Modulation of the CRL4CRBN E3 Ubiquitin Ligase by Thalidomide Analogs

Abstract

Thalidomide and its derivatives, lenalidomide and pomalidomide, are effective therapies for the hematopoietic malignancies multiple myeloma and del(5q) myelodysplastic syndrome. Their therapeutic properties are a result of their ability to induce the ubiquitination and proteasomal degradation of Ikaros (IKZF1), Aiolos (IKZF3), and Casein Kinase 1α (CK1α) by mediating the interaction of these proteins with Cereblon (CRBN), the substrate receptor for the CRL4CRBN E3 ubiquitin ligase. The following thesis details two projects which leverage high-throughput screens to further our understanding of this unique mechanism of action. In chapter 2 we utilize a genome-scale CRISPR-Cas9 screen to identify the cellular machinery which is required for lenalidomide-induced CRL4CRBN function and specifically focus on the cullin neddylation enzymes UBE2M and the COP9 signalosome, and the E2 ubiquitin-conjugating enzymes UBE2D3 and UBE2G1. In chapter 3 we utilize a combination of saturation mutagenesis, crystallography, and motif-based screening to characterize the C2H2 zinc finger degron in IKZF3 and identify novel targets of thalidomide analogs. In aggregate these studies provide evidence that therapeutic modulation of the CRL4CRBN ubiquitin ligase by thalidomide analogs requires multiple factors beyond the ligase itself and that these compounds induce the degradation of a greater than previously appreciated number of proteins which are recognized via C2H2 zinc finger degrons. These findings inform not only our understanding of thalidomide analogs, but also the growing field of targeted protein degradation.