In this study, Ito and colleagues from the Tokyo Institute of Technology delineate the mechanism whereby thalidomide causes phocomelia or amelia when administered to pregnant women. They showed that a carboxylic thalidomide derivative conjugated to inert beads precipitated two proteins from extracts of HeLa cells — cereblon (CRBN) and damaged DNA binding protein-1 (DDB1). To show specificity of the interactions, they demonstrated that CRBN binding was inhibited by free thalidomide and that recombinant CRBN could bind to the thalidomide beads. They then demonstrated that CRBN serves as a substrate receptor of E3 ubiquitin ligase cullin-4 (Cul4A) complexes containing DDB1, suggesting that DDB1 binds to thalidomide through its interaction with CRBN. This complex has E3 ubiquitin ligase activity, evidenced by auto-ubiquitination in a Cul4A- and DDB1-dependent fashion. Importantly, this activity was inhibited by thalidomide in wild-type cells, but not in cells with mutated CRBN binding sites that prevent thalidomide binding. These findings were confirmed in vivo in both zebrafish and chicks, since binding of thalidomide to CRBN homologous genes during embryogenesis also blocked ligase activity and conferred teratogenic phenotypes, again not observed when thalidomide binding sites were mutated. Downstream targets implicated in mediating these processes include fibroblast growth factors (fgf) 8 and 10, which are known to be responsible for limb outgrowth and which were reduced by thalidomide treatment. Conversely, overexpression of CRBN restored expression of fgf8 and 10 and abrogated the teratogenic effects. Most importantly, these investigators mapped the thalidomide binding site to a highly conserved C-terminal 104 amino acid region in CRBN.
Thalidomide was first used empirically to treat multiple myeloma (MM) based on its anti-angiogenic effects and has remarkable activity even in patients with advanced refractory disease. It is FDA-approved for initial treatment of MM and has also prolonged progression-free survival when used as a maintenance therapy. In addition to anti-angiogenic activity, preclinical studies have shown that thalidomide has modest direct MM cell cytotoxocity, abrogates binding of MM cells to bone marrow, inhibits constitutive and MM cell binding-induced transcription and secretion of cytokines, and augments host cytolytic T and NK cells with anti-MM activity. Which of these activities is most important in terms of mediating clinical benefit remains unknown. Nonetheless, next generation immunomodulatory drugs lenalidamide and pomalidamide more potently mediate these effects and have remarkable clinical activity even in thalidomiderefractory MM. However, each of these agents also causes teratogenicity in preclinical models, and therefore elaborate systems have been set up to assure that pregnant women do not receive these immunomodulatory drugs.
In Brief
This study is a major advance with important implications for clinical use of this class of immunomodulatory drugs. First, understanding thalidomide targets will allow for further characterization of its effects during embryogenesis. Second, it will facilitate delineation of the mechanisms of anti-tumor activity of thalidomide and, specifically, whether binding to CRBN is required for the aforementioned anti-MM cellular effects. If anti-tumor activity is preserved even in CRBN mutants that do not bind thalidomide, then the opportunity for synthesis of novel derivatives lacking CRBN binding for preclinical testing and eventual clinical application represents a major potential therapeutic advance. Already second-generation immunomodulatory drugs lenalidomide and pomalidamide have retained potent anti-MM clinical activity but lack the somnolence, neuropathy, and constipation attendant to thalidomide use, suggesting that altering structure to maintain potency but favorably impact adverse effects is possible. The current study, therefore, may lay the groundwork for synthesis of potent immunomodulatory drugs that lack CRBN binding and could have major implications for expansion of the spectrum of diagnoses and patients eligible for treatment with these agents.
Competing Interests
Dr. Anderson has served on the advisory board for Celgene.
