Differential Analysis of Cereblon Neosubstrates in Rabbit Embryos Using Targeted Proteomics

Abstract

Targeted protein degradation is the selective removal of a protein of interest through hijacking intracellular protein cleanup machinery. This rapidly growing field currently relies heavily on the use of the E3 ligase cereblon (CRBN) to target proteins for degradation, including the immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide which work through a molecular glue mechanism of action with CRBN. While CRBN recruitment can result in degradation of a specific protein of interest (e.g., efficacy), degradation of other proteins (called CRBN neosubstrates) also occurs. Degradation of one or more of these CRBN neosubstrates is believed to play an important role in thalidomide-related developmental toxicity observed in rabbits and primates. We identified a set of 25 proteins of interest associated with CRBN-related protein homeostasis and/or embryo/fetal development. We developed a targeted assay for these proteins combining peptide immunoaffinity enrichment and high-resolution mass spectrometry and successfully applied this assay to rabbit embryo samples from pregnant rabbits dosed with three IMiDs. We confirmed previously reported in vivo decreases in neosubstrates like SALL4, as well as provided evidence of neosubstrate changes for proteins only examined in vitro previously. While there were many proteins that were similarly decreased by all three IMiDs, no compound had the exact same neosubstrate degradation profile as another. We compared our data to previous literature reports of IMiD-induced degradation and known developmental biology associations. Based on our observations, we recommend monitoring at least a major subset of these neosubstrates in a developmental test system to improve CRBN-binding compound-specific risk assessment. A strength of our assay is that it is configurable, and the target list can be readily adapted to focus on only a subset of proteins of interest or expanded to incorporate new findings as additional information about CRBN biology is discovered.

Keywords: CRBN; IMiD; PRM; developmental biology; developmental toxicity; immunoaffinity; neosubstrate; rabbit embryo.

Graphical abstract

Fig. 1

Schematic diagram of experimental workflow.A, targeted MS workflow. B, timeline of animal dosing and collections for both targeted MS and developmental toxicity assessment. Figure created with BioRender.com. MS, mass spectrometry.

Fig. 2

Characterization of targeted MS assay performance. Reverse response curves were performed with heavy isotope-labeled peptides spiked into a common matrix sample and normalized to the endogenous peptide. A, in total, multiplexed acquisition for 16 peptides in IA and 20 peptides in the nonenriched runs were performed. Shown are example response curves for (B) SALL4 in the immunoenriched runs and (C) DTWD1 in the nonenriched runs. D, the number of points per peak was assessed for each endogenous peptide across 30 runs to ensure that sufficient data was collected to describe the peak (>10 points, dotted line on graph). MS, mass spectrometry.

Fig. 3

Abundance changes in selected CRBN neosubstrate proteins following IMiD dosing. Pregnant rabbits were dosed once daily from GD7-GD11 with thalidomide (180 mg/kg/day), pomalidomide (180 mg/kg/day), lenalidomide (20 mg/kg/day), or left undosed. Abundances of each protein target in each embryo were assessed approximately 24 h post final dose on GD11 and were grouped by mother (denoted as R1, R2, and R3) and graphed as percent of the average signal of the undosed embryos. Four embyros were analyzed for each mother except for the untreated controls where seven and five embryos were analyzed for R1 and R2, respectively. Individual embryo values are depicted as diamonds and the mean value per pregnant mother is represented by the bar graph. A, SALL4 shows differing responses to each IMiD with thalidomide having the greatest overall decrease in abundance levels. B, IKZF1 was decreased in rabbits dosed with pomalidomide. C, TP63 was observed to have a modest decrease following thalidomide exposure. D, DTWD1 showed the most robust decrease of all targets and was most impacted by pomalidomide. CRBN, Cereblon; ImiD, immunomodulatory drug.

Fig. 4

Heatmap summary of all targets following IMiD dosing. Shown are the average log₂ fold changes of each compound compared to the undosed embryos (n = 12 for all groups). Proteins marked with an asterisk are considered significant based on an FDR of 0.01 or less from a two-way ANOVA using litter as a covariate. FDR, false discovery rate; ImiD, immunomodulatory drug.

Fig. 5

Thalidomide-induced rabbit fetal dysmorphology. Representative images are shown of rabbit fetal external and skeletal abnormalities induced following in utero thalidomide exposure. A, arrow indicates absent forepaw digit with only small skin tag present. B, encircled region shows absent forepaw digits with only two of the normal five present. C, arrow indicates fusion of sacral centrum to caudal centrum. D, arrow denotes a short ulna. E, arrows point to bent long bones (bilateral femurs and fibula). Area marked with an asterisk is to note that hindpaw was present in animal but removed for image generation.