Fig 2: Transcriptional control of ORAI3.a Chromatin accessibility maps of naive CD4+ T cells from RA and PsA patients and from healthy individuals were generated by ATAC-seq. Accessible regions at ORAI3 TSS region (chr16:30,960,352–1215) and downstream of TSS (chr16:30,964,994–5367). b Accessible regions were analyzed for transcription factor (TF) motifs. Indicated TFs were silenced using siRNA transfected into HEK293T cells for 48 h, non-targeting siRNA was used as a negative control. ORAI3 transcripts were quantified by qPCR and normalized to β-actin. Data are presented as mean ± SEM from three independent experiments (n = 3). Knockdown efficiency is shown in Supplementary Fig. 6. c Purified CD4+ T cells were transfected with increasing concentrations of IKAROS siRNA smart pool for 48 h. ORAI3 transcription was analyzed by qPCR and normalized to β-actin. Results are triplicates from one of two independent experiments. Data are presented as mean ± SEM. d The sequence (860 bp) upstream of ORAI3 TSS was cloned into the PGL3-basic plasmid and transfected into HEK293T cells alone or with an IKAROS-expressing plasmid. Firefly luciferase activity relative to Renilla luciferase is shown. Data are presented as mean ± SEM from three independent experiments (n = 3). e ChIP assays of CD4+ T cells using anti-IKAROS antibodies and a primer set amplifying the indicated potential binding site. Data are presented as mean ± SEM from three independent experiments (n = 3). f Flow cytometry analysis of AA-induced Ca2+ influx in IKAROS siRNA smart pool- and control siRNA-transfected CD4+ naive T cells. Histograms are representative of two experiments. g Immunoblot analysis of AA-induced ERK phosphorylation in CD4+ naive T cells transfected with IKAROS siRNA smart pool; one of two experiments. Uncropped Western blots in Supplementary Fig. 10. h Flow cytometry analysis of AA-induced CD69 expression in purified naive CD4+ T cells transfected with IKAROS siRNA smart pool. Representative histograms and data from four experiments. Data in b, c were analyzed with one-way ANOVA followed by Tukey’s multiple comparison test with adjustment. Data in d, e, and h were analyzed with unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.

Fig 3: IKZF1 and IKZF3 utilize the N-terminal ZnF domain to associate with the activation and inhibition domain of RUNX1. a HEK293T cells were transfected with constructs encoding an empty vector (EV), FLAG-tagged IKZF3 wild-type (WT), or mutants. A schematic representation of IKZF3 mutants is shown. IKZF3 mutants that interact (+) or do not interact (−) with RUNX1 are shown. b Immunoblot analysis of FLAG-IKZF3 immunoprecipitation (IP). Immunocomplexes were probed with antibodies to the indicated proteins. c HEK293T cells were transfected with constructs encoding an empty vector (EV), FLAG-tagged IKZF3 wild-type (WT) or mutants as indicated. The cell extracts were subjected to anti-FLAG IP and the immunocomplexes were probed with antibodies to the indicated proteins. d Purified GST-tagged proteins as indicated were incubated with in vitro translated FLAG-tagged RUNX1. GST pull-downs were probed with anti-FLAG antibodies. Ponceau S staining shows the expressions of GST-proteins. e Schematic representation of RUNX1 mutants. RUNX1 mutants that interact (+) or do not interact (−) with IKZF3 are shown. f Immunoblot analysis of GST pull-downs. Purified GST-tagged proteins as indicated were incubated with in vitro translated FLAG-tagged RUNX1. GST pull-downs were probed with anti-FLAG antibodies. Ponceau S staining shows the expressions of GST-proteins. g Same as in f, except that the indicated GST-proteins were used. h HEK293T cells stably expressing IKZF3 were transfected with constructs encoding an empty vector (EV), FLAG-tagged RUNX1 wild-type (WT), or mutants as indicated. The whole cell lysates (WCL) were subjected to anti-FLAG IP and the immunocomplexes were probed with antibodies to the indicated proteins. i Schematic model of the interaction between IKZF1/3 and the AD and ID of RUNX1. Unless otherwise noted, immunoblots are representative of three independent experiments

Fig 4: F-3,6′-DP potently binds Cereblon but does not trigger degradation of the classical neo-substrates SALL4, Ikaros, and Aiolos. (A) A concentration-dependent evaluation of binding between IMiDs and cereblon by FRET assay provided an IC50 value of pomalidomide (Pom) and F-3,6′-DP, of 2.38 μM 2.66 μM respectively (N.B., the concentration-dependent curves extensively overlay each other). The thalidomide analog-mediated degradation of neo-substrates, SALL (B,C), Aiolos (D,F), and Ikaros (E,G) was evaluated by Western blotting, and the relative expression level of each neo-substrate was quantified.*, p < 0.05, **, p < 0.01 ****, p < 0.0001 vs. control (Con) value; #, p < 0.05, ##, p < 0.01, ####, p < 0.0001 vs. pomalidomide value (Tukey’s multiple comparisons test).

Fig 5: 3,6′-DP, 1,6′-DP and pomalidomide potently bind cereblon, but 3,6′-DP does not lower downstream neo-substrates, epitomized by Ikaros, Aiolos and SALL4. Binding (A) of pomalidomide (POM), 3,6′-DP and 1,6′-DP to cereblon provided IC50 values (B) of 2.37, 0.94 and 1.52 μM, respectively. Degradation of downstream neo-substrates Aiolos and Ikaros were evaluated in human MM.1S cells. (C) Amounts of 0.1 and 1.0 μM POM dramatically lowered Aiolos and Ikaros protein levels in human MM1S cells (normalized to GADPH), these same concentrations of 1,6′-DP less substantially lowered Aiolos (C) and Ikaros (D). Notably, 3,6′-DP had no significant action on protein degradation of Aiolos and Ikaros. (E) SALL4 protein levels were quantified in H9 cells challenged with thalidomide, pomalidomide, 1,6′- and 3,6′-DP (3 μM). All, with the exception of 3,6′-DP, significantly lowered SALL4 (normalized to β-actin). * p < 0.05, *** p < 0.001, **** p < 0.0001: treatments vs. control (i.e., DMSO/vehicle). # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001: treatments vs. POM of same concentration. + p < 0.05, ++ p < 0.01: 1,6′-DP vs. 3,6′-DP at same concentration (values: mean ± SEM, n = 4/group).