Compilation of the in vitro results and structural analysis of the CRBN mutations that demonstrated partial impact on CRBN function. (A) Immunoblotting results for CRBN neosubstrates Ikaros and ZFP91 and for transcription factor MYC for all CRBN mutations that demonstrated partial CRBN function. The blots for p.W386A and CRBNWT have been included as negative and positive control, respectively. Cells were drug treated (10 μM Len, 1 μM Pom, 0.1 μM Iber, 0.01 μM Mezi, or DMSO) and harvested 24 hours after IMiD/CELMoD treatment for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/western blotting. Left panel: data shown are representative of 3 biological repeats. Right panel: optical densitometry quantification of the 3 biological replicates. (B) GI50 cell viability results, displayed per compound, for all cell lines whose CRBN mutation demonstrated partial CRBN function in comparison with p.W386A (negative control) and CRBNWT (positive control). All cell lines were treated with DMSO or increasing concentrations of Len (maximum concentration at 20 μM), Pom (maximum concentration 8 μM), Iber (maximum concentration 2 μM), and Mezi (maximum concentration 1 μM) for 5 days. Cell viability was determined using the CellTiter-Blue assay. Results are the average ± SEM of n = 3 biological replicates. (C-F) Spatial localization of the CRBN mutations that demonstrated an intermediate/variable effect on CRBN function with respect to the IMiDs and neosubstrate-binding sites. The presented figure is a composite figure generated by overlaying our high-resolution crystallographic CRBN/DDB1 structure bound to Len (where CRBN is present in its closed conformation, PDB code 9JFX), with the publicly available crystallographic structure of neosubstrate Ikaros bound to CRBN/DDB1/Pom complex (where CRBN is present in its open conformation, PDB code 6H0F12). CRBN is represented in gold, Ikaros in orange, DDB1 in gray, Len in cyan, the 3 Trp defining the IMiD’s binding pocket in light blue, Zn2+ atoms as gray spheres, and the position of the mutations in magenta. Figures were generated with Pymol.25 (C) Localization of CRBN Cys326, mutated to Gly in patients. (D) Localization of CRBN Pro352, mutated to Ser in patients. (E) Localization of CRBN Cys366, mutated to Tyr in patients. (F) Localization of CRBN Phe381, mutated to Ser in patients.

Compilation of the in vitro results and structural analysis of the CRBN mutations that demonstrated partial impact on CRBN function. (A) Immunoblotting results for CRBN neosubstrates Ikaros and ZFP91 and for transcription factor MYC for all CRBN mutations that demonstrated partial CRBN function. The blots for p.W386A and CRBNWT have been included as negative and positive control, respectively. Cells were drug treated (10 μM Len, 1 μM Pom, 0.1 μM Iber, 0.01 μM Mezi, or DMSO) and harvested 24 hours after IMiD/CELMoD treatment for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/western blotting. Left panel: data shown are representative of 3 biological repeats. Right panel: optical densitometry quantification of the 3 biological replicates. (B) GI50 cell viability results, displayed per compound, for all cell lines whose CRBN mutation demonstrated partial CRBN function in comparison with p.W386A (negative control) and CRBNWT (positive control). All cell lines were treated with DMSO or increasing concentrations of Len (maximum concentration at 20 μM), Pom (maximum concentration 8 μM), Iber (maximum concentration 2 μM), and Mezi (maximum concentration 1 μM) for 5 days. Cell viability was determined using the CellTiter-Blue assay. Results are the average ± SEM of n = 3 biological replicates. (C-F) Spatial localization of the CRBN mutations that demonstrated an intermediate/variable effect on CRBN function with respect to the IMiDs and neosubstrate-binding sites. The presented figure is a composite figure generated by overlaying our high-resolution crystallographic CRBN/DDB1 structure bound to Len (where CRBN is present in its closed conformation, PDB code 9JFX), with the publicly available crystallographic structure of neosubstrate Ikaros bound to CRBN/DDB1/Pom complex (where CRBN is present in its open conformation, PDB code 6H0F12). CRBN is represented in gold, Ikaros in orange, DDB1 in gray, Len in cyan, the 3 Trp defining the IMiD’s binding pocket in light blue, Zn2+ atoms as gray spheres, and the position of the mutations in magenta. Figures were generated with Pymol.25 (C) Localization of CRBN Cys326, mutated to Gly in patients. (D) Localization of CRBN Pro352, mutated to Ser in patients. (E) Localization of CRBN Cys366, mutated to Tyr in patients. (F) Localization of CRBN Phe381, mutated to Ser in patients.

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