Characterization Of RING Finger E3-Ubiquitin Ligase Cereblon In Hematopoietic Regulation

Cereblon is a RING-domain E3 ubiquitin ligase (UbL) that is the direct protein target for thalidomide and lenalidomide. Inhibition of this molecule mediates antiproliferative activity in myeloma cells and in activated B-cell-like subtype diffuse large B-cell lymphoma. In addition to its anticancer properties, lenalidomide potentiates T-cell effector function through the engagement of the CD28-mediated co-stimulatory pathway by a CRBL-dependent mechanism that is poorly characterized. Human and animal studies show CRBL to be ubiquitously expressed in the hematopoietic compartment and in neurons, where a nonsense mutation causes a mild autosomal recessive non-syndromic intellectual disability. Ubiquitin ligase activity underlies suppression of growth factor signaling and maintains homeostasis within the hematopoietic compartment. C-Cbl, cbl-b, GRAIL, and ITCH represent four partially overlapping E3-UbL regulators of cytokine signaling and hematopoietic regulation. To understand the role of CRBL in hematopoetic development and function, we studied a CRBL-deficient mouse strain. All mice used in this study were bred and maintained under specific pathogen-free conditions according to institutional guidelines. The strategy used to create germline crbn deletion (crbl-/-) was described previously, although these mice were not investigated for aberrations in hematopoietic development. First, crbl-/- mice were viable, fertile and normal in appearance without limb malformations. Consistent with a role for CRBL in the hematopoietic development, crbl-/- mice exhibited marked changes in their hematopoietic profiles, including lymphoid hyperplasia, a markedly expanded peripheral white blood cell count and neutrophil expansion. In the T-cell compartment, splenic lymphocytes numbers were increased with demargination of the T-cells into B-cell zones by immunohistochemical staining. Double-negative thymocytes ratio were altered with an accumulation of DN1 and DN3 cells and lower DN4. In contrast, differentiated cell lineages such as mature T-cells, including CD4 and CD8 single-positive (SP) thymocytes, SP peripheral T-cells and naïve and memory T-cells were similar in number to wild-type littermates. The mature T-cells, similar to Cbl-b KO mice showed superior proliferation and IL-2 production in the absence of CD28 co-ligation. Collectively, these histological changes are indicative of a role for crbl in negatively regulating cytokine and receptor signaling events that control cell proliferation or survival. Interestingly, this phenotype overlaps partially with c-cbl and cbl-b KO mice suggesting that they could be coordinately regulating similar pathways. In summary, our findings demonstrate a novel role for crbl, the molecular target of thalidomide and lenalidomide in hematopoiesis and suggest that this molecule may act by an unknown mechanism in concert with other E3 UbLs involved in cytokine receptor signaling. This study provides the first characterization of crbl genetic deficiency in a murine model and demonstrates a unique regulatory relationship between CRBL and other known E3 UbLs.

McDaniel:Celgene: Employment.