Thalidomide, multiple myeloma, Protein-Protein interactions, and drug discovery
Thalidomide is a notorious drug that was approved in Europe in the late 1950s for use as a sedative, but was withdrawn in the early 1960s after the drug caused thousands of devastating birth defects. The FDA did not approve thalidomide at that time. However, beginning in the late 1990s, thalidomide has been repurposed and rehabilitated, provided that proper precautions are maintained to prevent its use in pregnant women and women who may become pregnant.
Currently, thalidomide (under the brand name Thalomide) is marketed by Celgene (Summit, NJ) mainly as a treatment for multiple myeloma (MM) and of a certain form of leprosy. Celgene has also been developing derivatives of thalidomide, the most important of which are lenalidomide (Celgene’s Revlimid) and pomalidomide (Celgene’s Pomalyst). All three agents are now approved in the U.S. and in Europe. Although lenalidomide and pomalidomide are more potent in treating MM and have fewer adverse effects than thalidomide, they are still teratogenic (as determined by animal studies), and are available only in a restricted distribution setting to avoid their use during pregnancy.
Celgene calls thalidomide and its derivatives “immunomodulatory drugs” (IMiDs). Until recently, their mechanism of action was poorly understood. IMiDs were found to have a wide range of activities, including antiangiogenic activity, induction of oxidative stress, upregulation of interleukin-2 (IL-2) production by activated T cells, inhibition of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), and stimulation of natural killer (NK) cells. It is thalidomide’s antiangiogenic activity that appears to be responsible for its teratogenic effects.
However, it was the antiangiogenic activity of thalidomide that gave rise to the hypothesis that this agent might be used to treat MM. MM is a B-cell malignancy that involves the proliferation of abnormal plasma cells, which accumulate in the bone marrow. In MM, the intimate interaction between the plasma cells and bone marrow stromal cells results in induction of the angiogenic factor vascular endothelial growth factor (VEGF) as well as the MM survival factor IL-6. Disruption of this interaction would reduce the induction of new blood vessels and of IL-6, thus decreasing tumor growth and survival. When tested against MM, thalidomide—and later lenalidomide and other IMiDs—were found to be effective in controlling MM, as predicted by the hypothesis.
However, as of 2010, researchers found that although IMiDs are indeed antiangiogenic, that is not the mechanism that explains their therapeutic effect. Now—in 2014—two papers were published in Science that expand upon that earlier effort and identify that pathway by which IMiDs work against MM. These studies were by Krönke et al. and Lu et al. The studies were led, respectively, by Benjamin L. Ebert, M.D., Ph.D. and William G. Kaelin Jr., M.D., both at the Dana-Farber Cancer Institute (Boston, MA). These two papers were accompanied by a brief Perspective by A. Keith Stewart, M.B., CH.B., of the Mayo Clinic (Scottsdale, AZ), in the same issue of Science (17 January, 2014).
The key to understanding the pathway by which lenalidomide (the drug that was used in both of the 2014 research studies) and other IMiDs work against MM is the finding that that they bind to an intracellular protein known as cereblon (CRBN). In a 2010 study, Astellas researchers and their academic collaborators demonstrated that thalidomide binds to zebrafish CRBN. Treatment of zebrafish with CRBN morpholinos or thalidomide caused fin defects, reminiscent of the limb defects seen with thalidomide in the 1960s.
As also demonstrated in the 2010 study, CRBN forms an E3 ubiquitin ligase complex with three other proteins—damaged DNA binding protein 1 (DDB1), Cullin-4A (CUL4A), and regulator of cullins 1 (Roc1). The complex is known as the CRBN-CRL4 ubiquitin ligase.
E3 ubiquitin ligases carry out the terminal step of the ubiquitin pathway—specific attachment of ubiquitin (and via repeated steps, ubiquitin chains) to substrate proteins. Attachment of ubiquitin (and especially of ubiquitin chains) to substrate proteins can tag them for destruction by the proteasome.
Lu et al. and Krönke et al. showed that lenalidomide binding to CRBN results in the selective ubiquitination and proteasomal degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3); they are also known, respectively as Ikaros and Aiolos.
Although IKZF1 is highly expressed in early lymphoid progenitors, studies in mice have shown that IKZF3 is required for the generation of plasma cells, which are the physiologic counterparts of MM cells. Both Krönke et al. and Lu et al. studied the roles of IKZF1 and IKZF3 via RNAi knockdown and other methods. Inhibition of IKZF1 or IKZF3 expression inhibited growth of lenalidomide-sensitive MM cell lines, but lenalidomide-insensitive cell lines were not affected. Downregulation of either IKZF protein in these cell lines led to loss of the other. Downregulation of IKZF1 and IKZF3 resulted in a decrease in interferon regulatory factor 4 (IRF4) and IRF4 mRNA, consistent with IRF4 acting downstream of IKZF1 and/or IKZF3 in lenalidomide-sepsitive MM cells. Previous studies have shown that IRF4 inhibition is toxic for MM cells.
In addition to its effects on MM cells, lenalidomide treatment also upregulates IL-2 expression in T cells. Since IKZF3 binds the IL-2 gene promoter and represses IL-2 transcription in T cells, Lu et al. and Krönke et al. investigated whether lenalidomide’s effects on IL-2 expression in T cells might work via the CRBN-CRL4 ubiquitin ligase-IKZF1/3 pathway. They found that RNAi knockdown of CRBN abrogated the effect of lenalidomide on IL-2 expression. They further found that lenalidomide treatment caused marked decreases in IKZF1 and IKZF3 protein levels In primary human T cells. Finally, they showed that RNAi knockdown of IKZF3 or IKZF1 induced IL-2 expression and repressed further response to lenalidomide. These studies thus show that lenalidomide indeed works via the CRBN-CRL4 ubiquitin ligase-IKZF1/3 pathway to upregulate IL-2 in T cells.
Thus IMiDs, working via the CRBN-CRL4 ubiquitin ligase-IKZF1/3 pathway, downregulate IRF4 in MM cells, resulting in cell death. They also upregulate IL-2 in T cells. A diagram of the pathway is given in Dr. Stewart’s Perspective.
The studies of Krönke et al. and Lu et al. have greatly advanced our understanding of the mechanism of action of IMiDs in MM. As pointed out by Krönke et al., other B cell malignancies against which lenalidomide has activity, such as mantle cell lymphoma and chronic lymphocytic leukemia, also exhibit high IKZF3 expression. Celgene is testing lenalidomide against chronic lymphocytic leukemia and other cancers in the clinic, and the drug is approved for treatment of myelodysplastic syndromes in Europe, in addition to MM. So the recent studies of the CRBN-CRL4 ubiquitin ligase-IKZF1/3 pathway may also apply to other cancers for which lenalidomide is being developed.
Nevertheless, there are still gaps in our understanding of the mechanism of action of IMiDs. For example, the proteasomal inhibitor bortezomib (Millennium’s Velcade) is used to treat MM. Combination therapies of bortezomib and lenalidomide have shown efficacy in early clinical trials, and further trials are continuing. This creates an apparent paradox, because proteasomal blockade prevents the destruction of IKZF1 and IKZF3 by lenalidomide via the CRBN-CRL4 ubiquitin ligase-IKZF1/3 pathway. Lu et al. hypothesize that since proteasomal inhibition by bortezomib is incomplete with therapeutic dosing, this might allow sufficient destruction of IKZF1 and IKZF3 while retaining bortezomib’s other therapeutic effects. Alernatively, they hypothesize that IKZF1 and IKZF2, once polyubiquitylated, may be inactive or act as dominant-negatives.
Implications for drug discovery
The most immediate implications of these findings is that they might be used to discover novel, more effective and safer modulators of the CRBN-CRL4 ubiquitin ligase-IKZF1/3 pathway as therapies for MM and other B cell malignancies. Such efforts might include finding a non-teratogenic modulator of this pathway, since thalidomide-CRBN-mediated teratogenicity may be mediated by substrates other than Ikaros family proteins in different cellular lineages.
Moreover, the 2010 zebrafish study suggested that thalidomide’s teratogenic effects are due to a loss of function of cereblon. In contrast, the 2014 studies in MM indicate that the therapeutic effects of the IMiDs reflect a cereblon gain of function. This supports the possibility of finding non-teratogenic modulators of the CRBN-CRL4 ubiquitin ligase-IKZF1/3 pathway.
The studies of Krönke et al. and Lu et al. may have wider implications for the targeting of E3 ubiquitin ligases in drug discovery for other diseases. We have discussed the possibility of targeting E3 ubiquitin ligases in our 2012 book-length report, Advances in the Discovery of Protein-Protein Interaction Modulators, published by Informa’s Scrip Insights.
The ubiquitin system is a fundamental regulatory system in all eukaryotic cells, comparable in importance to protein phosphorylation. In recent years, researchers have discovered and developed numerous important agents that modulate protein phosphorylation pathways, namely the protein kinase inhibitors. However, there as yet are very few approved and experimental drugs that modulate the ubiquitin system. Most are proteasome inhibitors, which indirectly target this system. The approved agent, bortezomib, has achieved blockbuster status despite its nonspecificity and limited field of application.
Despite the central importance of the ubiquitin system, there are only a handful of compounds that directly target it in clinical trials.
The reason that drug discovery of ubiquitin system-targeting drugs has lagged behind, for example, the discovery and development of protein kinase inhibitors is that modulating the ubiquitin system involves targeting protein-protein interactions (PPIs). Nevertheless, our 2012 report discusses novel technologies and strategies that might be applied to the discovery of PPI modulators.
As discussed in our April 25, 2012 article on this blog, there has been new interest in the discovery of PPIs by leading biotech/pharma companies in recent years, motivated by the development of these technologies and of the increasing strategic importance of PPI modulator development.
As we discussed in our 2012 report, the greatest drug discovery opportunity in the ubiquitin cascade is in targeting E3 ubiquitin ligases. That is because as one moves down the ubiquitinylation cascade, the degree of specificity of the process increases. There are over 600 E3 ubiquitin ligases encoded in the human genome, each of which targets its own specific class of proteins. Moreover, the total number of ubiquitin cascade enzymes encoded by the human genome is greater than the number of protein kinases.
As discussed by Krönke et al., their study (and that of Lu et al.) reveals that the small-molecule drug lenalidomide modulates the activity of the CRBN-CRL4 ubiquitin ligase complex to increase ubiquitination of two transcription factors, IKZF1 and IKZF3. It does so by specific binding to one component of the system, cereblon. This was found serendipitously—not by either classical or advanced technologies for discovering PPI modulators. Moreover, the targets of the CRBN-CRL4 ubiquitin ligase, IKZF1 and IKZF3, are transcription factors that act by forming PPIs. They are also involved in the complex process of chromatin remodeling, and the nature of their interactions are poorly understood. They are therefore considered “undruggable.”
Nevertheless, researchers can screen for compounds that bind cereblon, and which thus modulate the CRBN-CRL4 ubiquitin ligase. Might it also be possible to screen for compounds that modulate one component of other E3 ubiquitin ligases, and thus increase the interactions between these ligases and their specific substrates? If so, this might provide a novel means to discover drugs that modulate the ubiquitin system.
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