Project Summary Developed in the 1950s, Thalidomide was used to treat morning sickness in the early stages of pregnancy, which led to the birth of thousands of children with severe birth defects and subsequent withdrawal from the market. Today, thalidomide and its analogs lenalidomide and pomalidomide (collectively known as IMiDs) are FDA approved drugs and effective treatments for hematologic malignancies such as multiple myeloma and Del(5q) MDS. But despite over 60 years of research, the mechanistic understanding of how these molecules cause the characteristic birth defects is only just beginning to unravel. Due to the wide use of IMiDs in the clinic, and the recent explosion in the development of targeted protein degradation therapies based often on the original thalidomide scaffold, it is more critical than ever that we understand the full range of potential activities that IMiDs provoke. Here, we propose to explore the broad range of potential activities that result from these IMiD-like molecules binding to the E3 ligase CUL4-RBX1-DDB1-CRBN (CRL4CRBN) and redirecting its activity towards different targets. Specifically, in aim 1, we will engineer an in vivo mouse model of Sall4 degradation to dissect how thalidomide-mediated degradation of Sall4 leads to teratogenicity. We will do this through a combination of expression analyses and developmental measurements to assess the affect that IMiD molecules have on the developing embryo. This will not only enable the study of the mechanism of teratogenic activity of these drugs but will also provide a critical model system for testing the toxicity of current and future versions of these molecules. Not only do these molecules induce a gain-of-function to target new proteins, but they also induce a loss-of-function and in aim 2, we propose to use novel mass spectrometry-based proteomics technology in combination with potent and selective tool compounds to explore the biological consequences of blocking CRBN activity. We have previously shown that we can expand the scope of IMiD targets through rational design of new IMiD-like molecules capable of degrading new proteins related to known targets. In aim 3, we propose to expand the target scope even further by developing novel chemistry to target structurally diverse proteins that have a strong therapeutic rationale, thus providing proof-of-concept for target expansion and providing chemical leads for new therapeutic targets. The overarching goal of this grant is to explore the full range of potential activities of the widely utilized CRL4CRBN ligase by identifying and exploring the mechanistic consequences of degrading its physiological and IMiD-induced targets, as well as seeking to expand its target space into currently undruggable territories.