Extended Data Fig. 5: CRBN DMS screen and validation of results.
From: High-throughput ligand diversification to discover chemical inducers of proximity
a, Flow cytometry following treatment in MV4;11 CRBN KO “parental” cells expressing ENL-TagBFP-P2A-mCherry. Histograms for SR-1114, rac-dHTC1 and DMSO treatments overlap as ENL degradation is CRBN-dependent. b, Reintroduction of wild-type CRBN restores ENL degradation. c, Reintroduction of CRBN with inactivating mutation (W380I) fails to mediate ENL degradation. d, Cells transduced with CRBN DMS library regained activity; roughly 70% of cells showing ENL degradation. e, MV4;11 CRBN KO cells expressing ENL-TagBFP-P2A-mCherry were transduced with a DMS library, which reintroduced CRBN with single amino acid mutations within a 10 Å radius of the thalidomide binding site. After these cells were treated with rac-dHTC1 (1 µM, 8 h), endpoint ENL levels were quantified by flow cytometry. Results are depicted indicating log2 fold change of ENLhigh over the unsorted pools. f, SR-1114 (same as e). g, Heatmap comparing CRBN DMS results for rac-dHTC1 and SR-1114. Arrows above indicate residues showing selectivity for dHTC1 (blue), SR-1114 (gray), or mixed (purple) that were selected for follow-up assays. h, Validation of altered sensitivity to ENL degraders by individual CRBN mutants. MV4;11 CRBN knockout cells expressing ENL-TagBFP-P2A-mCherry reporter with 1 µM of rac-dHTC1 or 1 µM of SR-1114 (8 h), assessed by flow cytometry (BFP/mCherry normalized to DMSO, 3 technical replicates, 2 independent experiments ± S.D.). i, Immunoblot analyses of ENL levels in MV4;11 CRBN knockout cells expressing ENL-TagBFP-P2A-mCherry. Wild-type or mutant CRBN was reintroduced by lentiviral expression to evaluate mutations that selectively impact the ability of dHTC1 or SR-1114 to degrade ENL (1 µM, 6 h). For ENL, the lower molecular weight band is endogenous protein and higher molecular weight band is reporter construct. Representative of 2 independent experiments.
