GCKi induces anti-MM effects by targeting IKZF1 via a CRBN-independent mechanism. MM.1S-tet-on-shGCK cells were infected by PCDH-Flag-IKZF1-WT (IKZF1^WT) or PCDH-Flag-IKZF1-Q146H (IKZF1^Q146H) lentivirus and sorted by green fluorescent protein after 3 days. (A) Cell lysates were analyzed by western blotting to confirm IKZF1^WT and IKZF1^Q146H expression. (B-C) Sorted cells were treated with LEN at 2, 4, or 8 μM (B) or TL4-12 at 5, 10, 15 μM (C) or dimethyl sulfoxide (DMSO; 0.01%) for 24 hours. Cell lysates were analyzed by western blotting. (D) Sorted cells were treated with LEN or TL4-12 at indicated concentrations for 5 days. Cell proliferation was detected by MTS. (E) Sorted cells were treated with 5-μM LEN, 5-μM TL4-12, or DMSO (0.01%) for 5 days. Treated cells were stained with Annexin V and 7-AAD for apoptosis analysis. (F-G) Sorted cells were treated with 400 ng/mL doxycycline (DOX) for 5 days to induce GCK knockdown (shGCK) or DMSO (0.01%) as control (CT). Cell proliferation was detected by MTS (F). ∗∗P < .01 (by 2-way analysis of variance [ANOVA]). Apoptosis assay was detected by Annexin V and 7-AAD staining (G).