Degradation of known thalidomide-derivative targets in Crbn^I391Vmice. (A-C) c-Kit⁺ hematopoietic stem and progenitor cells were isolated from Crbn^I391V/I391V or wild-type (WT) mice and treated in vitro with 1 µM lenalidomide, 1 µM pomalidomide, or DMSO vehicle for 12 hours. Protein abundance was assessed by liquid chromatography-mass spectrometry using TMT10 isobaric tagging reagents for quantification. Data are plotted as adjusted P value vs log₂ fold change; the full range of log₂ fold change values is shown as an inset. The horizontal line indicates an adjusted P value of .05 and proteins with a P value < .05 are shown in red. Log₂ fold change is the average of 2 biological replicates. Ikzf3 is not expressed in c-Kit⁺ hematopoietic stem and progenitor cells and was not detected in these experiments. (D) Posttranslational degradation of an IKZF3 reporter in Crbn^I391V/I391V or WT Hoxb8-transformed cells treated with lenalidomide, thalidomide, or pomalidomide in vitro for 6 hours. In this and other reporter constructs, an independently translated mCherry serves as a transcriptional control. (D-E) Posttranslational degradation of a ZFP91 flow reporter in Crbn^I391V/I391V Hoxb8-transformed cells treated in vitro for 12 hours. (F) Degradation of Ikzf3 and CK1α as assessed by western blot in c-Kit⁺ and T cells from Crbn^I391V/I391V,Crbn^I391V/+, or WT mice after 18 hours of in vitro treatment with the drug indicated. All samples on the T-cell blot were run on adjacent lanes on the same gel, but the order of lanes has been changed for clarity. Results are representative of 3 independent experiments. (G) In vivo treatment of Crbn^I391V/I391V mice with lenalidomide or thalidomide results in degradation of Ikzf1 and Ck1α in T cells after 12 hours. (H) T cells from Crbn^I391V/I391V and Crbn^I391V/+ mice have increased production of IL-2 by ELISA when treated with lenalidomide or pomalidomide for 24 hours, but no change in seen in WT cells. For WT cells, P value is not significant for all comparisons with DMSO. For Crbn^I391V/+ and Crbn^I391V/I391V, all comparisons with DMSO have a P value < .05. Statistical significance calculated with an unpaired Student t test and n ≥ 3. Results are representative of 2 independent experiments. Error bars are standard error of the mean. GFP, green fluorescent protein; IB, immunoblot; Len, lenalidomide; mIL, murine interleukin.