Maria Wang

Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs

The Cul4–Rbx1–DDB1–Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degradation. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are critical for antiproliferative effects.

Lenalidomide efficacy in activated B‐cell‐like subtype diffuse large B‐cell lymphoma is dependent upon IRF4 and cereblon expression

Durable responses with lenalidomide monotherapy have been reported in patients with non‐Hodgkin lymphoma. In relapsed/refractory diffuse large B‐cell lymphoma (DLBCL), higher responses were observed in the activated B‐cell‐like (ABC) subtype than in the germinal centre B‐cell‐like subtype. Herein, the molecular mechanisms involved in the differential efficacy of lenalidomide in DLBCL subtypes were investigated. Using DLBCL cell lines, lenalidomide treatment was found to preferentially suppress proliferation of ABC‐DLBCL cells in vitro and delay tumour growth in a human tumour xenograft model, with minimal effect on non‐ABC‐DLBCL cells. This tumouricidal effect was associated with downregulation of interferon regulatory factor 4 (IRF4), a hallmark of ABC‐DLBCL cells. IRF4 inhibition by lenalidomide induced downregulation of B‐cell receptor (BCR)‐dependent NF‐κB. Whereas IRF4‐specific small, interfering RNA mimicked the effects of lenalidomide reducing NF‐κB activation, IRF4 overexpression enhanced NF‐κB activation and conferred resistance to lenalidomide. These findings indicate the crucial role of IRF4 inhibition in lenalidomide efficacy in ABC cells. Furthermore, lenalidomide‐induced IRF4 downregulation required the expression of cereblon, a molecular target of lenalidomide. Taken together, these findings suggest that lenalidomide has direct antitumour activity against DLBCL cells, preferentially ABC‐DLBCL cells, by blocking IRF4 expression and the BCR‐NF‐κB signalling pathway in a cereblon‐dependent manner.

Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity.

Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblons central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.

CC-122, a pleiotropic pathway modifier, mimics an interferon response and has antitumor activity in DLBCL

Cereblon (CRBN), a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex, is the target of the immunomodulatory drugs lenalidomide and pomalidomide. Recently, it was demonstrated that binding of these drugs to CRBN promotes the ubiquitination and subsequent degradation of 2 common substrates, transcription factors Aiolos and Ikaros. Here we report that CC-122, a new chemical entity termed pleiotropic pathway modifier, binds CRBN and promotes degradation of Aiolos and Ikaros in diffuse large B-cell lymphoma (DLBCL) and T cells in vitro, in vivo, and in patients, resulting in both cell autonomous as well as immunostimulatory effects. In DLBCL cell lines, CC-122-induced degradation or short hairpin RNA-mediated knockdown of Aiolos and Ikaros correlates with increased transcription of interferon (IFN)-stimulated genes independent of IFN-α, -β, and -γ production and/or secretion and results in apoptosis in both activated B-cell (ABC) and germinal center B-cell DLBCL cell lines. Our results provide mechanistic insight into the cell-of-origin independent antilymphoma activity of CC-122, in contrast to the ABC subtype selective activity of lenalidomide.

Clinical and pharmacodynamic analysis of pomalidomide dosing strategies in myeloma: impact of immune activation and cereblon targets

In preclinical studies, pomalidomide mediated both direct antitumor effects and immune activation by binding cereblon. However, the impact of drug-induced immune activation and cereblon/ikaros in antitumor effects of pomalidomide in vivo is unknown. Here we evaluated the clinical and pharmacodynamic effects of continuous or intermittent dosing strategies of pomalidomide/dexamethasone in lenalidomide-refractory myeloma in a randomized trial. Intermittent dosing led to greater tumor reduction at the cost of more frequent adverse events. Both cohorts experienced similar event-free and overall survival. Both regimens led to a distinct pattern but similar degree of mid-cycle immune activation, manifested as increased expression of cytokines and lytic genes in T and natural killer (NK) cells. Pomalidomide induced poly-functional T-cell activation, with increased proportion of coinhibitory receptor BTLA(+) T cells and Tim-3(+) NK cells. Baseline levels of ikaros and aiolos protein in tumor cells did not correlate with response or survival. Pomalidomide led to rapid decline in Ikaros in T and NK cells in vivo, and therapy-induced activation of CD8(+) T cells correlated with clinical response. These data demonstrate that pomalidomide leads to strong and rapid immunomodulatory effects involving both innate and adaptive immunity, even in heavily pretreated multiple myeloma, which correlates with clinical antitumor effects. This trial was registered at www.clinicaltrials.gov as #NCT01319422.

Activity of lenalidomide in mantle cell lymphoma can be explained by NK cell‐mediated cytotoxicity

Lenalidomide is an immunomodulatory agent that has demonstrated clinical benefit for patients with relapsed or refractory mantle cell lymphoma (MCL); however, despite this observed clinical activity, the mechanism of action (MOA) of lenalidomide has not been characterized in this setting. We investigated the MOA of lenalidomide in clinical samples from patients enrolled in the CC‐5013‐MCL‐002 trial (NCT00875667) comparing single‐agent lenalidomide versus investigators choice single‐agent therapy and validated our findings in pre‐clinical models of MCL. Our results revealed a significant increase in natural killer (NK) cells relative to total lymphocytes in lenalidomide responders compared to non‐responders that was associated with a trend towards prolonged progression‐free survival and overall survival. Clinical response to lenalidomide was independent of baseline tumour microenvironment expression of its molecular target, cereblon, as well as genetic mutations reported to impact clinical response to the Bruton tyrosine kinase inhibitor ibrutinib. Preclinical experiments revealed lenalidomide enhanced NK cell‐mediated cytotoxicity against MCL cells via increased lytic immunological synapse formation and secretion of granzyme B. In contrast, lenalidomide exhibited minimal direct cytotoxic effects against MCL cells. Taken together, these data provide the first insight into the clinical activity of lenalidomide against MCL, revealing a predominately immune‐mediated MOA.

A Dual Color Immunohistochemistry Assay for Measurement of Cereblon in Multiple Myeloma Patient Samples.

Clinical interest in the measurement of Cereblon (CRBN), the primary target of the IMiDs immunomodulatory drugs lenalidomide and pomalidomide, has been fueled by its essential requirement for antitumor or immunomodulatory activity of both drugs in multiple myeloma (MM). However, limited analyses of clinical samples for CRBN gene expression or protein levels have utilized unvalidated reagents and assays, raising uncertainty about the interpretation of these results. We previously described a highly specific rabbit monoclonal antibody CRBN65 against 65-76 AA of human Cereblon. Here we describe a validated dual color bright-field Cereblon/CD138 immunohistochemical (IHC) assay utilizing CRBN65 and a commercial mouse monoclonal CD138 antibody. Sensitivity and specificity of the assay was determined and assay precision was shown for both cytoplasmic and nuclear Cereblon in MM bone marrow samples with coefficient of variation values of 5% and 2%, respectively. The dual IHC assay was effective for detecting a continuous range of Cereblon levels in 22 MM patient bone marrow core biopsies and aspirate clots, as shown by average cytoplasmic H-scores ranging from 63 to 267 and nuclear H-scores ranging from 17 to 250. Interpathologist comparison of MM sample H-scores by 3 pathologists demonstrated good concordance (R2=0.73). This dual assay demonstrated superior Cereblon IHC measurement in MM samples compared with the single IHC assay using a published commercial rabbit polyclonal Cereblon antibody and could be used to explore the potential utility of Cereblon as a biomarker in the clinic.

SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate

AbstractTargeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly ‘undruggable’ C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.Thalidomide-induced degradation of the transcription factor SALL4 in a cereblon-dependent manner provides an explanation for the teratogenic effects.

Cereblon loss and up-regulation of c-Myc are associated with lenalidomide resistance in multiple myeloma patients

Multiple myeloma (MM) patients who become refractory to anti-MM drugs have a very poor prognosis. Therefore, it is important to gain insight into the mechanisms of resistance to these drugs. Immunomodulatory drugs (IMiDs) have immune-stimulatory and anti-angiogenic properties as well as direct anti-

Abstract 1354: Comparison of pomalidomide dosing strategies in lenalidomide-refractory myeloma: Impact on clinical outcome, immune activation and cereblon targets

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA In preclinical and mostly in vitro studies, pomalidomide (Pom) has been shown to mediate direct anti-proliferative effects on tumor cells, as well as immune-modulatory effects on T cells, NK cells and monocytes. Cereblon (CRBN), a direct cellular target for Pom has been involved in the anti-proliferative effects in tumor cells via selective degradation of Ikaros (IKZF1) and Aiolos (IKZF3). Depletion of IKZF1/IKZF3 has also been implicated in Len-mediated amplification of anti-CD3-induced IL2 production in human T cells in culture. However the impact of pomalidomide on tumor proliferation and immune activation in vivo is unknown. Here we have evaluated the clinical and pharmacodynamic effects of continuous or intermittent dosing strategies of pomalidomide/dexamethasone in lenalidomide-refractory myeloma in a randomized trial. 39 eligible patients with relapsed myeloma were randomized to therapy with Pom/Dexamethazone (following Pom alone for cycle 1), utilizing either continuous Pom dosing (2 mg-28/28 days, cohort 1, n = 19) or an intermittent dosing schedule (4 mg-21/28 days, cohort 2, n = 20). Dexamethazone was administered at 40 mg weekly at cycle 2 and beyond. Intermittent dosing strategy, despite having frequent adverse events, led to greater tumor reduction. Both cohorts experienced similar event-free and overall survival. Both regimens led to a distinct pattern but similar degree of mid-cycle immune activation as manifest by increased expression of cytokines and lytic genes in T and NK cells. Pomalidomide induced polyfunctional T cell activation, with increased proportion of co-inhibitory receptor BTLA+ T cells and Tim-3+ NK cells. Baseline levels of cereblon, ikaros and aiolos protein in tumor cells using validated IHC assay on marrow biopsies, did not correlate with response or survival. Pomalidomide treatment led to a rapid decline in Ikaros in T and NK cells in vivo as measured by intranuclear flow staining, and therapy-induced activation of CD8+ T cells correlated with clinical response. These data demonstrate that pomalidomide leads to strong and rapid immunomodulatory effects involving both innate and adaptive immunity, even in heavily pre-treated MM, which correlate with clinical anti-tumor effects. Clinicaltrials.gov-[NCT01319422][1]. Citation Format: Rituparna Das, Kartik Sehgal, Lin Zhang, Rakesh Verma, Yanhong Deng, Mehmet Kocoglu, Juan Vasquez, Srini Koduru, Yan Ren, Maria Wang, Suzana Couto, Mike Breider, Donna Hansel, Stuart Seropian, Dennis Cooper, Anjan Thakurta, Xiaopan Yao, Kavita M. Dhodapkar, Madhav V. Dhodapkar. Comparison of pomalidomide dosing strategies in lenalidomide-refractory myeloma: Impact on clinical outcome, immune activation and cereblon targets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1354. doi:10.1158/1538-7445.AM2015-1354 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01319422&atom=%2Fcanres%2F75%2F15_Supplement%2F1354.atom