XPO1 Inhibition Disrupts Ribosomal Subunits Assembly and Induces Multiple Myeloma (MM) Cell Death

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3165-3165 ◽  
Author(s):  
Ines Tagoug ◽  
Paola Neri ◽  
Jiri Slaby ◽  
Jacquelyn Babich ◽  
Justin Simms ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Stress Response ◽  
Cell Lines ◽  
Nuclear Export ◽  
Ribosomal Proteins ◽  
Conflicts Of Interest ◽  
Mrna Levels ◽  
Ribosomal Subunits ◽  
Cytoplasmic Transport ◽  
Ribosomal Stress

Abstract Background Chromosomal region maintenance (CRM1), also known as exportin 1 (XPO1) plays an important role in the nuclear-cytoplasmic shuttling. The nuclear export receptor, XPO1, is considered as a regulator of subcellular distribution of several proteins involved in the regulation of centrosome duplication such as nucleophosmin (NPM), breast and ovarian cancer susceptibility protein 1 (BRCA1) and many tumor suppressor proteins (p53, p21, FOXO and pRB). Furthermore, XPO1 is required for the export of assembled ribosomal subunits (60S & 40S) from the nucleolus back into the cytoplasm. Inhibition of XPO1 triggers a ribosomal stress response that may result in the death of transformed cells with stressed ribogenesis. Silencing of XPO1 is reported to be synthetically lethal in MM cells, however the mechanisms that mediate this effect are not fully elucidated. Methods and Results To determine the effect of XPO1 inhibition in MM, cells were exposed to different doses of KPT330 (Karyopharm), a selective inhibitor of nuclear-cytoplasmic transport by irreversibly binding to the XPO1 cargo recognition site. Nanomolar concentrations of KPT330 (50-150 nM) induced apoptosis (Puma up-regulation and caspase 3 cleavage) and suppressed the proliferation of myeloma cell lines MM1S, OPM2 while KMS11 cells were more resistant. Mechanistically treatment with KPT330 up-regulated the expression of p53, as well as p21, p27 and MDM2 at the protein and RNA levels and significantly decreased the expression of c-Myc and IRF4. Cognizant of the role XPO1 in cytoplasmic-nuclear shuttling of ribosomal subunits, we reasoned that c-Myc downregulation and p53 induction in MM cells exposed to KPT330 results from ribosomal biogenesis stress. Therefore we analyzed the cellular co-localization of ribosomal proteins (RPL5, RPL11), c-Myc and MDM2 in presence of KPT330. A shifting of RPL11 and RPL5 from the nucleolus to the nucleoplasm and cytosol was observed in presence of KPT330 where they accumulated in ribosome-free cellular fractions. Co-immunoprecipitation studies showed that RPL11 and RPL5 released from the nucleolus bind MDM2 and c-Myc. This binding of RPL11 and RPL5 to MDM2 and c-Myc is known to suppress their function and expression. Therefore our data explain the suppression of MDM2 E3 ligase activity with p53 stabilization and reduction of c-Myc at the post-transcriptional levels. Study of ribosome fractions with sucrose gradients showed that in the presence of KPT330, 40S and the polysomes were completely suppressed while 60S and 80S subunits were significantly downregulated in OPM2 and MM1S cell lines. Consistent with the disruption of ribosomal function and the translational machinery, c-Myc mRNA levels were significantly decreased in 40S, 60S and 80S fractions after treatment with KPT330. Confirming the role of the ribosomal stress response in KPT330-mediated MM cells' death, silencing of ribosomal proteins RPL11 or RPL5 fully protected them from KPT330 cytotoxicity. Furthermore silencing RPL11 or RPL5, suppressed the effects of KPT330 on MDM2, p53, p21 and c-MYC. Conclusion Inhibition of XPO1 induces a perturbation in the ribosome subunits transfer, disruption of ribosomal assembly and the induction of a ribosomal stress response in MM cells. Perturbation on the nucleolar-cytoplasmic shuttling by KPT330 and the targeting of the translational factory represents a novel therapeutic approach in multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of N6-Methyladenosine (m6A) RNA Modification in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5601-5601
Author(s):  
Christian Bach ◽  
Magdalena Leffler ◽  
Cindy Flamann ◽  
Jan Kronke ◽  
Dimitrios Mougiakakos ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Messenger Rna ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Stop Codon ◽  
Mrna Levels ◽  
Molecular Abnormalities ◽  
Gene Expression Control

Abstract Multiple myeloma (MM) is considered a chronic and incurable disease due to its highly complex and heterogeneous molecular abnormalities. In recent years, integrating proteasome inhibitors and immunomodulatory drugs into MM frontline therapy has significantly improved treatment efficacy with a median overall survival (OS) being prolonged from 3-4 to 7 years. Despite this progress, patients refractory to the aforementioned agent classes display a median OS of only 9 months. Thus, the clinical necessity for developing novel therapeutic alternative approaches is self-evident. Methylation of N6-adenosine (m6A) is known to be important for diverse biological processes including gene expression control, translation of protein, and messenger RNA (mRNA) splicing. m6A regulatory enzymes consist of "writers" METTL3 and METTL14, "readers" YTHDF1 and YTHDF2, and "erasers" FTO and ALKBH5. However, the functions of m6A mRNA modification and the specific role of these enzymes in MM remain unknown. Here we report that METTL3, a key component of the m6A methyltransferase complex, is highly expressed in MM cell lines and in isolated patient's MM cells. In contrast, we found no significant differences in the expression of the m6A demethylases FTO and ALKBH5. Accordingly, compared to plasma cells from healthy donors, global PolyA+ RNA showed a significant increase in m6A content in patient's MM plasma cells. In MM cell lines, global m6A profiling by methylated RNA-immunoprecipitation sequencing revealed m6A peaks near the stop codon in mRNAs of multiple oncogenes including MAF and CCND1. Cross-linking immunoprecipitation showed that METTL3 bound to the m6A peak within MAF and CCND1 mRNA. Depletion of METTL3 by shRNA had little effect on global mRNA levels, but specifically reduced protein levels of c-Maf and Cyclin D1. Moreover, downregulation of METTL3 in several MM cell lines results in cell cycle arrest and apoptosis. Together, these results describe a role for METTL3 in promoting translation of a subset of oncogenes in MM and identify this enzyme as a potential therapeutic target for multiple myeloma. Disclosures No relevant conflicts of interest to declare.

BET Bromodomain Blockade Enhances Ikaros Inhibition By Lenalidomide Therapy Providing Additional Activity in In Vitro and In Vivo Models of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 308-308
Author(s):  
Carlos Fernández de Larrea ◽  
Tania Díaz ◽  
Vanina Rodriguez ◽  
Ester Lozano ◽  
Patricia Pérez-Galán ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Median Response ◽  
Daily Dose ◽  
In Cells

Abstract Introduction: Even with the use of novel drugs in patients with multiple myeloma (MM), relapse remains a challenge. Anti-myeloma activity of the immunomodulatory drug lenalidomide has been shown to rely on cereblon E3 ubiquitin ligase complex-dependent degradation of IRF4 and Ikaros, both required for MM cell survival. As these two factors are involved in the regulation of MYC transcription, we wanted to evaluate the effect of combining a MYC-interfering therapy with a lenalidomide and dexamethasone (Len/Dex) regimen. Methods: Seven MM cell lines (ARP-1, JJN-3, U266, MM.1S, MM.1R, RMPI-8226 and KMM.1) were exposed to the BET bromodomain inhibitor CPI203 in the presence or absence of a standard dose of Len/Dex, followed by MTT assay, flow cytometry, western blot and gene expression arrays. Significant gene signatures were identified using gene set enrichment analysis (GSEA) v2.0 (Broad Institute at MIT). These results were validated in primary cells derived from bone marrow of 9 patients with MM co-cultured with mesenchymal stromal cells plus IL-6, as well as in a SCID mouse xenotransplant model of MM. Mice were randomly assigned into cohorts of 5 mice each and received during 2 weeks (5 days on/2 days off) by intraperitoneal injection a twice daily dose of CPI203, daily dose lenalidomide plus twice weekly dexamethasone, the combination of both, or an equal volume of vehicle. Results: We found CPI203 to exert a dose-dependent cell growth inhibition in all the cell lines tested (median response: 62.5%) (Figure 1) characterized by G1 cell cycle arrest and a concomitant inhibition of MYC and Ikaros signaling. Most interestingly, these effects were potentiated by the addition of Len/Dex treatment, with CI values ranging from 0.08 to 0.51. In the GSEA with an increasing profile analysis, the gene sets signatures related with MYC appeared downregulated in cells treated with the drug, and this downregulation was higher in cells treated with the combination of the three drugs. Moreover, genes associated with Ikaros and IRF4 also became downregulated after three drug treatment; these findings were confirmed by western blot analysis. Similarly, in 9 MM primary-stroma co-cultures, the drug combination evoked a 53% reduction in cell proliferation (Figure 1), and was associated with basal Ikaros mRNA levels (p=0.04). Finally, in immunodeficient mice engrafted with MM cells, addition of CPI203 to Len/Dex therapy further decreased tumor burden, evidenced by a lower glucose uptake (Figure 2) and a simultaneous downregulation of MYC- and Ikaros-related genes by immunohistochemical analysis of the tumoral tissue after mice death. Conclusion: The combination of a BET bromodomain inhibitor CPI203 with a lenalidomide-based regimen may represent a promising, rationally based therapeutic approach for patients with MM. Disclosures No relevant conflicts of interest to declare.

Tissue Factor Is Frequently Expressed in Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2132-2132 ◽  
Author(s):  
Sameer Gupta ◽  
Tanisha R Hayes ◽  
Yuchuan Liu ◽  
Matthew T Hurford ◽  
Charalambos Solomides ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Tissue Factor ◽  
Cell Lines ◽  
Light Chain ◽  
Conflicts Of Interest ◽  
Mrna Levels ◽  
Bone Marrow Biopsies ◽  
Increased Risk ◽  
Mcf 7

Abstract Abstract 2132 Poster Board II-109 Tissue factor (TF) is a 47 kDa transmembrane glycoprotein that initiates blood coagulation when complexed with factor VIIa. TF is constitutively expressed in a variety of tumor cells and has been shown to have a role in cellular signaling, angiogenesis and solid tumor progression. However, the role of TF in the hematologic malignancies is poorly defined. Multiple myeloma (MM) is associated with an increased risk of venous thromboembolic disease. However, whether increased TF expression contributes to the hypercoagulable state associated with MM remains controversial. In this study, we determined the expression of TF on archived bone marrow biopsies and plasmacytomas, and human MM cell lines. Immunohistochemical staining of TF was carried out on paraffin-embedded specimens from eighteen advanced stage MM patients. Staining for TF expression was scored as 0 (null), 1+ (weak), 2+ (moderate) and 3+ (strong). TF expression for the MM cell lines (U266B1, MM1.RL and MM1.S) was carried out by semi-quantitative real time RT-PCR. TF mRNA levels were normalized to 18S ribosomal mRNA levels. The TF: 18S ratios were then compared to that of a low TF expressing human breast cancer cell line cell line, MCF-7. Paraprotein distribution in the MM patient specimens was: IgG kappa (7), IgG lambda (5), IgA kappa (3), lambda light chain (2) kappa light chain (1). Overall, TF expression was observed in 10/18 (56%) of the patient specimens. Six specimens stained 1+, and two each stained 2+ and 3+. Staining was mainly cytoplasmic and did not correlate with the type of secreted paraprotein. TF expression (relative to MCF-7) was 17.3, 1.97 and 0.77 for the U266B1, MM1.RL and MM1.S cell lines, respectively. Results from these studies suggest that TF is frequently expressed in MM cells and might contribute to the hypercoagulability associated with this disease. In addition, TF may play a role in MM cell progression. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 832-832 ◽  
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.

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

Blood ◽  
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.

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 ◽  
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.

P53 is Activated without RPL11 Upregulation in Diamond-Blackfan Anemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3432-3432
Author(s):  
Hong-Yan Du ◽  
M. Tarek Elghetany ◽  
Blanche P Alter ◽  
Akiko Shimamura
Keyword(s):  
Bone Marrow ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Mrna Levels ◽  
Protein Levels ◽  
Diamond Blackfan Anemia ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
P53 Activation ◽  
Ribosomal Stress

Abstract Abstract 3432 Diamond-Blackfan anemia (DBA) is an autosomal dominantly inherited bone marrow failure syndrome characterized by red cell aplasia, physical anomalies, and cancer predisposition. DBA is caused by mutations resulting in haploinsufficiency of genes encoding ribosomal proteins. p53 is activated in the erythroid lineage following reduction of ribosomal protein expression; however the mechanism whereby ribosomal stress results in p53 activation in DBA remains unclear. RPL11 has been proposed to play a central role in p53 activation following ribosomal stress. Reduced expression of individual small ribosomal subunit proteins in a tumor cell line resulted in increased translation of RPL11. Excess free RPL11 can bind and inactivate HDM2, an E3 ubiquitin ligase targeting p53 for degradation. The recent demonstration that cellular responses to ribosomal perturbations vary widely between different tissues raised the question of whether RPL11 upregulation contributes to p53 activation following ribosomal stress in hematopoietic progenitors. To address this question, we modeled DBA in human CD34+ cells. Since RPS19 is the most commonly mutated gene in DBA, we used lentiviral vectors expressing short hairpin RNAs to knock down RPS19 expression in primary human CD34+ cells. RPS19 protein levels were reduced to about 50% of control levels in a manner reflecting the haploinsufficient state in DBA. RPS19 depletion resulted in elevated p53 protein levels and increased mRNA levels of p21, a transcriptional target of p53. Total p53 mRNA levels and p53 mRNA translational activity remained unchanged consistent with a post-transcriptional mechanism for p53 activation. Although total RPL11 mRNA levels were not diminished following RPS19 depletion, RPL11 protein levels were significantly decreased consistent with post-transcriptional downregulation. Depletion of RPS19 in human CD34+ cells did not affect polysome loading of RPL11 mRNA. Reduction of additional ribosomal proteins also accompanied RPS19 knockdown consistent with coordinate regulation of multiple ribosomal protein levels. Corticosteroids, which improve anemia in the majority of DBA patients, did not prevent p53 activation, nor did this improve RPS19 or RPL11 protein levels. Expression of p53 was also assessed in bone marrow biopsy slides from 26 DBA patients with the following genotypes: RPS19 (18), RPS24 (2), RPS26 (2), RPS10 (1), RPS17 (1), RPS7 (1), and RPL11 (1). p53 was over-expressed in all but one patient (RPS26), and was clearly over-expressed in the DBA patient harboring the RPL11 mutation. In summary, we find that p53 activation in DBA does not require upregulation of RPL11 translation or elevated RPL11 protein levels. p53 activation persists in DBA caused by RPL11 deficiency. Corticosteroids do not improve ribosomal protein levels nor do they prevent p53 activation. Disclosures: No relevant conflicts of interest to declare.

Membrane Lipid Biosynthesis As a Potential Therapeutic Target in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1836-1836
Author(s):  
Carolyne Bardeleben ◽  
Alan Lichtenstein
Keyword(s):  
Multiple Myeloma ◽  
Er Stress ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Membrane Lipid ◽  
Dependent Manner ◽  
Kennedy Pathway ◽  
Phosphatidylcholine Synthesis

Abstract Abstract 1836 Phosphatidylcholine (PC) is the most prominent phospholipid in mammalian endoplasmic reticulum (ER) membranes. The rate-limiting step in PC synthesis through the Kennedy pathway is the conversion of phosphocholine + cytidine triphosphate (CTP) to cytidine diphosphocholine, (CDP)-choline, via the enzyme CTP:phosphocholine cytidylyltransferase (CCT) (see figure). Multiple myeloma (MM) cells may be particularly dependent on this biosynthetic reaction because of their high consistent level of ER stress and requirement to continuously replenish ER membranes. Indeed, CCT-null mice have a defect in differentiation of B lymphocytes to plasma cells and deficiencies in Ig synthesis. To test whether this pathway remains critical in survival of malignant MM cells, we exposed MM cell lines to an inhibitor shown to inhibit CCT activity, HexPC. HexPC induced apoptosis in all MM cell lines in a concentration- and time-dependent manner. The addition of lysophosphatidylcholine (LPC), presumably converted to PC independently of the Kennedy pathway, completely rescued MM cell apoptosis. In contrast, similar concentrations of LPC in the same cell lines could not rescue apoptosis induced by bortezomib. An additional intervention to inhibit phosphatidylcholine synthesis, namely inducing pyrimidine starvation, also resulted in MM cell apoptosis and down-regulation of CDP-choline levels. Apoptosis of MM cells induced by HexPC was associated with induction of ER stress as shown by enhanced phosphorylation of IRE1 and eIF-2alpha. This ER stress was also prevented when LPC was added to HexPC although LPC could not prevent similar ER stress induced by bortezomib. These results underscore the importance of this phosphatidylcholine synthesis pathway in MM cells and provide new targets for future therapy. Disclosures: No relevant conflicts of interest to declare.

Identification of Tight Junction Protein (TJP)-1 As a Modulator of Sensitivity to Doxorubicin and Cisplatin in Models of Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3962-3962
Author(s):  
Xing-Ding Zhang ◽  
Robert Z. Orlowski ◽  
Lin Yang
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Hela Cells ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Cancer Center ◽  
In Vivo Models ◽  
Junction Protein ◽  
Rpmi 8226

Abstract Abstract 3962 Background: Therapeutic advances in multiple myeloma have improved the outcomes of patients with this malignant plasma cell disorder, but the disease course is still strongly influenced by both innate, or primary, as well as acquired, or secondary mechanisms of drug resistance. Identification and validation of genes that may mediate these phenotypes is therefore of importance, since they could be useful prognostic markers, and also potential targets to overcome the emergence of resistance, or possibly preclude its emergence altogether. Methods: To identify non-redundant determinants of chemoresistance, we designed a robust, high-throughput RNA interference (RNAi) screen targeting 9610 human genes. The screen involved retroviral-mediated transduction first of HeLa cervical carcinoma cells with either the RNAi library, or with non-targeting retrovirus particles. After infection, cells were selected with puromycin, and treated with different concentrations of doxorubicin and cisplatin. Doxorubicin (Dox) treatment led to 33 surviving colonies from the cells transduced with the shRNA library, cisplatin (Cis) treatment led produced 22 surviving colonies, while non-targeting retrovirus-infected cells failed to form colonies after treatment. Screening was performed to identify the shRNA target gene(s) in each colony, and genes that were identified in both Dox- and Cis-treated HeLa cells, and that were expressed in myeloma cells, were selected for further study. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results: TJP1 (zona occludens (ZO)-1) was identified as one gene whose knockdown promoted survival in Dox- and Cis-treated HeLa cells, and which was expressed in myeloma cell lines and in primary plasma cells. To further examine its potential role in myeloma chemosensitivity, we performed mRNA and protein expression profiling in a panel of 11 cell lines and observed that TJP1 expression was silenced in 3 cell lines (ARP-1, INA-6, and MOLP-8), while it was moderately to highly expressed in 7 cell lines (including RPMI 8226, MM1.S, and U266). Comparing TJP1-positive MM1.S cells to TJP1-null MOLP-8 cells, the latter displayed a significantly higher median inhibitory concentration to Dox and Cis. Knockdown of TJP1 in RPMI 8226 and U266 cells, which produced a >75% target suppression, was sufficient to reduce the proportion of apoptotic cells in the sub-G1 fraction after treatment with Dox or Cis compared to control cells. Conversely, MOLP-8 cells transfected with human TJP1 cDNA exhibited an increase in the sub-G1 population in response to Dox and Cis treatment compared to vector controls. Conclusion: Taken together, these studies support the hypothesis that TJP1 expression mediates myeloma cell resistance to the DNA damaging agents doxorubicin and cisplatin. Further studies are underway to determine the mechanism by which TJP1 influences chemosensitivity, and to validate its impact using in vivo models. Disclosures: No relevant conflicts of interest to declare.

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