CRBN: A key E3 ligase receptor for targeted protein degradation.

• Gene Information: CRBN (Cereblon) is encoded on human chromosome 3 and functions as a substrate receptor within the CRL4 E3 ubiquitin ligase complex. It plays a central role in targeted protein degradation.
• Protein Expression: CRBN is broadly expressed across multiple tissues, including immune cells, hematopoietic tissues, and the central nervous system. Its expression level can influence cellular sensitivity to CRBN-recruiting degraders.
• Signaling Pathway: As part of the CRL4^CRBN complex, CRBN recruits substrate proteins for ubiquitination and proteasomal degradation. Binding of IMiDs or molecular glues can redirect CRBN to degrade neosubstrates such as IKZF1, IKZF3, and GSPT1.
• Model application : CRBN is a key target for IMiDs, molecular glues, and PROTACs that induce selective degradation of disease-associated proteins. CRBN-based degraders are being developed for cancer, autoimmune, and inflammatory diseases.

CRBN

• The CDS of human CRBN gene that encodes the full-length protein was inserted into the mouse Crbn exons 2-3. The B-hCRBN mice will express the human CRBN protein, while mouse Crbn will no longer be expressed.

• Mouse Crbn mRNA was detectable in wild-type C57BL/6 mice. Human CRBN mRNA was detectable only in homozygous B-hCRBN mice but not in wild-type mice.

Strain specific analysis of CRBN mRNA expression in wild-type C57BL/6 mice and homozygous B-hCRBN mice by RT-PCR. Heart, liver, spleen, lung, kidney, stomach, small intestine, colon and cortex RNA was isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hCRBN mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human CRBN primers. Sequencing of the short-form PCR products confirmed that the amplified sequences were consistent with database reference sequences.

• CRBN were detectable in both wild-type mice and homozygous B-hCRBN mice. The anti-CRBN antibody was cross-reactive between human and mouse.

Strain specific CRBN expression analysis in homozygous B-hCRBN mice by Western blot. The cortex, liver, spleen, lung, kidney, stomach, small intestine, colon, heart and ear were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCRBN mice (H/H) and then analyzed by western blot with anti-CRBN antibody (CST, #71810).

• The results show that Lenalidomide significantly up-regulates the production of IL-2 in B-hCRBN mice, but not in wild-type C57BL/6 mice.

Naive CD4+ T cells derived from B-hCRBN mice exhibited increased IL-2 secretion after treatment with Lenalidomide. Naive CD4+ T cells were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCRBN mice (H/H), then stimulated with DMSO or Lenalidomide (MCE, HY-A0003) in vitro for 24 hours. The supernatants were collected, and IL-2 production was measured by ELISA (Biolegend, 431004).

Growth curve of B-hCRBN mice. Three-week-old mice were grouped by sex (10 males and 10 females). Body weight was measured weekly for 13 weeks on the same day each week. The minimum and maximum body weights shown in the table were calculated from the mean ± SD. The growth curve follows a normal distribution, with approximately 68% of values falling within ± SD.

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of B-hCRBN mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of B-hCRBN mice are shown. Values are expressed as mean ± SD.

• No abnormalities were observed.

Organs of female B-hCRBN mice (10-week-old, n = 10).

• No abnormalities were observed.

Organs of male B-hCRBN mice (10-week-old, n = 10).

• No obvious abnormalities were observed in any organs examined (brain, heart, lung, liver, spleen, stomach, small intestine, large intestine, kidney, ovary, uterus ,testis).

Histopathological analysis of organs in B-hCRBN mice. Major organs from B-hCRBN mice were collected at 10 weeks of age and analyzed by H&E staining (male, n = 10; female, n = 10).

In vivo Efficacy of NEK7-molecular glue (by the client) in mouse peritonitis model. B-hCRBN mice were dosed orally, BID, with vehicle or compound, starting 24h prior to stimulation. On the stimulation day, animals received a final dose, followed 1h later by LPS (1mg/kg, i.p.), and nigericin (3mg/kg, i.p.) 3h after LPS. Two hours later, spleens and peritoneal lavage fluid were collected for analysis.

• The NEK7-molecular glue can efficiently induce degradation of the NEK7 and IL-1β proteins in B-hCRBN mice.

Analysis of spleen and peritoneal lavage fluid by WB and ELISA. (A) Spleen tissue lysates were collected and analyzed by western blot with anti-NEK7 antibody. Relative NEK7 protein levels were quantified by densitometric analysis and presented.(B) Peritoneal lavage fluid were collected and mouse IL-1β was analyzed by ELISA. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test.  *P < 0.05, **P < 0.01, ***P < 0.001.

In vivo toxicity of CC-885. (A) B-hCRBN mice and C57BL/6N mice were administered the CC-885 intraperitoneally once daily, mice were euthanized 11 hours after the final dose on the following day, Lymph node, liver and large intestine were collected for analysis.

• The results show that CC-885 exhibits marked toxicity exclusively in B-hCRBN mice, with no detectable toxicity observed in wild-type mice.

In vivo toxicity of CC-885. Lymph node, liver and large intestine were collected for analysis. (A) Percent survival. (B) Body weight and body weight change during the treatment. (C) Histopathological analysis. Scale bar: 100 μm.

In vivo toxicity of CC-885. B-hCRBN mice and C57BL/6N mice were administered the CC-885 intraperitoneally once daily, mice were euthanized 6 hours after the final dose on the following day, skeletal muscle, heart and blood were collected for analysis.

• CC-885 induced degradation of the GSPT1 protein in B-hCRBN mice (G4), but not in wild-type mice.
• CC-885 exhibits hematological toxicity in B-hCRBN mice, but not in wild-type mice.

Skeletal muscle, heart and blood were collected for analysis. Skeletal muscle and heart tissue lysates were collected from all group, then analyzed by western blot with anti-CRBN antibody (CST, #71810), anti-GSPT1 antibody (Abcam, ab234433) and anti-CK1α antibody (Abcam, ab302638). 40 μg total proteins were loaded for western blotting analysis. (A) Western blot analysis of GSPT1 protein expression. (B) Biochemical test and complete blood count (Additional hematological parameters are not shown.)