The Search for Endogenous Lenalidomide in Cell Lines

Abstract

Thalidomide and its derivatives lenalidomide and pomalidomide bind to cereblon (CRBN), the substrate adaptor of the CRL4(CRBN) E3 ubiquitin ligase and induce the recruitment and ubiquitination of specific target proteins, ultimately resulting in their degradation by the proteasome. Recent crystal structures demonstrate that thalidomide derivatives recruit substrates by acting as a “molecular glue,” bridging the protein interfaces of CRBN and recruited substrates including the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3). This mechanism is analogous to that of multiple classes of plant hormones, but endogenous compounds that bind to E3 ligases to recruit substrates have not been described in mammalian cells. We hypothesized that there might exist a similar system in mammalian cells wherein an endogenous small molecule would bind to CRBN to induce the recruitment and ubiquitination of IKZF1 and IKZF3. We sought a cellular context with proteasomal degradation of IKZF3 that was blunted by treatment with glutarimide, a small molecule competitive inhibitor of the CRBN thalidomide-binding pocket. We found that the T-cell acute lymphoblastic leukemia cell line Molt-4 met these criteria. Metabolites extracted from Molt-4 cells induced CRBN-dependent degradation of IKZF3 in reporter cell lines, suggesting activity due to a small molecule rather than a post-translational modification. Several biochemical assays confirmed direct interaction between the extracted metabolites and CRBN. Extracted metabolites induced the recruitment and ubiquitination of IKZF1 in vitro and also induced the degradation of additional recently described thalidomide analog substrates RNF166 and ZNF692. Extraction and purification methods were developed to isolate this activity, dubbed eLen. Finally, an unbiased screen of a cell line library was undertaken to identify additional cellular contexts in which this activity could be identified. We identified eLen production in the renal medullary carcinoma cell line Peds005TSusp, which could be altered by changes in the expression of the tumor suppressor SMARCB1.