Mezigdomide mediates its effects through potent IKAROS degradation. (A-B) Mass spectrometry in the OCI-AML2 and MV4;11 cell lines after 12 hours of treatment. The y-axis depicts log₂ fold change (FC) in abundance for each protein relative to DMSO (dotted line at threshold of ± 1.5-FC), and the x-axis depicts log₁₀P value (dotted line at significance threshold of 10⁻³). Proteins with log₂FC ≤ −1.5-fold that were shared in common after treatment with any of the 3 IMiDs/CELMoDs in each cell line, in addition to CK1α, are labeled. (C) Western blot in the MV4;11 cell line after 8 hours of treatment. (D) Western blot after 18 hours in MOLM-13 cells transduced with pLKO5-GFP (empty vector) and IKAROS overexpression (O/E) constructs: wild-type (WT) IKAROS or a nondegradable mutant IKAROS^Q146H O/E. (E-F) In MOLM-13 cells transduced with empty vector and IKAROS O/E constructs: (E) proliferation relative to DMSO control after 9 days of treatment, and (F) apoptosis assessment after 6 days of treatment, with percentage of cells staining positive for annexin and PI by flow cytometry. (E-F) Data pooled from 3 independent experiments; n = 12 for each condition; data represent mean + SD. Statistical analysis with 2-way ANOVA with Tukey multiple comparisons test within each cell line; ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001. (G) RNA-sequencing data for MOLM-13 cell line treated with DMSO, IBER 1000 nM, or MEZI 50 nM for 3 days (n = 3 technical replicates for DMSO, IBER, and MEZI). (Left) Correlation between IBER and MEZI gene expression changes; (middle and right) volcano plots depicting log₂FC (x-axis) and log₁₀P value (y-axis) for DMSO vs IBER or MEZI. Genes with log₂FC < −3 or > +3.5 depicted. (H) Bar code plots depicting gene set enrichment analysis results for gene sets after HOXA9 shRNA knockdown.