Crbn I391V is sufficient to confer in vivo sensitivity to thalidomide and its derivatives in mice

Abstract

Thalidomide and its derivatives, lenalidomide and pomalidomide, are clinically effective treatments for multiple myeloma and myelodysplastic syndrome with del(5q). These molecules lack activity in murine models, limiting investigation of their therapeutic activity or toxicity in vivo. Here, we report the development of a mouse model that is sensitive to thalidomide derivatives because of a single amino acid change in the direct target of thalidomide derivatives, cereblon (Crbn). In human cells, thalidomide and its analogs bind CRBN and recruit protein targets to the CRL4 E3 ubiquitin ligase, resulting in their ubiquitination and subsequent degradation by the proteasome. We show that mice with a single I391V amino acid change in Crbn exhibit thalidomide-induced degradation of drug targets previously identified in human cells, including Ikaros (Ikzf1), Aiolos (Ikzf3), Zfp91, and casein kinase 1a1 (Ck1α), both in vitro and in vivo. We use the model to demonstrate that the in vivo therapeutic activity of lenalidomide in del(5q) myelodysplastic syndrome can be explained by heterozygous expression of Ck1α in del(5q) cells. We found that lenalidomide acts on hematopoietic stem cells with heterozygous expression of Ck1α and inactivation of causes lenalidomide resistance. We further demonstrate that is sufficient to confer thalidomide-induced fetal loss in mice, capturing a major toxicity of this class of drugs. Further study of the model will provide valuable insights into the in vivo efficacy and toxicity of this class of drugs.

Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome

“…Sequence analysis reveals that mice and zebrafish have critical mutations in the ZnF2 domain of SALL4 ( Figure 5B ), which abrogate binding to hsCRBN in vitro ( Figure 4H ), and render mmSALL4 and drSALL4 insensitive to IMiD-mediated degradation in cells ( Figure 4G,I and Figure 4—figure supplement 1C ). In line with these findings, mice harboring a homozygous CRBN I391V knock-in allele, despite exhibiting degradation of mmIKZF1/3, mmZFP91, and mmCSNK1A1 Fink et al, 2018 , show increased miscarriage upon IMiD treatment compared with control mice; however, they do not exhibit IMiD-induced embryopathies resembling the human phenotype Fink et al, 2018 . We next sought to test whether exchange of the mmSALL4 ZnF2 domain for the hsSALL4 ZnF2 domain would be sufficient to enable mmSALL4 degradation in a human cell line (Kelly cells).…”

Rapid and deep-scale ubiquitylation profiling for biology and translational research

“…2), and in-solution TMT labeling-based methods. Because of these complexities and inefficiencies in using the SILAC quantification approach for ubiquitylation profiling in cells noted above, studies focused on identifying drug-induced substrates of E3 ubiquitin ligases are being carried out with increasing frequency by proteome profiling only rather than ubiquitin profiling 36,37 . The simplicity and speed of our on-antibody labeling approach opens the door to deep-scale ubiquitylation profiling to more directly identify the substrates of E3 ligases degraded by the proteasome degradation pathway.…”

Peptidic degron for IMiD-induced degradation of heterologous proteins

“…A strength or weakness of the IKZF3 degron system, depending upon the application, is that mouse cereblon does not engage IKZF3 when bound to an IMiD. However, a mouse with a humanized cereblon has been developed (21). Two recent studies showed that indisulam redirects an ubiquitin ligase containing the substrate adaptor DCAF15 to degrade RBM39, much as the IMiDs redirect the cereblon ligase to target IKZF1 and IKZF3 for destruction (22,23).…”