First administered in the late 1950s as a mild sedative, thalidomide led to the birth of thousands of children with multiple birth defects. Despite their teratogenicity, thalidomide and closely related immunomodulatory drugs (IMiDs) are now a mainstay of cancer treatment. Thalidomide binds to the Cullin RING E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (CRL4CRBN) and promotes ubiquitination and degradation of key therapeutic targets such as IKZF1/3 and Ck1 in a molecular glue like mechanism. CRL4CRBN was further shown to exert ubiquitin dependent or independent functions on GLUL, CD147-MCT1, MEIS2, and ZFP91, however, none of these targets can account, in full, for the desired therapeutic and undesired adverse effects of IMiDs.
The human genome encodes for over 700 C2H2 zinc-finger (ZnF) proteins, which share high homology with the known targets IKZF1, IKZF3, and ZFP91, and contain thousands of potential thalidomide-CRBN binding ZnF degrons. Many of these ZnF proteins are expressed at low levels or in a lineage specific manner, and are therefore hard to detect by most methods. To interrogate the potential target repertoire of IMiDs, we set out to develop novel mass spectrometry-based proteomics approaches and screen a panel of human cell lines covering multiple lineages.
We will present 1) novel mass spectrometry-based proteomics approaches that allow profiling of 7 individual compounds inducing protein degradation – such as IMiDs – in a single mass spectrometry experiment at a depth of > 10,000 proteins and > 160,000 unique peptides. 2) data on the currently unexplored substrate repertoire of IMiDs across lineages, which includes dozens of novel ZnF proteins being targeted by these drugs, several of which have been linked to human disease. 3) newly identified IMiD-dependent ZnF substrates that share amino acid residues that are critical for the interaction with CRL4CRBN, and IMiD-dependent degradation and will guide future development of novel ZnF targeting analogs.