Author notes

    • Dennis L. Buckley
    • , Joshiawa Paulk
    • , Amanda Souza
    •  & James E. Bradner

    Present address: Novartis Institutes for BioMedical Research, Cambridge, MA, USA

    • Georg E. Winter

    Present address: CeMM- Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

  1. These authors contributed equally: Behnam Nabet, Justin M. Roberts, Dennis L. Buckley.


  1. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA

    • Behnam Nabet
    • , Alan L. Leggett
    • , Jun Qi
    •  & Nathanael S. Gray
  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    • Behnam Nabet
    •  & Nathanael S. Gray
  3. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    • Justin M. Roberts
    • , Dennis L. Buckley
    • , Joshiawa Paulk
    • , Shiva Dastjerdi
    • , Annan Yang
    • , Michael A. Erb
    • , Matthew A. Lawlor
    • , Amanda Souza
    • , Thomas G. Scott
    • , Sarah Vittori
    • , Jennifer A. Perry
    • , Georg E. Winter
    •  & James E. Bradner
  4. Department of Medicine, Harvard Medical School, Boston, MA, USA

    • Jun Qi
    •  & James E. Bradner
  5. Laura and Isaac Perlmutter Cancer Center, NYU Langone Medical Center, New York, NY, USA

    • Kwok-Kin Wong


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B.N., J.M.R., and D.L.B. conceived and led the study under the supervision of N.S.G. and J.E.B. D.L.B. and S.D. designed and performed molecule synthesis. J.M.R. constructed the lentiviral and knock-in vector systems. B.N. and J.M.R. designed and performed BRD4 knock-in and target panel studies. B.N. designed and performed KRAS studies. J.M.R. and J.P. designed and performed AlphaScreen assays and IKZF1 dual luciferase assays. S.V. and G.E.W. constructed FKBP12 dual luciferase vectors and B.N., J.M.R., and S.V. performed experiments using these systems. B.N. designed and performed RNA-sequencing experiments and B.N., M.A.E., and M.A.L. performed bioinformatics analyses. B.N., A.Y., A.S., and K.-K.W. designed and performed mouse studies. A.L.L. and T.G.S. assisted in cellular experiments. J.A.P. provided technical advice and data interpretation. J.Q. contributed reagents and technical advice. B.N. and J.E.B. wrote the manuscript with input from all authors.

Competing interests

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.

Corresponding authors

Correspondence to Nathanael S. Gray or James E. Bradner.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Table 1–6, Supplementary Figures 1–15

  2. Life Sciences Reporting Summary

  3. Supplementary Note

    Synthetic procedures and characterization of dFKBP-1, dTAG-7, dTAG-13, dTAG-48, dTAG-51, bio-SLF, bio-Thal

  4. Supplementary Dataset 1

    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.

  5. Supplementary Dataset 2

    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.

  6. Supplementary Dataset 3

    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.

  7. Supplementary Dataset 4

    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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