Fig 2: 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 3: RUNXs inhibit CRBN-dependent IKZF1 and IKZF3 degradation. a Immunoblot analysis of whole cell lysates from OPM-1 cells treated with 1 μM lenalidomide for the indicated durations of time. b Same as in (a) except that OPM-1 cells were treated with lenalidomide for the indicated time points. c Immunoblot analysis of whole cell lysates from OPM-1 cells treated with at the indicated concentrations of lenalidomide for 36 h. d HEK293T cells were transfected with FLAG-CRBN, HA-IKZF1, and un-tagged RUNX1. Where indicated, cells were treated with 2 μM lenalidomide for 6 h before harvesting. The cell extracts were subjected to anti-FLAG IP and the immunocomplexes were probed with antibodies to the indicated proteins. A low exposure (l.e.) and high exposure (h.e.) are shown for IKZF1. e Same as in d except that HEK293T cells were transfected with FLAG-IKZF1 and un-tagged RUNX1. f Chromatin immunoprecipitations of IKZF1 in OPM-1 cells coupled with qRT-PCR using primers for IRF4 promoter under the indicated conditions. Red bar shows the distance between the primers and transcription start site of IRF4 (n = 2 independent experiments). g Levels of IRF4 and MYC mRNA were analyzed by qRT-PCR under the indicated conditions (mean ± s.d., n = 2 independent measurements). h Cell counts of GFP/Cherry-sorted OPM-1 RUNX1+/+ RUNX3+/+ or RUNX1−/−RUNX3−/− cells grown in media containing DMSO or 0.1 μM lenalidomide (mean ± s.d., n = 3 independent experiments, two-way ANOVA, n.s., not significant, ****P ≤ 0.0001). Unless otherwise noted, immunoblots are representative of three independent experiments

Fig 4: 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).

Fig 5: IKZF1 and IKZF3 physically associate with RUNX1 and RUNX3 in multiple myeloma. a Scatter plot of distributed normalized spectral abundance factor (dNSAF) in FLAG-immunoprecipitates (IP) from ARP-1 cells stably expressing FLAG-tagged IKZF1 and IKZF3 upon mass spectrometry analysis. For proteins with NSAF = 0, the lowest NSAF value was arbitrarily assigned. b List of peptides for the indicated proteins. EV empty vector. c Schematic model of IKZF1/3 interactors. For a complete list of interacting proteins see Supplementary Table 1. d The cell extracts of ARP-1 (left) and OPM-1 (right) cells stably expressing FLAG-tagged IKZF1 and IKZF3 were immunoprecipitated with an anti-FLAG resin and the immunocomplexes were probed with antibodies to the indicated proteins. Specificity of RUNX1 and RUNX3 antibodies was assessed using siRNAs against RUNX1 or RUNX3 (Supplementary Figure 1a). e HEK293T cells stably expressing IKZF3 were transfected with FLAG-RUNX1 or RUNX3. FLAG-immunoprecipitates were probed with antibodies to the indicated proteins. f HEK293T cells were transfected with FLAG-tagged IKZF1 or IKZF3. The cell extracts were subjected to anti-FLAG IP and the immunocomplexes were treated with Benzonase for 30 min where indicated. IPs were probed with antibodies to the indicated proteins. g 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. The red asterisk indicates GST-IKZF1, blue asterisk shows GST-IKZF3, and black asterisks show cleavage products. Unless otherwise noted, immunoblots are representative of three independent experiments