Figure 5. Functional CRBN Hotspots Show Degrader Selectivity and are Mutated in Refractory Multiple Myeloma Patients.

(A) Heatmap depicting differential log2 fold-enrichment of CRBN mutations normalized to maximum log2 fold-changes vs. DMSO between BET bromodomain targeting PROTAC dBET6 (500 nM, 7 d treatment) and the GSPT1 targeting molecular glue CC-90009 (500 nM, 7 d treatment). n = 3 independent measurements.

(B) Dose-resolved, normalized viability after 3 d treatment with CC-90009 and dBET6 in RKO CRBN^-/- cells with over-expression of CRBN^WT, CRBN^E377K, CRBN^N351D and CRBN^H397D. Mean ± s.e.m.; n = 3 independent treatments.

(C, F and H) Protein levels in RKO CRBN^-/- cells with over-expression of CRBN^WT, CRBN^N351D, CRBN^H397D or CRBN^H57D treated with DMSO, CC-90009 (50 nM, 6 h), dBET6 (15 nM, 2 h), dBET57 (240 nM, 2 h) or THAL-SNS-032 (200 nM, 2 h). Representative

images of n = 2 independent measurements.

(D) Cocrystal structure of dBET6 (left) and CC-90009 (right) in a ternary complex with CRBN and BRD4^BD2 (PDB 6BOY) or GSPT1 (PDB 6XK9) depicting PPIs of CRBN^N351 and the GSPT1.

(E) Dose-resolved, normalized viability after 3 d treatment with dBET57 in RKO CRBN^-/- cells with over-expression of CRBN^WT and CRBN^H397D. Mean ± s.e.m.; n = 3 independent treatments.

(G) Depiction of clonogenic assays via crystal violet staining. Cells were treated for 10 days at EC90 of the degrader (30 nM dBET6, 60 nM CC-90009). Representative of n = 2 independent measurements.

See also Extended Data Fig. 6.