Cereblon Expression Research Articles

Abstract 3625: Expression of CRBN binding protein AGO2 plays important roles for myeloma cell growth

Abstract Immunomodulatory drugs (IMiDs) are therapeutically active compounds that bind to the E3 ubiquitin ligase substrate recruiter cereblon (CRBN) and induce many cellular responses, including cytotoxicity. We had found that the expression of CRBN in multiple myeloma (MM) cells provided the basis for their responses to IMiDs. In this report, we have identified argonaute 2 (AGO2) as a new member of CRBN-downstream binding protein and found that: 1) the steady-state levels of CRBN in IMiD-sensitive cells are significantly higher than in IMiD-resistant cells; 2) the steady-state levels of AGO2 in IMiD-sensitive cells are significantly less compared to IMiD-resistant cells, suggesting that CRBN plays a role in regulating AGO2. AGO2 is a component of microRNA (miRNA)-induced silencing complexes and plays an important role in miRNA maturation and function. The matured miRNAs are considered as key regulators of almost all cellular pathways. Treatment of IMiD-sensitive MM cells with lenalidomide, an IMiD, significantly increased CRBN, decreased both AGO2 and miRNAs and inhibited cell growth. In contrast, although treatment of IMiD-resistant MM cells with lenalidomide moderately increased CRBN and decreased both AGO2 and miRNAs, this treatment did not significantly inhibit cell growth. However, silencing of AGO2 with small hairpin RNA significantly decreased both AGO2 and miRNAs and inhibited cell growth regardless of whether the cells are sensitive or resistant to IMiDs. Therefore, AGO2 could be considered as a new drug target for overcoming IMiDs resistance in MM cells. Citation Format: Qinqin Xu, Yuexian Hou, Paul Langlais, Patrick Erickson, James Zhu, Changxin Shi, Moulun Luo, Yuanxiao Zhu, Lawrence Mandarino, Keith Stewart, Xiu-Bao Chang. Expression of CRBN binding protein AGO2 plays important roles for myeloma cell growth. [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 3625. doi:10.1158/1538-7445.AM2015-3625

Association of CRBN Gene with Immunomodulatory Drug Resis- tance in Multiple Myeloma

Human CRBN (cereblon) gene is located on chromosome 3 at 3p26 and its encoding protein is a member of E3 ubiquitin ligase complex (composed of CRBN, DDB1, CUL4A and ROC1). The E3 ubiquitin ligase complex functions in the ubiquitin-proteasome protein degradation pathway and attaches polyubiquitin chains to substrate proteins for degradation via the protease complex. Currently, there are no standardized assays for CRBN gene and protein measurement although quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry and Western blot are widely used. CRBN has been identified as a direct target for immunomodulatory drugs (IMiD) and plays a significant role in anti-proliferation, pro-apoptotic effects, anti-angiogenic activities, immunomodulatory activities and intervention of cell surface adhesion molecules between myeloma cells and bone marrow stromal cells. Recently, clinical data show that majority of the multiple myeloma patients treated with IMiD develop drug-resistance over time by unknown mechanisms. Fortunately, various in vivo and in vitro studies have revealed that the decreased CRBN expression or CRBN deletion is associated with resistance to IMiD in treating multiple myeloma, and CRBN expression levels may have a prognostic significance. Furthermore, the most recently discovered protein IKZF1, IKZF3, IRF4, C/EBPβ and Wnt/catenin signaling pathways may also be closely related to IMiD resistance in myeloma.

Rational combination treatment with histone deacetylase inhibitors and immunomodulatory drugs in multiple myeloma.

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide (Len) and pomalidomide trigger anti-tumor activities in multiple myeloma (MM) by targetting cereblon and thereby impacting IZF1/3, c-Myc and IRF4. Histone deacetylase inhibitors (HDACi) also downregulate c-Myc. We therefore determined whether IMiDs with HDACi trigger significant MM cell growth inhibition by inhibiting or downregulating c-Myc. Combination treatment of Len with non-selective HDACi suberoylanilide hydroxamic acid or class-I HDAC-selective inhibitor MS275 induces synergic cytotoxicity, associated with downregulation of c-Myc. Unexpectedly, we observed that decreased levels of cereblon (CRBN), a primary target protein of IMiDs, was triggered by these agents. Indeed, sequential treatment of MM cells with MS275 followed by Len shows less efficacy than simultaneous treatment with this combination. Importantly ACY1215, an HDAC6 inhibitor with minimal effects on class-I HDACs, together with Len induces synergistic MM cytotoxicity without alteration of CRBN expression. Our results showed that only modest class-I HDAC inhibition is able to induce synergistic MM cytotoxicity in combination with Len. These studies may provide the framework for utilizing HDACi in combination with Len to both avoid CRBN downregulation and enhance anti-MM activities.

43 A PREDICTIVE MODEL OF RESPONSE TO ERYTHROPOIETIC STIMULATING AGENTS IN PATIENTS WITH MYELODYSPLASTIC SYNDROME

Downregulation of cereblon (CRBN) gene expression is associated with resistance to the immunomodulatory drug (IMiD) lenalidomide and poor survival outcomes in multiple myeloma patients. However, the importance of CRBN gene expression in patients with myelodysplastic syndrome (MDS) and its impact on lenalidomide therapy are not clear. In this study we are following up our earlier investigation (results were published in EJH). We are presenting data from larger sample of patients and in addition bringing the results from lower risk MDS patients without del(5q), marked as non-5q-, treated with lenalidomide. We evaluate cereblon expression in mononuclear cells isolated from peripheral blood (PB) [43 lower risk MDS with isolated 5q deletion (5q-), 53 non-5qand 32 healthy controls] and from bone marrow (BM) [27 lower risk MDS with 5q-, 38 non-5qlower risk MDS and 25 healthy controls] to gain insight into, firstly, the role of cereblon in lower risk MDS patients with or without 5q deletion, and secondly, into the mechanism of lenalidomide action. Patients with 5qlower risk MDS have the highest levels of CRBN mRNA in comparison with both lower risk MDS patients without del(5q) and healthy controls. Five non-5qanemic patients who were treated with lenalidomide showed all lower levels of CRBN mRNA. All these patients did not respond to the treatment. CRBN gene expression level was analyzed by using the reverse transcriptase quantitative “best coverage” TaqMan PCR assay with primers in exon 9 and 10 and probe on exon 9. This assay allowed the detection of the full-length CRBN transcript but not alternative splicing forms without exon 10 (binding site of IMiDs on CRBN). We found that a high level of full-length CRBN mRNA in both bone marrow and peripheral blood mononuclear cells of lower risk MDS with isolated 5q deletion corresponds to the efficacy of lenalidomide in these patients. Moreover, we found that patients who stopped responding to lenalidomide and whose disease progressed showed a sudden decrease of CRBN gene expression. Supported by the research grant NT/13836 from the IGA, Ministry of Health, Czech Republic.

Dual-Color CRBN/CD138 Immunohistochemistry Assay for Measuring Cereblon Protein in Samples from Patients with Multiple Myeloma

Background: Cereblon (CRBN) is required for the antiproliferative activity of the IMiDs® immunomodulatory drugs lenalidomide (LEN) and pomalidomide (POM) in multiple myeloma (MM), but its value as a biomarker of IMiD response remains unknown. We have shown a disconnect between CRBN mRNA and protein regulation, indicating that measurement of CRBN protein is critical. However, accurate quantification of CRBN protein levels is challenging due to scarcity of standardized and validated reagents and assays. Several publications have suggested that CRBN mRNA and/or protein levels detected with commercial reagents and assays correlate with LEN and/or POM treatment outcomes. In an effort to establish a standardized and validated approach for CRBN protein measurement in MM biopsies, we developed a dual bright-field immunohistochemistry (IHC) assay to measure CRBN levels in CD138+ MM cells.Methods: Using the highly specific CRBN65 rabbit monoclonal antibody against amino acids 65-76 of human cereblon, a CRBN/CD138 dual IHC assay scored using the H-score method was developed and validated. H-scores range from 0 to 300 and are a result of the sum of the products for the percentage of tumor cells (0%-100%) and the intensity of staining (0-3). Since CRBN is a nuclear and cytoplasmic protein, both nuclear and cytoplasmic H-scores were recorded for each sample evaluated with the assay. The dual IHC assay with CRBN65 antibody was used to evaluate the high CRBN–expressing DF15 MM cell line and the low CRBN–expressing DF15R MM cell line (made resistant to POM), as well as bone marrow core biopsies or aspirate clots from 22 patients with MM. For comparison, the same set of samples were stained using a commercially available CRBN antibody in a single IHC assay format. To evaluate interpathologist score variation, H-scores were recorded by 3 pathologists examining the samples independently. Interassay variation was evaluated by staining serial sections of biopsies from the same patient in 3 different experiments and calculating the coefficient of variation (CV) among scores produced for each patient by the same pathologist.Results: The assay was specific and able to detect high CRBN levels in DF15 cells (cytoplasmic score = 190; nuclear score = 150) and low CRBN levels in DF15R cells (cytoplasmic score = 20; nuclear score = 30). The dual IHC assay was able to detect different CRBN staining levels in biopsies and clots from patients with MM, with average cytoplasmic H-scores ranging from 67 to 240 and nuclear H-scores ranging from 17 to 250. Compared with our dual assay, the single IHC assay performed with a commercial CRBN antibody demonstrated lack of specificity, did not detect nuclear CRBN, and showed similar immunoreactivity in the DF15 and DF15R cells. In addition, the commercial CRBN antibody had a narrow range of detection for CRBN (estimated) in patients with MM (Table). Interpathologist comparison of MM biopsy H-scores from 3 pathologists demonstrated high concordance (r2 = 0.73). Assay precision was shown for both cytoplasmic and nuclear CRBN in MM bone marrow biopsies, with CV values of 2% and 5%, respectively.Conclusion: A dual CD138/CRBN IHC assay was developed to provide a reliable semiquantitative method to evaluate CRBN protein levels in both bone marrow core biopsies and clots from patients with MM that is specific, precise, and reproducible. The data highlight the requirement for standardization in the evaluation of CRBN expression. This assay will be used in future trials to assess the value of CRBN as a biomarker in MM and other hematologic malignancies.Table:Estimated CRBN detection in patients with MMSamplesCytoplasmic H-ScoreNuclear H-ScoreDual IHCSingle IHCaDual IHCSingle IHCaCell linesDF15 (CRBN high)1901201500DF15R (CRBN low)20120300Patient biopsiesMM16 (CRBN high)270200b2700MM20 (CRBN low)90100b100a Commercially available antibody for CRBN.b Values are estimated because tumor cell cannot be reliably identified. DisclosuresRen:Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corp: Employment, Equity Ownership. Couta:Celgene Corp: Employment, Equity Ownership. Hansel:Celgene Corp: Consultancy. Miller:Celgene Corp: Employment, Equity Ownership. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Bjorklund:Celgene Corp: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership. Breider:Celgene Corp: Employment, Equity Ownership.

Upregulation of Cereblon Expression By the DNA Methyltransferase Inhibitor Azacytidine Strongly Enhances Lenalidomide Cytotoxicity in Germinal Center B-Cell-like (GCB) and Activated B-Cell-like (ABC) Diffuse Large B-Cell Lymphoma (DLBCL)

▪INTRODUCTION: Lenalidomide monotherapy exerts clinical activity in relapsed/refractory Diffuse Large B-cell Lymphoma (DLBCL) with better response rate and progression-free survival being recorded in activated B-cell-like (ABC) rather than germinal center B-cell-like (GCB)-DLBCL. Reasons for such a difference are likely due to different expression of key molecules involved in mediating activity of Lenalidomide, such as Interferon regulatory factor 4(IRF4) and cereblon (CRBN). Evidences supporting the key role of DNA methylation and histone modifications in regulating genome stability and gene expression in DLBCL prompted us to investigate the capacity of Azacytidine in modulating Lenalidomide activity, thereby sensitizing GCB-DLBCL to Lenalidomide and enhancing Lenalidomide efficacy in ABC-DLBCL.METHODS: DLBCL cell lines with ABC (U-2932, RIVA) or GCB (SU-DHL4, SU-DHL6) genotype were used to investigate the effects of Lenalidomide and Azacytidine on cell growth and cell death. Western blotting (WB) and immunofluorescence analysis were used to assess modulating effects of the two-drug combination on molecular determinants of Lenalidomide activity. Additionally, we studied CRBN, IRF4 and CRBN binding proteins expression, such as Ikaros and Aiolos (IKZF1 and IKZF3) by real time polymerase chain reaction (RT-PCR) in response to drug treatment.RESULTS: Graded concentrations of Lenalidomide (0.1-100 µM) inhibited cell proliferation by 20% to 40% and increased cell death up to 30% to 40% in ABC-DLBCL cell lines, whereas had minimal effects on GCB-DLBCL cell lines. Untreated ABC-DLBCL but not GCB-DLBCL consistently showed a high expression of CRBN and IRF4. Upon Lenalidomide treatment (3 days) CRBN was significantly upregulated and IRF4 downregulated in ABC-DLBCL, but not GCB-DLBCL cells. Since DNA methylation regulates gene expression in DLBCL cell lines, we next examined whether Azacytdine could modulate CRBN and IRF4 expression and in turn enhance responsiveness to Lenalidomide. Exposure of both ABC- and GCB-DLBCL cell lines to Azacytidine (up to 72 hours) induced a marked increase of CRBN and IRF4 transcripts; addition of Lenalidomide strongly increased Azacytidine-induced increase of CRBN and significantly downregulated IRF4 expression; the combined treatment induced a marked downregulation of Ikaros and Aiolos protein levels. At the cellular level, the concomitant Azacytidine (10 μM)/Lenalidomide (10 μM) treatment inhibited in a synergistic manner the mean (±SEM) cell growth of both ABC-DLBCL (Lena: -16 ± 4%; AZA: -22 ± 2%; AZA/Lena: -70 ± 1%, P<0.001) and GCB-DLBCL (Lena: -17 ± 3%; AZA: -40 ± 4%; AZA/Lena: -82 ± 2%, P<0.001). Additionally, the two drug exposure was associated with a 3-fold decrease of S phase cells(Lena: 28 ± 2%; AZA: 22 ± 0.8%; AZA/Lena: 9 ± 1%, P<0.001); a marked p21 overexpression, and a 3- to 4-fold cell death increase (P<0.001) in both ABC- and GCB-DLBCL.CONCLUSIONS: Our results indicate that Azacytidine sensitizes GCB-DLBCL to the cytotoxic effects of Lenalidomide and enhances Lenalidomide efficacy against ABC-DLBCL resulting in synergistic anti-proliferative and pro-apoptotic effects in both ABC- and GCB-DLBCL cell lines. Cytotoxicity of the two drug combination is mediated by signaling events involving CRBN upregulation and IRF4 downregulation leading to CRBN-binding proteins downregulation. Azacytidine-dependent activation of CRBN and IRF4 expression allow to hypothesize a methylation-driven regulation of these genes. These results might provide a rationale for clinical studies using Azacytidine and Lenalidomide combination in ABC- and GCB-DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Immunomodulatory Drugs Are Efficacious Against Primary Effusion Lymphoma By Targeting IKZF1, IRF4 and MYC in a CRBN-Dependent Manner and Are Synergistic with BRD4 Inhibitors

Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma localized predominantly in body cavities. Kaposi’s sarcoma-associated herpes virus is the causative agent of PEL. PEL is an incurable malignancy and has extremely poor prognosis when treated with conventional chemotherapy. PEL cells universally express interferon regulatory factor 4 (IRF4). We found that shRNA-mediated knockdown of IRF4 is toxic to PEL. Further, we observed that FDA-approved immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show selective cytotoxicity towards PEL in a panel of cell lines comprising 11 hematological malignancies. The cytotoxic effect of IMiDs towards PEL was observed within their physiologically achievable concentrations and involved the downregulation of IRF4 and MYC. Genome-wide microarray examination (illumina) of PEL cells treated with lenalidomide followed by Gene Set Enrichment Analysis (http://www.broadinstitute.org/gsea) revealed activation of interferon (IFN) signaling. Further, recombinant IFNs α, β and γ are very effective and selective against PEL in a panel of cell lines tested. However, blocking the IFN pathway using specific blocking antibodies did not block the anti-proliferative potential of IMiDs towards PEL suggesting that activation of IFN pathway is not responsible for the cytotoxicity of IMiDs against PEL. Ikaros family proteins IKZF1 and IKZF3 are transcription factors which play crucial roles in immunity and cell-fate decisions. Recently, it has been shown that IMiDs selectively degrade these transcription factors (Kronke et al., and Lu et al., Science, 2014). In PEL, IKZF1 is strongly downregulated upon treatment with IMiDs. Additionally, shRNA-mediated knockdown of IKZF1 is toxic to PEL. Cereblon (CRBN) is a direct cellular binding target of IMiDs. IMiDs have no significant effect on the expression of CRBN in PEL and shRNA-mediated knockdown of CRBN (shCRBN) is not toxic to PEL. However, all the anti-PEL effects of IMiDs were blunted in cells expressing shCRBN. CRBN is a component and substrate receptor for Culling-RING ubiquitin ligase complex (CRL) (Ito et al., Science, 2010) and CRL activity depends on NEDDylation (Soucy et al., Nature, 2009). MLN4924, small molecule NEDD8-activating enzyme inhibitor phenocopied the effects of shCRBN on the anti-PEL activities of IMiDs suggesting that CRBN and its CRL activity is essential for the anti-proliferative activity of IMiDs in PEL. MYC is a downstream target of IMiDs in PEL but the role of MYC on the activity of IMiDs is not known. While investigating the role of MYC in IMiDs activity against PEL we found that shRNA-mediated knockdown of MYC enhanced the cytotoxicity of IMiDs suggesting a potential synergism between IMiDs and MYC inhibition. BRD4 inhibitors have been shown to block MYC expression (Delmore et al., Cell, 2011; Mertz et al., PNAS, 2011). We observed that lenalidomide is synergistic with all the BRD4 inhibitors (JQ-1, IBET151 and PFI-1) tested in PEL. Combined treatment of IMiDs with JQ-1 at low doses resulted in comprehensive downregulation of MYC and significant increase in the number of cells undergoing apoptosis as observed by AnnexinV staining and PARP cleavage. Knocking down BRD4 using specific shRNA also enhanced the cytotoxicity of IMiDs towards PEL suggesting that the synergism observed between IMiDs and BRD4 inhibition may not be limited only to the BRD4 inhibitors used in our study. Furthermore, combined administration of lenalidomide and JQ-1 significantly increased the survival of PEL bearing NOD.SCID mice in an orthotopic xenograft model. These results provide the basis for clinical testing of IMiDs alone and in combination with BRD4 inhibitors for PEL. DisclosuresGopalakrishnan:University of Southern California: Inventor on a patent application relevant to this work filed to US patent office (No. 62/031,053). Patents & Royalties. Chaudhary:University of Southern California: Inventor on a patent application relevant to this work filed to US patent office (No. 62/031,053). Patents & Royalties.

CC-122 Has Robust Anti-Proliferative Activity in a Primary Chronic Lymphocytic Leukemia (CLL) Co-Culture Model and Is Superior to Lenalidomide

Background: The Ikaros family zinc finger transcription factor, Aiolos, is upregulated in more than 80% of CLL patients with data suggesting that it may be an important driver of CLL survival and hence a bona fide target for therapeutic intervention. To date, transcription factors such as Aiolos have not been amenable to drug therapy. We and others have shown that the IMiDs™ immunomodulatory drugs such as Lenalidomide (LEN) bind Cereblon (CRBN), a substrate receptor of the cullin ring E3 ubiquitin ligase complex (CRL4CRBN) and promote ubiquitination and subsequent proteosomal degradation of lymphoid transcription factors Ikaros and Aiolos. Such insights have been critical for the design and development of next generation IMiD analogues. CC-122 is a novel non-phthalimide, first-in-class, PPM™ pleiotropic pathway modifier drug with anti-proliferative, anti-angiogenic, and immunomodulatory properties. CC-122 specifically targets CRBN and promotes degradation of Aiolos and Ikaros. We have now investigated the effects of LEN and CC-122 on CLL proliferation, and Aiolos and Ikaros degradation, in a cohort of primary CLL cases using an in vitro co-culture system based on CD154 expressing mouse fibroblasts and exogenous IL-21 that recapitulates the in vivo CLL microenvironment.Results: Forty CLL primary patient PBMC samples, selected to represent diverse prognostic subgroups, were cultured in media supplemented with LEN, CC-122, or control and assessed in a BrdU incorporation assay. Among these analyzed samples, four distinct ‘response’ subsets were identified. In subset 1 (n=10, 25% of cases), we found that CC-122 is superior to LEN in inhibiting proliferation (mean IC50: LEN 5.7µM; CC-122 1.2µM). Subset 2 (n=23; 57% of cases) was sensitive to CC-122, but unresponsive to LEN. In these cases, CC-122 inhibited proliferation by at least 50% at a concentration of ≤3µM despite the failure of LEN (IC50: >10 µM) to inhibit proliferation, highlighting a qualitative difference between the two agents. In subset 3, the anti-proliferative activities of CC-122 and LEN were comparable (n=2, 5% of cases). Finally, both CC-122 and LEN were inactive (up to 10µM) in a 4th subset (n=5; 12% of cases). We subsequently explored associations between CC-122 activity and known CLL prognostic risk factors in these samples. In 5 out of 6 cases with 17p deletion CC-122 inhibited proliferation (IC50: <0.3-0.8 mM). Similarly, in 4 out 6 cases harbouring an 11q- defect CC-122 was active (IC50: 0.8-3 µM). In general, the efficacy of either drug at inhibiting CLL cell proliferation did not segregate with established prognostic variables (e.g. IgVH mutation status, cytogenetic abnormalities, etc.), or previous treatment exposure (e.g. chlorambucil, fludarabine, alemtuzumab, etc.). Preliminary investigations of the CLL cells demonstrate wide variation in baseline Ikaros, Aiolos, and CRBN levels between individual cases as measured by either flow cytometry and/or western blot. We show that treatment of CLL cells with CC-122 or LEN results in variable degradation of Ikaros (LEN 0-100%; CC-122 55-100%) and Aiolos (LEN 72-100%; CC-122 97-100%) compared to untreated controls. Of note, CC-122 was consistently superior to LEN in promoting Aiolos and Ikaros degradation. In contrast, levels of CRBN, the molecular target of CC-122 and LEN, were unaffected by either compound. The predictive value of baseline Aiolos, Ikaros and CRBN expression, as well as their degradation, by CC-122 in relation to cellular response is currently being assessed.Conclusions: Taken together, these data demonstrate that CC-122 has robust anti-proliferative activity in primary CLL cells through a unique mechanism of selective degradation of Aiolos and Ikaros transcription factors. CC-122 is superior to LEN and is active across several prognostic backgrounds. Furthermore, we show significant activity of CC-122 even in cases that are non-responsive to LEN suggesting that CC-122 targets a novel pathway in CLL. These data suggest that further evaluations of CC-122 in pre-clinical and clinical studies, as a single agent or in combination with established or novel compounds (such as Btk and PI3Kd inhibitors), are warranted. DisclosuresBlocksidge:Celgene: Research Funding. Glenn:Celgene: Research Funding. Gandhi:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership. Kalakonda:Celgene: Research Funding.

IMiDs® Immunomodulatory Agents Regulate Interferon-Stimulated Genes through Cereblon-Mediated Aiolos Destruction in Multiple Myeloma (MM) Cells: Identification of a Novel Mechanism of Action and Pathway for Resistance

Numerous cellular effects have been attributed to IMiDs lenalidomide (LEN) and pomalidomide (POM) over the years. However, the zinc finger transcription factors Aiolos (IKZF3) and Ikaros (IKZF1) were identified only recently as substrates targeted for destruction in the presence of IMiDs compounds. LEN and POM bind directly to cereblon (CRBN), a substrate receptor of the cullin ring E3 ligase 4 (CRL4). Binding of these compounds to CRBN triggers the activation of CRL4CRBN, ubiquitylation and destruction of Aiolos and Ikaros.We systematically compared growth, gene expression, and signal transduction responses elicited by doxycycline-inducible, stable Aiolos knockdown or POM treatment in MM cell lines, and their POM-resistant counterparts. Upon exposure to POM, U266 MM cells undergo rapid degradation of Aiolos and Ikaros (90 min) and subsequent downregulation of IRF4 and Myc at 72 hr. At 72-96 hr, a decrease in proliferation and an increase in apoptosis are also observed. U266 MM cells selected in vitro for resistance to POM have lost CRBN expression and consequently no longer downregulate Aiolos and Ikaros after POM administration. Knockdown of Aiolos in these POM-resistant U266 MM cells is sufficient to inhibit their proliferative capacity by 50%, while Aiolos knockdown in POM-sensitive cells caused a greater inhibition of proliferation (90%). This suggests that MM cells with acquired resistance are still dependent on Aiolos for growth, but that a second mechanism may contribute to the antitumorigenic effect of Aiolos downregulation in POM-sensitive U266 cells.We have found that treatment with LEN or POM induces expression of antiviral response genes in MM cells. The induction of interferon (IFN)-stimulated genes (ISGs) such as DDX58, IFIT1, IFIT3, XAF1, ISG15, IFI44, and IFI27 are seen by qPCR in 8 hr of compound treatment (1.8- to 5-fold increase in transcript level) and this effect is further enhanced at 24 and 72 hr. Of note, this response is not accompanied by an increase in β IFN production. The IMiD compound-induced upregulation of the antiviral response correlates with CRL4-CRBN-mediated destruction of the lymphoid restricted transcription factor, Aiolos. In agreement with this, Aiolos knockdown by shRNA is sufficient to trigger a similar effect. These data suggest that Aiolos functions as a transcriptional repressor of ISGs, regulating the antiviral response. Consequently, Aiolos chromatin immunoprecipitation and sequencing (ChIP-Seq) experiments were performed, demonstrating that Aiolos binds near the transcription start site of numerous ISGs, including DDX58, IFIT1, ISG15, XAF1, IFI44, and IFI35. In addition, our data suggest that Aiolos co-binds with STAT and IRF family transcription factors and thereby co-regulates expression of these genes. STAT1 is part of the ISGF3 complex that drives ISG transcription upon viral infection. POM-resistant MM cells lacking CRBN expression do not have STAT1 activity and do not upregulate ISGs upon Aiolos knockdown, even though Myc and IRF4 are still being downregulated. In order to elucidate the relevance of the ISG expression in patients receiving IMiD treatment, we compared the gene expression profile of 12 patients after relapse or disease progression. Paired pre- and posttreatment samples from bone marrow-isolated CD-138 cells were evaluated with RNAseq and gene set enrichment analysis. We found an overall decrease in expression of ISGs, with significant negative enrichment of genes involved in IFN α, β, and γ signaling in relapsed patients. These data from clinical samples confirmed the importance and relevance of the ISGs in the response to IMiDs.In conclusion, our results indicate that Aiolos is a substrate of consequence in IMiD-sensitive MM cells, based on at least 2 pathways: driving the Myc-IRF4 feedback loop and repressing the antiviral pathway. Both in vitro and in vivo patient data suggest that one mechanism of IMiD resistance may be the abrogation of the STAT1 pathway resulting in subsequent blunting of the ISG induction. Finally, while upregulation of ISGs by IMiD treatment may serve as a relevant diagnostic marker of patient responsiveness to these drugs, these data highlight how response and resistance of the IMiD drugs are regulated by the interplay between complex pathway networks, suggesting that the measurement of only one component will not necessarily define the clinical course and outcomes for an individual patient. DisclosuresHavens:Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corp: Employment, Equity Ownership. Kang:Celgene Corp: Employment, Equity Ownership. Ortiz:Celgene Corp: Employment, Equity Ownership. Fontanillo:Celgene Corp: Employment, Equity Ownership. Amatangelo:Celgene Corporation: Employment, Equity Ownership. Lu:Celgene Corp: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Bahlis:Celgene Corp: Honoraria, Research Funding. Thakurta:Celgene Corp: Employment, Equity Ownership. Trotter:Celgene Corp: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene Corp: Employment. Chopra:Celgene Corp: Employment, Equity Ownership.

Cereblon (CRBN) Splice Isoform Lacking Exon 10 Attenuates Lenalidomide-Mediated Degradation of Aiolos and Is Upregulated in Immunomodulatory Drugs (IMiDs) Resistant Myeloma (MM) Patients

▪Background: IMiDs cytotoxicity in MM cells is mediated through their binding to CRBN within the cullin ring (CRL4) ligase. This binding triggers the ubiquitylation and proteasomal degradation of IKZF1/3. CRBN thalidmomide binding domain (TBD) was mapped to its C-terminus and the crystal structure of the CRBN-IMiDs bound complex identified several aa within exons 10 and 11 as essential for the IMiDs glutarimide ring binding to CRBN. Several groups including ours have reported that loss of CRBN is associated with resistance to IMiDs, however this does not appear to be the sole mechanism of resistance in primary MM cells. Furthermore, and while CRBN mutants (Y384A and W386A) are defective for IMiDs binding, acquisition of CRBN mutation is a rare event in MM patients suggesting alternative mechanisms of resistance. Thirteen splice variants of CRBN are reported (ensembl.org), however to date it is unclear whether these isoforms are expressed as proteins and their contribution to IMiDs resistance is yet to be defined.Methods and Results: In this study we investigated whether expression of full length CRBN (FL-CRBN) relative to its variants lacking the TBD and particularly the splice variant CRBN-005 (ENST00000424814) lacking exon 10, contribute to IMiDs resistance. RNA-seq analysis was performed on CD138 sorted cells in 15 paired patients samples obtained sequentially prior to lenalidomide treatment initiation and after development of resistance. Transcriptome sequence data was generated by RNA-seq with a minimum of 70x106 reads per sample. Filtered Fastq files were processed with the splice aligner TopHat against hg19. Of interest, splice isoforms of CRBN including isoforms lacking exon 10 and to a lesser extent exon 8 were identified in nearly all patients, albeit with at variable frequency. Splicing almost universally involved the full length of exon 10 including aa W382 and H378 that are now recognized to bind the 2 carbonyls residues on the IMiDs glutarimide ring and hence required for IMiDs binding to CRBN. Of note, mutation analysis of these 30 samples using the GATK RNAseq pipeline did not identify any mutations within CRBN exons 10 or 11. Furthermore, exome sequencing of CD138 cells from 10 additional lenalidomide resistant patients did not identify any CRBN SNVs or indels confirming the rarity of this event. In order to assess the contribution of FL-CRBN transcript and/or its splice variant (CRBN-005) to IMiDs sensitivity, we first confirmed by qRT-PCR (n=26, amplicons with 2 sets of primers overlapping exons 8-9 and exons 10-11) that low pre-treatment CRBN levels was significantly associated with shorter PFS (p=0.008) to lenalidomide. We next compared FL-CRBN (probe spanning exons 10-11) and CRBN-005 (Taqman probe spanning exons 9-11 junction) mRNA expression (qRT-PCR) in paired samples (n=21 patients - 42 pairs) collected immediately pre-treatment and at the time of progression post-lenalidomide. In 9/21 (42.8%), a significant reduction (2-ΔΔCT < 0.75) in the FL-CRBN amplicon levels was observed between the paired pre- and post-treatment samples. The ratio of spliced CRBN-005 to full length CRBN (CRBN-005 / FL-CRBN) was significantly higher (1.3 to 54 fold) at the time of relapse in 11/21 (52.3%), including 5 patients where CRBN-FL transcript levels were unchanged. Lastly, to confirm whether CRBN-005 expresses a stable protein and to evaluate its role in IMiDs resistance, we cloned spliced CRBN-005 isoform (Δ10-CRBN) or full length CRBN (WT-CRBN) into pcDNA3 plasmid and transfected them in HEK293T cells. The Δ10-CRBN and WT-CRBN plasmids expressed a ~ 45 and 51 kDa proteins respectively that were detectable by western blotting with CRBN65 antibody (Celgene). Functionally, we co-transfected HEK293T cells with a lentiviral plasmid expressing Aiolos and the Δ10-CRBN or WT-CRBN plasmids. While treatment of WT-CRBN expressing cells with lenalidomide resulted in full loss of Aiolos, the expression of Δ10-CRBN significantly mitigated this effect.Conclusions: Study of the transcriptome of paired pre- and post-IMIDs in myeloma primary cells confirms the expression of CRBN-005 splice isoform lacking the IMiDs binding domain and reveals its enrichment in a subset of IMiDs resistance patients. Functionally we have demonstrated a novel mechanism of IMiDs resistance where the spliced isoform CRBN-005 acts as a dominant negative blocking IMiDs binding the CRL4 E3 ligase. DisclosuresBahlis:Celgene: Honoraria, Research Funding.

IMiD® Compounds Affect the Hematopoiesis Via CRBN Dependent Degradation of IKZF1 Protein in CD34+ Cells

Introduction: Lenalidomide (LEN) and pomalidomide (POM) are derivatives of thalidomide (IMiD® compounds) and currently used to treat multiple myeloma (MM) and B-cell malignancies. We have shown before that IMiD® compounds shift lineage commitment of CD34+ cells towards myeloid development by affecting critical transcription factors such as GATA1 and PU.1 with concomitant inhibition of cell maturation resulting in anemia and neutropenia. Nonetheless the underlying pathomechanism is still unknown. Recently, IMiD® compounds were shown to bind to cereblon (CRBN) in MM cells, which is the substrate recognition component of cullin-dependent ubiquitin ligase and LEN leads to ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IZKF3 by the CRBN-CRL4 ubiquitin ligase. We investigated here the role of CRBN, IKZF1 and IKZF3 in IMiD®-induced effects on lineage commitment and maturation of CD34+ cells.Methods and Results: By western blot analysis we found that CRBN and IKZF1, but not IKZF3 are expressed in CD34+ cells. Treatment of CD34+ cells with LEN and POM for only 1h almost completely decreased the expression of IKZF1 without affecting CRBN protein expression. By using a thalidomide analog bead assay, we found that IMiD® compounds directly bind CRBN in CD34+ cells. In contrast to our protein studies, IKZF1-mRNA level was not altered in real-time PCR, suggesting that IMiD® compounds regulate IKZF1 at post-transcriptional level. Treatment with proteasome inhibitors MG132, PS341 or MLN4924 which function as cullin-dependent ubiquitin ligase inhibitors blocked LEN and POM induced IKZF1 degradation, confirming that IMiD® compounds induce ubiquitination and subsequent protein degradation of endogenous IKZF1. Next we generated lentiviral constructs to knockdown the expression of CRBN in CD34+ cells. Knockdown of CRBN in CD34+ cells induced resistance to POM-induced IKZF1 downregulation and subsequently reversed the POM-induced lineage shift in colony-formation assays, suggesting that POM-induced degradation of IKZF1 in HSC requires CRBN.Knowing that PU.1 and GATA-1 are critically involved in the IMID-induced lineage shift in CD34+ cells we demonstrated in chromatin immunoprecipitation assays that IKZF1 binds to promoter regions of PU.1 and GATA-1, suggesting that PU.1 and GATA-1 are direct downstream targets of IKZF1 in CD34+ cells .Conclusion: Our findings show that CRBN and IKZF1 mediate the effects of IMiD® compounds on hematopoietic progenitors. IMiD® compounds promote CRBN dependent degradation of IKZF1 protein in CD34+ cells that subsequently decreases transcription factors such as GATA1 and PU.1 which are critical for development and maturation of neutrophils and erythrocytes as well as thrombocytes. Our findings that IMiD® compounds mediate their effects via CRBN and IKZF1 provide for the first time the pathomechanism how LEN and POM affect hematopoiesis and induce neutropenia, thrombocytopenia as well as anemia. DisclosuresLentzsch:Bristol Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Research Funding.

Cereblon (CRBN) Gene Polymorphisms Predict Clinical Response and Progression-Free Survival in Multiple Myeloma Patients Treated with Lenalidomide: A Pharmacogenetic Study of Immense Consortium

Background: Cereblon (CRBN), a subunit of cullin 4-based E3 ubiquitine ligase complex, has been characterized as the main immunomodulatory drugs (IMiDs), thalidomide, lenalidomide and pomalidomide, binding protein that is crucial for their antiproliferative and immunomodulatory properties. Several recent reports have suggested that CRBN gene and protein expression or alternative splicing affect sensitivity to IMiDs in multiple myeloma (MM). It can be speculated that CRBN expression level, alternative splicing or CRBN-E3 ubiquitine ligase substrate specificity in MM and microenvironment cells may be partially determined by inherited genetic background including functional single nucleotide polymorphisms (SNPs).Aim: The objective of this study was to verify whether naturally occurring SNPs in CRBN encoding CRBNgene (locus chromosome 3p26.2) may influence outcome of lenalidomide-based therapy in patients with MM.Methods: We genotyped 14 tagging SNPs in CRBN gene in 169 Polish Caucasian patients with refractory/relapsed MM treated with lenalidomide-based therapy. Among these patients 151 (89%) received lenalidomide/dexamethasone (Len-Dex) regimen while the remaining patients were treated with lenalidomide-based three drug combinations. Genomic DNA samples were collected and genotyped in the context of the IMMEnSE consortium. The influence of CRBNallelic variants on probability to achieve clinical response (at least partial response, PR) to lenalidomide-based therapy, achievement of complete response (CR), duration of progression-free survival (PFS) and overall survival (OS) were tested in regard to established clinical and laboratory predictive factors using logistic regression and proportional-hazards regression models. For all genotyped SNPs co-dominant, dominant and recessive inheritance modes were tested.Results: We found that carriers of minor alleles of two studied SNPs, namely CRBN rs1714327G>C and CRBN rs1705814T>C significantly associated with lower probability of achievement of clinical response (≥PR) to lenalidomide-based therapy using dominant inheritance model (OR=0.25, 95%CI 0.10-0.63; p=0.0033, Bonferroni corrected p=0.019 and OR=0.21, 95%CI 0.07-0.61; p=0.0041, Bonferroni corrected p=0.024, respectively). Moreover, in concordance with these findings one of these two SNPs, namely CRBN rs1705814T>C, was also significantly associated with shorter PFS in the analyzed group of lenalidomide treated MM patients (OR=2.49; 95%CI 1.31-4.74; P=0.0054, Bonferroni corrected p=0.032). In contrast, none of the tested allelic variants of CRBN showed significant influence on OS.Conclusions: Taken together, our observations suggest that selected germline CRBN SNPs (rs1714327G>C and rs1705814T>C) affect lenalidomide efficacy in relapsed/refractory MM. Further studies are needed to explain functional mechanisms underlying these associations as well as to establish whether CRBN genetic variants may be useful as potential biomarkers for IMiDs-based therapy.Acknowledgments: This work was supported by the grants of Polish Young Heamatologists Club and Polish Myeloma Consortium. The work of Pawe³ Gaj was supported by a grant from the European Commission 7th Framework Programme: FP7-REGPOT-2012-CT2012-316254-BASTION DisclosuresNo relevant conflicts of interest to declare.

High level of full-length cereblon mRNA in lower risk myelodysplastic syndrome with isolated 5q deletion is implicated in the efficacy of lenalidomide.

Downregulation of cereblon (CRBN) gene expression is associated with resistance to the immunomodulatory drug lenalidomide and poor survival outcomes in multiple myeloma (MM) patients. However, the importance of CRBN gene expression in patients with myelodysplastic syndrome (MDS) and its impact on lenalidomide therapy are not clear. In this study, we evaluate cereblon expression in mononuclear cells isolated from bone marrow [23 lower risk MDS patients with isolated 5q deletion (5q-), 37 lower risk MDS patients with chromosome 5 without the deletion of long arms (non-5q-), and 24 healthy controls] and from peripheral blood (38 patients with 5q-, 52 non-5q- patients and 25 healthy controls) to gain insight into, firstly, the role of cereblon in lower risk MDS patients with or without 5q deletion and, secondly, into the mechanisms of lenalidomide action. Patients with 5q- lower risk MDS have the highest levels of CRBN mRNA in comparison with both lower risk MDS without the deletion of long arms of chromosome 5 and healthy controls. CRBN gene expression was measured using the quantitative TaqMan real-time PCR. High levels of CRBN mRNA were detected in all lenalidomide responders during the course of therapy. A significant decrease of the CRBN mRNA level during lenalidomide treatment is associated with loss of response to treatment and disease progression. These results suggest that, similar to the treatment of MM, high levels of full-length CRBN mRNA in lower risk 5q- patients are necessary for the efficacy of lenalidomide.

Cereblon - a new target of therapy in the treatment of multiple myeloma

Treatment of multiple myeloma (MM), currently an incurable disease, aims to achieve complete remission. Immunomodulatory drugs (IMiDs), represented by thalidomide, are one class of very effective drugs. However, the mechanism of IMiDs action is not yet completely understood. Recent research suggests that cereblon (CRBN) plays an important role in mediating anti-tumor effects of IMiDs; therefore, our review focuses on this protein. CRBN is a substrate receptor of Cul4- E3 ubiquitin ligase complex, and thus recognizes proteins destined for degradation. Bind-ing of CRBN and IMiDs inhibits function of the entire ubiquitin proteasome complex which partly explains their anti-tumor effects. In addition, a correlation between CRBN gene expression and effectiveness of treatment in MM patients treated with IMiDs was confirmed. These findings suggest that CRBN expression could possibly serve as a bio-marker to predict response to IMiD in MM patients.

Functional Effects of a Pathogenic Mutation in Cereblon (CRBN) on the Regulation of Protein Synthesis via the AMPK-mTOR Cascade

Initially identified as a protein implicated in human mental deficit, cereblon (CRBN) was recently recognized as a negative regulator of adenosine monophosphate-activated protein kinase (AMPK) in vivo and in vitro. Here, we present results showing that CRBN can effectively regulate new protein synthesis through the mammalian target of rapamycin (mTOR) signaling pathway, a downstream target of AMPK. Whereas deficiency of Crbn repressed protein translation via activation of the AMPK-mTOR cascade in Crbn-knock-out mice, ectopic expression of the wild-type CRBN increased protein synthesis by inhibiting endogenous AMPK. Unlike the wild-type CRBN, a mutant CRBN found in human patients, which lacks the last 24 amino acids, failed to rescue mTOR-dependent repression of protein synthesis in Crbn-deficient mouse fibroblasts. These results provide the first evidence that Crbn can activate the protein synthesis machinery through the mTOR signaling pathway by inhibiting AMPK. In light of the fact that protein synthesis regulated by mTOR is essential for various forms of synaptic plasticity that underlie the cognitive functions of the brain, the results of this study suggest a plausible mechanism for CRBN involvement in higher brain function in humans, and they may help explain how a specific mutation in CRBN can affect the cognitive ability of patients.

Functional effects of a pathogenic mutation in Cereblon on the regulation of protein synthesis via the AMPK‐mTOR cascade (LB223)

Initially identified as a protein implicated in human mental deficit, cereblon (CRBN) was recently recognized as a negative regulator of adenosine monophosphate‐activated protein kinase (AMPK) in vivo and in vitro. Here, we present results showing that CRBN can effectively regulate new protein synthesis through the mammalian target of rapamycin (mTOR) signaling pathway, a downstream target of AMPK. Whereas deficiency of Crbn repressed protein translation via activation of the AMPK‐mTOR cascade in Crbn‐knockout mice, ectopic expression of the wild‐type CRBN increased protein synthesis by repressing endogenous AMPK. Unlike the wild‐type CRBN, a mutant CRBN found in human patients, which lacks the last 24 amino acids, failed to rescue mTOR‐dependent repression of protein synthesis in Crbn‐deficient mouse fibroblasts. These results provide the first evidence that Crbn can activate the protein synthesis machinery through the mTOR signaling pathway by inhibiting AMPK. In light of the fact that protein synthesis regulated by mTOR is essential for various forms of synaptic plasticity that underlie the cognitive functions of the brain, the results of this study suggest a plausible mechanism for CRBN involvement in higher brain function in humans, and they may help explain how a specific mutation in CRBN can affect the cognitive ability of patients.Grant Funding Source: Supported by Ministry for Health and Welfare [HI13C1412] and Ministry of Science, ICT &amp; Future Planning of Korea [2007‐0056157] [2011‐0028665] of Korea.

What Determines the Response to Immunomodulatory Therapy in Multiple Myeloma?

Zhu et al. demonstrated that cereblon (CRBN) is essential for IMiDs activity, and low levels of CRBN correlate with poor drug response, and CRBN expression in MM cells may help to distinguish MM patients that will or will not benefit from thalidomide [2]. In the present study we evaluated CRBN and IL-6R expressions and their impact on clinical efficacy of dexamethasone–thalidomide therapy in Multiple Myeloma (MM) patients, in addition to their association with other clinical and prognostic parameters. Forty-six newly diagnosed MM patients were enrolled in the study. We measured CRBN expression prior to therapy initiation by real-time polymerase chain reaction in 46 Bone Marrow (BM) aspiration samples of patients and 15 controls. In addition, IL-6R expression was evaluated on BM biopsies of patients and controls by Immunohistochemistry (IHC). Median CRBN expression in 46 BM samples of MM patients was significantly higher than in controls (P < 0.001).Among established prognostic parameters, International Staging System (ISS), serum Beta-2-Microglobulin (B2M), and serum albumin correlated reversely with CRBN expression. Strong IL-6R expression was significantly higher in patients than in controls.IL-6R expression was significantly associated with poor response to treatment (P < 0.001), B2M(P = 0.032),and ISS(P = 0.028).Strong intensity expression was associated with low CRBN expression(P = 0.001).In conclusion, CRBN expression may provide a biomarker to predict response to IMiDs in patients with MM and its high expression can serve as a marker of good prognosis. Strong IL-6R expression is associated with poor response to therapy in multiple myeloma patients and can be used as a prognostic marker [3].

High Expression Of Cereblon (CRBN) Is Associated With Achievement Of Complete Response To Thalidomide Plus Fludarabine Regimen In Chronic Lymphocytic Leukemia

Cereblon (CRBN) has recently been identified as a target for immunomodulatory drugs (IMiDs), thalidomide, lenalidomide and pomalidomide, and its downregulation has been linked to resistance to IMiDs in multiple myeloma (MM). However, little is known on the role of CRBN as a resistance/response biomarker in other diseases in which treatment with IMiDs is investigated, such as chronic lymphocytic leukemia (CLL). Although lenalidomide has recently proven to be more promising drug in CLL, we had previously performed a clinical trial that assessed efficacy of thalidomide (100mg p.o on days 1-28) combined with fludarabine (25mg/m2 i.v. on days 7–11) given for six 4-week cycles in patients with CLL. The objective of the present study was to correlate retrospectively pre-treatment CRBN expression in CLL cells with response to thalidomide/fludarabine regimen as well as to cellular and molecular changes that were monitored in vivo after initial 7-day thalidomide monotherapy. CRBN and interferon regulatory factor 4 (IRF4) expressions were studied by quantitative RT-PCR using mRNA extracted from frozen pre-treatment CLL cells. Tumor necrosis factor (TNF) and TNF receptor expression were analyzed by ELISA. T-cell populations were estimated using flow cytometry during the clinical part of the study. Frozen biological pre-treatment samples were available for 27 out of 40 patients included to the thalidomide/fludarabine clinical trial. First, we verified that CRBN expression was independent from all established prognostic parameters in CLL including IGVH mutational status, high-risk FISH, ZAP-70 and CD38 expression and beta-2 microglobulin level (p>0.05 for all parameters). Subsequently, we analyzed the CRBN expression in regard to response and CLL prognosis. Interestingly, we found that patients with CRBN expression in the highest quartile tended to have significantly higher probability of complete response (CR) (50% vs. 10%, Fisher exact test p=0.56). However, although median CRBN expression was highest in patients with CR (median=0.80), it was lower in patients achieving partial remission (PR) (0.24) than in patients who did not responded (0.50), and the response categories did not correlate directly with CRBN expression in CLL cells, (r=0.07, p=0.71). Furthermore, no significant association was found between CRBN expression above and below median and duration of response after thalidomide/fludarabine treatment (28 vs. 29 month, p=0.88). Interestingly, in contrast to previous data in MM, we found that CRBN expression did not correlate with IRF4 expression (r=0.25, p=0.22). Furthermore, analysis of molecular and cellular parameters assessed after initial 7-day thalidomide monotherapy including the changes in TNF levels and regulatory T-cell populations did not reveal any significant relation to basal CRBN levels. In conclusion, we found that high pre-treatment CRBN expression in CLL cells is associated with CR achievement on thalidomide-containing chemotherapy. However, in contrast to published data on MM, CRBN expression in CLL cells does not correlate directly with CLL prognosis or molecular and cytological effects caused by thalidomide. Our results may indicate complex mechanisms of IMiDs activity in CLL and that basal CRBN levels in CLL microenvironment and immune effector T and NK cells may be more important for CLL outcome. Disclosures:Off Label Use: The presentation is a retrospective analysis of a clinical trial on thalidomide that is not approved in CLL. Currently, another IMiD, lenalidomid, is being investigated in CLL with promising activity,and the data from this retrospective study may add to understanding on its action. Robak:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees.

High Cereblon Expression In Lower Risk Myelodysplastic Syndromes With 5q Deletion Is Associated With The Efficacy Of Lenalidomide

IntroductionThe binding of immunomodulatory drugs (IMiDs), including lenalidomide, to CRBN is associated with cytotoxicity of IMiDs used in the treatment of multiple myeloma, myelodysplastic syndromes (MDS) and lymphomas. Cereblon (CRBN) is named for its putative role in cerebral development, especially in memory and learning. CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), cullin-4 (CUL4) and regulator of cullin 1 (ROC1). This complex regulates DNA repair, DNA replication and transcription. CRBN is a primary target of thalidomide teratogenicity (Ito et al., Science 2010; 327:1345-1350). Down-regulation of the CRBN expression is associated with the development of marked IMiDs resistance in human multiple myeloma cells (Zhu et al., Blood 2011; 118: 4771-4779; Lopez Girona et al., Leukemia 2012; 26: 2326-2335; Heintel et al., Br J Haematol 2013; 161: 695-700; Broyl et al., Blood 2013; 121: 624-627; Lodé et al., Br J Haematol 2013; Jul 17. doi: 10.1111/bjh.12478). CRBN expression is thus required for the antimyeloma activity of IMiDs. AimsTo gain insight into the role of CRBN in lower risk myelodysplastic syndromes with or without 5q deletion and into the mechanisms of lenalidomide action, we study CRBN expression in these two groups of lower risk MDS patients and in healthy controls. We further study the expression of DDB1 and IRF4 (interferon regulatory factor 4, one of target genes of CRBN). MethodsMononuclear cells were isolated both from bone marrow [23 low risk MDS patients with 5q- deletion, 37 low-risk MDS patients with normal chromosome 5 (non5q-) and 24 healthy controls] and from peripheral blood [38 patients with 5q- , 52 non 5q- patients and 25 healthy controls] by Ficoll-Paque PLUS gradient separation, and washed with phosphate-buffered saline. The rest of the red cells were lysed. Total RNA was isolated using RNA isolation solvent and complementary DNA was synthesized from total RNA using SuperScript II reverse transcriptase. Relative levels of the CRBN, DDB1 and IRF4 mRNAs were determined by TaqMan-based quantitative real-time PCR and by calculation to the level of housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The experiments were performed in duplicate. Informed consent was obtained from all patients and healthy controls. Evaluation of 2-ddCt indicates the fold change in gene expression relative to the control. ResultsThe median of CRBN mRNA levels in total RNA isolated from peripheral blood mononuclear cells was the highest (3.6) in lower risk MDS with 5q deletion, lower (1.5) in non5q- MDS and lowest (1.2) in healthy controls. Similar results were obtained in bone marrow mononuclear cells (medians 3.3 in 5q- syndrome, 2.2 in non5q- MDS and 1.3 in healthy controls). The differences between the 3 groups were statistically significant (p˂0.05, Mann-Whitney test) with the exception of non5q- MDS in comparison with healthy controls in mononuclear cells of peripheral blood. Very similar results were obtained in the analysis of DDB1 and IRF4 mRNAs levels, which correlated with CRBN levels. We analyzed CRBN mRNA levels before and in the course of the lenalidomide treatment of 5q- low risk MDS (6 patients). We obtained high CRBN mRNA levels before and during the treatment by lenalidomide in all lenalidomide responders (4 patients). We found a sharp decrease of CRBN mRNA in mononuclear cells of bone marrow in two patients who stopped responding to lenalidomide therapy and subsequently progressed to higher risk MDS. A/G polymorphism located at -29 nt of the 5x UTR of CRBN does not act as a biomarker of response to treatment with lenalidomide in 5q- syndrome. Conclusions5q- low risk MDS patients have the highest levels of CRBN mRNA in comparison to lower risk myelodysplastic syndromes with normal chromosome 5 and to healthy controls. DDB1 and IRF4 mRNAs levels correlate with CRBN levels. High levels of CRBN mRNA were detected in all lenalidomide responders during the course of the therapy. Significant decrease of CRBN levels during treatment by lenalidomide is associated with loss of response to treatment and disease progression. Similarly to the treatment of multiple myeloma, these results suggest that high levels of CRBN mRNA are necessary for the efficacy of lenalidomide in low risk 5q- patients.Supported by the MH CR grant NT/13836-4/2012, PRVOUK-27/LF1/2 and by the MH CR project for development of research institute (UHKT). Disclosures:No relevant conflicts of interest to declare.

Responsiveness Of Cytogenetically Discrete Human Myeloma Cell Lines To Lenalidomide: Lack Of Correlation With Cereblon and Interferon Regulatory Factor 4 Expression Levels

The introduction of novel immunomodulatory drugs (IMiDs), such as lenalidomide, has drastically improved the survival of patients with multiple myeloma (MM). While it has been shown that patients with specific cytogenetic subtypes, namely t(4;14), have the best outcomes when treated with bortezomib-based regimens, the relationship between cytogenetic subtypes and response to IMiDs remains unclear. Using DNA synthesis and apoptosis assays, we investigated the relationship between cytogenetic subtype and lenalidomide response in a representative panel of human myeloma cell lines (HMCLs). Additionally, we examined HMCL protein expression levels of the lenalidomide target cereblon (CRBN) and its putative downstream target interferon regulatory factor 4 (IRF4), both of which have previously been shown to be predictive of lenalidomide response in HMCLs. Our results reveal that response to lenalidomide did not correlate with specific cytogenetic translocations. There were, however, distinct groups of lenalidomide-responsive and non-responsive HMCLs, as defined by inhibition of cellular proliferation; notably, all of the hyperdiploid HMCLs fell into the latter category. Repeated dosing of lenalidomide significantly lowered the IC50 of the responsive HMCL ALMC-1 (IC50 = 2.6 μM versus 0.005 μM, p=0.004), but did not have an effect on the IC50 of the non-responsive HMCL DP-6 (p>0.05). Moreover, no association was found between lenalidomide responsiveness and CRBN or IRF4 expression levels. In summary, our data indicate that lenalidomide sensitivity is independent of cytogenetic subtype in HMCLs. Additionally, while CRBN and IRF-4 have been shown to be associated with response to lenalidomide in patients, these findings do not translate back to HMCLs, which could be attributable to factors present in the bone marrow microenvironment. These results suggest that the development of a new treatment paradigm based on cytogenetic subtype of MM alone may be insufficient, and that additional markers may need to be incorporated. Furthermore, the disconnect between our findings of expression of CRBN and IRF-4 in lenalidomide-treated HMCLs and patient data highlights the need for further investigation into the role of the bone marrow microenvironment on the regulation of these previously demonstrated markers of lenalidomide response. Disclosures:No relevant conflicts of interest to declare.