Inactivation of SENP8 confers resistance to LEN, but not POM. (A) Representative western blot analysis confirms efficient CRISPR-induced inactivation of SENP8 in BC-3/Cas9 cells by 2 independent SENP8-specific sgRNAs (sg1, sg2). sgAAVS1 served as negative control guide, GAPDH served as loading control. (B-C) Growth curve analyses of SENP8-inactivated BC-3/Cas9 cells after treatment with 5 µM LEN (B) or 1 µM POM (C). BC-3/Cas9 cells transduced with sgAAVS1 were included as a CM-sensitive negative control, whereas previously described clonal CRBN-inactivated BC-3 cells (CRBN KO) served as a positive control for complete CM resistance. Absolute live cell numbers were normalized to corresponding DMSO vehicle-treated cells at each passage (represented by the dotted line). These experiments were performed in parallel with UBE2G1-inactivated cells shown in Figure 3B-C, and thus share common negative and positive controls for several or all replicates (n = 3; error bars represent SEM). (D-E) Representative western blot analyses of IKZF1, IKZF3, CK1α, and IRF4 at indicated points confirm incomplete neosubstrate degradation and IRF4 downregulation in SENP8-inactivated BC-3/Cas9 cells compared with sgAAVS1 transduced BC-3/Cas9 control cells upon treatment with 5 µM LEN (D) or 1 µM POM (E). Lysates are matched with growth curves shown in panels B and C, respectively. GAPDH served as loading control. The asterisk marks a nonspecific band. (F) Dose-response analysis of LEN toxicity in Cas9-expressing PEL cell lines BC-3, BCBL-1, BC-1, and MM cell lines KMS12-BM and MM.1S on inactivation of SENP8. sgAAVS1- or sgCRBN-transduced cell lines served as negative or positive controls for drug resistance, respectively. Readout of the assay was on day 7 (BC-3, BCBL-1, and MM.1S) or day 9 (BC-1, KMS12-BM) into CM treatment. Statistical analyses in panels B and C were performed by unpaired Student t tests comparing specified conditions to corresponding sgAAVS1 controls. *P < .05; **P < .01; ***P < .001; ****P < .0001.