Nek2 Stabilization By Usp7 Leads to Activation of NF-Kb in Multiple Myeloma

Abstract NIMA (Never In Mitosis Gene A)-Related Kinase 2 (Nek2), a centrosomal Serine/Threonine kinase, is a key player in numerous malignancies. Overexpression of Nek2 has been related to many cancers including Multiple Myeloma (MM). In MM, Nek2 is one of the chromosomal instability genes associated with drug resistance and disease relapse. However, very little is known about the mechanisms that lead to these Nek2-driven disparities. Here, we show that the Ubiquitin Specific Peptidase 7 (USP7) stabilizes Nek2 leading to activation of NF-kb pathway. Using gene expression profile (GEP) data from patients and cell lines we discovered that Nek2 overexpression leads to increases of several targets of the NF-kb pathway. We, thus, hypothesize that Nek2 is activating NF-kb. To address this, we overexpressed Nek2 and tested the classic canonical NF-kb hallmarks proteins by western blotting. Nek2 overexpression led to an increase in phosphorylation of IKK, activator of NF-kb, and to decrease levels of IKb-alpha, a negative regulator of the pathway. Nek2 overexpression also increased nuclear and phosphorylated p65 on residue S536, known as active transcriptional site. To further confirm that Nek2 is activating canonical NF-kb luciferase assay was performed. The luciferase reporter is driven by a p65 promoter and in cells overexpressing Nek2 luciferase levels were increased. To characterize Nek2 interacting partners a tandem affinity purification/mass spectrometry (TAP/MS) approach was performed. We found that Nek2 binds to Usp7, a deubiquitinase overexpressed in numerous cancers. This led to hypothesize that that Nek2, a known target of the ubiquitin proteasome system, is being stabilized by the Usp7 contributing to its overexpression and the increased activation of the NF-kb pathway. To test our hypothesis, we treated different cancer cell lines with the commercially available Usp7 inhibitor, P5091, or silenced the protein using shRNA. In both case, we found a reduction in Nek2 protein level. Additionally, we overexpressed Usp7 and Nek2 increased confirming that Usp7 stabilizes Nek2. To further show that Usp7 stabilizes Nek2 by de-ubiquitination, we overexpressed Usp7 and analyzed Nek2 ubiquitination after immunoprecipitation. When Usp7 was overexpressed no ubiquitination of Nek2 was detected. Finally, by using GEP data from MM patients, we found that individuals who overexpressed Nek2 along with an active NF-kb signature have worst event free survival as well as overall survival, indicating Nek2 overexpression leading to increased NF-kb signature has clinical significance. Disclosures No relevant conflicts of interest to declare.

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Epigenetic Silencing Of Mir-137 Contributes To The Proliferation Of Multiple Myeloma By Targeting AURKA

Blood ◽

10.1182/blood.v122.21.3144.3144 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3144-3144

Author(s):

Yu Qin ◽

Fei Li ◽

Gang An ◽

Mu Hao ◽

Meirong Zang ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cpg Island ◽

Methylation Status ◽

Ectopic Expression ◽

Mrna Translation ◽

Negative Control ◽

Mitogen Activated Protein Kinase ◽

Luciferase Reporter

Abstract Background MicroRNAs are non-coding small RNAs that modulate protein expression and are implicated in the pathogenesis of much kind of cancers. miR-137 was reported to act as a tumor suppressor in different cancers. In the present study, we describe the epigenetic regulation of miR-137 and its contribution in Multiple Myeloma (MM). Methods and Results Real-time RT-PCR was used to screen the expression levels of miR-137 in MM cell lines and CD138+ cell sorted from MM patients, which confirmed the downregulation of miR-137 in MM cell lines and patients, and the expression of cell lines increasing treated with epigenetic drugs 5-aza-dC. The methylation status of miR-137 CpG island was determined by bisulfite pyrosequencing and methylation specific polymerase chain reaction (MSP). Methylation of the miR-137 CpG island was frequently observed in MM cell lines and patients but not in healthy donor and MGUS. Cck-8 assay showed transfection of miR-137 precursor in MM cells significantly inhibited cell proliferation and increased cell drug sensitivity. Importantly, Ectopic expression of miR-137 in MM cells inhibited phosphorylation of mitogen-activated protein kinase (MAPK/ERK). To further identify miR-137 targets, we used bioinformatics analysis and confirmed using a luciferase reporter assay. To validate AURKA as miR-137 target, we cloned the 3' UTR sequence of human AURKA into the luciferase-expressing vector psiCHECK. 293Tcells were transiently transfected with this construct in the presence of pre-miR-137 or a scrambled oligonucleotide acting as a negative control. As reported in luciferase plate reader, miR-137 significantly reduced luciferase activity compared with the scrambled control miRNA. This indicated that miR-137 binds to the 3'UTR of AURKA and impairs its mRNA translation. Furthermore, NCI-H929 cells were transfected with AURKA shRNA vector psiHIV-mH1-AURK and westernblot showed phosphorylation of ERK was also significantly decreased. Conclusion miR-137 was epigenetic Silenced and targeted AURKA expression to contribute to the proliferation through MAPK/ERK pathway in MM. Disclosures: No relevant conflicts of interest to declare.

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MiR-137 Contributes to Drug Susceptibility and Chromosomal Instability of Multiple Myeloma through Targeting AURKA

Blood ◽

10.1182/blood.v124.21.5715.5715 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5715-5715

Author(s):

Yu Qin ◽

Fei Li ◽

Xiaoqi Qin ◽

Gang An ◽

Mu Hao ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Chromosomal Instability ◽

Drug Susceptibility ◽

Soft Agar ◽

Scid Mice ◽

Luciferase Assay ◽

Luciferase Reporter ◽

Cgh Array ◽

Apoptosis Assay

Abstract Background: MicroRNAs are non-coding small RNAs that modulate protein expression and implicated in the pathogenesis of much kind of cancers, including multiple myeloma (MM). Previous study revealed that mic-137 was significant down regulated in MM patients compared with health donors. The purpose of this study is to investigate how miR-137 involved in pathogenesis and drug resistance in MM and its potential as prognostic biomarker. Materials and Methods: Real-time RT-PCR was performed to identify the expression of miR-137 in 6 MM cell lines and CD138+ cell sorted from 21 MM patients and 10 healthy donors. The methylation status of miR-137 CpG island was determined by bisulfite pyrosequencing, methylation specific polymerase chain reaction （MSP）and bisulfite sequencing PCR (BSP). The functional roles of miR-137 in MM were characterized by CCK-8 assay, soft agar clonogenic- formation, standard apoptosis assay and CGH array (CytoScan"HD Array, Affymetrix). Effect of miR-137 on MM progression in vivo was assessed in the NOD/SCID mice models. In addition, to further identify miR-137 targets, we used bioinformatics analysis and confirmed by luciferase reporter assay. In addition, a total of 19 paired sequential samples with GEP and clinical data, obtained from published studies, were analyzed. Results: The expression of miR-137 was down regulated in all 6 MM cell lines and MM patients (p=0.0017). Further study revealed that methylation of the miR-137 CpG islands was frequently observed in MM cell lines (p<0.0001) and patients (p=0.004) compared with healthy donor by MSP and BSP. Functional study of Cck-8 assay, soft agar clonogenic assay and standard apoptosis assay showed miR-137-OE NCI-H929 significantly inhibited cell proliferation and increased cell drug sensitivity compared with NCI-H929-EV in vitro. Meanwhile, the tumorigenicity of miR-137 overexpressed NCI-H929 reduces tumor volume in xenograft models at day 20 and prolonged the survival of NOD-SCID mice following tail vein injection of miR-137-OE NCI-H929 compared with control (p=0.0198). Overexpression of miR-137 inactivates both AKT and MAPK/ERK Signaling pathway by up-regulating p53 and down-regulating pAKT. Interestingly, CGH-array analysis indicated miR-137 overexpression could decrease chromosomal instability in NCI-H929, such as gain at 1q21, loss at 12p13.31 and 14q22.2 etc. To explore the mechanism of miR-137 involved in chromosome instability, our results demonstrated miR-137 mainly through up regulated AURKA expression and luciferase assay confirmed miR-137 targeted at position 374-380 of AURKA 3’UTR. Conclusion: Our data suggested that miR-137 was epigenetic silenced and targeted AURKA expression to contribute to drug susceptibility and chromosomal Instability in MM. Future studies will be performed on how miR-137 targets AURKA linked to the p53 pathway and evaluating miR-137 as prognostic biomarkers and targets for treatment in MM. Disclosures No relevant conflicts of interest to declare.

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Super-Enhancer Profiling Identifies Novel Oncogenes and Therapeutic Targets in Multiple Myeloma

Blood ◽

10.1182/blood-2019-124252 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 362-362

Author(s):

Jianbiao Zhou ◽

Yunlu Jia ◽

Tze King Tan ◽

Tae-Hoon Chung ◽

Takaomi Sanda ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Plasma Cell ◽

Conflicts Of Interest ◽

Cell Cancer ◽

Ectopic Expression ◽

Therapeutic Targets ◽

Physical Interaction ◽

Rna Seq ◽

Normal Plasma

Background: Multiple myeloma (MM) is an aggressive neoplastic plasma cell cancer characterized by diversely cytogenetic abnormalities. MM can be divided into subtypes with immunoglobulin heavy chain (IGH) gene translocations involving CCND1-3, FGFR3/MMSET, MAFs and hyperdiploid myeloma containing trisomies of several odd numbered chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. Although several new drugs have been introduced into clinic, treatment for MM patients remains challenge and refractory/resistant to therapy is often seen. Thus, a better understanding of the molecular pathogenesis of MM can lead to generate new prognostic classification and identify new therapeutic targets. Super-enhancers (SEs) are defined as large clusters of cis-acting enhancers, marked by high level bindings of acetylation of histone H3 lysine 27 (H3K27ac) and mediator complex. SEs have been shown to control genes for maintaining cellular identity and also key tumor drivers in various malignancies. Methods: H3K27Ac ChIP-seq and RNA-seq were performed on primary MM patient samples, MM cell lines. Normal plasma cells and lymphoma cell lines were served as controls. We systematically compared SEs and their associated genes of normal and cancerous tissue. THZ1, a CDK7 inhibitor, was used to efficiently down-regulate SE-associated genes. Combinatory analysis of THZ1-sensitive and SE-associated gene revealed a number of promising MM oncogenes. CRISPR/Cas9 technology and ectopic expression experiments in conjunction with cellular functional assays were performed to determine the effects of candidate SE-genes on MM cells. Circularized chromatin conformation capture followed by sequencing (4C-seq) was applied to explore the direct contact of SE and promoter. Results: SE analysis uncovered some cell lineage-specific transcription factors (TFs) and known oncogenes in MM. Several key TFs, including IRF4, PRDM1, MYC and XBP1, were identified in most MM samples, confirming the origin of MM cells. These data reinforce the concept that SE establishment is a key component of MM biology. The acquisition of SEs around oncogene drivers is widely observed during tumorigenesis. ST3GAL6 and ADM were two known oncogenic drivers in myeloma cells, which were associated with super-enhancers in all MM samples but not in normal plasma cell and lymphoma cells. We also found SE constituents for multiple subtype-specific key oncogenes such as CCND1 in t(11;14) cells, C-MAF in t(14;16) cells, and NSD2 and FGFR3 in t(4;14) cells. Furthermore, THZ1 showed prominent anti-neoplastic effect against MM cells. SE-associated genes were more sensitive to THZ1 compared with those genes associated with typical enhancers (TEs). By overlapping THZ1-sensitve gene with SE-associated genes, we identified a number of novel MM oncogenes, including MAGI2, EDEM3, HJURP, LAMP5, MBD1 and UCK2 as a potential druggable kinase. The expression level of MAGI2 and HJURP confers poor prognosis in several MM datasets. MAGI2 silencing in MM cells decreased cell proliferation and induced apoptosis. qRT-PCR and Western blot analysis confirmed the overexpression of HJURP in t(4;14) cells relative to non-t(4;14) MM cells. Furthermore, 4C-seq analysis revealed the physical interaction between HJURP-SE and promoter and THZ1 treatment diminished this interaction. Motif search at SE constituents revealed a highly significant enrichment of NSD2 recognition. Significant reduction of NSD2 binding at HJURP-SE region was observed in KMS11 infected with NSD2-specific shRNAs. Interestingly, blocking SE sites by CRISPR/Cas9i or silencing HJURP by shRNA led to decreased HJURP expression and cell apoptosis, whereas overexpression of this gene promoted cell growth. Taken together, our data demonstrated that HJURP is a novel SE-associated oncogene in t(4;14) MM. Conclusions: Our integrative approaches by combing H3K27Ac ChIP-seq, RNA-seq and THZ1-sensitive transcript defined the landscape of SE and identified SE-associated novel oncogenes, as well as lineage-specific TFs in MM. Furthermore, we also revealed subtype-specific SE-driving oncogenic program in MM. Taken together, these results not provide novel insight into the MM pathology, but also offer novel, potential therapeutic targets, such as MAGI2, and HJURP for the treatment of MM patients. Disclosures No relevant conflicts of interest to declare.

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CD40 activation mediates p53-dependent cell cycle regulation in human multiple myeloma cell lines

Blood ◽

10.1182/blood.v95.3.1039.003k02_1039_1046 ◽

2000 ◽

Vol 95 (3) ◽

pp. 1039-1046 ◽

Cited By ~ 42

Author(s):

G. Teoh ◽

Y.-T. Tai ◽

M. Urashima ◽

S. Shirahama ◽

M. Matsuzaki ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Growth Arrest ◽

Myeloma Cell ◽

Luciferase Reporter ◽

Temperature Sensitive ◽

Multiple Myeloma Cell ◽

Cd40 Activation ◽

Human Multiple Myeloma ◽

Rpmi 8226

It has been reported that the activation of multiple myeloma (MM) cells by CD40 induces proliferation, growth arrest, and apoptosis. To determine whether the biologic sequelae of CD40 activation in MM cells depends on p53 function, we identified temperature-sensitive p53 mutations in the RPMI 8226 (tsp53E285K) and the HS Sultan (tsp53Y163H) MM cell lines. These cells were then used as a model system of inducible wtp53-like function because wild-type-like p53 is induced at permissive (30°C) but not at restrictive (37°C) temperatures. Using p21-luciferase reporter assays, we confirmed that CD40 induces p53 transactivation in RPMI 8226 and HS Sultan cells cultured under permissive, but not restrictive, conditions. Furthermore, CD40 activation of these MM cells under permissive, but not restrictive, temperatures increased the expression of p53 and p21 mRNA and protein. Importantly, CD40 activation induced the proliferation of RPMI 8226 and HS Sultan cells at restrictive temperatures and growth arrest and increased subG1 phase cells at permissive temperatures. These data confirmed that CD40 activation might have distinct biologic sequelae in MM cells, depending on their p53 status.

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USP7 inhibits Wnt/β-catenin signaling through promoting stabilization of Axin

Nature Communications ◽

10.1038/s41467-019-12143-3 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 14

Author(s):

Lei Ji ◽

Bo Lu ◽

Raffaella Zamponi ◽

Olga Charlat ◽

Robert Aversa ◽

...

Keyword(s):

Osteoblast Differentiation ◽

Adipocyte Differentiation ◽

Ubiquitin Proteasome System ◽

Cellular Systems ◽

Negative Regulator ◽

Scaffolding Protein ◽

Complex Stability ◽

Genetic Ablation ◽

Ubiquitin Proteasome ◽

Potent Negative Regulator

Abstract Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/β-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/β-catenin signaling.

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Ubiquitination and Ubiquitin-Like Modifications in Multiple Myeloma: Biology and Therapy

Cancers ◽

10.3390/cancers12123764 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3764

Author(s):

Matthias Wirth ◽

Markus Schick ◽

Ulrich Keller ◽

Jan Krönke

Keyword(s):

Multiple Myeloma ◽

Translational Regulation ◽

Proteasome Inhibitors ◽

Ubiquitin Proteasome System ◽

Organ Damage ◽

Post Translational Modifications ◽

Damage Repair ◽

Ubiquitin Proteasome ◽

New Treatments ◽

Effective Drugs

Multiple myeloma is a genetically heterogeneous plasma cell malignancy characterized by organ damage and a massive production of (in-)complete monoclonal antibodies. Coping with protein homeostasis and post-translational regulation is therefore essential for multiple myeloma cells to survive. Furthermore, post-translational modifications such as ubiquitination and SUMOylation play key roles in essential pathways in multiple myeloma, including NFκB signaling, epigenetic regulation, as well as DNA damage repair. Drugs modulating the ubiquitin–proteasome system, such as proteasome inhibitors and thalidomide analogs, are approved and highly effective drugs in multiple myeloma. In this review, we focus on ubiquitin and ubiquitin-like modifications in the biology and current developments of new treatments for multiple myeloma.

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Use of Ubiquitin-Proteasome System Profiling for Differentiating Between Various Leukemic Processes.

Blood ◽

10.1182/blood.v114.22.4712.4712 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4712-4712

Author(s):

Ke Zhang ◽

Hagop M. Kantarjian ◽

Wanlong Ma ◽

XI Zhang ◽

Xiuqiang Wang ◽

...

Keyword(s):

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Blast Crisis ◽

Chronic Phase ◽

Direct Analysis ◽

Cell Types ◽

Ubiquitin Proteasome System ◽

Lymphocytic Leukemia ◽

Ubiquitin Proteasome

Abstract Abstract 4712 The ubiquitin-proteasome system (UPS) plays a major role in cell homeostasis in normal and neoplastic states. Expression and function of the UPS system vary with the specific characteristics of individual cell types, suggesting that determination of UPS “signatures” could be useful in identifying various cell populations. Since direct analysis of cancer cells is often problematic, even in hematologic diseases, we explored the potential of using UPS signatures in plasma to differentiate between various leukemias. We first analyzed plasma UPS profiles of patients with acute myeloid leukemia (AML; n=111), acute lymphoblastic leukemia (ALL; n=29), advanced myelodysplastic syndrome (MDS; n=20), chronic lymphocytic leukemia (CLL; n=118), or chronic myeloid leukemia (CML; n=128; 46 in accelerated/blast crisis [ACC/BL], 82 in chronic phase), and 85 healthy control subjects. Plasma levels of proteasome, ubiquitin (poly-ubiquitin), and the 3 proteasome enzymatic activities (chymotrypsin-like [Ch-L], caspase-like [Cas-L], trypsin-like [Tr-L]) were measured. Specific activities were calculated by normalizing each of the 3 enzyme activities to the levels of proteasome protein in plasma (Ch-L/p, Cas-L/p, and Tr-L/p). These 8 variables were used in multivariate logistic regression models to differentiate between leukemic processes. UPS signatures provided clear differentiation between patients with a leukemic process and normal controls (AUC=0.991), using 6 different variables (Tr-L/P, Ch-L, Ch-L/p, Cas-L, Cas-L/P, ubiquitin). Distinguishing between acute (AML, ALL, MDS) and chronic (CML, CLL) processes was less efficient (AUC=0.853 using Tr-L, Tr-L/P, Cas-L/P, Ch-L/P, proteasome, Ch-L), likely due to the high proportion (36%) of CML patients in ACC/BL phase. However, UPS signatures generally yielded powerful differentiation between individual leukemias (Table). MDS was not well differentiated from AML (AUC=0.791), reflecting the significant biological overlap of these diseases. These data support the potential usefulness of the UPS profile to aid in the differential diagnosis of various leukemias. Disclosures: No relevant conflicts of interest to declare.

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HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.832.832 ◽

2009 ◽

Vol 114 (22) ◽

pp. 832-832 ◽

Cited By ~ 1

Author(s):

Michael A Chapman ◽

Jean-Philippe Brunet ◽

Jonathan J Keats ◽

Angela Baker ◽

Mazhar Adli ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Histone Modification ◽

Cell Lines ◽

Therapeutic Target ◽

Copy Number ◽

Conflicts Of Interest ◽

Specific Expression ◽

Aberrant Expression ◽

High Level

Abstract Abstract 832 We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM. Disclosures: No relevant conflicts of interest to declare.

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Transcriptional Silencing of the Wnt-Antagonist Dickkopf-1 (DKK1) by Promoter Methylation Unleashes Aberrant Wnt Signaling In Advanced Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.1919.1919 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1919-1919

Author(s):

Kinga A Kocemba ◽

Richard W Groen ◽

Harmen van Andel ◽

Karene Mahtouk ◽

Marie Jose Kersten ◽

...

Keyword(s):

Multiple Myeloma ◽

Wnt Signaling ◽

Cell Lines ◽

Wnt Pathway ◽

Conflicts Of Interest ◽

Cpg Island ◽

Nuclear Accumulation ◽

Aberrant Methylation ◽

Dkk1 Expression ◽

Osteolytic Bone Disease

Abstract Abstract 1919 Aberrant activation of the Wnt/β-catenin pathway is implicated in driving the formation of various human cancers. Recent studies indicate that the Wnt pathway plays at least two distinct roles in the pathogenesis of multiple myeloma (MM): i) Aberrant, presumably autocrine, activation of canonical Wnt signaling in MM cells promotes tumor proliferation and metastasis; ii) Overexpression of the Wnt inhibitor Dickkopf1 (DKK1), contributes to osteolytic bone disease by inhibiting osteoblast differentiation. Since DKK1 itself is a target of TCF/β-catenin mediated transcription, these findings suggests the presence of a negative feedback loop in MM, in which DKK1 acts as a potential tumor suppressor. In line with this hypothesis, we show here that DKK1 expression is lost in most MM cell lines and in a subset of patients with advanced MM. This loss is correlated with activation of the Wnt pathway, as demonstrated by increased nuclear accumulation of β-catenin. Analysis of the DKK1 promoter revealed CpG island methylation in several MM cell lines as well as in MM cells from patients with advanced MM. Moreover, demethylation of the DKK1 promoter restores DKK1 expression, which results in inhibition of β-catenin/TCF-mediated gene transcription in MM lines. Taken together, our data identify aberrant methylation of the DKK1 promoter as a cause of DKK1 silencing in advanced stage MM, which may play an important role in the progression of MM by unleashing Wnt signaling. Disclosures: No relevant conflicts of interest to declare.

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Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells

Blood ◽

10.1182/blood.v116.21.1915.1915 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1915-1915

Author(s):

Unn-Merete Fagerli ◽

Thorsten Stühmer ◽

Toril Holien ◽

Randi Utne Holt ◽

Ove Bruland ◽

...

Keyword(s):

Multiple Myeloma ◽

Growth Factors ◽

Cell Lines ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Transcriptional Response ◽

Myeloma Cell ◽

Myeloma Cells ◽

Growth And Survival ◽

Stat3 Phosphorylation

Abstract Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

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