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The dTAG system for immediate and target-specific protein degradation

Abstract

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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Fig. 1: Heterobifunctional dTAG molecules engage and dimerize FKBP12F36V and CRBN in biochemical assays.
Fig. 2: dTAG-7 and dTAG-13 selectively degrade FKBP12F36V in a CRBN-dependent manner in cells.
Fig. 3: Selective pharmacological degradation of endogenously tagged BRD4.
Fig. 4: Rapid degradation of nuclear and cytoplasmic FKBP12F36V fusion chimeras.
Fig. 5: KRASG12V degradation rapidly reverses the deregulated proteomic and transcriptional signaling program of transformed cells.
Fig. 6: Evaluation of rapid and reversible degradation in vivo.

References

  1. 1.

    Winter, G. E. et al. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381 (2015).

    CAS  Article  Google Scholar 

  2. 2.

    Lu, J. et al. Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4. Chem. Biol. 22, 755–763 (2015).

    CAS  Article  Google Scholar 

  3. 3.

    Zengerle, M., Chan, K. H. & Ciulli, A. Selective small molecule induced degradation of the BET bromodomain protein BRD4. ACS Chem. Biol. 10, 1770–1777 (2015).

    CAS  Article  Google Scholar 

  4. 4.

    Winter, G. E. et al. BET bromodomain proteins function as master transcription elongation factors independent of CDK9 recruitment. Mol. Cell 67, 5–18.e19 (2017).

    CAS  Article  Google Scholar 

  5. 5.

    Bondeson, D. P. et al. Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 11, 611–617 (2015).

    CAS  Article  Google Scholar 

  6. 6.

    Remillard, D. et al. Degradation of the BAF complex factor BRD9 by heterobifunctional ligands. Angew. Chem. Int. Edn. Engl 56, 5738–5743 (2017).

    CAS  Article  Google Scholar 

  7. 7.

    Bonger, K. M., Chen, L. C., Liu, C. W. & Wandless, T. J. Small-molecule displacement of a cryptic degron causes conditional protein degradation. Nat. Chem. Biol. 7, 531–537 (2011).

    CAS  Article  Google Scholar 

  8. 8.

    Neklesa, T. K. et al. Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins. Nat. Chem. Biol. 7, 538–543 (2011).

    CAS  Article  Google Scholar 

  9. 9.

    Buckley, D. L. et al. HaloPROTACS: Use of small molecule PROTACs to induce degradation of HaloTag fusion proteins. ACS Chem. Biol. 10, 1831–1837 (2015).

    CAS  Article  Google Scholar 

  10. 10.

    Chung, H. K. et al. Tunable and reversible drug control of protein production via a self-excising degron. Nat. Chem. Biol. 11, 713–720 (2015).

    CAS  Article  Google Scholar 

  11. 11.

    Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. & Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods 6, 917–922 (2009).

    CAS  Article  Google Scholar 

  12. 12.

    Banaszynski, L. A., Chen, L. C., Maynard-Smith, L. A., Ooi, A. G. & Wandless, T. J. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126, 995–1004 (2006).

    CAS  Article  Google Scholar 

  13. 13.

    Shoulders, M. D., Ryno, L. M., Cooley, C. B., Kelly, J. W. & Wiseman, R. L. Broadly applicable methodology for the rapid and dosable small molecule-mediated regulation of transcription factors in human cells. J. Am. Chem. Soc. 135, 8129–8132 (2013).

    CAS  Article  Google Scholar 

  14. 14.

    Zhou, Q. et al. A chemical genetics approach for the functional assessment of novel cancer genes. Cancer Res. 75, 1949–1958 (2015).

    CAS  Article  Google Scholar 

  15. 15.

    Clackson, T. et al. Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc. Natl. Acad. Sci. USA 95, 10437–10442 (1998).

    CAS  Article  Google Scholar 

  16. 16.

    Roberts, J. M. & Bradner, J. E. A bead-based proximity assay for BRD4 ligand discovery. Curr. Protoc. Chem. Biol. 7, 263–278 (2015).

    Article  Google Scholar 

  17. 17.

    Douglass, E. F. Jr., Miller, C. J., Sparer, G., Shapiro, H. & Spiegel, D. A. A comprehensive mathematical model for three-body binding equilibria. J. Am. Chem. Soc. 135, 6092–6099 (2013).

    CAS  Article  Google Scholar 

  18. 18.

    Lu, G. et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343, 305–309 (2014).

    CAS  Article  Google Scholar 

  19. 19.

    Krönke, J. et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305 (2014).

    Article  Google Scholar 

  20. 20.

    Schneekloth, J. S. Jr. et al. Chemical genetic control of protein levels: selective in vivo targeted degradation. J. Am. Chem. Soc. 126, 3748–3754 (2004).

    CAS  Article  Google Scholar 

  21. 21.

    Anand, P. et al. BET bromodomains mediate transcriptional pause release in heart failure. Cell 154, 569–582 (2013).

    CAS  Article  Google Scholar 

  22. 22.

    Bradner, J. E., Hnisz, D. & Young, R. A. Transcriptional addiction in cancer. Cell 168, 629–643 (2017).

    CAS  Article  Google Scholar 

  23. 23.

    Delmore, J. E. et al. BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146, 904–917 (2011).

    CAS  Article  Google Scholar 

  24. 24.

    Deshpande, A. J., Bradner, J. & Armstrong, S. A. Chromatin modifications as therapeutic targets in MLL-rearranged leukemia. Trends Immunol. 33, 563–570 (2012).

    CAS  Article  Google Scholar 

  25. 25.

    Lovén, J. et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 153, 320–334 (2013).

    Article  Google Scholar 

  26. 26.

    Shortt, J., Ott, C. J., Johnstone, R. W. & Bradner, J. E. A chemical probe toolbox for dissecting the cancer epigenome. Nat. Rev. Cancer 17, 160–183 (2017).

    CAS  Article  Google Scholar 

  27. 27.

    Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067–1073 (2010).

    CAS  Article  Google Scholar 

  28. 28.

    Tanaka, M. et al. Design and characterization of bivalent BET inhibitors. Nat. Chem. Biol. 12, 1089–1096 (2016).

    CAS  Article  Google Scholar 

  29. 29.

    Sakuma, T., Nakade, S., Sakane, Y., Suzuki, K. T. & Yamamoto, T. MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nat. Protoc. 11, 118–133 (2016).

    CAS  Article  Google Scholar 

  30. 30.

    Stephen, A. G., Esposito, D., Bagni, R. K. & McCormick, F. Dragging ras back in the ring. Cancer Cell 25, 272–281 (2014).

    CAS  Article  Google Scholar 

  31. 31.

    Cox, A. D., Fesik, S. W., Kimmelman, A. C., Luo, J. & Der, C. J. Drugging the undruggable RAS: mission possible? Nat. Rev. Drug Discov. 13, 828–851 (2014).

    CAS  Article  Google Scholar 

  32. 32.

    Feramisco, J. R., Gross, M., Kamata, T., Rosenberg, M. & Sweet, R. W. Microinjection of the oncogene form of the human H-ras (T-24) protein results in rapid proliferation of quiescent cells. Cell 38, 109–117 (1984).

    CAS  Article  Google Scholar 

  33. 33.

    Shih, C., Padhy, L. C., Murray, M. & Weinberg, R. A. Transforming genes of carcinomas and neuroblastomas introduced into mouse fibroblasts. Nature 290, 261–264 (1981).

    CAS  Article  Google Scholar 

  34. 34.

    Stacey, D. W. & Kung, H. F. Transformation of NIH 3T3 cells by microinjection of Ha-ras p21 protein. Nature 310, 508–511 (1984).

    CAS  Article  Google Scholar 

  35. 35.

    McAlister, G. C. et al. Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses. Anal. Chem. 84, 7469–7478 (2012).

    CAS  Article  Google Scholar 

  36. 36.

    McAlister, G. C. et al. MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal. Chem. 86, 7150–7158 (2014).

    CAS  Article  Google Scholar 

  37. 37.

    Erickson, B. K. et al. Evaluating multiplexed quantitative phosphopeptide analysis on a hybrid quadrupole mass filter/linear ion trap/orbitrap mass spectrometer. Anal. Chem. 87, 1241–1249 (2015).

    CAS  Article  Google Scholar 

  38. 38.

    Smeal, T., Binetruy, B., Mercola, D. A., Birrer, M. & Karin, M. Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature 354, 494–496 (1991).

    CAS  Article  Google Scholar 

  39. 39.

    Sears, R. et al. Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes Dev. 14, 2501–2514 (2000).

    CAS  Article  Google Scholar 

  40. 40.

    Chin, Y. R. & Toker, A. The actin-bundling protein palladin is an Akt1-specific substrate that regulates breast cancer cell migration. Mol. Cell 38, 333–344 (2010).

    CAS  Article  Google Scholar 

  41. 41.

    Gilmartin, A. G. et al. GSK1120212 (JTP-74057) is an inhibitor of MEK activity and activation with favorable pharmacokinetic properties for sustained in vivo pathway inhibition. Clin. Cancer Res. 17, 989–1000 (2011).

    CAS  Article  Google Scholar 

  42. 42.

    Robert, C. et al. Improved overall survival in melanoma with combined dabrafenib and trametinib. N. Engl. J. Med. 372, 30–39 (2015).

    Article  Google Scholar 

  43. 43.

    Nabet, B. et al. Deregulation of the Ras-Erk signaling axis modulates the enhancer landscape. Cell Rep. 12, 1300–1313 (2015).

    CAS  Article  Google Scholar 

  44. 44.

    Natsume, T., Kiyomitsu, T., Saga, Y. & Kanemaki, M. T. Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Rep. 15, 210–218 (2016).

    CAS  Article  Google Scholar 

  45. 45.

    Weintraub, A. S. et al. YY1 is a structural regulator of enhancer-promoter loops. Cell 171, 1573–1588.e28 (2017).

    CAS  Article  Google Scholar 

  46. 46.

    Bisgrove, D. A., Mahmoudi, T., Henklein, P. & Verdin, E. Conserved P-TEFb-interacting domain of BRD4 inhibits HIV transcription. Proc. Natl. Acad. Sci. USA 104, 13690–13695 (2007).

    CAS  Article  Google Scholar 

  47. 47.

    Schröder, S. et al. Two-pronged binding with bromodomain-containing protein 4 liberates positive transcription elongation factor b from inactive ribonucleoprotein complexes. J. Biol. Chem. 287, 1090–1099 (2012).

    Article  Google Scholar 

  48. 48.

    Banaszynski, L. A., Sellmyer, M. A., Contag, C. H., Wandless, T. J. & Thorne, S. H. Chemical control of protein stability and function in living mice. Nat. Med. 14, 1123–1127 (2008).

    CAS  Article  Google Scholar 

  49. 49.

    Erb, M. A. et al. Transcription control by the ENL YEATS domain in acute leukaemia. Nature 543, 270–274 (2017).

    CAS  Article  Google Scholar 

  50. 50.

    Huang, H. T. et al. MELK is not necessary for the proliferation of basal-like breast cancer cells. eLife 6, e26693 (2017).

    Google Scholar 

  51. 51.

    Yang, X. et al. A public genome-scale lentiviral expression library of human ORFs. Nat. Methods 8, 659–661 (2011).

    CAS  Article  Google Scholar 

  52. 52.

    Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    CAS  Article  Google Scholar 

  53. 53.

    Trapnell, C. et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    CAS  Article  Google Scholar 

  54. 54.

    Lovén, J. et al. Revisiting global gene expression analysis. Cell 151, 476–482 (2012).

    Article  Google Scholar 

  55. 55.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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.

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Authors

Contributions

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.

Corresponding authors

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

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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.

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Supplementary information

Supplementary Text and Figures

Supplementary Table 1–6, Supplementary Figures 1–15

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Supplementary Note

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

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.

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.

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.

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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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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