Figure 1.
CC-90009, a GSPT1-selective cereblon modulator, induces cereblon-and GSPT1-dependent anti-AML activity. (A) The chemical structures of CC-90009, CC-885, and lenalidomide (LEN) with the glutarimide ring shown in red. (B) The antiproliferative effect of CC-90009 in AML cell lines. Cells were incubated with DMSO or CC-90009 at the indicated concentrations. At day 3, cell proliferation was assessed by the Cell-Titer Glo (CTG) assay. (C-D) The effect of CC-90009 on cell viability of leukemic cells (C) and normal lymphocytes (D) in bone marrow aspirates of AML patients. Cells were treated with DMSO or increasing concentrations of CC-90009 for 48 hours. Cells were then stained with fluorescently labeled antibodies and Annexin-V, followed by flow cytometry to determine the cell number of live leukemic cells and lymphocytes. Data shown in panels C and D are presented as the percentage of viable cell counts in CC-90009-treated patient samples relative to DMSO controls. (E) Volcano plot of differentially abundant proteins in response to CC-90009 treatment relative to DMSO control. KG1 cells were treated with DMSO or 100 nM CC-90009 for 4 hours and subjected to tandem mass tag proteomics analysis. The x-axis indicates the log2–fold change of CC-90009 vs DMSO control for each protein. P values were corrected for multiple hypothesis testing using the Benjamini-Hochberg method to arrive at an adjusted P value (adj-P; also known as a false discovery rate). The y-axis is the -log10 (adj-P) values indicating statistical significance such that proteins lying above the dotted red line are statistically significant findings with adj-P <.05. (F) Immunoblot analysis of KG1 and U937 cells treated with DMSO or CC-90009 for 4 hours. Where indicated, cells were pretreated with mortezomib or MLN4924 or 30 minutes. (G-H) Immunoblot analysis (G) and cell proliferation (H) of U937-Cas9 parental cells or cells stably transduced with lentiviral vectors expressing a nontargeting sgRNA (sgNT-1), an sgRNA targeting a noncoding region (sgNC-8), or an sgRNA targeting CRBN (sgCRBN-8). Cells were treated with DMSO or CC-90009 at indicated concentrations. Crystal structure of GSPT1 in complex with cereblon, DDB1 and CC-90009. (I) Surface representation of the complex with DDB1 shown in purple, cereblon in blue, and GSPT1 in orange. The position of CC-90009 is shown with an arrow. (J) GSPT1 interaction with cereblon is mediated by a β-hairpin loop. Hydrogen bond interactions between cereblon and the GPST1 β-hairpin are shown as yellow dashes. (K-L) Immunoblot analysis (K) and cell proliferation (L) of U937 parental cells or cells stably expressing HA-GSPT1-G575N. Cells were treated with DMSO or CC-90009 at the indicated concentrations. Data in panels B-D, G, and L are shown as mean ± standard deviation (SD), n = 3 technical replicates. Result shown in all figure panels is representative of 3 biological replicates.

CC-90009, a GSPT1-selective cereblon modulator, induces cereblon-and GSPT1-dependent anti-AML activity. (A) The chemical structures of CC-90009, CC-885, and lenalidomide (LEN) with the glutarimide ring shown in red. (B) The antiproliferative effect of CC-90009 in AML cell lines. Cells were incubated with DMSO or CC-90009 at the indicated concentrations. At day 3, cell proliferation was assessed by the Cell-Titer Glo (CTG) assay. (C-D) The effect of CC-90009 on cell viability of leukemic cells (C) and normal lymphocytes (D) in bone marrow aspirates of AML patients. Cells were treated with DMSO or increasing concentrations of CC-90009 for 48 hours. Cells were then stained with fluorescently labeled antibodies and Annexin-V, followed by flow cytometry to determine the cell number of live leukemic cells and lymphocytes. Data shown in panels C and D are presented as the percentage of viable cell counts in CC-90009-treated patient samples relative to DMSO controls. (E) Volcano plot of differentially abundant proteins in response to CC-90009 treatment relative to DMSO control. KG1 cells were treated with DMSO or 100 nM CC-90009 for 4 hours and subjected to tandem mass tag proteomics analysis. The x-axis indicates the log2–fold change of CC-90009 vs DMSO control for each protein. P values were corrected for multiple hypothesis testing using the Benjamini-Hochberg method to arrive at an adjusted P value (adj-P; also known as a false discovery rate). The y-axis is the -log10 (adj-P) values indicating statistical significance such that proteins lying above the dotted red line are statistically significant findings with adj-P <.05. (F) Immunoblot analysis of KG1 and U937 cells treated with DMSO or CC-90009 for 4 hours. Where indicated, cells were pretreated with mortezomib or MLN4924 or 30 minutes. (G-H) Immunoblot analysis (G) and cell proliferation (H) of U937-Cas9 parental cells or cells stably transduced with lentiviral vectors expressing a nontargeting sgRNA (sgNT-1), an sgRNA targeting a noncoding region (sgNC-8), or an sgRNA targeting CRBN (sgCRBN-8). Cells were treated with DMSO or CC-90009 at indicated concentrations. Crystal structure of GSPT1 in complex with cereblon, DDB1 and CC-90009. (I) Surface representation of the complex with DDB1 shown in purple, cereblon in blue, and GSPT1 in orange. The position of CC-90009 is shown with an arrow. (J) GSPT1 interaction with cereblon is mediated by a β-hairpin loop. Hydrogen bond interactions between cereblon and the GPST1 β-hairpin are shown as yellow dashes. (K-L) Immunoblot analysis (K) and cell proliferation (L) of U937 parental cells or cells stably expressing HA-GSPT1-G575N. Cells were treated with DMSO or CC-90009 at the indicated concentrations. Data in panels B-D, G, and L are shown as mean ± standard deviation (SD), n = 3 technical replicates. Result shown in all figure panels is representative of 3 biological replicates.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal