scholarly journals DNA Methylation in Multiple Myeloma Is Weakly Associated with Gene Transcription

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1285-1285
Author(s):  
Sungwon Jung ◽  
Kim Seungchan ◽  
Molly Gale ◽  
Rafael Fonseca ◽  
John Carpten ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Multiple Myeloma ◽  
Pearson Correlation ◽  
Enrichment Analysis ◽  
Epigenetic Mechanism ◽  
Methylation Data ◽  
Rt Pcr ◽  
Number Of Genes ◽  
Unique Genes

Abstract Abstract 1285 Introduction: Recent studies have now demonstrated that both genic and global hypomethylation characterizes the multiple myeloma (MM) epigenome. We previously conducted a study to measure changes in DNA methylation at approximately 1500 CpG (GoldenGate Methylation BeadArray, Illumina) loci in 193 MM samples used in the Multiple Myeloma Research Consortium Genomics Initiative. The purpose of this study was to determine the extent to which DNA methylation changes in MM correlate to changes at the gene expression level. Methods: To evaluate this correlation we performed three independent analyses. In the first approach we analyzed matching gene expression and DNA methylation data using a Pearson correlation to assess the linear relationship between the datasets. In the second approach methylation and gene expression data from 193 matching samples were quantile-normalized and standardized across genes and samples. Methylation data was then discretized into three groups (methylated, unchanged, unmethylated) according to the mean and standard deviation of the values for each probe on the array. We then computed a t-test to compare the gene expression differences for each gene represented on the methylation array between samples in the methylated and unmethylated groups. We also performed a class enrichment analysis to assess DNA methylation/gene expression correlations that might be associated with hyperdiploidy or molecular subtype. In the third approach we performed RT-PCR and methylation pyrosequencing to validate results in a subset of samples used for the analysis and in an independent cohort comprised of 50 MM samples. Results: By Pearson correlation, only 31 (2.1%) CpG loci (corresponding to 24 unique genes) had a gene expression probe with a statistically significant correlation. Using the discretization approach, we identified 382 loci (25.3%, 309 unique genes) with methylation-expression correlations. Although we identified more correlations by discretizing the data, methylated and unmethylated samples were represented by an average of only 30 and 27 samples per group. We identified correlations where the directionality of methylation and expression values were either in the opposite (negative) or same direction (positive). Among the genes identified, the tumor suppressor genes CDKN2A and DLC1 interestingly demonstrated a positive correlation (methylated and expressed) and the IGF1R and IL17RB genes were negatively correlated (unmethylated and expressed). A class enrichment analysis revealed that samples with DLC1, CDKN2A, IGF1R or IL17RB methylation were associated with hyperdiploid MM. Conversely, samples that were unmethylation for these genes were generally non-hyperdiploid. These findings were validated by RT-PCR and methylation pyrosequencing. Conclusion: Overall, our findings demonstrated, albeit in a limited number of genes, that DNA methylation changes are weakly associated to gene expression. These data suggest that CpG methylation may have other functional consequences such as predisposing the genome to global gene transcriptional changes or chromosomal instability. While future studies are needed to determine the exact role of DNA methylation, we identified a number of genes regulated by an epigenetic mechanism with important clinical and biological implications to MM and warrant further study. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Stage-specific differential DNA methylation data analysis during human erythropoiesis in chromosome 16

2018 ◽  
Vol 100 ◽  
Author(s):  
Najyah A. Garoot ◽  
Byung Guk Kim
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Data Analysis ◽  
Empirical Bayes ◽  
Intermediate Phase ◽  
Enrichment Analysis ◽  
Methylation Pattern ◽  
Methylation Data ◽  
Single Chromosome ◽  
Methylation Patterns

AbstractPrevious studies have generated controversial findings regarding the correlation between DNA methylation in the human genome and gene expression. Some reports have indicated that promoter methylation is negatively correlated with gene expression levels; however, in some cases, a poor or positive correlation was reported. Most previous findings were based on general trends observed with whole-genome data analysis. Here, we present a novel chromosome-specific statistical analysis design of empirical Bayes differential tests for five phases of erythroid development. To better understand the common methylation patterns of differentially methylated regions (DMRs) during specific stages, we defined differential phases for each CpG locus, based on a maximum log2 fold change. Analyzing hypermethylated and hypomethylated CpG loci separately showed variations in methylation patterns during erythropoiesis in the gene body, promoter and enhancer regions. Hypomethylated DMRs showed stronger associations with erythroid-specific enhancers at the differentiation start phase and with exons in the intermediate phase. To investigate the hypomethylated DMRs further, transcription factor binding site-enrichment analysis was conducted. This analysis highlighted novel transcription factors during each differentiation stage that were not detected by previous differential methylation data analysis. In contrast, hypermethylated DMRs showed a consistent methylation pattern over the different genomic regions. Thus, a closer examination of DNA methylation patterns in a single chromosome during each developmental stage can contribute to verify the association nature between gene expression and DNA methylation.

Abstract P098: An Epigenome-wide Study of Obesity in African American Youth and Young Adults: Novel Findings, Replication in Neutrophils and Relationship With Gene Expression

Circulation ◽  
2017 ◽  
Vol 135 (suppl_1) ◽  
Author(s):  
Xiaoling Wang ◽  
Yue Pan ◽  
Haidong Zhu ◽  
Guang Hao ◽  
Xin Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Older Adults ◽  
Dna Methylation ◽  
Young Adults ◽  
Methylation Data ◽  
Middle Aged ◽  
Cpg Sites ◽  
Genome Wide ◽  
Obesity Status ◽  
Youth And Young Adults

Background: Several large-scale epigenome wide association studies on obesity-related DNA methylation changes have been published and in total identified 46 CpG sites. These studies were conducted in middle-aged and older adults of Caucasians and African Americans (AAs) using leukocytes. To what extend these signals are independent of cell compositions as well as to what extend they may influence gene expression have not been systematically investigated. Furthermore, the high prevalence of obesity comorbidities in middle-aged or older population may hide or bias obesity itself related DNA methylation changes. Methods: In this study of healthy AA youth and young adults, genome wide DNA methylation data from leukocytes were obtained from three independent studies: EpiGO study (96 obese cases vs. 92 lean controls, aged 14-21, 50% females, test of interest is obesity status), LACHY study (284 participants from general population, aged 14-18, 50% females, test of interest is BMI), and Georgia Stress and Heart study (298 participants from general population, aged 18-38, 52% females, test of interest is BMI) using the Infinium HumanMethylation450 BeadChip. Genome wide DNA methylation data from purified neutrophils as well as genome wide gene expression data from leukocytes using Illumina HT12 V4 array were also obtained for the EpiGO samples. Results: The meta-analysis on the 3 cohorts identified 76 obesity related CpG sites in leukocytes with p<1х10 -7 . Out of the 46 previously identified CpG sites, 36 can be replicated in this AA youth and young adult sample with same direction and p<0.05. Out of the 107 CpG sites including the 36 replicated ones and the 71 newly identified ones, 71 CpG sites (66%) had their relationship with obesity replicated in purified neutrophils (p<0.05). The analysis on the cis regulation of the 107 CpG sites on gene expression showed that 59 CpG sites had at least one gene within 250kb having expression difference between obese cases and lean controls. Furthermore, out of the 59 CpG sites, 6 showed significantly negative correlations and 1 showed significantly positive correlation with the differentially expressed genes. These CpG sites located in SOCS3, CISH, ABCG1, PIM3 and PTGDS genes. Conclusion: In this study of AA youth and young adults, we identified novel CpG sites associated with obesity and replicated majority of the CpG sites previously identified in middle-aged and older adults. For the first time, we showed that majority of the obesity related CpG sites identified from leukocytes are not driven by cell compositions and provided the direct link between DNA methylation-gene expression-obesity status for 7 CpG sites in 5 genes.

scholarly journals The Expression Level of mRNA, Protein, and DNA Methylation Status of FOSL2 of Uyghur in XinJiang in Type 2 Diabetes

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Jun Li ◽  
Siyuan Li ◽  
Ying Hu ◽  
Guolei Cao ◽  
Siyao Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Type 2 Diabetes ◽  
Dna Methylation ◽  
Methylation Status ◽  
Methylation Data ◽  
Biochemical Indicators ◽  
Expression Levels ◽  
Protein Levels ◽  
Cpg Sites

Objective. We investigated the expression levels of both FOSL2 mRNA and protein as well as evaluating DNA methylation in the blood of type 2 diabetes mellitus (T2DM) Uyghur patients from Xinjiang. This study also evaluated whether FOSL2 gene expression had demonstrated any associations with clinical and biochemical indicators of T2DM. Methods. One hundred Uyghur subjects where divided into two groups, T2DM and nonimpaired glucose tolerance (NGT) groups. DNA methylation of FOSL2 was also analyzed by MassARRAY Spectrometry and methylation data of individual units were generated by the EpiTyper v1.0.5 software. The expression levels of FOS-like antigen 2 (FOSL2) and the protein expression levels were analyzed. Results. Significant differences were observed in mRNA and protein levels when compared with the NGT group, while methylation rates of eight CpG units within the FOSL2 gene were higher in the T2DM group. Methylation of CpG sites was found to inversely correlate with expression of other markers. Conclusions. Results show that a correlation between mRNA, protein, and DNA methylation of FOSL2 gene exists among T2DM patients from Uyghur. FOSL2 protein and mRNA were downregulated and the DNA became hypermethylated, all of which may be involved in T2DM pathogenesis in this population.

scholarly journals Uncovering Driver DNA Methylation Events in Nonsmoking Early Stage Lung Adenocarcinoma

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Xindong Zhang ◽  
Lin Gao ◽  
Zhi-Ping Liu ◽  
Songwei Jia ◽  
Luonan Chen
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Lung Adenocarcinoma ◽  
Early Stage ◽  
Methylation Data ◽  
Driver Genes ◽  
Differential Network Analysis ◽  
Differential Network ◽  
Aberrant Dna Methylation ◽  
Never Smokers

As smoking rates decrease, proportionally more cases with lung adenocarcinoma occur in never-smokers, while aberrant DNA methylation has been suggested to contribute to the tumorigenesis of lung adenocarcinoma. It is extremely difficult to distinguish which genes play key roles in tumorigenic processes via DNA methylation-mediated gene silencing from a large number of differentially methylated genes. By integrating gene expression and DNA methylation data, a pipeline combined with the differential network analysis is designed to uncover driver methylation genes and responsive modules, which demonstrate distinctive expressions and network topology in tumors with aberrant DNA methylation. Totally, 135 genes are recognized as candidate driver genes in early stage lung adenocarcinoma and top ranked 30 genes are recognized as driver methylation genes. Functional annotation and the differential network analysis indicate the roles of identified driver genes in tumorigenesis, while literature study reveals significant correlations of the top 30 genes with early stage lung adenocarcinoma in never-smokers. The analysis pipeline can also be employed in identification of driver epigenetic events for other cancers characterized by matched gene expression data and DNA methylation data.

scholarly journals Specific Histone Tail Modification and Not DNA Methylation Is a Determinant of Herpes Simplex Virus Type 1 Latent Gene Expression

2004 ◽  
Vol 78 (3) ◽  
pp. 1139-1149 ◽  
Author(s):  
Nicole J. Kubat ◽  
Robert K. Tran ◽  
Peterjon McAnany ◽  
David C. Bloom
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Epigenetic Mechanism ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT During herpes simplex virus type 1 (HSV-1) latency, gene expression is tightly repressed except for the latency-associated transcript (LAT). The mechanistic basis for this repression is unknown, but its global nature suggests regulation by an epigenetic mechanism such as DNA methylation. Previous work demonstrated that latent HSV-1 genomes are not extensively methylated, but these studies lacked the resolution to examine methylation of individual CpGs that could repress transcription from individual promoters during latency. To address this point, we employed established models to predict genomic regions with the highest probability of being methylated and, using bisulfite sequencing, analyzed the methylation profiles of these regions. We found no significant methylation of latent DNA isolated from mouse dorsal root ganglia in any of the regions examined, including the ICP4 and LAT promoters. This analysis indicates that methylation is unlikely to play a major role in regulating HSV-1 latent gene expression. Subsequently we focused on differential histone modification as another epigenetic mechanism that could regulate latent transcription. Chromatin immunoprecipitation analysis of the latent HSV-1 DNA repeat regions demonstrated that a portion of the LAT region is associated with histone H3 acetylated at lysines 9 and 14, consistent with a euchromatic and nonrepressed structure. In contrast, the chromatin associated with the HSV-1 DNA polymerase gene located in the unique long segment was not enriched in H3 acetylated at lysines 9 and 14, suggesting a transcriptionally inactive structure. These data suggest that histone composition may be a major regulatory determinant of HSV latency.

Study on DNA Methylation Status of WT1 Gene in Its Promoter Region in Hematologic Neoplasm Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4386-4386
Author(s):  
Ye Zhao ◽  
Zi-xing Chen ◽  
Shao-yan Hu ◽  
Jian-nong Cen
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Cell Lines ◽  
Promoter Region ◽  
U937 Cell ◽  
Methylation Status ◽  
Gene Promoter ◽  
Epigenetic Mechanism ◽  
Wt1 Gene ◽  
Gene Promoter Region

Abstract The methylation at CpG island in the promoter region of a gene is one of the important epigenetic mechanism which regulates the gene activity. To study the DNA methylation pattern of WT1 gene promoter region within hematologic neoplastic cell lines and its correlation with WT1 gene expression by using the PCR-based methods. RT-PCR and Methylation-specific PCR were performed to study the WT1 gene expression in 8226, HL-60, Jurkat, K562, KG-1, NB4, Raji, SHI-1, U266 and U937 cell lines and the DNA methylation status in promoter region of WT1 gene. After treatment of U937 cell line by 5-aza-CdR, a demethylation inducing agent, the changes of WT1 gene expression level and the methylation status in its promter region in U937 cells was determined. Our Results showed that HL-60, K562, KG-1, NB4, SHI-1 cell lines demonstrated higher level of WT1 expression, while extremely low level was found in 8226, Jurkat, Raji, U266 and U937. The DNA hypermethylation in WT1 gene promoter region was identified in 8226, Jurkat, Raji, U266 and U937 cell lines. The WT1 gene expression in U937 was markedly enhanced after treatment with 5-aza-CdR in company with the decrease of methylated level and the increase of unmethylated level in its promoter region. These results indicate that modulation of the DNA methylation in WT1 promoter region is one of the epigenetic mechanisms to regulate its expression.

Peripheral Blood Cells from Multiple Myeloma Patients Show Increased Osteoclast and Decreased Osteoblast Gene Expression.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5098-5098
Author(s):  
Melinda S. Gordon ◽  
Ariana M. Berenson ◽  
Charles B. Drucker ◽  
Matthew Katz ◽  
Hee Jin Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Alkaline Phosphatase ◽  
Bone Disease ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Bisphosphonate Treatment ◽  
Rt Pcr ◽  
Circulating Cells ◽  
Osteolytic Bone Disease

Abstract Bone resorption leading to osteolytic bone disease is characteristic of multiple myeloma (MM). Recent studies show the presence of bone-resorbing osteoclasts and bone-forming osteoclasts in the circulation, and these cells may correlate with bone disease and change with anti-bone resorptive therapies. We have investigated whether there is an imbalance in the expression of osteoblast and osteoclast genes in the peripheral blood mononuclear cells (PBMCs) from MM patients relative to normal age-matched controls and the effect of bisphosphonate treatment on the expression of these genes. We analyzed the expression of a panel of osteoblast-related (bone alkaline phosphatase [bone AP], bone morphogenic protein 2 [BMP2], collagen I and osteocalcin) and osteoclast-related (b3 integrin, calcitonin, receptor for activation of nuclear factor kappa B [RANK] and tartrate-resistant alkaline phosphatase [TRAP]) genes by semi-quantitative RT-PCR on total RNA isolated from PBMCs obtained following density gradient separation. We demonstrated that the expression of the osteoblast-related gene BMP2 was reduced in eight of nine MM patients when compared with normal donors. In marked contrast, three osteoclast-related genes, b3 integrin, RANK and TRAP, were more highly expressed in all nine MM patients compared to the normal donors; only calcitonin expression was similar to the control subjects. Interestingly, patients receiving bisphosphonate treatment appeared to show increased osteoblast gene expression with higher amounts of bone AP, BMP2 and osteocalcin RNA compared to the patients not receiving anti-bone resorptive therapy. However, there was no alteration in the level of the RNA in any of the four osteoclast genes compared to patients not receiving anti-bone resorptive therapy. We are extending our analysis to a larger panel of MM patients in order to determine the relationship between these circulating cells and bone disease, overall clinical status and change in their levels with anti-bone resorptive therapy. In addition, we are also investigating whether there exist larger and smaller numbers of circulating osteoclasts and osteoblasts, respectively, in MM patients, or whether these circulating cells show alteration of their expression of these genes. Our semi-quantitative RT-PCR results are being correlated with immunohistochemical staining results from osteoblast and osteoclast markers obtained on PBMCs from MM and normal subjects. These studies provide evidence that the number of circulating osteoblasts and osteoclasts is altered in patients with MM, and also may suggest that bisphosphonate therapy may also be associated with changes in these cell populations.

Proteomic Signature Predicting Achievement of Very Good Partial Response In Patients with Multiple Myeloma Based On Complementary Label-Free and iTRAQ Quantitative Proteome Analysis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1902-1902
Author(s):  
Dominik Dytfeld ◽  
Malathi Kandarpa ◽  
John R Strahler ◽  
Dattatreya Mellacheruvu ◽  
Suchitra Subramani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Western Blot ◽  
Differential Expression ◽  
Proteomic Analysis ◽  
Quantitative Proteomics ◽  
Proteomic Profile ◽  
Differentially Expressed Proteins ◽  
Label Free ◽  
Rt Pcr

Abstract Abstract 1902 Introduction: Multiple myeloma (MM) remains mostly incurable. Novel therapies have improved response rates, which are now reaching 100%. More importantly, number of recent studies showed that the depth of response, e.g. achievement of at least 90% reduction of the disease (≥VGPR) is associated with longer disease control. Therefore, improving VGPR rates and establishing predictors of VGPR to a given regimen may be an important clinical goal. High throughput quantitative proteomics may offer greater insight into the actual biology of the malignant cell than genome analysis and therefore, may be more useful in the development of personalized therapy. The objective of this study is to establish a proteomic signature predicting achievement of at least VGPR to initial treatment with bortezomib (Velcade®), pegylated liposomal doxorubicin, and dexamethasone (VDD). We previously reported preliminary proteomic profile of malignant plasma cells (PCs) obtained from a set of naïve MM pts enrolled in the VDD trial (Dytfeld et al., ASH 2009). Here we present the results of differential proteomic analysis of MM PCs of all available samples from the frontline VDD study (≥VGPR vs. <VGPR) using two independent and complementary quantitative proteomic platforms. We also compared the proteomic profile with gene expression data. Preliminary validation of the biomarkers of response prediction is presented. Methods: PCs were acquired from pre-treatment bone marrow specimens after obtaining informed consent from patients (pts), and were thereafter enriched with a RosetteSep® negative selection kit. Quantitative proteomic analysis of PCs from 17 naïve pts with MM from the VDD study was performed using iTRAQ approach in 8-plex variant. To increase confidence of analysis, label-free quantitative proteomics (LF) based on spectra counting was conducted on PCs from 12 pts. In iTRAQ experiments, proteins were processed with reagents according to the manufacturer's protocol followed by SCX fractionation and LC-MS/MS analysis (4800 Plus MALDI TOF/TOF). Peptides from the MM1S cell line were used as a reference. The data were analyzed using ProteinPilot™. For LF analysis, proteins were fractionated before trypsin digestion on Bis-Tris-Gel and subsequently run on LC-ESI-MS/MS on a linear trap mass spectrometer (LTQ Orbitrap). A database search was carried out using X!Tandem followed by Trans-proteomic Pipeline. At least 1.5-fold difference in expression in both platforms was used as a cut-off value. To correlate proteomics with gene expression of dysregulated proteins of interest, mRNA levels were analyzed by quantitative real time PCR (RT-PCR). Validation of proteomic findings on proteins of interest was performed using Western Blot. Results: We identified a total of 894 proteins in 3 iTRAQ experiments with high confidence (FDR<1%) and 1058 proteins by LF approach. Based on iTRAQ analysis, 20 proteins were found up-regulated in samples from pts with ≥VGPR (8 out of 17 pts) while 14 were down- regulated. Using LF approach, 284 proteins were elevated in the ≥VGPR group (6 out of 12 pts) while 315 proteins were down-regulated. Both iTRAQ and LF methods showed 15 differentially expressed proteins in common and 14 of them showed identical up or down trends. Interestingly, among differentially expressed proteins, there were proteins involved in proteasome activation (PSME1 and TXNL1), protection against oxidative stress (TXN and TXNDC5), glucose and cholesterol metabolism (TP1, APOA1 and ACAT1) and apoptosis (MX1). RT-PCR performed on a subset of genes confirmed the trend in differential expression between pts with ≥VGPR and <VGPR for TXNDC5 and PSME1. No change in mRNA expression levels was observed in TXN, APOA1, TPI1 and MX1while the trend in expression was reversed for ACAT1. Western blot analysis performed to date validated differential expression of PSME1. Conclusions: We present patient-derived proteomic characteristics of MM cells using two independent proteomic platforms. As a proof of concept, analysis of PCs obtained from pts enrolled in the frontline VDD study shows differential expression of 34 proteins in pts who achieved ≥VGPR vs. pts with <VGPR. Correlation with gene expression and further validation and functional analysis are in progress. This study was supported by a grant from the Multiple Myeloma Research Foundation. Disclosures: Jakubowiak: Millennium, Celgene, Bristol-Myers Squibb, Johnson & Johnson Ortho-Centocor: Honoraria; Millennium, Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millennium, Celgene, Centocor-Ortho Biotech: Speakers Bureau.

TRAF6 Dominant Negative Peptides Block Intracellular Signaling Resulting in Anti-Myeloma and Anti-Bone Resorptive Effects in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2919-2919
Author(s):  
Mingjie Li ◽  
Marissa P Dreyer ◽  
Cameryn P Ahles ◽  
David Ramirez ◽  
Cydney M Nichols ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Tumor Cells ◽  
Intracellular Signaling ◽  
Conflicts Of Interest ◽  
Cell Formation ◽  
Osteoclast Formation ◽  
Dominant Negative ◽  
Rt Pcr

Abstract Abstract 2919 Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating the NF-kB and JNK signal transduction pathways; and, thus, is likely to promote tumor cell proliferation and osteoclast formation. We have previously reported inhibition of cell proliferation and increase of apoptosis in multiple myeloma (MM) cells through regulation of these intracellular pathways through silencing of TRAF6 C-domain mRNA. To determine TRAF6 protein expression in fresh MM tumor cells, we performed an immunofluorescence assay (IFA). The results showed that expression of this factor in tumor cells from bone marrow (BM) from MM patients with progressive disease is higher than in cells from patients with monoclonal gammopathies without disease progression or normal controls. We further examined the effects of TRAF6 negative dominant peptides on intracellular signaling pathways. Briefly, cells from the RPMI8226 or MM1s MM cell lines or primary MM BM samples were treated with or without TRAF6 inhibition peptide for 24 hours and then stimulated with either IGF1 (30ng/ml) or IL1 β (20ng/ml) for 30 minutes. The cells were lysed and Western blot analysis performed to determine protein phosphorylation and RT-PCR for gene expression. TRAF6 has been found to be an E3 ligase for Akt ubiquitination. We found that IGF1 increased the phosphorylation of AKT and treatment with TRAF6 inhibition peptide markedly decreased its phosphorylation compared to treatment with a control peptide in RPMI8226 and primary MM tumor cells. Downstream of AKT, C-Raf phosphorylation was also significantly reduced with treatment with TRAF6 inhibition peptide. Notably, cyclin D gene expression in MM tumor cells treated with TRAF6 inhibition peptide was reduced as determined with an RT-PCR. In contrast, the gene expression of mTOR was increased in RPMI8226 cells treated with TRAF6 inhibition peptide whereas there was no change in its expression in MM1s and primary MM tumor cells. It is quite possible that the increase in mTOR expression in RPMI8226 cells may act as a negative feedback which results from blockage of the ubiquitination of TRAF6. We further examined the effect of the TRAF6 inhibition peptide on NF-kB and JNK signaling as determined through evaluation of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. Phospho-NF-kB protein was reduced and phosphorylation of JNKK was clearly decreased with exposure to the TRAF6 inhibition peptide. We examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. Total endogenous c-Jun is reduced following exposure of RPMI8226 cells to the TRAF6 inhibition peptide. Consistent with our past findings, TRAF6 inhibition peptide significantly inhibited osteoclast formation from CD14+ induced by RANKL and M-CSF with in a concentration dependent fashion whereas control peptides showed no effects on osteoclast formation. In addition, inhibition of the TRAF6 signaling blocked not only myeloma cell proliferation induced by AKT and NF-kB activation but also osteoclast cell formation mediated through transcription at TRE/AP-1 elements. The study has been extended to our SCID-hu murine model of human myeloma. Disclosures: No relevant conflicts of interest to declare.

Cytogenetic Analysis of Multiple Myeloma Identifies Cytogenetic Alterations Implicated in Disease Complexity and Progression

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3360-3360
Author(s):  
Erik Wendlandt ◽  
Guido J. Tricot ◽  
Benjamin Darbro ◽  
Fenghuang Zhan
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cathepsin B ◽  
Copy Number ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Enrichment Analysis ◽  
Copy Number Variations ◽  
Expression Arrays ◽  
Gene Expression Arrays

Abstract Background: Multiple myeloma is the second most common blood borne neoplasia, accounting for nearly 10% of all diagnosed hematologic malignancies and has a disproportionately high incidence in elderly populations. Here we explored copy number variations using the high fidelity CytoScan HD arrays to develop a detailed map of copy number variations and identify novel mediators of disease progression. The results from CytoScan HD microarrays provide a detailed view of the entire genome with a resolution up to 25kb. Furthermore, 750,000 single-nucleotide polymorphisms are included and the array provides information about loss of heterozygosity and uniparental disomy. Materials and methods: CytoScan HD arrays were performed on 97 myeloma patient samples to identify cytogenetic regions important to the development and progression of the disease. Gene expression profiles from 351 patients were analyzed to identify genes with a change in gene expression of 1.5 fold or more. Data from CytoScan and gene expression arrays was combined to perform chromosomal positional enrichment analysis to identify cytogenetic driver lesions, or lesions that provide a small, but significant growth and survival advantage to the cell. Furthermore, Kaplan-Meier, log-rank test and Hazard ratio analyses were performed to identify gene within the driver lesions that have a significant impact on survival when dysregulated. Results: The results from the CytoScan HD analysis closely mirrored what has been shown by FISH and SNP arrays, with gains to the odd numbered chromosomes, specifically 3, 5, 7, 9, 11, 15 and 17 as well as losses to chromosomes 1p and 13. Interestingly, we identified gains to a small region within chromosome 8p, contrary to published reports demonstrating a large scale loss of this region. We identified numerous genes within this region that are important for survival and their overexpression resulted in a decreased progression free survival. For example, Cathepsin B (CTSB) is encoded for in chromosome 8p22-p21 with an increased gene expression of at least 1.5 fold over normal controls, among others. Furthermore, Cathepsin B, a cysteine protease, has been linked to cancer of the ileum, suggesting that a similar role may be present within myeloma. We then integrated the 97 copy number profiles results with 351 myeloma gene expression profiles to identify cytogenetic driver lesions in myeloma important for disease development, progression and poor clinical outcome. Chromosomal positional enrichment analysis was employed to identify global myeloma cytogenetic driver aneuploidies as well as develop unique cytogenetic copy number profiles. Our results identified portions of chromosomes 1q, 3, 8p, 9, 13q and 16q, among others, as important driver lesions with changes to these regions providing growth advantages to the cell. Furthermore, our analysis identified five unique cytogenetic classifications based on common cytogenetic lesions. We continue to explore these driver regions to identify lesions important for the oncogenic properties of the larger regions. Conclusion: The data presented here represents a novel and highly sensitive approach for the identification of novel copy number variations and driver lesions. Furthermore, correlations between copy number variations and gene expression arrays identified novel targets important for disease progression and patient survival. CytoScan HD arrays in conjunction with gene expression analysis provided a high resolution image of important cytogenetic lesions in myeloma and identified potentially important therapeutic targets for drug development. Further work is needed to validate our findings and determine the therapeutic efficacy of the identified targets. Disclosures No relevant conflicts of interest to declare.

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