Investigating the Molecular Mechanisms of Action of Lenalidomide and Other Immunomodulatory Derivatives

Abstract

Background: Thalidomide was first released as a sedative and anti-emetic by a German pharmaceutical company in the 1950s. After its withdrawal from the market due to severe teratogenicity, thalidomide and its novel iMiD analogs were repurposed decades later for a variety of new clinical indications, including the treatment of erythema nodosum leprosum, multiple myeloma, and del(5q)-MDS. Recent work has demonstrated that iMiDs act by modulating the substrate specificity of the CRBN-CRL4 E3 ubiquitin ligase. Selective degradation of IKZF1/IKZF3 and CK1α by this complex in the presence of iMiDs leads to therapeutic activity against multiple myeloma and del(5q)-MDS, respectively. In spite of these advances, our molecular understanding of the pleiotropic effects of iMiDs remains limited. Here, we address two distinct aspects of iMiD biology that warrant further investigation: (1) substrate recognition by the iMiD-CRBN complex via degron motifs and (2) the molecular mechanism of iMiD-induced TNF-α inhibition. Methods: Characterization of iMiD-sensitive degron motifs was performed using a flow cytometry-based fluorescent reporter assay. In brief, HEK293T cells were transfected with GFP-fusion constructs expressing wild-type and mutant degrons of IKZF3 and ZFP91. Substrate degradation in the presence of iMiD treatment was assessed by quantifying GFP reporter expression via flow cytometry. Comparison of a Drug:DMSO GFP ratio between wild-type and mutant degrons was used to determine the effects of single amino acid substitutions on iMiD-induced substrate degradation. The mechanism of TNF-α inhibition by iMiDs was investigated by generating CRISPR/Cas9-mediated single cell knockout clones in the pro-monocytic U937 cell line. Targeted genes were components of the CRBN-CRL4 complex and its associated substrates including CRBN, IKZF1, IKZF3, CSNK1A1, ZFP91, ZNF692, and RNF166. Knockout clones were analyzed by deep sequencing to confirm the presence of inactivating out-of-frame indels. In parallel with non-targeting sgRNA clones, validated knockout clones were treated with PMA to induce macrophagic differentiation, subjected to iMiD treatment, and then stimulated with LPS. TNF-α levels were measured by ELISA to determine any changes resulting from genetic inactivation of candidate targets. Results: Degron mutagenesis studies revealed the presence of critical amino acid residues located in zinc-finger domain 2 of IKZF3 including Q147, C148, G152, A153, and H164. Similarly, amino acids Q401 and G406 in zinc-finger domain 4 were identified as critical residues for the ZFP91 degron. In addition, ZFP91 was validated as a CRBN-CRL4 substrate by Western immunoblotting. Lastly, preliminary TNF-α studies performed to phenotypically characterize CRBN knockout U937 clones suggested a plausible CRBN-dependent mechanism for this iMiD effect, although the results were limited by the presence of clonal heterogeneity. Conclusions: In this study, degron characterization via a fluorescent reporter assay led to the identification of shared amino acid motifs among CRBN-CRL4 substrates that are necessary for conferring sensitivity to iMiD-inducible degradation. Future work employing structural biology approaches would be informative in elucidating the structural determinants of novel zinc-finger substrate recognition by the iMiD-CRBN complex. On the other hand, efforts to dissect the mechanism of iMiD-induced TNF-α inhibition were not fully conclusive. Although the results suggest a plausible CRBN-dependent mechanism, our experimental model warrants further refinement to overcome the limitations encountered in the present study. As an alternative approach, an unbiased proteomics experiment conducted in a monocytic system should be considered to identify novel lineage-specific targets that may provide insight into this elusive mechanism.