Int J Hematol (2016) 104:293–299 DOI 10.1007/s12185-016-2073-4 PROGRESS IN HEMATOLOGY Mechanisms of action of novel drugs in multiple myeloma and those responsible for the acquired resistance Cereblon and its downstream substrates as molecular targets of immunomodulatory drugs Takumi Ito1,2 · Hiroshi Handa1 Received: 15 June 2016 / Revised: 19 July 2016 / Accepted: 19 July 2016 / Published online: 26 July 2016 © The Japanese Society of Hematology 2016 Abstract Thalidomide was first developed as a sedative History of immunomodulatory drugs (IMiDs) around 60 years ago, but exhibited teratogenicity, leading to serious defects such as limb deformities. Nevertheless, Immunomodulatory drugs (IMiDs) are a new class of anti- thalidomide is now recognized as a therapeutic drug for the cancer drugs for which the parent molecule is thalidomide. treatment of Hansen’s disease and myeloma. Immunomod- Thalidomide (Fig. 1) was developed as a sedative in 1950s ulatory drugs (IMiDs), a new class of anti-cancer drug by the German pharmaceutical company Grunenthal. derived from thalidomide, have also been developed and Experiments using rodents initially suggested it to be safe exert potent anti-cancer effects. Although the molecular for use in humans, and the drug was sold over 40 countries, mechanism of thalidomide and IMiDs remained unclear for including Japan. However, as is widely known, thalidomide a long time, cereblon, a substrate receptor of the CRL4 E3 was found to have serious teratogenic effects. Use during ubiquitin ligase was identified as a primary direct target by pregnancy is associated with developmental defects of the a new affinity technique. A growing body of evidence sug- limbs and ears. In the 1960s, thalidomide treatment resulted gests that the effect of IMiDs on myeloma and other can- in thousands of children born with phocomelia or amelia. cer cells is mediated by CRBN. Each IMiD binds to CRBN It is estimated that more than 10,000 children worldwide and alters the substrate specificity of the CRBN E3 ubiq- have been affected by thalidomide teratogenicity [1–7]. uitin ligase complex, resulting in breakdown of intrinsic Due to these serious side effects, thalidomide was downstream proteins such as Ikaros and Aiolos. Here we withdrawn from the market in the early 1960s. However, give an overview of the current understanding of mecha- research on thalidomide continued behind the scenes, and it nism of action of IMiDs via CRBN and prospects for the was found to exert favorable effects against erythema nodo- development of new drugs that degrade protein of interest. sum leprosum (a form of Hansen’s disease), as well as to have anti-inflammatory and anti-angiogenic effects [8–10]. Keywords Cereblon · Thalidomide · IMiDs · Ubiquitin · In 1999 it was also found to exert highly favorable effects Myeloma against multiple myeloma [11], a type of blood cancer, and in 2006 the use of thalidomide was approved by the Food and Drug Administration (FDA) in the United States for the treatment of multiple myeloma. This drug was subse- quently approved in Japan, and has been sold in this coun- try since 2008. However, due to its teratogenicity, the use * Hiroshi Handa of thalidomide is strictly controlled by the System for Tha- hhanda@tokyo‑med.ac.jp lidomide Education and Prescribing Safety (S.T.E.P.S) in 1 Department of Nanoparticle Translational Research, the US and the Thalidomide Education and Risk Manage- Tokyo Medical University, 6‑1‑1, Shinjuku, Shinjuku‑ku, ment System (TERMS) in Japan [12, 13]. Tokyo 160‑8402, Japan Numerous thalidomide derivatives have been developed 2 PRESTO, JST, 4‑1‑8, Honcho, Kawaguchi, and are collectively referred to as IMiDs. Of these, lenalid- Saitama 332‑0012, Japan omide and pomalidomide (Fig. 1) show potent anti-cancer 1 3 294 T. Ito, H. Handa O O NH2 O O O O O O NH NH NH NH N O N O N O N O N O O NH2 NH2 Phthalimide Glutarimide Thalidomide Lenalidomide Pomalidomide CC-122 Fig. 1 Structure of IMiDs. Thalidomide is composed of a glutarim- lidomide lacking a carbonyl group of pthalidmide. Poamlidomide is ide ring and a pthalimide ring. Lenalidomide and pomalidomide are 4-amino analog of thalidomide. Although CC-122 possesses a glutar- very similar to thalidomide. Lenalidomide is 4-amino analog of tha- imide ring, the structure is quite different from other IMiDs activity [3, 14]. The use of these IMiDs has been approved We first covalently bound a thalidomide derivative onto by the FDA for the treatment of multiple myeloma. Lena- the surface of Handa beads (Fig. 2), and performed affin- lidomide (but not pomalidomide) is shown to be specifi- ity purification using these beads and cell lysates of various cally effective for the treatment of myelodysplastic syn- tissues to identify proteins specifically binding to thalido- drome del(5q) (5q syndrome). mide. As described above, thalidomide is known to exert a − variety of effects, which suggest that it binds to multiple protein targets. However, unexpectedly, we identified only Cereblon, a direct target of thalidomide two proteins that specifically bind to thalidomide among the various lysates tested. Mass spectrometry analysis iden- The mechanisms underlying the therapeutic actions and tified these proteins to be damage-specific DNA binding adverse effects of thalidomide remained a mystery for protein 1 (DDB1) and cereblon (CRBN). Binding analyses many years, as the target of thalidomide, i.e., the proteins to using affinity beads and DDB1 and CRBN recombinant which it directly binds to in vivo to exert its pharmacologi- proteins demonstrated that CRBN binds directly to thalid- cal effects, were unknown. omide, whereas DDB1 forms a complex with CRBN, and In previous work, our group developed a technique hence binds indirectly to thalidomide. using magnetic nanoparticles (known as Handa beads) by Cereblon, the direct target of thalidomide, is a 442- which proteins [including target protein(s) of thalidomide] amino acid protein that is evolutionary conserved from that bind specifically to a chemical compound can be affin- plants to humans. However, the cellular function of CRBN ity purified in a single step. The diameter of these beads was largely unknown at the time we identified it as a can- is ~200 nm, which allows to them have numerous linkers didate binding partner of thalidomide. An important clue for ligand immobilization (Fig. 2). Using this technique, we to understanding the function of CRBN was DDB1. DDB1 have been identifying the targets of many drugs and natural has been shown to be an adaptor subunit of the E3 ubiqui- compounds [15], and during this work we became inter- tin ligase complex formed together with Cul4, Roc1, and a ested in identifying the target of thalidomide. substrate receptor [16–18]. This type of E3 ubiquitin ligase Co-polymer of Styrene and Glycidyl methacrylate Poly-Glycidyl methacrylate Magnete (Fe3O4) 200 nm Fig. 2 Handa beads. Electron microscopic image of Handa beads (left). Structure of thalidomide-immobilized Handa beads (right) 1 3 Cereblon and its downstream substrates as molecular targets of immunomodulatory drugs 295 complex is known as Cullin-RING ligase 4 (CRL4). CRBN was shown that the expression change of c-myc, IRF4, and competed with DDB2, another substrate receptor, for bind- p21 by IMiDs are mediated by CRBN. These results sug- ing to DDB1. By demonstrating its similarly to other sub- gest that CRBN is essentially required for the anti-cancer strate receptors, including DDB2, we found that CRBN effects of IMiDs. ubiquitinates itself (autoubiquitination). We suggest that The next issue was the substrate of CRBN, which was CRBN itself functions as a substrate receptor. Furthermore, associated with the anti-cancer effects of IMiDs. Ebert’s we found that thalidomide inhibits the autoubiquitination gourp and Kaelin Jr’s group independently demonstrated of CRBN. We concluded that CRBN is a direct target of that when lenalidomide binds to CRBN, a new function of thalidomide. CRBN is induced, namely, to ubiquitinate and degrade Ika- Next we examined whether CRBN was involved in tera- ros (IKZF1) and Aiolos (IKZF3), which are transcription togenic effects of thalidomide. We chose zebrafish (Danio factors that are known to be responsible for the maturation rerio) as a primary animal model. Zebrafish exhibit rapid of B cells [25, 26]. Concurrently, our group, in collabora- growth and have transparent bodies, so thalidomide defects tion with Celgene, demonstrated that in T cells, the ubiq- are easier to observe and analyze. Thalidomide-treated uitination and degradation of Ikaros and Aiolos induced by embryos showed developmental defects in their pectoral fins IMiDs leads to the enhanced expression of IL-2 [27]. These and otic vesicles (ear), similar to the teratogenic effects seen results demonstrated at the molecular level that IMiDs are in humans. To demonstrate that CRBN is the bona fide tar- excellent anti-cancer drugs, exerting anti-cancer effects in get of thalidomide in its teratogenic activity, we constructed B cell derived cancer cells as well as inducing the activity a thalidomide-binding-deficient mutant (Y384A and of T cells. W386A) of CRBN by genetic engineering techniques. We Subsequently it was found that even when thalidomide is expressed this mutant in embryos in zebrafish and chicks bound to CRBN, the ubiquitination and degradation of Ika- (Gallus gallus), and treated the resulting embryos with ros and Aiolos are weakly induced, resulting in a new issue. thalidomide. When wild-type CRBN was expressed, both In other words, although when IMiDs bind to CRBN, their animals showed developmental defects, however, when the common substrates Ikaros and Aiolos are ubiquitinated and CRBN mutant was expressed, these developmental defects degraded, the question arose as to whether there could be were largely suppressed. We concluded that CRBN is a specific substrates for each IMiD. bona fide target for thalidomide teratogenicity [19].