Fig. 3. Compound 9 promotes proteasomal degradation of BET proteins via the CRL^DCAF11 complex.

a Schematic of FACS-based BRD4 protein stability CRISPR screen assay. b, c Identification of genes required for compound 9-mediated degradation of BRD4. b FACS-based CRISPR screens for regulators of BRD4 degradation induced by 9. Average gene-level fold-changes and non-adjusted one-sided p-values of BRD4^HIGH and BRD4^LOW cell populations compared to BRD4^MID fraction were calculated using MAGeCK⁴⁶. Essential control genes (BRD4^LOW) and 20S proteasome subunits, COP9 signalosome subunits and E1 or E2 ubiquitin ligases (BRD4^HIGH) inside the scoring window (p-value < 0.01, fold-change > 1.5) are labelled. c Heatmap of selected screen hits from Fig. 3b and Supplementary Fig. 4a, b. Gene-level score was calculated as Log₂ (fold change) * Log₁₀ (p-value). d, e BRD2 and DCAF11 protein levels in Hep G2 cells transfected with non-targeting (NT) siRNA or DCAF11 siRNA, followed by 9 addition. d Quantification of the relative BRD2 levels. Data are mean values ± SD (n = 3 biological replicates). e Quantification of the relative DCAF11 levels. Data are mean values ± SD (n = 3 biological replicates). f DCAF11 levels in wild type (WT) KBM7 cells and DCAF11 knockout (KO1 and KO2) KBM7 cells. Representative result of n = 3. g BRD2 levels in WT and DCAF11 knockout (KO1 and KO2) KBM7 cells treated with compound 9 at 3 μM, 10 at 10 μM and 11 at 10 μM, for 6 h. Data are representative of n = 3. h BRD2 levels in Dox-inducible DDB1 knockout KBM7 cells pretreated with 0.5 µg/mL Dox for 3 days followed by addition of 9 at 3 μM for another 4 h. Representative result of n = 3. i BRD2 and RBX1 levels in MDA-MB-231 cells with NT or RBX1 siRNA followed by 9 addition. Representative result of n = 3. j BRD2 co-immunoprecipitation. FLAG-BRD2 protein was enriched by anti-FLAG beads and samples were analyzed for DCAF11. Representative result of n = 2. k Structure of biotinylated affinity probe 14. l Affinity-based enrichment by pulldown in Hep G2 cells overexpressing DCAF11. Protein levels were analyzed by WB. Representative result of n = 2.