Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Acute pharmacological degradation of Helios destabilizes regulatory T cells

Abstract

The zinc-finger transcription factor Helios is critical for maintaining the identity, anergic phenotype and suppressive activity of regulatory T (Treg) cells. While it is an attractive target to enhance the efficacy of currently approved immunotherapies, no existing approaches can directly modulate Helios activity or abundance. Here, we report the structure-guided development of small molecules that recruit the E3 ubiquitin ligase substrate receptor cereblon to Helios, thereby promoting its degradation. Pharmacological Helios degradation destabilized the anergic phenotype and reduced the suppressive activity of Treg cells, establishing a route towards Helios-targeting therapeutics. More generally, this study provides a framework for the development of small-molecule degraders for previously unligandable targets by reprogramming E3 ligase substrate specificity.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Anilinomaleimides induce Helios degradation.
Fig. 2: ALV1 accommodates key H141 residue of IKZF2.
Fig. 3: Acute degradation of Ikaros family transcription factors in human Treg cells.
Fig. 4: Pharmacological degradation of Helios destabilizes human Treg cells ex vivo.

Data availability

Source data are provided with this paper. All other data supporting the findings in this study are provided in the main text and supplementary materials. Structural coordinates are deposited in the Protein Data Bank and available under accession number 7LPS. MS raw data files have been deposited in the PRIDE Archive (PXD016168 and PXD023691) for ALV1 and ALV2. The Uniprot human database was used for proteomics analysis.

Code availability

The code necessary to reproduce the statistical analysis for quantitative proteomics can be found at https://github.com/fischerlab/.

References

  1. Sakaguchi, S., Yamaguchi, T., Nomura, T. & Ono, M. Regulatory T cells and immune tolerance. Cell 133, 775–787 (2008).

    Article  CAS  Google Scholar 

  2. Tanaka, A. & Sakaguchi, S. Regulatory T cells in cancer immunotherapy. Cell Res. 27, 109–118 (2017).

    Article  CAS  Google Scholar 

  3. Onizuka, S. et al. Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor α) monoclonal antibody. Cancer Res. 59, 3128–3133 (1999).

    CAS  PubMed  Google Scholar 

  4. Sakaguchi, S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22, 531–562 (2004).

    Article  CAS  Google Scholar 

  5. Nakagawa, H. et al. Instability of Helios-deficient Tregs is associated with conversion to a T-effector phenotype and enhanced antitumor immunity. Proc. Natl Acad. Sci. USA 113, 6248–6253 (2016).

    Article  CAS  Google Scholar 

  6. Kim, H. J. et al. Stable inhibitory activity of regulatory T cells requires the transcription factor Helios. Science 350, 334–339 (2015).

    Article  CAS  Google Scholar 

  7. Yates, K., Bi, K., Haining, W. N., Cantor, H. & Kim, H. J. Comparative transcriptome analysis reveals distinct genetic modules associated with Helios expression in intratumoral regulatory T cells. Proc. Natl. Acad. Sci. USA 115, 2162–2167 (2018).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Kronke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523, 183–188 (2015).

    Article  CAS  Google Scholar 

  11. An, J. et al. pSILAC mass spectrometry reveals ZFP91 as IMiD-dependent substrate of the CRL4CRBN ubiquitin ligase. Nat. Commun. 8, 15398 (2017).

    Article  CAS  Google Scholar 

  12. Donovan, K. A. et al. Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome. eLife 7, e38430 (2018).

  13. Matyskiela, M. E. et al. SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate. Nat. Chem. Biol. 14, 981–987 (2018).

    Article  CAS  Google Scholar 

  14. Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase. Nature 535, 252–257 (2016).

    Article  CAS  Google Scholar 

  15. John, L. B. & Ward, A. C. The Ikaros gene family: transcriptional regulators of hematopoiesis and immunity. Mol. Immunol. 48, 1272–1278 (2011).

    Article  CAS  Google Scholar 

  16. Fan, Y. & Lu, D. The Ikaros family of zinc-finger proteins. Acta Pharm. Sin. B 6, 513–521 (2016).

    Article  Google Scholar 

  17. Powell, C. E. et al. Selective degradation of GSPT1 by cereblon modulators identified via a focused combinatorial library. ACS Chem. Biol. 15, 2722–2730 (2020).

    Article  CAS  Google Scholar 

  18. Petzold, G., Fischer, E. S. & Thoma, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligase. Nature 532, 127–130 (2016).

    Article  CAS  Google Scholar 

  19. Nowak, R. P. et al. Plasticity in binding confers selectivity in ligand-induced protein degradation. Nat. Chem. Biol. 14, 706–714 (2018).

    Article  CAS  Google Scholar 

  20. Sievers, Q. L. et al. Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 362, eaat0572 (2018).

  21. Baine, I., Basu, S., Ames, R., Sellers, R. S. & Macian, F. Helios induces epigenetic silencing of IL2 gene expression in regulatory T cells. J. Immunol. 190, 1008–1016 (2013).

    Article  CAS  Google Scholar 

  22. Haslett, P. A., Corral, L. G., Albert, M. & Kaplan, G. Thalidomide costimulates primary human T lymphocytes, preferentially inducing proliferation, cytokine production, and cytotoxic responses in the CD8+ subset. J. Exp. Med. 187, 1885–1892 (1998).

    Article  CAS  Google Scholar 

  23. Gandhi, A. K. et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4CRBN. Br. J. Haematol. 164, 811–821 (2014).

    Article  CAS  Google Scholar 

  24. Corral, L. G. et al. Differential cytokine modulation and T cell activation by two distinct classes of thalidomide analogues that are potent inhibitors of TNF-α. J. Immunol. 163, 380–386 (1999).

    CAS  PubMed  Google Scholar 

  25. Fink, E. C. et al. CrbnI391V is sufficient to confer in vivo sensitivity to thalidomide and its derivatives in mice. Blood 132, 1535–1544 (2018).

    Article  CAS  Google Scholar 

  26. Gokhale, A. S., Gangaplara, A., Lopez-Occasio, M., Thornton, A. M. & Shevach, E. M. Selective deletion of Eos (Ikzf4) in T-regulatory cells leads to loss of suppressive function and development of systemic autoimmunity. J. Autoimmun. 105, 102300 (2019).

    Article  CAS  Google Scholar 

  27. Pan, F. et al. Eos mediates Foxp3-dependent gene silencing in CD4+ regulatory T cells. Science 325, 1142–1146 (2009).

    Article  CAS  Google Scholar 

  28. Donovan, K. A. et al. Mapping the degradable kinome provides a resource for expedited degrader development. Cell 183, 1714–1731 (2020).

    Article  CAS  Google Scholar 

  29. Sebastian, M. et al. Helios controls a limited subset of regulatory T cell functions. J. Immunol. 196, 144–155 (2016).

    Article  CAS  Google Scholar 

  30. Abdulrahman, W. et al. A set of baculovirus transfer vectors for screening of affinity tags and parallel expression strategies. Anal. Biochem. 385, 383–385 (2009).

    Article  CAS  Google Scholar 

  31. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl Acad. Sci. USA 109, E690–E697 (2012).

    Article  CAS  Google Scholar 

  32. Cavadini, S. et al. Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome. Nature 531, 598–603 (2016).

    Article  CAS  Google Scholar 

  33. McCoy, A. J. et al. Phasertng: directed acyclic graphs for crystallographic phasing. Acta Crystallogr. D Struct. Biol. 77, 1–10 (2021).

    Article  CAS  Google Scholar 

  34. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    Article  CAS  Google Scholar 

  35. Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D Biol. Crystallogr. 68, 368–380 (2012).

    Article  CAS  Google Scholar 

  36. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  37. Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013).

    Article  Google Scholar 

  38. Carpenter, A. E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).

    Article  Google Scholar 

  39. 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).

    Article  CAS  Google Scholar 

  40. R: a language and environment for statistical computing. (R Foundation for Statistical Computing, 2014).

  41. Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    Article  Google Scholar 

Download references

Acknowledgements

This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6M detector on the 24-ID-C beamline is funded by an NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract number DE-AC02-06CH11357. Software used in this project was curated by SBGrid. We gratefully acknowledge the generous financial support of the following sources: NIH grant NCI R01CA214608 (E.S.F.), Damon Runyon Cancer Research Fellowship DRG-2270-16 (E.S.W.) and the Damon Runyon-Rachleff Innovator Award DRR-50-18 (E.S.F.).

Author information

Authors and Affiliations

Authors

Contributions

E.S.W. performed the immunoblotting, flow cytometry and pharmacodynamic experiments. A.L.V. designed and synthesized all compounds. J.C.Y. and R.P.N. designed and constructed and J.C.Y. performed the biochemical TR-FRET and cellular reporter assays. R.P.N. and J.C.Y. conducted protein purification and crystallization, and R.P.N. collected, processed and refined X-ray data. K.A.D. and N.A.E. conducted the MS experiments. H.Y. helped with protein purification and TR-FRET assays. K.H.N., P.H.L. and P.C.G. performed the pharmacodynamic experiments. E.S.W., A.L.V. and R.P.N. wrote the manuscript. N.S.G. and E.S.F. supervised all aspects of the project. All authors read, revised and approved the manuscript.

Corresponding authors

Correspondence to Nathanael S. Gray or Eric S. Fischer.

Ethics declarations

Competing interests

N.S.G. is an equity holder and scientific advisor for Syros, Soltego (board member), C4, B2S, Petra, Allorion, Inception and Jengu. E.S.F. is an equity holder and scientific advisor for C4 Therapeutics, Jengu (board member), Neomorph and Civetta Therapeutics and is a consultant to Novartis, Sanofi, AbbVie, Pfizer, Astellas, EcoR1 capital and Deerfield. The Fischer lab receives or has received research funding from Novartis, Ajax and Astellas not related to this work. E.S.W., A.L.V., R.P.N., J.C.Y., K.A.D., N.S.G. and E.S.F. are inventors on a patent covering the compounds described in this paper.

Additional information

Peer review information Nature Chemical Biology thanks Zoran Rankovic and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Critical histidine residue in Helios regulates sensitivity to imide-induced degradation.

a, Sequence alignment of the second zinc-finger domain of Ikaros family proteins, with the residue that controls sensitivity to IMiD-induced degradation (glutamine residue in IKZF1/3, histidine in IKZF2/4) highlighted in cyan. b, Immunoblot from Jurkat cells treated as indicated. c, Immunoblot from Jurkat cells treated as indicated for 16 h. Loss of Helios abundance may be secondary to defects in translation and subsequent initiation of programmed cell death. Data in b, c are representative of n = 2 independent experiments. Uncropped gels for c are included as Source Data – Extended Fig. 1.

Source data

Extended Data Fig. 2 Novel imide analogs with anilinomaleimide cores bind CRBN in cells to induce Helios degradation.

a, Chemical structure of lenalidomide and ALV-02-146-03 (1), with the distinct isoindolinone and anilinomaleimide cores, respectively, highlighted. b, Cellular CRBN engagement assay for lenalidomide, ALV1, ALV2, and ALV-02-146-03. Data reported as n = 2 independent replicates. c, Quantitative assessment of cellular degradation using IKZF1-, IKZF2-, or GSPT1-EGFP reporter assay. Cells stably expressing EGFP fusions and mCherry were treated for 5 h with increasing concentrations of ALV1, ALV2, CC-885 or lenalidomide, and EGFP and mCherry fluorescence was quantified, with half degradation constants (DC50) and maximum percentage degradation (DCmax). Data reported as mean ± SD of n = 3 biologically independent samples and are representative of n = 2 independent experiments.

Extended Data Fig. 3 Selectivity profile of ALV1 and ALV2.

Multiple sequence alignment of zinc finger domains of the proteins downregulated in Fig. 1f. The glycine residue indicated by the red arrow is a key determinant of imide dependent degradation and is present at least once in all downregulated proteins.

Extended Data Fig. 4 Helios degradation promotes IL-2 secretion.

Jurkat cells were pre-treated with 1 µM of the indicated compounds for 18 h and then activated with α-CD3/CD28 antibodies for 24 h. Data is presented as mean ± SD of n = 3 (for untreated) and n = 4 (for stimulated) biologically independent samples and are representative of n = 2 independent experiments. Significance was assessed by two-way ANOVA with Bonferroni’s correction for multiple comparisons.

Extended Data Fig. 5 Acute Helios degradation in CrbnI391V/I391V but not wildtype murine Tregs.

Representative histograms and quantification of (a) wildtype or (b) CrbnI391V/I391V splenocytes treated with 1 µM of the indicated compounds for 16 h. Data is presented as mean ± SD of n = 3 biologically independent samples.

Extended Data Fig. 6 Acute Helios degradation in destabilizes murine Tregs.

a, Histogram of Helios levels in CD4 + Foxp3+ Tregs after treatment ex vivo with ALV2 (2 µM) or DMSO vehicle in the presence of 5 ng/ml IL-2 + 20 ng/ml IL-4 for 4 d. Data is representative of n = 2 biologically independent experiments. b, FACS plot and quantification of IFNγ + CD4 + Foxp3+ Tregs after PMA/ionomycin stimulation. Data is presented as mean ± SD of n = 3 biologically independent samples and is representative of n = 2 independent experiments, and significance was assessed by two-way ANOVA with Bonferroni’s correction for multiple comparisons. c, Representative histograms and quantification of Ikaros and Helios levels gated on splenic CD4 + FoxP3+ Tregs after treatment of CrbnI391V/I391V mice with vehicle (10% DMSO/50% PEG400/40% water) or ALV2 (100 mg/kg BID via intraperitoneal injection daily) for 7 d. Data is presented as mean ± SD of n = 4 biologically independent mice, and p values are derived from a two-tailed t test.

Extended Data Fig. 7 FACS gating strategy.

Gating strategy used to identify different immune cell populations related to a, Extended Data Figs. 5, 6c; b, Extended Data Fig. 6a,b; c, Fig. 3a; d, Fig. 4a,b; and e, Fig. 4c. FMO controls were used to set positive and negative gates as indicated.

Supplementary information

Supplementary Information

Supplementary Tables 1–3 and Note

Reporting Summary

Supplementary Dataset

Proteomics hitlist

Source data

Source Data Fig. 1

Unprocessed Western blots.

Source Data Extended Data Fig. 1

Unprocessed Western blots.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, E.S., Verano, A.L., Nowak, R.P. et al. Acute pharmacological degradation of Helios destabilizes regulatory T cells. Nat Chem Biol 17, 711–717 (2021). https://doi.org/10.1038/s41589-021-00802-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-021-00802-w

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing