Figure 2. CC-885 treatment impairs translation termination, activates the internal stress response pathway, and results in TP53-independent cell death.

(A) Design of fluorescent translation termination reporter (position of the stop codon is indicated in red). (B) GFP/BFP ratio in K562 cells expressing reporters with BFP stop codons following 72 hours of exposure to G418 (ratio expressed relative to DMSO). Results are combined from 4 independent replicates performed in triplicate (Kruskal-Wallis test). (C) GFP/BFP ratio in K562 cells expressing reporters with BFP stop codons following 72 hours of exposure to CC-885 (ratio expressed as fold change, indicated by depth of red color, relative to DMSO). Results are combined from 4 independent replicates performed in triplicate. Statistical analysis using Wilcoxon’s test is given in Supplemental Table 2. (D and E) RNA-Seq results in MOLM13 cells following 6 hours of treatment with 1 μM CC-885 (D) and 1 μM CC-90009 (E). (F) Correlation of gene expression changes in MOLM13 cells following treatment with CC-885 or CC-90009. (G) Volcano plot of proteomics data comparing native HEK293T cells with cells overexpressing GSPT1^G575N following 18 hours of treatment with 1 μM CC-885. Protein abundance is represented by tandem mass tag (TMT) reporter ion intensity ratios relative to average of DMSO-treated native 293T samples, all measured in duplicate. Significance of a 2-sample modT test between each condition is shown on the y axis. Relative change in abundance of proteins between conditions is expressed as fold change on the x axis. (H–J) Cell viability of MOLM13 cells that are TP53 WT or TP53 knockout or have endogenous expression of mutant TP53 following treatment with cytarabine (AraC) (H), daunorubicin (Dauno) (I), or CC-885 (J). CellTiter-Glo luminescent assay 72 hours after treatment; relative luminescent units (RLU) relative to DMSO (graphs represent combined data from 3 biological replicates performed in technical triplicate; symbols represent mean ± SEM).