Degradation of GSPT1 causes TP53-independent cell death in leukemia while sparing normal hematopoietic stem cells
Rob S. Sellar, … , Chun-Wei Chen, Benjamin L. Ebert
Rob S. Sellar, … , Chun-Wei Chen, Benjamin L. Ebert
Published June 28, 2022
Citation Information: J Clin Invest. 2022;132(16):e153514. https://doi.org/10.1172/JCI153514.
View: Text | PDF
Research Article Hematology

Degradation of GSPT1 causes TP53-independent cell death in leukemia while sparing normal hematopoietic stem cells

Abstract

Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early-phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR/Cas9 screens implicated decreased translation initiation as protective following GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to the effects of GSPT1 degradation. We defined 2 Crbn amino acids that prevent Gspt1 degradation in mice, generated a knockin mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant acute myeloid leukemia.

Authors

Rob S. Sellar, Adam S. Sperling, Mikołaj Słabicki, Jessica A. Gasser, Marie E. McConkey, Katherine A. Donovan, Nada Mageed, Dylan N. Adams, Charles Zou, Peter G. Miller, Ravi K. Dutta, Steffen Boettcher, Amy E. Lin, Brittany Sandoval, Vanessa A. Quevedo Barrios, Veronica Kovalcik, Jonas Koeppel, Elizabeth K. Henderson, Emma C. Fink, Lu Yang, Anthony Chan, Sheela Pangeni Pokharel, Erik J. Bergstrom, Rajan Burt, Namrata D. Udeshi, Steven A. Carr, Eric S. Fischer, Chun-Wei Chen, Benjamin L. Ebert

×

Figure 2

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

Options: View larger image (or click on image) Download as PowerPoint
CC-885 treatment impairs translation termination, activates the internal...
(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 GSPT1G575N 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).