Identification of genes and pathways modulating the response to CC-90009 via CRIPSR/Cas9 screen. (A) Schematic showing the design of the genome-wide CRISPR screen to identify molecular determinants of CC-90009 response. At day 3 posttransduction, cells were treated with 10 µM CC-90009 or DMSO vehicle control for an additional 9 days, followed by amplification of sgRNA coding regions and next-generation sequencing. The log₂ fold change (log₂FC) in sgRNA read count in the CC-90009-treated sample as compared with DMSO control was designated as the enrichment score, and the average log₂FC value of all sgRNAs for a gene of interest was used to quantify the effect of gene knockout on CC-90009 response. (B) Cell proliferation curve of U937-Cas9 cells transduced with the lentiviral sgRNA library and treated with DMSO or CC-90009. Three days posttransduction, cells were treated with DMSO or 10 µM CC-90009 for 9 days. (C) Pathway enrichment analysis of genes enriched by CC-90009 treatment with log₂FC >2 and false discovery rate (FDR) <0.05 relative to DMSO control. The color and size of the dots represent adjusted significance level and gene ratio, respectively. Gene ratio refers to the number of input genes annotated to an individual pathway as a ratio of all input genes annotated to any Reactome pathway. (D) Scatter plot of 78 genes significantly enriched by CC-90009 (log₂FC >2 and FDR <0.05). X-axis, CC-90009 enrichment score shown as log₂FC (T12_CC-90009 vs T12_DMSO); y-axis, gene essentiality score shown as log₂FC (T12_DMSO vs T3_DMSO). Some of these genes were grouped into 10 functional modules with different color coding. (E,F) Log₂FC values of sgRNAs targeting CC-90009 enriched genes in the functional modules as indicated. Background shown in dark blue represents the log₂FC values of all sgRNAs in the library. Each solid line with a color representing a functional module indicates the log₂FC value of an individual sgRNA. Well-characterized genes known to be essential for the activity of the cereblon E3 ligase complex (E); novel genes that regulate the response to CC-90009 with no clear mechanistic understanding (F). Note that in panels E and F, multiple sgRNAs targeting each of these top-ranked genes were significantly enriched by CC-90009, strongly supporting the on-target gene knockout effect.