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. 2021 Jun;17(6):711-717.
doi: 10.1038/s41589-021-00802-w. Epub 2021 May 25.

Acute pharmacological degradation of Helios destabilizes regulatory T cells

Eric S Wang #  1   2 , Alyssa L Verano #  1   2 , Radosław P Nowak #  1   2 , J Christine Yuan #  1   2 , Katherine A Donovan  1   2 , Nicholas A Eleuteri  1 , Hong Yue  1   2 , Kenneth H Ngo  3 , Patrick H Lizotte  3 , Prafulla C Gokhale  3 , Nathanael S Gray  4   5 , Eric S Fischer  6   7
Affiliations

Acute pharmacological degradation of Helios destabilizes regulatory T cells

Eric S Wang et al. Nat Chem Biol. 2021 Jun.

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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Figures

Extended Data Fig. 1
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 16h. 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 Figure 1.
Extended Data Fig. 2
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 5h 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
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
Extended Data Fig. 4. Helios degradation promotes IL-2 secretion
Jurkat cells were pre-treated with 1 μM of the indicated compounds for 18h and then activated with α-CD3/CD28 antibodies for 24h. 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
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 16h. Data is presented as mean ± SD of n = 3 biologically independent samples.
Extended Data Fig. 6
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 4d. 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 7d. 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
Extended Data Fig. 7. FACS gating strategy
Gating strategy used to identify different immune cell populations related to (a) Extended Data Fig. 5, 6c; (b) Extended Data Figure 6a, b; (c) Figure 3a; (d) Figure 4a, b; and (e) Figure 4c. FMO controls were used to set positive and negative gates as indicated.
Figure 1:
Figure 1:. Novel anilinomaleimides induce Helios degradation
a, Chemical structures of ALV1, ALV2, lenalidomide, and CC-885. b, CRBN-binding TR-FRET assay. Displacement of BODIPY-lenalidomide tracer by titration of lenalidomide, ALV1 and ALV2 to DDB1ΔB-CRBNbiotinylated and Tb-streptavidin. Data is presented as mean ± SD of n = 3 biologically independent samples. c, CRBN-IKZF2 TR-FRET assay. Titration of lenalidomide, CC-885, ALV1 or ALV2 to DDB1ΔB-CRBNSPYCATCHER-BODIPY, Terbium-Streptavidin and biotinylated IKZF2. Data is presented as mean ± SD of n = 3 biologically independent samples and is representative of n = 3 independent experiments. d, Representative immunoblots and quantification from n = 3 independent experiments of wildtype or Crbn−/− Jurkat cells treated as indicated for 4h. e, Immunoblots from Jurkat cells co-treated with carfilzomib (1 μM) and the indicated compounds for 4h. Data is representative of two independent experiments. f, Quantitative proteomics profiles (7972 total proteins) of wildtype Jurkat cells treated for 4h with 1 μM of ALV1 or ALV2. Significant changes were assessed using a moderated t-test as implemented in Bioconductor’s Limma package. Uncropped gels for d, e are included as Source Data - Figure 1.
Figure 2:
Figure 2:. ALV1 accommodates key histidine 141 residue of IKZF2
a, Cartoon representation of DDB1ΔB-CRBN-ALV1-IKZF2ZnF2 highlighting the domain architecture. b, CRBN ALV1 binding site. The ALV1 Fo-Fc map contoured at 3.0 σ is shown as green mesh. c, Overlay of IKZF1ZnF1 (PDB: 6H0F) and IKZF2ZnF2 based on structure alignment of CTD of CRBN reveals 0.7 Å translation between the zinc fingers. IKZF2 H141 and corresponding IKZF1 Q146 residues are shown in stick representation. d, Overlay of the CC-885-GSPT1 complex (PDB: 5HXB) with the ALV1-IKZF2ZnF2 complex. Distances between GSPT1 Q534, V536 and ALV molecules are shown with dashed lines and measured in Å.
Figure 3:
Figure 3:. Acute degradation of Ikaros family transcription factors in human Tregs
a, Representative histograms and quantification of ex vivo expanded human Tregs treated with 1 μM of the indicated compounds for 24h. Data is presented as mean ± SD of n = 4 biologically independent samples and is representative of n = 3 independent experiments. b, Quantitative proteomics profiles (6808 total proteins) of human Tregs treated for 4h with 1 μM of ALV1 or ALV2. Significant changes were assessed using a moderated t-test as implemented in Bioconductor’s Limma package. c, Relative abundance of indicated transcription factors in human Tregs treated for 4h with 1 μM of ALV1 or ALV2. Data is presented as mean ± SD of n = 3 biologically independent samples.
Figure 4.
Figure 4.. Pharmacological degradation of Helios destabilizes human Tregs ex vivo
Representative FACS plots and quantification of (a) % IL2+ or (b) % IFNγ+ cells within CD4+ Foxp3+ Tregs after PMA/ionomycin stimulation. Data is presented as mean ± SD of n = 4 biologically independent samples and is representative of n = 3 independent experiments. Significance was assessed by two-way ANOVA with Bonferroni’s correction for multiple comparisons. c, Representative cell division profiles and quantification of proliferating CD3+ Tresp cells incubated with hTregs and ALV2 or vehicle control. Data is presented as mean ± SD of n = 4 biologically independent samples and is representative of n = 2 independent experiments. Significance was assessed by two-way ANOVA with Bonferroni’s correction for multiple comparisons (1:8, p = 0.0048; 1:4, p = 0.0008; 1:2, p < 0.0001).
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