Illumina HT-12 Microarrays Research Articles

A co-formulation of interferons alpha2b and gamma distinctively targets cell cycle in the glioblastoma-derived cell line U-87MG

BackgroundHeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, which has shown its clinical superiority over individual interferons in basal cell carcinomas. In glioblastoma (GBM), HeberFERON has displayed promising preclinical and clinical results. This led us to design a microarray experiment aimed at identifying the molecular mechanisms involved in the distinctive effect of HeberFERON compared to the individual interferons in U-87MG model.MethodsTranscriptional expression profiling including a control (untreated) and three groups receiving α2b-interferon, γ-interferon and HeberFERON was performed using an Illumina HT-12 microarray platform. Unsupervised methods for gene and sample grouping, identification of differentially expressed genes, functional enrichment and network analysis computational biology methods were applied to identify distinctive transcription patterns of HeberFERON. Validation of most representative genes was performed by qPCR. For the cell cycle analysis of cells treated with HeberFERON for 24 h, 48 and 72 h we used flow cytometry.ResultsThe three treatments show different behavior based on the gene expression profiles. The enrichment analysis identified several mitotic cell cycle related events, in particular from prometaphase to anaphase, which are exclusively targeted by HeberFERON. The FOXM1 transcription factor network that is involved in several cell cycle phases and is highly expressed in GBMs, is significantly down regulated. Flow cytometry experiments corroborated the action of HeberFERON on the cell cycle in a dose and time dependent manner with a clear cellular arrest as of 24 h post-treatment. Despite the fact that p53 was not down-regulated, several genes involved in its regulatory activity were functionally enriched. Network analysis also revealed a strong relationship of p53 with genes targeted by HeberFERON. We propose a mechanistic model to explain this distinctive action, based on the simultaneous activation of PKR and ATF3, p53 phosphorylation changes, as well as its reduced MDM2 mediated ubiquitination and export from the nucleus to the cytoplasm. PLK1, AURKB, BIRC5 and CCNB1 genes, all regulated by FOXM1, also play central roles in this model. These and other interactions could explain a G2/M arrest and the effect of HeberFERON on the proliferation of U-87MG.ConclusionsWe proposed molecular mechanisms underlying the distinctive behavior of HeberFERON compared to the treatments with the individual interferons in U-87MG model, where cell cycle related events were highly relevant.

Analysis of Adct-602 Pre-Clinical Activity in B-Cell Lymphoma Models and Identification of Potential Biomarkers for Its Activity

Analysis of Adct-602 Pre-Clinical Activity in B-Cell Lymphoma Models and Identification of Potential Biomarkers for Its Activity

Differential gene expression analysis in blood of first episode psychosis patients

BackgroundPsychosis is a condition influenced by an interaction of environmental and genetic factors. Gene expression studies can capture these interactions; however, studies are usually performed in patients who are in remission. This study uses blood of first episode psychosis patients, in order to characterise deregulated pathways associated with psychosis symptom dimensions. MethodsPeripheral blood from 149 healthy controls and 131 first episode psychosis patients was profiled using Illumina HT-12 microarrays. A case/control differential expression analysis was performed, followed by correlation of gene expression with positive and negative syndrome scale (PANSS) scores. Enrichment analyses were performed on the associated gene lists. We test for pathway differences between first episode psychosis patients who qualify for a Schizophrenia diagnosis against those who do not. ResultsA total of 978 genes were differentially expressed and enriched for pathways associated to immune function and the mitochondria. Using PANSS scores we found that positive symptom severity was correlated with immune function, while negative symptoms correlated with mitochondrial pathways. ConclusionsOur results identified gene expression changes correlated with symptom severity and showed that key pathways are modulated by positive and negative symptom dimensions.

Inhibition of Pro-Survival Pathways in DLBCL Cells By Functional Lipoprotein-like Nanoparticles

Introduction: Diffuse large B cell lymphoma (DLBCL) cells have an increased demand for cholesterol and cholesteryl esters to maintain membrane-anchored pro-survival signaling pathways, such as B cell receptor (BCR) signaling. In addition to BCR-mediated stimulation of de novo cholesterol synthesis, another mechanism employed by lymphoma cells to maintain cholesterol and cholesteryl ester homeostasis is by binding cholesterol-rich HDLs via the high-affinity receptor, scavenger receptor type B-1 (SCARB1). We previously reported that, unlike normal B cells, DLBCL cells highly express SCARB1, and that synthetic, cholesterol-poor high-density lipoprotein (HDL)-like nanoparticles (HDL NPs) exquisitely target SCARB1, deplete cellular cholesterol, and induce lymphoma cell death in vitro and in vivo. Given the critical role of cholesterol in maintaining proper membrane organization for BCR-mediated intracellular signaling, we hypothesized that HDL NP binding to SCARB1 and subsequent cellular cholesterol depletion modulates the organization of the cell membrane, reduces intracellular signaling downstream of membrane-anchored pathways like the BCR, and modulates the expression of genes relevant to cholesterol synthesis. Collectively, these mechanisms potently induce lymphoma cell death.Methods: We focused on germinal center (GC) DLBCL and Burkitt's Lymphoma (BL), due to the previously reported sensitivity of the SUDHL4 (GC DLBCL) and Ramos (BL) cell lines to HDL NP-induced cell death. In addition to SUDHL4 and Ramos, we tested the GC DLBCL cell line SUDHL6 and the BL cell lines Raji, Daudi and Namalwa. HDL NPs were synthesized, purified and characterized using standard protocols. Cell viability was quantified using the MTS assay, and total cholesterol levels were measured using the Amplex Red cholesterol assay. A blocking antibody against SCARB1 was used to inhibit binding of HDL NPs to the receptor. Confocal microscopy was used to visualize changes in membrane organization of SUDHL4 and Ramos cells following HDL NP treatment. Changes in the phosphorylation of signaling kinases (e.g. AKT, ERK1/2) following HDL NP treatment was measured using phospho-kinase arrays, phos-flow analysis, and western blot assays. RNA was harvested from cells treated with the HDL NPs for 48 hours and analyzed using Illumina's HT-12 microarray and RT-qPCR. Protein expression changes were measured using western blot assays.Results: HDL NPs potently induced cell death in all of the GC DBLCL and BL cell lines, all of which expressed SCARB1, with HDL NP IC50 values between 1 ~ 5 nM. As expected, HDL NP-induced cell death correlated with reduced total cellular cholesterol levels. Inhibition of HDL NP binding to SCARB1 by antibody blockade protected cells from HDL NP-induced cell death. HDL NP treatment induced a reorganization of the plasma membrane in SUDHL4 and Ramos cells, resulting in clustering of SCARB1, a phenomenon not seen in cholesterol-rich human HDL or saline controls. HDL NP treatment led to a decrease in the phosphorylation of a number of kinases downstream of BCR signaling, including AKT, ERK1/2, LCK and LYN over time. HDL NPs demonstrated synergy with small molecule inhibitors of various signaling kinases, such as ATK (inhibitor = GDC-0068) and SYK (inhibitor = R406), in Ramos and SUDHL4 cells. By microarray analysis and RT-qPCR, HDL NPs up-regulated a number of cholesterol biosynthesis genes, as well as the cell cycle inhibitor p21 and the pro-apoptotic protein APOPT1.Conclusions: These data demonstrate that HDL NPs bind SCARB1, altering the organization of the plasma membrane and reducing cellular cholesterol levels. Collectively, this results in decreased membrane-anchored pro-survival signaling, changes in gene expression pathways, and, ultimately, lymphoma cell death. Enhancing cholesterol depletion strategies, such as the combination of the targeted cholesterol depletion agent HDL NP with small molecule inhibitors of signaling kinases (e.g. AKT and SYK inhibitors), represents a novel and targeted therapeutic strategy for DLBCL and may be broadly applicable to other malignancies dependent on cholesterol homeostasis and membrane-anchored signaling pathways. DisclosuresKarmali:Gilead: Speakers Bureau; AstraZeneca: Speakers Bureau. Thaxton:AuraSense: Other: Co-founder of the biotech company AuraSense.

Abstract WMP45: Gene Expression Profile of Cervical Artery Dissection by Time of Event (CADEX Study)

Introduction: Cervical artery dissection (CeAD) of the carotid and vertebral arteries is a prevalent cause of stroke in young adults. While the pathogenesis of incident CeAD remains poorly understood, clinical and genetic associations suggest a systemic arteriopathy at time of event. Hypothesis: Individuals suffering CeAD express a distinct gene expression profile at time of event compared to (1) age and sex-matched controls and (2) convalescent profiles within the same individual at a later time point. Methods: We enrolled 37 consecutive patients with carotid or vertebral artery dissection (with and without ischemic stroke) from 2013-2016, excluding concurrence of major trauma. Cases were age and sex matched to non-CeAD ischemic stroke controls (n=16) and healthy controls (n=11). Whole blood samples were collected within four weeks from time of event, and again at 3 and 6-month follow-up. We used IlluminaHT-12 microarrays to assess differential gene expression at time of CeAD relative to controls, and relative to gene expression at >3 month follow-up within CeAD cases. Mixed effects regression models included relevant covariates of age, sex, race/ethnicity, time of enrollment, and occurrence of stroke. We used a False Discovery Rate (FDR) cutoff of 5% to account for multiple testing as well as a 1.5 fold change cutoff to identify robust, differential gene expression. Results: We identified 538 differentially expressed genes between CeAD patients and healthy controls with 30 of these genes reaching our predetermined 1.5x fold change limit. Within CeAD cases, 1,238 genes showed differential expression at a 5% FDR compared to time points at 3-6 month follow-up, with 31 genes showing at least a 1.5 fold change. Compared to controls and convalescent profiles, 12 genes were significant using the 5% FDR and 1.5 fold change cutoffs. Preliminary gene-ontology analysis indicate the largest differential expression for genes associated with oxygen and gas transport. Conclusion: In this consecutive cohort of individuals with CeAD, we identified a distinct gene expression profile associated with incident dissection. These data are limited by small sample size and results are hypothesis generating. Replication in larger, independent cohorts is warranted.

Activation of Type I Interferon Signal Pathway in Patients with Multiple Sclerosis by the Russian Analog of β-Interferon-1b (transcriptional profiling data)

The introduction of Russian analogs of β-interferon (β-IFN) produced by recombinant strains of E. coli (β-IFN-1b) into medical practice in Russia for the pathogenetic treatment of multiple sclerosis (MS) requires, in accordance with contemporary standards, confirmation by transcriptome analysis that they activate the main signal pathways involved in the mechanism of action of β-IFN. In the present report we present such an analysis for Infibeta (produced by Generium). Whole-genome transcription profiling using an Illumina HT-12 microarray was performed to identify genes showing differential expression in peripheral blood mononuclear cells from MS patients in response to exposure to this agent. Comparison of the levels of gene expression in MS patients not previously treated with any kind of immunomodulatory therapy before and 10 h after the first dose of agent identified 490 genes whose expression showed statistically significant changes, of which 191 genes showed increases in expression (up-regulated genes) and 299 genes showed decreases in expression (down-regulated genes). These genes included genes from the inflammation system, the innate and adaptive immune systems, apoptosis, signal transmission, transcription, translation, and degradation. Analysis of the overall expression of sets (groups) of genes (gene set analysis) yielded data which may provide evidence that IFN type I signal pathway genes, as well as IFN-induced genes, are involved in patients’ responses to the agent. Thus, analysis by transcriptional profiling led to the conclusion that the mechanism of the immunomodulatory action of Infibeta is identical to that described for original β-IFN formulations.

Expression quantitative trait loci (eQTL) mapping in Puerto Rican children.

BackgroundExpression quantitative trait loci (eQTL) have been identified using tissue or cell samples from diverse human populations, thus enhancing our understanding of regulation of gene expression. However, few studies have attempted to identify eQTL in racially admixed populations such as Hispanics.MethodsWe performed a systematic eQTL study to identify regulatory variants of gene expression in whole blood from 121 Puerto Rican children with (n = 63) and without (n = 58) asthma. Genome-wide genotyping was conducted using the Illumina Omni2.5M Bead Chip, and gene expression was assessed using the Illumina HT-12 microarray. After completing quality control, we performed a pair-wise genome analysis of ~15 K transcripts and ~1.3 M SNPs for both local and distal effects. This analysis was conducted under a regression framework adjusting for age, gender and principal components derived from both genotypic and mRNA data. We used a false discovery rate (FDR) approach to identify significant eQTL signals, which were next compared to top eQTL signals from existing eQTL databases. We then performed a pathway analysis for our top genes.ResultsWe identified 36,720 local pairs in 3,391 unique genes and 1,851 distal pairs in 446 unique genes at FDR <0.05, corresponding to unadjusted P values lower than 1.5x10-4 and 4.5x10-9, respectively. A significant proportion of genes identified in our study overlapped with those identified in previous studies. We also found an enrichment of disease-related genes in our eQTL list.ConclusionsWe present results from the first eQTL study in Puerto Rican children, who are members of a unique Hispanic cohort disproportionately affected with asthma, prematurity, obesity and other common diseases. Our study confirmed eQTL signals identified in other ethnic groups, while also detecting additional eQTLs unique to our study population. The identified eQTLs will help prioritize findings from future genome-wide association studies in Puerto Ricans.

Immune correlates of varlilumab (CDX-1127) treated cancer patients are consistent with CD27 costimulatory activity

Varlilumab is a human IgG1 agonist anti-CD27 antibody designed to activate T cells through CD27 costimulation. Preclinical studies have shown that Varlilumab efficiently activates human T cells when combined with T cell receptor stimulation, enhances antigen specific CD8 T cell responses in human CD27 transgenic mice when combined with vaccination, and mediates anti-tumor activity in these mice when challenged with syngeneic tumors. A multi-dose, dose-escalation/expansion trial of Varlilumab in patients with advanced solid tumors (n = 56) or lymphoma (n = 24) has demonstrated a good safety profile with no MTD reached through the 10 mg/kg dose level. Evidence of clinical activity includes a complete response in a treatment-refractory Stage IV Hodgkin’s patient (remains in remission at 12.9 months); a renal cell carcinoma (RCC) patient with a partial response (ongoing at 3.8 months); an additional RCC patient with extended stable disease (ongoing at 22.4+ months); and 12 additional patients with stable disease (range: 2.7+ to 14 months). We performed extensive correlative immune monitoring from serum and peripheral blood cells, particularly in melanoma and RCC patients. Differential gene expression analysis was assessed preand post-treatment by hybridizing RNA isolated from whole blood PAXgene RNA tubes onto Illumina HT-12 microarrays. Transcriptomic analysis showed significant impact of Varlilumab on immunological pathways, including T cell receptor signaling that was observed at early time points (day 2 and day 8 post treatment). Interestingly, these changes were most significant at low dose levels (0.1 and 0.3 mg/kg), however, a larger sample size would be required to confirm these results. Varlilumab administration was associated with enhanced T cell activity by evaluation of activation markers and functional analysis using IFN-g Elispot. In particular, evaluation of response to peptides derived from melanoma antigens in selected patients showed evidence that Varlilumab promoted CD8 T cell function against melanoma antigens. Specifically, expanded responses were detected to epitopes from gp100 and MART-1, while de novo responses to epitopes MAGE-A1, NY-ESO-1 and gp100 were evident. These responses were confirmed by MHC-multimer staining. These data, and our previous analyses demonstrating a transient increase in pro-inflammatory cytokines (IP-10, IL-6, MCP-1), up-regulation of HLA-DR expression on T cells, increase in NK cell numbers, and decrease in regulatory T cells in response to Varlilumab treatment, show a pattern consistent with CD27 costimulation. These results, together with our preclinical data, provide the rationale for initiating clinical studies of Varlilumab in combination with therapies such as vaccines, checkpoint inhibitors and targeted therapies.

Gene expression profiles associated with acute myocardial infarction and risk of cardiovascular death

BackgroundGenetic risk scores have been developed for coronary artery disease and atherosclerosis, but are not predictive of adverse cardiovascular events. We asked whether peripheral blood expression profiles may be predictive of acute myocardial infarction (AMI) and/or cardiovascular death.MethodsPeripheral blood samples from 338 subjects aged 62 ± 11 years with coronary artery disease (CAD) were analyzed in two phases (discovery N = 175, and replication N = 163), and followed for a mean 2.4 years for cardiovascular death. Gene expression was measured on Illumina HT-12 microarrays with two different normalization procedures to control technical and biological covariates. Whole genome genotyping was used to support comparative genome-wide association studies of gene expression. Analysis of variance was combined with receiver operating curve and survival analysis to define a transcriptional signature of cardiovascular death.ResultsIn both phases, there was significant differential expression between healthy and AMI groups with overall down-regulation of genes involved in T-lymphocyte signaling and up-regulation of inflammatory genes. Expression quantitative trait loci analysis provided evidence for altered local genetic regulation of transcript abundance in AMI samples. On follow-up there were 31 cardiovascular deaths. A principal component (PC1) score capturing covariance of 238 genes that were differentially expressed between deceased and survivors in the discovery phase significantly predicted risk of cardiovascular death in the replication and combined samples (hazard ratio = 8.5, P < 0.0001) and improved the C-statistic (area under the curve 0.82 to 0.91, P = 0.03) after adjustment for traditional covariates.ConclusionsA specific blood gene expression profile is associated with a significant risk of death in Caucasian subjects with CAD. This comprises a subset of transcripts that are also altered in expression during acute myocardial infarction.

A systematic experimental evaluation of microRNA markers of human bladder cancer

Background: MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression. They are aberrantly expressed in many human cancers and are potential therapeutic targets and molecular biomarkers.Methods: In this study, we for the first time validated the reported data on the entire set of published differential miRNAs (102 in total) through a series of transcriptome-wide experiments. We have conducted genome-wide miRNA profiling in 17 urothelial carcinoma bladder tissues and in nine normal urothelial mucosa samples using three methods: (1) An Illumina HT-12 microarray hybridization (MA) analysis (2) a suppression-subtractive hybridization (SSH) assay followed by deep sequencing (DS) and (3) DS alone.Results: We show that DS data correlate with previously published information in 87% of cases, whereas MA and SSH data have far smaller correlations with the published information (6 and 9% of cases, respectively). qRT-PCR tests confirmed reliability of the DS data.Conclusions: Based on our data, MA and SSH data appear to be inadequate for studying differential miRNA expression in the bladder.Impact: We report the first comprehensive validated database of miRNA markers of human bladder cancer.

Relation between smoking history and gene expression profiles in lung adenocarcinomas

BackgroundLung cancer is the worldwide leading cause of death from cancer. Tobacco usage is the major pathogenic factor, but all lung cancers are not attributable to smoking. Specifically, lung cancer in never-smokers has been suggested to represent a distinct disease entity compared to lung cancer arising in smokers due to differences in etiology, natural history and response to specific treatment regimes. However, the genetic aberrations that differ between smokers and never-smokers’ lung carcinomas remain to a large extent unclear.MethodsUnsupervised gene expression analysis of 39 primary lung adenocarcinomas was performed using Illumina HT-12 microarrays. Results from unsupervised analysis were validated in six external adenocarcinoma data sets (n=687), and six data sets comprising normal airway epithelial or normal lung tissue specimens (n=467). Supervised gene expression analysis between smokers and never-smokers were performed in seven adenocarcinoma data sets, and results validated in the six normal data sets.ResultsInitial unsupervised analysis of 39 adenocarcinomas identified two subgroups of which one harbored all never-smokers. A generated gene expression signature could subsequently identify never-smokers with 79-100% sensitivity in external adenocarcinoma data sets and with 76-88% sensitivity in the normal materials. A notable fraction of current/former smokers were grouped with never-smokers. Intriguingly, supervised analysis of never-smokers versus smokers in seven adenocarcinoma data sets generated similar results. Overlap in classification between the two approaches was high, indicating that both approaches identify a common set of samples from current/former smokers as potential never-smokers. The gene signature from unsupervised analysis included several genes implicated in lung tumorigenesis, immune-response associated pathways, genes previously associated with smoking, as well as marker genes for alveolar type II pneumocytes, while the best classifier from supervised analysis comprised genes strongly associated with proliferation, but also genes previously associated with smoking.ConclusionsBased on gene expression profiling, we demonstrate that never-smokers can be identified with high sensitivity in both tumor material and normal airway epithelial specimens. Our results indicate that tumors arising in never-smokers, together with a subset of tumors from smokers, represent a distinct entity of lung adenocarcinomas. Taken together, these analyses provide further insight into the transcriptional patterns occurring in lung adenocarcinoma stratified by smoking history.

Effect of serum amyloid A1 treatment on global gene expression in THP-1-derived macrophages

To investigate the effect of serum amyloid A1 (SAA1) on global gene expression in macrophages derived from THP-1 monocytes. Global genetic expression in THP-1-derived macrophages was determined using Illumina HT-12 microarray chips and the results were validated by real-time PCR. Cytokine levels in cellular supernatant were quantified by ELISA. In total, 55 genes were upregulated with fold difference greater than two when THP-1-derived macrophages were incubated with SAA1 for 8h. SAA1 is a strong cytokine inducer with significant upregulation of chemokines CCL1, CCL3, and CCL4 and this was confirmed by both real-time PCR and ELISA quantification. SAA1 also promotes the upregulation of genes involved in phagocytosis, anti-apoptosis, and tissue remodeling. SAA1 appears to play an important role during the immune response and in chronic inflammatory diseases through the stimulation of genes involved in cytokine production, phagocytosis, and anti-apoptosis.

P4-01-09: Reduced CYBA Expression Leads to the Development of Fulvestrant Resistance in a Breast Cancer Cell Line.

Abstract Background Acquired resistance to endocrine therapy is an important problem in the management of advanced breast cancer. In this study we used a cell line model to investigate the evolution of fulvestrant resistance. We examined gene expression and promoter methylation in estrogen-dependent MCF7 cells and compared two derived cell lines, estrogen-hypersensitive LCC1 and fulvestrant-resistant LCC9, using array technology. Thus we determined the epigenetic changes associated with the evolution of estrogen independence and fulvestrant resistance, which revealed a role for CYBA/p22phox in the acquisition of resistance. Material and Methods: Estrogen-dependent gene expression was measured in MCF7, LCC1 and LCC9 cells using Illumina HT-12 microarrays. In parallel, differential methylation between MCF7, LCC1 and LCC9 cells was examined using Illumina Infinium methylation 27 arrays. Results: Despite marked physiological differences between MCF7, LCC1 and LCC9 cells there were few genes which were both differentially methylated and differentially expressed. However, CYBA was underexpressed in LCC1 (p&amp;lt;10−5) and LCC9 (p&amp;lt;10−4) and had a methylated promoter in LCC1 (p&amp;lt;10−5) and LCC9 (p&amp;lt;10−4), with reduced expression correlated to increased estrogen independence. This gene codes for the protein p22phox, a critical component of membrane NADPH oxidase. We verified expression by qRT-PCR and Western blot, and methylation by CpG island bisulphite sequencing, demonstrating significant and biologically relevant changes. Fulvestrant resistant LCC9 cells exhibited a 20-fold reduction in CYBA expression compared to MCF7, corresponding to undetectable levels of p22phox. Due to its role in the generation of reactive oxygen species (ROS), we investigated the cellular response to oxidant damage, finding that LCC9 cells showed reduced sensitivity to oxidant damage when compared to MCF7 (p&amp;lt;10−7), in keeping with impaired NADPH oxidase function. We then treated MCF7 cells with a ROS scavenger, N-acetyl cysteine, which reduced their sensitivity to fulvestrant (p&amp;lt;10-13). Using siRNA, we knocked down CYBA expression, which conferred slight fulvestrant resistance on MCF7 cells, mitigating fulvestrant-induced growth arrest (p&amp;lt;10−7). Discussion: The striking differences in methylation in the CYBA promoter and gene expression between these cell lines lead us to conclude that the repression of this gene represents an important step in the development of fulvestrant resistance. As these cells develop estrogen independence and fulvestrant resistance, CYBA/p22phox levels drop, making the cells more resistant to oxidant damage caused by estrogen deprivation and, ultimately, providing a selective advantage against fulvestrant-induced growth arrest and cell death. CYBA/p22phox may represent a novel druggable target in breast cancer, either in the prevention of the acquisition of fulvestrant resistance, or the further treatment of resistant tumours. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-09.

P53-Independent Anti-Myeloma Activity of Prima-1met

Abstract 1826 Background:Although mutation/deletion of p53 is relatively rare (∼10%) in multiple myeloma (MM), this subset of patients are resistant to current therapies with very poor prognosis. Thus, improved therapeutic strategy is urgently required for this high risk group of patients. Prima-1met (p53 reactivation and induction of massive apoptosis), a methylated derivative and more active analog of prima-1, is a small molecule which restores tumor suppressor function to mutant p53 and has shown to induce cytotoxic effects and apoptosis in various human tumor cells harboring mutant p53. However, anti-myeloma activity of prima-1met is unknown. Experimental design:6 human MM cell lines, primary MM samples from 7 newly diagnosed patients, bone marrow mononuclear cells (BMNCs) and peripheral blood mononuclear cells (PBMNCs) from 3 healthy donors were used for this study. MM.1S and H929 cell lines harbour wild type p53; 8266, U266, and LP1 express mutant p53; and 8226R5 does not express p53 (p53 null). The cells were treated with different dose (2.5-100 μM) of prima-1met for different time periods. The therapeutic effect of prima-1met was studied in these cells using MTT cell viability assay, flow cytometry (FCM), qRT-PCR, and Western blot (WB) analysis. Results:Prima-1met efficiently inhibited the viability of MM cell lines irrespective of p53 status. Similarly, prima-1met induced a dose-dependent cytotoxic response in 5 of the 7 patient samples tested. However, prima-1met only showed limited cytotoxicity toward normal BMNCs or PBMNCs suggesting a preferential killing of MM cells by prima-1met. We next examined whether the declining in the viability of the cells was due to the apoptosis induction by prima-1met. Depending on the cell types, 48 hrs after treatment with 100 μM prima-1met 35–70% Annexin V-positive cells was determined by FCM. The cytotoxic effect of prima-1met was associated with activation of caspase-3, PARP cleavage, up-regulation of Noxa and c-Jun without significant modulation of p53 level. Treatment of cells with prima-1met induced the activation of caspase-8 but not -9 suggesting the association of extrinsic pathway. Furthermore, the pan-caspase inhibitor, Z-VAD-FMK, inhibited the activation of caspase-3 indicating that prima-1met-induced apoptosis in MM cells is caspase-dependent. The apoptotic effect of prima-1met was not significantly affected in H929 cells transfected with p53 siRNA confirming that prima-1met exerts anti-myeloma activity in a p53-independent manner. To further explore the molecular mechanisms associated with prima-1met-induced apoptosis, RNA from DMSO-treated and prima-1met-treated MM cells (MM.1S, U266 and 8266R5) was profiled by Illumina HT-12 microarray and differentially expressed genes were analysed. A significant number of genes associated with stress response such as HASP1B, HSPA6, FOS, ATF3, MYC and EGR-1 were modulated in all 3 cell lines upon prima-1met treatment. Of note, EGR-1 is highly up-regulated (7 to 21-fold) in all 3 cell lines. EGR-1 is a member of the immediate-early gene family and plays a pivotal role in the regulation of cell growth, differentiation and apoptosis. Overexpression of EGR-1 has shown to down-regulate anti-apoptotic protein survivin and induction of apoptosis in myeloma cells. The role of EGR-1 and other relevant stress response genes associated with p53-independent apoptosis induced by prima-1met is currently under investigation.In addition, we found a strong synergistic effect in MM cells harbouring either wild type or mutant p53 when prima-1met was combined with dexamethasone (DXM) or doxorubicin (DOXO). We examined cell cytotoxicity of the combination by using prima-1met and DOXO/DXM at concentrations lower than their maximal cytotoxic concentrations. The combination of 2 μM prima-1met and 0.5 μM DOXO/DXM produced a synergistic response (CI=0.82-0.87) in H929 cells. The combination of 10 μM prima-1met and 2.0 μM DOXO displayed a synergistic cytotoxic response (CI=0.68) in U266 cells. Conclusion:This study is the first to show the potential p53-indpendent anti-myeloma activity of prima-1met. Our results provide the framework for the clinical evaluation of prima-1met either alone or in combination with other chemotherapeutic agents which may offer a novel and more efficient therapeutic strategy for the treatment of MM patients carrying either wild type or mutant p53. Disclosures:No relevant conflicts of interest to declare.

Genes Regulated by miRNA in Endocrine-Resistant Breast Tumours.

Abstract RationaleChange in genes expression is one of the major molecular drivers of tumour growth. Mechanisms controlling genes expression include transcriptional factors, mutations, aberrations in gene copy number, epigenetic mechanisms and micro-RNA (miRNA) regulation. The last mechanism has been discovered recently and there is a need to investigate its involvement in genes regulation in endocrine resistant breast tumours.Methods and ResultsTo identify genes regulated by miRNA in endocrine resistant breast tumours we collected frozen biopsies from a series of 51 tumours growing on endocrine treatment (aromatase inhibitors or tamoxifen, post-menopausal patients). Gene expression profiles were obtained using Illumina HT-12 micro-arrays; miRNA expression profiles were obtained using Illumina DASL® Sentrix Array Matrix.Unsupervised hierarchical clustering using 50 most variable miRNAs split tumours into two major clusters (25 and 26 tumours each). We suggest that genes differentially expressed between these two clusters can be potentially regulated by miRNA. The top differentially expressed genes included SAA1, VTCN1, SLC40A1, FBLN1, COL17A1, HDC and CD59.An alternative bioinformatics approach was based on correlations between mRNA and miRNA in the studied tumours. Initially we identified 66 genes negatively correlated with at least one miRNA (r&amp;lt;-0.6). Then we screened the correlated pairs to identify those in which miRNA has target sequence within the corresponding gene. This approach yielded just 2 genes: RAG1 and USP19: RAG1 being potentially regulated by mir92 and USP19 regulated by miR15a.DiscussionEndocrine resistance is a major obstacle in treatment of many breast cancer patients. Molecular mechanisms of the resistance are yet unclear. The abstract presents a sub-group analysis within a larger study, aimed to investigate molecular mechanisms underlying endocrine resistance at both mRNA and miRNA levels. Most of the identified potential miRNA-regulated genes have not been previously reported in the context of breast cancer biology. On our knowledge, this study represents the first systematic attempt to identify genes regulated by miRNA in endocrine-resistant breast cancers. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5149.