Inhibition of the CRBN-DDB1-CUL4-ROC1 E3 Ubiquitin Ligase Mediates the Anti-Proliferative and Immunomodulatory Properties of Lenalidomide

Abstract 919

Lenalidomide is a highly effective drug for the treatment of del(5q) MDS and multiple myeloma, and its use in a range of other conditions is being actively explored. Despite its increasing use for the treatment of malignancies, the precise mechanism of action of lenalidomide has not been established. We sought to identify the direct protein targets of lenalidomide using a quantitative, mass spectrometry-based proteomic approach we developed.

Using a validated derivative of lenalidomide immobilized to beads, we identified DDB1 as a target of the drug by affinity enrichment of protein binders and analysis by high performance LC-MS/MS. DDB1, together with CRBN, CUL4A, and ROC1, forms an E3 ubiquitin ligase known as CRBN-CRL4. We confirmed that members of the complex bind to the immobilized lenalidomide derivative, and could be competed off with soluble lenalidomide, further supporting the role of CRBN-CRL4 in the actions of lenalidomide.

CRL4 targets multiple proteins for ubiquitination and subsequent proteasomal degradation, including the cell cycle regulators CDKN1A (p21) and CDKN1B (p27), as well as the DNA licensing factor CDT1. We hypothesized that lenalidomide disrupts the ubiquitination of these and other proteins, leading to increased levels of the respective targets. We found that treatment of the lenalidomide sensitive cell line MM1S and NCI-H929 increased protein levels of p21, p27 and CDT1 in a dose and time dependent manner. Furthermore, overexpression of these three targets led to growth inhibition. Similarly, knockdown of DDB1, CUL4A, ROC1 and CRBN by lentiviral shRNAs increased p21 and p27 protein levels and inhibited growth of these cell lines.

Lenalidomide is also known to increase IL-2, promote erythropoiesis and inhibit TNF-alpha. We found that in activated primary human T cells, shRNA knockdown of DDB1 recapitulated the stimulatory effects of lenalidomide on IL-2 expression levels and release. We also found that shRNA knockdown of DDB1 and CRBN recapitulated the pro-erythropoietic effects of the drug with an increase in the number of colony-forming units-erythroid (CFU-E) compared to control knockdown. Experiments studying the effects on TNF-alpha are underway.

To further establish that the CRBN-CRL4 complex is the target of lenalidomide, we tested a previously published mutant form of CRBN which prevents binding of the drug to the complex. Ectopic expression of this mutant CRBN conferred resistance to lenalidomide induced cell death to multiple myeloma cells. It also resulted in the loss of CFU-E production by lenalidomide.

To gain further insight into how lenalidomide might disrupt the function of the CRBN-CRL4 complex, we did immunoprecipitation against CRBN with or without the drug and found that lenalidomide disrupts the formation of the complex by preventing binding of ROC1, the adaptor protein to ubiquitin charged E2 conjugating enzyme. Using in vivo and in vitro ubiquitination assays, we also demonstrated that lenalidomide inhibits the auto-ubiquitination of CRBN. We are currently performing a ubiquitin profiling experiment to identify other protein targets that might be affected by the disruption of the CRBN-CRL4 complex by lenalidomide.

Our study establishes that lenalidomide's antiproliferative and immunomodulatory properties rely on binding to CRBN-CRL4 and inhibiting its function as ubiquitin ligase. Ito et al. showed 2010 that the same mechanism is also responsible for the teratogenic effects of thalidomide. The characterization of lenalidomide as a specific E3 ubiquitin ligase inhibitor will provide insight into the mechanism of therapeutic efficacy in MDS and multiple myeloma, and serves as a proof-of-concept that selective ubiquitin ligases are efficacious targets for cancer therapy.