YBLRE-00387; No of Pages 6 Blood Reviews xxx (2015) xxx–xxx Contents lists available at ScienceDirect Blood Reviews journal homepage: www.elsevier.com/locate/blre REVIEW Cereblon binding molecules in multiple myeloma K.M. Kortüm, Y.X. Zhu, C.X. Shi, P. Jedlowski, A.K. Stewart ⁎ Mayo Clinic in AZ, Department of Hematology, USA a r t i c l e i n f o Available online xxxx Keywords: Multiple myeloma Cereblon binding molecules IMiDs Cereblon Ikaros Aiolos Lenalidomide Thalidomide Pomalidomide a b s t r a c t Immunomodulation is an established treatment strategy in multiple myeloma with thalidomide and its derivatives lenalidomide and pomalidomide as its FDA approved representatives. Just recently the method of action of these cereblon binding molecules was deciphered and results from large phase 3 trials confirmed the backbone function of this drug family in various combination therapies. This review details the to-date knowledge concerning mechanism of IMiD action, clinical applications and plausible escape mechanisms in which cells may become resistant/refractory to cereblon binding molecule based treatment. Published by Elsevier Ltd. 1. Introduction 2. Cereblon in IMiD action A decade ago the FDA approval of the immunomodulator thalidomide marked a breakthrough in the treatment of multiple myeloma (MM), a malignant plasma cell dyscrasia, which had faced decades of stagnation since Melphalan therapy was introduced in the 1960s. Thalidomide was initially approved and prescribed as a sedative for pregnancy related nausea in the early 1950s. Its thitherto unknown teratogenic effects caused death and severe limb malformation in several thousand children before it was retracted from the market. In 1999 the first reports about anti-MM activity of single agent thalidomide were published [1], and the era of “novel” agents in MM therapy began. Derivatives of thalidomide were developed, aiming for higher potency and fewer side effects and FDA approval was granted to lenalidomide in 2006 and pomalidomide in 2013. Today, immunomodulatory agents are the backbone for various treatment regimens in multiple myeloma. They are used in younger and elderly patients, in upfront, relapse and maintenance therapy and significantly contributed to remarkable survival extension and to long-term disease control/even cure that can be achieved today in a subset of patients. This review will focus on thalidomide and its derivatives lenalidomide and pomalidomide; their role in the treatment of multiple myeloma and their method of action that recently has just been revealed. The mechanisms that associate with anti-myeloma activity of immunomodulators (IMiDs) remained unknown until a seminal breakthrough was made in 2010 from Dr. Ito's group in the study of thalidomide mediated teratogenicity [2]. In this study, THAL was found to bind to the protein called cereblon. Cereblon is a protein that in humans is encoded by the CRBN gene. Mutations in the CRBN gene are associated with autosomal recessive nonsyndromic mental retardation possibly as a result of dysregulation of calcium-activated potassium channels in the brain. Cereblon forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), cullin-4A (CUL4A), and regulator of cullins 1 (ROC1). Cereblon is speculated to bind substrate proteins in this complex, which together ubiquitinate substrates, targeting them for proteolysis. We and other groups demonstrated that cereblon is required for anti-MM activity of IMiDs [3–5]. Low CRBN expression was found to correlate with IMiD drug resistance in MM cell lines and primary MM cells. Clinical correlative studies have observed a positive association between CRBN and response to THAL maintenance and upfront lenalidomide (LEN) and dexamethasone (DEX) therapy [6,7]. Our recent study further indicated that CRBN expression is highly predictive of response and survival outcomes following pomalidomide (POM) therapy [8]. Furthermore, expression of cereblon protein assessed by immunohistochemical staining in myeloma cells is associated with superior response of THAL- and LEN-based treatment [9]. The downstream signaling of CRBN in IMiDs mediated-myeloma cytotoxicity was demonstrated to associate with downregulation of IRF4 and MYC. To identify the intermediaries and facilitators of cell ⁎ Corresponding author at: Mayo Clinic in Arizona, Vasek and Anna Maria Polak Professorship in Cancer Research, Consultant, Division of Hematology/Oncology, USA. Tel.: +1 480 301 8335; fax: +1 480 301 8387. E-mail address: Stewart.Keith@mayo.edu (A.K. Stewart). http://dx.doi.org/10.1016/j.blre.2015.03.003 0268-960X/Published by Elsevier Ltd. Please cite this article as: Kortüm KM, et al, Cereblon binding molecules in multiple myeloma, Blood Rev (2015), http://dx.doi.org/10.1016/ j.blre.2015.03.003 2 K.M. Kortüm et al. / Blood Reviews xxx (2015) xxx–xxx death following CRBN and IMiD interaction that ultimately induce IRF4 and MYC downregulation, we and other groups used various approaches to identify CRBN binding proteins that are relevant to MM cytotoxicity. Two independent research groups recently reported that, by binding to CRBN, LEN potentiates the ubiquitination and proteolysis of 2 specific proteins, IKZF1 and IKZF3 [9,10]. IKZF1 and IKZF3 are important transcription factors for B cell differentiation. Knockdown of IKZF1 and IKZF3 in myeloma cells induced myeloma cell cytotoxicity and downregulation of IRF4. A single amino acid substitution of IKZF3 conferred resistance to LEN-induced degradation and rescued LENinduced inhibition of cell growth, suggesting that repression of these transcription factors is a likely mechanism for LEN activity in this disease. The same group also demonstrated that LEN induced-IL2 production in T cells is due to CRBN mediated depletion of IKZF3, suggesting that IKZF1 and IKZF3 depletion may also contribute to indirect anti-myeloma effects of IMiDs (Fig. 1). Our recent study identified that numerous novel CRBN binding proteins altered in MM cells after IMiD treatment and confirmed recent work implicating the Ikaros transcription factors as important members of this group [11]. We also demonstrated that expression of IKZF1 correlates with significant differences in response and overall survival of IMiD therapy. In addition to Ikaros family members, our mass spectrometry data identified other interesting proteins, which may bind the CRBN complex and are downregulated by IMiDs [11]. Several binding partners involved in metabolism, nuclear transport and protein folding such as KPNA2, NUP153 and DNAJA2, support other changes mediated by multiple proteins in the CRBN complex (Table 1 list 10 proteins identified from CRBN complex by two different methods in our study). IMiDs have a broad variety of clinically usable actions, including significant single agent activity in MM in all three drugs [12]. Most prominently IMiDs affect the immune system, which led to their denomination as “immunomodulators”. However, in the light of the recent discoveries, the term “cereblon binding molecules (CBMs)” seems to be more accurate. Table 1 Cereblon binds a multitude of proteins. We used an anti-CRBN antibody to immunoprecipitate CRBN and its associated proteins as well as Ni charged (Ni1) beads and identified 10 CRBN-associated proteins shared between the chosen methods. CRBN complex associated proteins identified by co-IP and pull-down assays (Zhu et al.) CUL4A DDB1 IKZF1 KPNA2 LTF PFKL PRKAR2A CRBN IKZF3 RANGAP1 SHMT2 human proteins, most likely explaining lack of rodent responsiveness to IMiDs [13]. THAL and it's FDA approved analogues LEN and POM share a common phthalimide and a glutarimide ring and differ in not more than an additional amino or a carbonyl group (Fig. 2). However these minor structural alterations lead to significant differences in clinical behavior, with increased potency from THAL to its derivatives [16,17]. In a luciferase based experiment POM exhibited increased levels of IKZF1 knockdown compared to LEN and LEN increased levels compared to THAL [14]. This corresponds well with the clinically observed increased potency and specificity in the THAL analogues. Of interest differences in the CRBN binding affinity between the drugs were rather low, not explaining the differences in cellular potency, however differences in functional groups at the phthaloyl C4, C5 and C6 positions were demonstrated to alter IKZF1 degradation capacity [14]. 3. Chemical structure 4. Clinical properties Recently, the crystal structure of DDB1-CRBN E3 ubiquitin ligase in complex with THAL/LEN has been described [13,14]. CBMs bind at the C terminus of CRBN in a small hydrophobic pocket composed of three tryptophan residues (Trp380, Trp386 and Trp400) [13] and mutations in these residues have been demonstrated to cause CBM resistance [2, 10,15]. This thalidomide binding domain (TBD) is highly conserved across species, however four amino acids differ between mouse and 4.1. Angiogenesis Multiple myeloma is characterized by an increased bone marrow angiogenesis [18], based on various factors, most prominently by increased Vascular Endothelial Growth Factor (VEGF) and BasicFibroblast Growth Factor-2 (bFGF) levels, but also mediated by Angiopoietins, Metalloproteases, Osteopontin, Hepatocyte Growth Fig. 1. The CBM action is mediated via CRBN. IMiDs bind to CRBN and cause proteasomal degradation of IKZF1 and IKZF3, consecutively downregulating IRF4 and Myc resulting in MM associated cytotoxicity. Fig. 2. Chemical structure of CBMs. All three drugs share a phthalimide and a glutarimide ring and differ only in a carboxyl and amino group. Please cite this article as: Kortüm KM, et al, Cereblon binding molecules in multiple myeloma, Blood Rev (2015), http://dx.doi.org/10.1016/ j.blre.2015.03.003 K.M. Kortüm et al. / Blood Reviews xxx (2015) xxx–xxx Factor (HGF), Syndecan-1, Heparanase, Interleukin 6 and Interleukin 8 [18]. The known anti-angiogenic properties of THAL provided the rationale to investigate its efficacy in MM. In fact THAL is the most potent angiogenesis inhibitor in of all CBMs and was shown to abrogate the small vessel building in the bone marrow [19]. However, it is not clear whether this angiogenesis inhibition contributes to the overall tumor effect of CBMs in MM. 4.2. Anti-inflammation CBMs provide anti-inflammatory properties by decreasing TNF alpha levels from LPS stimulated monocytes (derivatives exceed the THAL capacity by more than 50,000 fold) [20,21]. Of note, TNF-alpha secretion is induced from MM cells, however quantities are low and this paradoxical effect is by far compensated by the decreased production in the bone marrow environment [22]. Furthermore the proinflammatory factors IL1, IL6 and IL12 and Cox 2 are blocked and levels of antiinflammatory cytokines, including IL10 [20,23,24] are increased. These properties can be utilized in the treatment of leprosy [25], Morbus Castleman [26], a lymphoproliferative disease mainly driven by IL6 hypersecretion, in autoimmune diseases, including rheumatoid arthritis [27], lupus erythematodes [28] and Behcet's disease [29]. 4.3. Bone marrow environment and cell–cell contacts Multiple myeloma cells are dependent on the bone marrow environment [30] and cell adhesion-mediated drug resistance (CAMDR) can be established by various tumor bone marrow interactions, including activation of Wnt/b-catenin signaling [31] and upregulated CD44 [32]. CBMs can alter this cross talk between MM and bone marrow stroma cells (BMSCs) [33,34], by downregulation of various cell surface adhesion molecules [35–43] leading to reduced cell–cell interactions and altered cell migration [44]. CBMs decrease the osteoclast (OC) growth and survival factor MIP-1α [45] and inhibit OC activation by BAFF and APRIL inhibition [33]. Furthermore CBMs reduce MM induced bone disease by blocking osteoclastogenesis by the downregulation of PU.1 that consecutively completely abrogates Receptor Activator of Nuclear factor kappa-B Ligand (RANKL), which is a central regulator of bone remodeling [46,47]. 4.4. Cell proliferation CBM based degradation of IKZF1 and IKZF3 in myeloma cells induce direct myeloma cell cytotoxicity by downregulation of IRF4 and MYC [10,11]. Furthermore CBMs anti-proliferative properties are based on the ability to increase expression of tumor suppressor genes, including Egr1, 2 and 3, p15, p16, p21 and p27, leading to cell cycle arrest by cyclin-dependent kinases 2, 4, and 6 inhibition [48–50]. Extrinsic and intrinsic apoptosis is induced by the activation of caspases 3, 8 and 9 [51–54], further enhanced by the decrease of anti-apoptotic proteins, including cellular inhibitor of apoptosis protein 2 (cIAP2), FLICE inhibitor protein (FLIP) [55,56] and critical MM growth and survival factors, including IL-6, VEGF and IGF-1 [38,57,58]. 4.5. Immunomodulatory effects In MM the immune system harbors humoral and cellular immune defects including T-cell, macrophage, natural killer (NK) cells and dendritic cell abnormalities [59–63]. CBMs are able to recuperate disturbed immune function [64–66]. Barely functional antigen-presenting dendritic cells (DCs), frequent in MM [67–69], regain antigen presentation capacity and additional DCs are generated from mononuclear cells [70]. Innate immune function is improved by increasing the number of NK cells [71] and their cell-mediated and monocyte-mediated antibody-dependent cellular cytotoxicity [72–74]. Impaired T-cell function is reversed by CBMs including cytoskeletal 3 reorganization of the immunological synapse [75–77], T-cell activation [78], by activating cytokine production and the degradation of the negative IL2 regulators Ikaros and Aiolos [10,11,79]. Enhanced activation of the transcription factor AP-1 further contributes to increased production of IL-2 from activated CD4+ and CD8+ T-cells, IFN gamma from TH1 cells, and IL5 and IL10 from TH2 cells [20,80,81]. Furthermore, CD28, a major costimulatory molecule on T cells, is activated and its counterpart, cytotoxic T-lymphocyte antigen ((CTLA)-4 or CD 152) is downregulated, thus reducing effects on T-cells are diminished [82]. Increased levels of IL2 from CD4+ and CD8+ peripheral T cells furthermore improve lytic capacities of cytotoxic T lymphocytes [71] and reduce suppressive effects of regulatory T cells (T-regs) [74,83]. T-regs are important regulators of the immune surveillance and their dysfunction leads to disturbed self tolerance including autoimmune diseases and cancer [84]. T-regs are present in increased quantity in MM [85–87], however, controversy exists in the literature about the number and functionality of T-regs in MM patients. In CLL a decrease in number of T-regs by THAL was reported [88] and after allogeneic transplantation CBM induced increase of CD4+ Foxp3+ T-regs in MM patients [89] and increased CD8 T-reg cells were counted after LEN-DEX treatment [90]. Of interest, immune reaction and subsequent tumor cell rejection was observed in melanoma due to the depletion of regulatory T-cells, which had previously evaded immune surveillance [91]. CBMs are able to inhibit T-reg proliferation and its immune suppressor-function [83,92], however other regulatory cells, such as myeloid-derived immune suppressor cells (MDSCs) also contribute to tumor cell immune evasion. These cells are not necessarily susceptible to CBM treatment; reports show alteration in cytokines, but no change in frequency or in their immune suppressive function [93]. T-regs are an intriguing target for cancer treatment and could be a pivotal in the development of cancer immunotherapy using CBMs, however further investigation is needed. 5. CBM treatment in MM CBMs provide the backbone of many regimens in the treatment of MM, including newly diagnosed and relapsed patients, young and elderly; it is given before and after transplant as well as a single agent or in combination with others. In recent years the use of THAL has declined in favor of its derivatives, due to their higher potency and more favorable side effect profile. However a combination of Melphalan THAL and Prednisolone is still considered standard of care for elderly patients in many countries outside the Unites States. CBMs clinically show synergistic effects with multiple compounds, including the first and second-generation proteasome inhibitors Bortezomib [51,94,95] and Carfilzomib [96–99]. This clinical observation can hardly be explained if the CBM anti-MM function is based on the proteasomal degradation of IKZF, which is reversibly (Bortezomib) or irreversibly (Carfilzomib) blocked in these combinations [100]. However, unpublished data from our lab suggest a therapeutic window in which both drugs work. Differences in the administration schedule might also allow dual action, if the proteasome inhibition is not complete or does not persist over the whole treatment cycle. Alternatively, polyubiquitination of IKZF1/3 alone might sufficiently inactivate IKZF1/3 function, thus enabling proteasomal independent CBM anti-MM properties. 6. Side effect profile Common side effects of CBMs include sedation, teratogenic effects and the risk of venous thromboembolism (VTE), especially when used in combination with anthracyclines and DEX [101]. Additionally an increase of secondary malignancies has been observed in patients treated with prolonged CBM therapy in combination with additional DNAdamaging agents e.g. Melphalan. The underlying mechanism behind these secondary malignancies has not yet been revealed, however, it is generally agreed that the increased risk is comparably low and is Please cite this article as: Kortüm KM, et al, Cereblon binding molecules in multiple myeloma, Blood Rev (2015), http://dx.doi.org/10.1016/ j.blre.2015.03.003 4 K.M. Kortüm et al. / Blood Reviews xxx (2015) xxx–xxx counterbalanced by the survival benefits gained by the CBM treatment [102]. One possibility is that the CBM depletion of tumor suppressor Ikaros and perhaps other proteins provides a secondary signal in malignancy development. The side effect profile of CBMs has generally improved from the introduction of THAL through its more recent derivatives e.g. irreversible peripheral neuropathy is dose dependently caused by THAL, but not by LEN and POM. Additionally severity of constipation and the grade of sedation are decreased among LEN and POM. The teratogeneic effects, however, are mediated by CRBN, as are the MM cytotoxic effects, consequently present in THAL and all its derivatives. In order to be non-teratogeneic, a CBM would need to target downstream of CRBN or use alternative pathways. 7. CBM resistance Resistance to single agent CBMs in MM is critical; around 30%–50% of patients are unresponsive to single agent CBM treatment [103,104] and almost all patients gain drug resistance over prolonged time of treatment. The mechanisms are only partly understood and may vary between the single drugs. OCI-MY5 is a CBM resistant cell line due to low CRBN expression levels. It regains sensitivity to CBMs when wild type CRBN is introduced [11]. Similarly, MM patients with low CRBN levels do not respond to POM and DEX based treatment [8], low IKZF1 levels correlate with decreased overall survival [11] and mutant IKZF3 can confer resistance to LEN by preventing LEN-induced degradation of IKZF3 [10]. Of note, CBM resistant cells may have normal CRBN or IKZF levels, suggesting alternative ways to circumvent IKZF breakdown or upregulate IKZF proteins in response to CBMs [105]. Further pathway related mechanisms of CBM resistance include the upregulation of WNT [31] or may be located downstream of CRBN e.g. in FR4, a CBM resistant cell line, a translocation in the IRF4 gene leads to upregulated levels of IRF4 [11] preventing CBM-induced MM cytotoxicity. CRBN mutations have been reported in a CBM resistant MM patient [3], however, early this year 2 large sequencing trials were published that did not show high frequency of mutations in CRBN or CRBN pathway related genes [106–108]. However, these investigations were done on highly diverse populations, lacking a significant number of CBM resistant patients. Therefore more patients of specifically CBM resistant cohorts should be sequenced to better estimate the impact of CRBN pathway related mutations as a cause of CBM resistance. Of interest, a subgroup of patients resistant to THAL may respond to LEN or POM treatment and patients refractory to LEN may respond to POM [109–114]. These observations, however, can be at least partially attributed to differences in potency on CRBN mediated cytotoxicity, however it is very likely that additional factors exist further differentiating their anti-MM properties [105] and resistance mechanisms. More investigation is needed. 8. Summary The era of novel agents revolutionized MM treatment in the recent years and CBMs have contributed significantly to improve response rates and extend survival times in MM therapy. It is to hope that the recent revelation of the CBM action and the recent description of the crystal structure of CRBN will promote further advances including better understanding of drug resistance mechanisms and the development of more potent and less toxic, in particular non-teratogeneic IMiD like drugs that are safe for long-term treatment. 9. Practice points • Immunomodulation is an efficient treatment strategy in multiple myeloma. • Three cereblon binding molecules (thalidomide, lenalidomide and pomalidomide) are currently FDA approved immunomodulating agents for the treatment of multiple myeloma. • Their common method of anti-MM action is the degradation of the transcription factors Ikaros and Aiolos that leads to the downregulation of IRF4 and Myc. • All available IMiDs share teratogeneic side effects. 10. Research agenda • To decipher the paradox wherein proteasome inhibitors clinically synergize with CBM therapy despite a working proteasome being required for CBM action. • To further elucidate resistance mechanisms and establish a predictive test for CBM sensitivity. • To develop non-teratogeneic IMiDs. Conflict of interest statement KMK, YXZ, CXS, and PJ have nothing to disclose. AKS: consultation for Celgene, Novartis, Array BioPharma, BMS and Sanofi Aventis. 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