Frequent Involvement of PVT1 in Multiple Myeloma Carrying 8q24 Rearrangement and Identification of Novel PVT1-NBEA Chimeric Gene,

Abstract Abstract 3917 The 8q24 rearrangement has been identified in 3.5–5.0 % of multiple myeloma (MM) patients with conventional cytogenetic analyses, such as G-banding, and in 9.5–20% of MM patients with fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) techniques. Of note, 8q24 rearrangements accompany with advanced MM patients and MM cell lines more frequently. Chromosomal translocation of the immunoglobulin gene (Ig), such as t(8;14)(q24;q32) and t(8;22)(q24;q11), occur in approximately 25% in MM with 8q24 rearrangements, while non-Ig chromosomal loci, including 1p13, 1p21–22, 6p21, 6q12–15, 13q14, and 16q22, have been also identified as translocation partners where no candidate genes have been delineated so far. In this study, we precisely investigate molecular features of chromosomal 8q24 rearrangements to access pathophysiology of MM. FISH, SKY, and RT-PCR analyses were performed as described previously. DNA gain and loss assay based on oligonucleotide array (GeneChip Human Mapping 50K, 250K, or 6.0 array, Affymetrix) was performed on the genomic DNA extracted from MM cells. Oligonucleotide array data was analyzed using the CNAG3.0 or 3.3 programs. Various types of 8q24 rearrangements were detected in 12 (22.2 %) of 54 MM patients and 8 (72.7 %) of 11 MM cell lines by means of FISH procedure. A breakpoint cluster of approximately 360 kb region containing myelocytomatosis oncogene (MYC) and plasmacytoma variant translocation 1 (PVT1) genes at 8q24 was divided into three subclusters according to rearranged region, that is, PVT1 region, region which is centromerically adjacent to PVT1 (including MYC), and region which is 120kb centromeric to MYC. Seven of 12 patient-derived cells and 5 of 8 cell lines with 8q24 abnormalities showed PVT1 rearrangements with various partner loci, such as 4p16, 4q13, 13q13, 14q32, or 16q23–24 in SKY combined with FISH analyses (SKY-FISH). Oligonucleotide array combined with SKY analysis delineated the candidate genes within partner loci of 8q24 rearrangements by mapping boundary breakpoints of copy number gains and losses, identifying MMSET, EPHA5, NBEA, and WWOX as candidate genes at 4p16, 4q13, 13q13, and 16q23–24, respectively. We identified the novel chimeric gene PVT1-NBEA in AMU-MM1 cell line with t(8;13)(q24;q13). The PVT1-NBEA fusion transcript in which PVT1 exon 1 was fused to NBEA exon 3 was accompanied with overexpression of abnormal NBEA lacking its part of N-terminus. In conclusion, PVT1 rearrangement may play pivotal roles in pathophysiology of MM harboring 8q24 abnormalities. Disclosures: No relevant conflicts of interest to declare.

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Adenylate Kinase 2 Is a Selective Dependency in NSD2-High Multiple Myeloma

Blood ◽

10.1182/blood-2020-141266 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-31

Author(s):

Amin Sobh ◽

Charlotte Kaestner ◽

Jianping Li ◽

Alberto Riva ◽

Richard Lynn Bennett ◽

...

Keyword(s):

Multiple Myeloma ◽

Er Stress ◽

Candidate Genes ◽

Cell Lines ◽

Adenylate Kinase ◽

Conflicts Of Interest ◽

Essential Gene ◽

Gene Knockout ◽

A Genome

Background: Multiple myeloma (MM)-associated t (4;14) chromosomal translocation leads to overexpression of NSD2, the histone H3 lysine 36 specific methyltransferase. t(4;14) MM patients have a high risk of relapse and NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance in MM cells. The lack of a specific and potent NSD2 inhibitors mandates finding alternative strategies for treating NSD2-high MM. Aim: This study aims to test the hypothesis that NSD2 overexpression in MM cells generates cellular vulnerabilities that can be therapeutically exploited for treatment of t (4;14) MM. Methods: We conducted a genome wide CRISPR-based loss-of-function genetic screen using the human Brunello library in isogenic NSD2-high (NTKO) and NSD2-low (TKO) KMS-11 derived MM cells to define genes whose loss is selectively detrimental to cells with NSD2 overexpression. The cellular dependency of each identified candidate was then investigated across hundreds of human cell lines using the Cancer Dependency Map portal (www.Depmap.org). Candidate genes were validated using CRISPR-Cas9 gene knockout and shRNA knockdown of individual target genes followed by in vitro competitive growth assays and cell viability assays. Results: Our study revealed multiple candidate genes with increased dependency in NSD2-high cells including the adenine nucleotide regulator Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible conversion of ADP to AMP and ATP and can thus modulates energy balance within the cell. Dependency map analysis showed that AK2 is not a commonly essential gene. The top enriched lineages with AK2 dependency included MM with notable representation of t(4;14)-positive MM cell lines. The increased dependency of NTKO and other t (4;14) MM cells on AK2 was confirmed by in vitro competition assays. Disruption of AK2 in TKO cells had a minimal effect on cellular fitness but the dependency on AK2 was restored upon engineered overexpression of NSD2 in these cells. In addition, NSD2-high cells displayed higher sensitivity to the proteasome inhibitor bortezomib than NSD2-low cells suggesting elevated levels of endoplasmic reticulum (ER) stress in cells overexpressing NSD2. Elevated ER stress necessitates increased levels of ATP to refold proteins and could underlie the increased dependency of NSD2-high cells on AK2. Notably, suppression of AK2 increased bortezomib sensitivity in t (4;14) MM cell lines. Conclusions: Our findings indicate that NSD2 high t(4;14) MM may have a vulnerability due to increased proteostatic stress. Accordingly, AK2 inhibition could be used in combination with proteasome inhibitors to treat MM patients with t (4;14) translocations by inducing the accumulation of lethal levels of unfolded proteins. 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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Detection of t(4;14)(p16.3;q32) Chromosomal Translocation in Multiple Myeloma by Double-Color Fluorescent In Situ Hybridization

Blood ◽

10.1182/blood.v94.2.724 ◽

1999 ◽

Vol 94 (2) ◽

pp. 724-732 ◽

Cited By ~ 47

Author(s):

Palma Finelli ◽

Sonia Fabris ◽

Savina Zagano ◽

Luca Baldini ◽

Daniela Intini ◽

...

Keyword(s):

Multiple Myeloma ◽

In Situ Hybridization ◽

Cell Lines ◽

Fluorescent In Situ Hybridization ◽

Chromosomal Translocation ◽

Fish Analysis ◽

Interphase Nuclei ◽

Igh Locus ◽

Normal Controls

Abstract Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at chromosome 14q32 represent a common mechanism of oncogene activation in lymphoid malignancies. In multiple myeloma (MM), variable chromosome partners have been identified by conventional cytogenetics, including the 11q13, 8q24, 18q21, and 6p21 loci. We and others have recently reported a novel, karyotypically undetectable chromosomal translocation t(4;14)(p16.3;q32) in MM-derived cell lines, as well as in primary tumors. The 4p16.3 breakpoints are relatively scattered and located less than 100 kb centromeric of the fibroblast growth factor receptor 3 (FGFR3) gene or within the recently identified WHSC1 gene, both of which are apparently deregulated by the translocation. To assess the frequency of the t(4;14)(p16.3;q32) translocation in MM, we performed a double-color fluorescent in situ hybridization (FISH) analysis of interphase nuclei with differently labeled probes specific for the IGH locus (a pool of plasmid clones specific for the IGH constant regions) or 4p16.3 (yeast artificial chromosome (YAC) 764-H1 spanning the region involved in breakpoints). Thirty MM patients, the MM-derived cell lines KMS-11 and OPM2, and six normal controls were examined. The identification of a t(4;14) translocation, evaluated as the presence of a der(14) chromosome, was based on the colocalization of signals specific for the two probes; a cutoff value of 15% (mean + 3 standard deviation [SD]) derived from the interphase FISH of the normal controls (range, 5% to 11%; mean ± SD, 8.16 ± 2.2) was used for the quantification analysis. In interphase FISH, five patients (one in clinical stage I, two in stage II, one in stage III, and a plasma cell leukemia) were found to be positive (≈15%). FISH metaphases with split or colocalized signals were detected in only two of the translocated cases and confirmed the pattern found in the interphase nuclei. Furthermore, in three of the five cases with the translocation, FISH analysis with the IGH joining probe (JH) showed the presence of the reciprocal product of the translocation [der(4) chromosome]. Overall, our study indicates that the t(4;14)(p16.3;q32) chromosomal translocation is a recurrent event in MM tumors and may contribute towards the detection of this lesion and our understanding of its pathogenetic and clinical implications in MM.

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Molecular Analysis of 11q13 Breakpoints in Multiple Myeloma

Blood ◽

10.1182/blood.v93.4.1330 ◽

1999 ◽

Vol 93 (4) ◽

pp. 1330-1337 ◽

Cited By ~ 59

Author(s):

Domenica Ronchetti ◽

Palma Finelli ◽

Raffaella Richelda ◽

Luca Baldini ◽

Mariano Rocchi ◽

...

Keyword(s):

Multiple Myeloma ◽

Cyclin D1 ◽

Cell Line ◽

Cell Lines ◽

Germ Line ◽

Chromosomal Translocation ◽

Primary Tumors ◽

Line Region ◽

Mantle Cell ◽

U266 Cell

Abstract The t(11;14)(q13;q32) chromosomal translocation, which is the hallmark of mantle cell lymphoma (MCL), is found in approximately 30% of multiple myeloma (MM) tumors with a 14q32 translocation. Although the overexpression of cyclin D1 has been found to be correlated with MM cell lines carrying the t(11;14), rearrangements of theBCL-1/cyclin D1 regions frequently involved in MCL rarely occur in MM cell lines or primary tumors. To test whether specific 11q13 breakpoint clusters may occur in MM, we investigated a representative panel of primary tumors by means of Southern blot analysis using probes derived from MM-associated 11q13 breakpoints. To this end, we first cloned the breakpoints and respective germ-line regions from a primary tumor and the U266 cell line, as well as the germ-line region from the KMS-12 cell line. DNA from 50 primary tumors was tested using a large panel of probes, but a rearrangement was detected in only one case using the KMS-12 breakpoint probe. Our results confirm previous findings that the 11q13 breakpoints in MM are scattered throughout the 11q13 region encompassing the cyclinD1 gene, thus suggesting the absence of 11q13 breakpoint clusters in MM.

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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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Membrane Lipid Biosynthesis As a Potential Therapeutic Target in Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.1836.1836 ◽

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.

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

Blood ◽

10.1182/blood.v120.21.3962.3962 ◽

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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Celastrol Overcomes Chemoresistance in Vitro and Potentiates the Effect of Bortezomib Against Human Multiple Myeloma in Nude Mice Mode

Blood ◽

10.1182/blood.v120.21.5017.5017 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5017-5017

Author(s):

Radhamani Kannaiyan ◽

Manu Kanjoormana Aryan ◽

Muthu K Shanmugam ◽

Feng Li ◽

Gautam Sethi

Keyword(s):

Multiple Myeloma ◽

B Cell ◽

Cell Lines ◽

Nude Mice ◽

Tumor Volume ◽

Corn Oil ◽

Conflicts Of Interest ◽

Chemotherapeutic Agents ◽

Antitumor Effects ◽

Group I

Abstract Abstract 5017 Introduction: Multiple myeloma (MM) is a B cell malignancy characterized by clonal proliferation of B cell in the bone marrow with low proliferative index. Despite the advent of novel therapeutics in addition to conventional chemotherapeutics, MM remains incurable because of the development of chemoresistance. Persistent activation of NF-κB/STAT3 signaling pathways and deregulation of apoptosis is considered to play an important role in the development of chemoresistance. The use of anticancer drugs derived from natural sources may be able to overcome resistance without some of the debilitating side effects of conventional chemotherapy. Celastrol is one such compound that has gained substantial attention recently for its anti-inflammatory and anticancer activities and is derived from the Chinese medicinal plant ‘Tripterygium wilfordii. We have demonstrated that celastrol overcomes the chemoresistance and induce apoptosis in MM cells by inhibiting NF-κB and STAT 3 pathways cell lines sensitive and resistant to various chemotherapeutic agents and Bortezomib. Our experimental findings have indicated that celastrol in combination with bortezomib/thalidomide can inhibit proliferation, induce apoptosis and overcome chemoresistance in MM cells in synergistic manner. We also observed that celastrol inhibited the activation of NF-κB and STAT3 and downregulated the expression of various genes involved in MM proliferation, survival and angiogenesis. Materials and Methods: Male athymic balb/c nude mice were implanted with 2×106 cells with either Human MM U266 cell lines subcutaneously. When tumors have reached more than 0. 3 cm in diameter, the mice were randomized into four groups. Group I (control) received corn oil 100 ul i. p. for five days a week, group II received 0. 25 mg/kg celastrol in 100ul corn oil for five days a week, group III received 0. 25 mg/kg bortezomib in 100 ul corn oil i. p. weekly and group IV received 0. 25mg/kg celastrol in 100 ul corn oil i. p. 5 days a week and 0. 25 mg/kg bortezomib in 100 ul corn oil i. p. weekly for 3 consecutive weeks. The tumor volume and body weight of the mice were monitored twice a week for the duration of the experiment. On completion of the treatment period, mice were euthanized by i. p. phentobarbital (40 mg/kg b. w) followed by cervical dislocation and then tumors were dissected and diameters measured. The tumor volume was calculated using the formula [L × W2]/2, where W and L are the width (short diameter) and the length (long diameter) of the tumor and the tumors were subjected to histological examination. Results: In the MM xenograft mice model, we observed that celastrol potentiated the antitumor effects of bortezomib and this correlated with significant suppression of NF-κB, STAT3, COX-2 and VEGF which was demonstrated by IHC. Overall, our data indicates that celastrol could be a potential therapeutic agent for the treatment of MM, especially in combination with the novel anti-myeloma agents. Disclosures: No relevant conflicts of interest to declare.

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