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Acute pharmacological degradation of Helios destabilizes regulatory T cells

Nature Chemical Biology volume 17, pages 711–717 (2021)Cite this article

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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.

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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.

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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/.

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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.).

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Author notes

  1. These authors contributed equally: Eric S. Wang, Alyssa L. Verano, Radosław P. Nowak, J. Christine Yuan.

Authors and Affiliations

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

    Eric S. Wang, Alyssa L. Verano, Radosław P. Nowak, J. Christine Yuan, Katherine A. Donovan, Nicholas A. Eleuteri, Hong Yue, Nathanael S. Gray & Eric S. Fischer

  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Eric S. Wang, Alyssa L. Verano, Radosław P. Nowak, J. Christine Yuan, Katherine A. Donovan, Hong Yue, Nathanael S. Gray & Eric S. Fischer

  3. Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, MA, USA

    Kenneth H. Ngo, Patrick H. Lizotte & Prafulla C. Gokhale

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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.

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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.

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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.

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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

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