DDI2 KO is cytotoxic in MM and sensitized to PI treatment by impairing de novo proteasome subunit biogenesis. (A) Relative changes in viability of DDI2-KO AMO1, H929, and KMS20 cells (blue bars) compared with cells subjected to RNP with a nontargeting (NT) gRNA (red bars). The average of 3 independent biological experiments is shown. (B) Relative changes in viability in NRF1-KO AMO1, H929, and KMS20 cells (blue bars) compared with cells subjected to RNP with an NT gRNA (red bars). The average of 3 independent biological experiments is shown. (C-D) Western blotting showing DDI2 (C) or NRF1 (D) KO in cells used for the growth competitive assay in (A) and (B), respectively. GAPDH and α-tubulin served as loading controls, respectively. FL, full length; P, processed. (E-G) Percentage of living (Annexin V⁻/PI⁻ on flow cytometry) DDI2 KO (blue line) H929 (E), AMO1 (F), and KMS20 (G) cells after 48 hours of treatment with the indicated dose of carfilzomib. Data were normalized against DDI2 WT cells (red line). The average of 3 independent experiments is shown. (H) Real-time PCR showing expression of messenger RNA coding for proteasome subunits PSMA7, PSMB5, PSMB6, and PSMD11 in DDI2 WT vs DDI2-KO KMS20 cells treated with DMSO (red bars) or a sublethal dose of carfilzomib (CFZ, blue bars). For each gene, RNA level in WT DDI2 cells treated with DMSO was used as control for normalization. The P value was calculated for each paired condition (same proteasome subunit and same treatment) for DDI2 WT vs KO. One representative experiment of 3 biological replicates, each with triplicate conditions, is shown. *P < .05, **P < 0.01, ***P < .001, ****P < .0001.