Cereblon Expression Research Articles

Enhanced homeostatic sleep response and decreased neurodegenerative proteins in cereblon knock-out mice

Energy homeostasis and sleep have a bidirectional relationship. Cereblon (CRBN) regulates energy levels by ubiquitinating the AMP-activated protein kinase(AMPK), an energy sensor. However, whether CRBN participates in sleep is unclear. Here, we examine sleep–wake patterns in Crbn+/+ and Crbn−/− mice during 24-h baseline, 6-h sleep deprivation (SD), and following 6-h recovery sleep (RS). At baseline, overall sleep patterns are similar between genotypes. However, SD decreases CRBN expression in Crbn+/+ mice and increases phospho-Tau, phospho-α-synuclein, DNAJA1 (DJ2), and DNAJB1 (DJ1) in both genotypes, with Crbn−/− mice showing a lesser extent of increase in p-Tau and p-α-synuclein and a higher level of heat shock protein 70 (HSP70), DJ2, and DJ1. During RS, Crbn−/− mice show increased slow-wave activity in the low-delta range (0.5–2.5 Hz), suggesting higher homeostatic sleep propensity associated with AMPK hyperactivation. By illuminating the role of CRBN in regulating sleep–wake behaviors through AMPK, we suggest CRBN as a potential therapeutic target for managing sleep disorders and preventing neurodegeneration.

The Relationship Between the Expression of CRBN in Peripheral Blood and the Severity and Prognosis of Adult Sepsis.

To investigate the correlation between the expression of cereblon (CRBN) protein in peripheral blood and the severity and prognosis of sepsis. A total of 130 patients with sepsis admitted to our hospital were selected as the observation subjects (sepsis group). The patients were divided into mild group, moderate group and severe group according to their conditions. The patients were divided into survival group and death group according to their living conditions within 28 days after admission. 130 health individuals were selected as the control group. The levels of CRBN mRNA, CRP and PCT in peripheral blood were detected. The levels of serum CRBN mRNA, CRP, and PCT in patients with sepsis were higher than those in the control group (P<0.05); As the condition worsens, the levels of CRBN mRNA, CRP, and PCT gradually increase, and there are statistically significant differences among patients with mild, moderate, and severe sepsis; Correlation analysis showed that the expression of CRBN mRNA in sepsis patients was positively correlated with CRP, PCT levels, APACHE II score and SOFA score (P<0.05); the 28-day cumulative survival rate of patients with high CRBN mRNA expression was significantly lower than that of patients with low CRBN mRNA expression (P<0.05); compared with the survival group, the levels of serum CRBN mRNA, CRP and PCT in the death group were significantly higher (P<0.05); the AUC of death in sepsis patients diagnosed by CRBN mRNA, CRP and PCT was 0.961, the combined diagnostic efficacy was higher than that of single detection (P<0.05). The expression level of CRBN in the peripheral blood of patients with sepsis is increased, which is related to the severity and prognosis of the patients. The combination of CRP and PCT has certain diagnostic value for the death of sepsis patients.

High levels of CRBN isoform lacking IMiDs binding domain predicts for a worse response to IMiDs-based upfront therapy in newly diagnosed myeloma patients

In recent years, the immunoderivative (IMiD) agents have been extensively used for the treatment of multiple myeloma (MM). IMiDs and their newer derivatives CRBN E3 ligase modulator bind the E3 ligase substrate recognition adapter protein cereblon (CRBN), which has been recognized as one of the IMiDs’ direct target proteins, and it is essential for the therapeutic effect of these agents.High expression of CRBN was associated with improved clinical response in patients with MM treated with IMiDs, further confirming that the expression of IMiDs’ direct target protein CRBN is required for the anti-MM activity. CRBN’s central role as a target of IMiDs suggests potential utility as a predictive biomarker of response or resistance to IMiDs therapy. Additionally, the presence of alternatively spliced variants of CRBN in MM cells, especially those lacking the drug-binding domain for IMiDs, raise questions concerning their potential biological function, making difficult the transcript measurement, which leads to inaccurate overestimation of full-length CRBN transcripts. In sight of this, in the present study, we evaluated the CRBN expression, both full-length and spliced isoforms, by using real-time assay data from 87 patients and RNA sequencing data from 50 patients (n = 137 newly diagnosed MM patients), aiming at defining CRBN’s role as a predictive biomarker for response to IMiDs-based induction therapy. We found that the expression level of the spliced isoform tends to be higher in not-responding patients, confirming that the presence of a more CRBN spliced transcript predicts for lack of IMiDs response.

Incidence of cereblon protein in intensive care patients: a cross-sectional study.

Sepsis remains a common cause of death. The cereblon (CRBN) protein, which is involved in important cellular processes, plays a role in sepsis. This cross-sectional study aimed to show the CRBN protein expression and its effects on patients in the intensive care unit. Samples were taken by deep tracheal aspiration from patients. The presence of CRBN was pathologically investigated using immunohistochemical tests and polyclonal antibodies against CRBN. The relationship between gender, sepsis, steroid, survival and the presence of CRBN was examined. Respiratory and neurologic diseases were the most common reasons for admission. Acinetobacter was the most frequent microorganism. In patients with more than normal inflammatory cells, a negative correlation was observed between CRBN expression and leukocyte rate (p=0.031). In patients with CRBN, there was no correlation between steroid and mortality, APACHE/Glasgow score, hospital stay length, and ICU. Although the prognosis for sepsis is better in CRBN-deficiency animals, the presence of CRBN in humans does not affect it. In our study, CRBN decreased as inflammatory cells increased in the patient's aspiration material. The response to steroids, an immunomodulator, did not change with the presence of the immunomodulator target molecule, CRBN. Therefore, using immunomodulators in the treatment of sepsis should be reconsidered.

Enhancement of the SESN2-SHP cascade by melatonin ameliorates hepatic gluconeogenesis by inhibiting the CRBN-BTG2-CREBH signaling pathway

Melatonin is involved in the regulation of various biological functions. Here, we explored a novel molecular mechanism by which the melatonin-induced sestrin2 (SESN2)-small heterodimer partner (SHP) signaling pathway protects against fasting- and diabetes-mediated hepatic glucose metabolism. Various key gene expression analyses were performed and multiple metabolic changes were assessed in liver specimens and primary hepatocytes of mice and human participants. The expression of the hepatic cereblon (CRBN) and b-cell translocation gene 2 (BTG2) genes was significantly increased in fasting mice, diabetic mice, and patients with diabetes. Overexpression of Crbn and Btg2 increased hepatic gluconeogenesis by enhancing cyclic adenosine monophosphate (cAMP)-responsive element-binding protein H (CREBH), whereas this phenomenon was prominently ablated in Crbn null mice and Btg2-silenced mice. Interestingly, melatonin-induced SESN2 and SHP markedly reduced hepatic glucose metabolism in diabetic mice and primary hepatocytes, and this protective effect of melatonin was strikingly reversed by silencing Sesn2 and Shp. Finally, the melatonin-induced SESN2-SHP signaling pathway inhibited CRBN- and BTG2-mediated hepatic gluconeogenic gene transcription via the competition of BTG2 and the interaction of CREBH. Mitigation of the CRBN-BTG2-CREBH axis by the melatonin-SESN2-SHP signaling network may provide a novel therapeutic strategy to treat metabolic dysfunction due to diabetes.

Abstract 481: Investigating molecular glues as a new therapeutic approach to high-risk neuroblastoma

Abstract High-risk neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Despite extensive study, treatments remain highly toxic and are often insufficient to cure. Thus, there is an urgent need to develop more effective and less toxic treatments for children with NB. NB commonly displays reliance on a relatively small cohort of transcription factors (TFs) and epigenetic proteins to drive cell growth, however, many of these targets are inaccessible with conventional small molecules. In recent years, new modalities such as targeted protein degradation (TPD) have emerged. TPD may permit small molecule degradation of traditionally “undruggable” targets, such as TFs. This makes TPD a particularly attractive modality for defining new therapeutic strategies for high-risk NB. One category of TPD agents are molecular glues (MGs). MGs are monovalent small molecules capable of simultaneously binding to an E3 ligase receptor such as CRBN (cereblon), and a target protein of interest. Importantly, in comparison to conventional protein inhibitors, MGs do not require a conventional binding pocket on the protein of interest to bind to it, and can be utilized in sub-stoichiometric concentrations. Thus, this may result in the recruitment, polyubiquitination, and subsequent proteasomal degradation of proteins previously thought to be “undruggable.” Here, we demonstrate that high-risk NB cells are critically sensitive to a cluster of previously unknown MGs. We performed high-throughput screening of a proprietary library (&amp;gt;4000 unique compounds) of MGs in two MYCN-amplified NB cell lines, Kelly and MHHNB11. These two cell lines demonstrate a strikingly close correlation in Cell-Titer Glo-based growth response to MGs, as compared with an outgroup of other MG-responsive cancer cell lines. We identified a unique subcluster of 112 common MGs that display &amp;gt;70% growth inhibition in both cell lines. To identify the mechanism of cell growth inhibition, we established Kelly cells with stable knockout of CRBN, and coupled these with screening to confirm that the majority of these MGs (84%; 94/112) require CRBN expression. To rule out common off-target effects of MGs, candidate compounds were then tested in cells transgenically expressing either wild-type or degradation-resistant GSPT1 (G575N) constructs, revealing a subset of MGs retaining cytotoxicity without effecting GSPT1 protein levels. Current studies are focused on determining the targets of these high confidence MGs, as a foundation for preclinical in vivo studies. These studies will identify new compounds with defined mechanisms of action, with an ultimate goal of developing new treatments that are more effective and less toxic for children with NB. Citation Format: Ian Delahunty, Gisele Nishiguchi, Yang Zhang, Abigail Fish, Anand Mayasundari, Jun Qi, Brian Abraham, Anang Shelat, Zoran Rankovic, Adam Durbin. Investigating molecular glues as a new therapeutic approach to high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 481.

Multiomics Analysis of IMiD/CELMoD Resistant Multiple Myeloma Models Uncovers Novel and Targetable Vulnerabilities in the SREBP Lipid Synthesis Pathway

Background Acquired resistance to immunomodulatory drugs (IMiDs)/cereblon (CRBN) E3 ligase modulators (CELMoDs) is a major challenge in multiple myeloma (MM) treatment. These drugs bind to the CRBN component of the CRL4CRBN E3 ubiquitin ligase and designate a new set of substrates for degradation via the proteasome. Generation of resistance is frequently associated with decreased CRBN expression and this is due to genetic alteration in ~1/3rd of patients. Alternative drivers of the low CRBN state, and other mechanisms of resistance, need to be elucidated. To tackle this complex problem, MM models with acquired IMiD/CELMoD resistance were generated and multiomics analysis performed. Methods IMiD/CELMoD resistant human MM cell lines were generated by treating MM1s and H929 cells with lenalidomide (Len), pomalidomide (Pom) or iberdomide (Iber) at ~10x GI50 concentration for ~12 weeks until resistance was achieved. Resistant cell lines and controls were characterised by whole exome sequencing (WES), RNA-Seq and proteomics. A genome-wide loss-of-function CRISPR screen (Brunello library) was carried out in Iber-resistant MM1s; gene effect scores were calculated with the Chronos model and compared to parental MM1s using data from the Broad Institute DepMap portal. Pathway analysis was performed using g:Profiler. The Membrane Bound Transcription Factor Peptidase, Site 1 (MBTPS1) inhibitor PF-429242 was used to inhibit activation of the Sterol Regulatory-Element Binding Protein (SREBP) pathway and the effect on cell viability measured using CellTiter-Blue®. The Multiple Myeloma Research Foundation CoMMpass database was used to explore the correlation between mRNA expression of SREBP pathway genes in newly diagnosed patients and progression-free survival (PFS). Results All models were resistant to the IMiD/CELMoD with which they were generated (to 20-100x the GI50 concentration) and exhibited cross-resistance to other IMiDs/CELMoDs. Functional assays showed that well-characterised effects of IMiD/CELMoD treatment, e.g. degradation of Ikaros/Aiolos, were abrogated. WES showed Pom-resistant MM1S and Pom- and Iber-resistant H929 cells had acquired mutations in CRBN predicted to have a high impact on function. Len-resistant H929 cells had new copy number loss at the CRBN locus but Len- and Iber-resistant MM1s cells had no genetic changes in CRBN. CRBN protein expression was reduced in all resistant lines compared to control (log2 fold changes (FCs) by proteomics ranging from -0.25 to -1.95, adj p <0.05). Together these models display diverse resistance mechanisms, reflecting the clinical picture. Proteomic analysis of the resistant lines identified key changes in the SREBP pathway. The proteomes of the resistant lines were heterogeneous and the only pathway with common significant enrichment was SREBP/fatty acid metabolism (on analysis performed per cell line of proteins with significantly decreased expression). Stearoyl-CoA Desaturase (SCD), a key effector of the SREBP pathway, was one of only 5 proteins with significantly altered expression in all 6 cell lines compared to control. SCD had log2FCs ranging from -0.37 to -1.50 (adj p <0.05). Other key pathway members are shown in Figure 1. A genome-wide CRISPR screen using Iber-resistant MM1s cells identified potential new dependencies (gene effect score <-1 in resistant cells and >-0.5 in parental cells) in 47 genes including SCD and MBTPS1. MBTPS1 is critical for activation of the SREBP pathway and demonstrated one of the largest changes in gene effect (-1.4 vs -0.3). The activity of PF-429242, an inhibitor of MBTPS1, was explored in the resistant lines. A significant difference in GI50 between Iber-resistant H929 cells and control was found (1uM and >10uM respectively) with greater activity in the resistant cells. The same pattern was observed with the other resistant H929 cell lines, but not the resistant MM1s. Other pathway inhibitors are being explored. The expression of genes encoding SREBP pathway components was explored in the CoMMpass dataset. High SCD or MBTPS1 mRNA expression was associated with significantly worse PFS (logrank p<0.01), providing a further rationale for targeting the pathway. Conclusions Models representing multiple different IMiD/CELMoD resistance mechanisms have unified alterations in the SREBP pathway, highlighting a role in resistance biology and potential novel and targetable vulnerabilities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mechanistic Studies and a Retrospective Cohort Study: The Interaction between PPAR Agonists and Immunomodulatory Agents in Multiple Myeloma.

Our previous study demonstrated that peroxisome proliferator-activated receptor (PPAR) agonists downregulated cereblon (CRBN) expression and reduced the anti-myeloma activity of lenalidomide in vitro and in vivo. We aimed to determine whether DNA methylation and protein degradation contribute to the effects of PPAR agonists. CRBN promoter methylation status was detected using methylation-specific polymerase chain reaction. The CRBN protein degradation rate was measured using a cycloheximide chase assay. Metabolomic analysis was performed in multiple myeloma (MM) cells treated with PPAR agonists and/or lenalidomide. Our retrospective study determined the effect of co-administration of PPAR agonists with immunomodulatory drugs on the outcomes of patients with MM. CpG islands of the CRBN promoter region became highly methylated upon treatment with PPAR agonists, whereas treatment with PPAR antagonists resulted in unmethylation. The CRBN protein was rapidly degraded after treatment with PPAR agonists. Lenalidomide and fenofibrate showed opposite effects on acylcarnitines and amino acids. Co-administration of immunomodulatory drugs and PPAR agonists was associated with inferior treatment responses and poor survival. Our study provides the first evidence that PPAR agonists reduce CRBN expression through various mechanisms including inducing methylation of CRBN promoter CpG island, enhancing CRBN protein degradation, and affecting metabolomics of MM cells.

EP300 Selectively Controls the Enhancer Landscape of MYCN-Amplified Neuroblastoma.

EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2β. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective in vivo in a CRBN-dependent manner. This article is highlighted in the In This Issue feature, p. 587.

Evaluation of the Prognostic Significance of Cereblon Protein Expression in Multiple Myeloma.

Considerable progress has been made in the treatment of multiple myeloma (MM) patients with the development of various new agents that increased survival rates over the past fifteen years. Cereblon (CRBN) plays an important role in mediating the antitumor effects of immunomodulatory drugs (IMiDs) among these new agents. The aim of our study is to investigate immunohistochemically (IHC) cereblon protein expression status in MM. Immunohistochemically, CRBN expression and its relationship with various prognostic factors were evaluated in bone marrow biopsies of 96 patients with MM in a single centre. Cytoplasmic and nuclear CRBN expression was detected in all neoplastic cells. While a complete or partial response to treatment was obtained in 45 patients, the disease was stable in 13 and progressive in 17 patients. Survival was longer in those treated with IMiD-containing regimens (p = 0.044). Both the survival rate (p = 0.013) and the survival time were significantly increased (p = 0.023) in those who received the treatment protocol containing protease inhibitors. A significant relationship was found between the treatment protocol and treatment response in the chi-squared analysis (p = 0.008). Although the longest survival time - though not statistically significant - was detected in the group treated with protease inhibitors (log rank, p = 0.217). The survival analysis revealed the presence of a relationship between IgG and IgA positivity and survival. In this study, the survival time of the patients who received treatment regimens containing protease inhibitors and IMiD was longer, independent of the presence of strong nuclear CRBN expression. The survival rate was significantly higher in those who used IMiD and protease inhibitors in combination. Since the survival rate was found to be increased in IgG positive cases and we thought that evaluation of immunoglobulin tissue expression in MM cases can provide prognostic prediction.

Prediction Model for Cereblon Expression in Bone Marrow Plasma Cells Based on Blood Markers in Multiple Myeloma Patients.

BackgroundCereblon (CRBN) is a direct target of immunomodulatory drugs (IMiDs) and is known to be sensitive and responsive to IMiD therapy. We evaluated CRBN expression in bone marrow plasma cells and analyzed whether CRBN expression was associated with multiple myeloma prognosis. Lastly, we developed a nomogram model for predicting high CRBN expression based on clinically significant blood markers.MethodsWe evaluated 143 multiple myeloma patients (internal dataset) who underwent bone marrow examinations. For evaluating the prognostic ability of the nomogram model, two external cohorts (235 patients in external dataset 1 and 156 patients in external dataset 2) were analyzed. The expression of CRBN in bone marrow aspirate samples was evaluated using immunohistochemistry. High CRBN expression was defined as the study-defined H-score ≥6.ResultsIn the high CRBN group, the median progression-free survival (PFS) and overall survival (OS) of patients receiving the IMiD-based therapy and non-IMiD therapy were 29 and 10 months for PFS, and NR (not reached) and 54 months for OS, respectively. IMiD-based therapy was significantly associated with better PFS and OS outcomes. High CRBN expression was independently predicted by female sex, high serum free-light chain (FLC) ratio, higher serum M-protein level, and higher β2-microglobulin level. Based on these results, we constructed a new nomogram model to predict high CRBN expression and the effectiveness of IMiD therapy in multiple myeloma.ConclusionThis nomogram could improve the prognostic evaluation of myeloma patients exhibiting high CRBN expression treated with IMiD therapy and might help provide personalized treatment strategies to clinicians.

A Molecular Analysis of Cereblon-Related Immunomodulatory Drug Resistance in Commpass Multiple Myeloma Patients

Immunomodulatory drugs (IMiDs) are commonly used in first-line therapy to treat multiple myeloma (MM), a plasma cell malignancy. Previous studies have shown that cereblon (CRBN) is critical for the efficacy of IMiDs, such as lenolidomide, pomalidomide and thalidomide, in MM. In a small cohort of 9 patients with lenalidomide-resistant MM, CRBN expression decreased 20-90% after treatment. In a MM cell line, acquired deletion of CRBN was the primary genetic event conferring IMiD resistance. Although CRBN expression is copy number (CN) dependent, CN aberrations affecting CRBN are thought to be rare, with less than 2% of patients exhibiting monoallelic deletion of CRBN. We analyzed clinical and molecular data from the Multiple Myeloma Research Foundation (MMRF) CoMMpass trial (NCT145429) IA11 release to better understand the molecular basis underlying CRBN-related IMiD resistance.IMiDs were used in first-line therapy for 75% (728/971) of patients in the CoMMpass cohort. We identified five CRBN mutations in 5/891 (<1%) patients with available whole exome sequencing data at baseline, before therapeutic intervention. Four baseline CRBN mutations were nonsynonymous; two occurred in exon 3, and one each in exon 10 and 11, which are involved in forming the IMiD binding pocket. None of the four patients with both follow up data and CRBN mutations at baseline had relapsed, with follow up ranging from 18-30 months. Despite IMiD treatment in 68/84 (81%) patients between serial timepoints, no patient developed a CRBN mutation at relapse, suggesting that acquired mutations in CRBN do not play a large role in the development of IMiD resistance in treated patients.RNAseq data was available for 32 serial patients of which 23 (72%) were IMiD treated. Only a subset of IMiD-treated patients (13/23, 57%) exhibited a decrease (ranging from 5-75%) in CRBN expression at relapse, however the decrease from baseline (mean = 41.4 transcripts per million (TPM)) to relapse (mean = 26.8 TPM) was significant (p=0.004) in this group. We analyzed the change in transcript expression of nine noncoding and four protein-coding CRBN transcripts from baseline to relapse in this patient subset and found four differentially expressed non-coding transcripts and two differentially expressed protein coding transcripts; CRBN-001 ENST00000231948 (17.4 versus 10.7 mean TPM; p = 0.04) which codes the full length CRBN protein, and CRBN-010 ENST00000450014 (3.3 versus 2.1 mean TPM; p = 0.02) which is missing exons 10 and 11. Reduced expression of CRBN-001 plays the greatest role in the overall reduction of CRBN and since it contains the IMiD binding motifs, likely also plays a greater role in CRBN resistance in IMiD-treated patients. Of the 13 patients who exhibited a decrease in CRBN expression at relapse, 10 had whole genome sequencing (WGS) CN data at both timepoints and 2/10 (20%) patients experienced CRBN CN loss from baseline to relapse. This suggests that CRBN CN loss may account for acquired IMiD resistance in a larger subset of patients than previously reported.Across the cohort, 14/871 (2%) patients with WGS data exhibit CRBN CN loss at baseline, while 342/871 (39%) patients exhibit CRBN CN gain, primarily whole chromosome gains related to trisomies of chr3 in a subset of hyperdiploid patients at baseline. Survival analyses revealed that IMiD-treated patients with CRBN CN gains at baseline exhibit better progression free survival (PFS) (median = 43 months) and overall survival (OS) (median = 55 months) than non IMiD-treated patients with CRBN CN gains (median PFS = 24 months, p<0.0001, median OS not reached, p<0.001). Although not significant, IMiD-treated patients with CRBN CN gains exhibit better PFS than IMiD-treated CN-normal patients (median = 39 months), suggesting that patients with CRBN CN gains at baseline may benefit from an IMiD based treatment regimen. Overall, these results highlight that chr3/CRBN CN gain can be a predictor of good outcome in IMiD treated patients and demonstrate the clinical benefit of obtaining CRBN CN and expression data throughout a MM patient's disease course. DisclosuresNo relevant conflicts of interest to declare.

Correlative Analyses of Cereblon (CRBN), cMYC, IRF4, BLIMP1, and XBP1 with Clinical Outcomes in Stratus (MM-010), a Phase 3b Study of Pomalidomide (POM) + Low-Dose Dexamethasone (LoDEX) in Patients (pts) with Relapsed/Refractory Multiple Myeloma (RRMM)

Correlative Analyses of Cereblon (CRBN), cMYC, IRF4, BLIMP1, and XBP1 with Clinical Outcomes in Stratus (MM-010), a Phase 3b Study of Pomalidomide (POM) + Low-Dose Dexamethasone (LoDEX) in Patients (pts) with Relapsed/Refractory Multiple Myeloma (RRMM)

Higher Expression of Nuclear Cereblon in Bone Marrow Biopsies of Patients with Multiple Myeloma Included in the HOVON-87/Nmsg-18 Trial May be Associated with Longer PFS and OS

IntroductionResponse to treatment in patients with multiple myeloma (MM) is variable. With increasing possibilities of treatment regimens, predictive factors for response are important. Immune modulating agents (IMiDs) require Cereblon (CRBN) for activity. Therefore, the aim of this study is to identify the genes of the CRBN pathway which predict the response to therapy with IMiDs. In this abstract the first results are presented.MethodsParaffin embedded bone marrow biopsies from patients included in HOVON-87/NMSG-18 trial obtained at inclusion were used for this study. In this trial elderly patients with MM were randomized between treatment with Melphalan-Prednisone (MP)-Thalidomide followed by thalidomide maintenance versus MP-Lenalidomide followed by lenalidomide maintenance (Zweegman et al. Blood 2016;127:1109-1116).Bone marrow biopsies were stained with a fully automated dual color, bright-field immunohistochemical (IHC) assay for CRBN, its neosubstrates Ikaros and Aiolos and the downstream targets IRF4 and c-MYC. CD138 was used to identify MM plasma cells in the bone marrow samples. For CRBN, both nuclear and cytoplasmic staining was evaluated. The distribution and intensity of the immunostaining was assessed using the H-score. The H-scores were calculated using the following formula: [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)] and range from 0-300 (0-600 for combined cytoplasmic-nuclear CRBN H-score). For the Cox regression analysis H-scores were corrected by dividing these by a factor 100: hazard rates were considered per 100 points increase of the H-score. 113 bone marrow samples were selected so far. Protein levels were compared between patients with complete response (CR) or very good partial response (VGPR) vs partial response (PR) and no change (NC). Statistical analysis was done using univariate Cox regression analysis for progression free survival (PFS) and overall survival (OS), and Mann-Whitney test for comparing response groups. Kaplan-Meier survival curves were generated to illustrate survival.ResultsSo far, bone marrow samples obtained at diagnosis from 34 patients were evaluated. Fourteen patients were treated in the Thalidomide arm vs 20 patients in the Lenalidomide arm. Median age was 72 years [range 60-84]. ISS stages I/II/III were 38%/47%/15% respectively. At the time of analysis, median follow up of the 16 patients still alive was 53 months [range 18-85 months]. Best response on protocol treatment was sCR in 12%, CR in 9%, VGPR in 35%, PR in 29% and NC in 15%.Comparing protein expression across the response groups showed a trend of higher levels of combined cytoplasmic and nuclear CRBN in patients who responded better (sCR/CR/VGPR; median H-score: 360 (90-544)) compared to patients with a worse response (PR/NC; median H-score: 273 (185-471)) Mann-Whitney p-value=0.09). In addition, a trend for increased Aiolos expression to be associated with poorer response was found (Mann-Whitney p=0.08; median H-score sCR/CR/VGPR: 251 (162-260) vs PR/NC: 254 (221-267)).Higher H-score of nuclear staining of CRBN was associated with a longer PFS and OS, with a hazard ratio (HR) of 0.47 for PFS (95% confidence interval (95% CI)=0.24-0.92, p=0.027) and a HR of 0.42 for OS (95% CI=0.19-0.91; p=0.028). Using the median nuclear CRBN expression as an arbitrary cut-off, a significant separation of the lines representing high and low expression could be found for PFS, but not for OS (Figure 1). None of the other markers were associated with survival in the current limited set.Six patients had high-risk FISH (deletion of 17p and/or translocation t(4;14) and/or translocation t(14;16)) out of 24 patients with known FISH status. These patients did not demonstrate a higher or lower expression of any of the markers evaluated, and showed no association with PFS (log rank p=0.2) but a clear association with shorter OS (log rank p=0.008). As expected, no association between arm and survival was seen.ConclusionsIn this study we demonstrate that higher expression of nuclear CRBN in myeloma cells in bone marrow of patients with MM was associated with a superior PFS and OS. These are early data from an ongoing analysis; a total of 113 bone marrow samples were selected up to now. [Display omitted] DisclosuresCouto:Celgene: Employment, Equity Ownership. Ren:Celgene: Employment, Equity Ownership. Wang:Celgene: Employment, Equity Ownership. Qian:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership. Broyl:Amgen: Honoraria; Janssen: Honoraria; Celgene: Honoraria. Zweegman:Amgen: Other: advisory board participation; Takeda: Other: advisory board participation, Research Funding; Janssen: Other: advisory board participation, Research Funding; Celgene: Other: advisory board participation, Research Funding. Sonneveld:Celgene, Amgen, Janssen, Karyopharm, Takeda: Consultancy, Honoraria, Research Funding; Celgene Corporation, Amgen, Janssen, Karyopharm, PharmaMar, SkylineDx: Honoraria; Celgene Corporation, Amgen, Janssen, Karyopharm, SkylineDx, PharmaMar: Consultancy.

Pan-PIM Kinase Inhibitors Enhance Lenalidomide's Anti-Myeloma Activity Via Cereblon-IKZF1/3 Cascade

Pan-PIM Kinase Inhibitors Enhance Lenalidomide's Anti-myeloma Activity Via Cereblon-IKZF1/3 CascadeYonggang Sha1, Jing Zheng2, Jianda Hu2+, and Yubin Kang1+1Division of Hematologic Malignancies and Cellular Therapy, Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA; 2Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, China+ Corresponding author: Yubin Kang, MD, Email: yubin.kang@duke.edu; and Jianda Hu, Email: drjiandahu@163.com.ABSTRACTPurpose: Multiple myeloma (MM) remains an incurable disease, and continuous efforts are required to develop novel therapeutic agents and novel drug combinations with more effective anti-myeloma activity. Immunomodulatory agents (IMiDs) including lenalidomide are one of the mainstays in the treatment of MM. It is important to identify and develop novel agents that can work with IMiDs for enhanced anti-myeloma effects. Here, we determined anti-myeloma effect of pan-PIM inhibitors and the synergistic anti-myeloma effects of pan-PIM inhibitors with IMiDs (lenalidomide).Experimental Design: The anti-myeloma activities of pan-PIM inhibitors (SGI1776 and CX6258) and the synergistic anti-myeloma effects of pan-PIM inhibitors with lenalidomide were determined in human myeloma cells, transplanted VK*MYC myeloma mice, and myeloma xenograft mouse models. Western blot, protein stability assay, shRNA specific gene knockdown, and ubiquitination assay were performed to determine the molecular mechanisms involved.Results: Pan-PIM inhibitors (SGI1776 and CX6258) induced the apoptosis of MM cells and exhibited significant anti-myeloma activities in human myeloma cells and in transplanted VK*MYC myeloma mice. Combination of PIM inhibitors with lenalidomide showed synergistic anti-myeloma effects without additional hematological or liver toxicities in vivo in myeloma xenograft mouse models. The treatments of pan-PIM inhibitors promoted ubiquitination and degradation of IKZF1 and IKZF3, the two transcription factors important for the survival of myeloma cells, and combining Pan-PIM inhibitors with lenalidomide leds to more effective degradation of IKZF1 and IKZF3 in vitro in human multiple myeloma cells and in vivo in myeloma xenograft mouse model. We further demonstrated that treatments with pan-PIM inhibitors resulted in an increased expression of Cereblon, and knockdown of Cereblon with shRNA abolished the effects of pan-PIM inhibitors on IKZF1 and IKZF3, demonstrating a central role of Cereblon in Pan-PIM inhibitor-mediated IKZF1/3 degradation.Conclusions: We demonstrated that pan-PIM inhibitors (SGI1776 and CX6258) exhibited significant anti-myeloma activities and combination of pan-PIM inhibitors with lenalidomide showed synergistic anti-myeloma effects without additional hematological or liver toxicities. Mechanistically, pan-PIM kinase inhibitors enhance lenalidomide's anti-myeloma activity via Cereblon-IKZF1/3 Cascade. Our study provides molecular rationale and justification for clinical trials combining pan-PIM inhibitors and lenalidomide for the treatment of myeloma. DisclosuresNo relevant conflicts of interest to declare.

The CRBN, CUL4A and DDB1 Expression Predicts the Response to Immunomodulatory Drugs and Survival of Multiple Myeloma Patients

Immunomodulatory drugs (IMiDs) are effective in the treatment of multiple myeloma (MM), myelodysplastic syndrome with deletion of chromosome 5q and other haematological malignancies. Recent studies showed that IMiDs bind to cereblon (CRBN), a substrate receptor of the CRL4–CRBN complex, to induce the ubiquitination and degradation of IKZF1 and IKZF3 in MM cells, contributing to their anti-myeloma activity. We aimed to determine whether the CRL4–CRBN complex proteins’ expression predicts the prognosis of MM patients treated with IMiDs. Here, we evaluated the expression of CRL4–CRBN complex proteins and their downstream targets with immunohistochemistry (IHC) staining in 130 bone marrow samples from MM patients treated with thalidomide or lenalidomide-based regimens. We found that the expression of CRBN and CUL4A was associated with the superior IMiD-based treatment response (p = 0.007 and p = 0.007, respectively). Moreover, the CUL4A expression was associated with improved PFS (HR = 0.66, 95% CI 0.44–0.99; p = 0.046) and DDB1 expression showed a negative impact on OS both in the univariate (HR = 2.75, 95% CI 1.65–4.61; p = 0.001) and the multivariate (HR 3.67; 95% CI 1.79–7.49; p < 0.001) analysis. Overall, our data suggest that the expression of DDB1, CUL4A and CRBN assessed by IHC predicts the clinical course of MM patients and identifies patients with a high probability of responding to IMiD-based therapy.

Regulation of AMPK Activity by CRBN Is Independent of the Thalidomide-CRL4CRBN Protein Degradation Axis.

Cereblon (CRBN), a primary target of immune-modulatory imide drugs (IMiDs), functions as a substrate receptor in the CUL4-RBX1-DDB1-CRBN (known as CRL4CRBN) E3 ubiquitin ligase complex. Binding of IMiDs to CRBN redirects the CRL4CRBN E3 ubiquitin ligase to recruit or displace its substrates. Interaction between CRBN and the AMPK α subunit leads to CRL4CRBN-dependent degradation of the γ subunit and inhibits AMPK activity. However, the effect of thalidomide on the function of CRBN as a negative regulator of AMPK through interaction with the α subunit remains unclear. Here, we show that thalidomide does not affect AMPK activation or the binding affinity between CRBN and the AMPK α subunit. Thalidomide had no effect on AMPK activity independent of CRBN expression. The N-terminal region and C-terminal tail of CRBN, which is distinct from the IMiD binding site, were critical for interaction with the AMPK α subunit. The present results suggest that CRL4CRBN negatively regulates AMPK through a pathway independent from the CRBN-IMiD binding region.

Cereblon Regulates the Proteotoxicity of Tau by Tuning the Chaperone Activity of DNAJA1.

Protein aggregation can induce explicit neurotoxic events that trigger a number of presently untreatable neurodegenerative disorders. Chaperones, on the other hand, play a neuroprotective role because of their ability to unfold and refold abnormal proteins. The progressive nature of neurotoxic events makes it important to discover endogenous factors that affect pathologic and molecular phenotypes of neurodegeneration in animal models. Here, we identified microtubule-associated protein tau, and chaperones Hsp70 (heat shock protein 70) and DNAJA1 (DJ2) as endogenous substrates of cereblon (CRBN), a substrate-recruiting subunit of cullin4-RING-E3-ligase. This recruitment results in ubiquitin-mediated degradation of tau, Hsp70, and DJ2. Knocking out CRBN enhances the chaperone activity of DJ2, resulting in decreased phosphorylation and aggregation of tau, improved association of tau with microtubules, and reduced accumulation of pathologic tau across brain. Functionally abundant DJ2 could prevent tau aggregation induced by various factors like okadaic acid and heparin. Depletion of CRBN also decreases the activity of tau-kinases including GSK3α/β, ERK, and p38. Intriguingly, we found a high expression of CRBN and low levels of DJ2 in neuronal tissues of 5XFAD and APP knock-in male mouse models of Alzheimer's disease. This implies that CRBN-mediated DJ2/Hsp70 pathway may be compromised in neurodegeneration. Being one of the primary pathogenic events, elevated CRBN can be a contributing factor for tauopathies. Our data provide a functional link between CRBN and DJ2/Hsp70 chaperone machinery in abolishing the cytotoxicity of aggregation-prone tau and suggest that Crbn-/- mice serve as an animal model of resistance against tauopathies for further exploration of the molecular mechanisms of neurodegeneration.

BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL.MethodsExpression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP).ResultsBRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo.ConclusionsBRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

Multiomic Analysis of Cereblon Expression and Its Prognostic Value in Kidney Renal Clear Cell Carcinoma, Lung Adenocarcinoma, and Skin Cutaneous Melanoma

Cereblon (CRBN) is a component of the E3 ubiquitin ligase complex that plays crucial roles in various cellular processes. However, no systematic studies on the expression and functions of CRBN in solid tumors have been conducted to date. Here, we analyzed CRBN expression and its clinical value using several bioinformatic databases. CRBN mRNA expression was downregulated in various cancer types compared to normal cells. Survival analysis demonstrated that overall survival was significantly positively correlated with CRBN expression in some cancer types including lung adenocarcinoma (LUAD), kidney renal clear cell carcinoma (KIRC), and skin cutaneous melanoma (SKCM). CRBN expression was downregulated regardless of clinicopathological characteristics in LUAD and KIRC. Analysis of genes that are commonly correlated with CRBN expression among KIRC, LUAD, and SKCM samples elucidated the potential CRBN-associated mechanisms of cancer progression. Overall, this study revealed the prognostic value of CRBN and its potential associated mechanisms, which may facilitate the development of anti-cancer therapeutic agents.