Potency of mezigdomide is related to increased depth, rate, and duration of IKAROS degradation. (A,C) Mass spectrometry in the OCI-AML2 and MV4;11 cell lines after 3 hours of treatment. The y-axis depicts log₂ 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 3- or 12-hour treatment with any of the 3 IMiDs/CELMoDs in each cell line, in addition to CK1α, are labeled. (B,D) Western blot in the OCI-AML2 cell line after 3 hours of treatment and in the MV4;11 cell line after 90 minutes, 5 hours, and 24 hours of treatment. (E) Western blot in the MV4;11 cell line after treatment (tx) for 15 hours, followed by large volume washing of cells (3 washes), and culturing cells for 6 and 24 more hours before cell pellet collection. (F-G) Experiments performed in the MOLM-13 cell line transduced with IKZF1-eGFP-IRES-mCherry degradation reporter vector. (F) Cells treated for 4.5 and 12 hours and eGFP/mCherry ratio relative to DMSO control calculated. Cells plated in triplicate with each data point representing mean ± SD. (G) Cells treated for 12 hours followed by extensive washing (7 washes that lasted from hour 12-13 in duration) and then assessing eGFP recovery thereafter. Representative of 3 experiments, with each data point representing mean of technical triplicates ± SD. (H) Proliferation (cell count) relative to DMSO after 10 days from initial treatment comparing continuous treatment (left) with 18-hour treatment followed by washout (right) in MV4;11 cell line. Data depicted pooled from 3 independent experiments, each of which had at least 5 technical replicates for n > 15 per bar; bars represent mean + SD. Statistical analysis with 2-way ANOVA with Tukey multiple comparisons test. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.