shRNA screening in MM cell lines identifies IDH2 gene as synthetic lethal to the proteasome inhibitor carfilzomib. (A) Experimental design of the shRNA screen to identify genes conferring sensitivity to CFZ in MM cells. KMM-1^PIR cells were infected with 684 shRNAs targeting 152 cancer driver genes (day −3) and incubated in presence or absence of puromycin (day −2). KMM-1^PIR cells were then split and treated with 2.5 nM CFZ or with control diluent (DMSO) (day 0). Growth rate was calculated at day 3 and 7 posttreatment (supplemental Table 5), and positive hits selected according to the z score. Top 24 selected genes were validated in a secondary screening performed in U266^PIR cells. (B) Representation of the z score (y-axis) for every shRNA (x-axis) calculated on growth rate reduction for each shRNA. Red box highlights candidates with z score below −0.8 (day 7) (supplemental Table 6). (C) Correlation between percentage of gene silencing and percentage of growth inhibition in presence of CFZ for top 3 candidate genes (IDH2, KDM1A, and SOX2) in U266^PIR cells. (D) KMM-1^PIR, (E) U266^PIR, (F) KMM-1, and (G) U266 cell lines were transduced with the empty vector or shRNAs targeting IDH2 (shIDH2_A4, shIDH2_A6) and treated with CFZ (KMM-1^PIR and U266^PIR, 5 nM; KMM-1 and U266, 2.5 nM) or DMSO every 48 hours. Cell viability was measured by TMRM staining-flow cytometry 96 hours posttreatment for KMM-1^PIR and U266^PIR and 48 hours posttreatment for KMM-1 and U266. Data are the means ± standard deviation (SD) of 3 independent experiments (*P < .05; **P < .01). TMRM, tetramethylrhodamine.