Whole-genome Expression Arrays Research Articles

Preimplantation Endometrial Transcriptomics in Natural Conception Cycle of the Rhesus Monkey

There is no report on preimplantation phase endometrial transcriptomics in natural conception cycles of primates. In the present study, the whole-genome expression array of endometrium on Days 2, 4, and 6 post-ovulation (pov) in proven natural conception (Group 1; n = 12) and non-mated, ovulatory (Group 2; n = 12) cycles of rhesus monkeys was examined, compared, and validated. Of fifteen (15) genes showing differential expression (>2-fold; pFDR < 0.05), six genes (CHRND, FOXD3, GJD4, MAPK8IP3, MKS1, and NUP50) were upregulated, while eight genes (ADCY5, ADIPOR1, NNMT, PATL1, PIGV, TGFBR2, TOX2, and VWA5B1) were down regulated on Day 6 pov as compared to Day 2 pov in conception cycles. On Day 6 pov, four genes (ADCY5, NNMT, TOX2, and VWA5B1) were down regulated, and AVEN was upregulated in conception cycles compared with the non-conception cycle. These observations were orthogonally validated at protein expression level. Group-specifically expressed unique genes in conception cycles influence the process of induction of immune-tolerance, while the genes expressed in both groups influence processes of protein targeting and metabolism. A triad of timed-actions of progesterone, seminal plasma, and preimplantation embryo putatively regulate several input molecules to CREB, NF-kB, and STAT regulatory networks during secretory phase towards evolution of endometrial receptivity in the rhesus monkey.

Serum from Older Adults Increases Apoptosis and Molecular Aging Markers in Human Hippocampal Progenitor Cells.

Age-related alteration in neural stem cell function is linked to neurodegenerative conditions and cognitive decline. In rodents, this can be reversed by exposure to a young systemic milieu and conversely, the old milieu can inhibit stem cell function in young rodents. In this study, we investigated the in vitro effect of the human systemic milieu on human hippocampal progenitor cells (HPCs) using human serum from early adulthood, mid-life and older age. We showed that neuroblast number following serum treatment is predictive of larger dentate gyrus, CA3, CA4 and whole hippocampus volumes and that allogeneic human serum from asymptomatic older individuals induced a two-fold increase in apoptotic cell death of HPCs compared with serum from young adults. General linear models revealed that variability in markers of proliferation and differentiation was partly attributable to use of antihypertensive medication and very mild cognitive decline among older subjects. Finally, using an endophenotype approach and whole-genome expression arrays, we showed upregulation of established and novel ageing molecular hallmarks in response to old serum. Serum from older subjects induced a wide range of cellular and molecular phenotypes, likely reflecting a lifetime of environmental exposures. Our findings support a role for the systemic enviroment in neural stem cell maintenance and are in line with others highlighting a distinction between neurobiological and chronological ageing. Finally, the herein described serum assay can be used by future studies to further analyse the effect of environmental exposures as well as to determine the role of the systemic environment in health and disease.

Downregulation and Aberrant Localization of Forkhead Box J1 in Allergic Nasal Mucosa

Background: Forkhead box J1 (FOXJ1) plays pivotal roles in motile cilia formation. However, it remains unclear whether abnormal expression or localization of FOXJ1 in nasal mucosa tissues is associated with allergic rhinitis (AR), in which impaired mucociliary clearance is implicated. Objective: We sought to investigate the expression and localization of FOXJ1 in inferior turbinate from patients with AR and controls. Methods: We assayed mRNA levels of FOXJ1, DNAI1, DNALI1, and DNAH9 by using whole-genome expression array and quantitative real-time polymerase chain reaction. We elucidated the localization of FOXJ1 by using immunofluorescence assays in paraffin sections and primary single cells. Four patterns of FOXJ1 localization (normal, N; intermediate, I; mislocalization, M; absence, A) were defined. We developed a semiquantitative scoring system to elucidate their localization in 5 areas per paraffin section, with individual sections being assigned a score between 0 and 2. Results: The mRNA levels of FOXJ1, DNAI1, DNALI1, and DNAH9 were significantly reduced in patients with AR compared with controls (all p < 0.05). The median (1st and 3rd quartile) of the FOXJ1 score was 0.4 (0.0 and 0.85) in patients with AR, and 0.2 (0.0 and 0.4) in controls (p < 0.05). For primary cytospin samples, the mean percentages of FOXJ1 localization patterns N, I, M, and A were 46.7, 10.0, 30.0, and 26.7% in patients with AR, and 82.5, 5.0, 5.0, and 7.5% in controls, respectively (p < 0.05). Conclusion: Downregulation and aberrant localization of FOXJ1 may be crucial characteristics of the allergic nasal mucosa.

Abstract 1994: Defining peritumoral and tumoral stromal gene signatures associated with response to neoadjuvant chemotherapy in breast cancer

Abstract Neoadjuvant chemotherapy (NACT) is used in breast cancer to decrease tumor and lymph node burden and the extent of surgery. However, response is variable and prediction poor. Stromal cells are involved in breast cancer progression and drug resistance although very little is known about the mechanisms. Our research aims to characterize breast cancer-associated fibroblasts (CAFs) and peritumoral fibroblasts (PTFs) that predict response to NACT and could be used to influence treatment choices. Pre-NACT core biopsies from cancer-associated and peritumoral tissues were collected under ultrasound guidance. To date, mRNAs from early passage (passage ≤ 5) fibroblasts isolated from cores of 23 matched pairs (CAFs and PTFs) (8 triple-negative (TNBC), 7 ER+Her2+ and 8 ER+Her2- tumors) and an additional 8 PT samples (4 TNBC, 1 ER+Her2+ and 3 ER+Her2-) have been analysed by whole-genome expression array (Illumina beadchip HT12v4). Data were analysed with Partek® Genome SuiteTM 6.6 and Pathway softwares (Partek Inc., St. Louis, MO, USA). Differentially expressed genes (DEGs) were identified (t-test, p-value ≤ 0.05, fold-change (FC) ≥ 1.3) between CAFs and PTFs and between NACT responders (Residual Cancer Burden (RCB) 0/1) and non-responders (RCB 2/3). No significant difference between CAFs and PTFs was seen by principal component analysis and t-test. PTFs showed similar mRNA levels of major myofibroblast markers (e.g. FAP, ASMA) with only FSP1 being lower in PTFs compared to CAFs (FC = -1.4, p-value = 0.023), suggesting that PTFs, like CAFs, are activated myofibroblasts. We then tried to identify a gene signature in CAFs or PTFs that could predict response to NACT. Comparing RCB 0/1 vs RCB 2/3 resulted in very few DEGs due to cancer subtype variation in both CAFs and PTFs. Similar to other studies, only 2/19 patients in our cohort with ER+ tumours achieved RCB 0/1, preventing analysis of these samples. However, 45% (5/11) TNBC responded to NACT and we identified 566 DEGs between RCB 0/1 and RCB 2/3 CAFs, with changes in metabolism, Pi3K/AKT pathway, proteoglycan synthesis or ECM-receptor interaction. For PTFs, 186 DEGs were identified, with changes in Wnt signalling and ECM-receptor pathways. Microarray data from our uniquely large sample set of 23 pairs of CAFs and PTFs from breast cancer patients suggest that PTFs have a similar gene expression profile to CAFs. Although others have demonstrated that CAF phenotype is breast cancer subtype dependent, we are the first to show similar observations in PTFs. Finally, we identified gene signatures from both CAFs and PTFs that relate to NACT response in TNBC. We are currently in the process of confirming the most highly dysregulated genes by qPCR and/or immunohistochemistry analysis. Citation Format: Celine Pourreyron, Violet Warwick, Patsy Whelehan, Philip Coates, Lee Jordan, Colin Purdie, Sarah Vinnicombe, Alastair Thompson, Andrew Evans, Frances Fuller-Pace. Defining peritumoral and tumoral stromal gene signatures associated with response to neoadjuvant chemotherapy in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1994. doi:10.1158/1538-7445.AM2017-1994

Apomorphine-induced pathway perturbation in MPP&lt;sup&gt;+&lt;/sup&gt;-treated SH-SY5Y cells

Apomorphine (APOM) is a non-selective dopamine agonist for Parkinson’s disease (PD). It also offers protection against oxidative stress. Thus, it has been used for treating advanced PD patients who do not respond to levodopa or other dopamine agonists. However, side effects such as orthostatic hypotension, nausea, and fibrotic nodules at the site of APOM injection have been reported after long-term use of APOM in PD patients. To secure the use of APOM for PD treatment without side effect, it is essential to understand the molecular mechanism involved in the action of APOM in PD. In this study, gene expression profile changes by APOM in a PD cell model, i.e., MPP+-treated SH-SY5Y cells, were measured at six time points (0, 3, 6, 9, 12, and 24 h) after APOM treatment using a commercial whole-genome expression array. A total of 2249 genes showed significant and differential expression profile. Pathways significantly affected by APOM were estimated using signaling pathway impact analysis (SPIA). In addition, differentially regulated regions within each affected pathway were identified with covariance analysis using a structure equation model.

Abstract 4435: Genome-scale DNA methylation changes delineate uterine leiomyoma subgroups

Abstract Symptomatic uterine leiomyomas (ULs) occur in as many as 20% of women. These bening lesions are a major burden to women's health by causing health complications such as abnormal bleeding, pain and infertility, and they are a common cause of hysterectomy that is at present the only curative treatment. Similar to other solid tumors, genetic and epigenetic changes are likely to have value as predictors of UL clinical outcome, as well as response to treatment. Recent findings from us and others have provided evidence for existence of at least three distinct molecular UL subgroups, each displaying a characteristic genetic driver aberration; MED12 mutation, HMGA2 activation or FH mutation. We conducted DNA methylation analysis of bisulfite sequencing data from 59 tumors and 38 normal myometrium samples on targeted genomic regions (84.5Mb) such as CpG islands, shores and shelves, GENCODE promoters and DNaseI hypersensitive sites from ENCODE data using Agilent SureSelect methyl-seq target enrichment system. The studied samples consisted of 17 MED12-mutated, 11 HMGA2-upregulated and 6 FH-mutated tumors. Whole-genome sequencing and expression array profiling had been performed previously from the same specimens (Mehine, Kaasinen, et al. 2014). We developed a data processing pipeline that makes use of: (i) Trim Galore tool for quality control of sequencing data (ii) Bismark for alignment and DNA methylation calling (Krueger &amp; Andrews 2011). Hierarchical clustering of all detected CpG sites revealed that MED12- and FH-mutated tumors cluster according to the driver changes, and HMGA2-upregulated tumors cluster in two separate branches. Global hypermethylation profile was detected in FH-mutated tumors, which is compatible with previous reports in paragangliomas (Letouzé et al. 2013). Four tumors with COL4A5/COL4A6 aberration did not display uniform DNA methylation pattern and four tumors without known genetic driver changes clustered among normal myometrium samples; one of these tumors harbors a somatic TP53 rearrangement created through massive chromothripsis rearrangement event. As myometrium samples showed uniform global DNA methylation by clustering clearly separate from majority of the tumors, statistically powerful comparison of UL subgroups with different genetic background to myometrium samples was enabled. Furthermore, integration of differentially methylated regions with significant expression changes allowed us to characterize genes which are epigenetically activated or silenced in different UL subgroups. All ULs displayed increased methylation of genes enriched in developmentally related biological processes. Our results provide strong evidence that the known UL subgroups are distinguishable by global DNA methylation profiling. The role of the identified epigenetically activated or silenced genes should be studied further to allow better understanding of the biological processes related to UL development and clinical outcome. Citation Format: Eevi Kaasinen, Amjad Alkodsi, Miika Mehine, Hanna-Riikka Heinonen, Netta Mäkinen, Mervi Aavikko, Kati Kampjärvi, Minna Taipale, Pia Vahteristo, Rainer Lehtonen, Lauri A. Aaltonen. Genome-scale DNA methylation changes delineate uterine leiomyoma subgroups. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4435.

Abstract 2119: Acquired nintedanib resistance in FGFR1-driven small cell but not non-small cell lung cancer is mediated by ABCB1

Abstract Lung cancer accounts for the highest number of cancer-related deaths worldwide. Patient outcome is dismal and approval of targeted compounds is yet restricted to non-small cell lung cancer (NSCLC) histology. In defined subgroups of NSCLC, but also of small cell lung cancer (SCLC), genetically amplified fibroblast growth factor receptor 1 (FGFR1) is an oncogenic driver and inhibition of the FGFR1 signaling axis exerts potent antitumor effects. The FGFR/PDGFR/VEGFR inhibitor (TKI) nintedanib has recently been approved for second-line treatment of lung adenocarcinoma (AC) and is also being investigated in clinical trials for the treatment of SCLC. Despite initial treatment success, tumor recurrence due to acquired drug resistance is anticipated. We therefore aimed at characterizing the molecular mechanisms underlying resistance development of FGFR1-driven lung cancer against nintedanib. One SCLC line (DMS114) and two NSCLC squamous cell carcinoma (SCC) lines (NCI-H1703, NCI-H520) driven by FGFR1 based on gene amplification were selected for nintedanib resistance in vitro. Cytotoxicity was evaluated by MTT and FACS. Intracellular responses to drug exposure were analyzed by qPCR and Western Blot. To compare transcription levels of parental cells with their selected sublines, whole-genome gene expression array was performed. Cross-cytotoxicity profiles were determined by a large-scale anticancer compound screen. ATP-binding cassette (ABC) transporter activity was analyzed by calcein AM efflux- and ATPase assay, intracellular Nintedanib levels were determined by liquid chromatograpy-mass spectrometry (LC-MS). Chronic exposure to the FGFR/PDGFR/VEGFR inhibitor nintedanib led to expression of the ABCB1 multidrug-resistance (MDR) efflux pump in the SCLC cell line DMS114 but not in the NSCLC SCC cell lines NCI-H1703 and NCI-H520. In the nintedanib-selected subline of DMS114 (DMS114/NIN), the FGFR1 signaling axis remained active. Insensitivity of DMS1114/NIN towards Nintedanib was mediated by efflux via ABCB1, revealing Nintedanib as a substrate for this efflux pump. Ponatinib, another non-ABCB1-substrate FGFR inhibitor, retained strong cytotoxic activity. Additionally, DMS114/NIN cells exerted profound and ABCB1-dependent collateral sensitivity against the MDR-selective lanthanum compound KP772. ABCB1 needs to be considered as a factor underlying intrinsic and acquired nintedanib resistance. On one hand, in second-line therapy, chemotherapy-induced MDR might render tumors resistant to Nintedanib. On the other hand, in first-line treatment, high ABCB1 expression in cancer cells or the microvasculature e.g. in clear-cell renal carcinoma or CNS tumors might account for intrinsic Nintedanib resistance. Switching to MDR-selective anticancer compounds or to non-ABCB1-substrate small molecule TKIs in FGFR/PDGFR/VEGFR-driven tumors might be an option to circumvent nintedanib resistance. Citation Format: Bernhard Englinger, Daniela Lötsch, Christine Pirker, Sushilla van Schoonhoven, Bernd Boidol, Charles Lardeau, Stefan Kubicek, Pal Szabó, Gergely Szakacs, Walter Berger. Acquired nintedanib resistance in FGFR1-driven small cell but not non-small cell lung cancer is mediated by ABCB1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2119.

Abstract LB-323: The genomic landscape of non-muscle-invasive bladder cancer: implications for molecular classification and treatment

Abstract Non-muscle-invasive bladder cancers (NMIBC) and muscle-invasive bladder cancers (MIBC) show distinct molecular and clinical features and are considered to follow different pathogenesis pathways. MIBC commonly metastasise (&amp;gt;50%) and have poor prognosis whereas NMIBC, particularly low-stage low-grade tumors, rarely progress to invade muscle (&amp;lt;5%). Patients diagnosed with NMIBC frequently suffer disease recurrence, demanding long-term disease monitoring and repeated resection of recurrences. Large MIBC cohorts have been extensively interrogated on a genome-wide scale but relatively few NMIBC have been studied at this level. To address this we determined the mutational landscape of 82 non-muscle-invasive tumors (stage Ta grade 2) using whole-exome and targeted deep sequencing. Whole-exome sequencing identified an average of 124±79 synonymous and non-synonymous somatic mutations (single nucleotide substitutions and indels) per sample, giving mean and median somatic mutation rates of 2.41 and 1.64 per megabase, respectively. Overall, 47% of nucleotide substitutions were C&amp;gt;T transitions, followed by C&amp;gt;G transversions (24.8%). Similar to MIBC, APOBEC mutagenesis was the strongest source of mutation in NMIBC with 75% of samples showing up to 5-fold enrichment of the APOBEC mutation signature. Significantly mutated genes included genes implicated in epigenetic regulation and several genes that have not previously been reported as significantly mutated in bladder cancer. Comparison of mutation frequencies in NMIBC with those found in MIBC revealed both known differences (absence of TP53 mutations and high frequency of FGFR3, PIK3CA and STAG2 mutations in NMIBC) and novel findings including a higher frequency of mutations in chromatin-state regulators in NMIBC. Notably CDKN1A, RB1, ERCC2, ERBB3 and FBXW7, which are mutated in &amp;gt;10% of MIBC were not significantly mutated in TaG2 tumors, further delineating the distinct pathogenesis pathways of NMIBC and MIBC. Whole-genome expression array profiling and immunohistochemistry using a panel of markers were carried out to further examine the molecular features of NMIBC samples and define potentially clinically relevant subtypes. These data provide a detailed view of the genomic landscape of NMIBC that highlights chromatin modification as a key area for consideration in the development of potential future therapeutic approaches to the treatment of patients with non-muscle-invasive disease. Citation Format: Carolyn D. Hurst, Olivia Alder, Fiona M. Platt, Alastair Droop, Lucy F. Stead, Julie E. Burns, George J. Burghel, Sunjay Jain, Leszek J. Klimczak, Helen Lindsay, Jo-An Roulson, Claire F. Taylor, Helene Thygesen, Angus J. Cameron, Anne J. Ridley, Helene R. Mott, Dmitry A. Gordenin, Margaret A. Knowles. The genomic landscape of non-muscle-invasive bladder cancer: implications for molecular classification and treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-323.

Males are from Mars, and females are from Venus: sex-specific fetal brain gene expression signatures in a mouse model of maternal diet-induced obesity

Maternal obesity is associated with adverse neurodevelopmental outcomes in children, including autism spectrum disorders, developmental delay, and attention-deficit hyperactivity disorder. The underlying mechanisms remain unclear. We previously identified second-trimester amniotic fluid and term cord blood gene expression patterns suggesting dysregulated brain development in fetuses of obese compared with lean women. We sought to investigate the biological significance of these findings in a mouse model of maternal diet-induced obesity. We evaluated sex-specific differences in fetal growth, brain gene expression signatures, and associated pathways. Female C57BL/6J mice were fed a 60% high-fat diet or 10% fat control diet for 12-14 weeks prior to mating. During pregnancy, obese dams continued on the high-fat diet or transitioned to the control diet. Lean dams stayed on the control diet. On embryonic day 17.5, embryos were weighed and fetal brains were snap frozen. RNA was extracted from male and female forebrains (10 per diet group per sex) and hybridized to whole-genome expression arrays. Significantly differentially expressed genes were identified using a Welch's t test withthe Benjamini-Hochberg correction. Functional analyses were performed using ingenuity pathways analysis and gene set enrichment analysis. Embryos of dams on the high-fat diet were significantly smaller than controls, with males more severely affected than females (P= .01). Maternal obesity and maternal obesity with dietary change in pregnancy resulted in significantly more dysregulated genes in male vs female fetal brains (386 vs 66, P < .001). Maternal obesity with and without dietary change in pregnancy was associated with unique brain gene expression signatures for each sex, with an overlap of only 1 gene. Changing obese dams to a control diet in pregnancy resulted in more differentially expressed genes in the fetal brain than maternal obesity alone. Functional analyses identified common dysregulated pathways in both sexes, but maternal obesity and maternal dietary change affected different aspects of brain development in males compared with females. Maternal obesity is associated with sex-specific differences in fetal size and fetal brain gene expression signatures. Male fetal growth and brain gene expression may be more sensitive toenvironmental influences during pregnancy. Maternal diet during pregnancy has a significant impact on the embryonic brain transcriptome. It is important toconsider both fetal sex and maternal diet when evaluating the effects ofmaternal obesity on fetal neurodevelopment.

Characterization of a Gene Expression Signature in Normal Rat Prostate Tissue Induced by the Presence of a Tumor Elsewhere in the Organ

Implantation of rat prostate cancer cells into the normal rat prostate results in tumor-stimulating changes in the tumor-bearing organ, for example growth of the vasculature, an altered extracellular matrix, and influx of inflammatory cells. To investigate this response further, we compared prostate morphology and the gene expression profile of tumor-bearing normal rat prostate tissue (termed tumor-instructed/indicating normal tissue (TINT)) with that of prostate tissue from controls. Dunning rat AT-1 prostate cancer cells were injected into rat prostate and tumors were established after 10 days. As controls we used intact animals, animals injected with heat-killed AT-1 cells or cell culture medium. None of the controls showed morphological TINT-changes. A rat Illumina whole-genome expression array was used to analyze gene expression in AT-1 tumors, TINT, and in medium injected prostate tissue. We identified 423 upregulated genes and 38 downregulated genes (p<0.05, ≥2-fold change) in TINT relative to controls. Quantitative RT-PCR analysis verified key TINT-changes, and they were not detected in controls. Expression of some genes was changed in a manner similar to that in the tumor, whereas other changes were exclusive to TINT. Ontological analysis using GeneGo software showed that the TINT gene expression profile was coupled to processes such as inflammation, immune response, and wounding. Many of the genes whose expression is altered in TINT have well-established roles in tumor biology, and the present findings indicate that they may also function by adapting the surrounding tumor-bearing organ to the needs of the tumor. Even though a minor tumor cell contamination in TINT samples cannot be ruled out, our data suggest that there are tumor-induced changes in gene expression in the normal tumor-bearing organ which can probably not be explained by tumor cell contamination. It is important to validate these changes further, as they could hypothetically serve as novel diagnostic and prognostic markers of prostate cancer.

Establishment and Drug Sensitivity, Gene Expression and Epigenetic Characterization of a Pediatric Leukemia Cell Line (POETIC1) with Primary Resistance to Decitabine

Introduction: Epigenetic alterations leading to the silencing of key tumor suppressor genes by promoter hypermethylation have been implicated in the pathogenesis of a number of malignancies, including MDS and AML. Currently, the prototypical DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (decitabine) has been studied in a number of diverse protocols as an anti-leukemic agent. However, the pattern of non-responsiveness to decitabine appears to be complex and multifactorial with some patients showing primary resistance whereas others develop resistance following initial responsiveness. To further understand the molecular mechanisms that define growth regulatory networks in pediatric AML, we have established and performed initial characterization using primary blasts that showed increased cell survival and proliferation in the presence of decitabine.Methods: Bone marrow leukemic blasts from a relapsed pediatric AML patient, who received only conventional chemotherapy, were obtained following local REB approval and informed parental consent. Upon in vitro culture to identify effective therapeutic agents, enhanced cell survival was noted in wells containing decitabine (1uM) compared to untreated cells. This cell population was further expanded in higher concentrations of decitabine that tolerated concentrations higher than 10 uM. These cells were then clonally expanded, and the resulting cell line designated POETIC1, was screened in growth inhibition assays against a panel of 142 pharmaceutical pipe-line agents that target known growth regulatory pathways and signaling molecules. The original primary leukemic cells and normal lymphocytes were used as control. Gene expression analyses were carried out using humanHT-12 v4 Expression BeadChip whole-genome expression arrays, normalized and analyzed using the Illumina BeadStudio Software. The distribution and plasticity, and quantity of DNA methylation were studied using the Illumina Infinium Human Methylation BeadChip Assay.Results: POETIC1 cells showed a differential drug sensitivity in approximately 20% of the agents tested. This includes enhanced susceptibility to agents that interfere with cell cycle regulation such as aurora kinase inhibitors, PLK, HDAC inhibitors and agents that targeted mTOR and proteasome activities. Transcriptome profiling revealed that 399 genes were down-regulated and 977 were up-regulated in the leukemia cells, compared to normal controls. POETIC 1 cells had significantly up-regulated DNA repair, cell cycle, oxidative phosphorylation and many other pro-survival pathways. Pathway analysis revealed that up-regulated genes belonged to cell cycle control and pro-survival signalling pathways including genes encoding for cyclins A and B, Cdc 7, Cdc 20 among others. They also had down-regulated genes relating to apoptosis, endocytosis and cell differentiation pathways. Global DNA methylome analysis revealed profound genome-wide deregulation of DNA methylation in POETIC 1 cells with a large number of genes were differentially methylated, including those involved in the control of cell cycle, oxidative phosphorylation, apoptosis and DNA repair pathways.Discussion: Our findings indicate aberrant cell cycle and metabolic pathways in leukemia cells with primary resistance to decitabine. The POETIC1 cell line, provides a critical experimental tool to investigate the role of epigenetic alterations in leukemogenesis as well as the molecular and physiological mechanisms that define primary resistance to methyltransferase inhibitors and facilitate the identification of novel therapeutic agents for refractory disease in future clinical studies. DisclosuresNo relevant conflicts of interest to declare.

Identification and Validation of Genetic Variants that Influence Transcription Factor and Cell Signaling Protein Levels

Many genetic variants associated with human disease have been found to be associated with alterations in mRNA expression. Although it is commonly assumed that mRNA expression changes will lead to consequent changes in protein levels, methodological challenges have limited our ability to test the degree to which this assumption holds true. Here, we further developed the micro-western array approach and globally examined relationships between human genetic variation and cellular protein levels. We collected more than 250,000 protein level measurements comprising 441 transcription factor and signaling protein isoforms across 68 Yoruba (YRI) HapMap lymphoblastoid cell lines (LCLs) and identified 12 cis and 160 trans protein level QTLs (pQTLs) at a false discovery rate (FDR) of 20%. Whereas up to two thirds of cis mRNA expression QTLs (eQTLs) were also pQTLs, many pQTLs were not associated with mRNA expression. Notably, we replicated and functionally validated a trans pQTL relationship between the KARS lysyl-tRNA synthetase locus and levels of the DIDO1 protein. This study demonstrates proof of concept in applying an antibody-based microarray approach to iteratively measure the levels of human proteins and relate these levels to human genome variation and other genomic data sets. Our results suggest that protein-based mechanisms might functionally buffer genetic alterations that influence mRNA expression levels and that pQTLs might contribute phenotypic diversity to a human population independently of influences on mRNA expression.

Genome-wide analysis of DNA methylation, copy number variation, and gene expression in monozygotic twins discordant for primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an uncommon autoimmune disease with a homogeneous clinical phenotype that reflects incomplete disease concordance in monozygotic (MZ) twins. We have taken advantage of a unique collection consisting of genomic DNA and mRNA from peripheral blood cells of female MZ twins (n = 3 sets) and sisters of similar age (n = 8 pairs) discordant for disease. We performed a genome-wide study to investigate differences in (i) DNA methylation (using a custom tiled four-plex array containing tiled 50-mers 19,084 randomly chosen methylation sites), (ii) copy number variation (CNV) (with a chip including markers derived from the 1000 Genomes Project, all three HapMap phases, and recently published studies), and/or (iii) gene expression (by whole-genome expression arrays). Based on the results obtained from these three approaches we utilized quantitative PCR to compare the expression of candidate genes. Importantly, our data support consistent differences in discordant twins and siblings for the (i) methylation profiles of 60 gene regions, (ii) CNV of 10 genes, and (iii) the expression of 2 interferon-dependent genes. Quantitative PCR analysis showed that 17 of these genes are differentially expressed in discordant sibling pairs. In conclusion, we report that MZ twins and sisters discordant for PBC manifest particular epigenetic differences and highlight the value of the epigenetic study of twins.

Impact of Cigarette Smoke on the Human and Mouse Lungs: A Gene-Expression Comparison Study

Cigarette smoke is well known for its adverse effects on human health, especially on the lungs. Basic research is essential to identify the mechanisms involved in the development of cigarette smoke-related diseases, but translation of new findings from pre-clinical models to the clinic remains difficult. In the present study, we aimed at comparing the gene expression signature between the lungs of human smokers and mice exposed to cigarette smoke to identify the similarities and differences. Using human and mouse whole-genome gene expression arrays, changes in gene expression, signaling pathways and biological functions were assessed. We found that genes significantly modulated by cigarette smoke in humans were enriched for genes modulated by cigarette smoke in mice, suggesting a similar response of both species. Sixteen smoking-induced genes were in common between humans and mice including six newly reported to be modulated by cigarette smoke. In addition, we identified a new conserved pulmonary response to cigarette smoke in the induction of phospholipid metabolism/degradation pathways. Finally, the majority of biological functions modulated by cigarette smoke in humans were also affected in mice. Altogether, the present study provides information on similarities and differences in lung gene expression response to cigarette smoke that exist between human and mouse. Our results foster the idea that animal models should be used to study the involvement of pathways rather than single genes in human diseases.

Interference Of The Tumor Supportive Effects Of BCL2 and MCL1 Sensitize Malignant Plasma Cells To The Lethal Effects Of Lenalidomide and Dexamethasone Regimen: An Important Clinical Path For BCL2 Targeting Drugs

BackgroundMultiple myeloma (MM) is the second most common hematologic malignancy in the U.S. and is characterized by increased immunoglobulin production and infiltration of the bone marrow by malignant plasma cells. Myeloma cell growth is supported by both the elements present in the bone marrow microenvironment as well as deregulation of internal cellular systems associated with proliferation and apoptosis. Defective programmed cell death by BCL2 or MCL1 upregulation is observed in >80% of myeloma cases and is associated with an aggressive clinical course. Remarkably, there is no approved drug with the ability to target BCL2 or MCL1. Various strategies to mitigate the effects of increased BCL2 functionality, via small molecule inhibitors or BCL2-specific antisense oligonucleotides have been previously examined in MM. These studies revealed that 1) apart from BCL2, additional anti-apoptotic members belonging to the BCL2 family (i.e. MCL1, BCL-xL) play integral roles in maintenance of the myeloma cell phenotype, 2) drugs, which target only BCL2 offer limited therapeutic advantage and 3) given that survival factors engaged in MM are multifactorial, perhaps isolated targeting of the BCL2 pathways may not be sufficient. We therefore hypothesized that the clinical development of drugs that target various antiapoptotic BCL2 family members will require a partnership with established anti-MM regimens whose activity can potentially be further enhanced with correction of the apoptotic response system. AimSince lenalidomide and dexamethasone (LD) is an established therapy for plasma cell cancers, we investigated if the pan-BCL2 inhibitor AT-101 (BH3 mimetic), which has high binding specificity for BCL2, MCL1 and BCL-xL, can be an effective therapeutic partner to enhance anti-MM effects of LD. MethodsHuman MM (KMS11, U266, OPM2) and WM (BCWM.1) cell lines along with their corresponding bortezomib resistant (BR) clones, (KMS11/BR, U266/BR, OPM2/BR and BCWM.1/BR) which were developed in our laboratory were used in this study. For gene expression profiling, the Illumina HumanHT-12 v3 whole-genome gene expression array and Nanostring nCounter mRNA quantification assays (NanoString, Seattle, WA) were utilized. Statistical analysis was conducted using R-based packages and the MeV software (TIGR). Apoptosis was measured by annexin-v/PI staining, and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiles were ascertained by western blot. ResultsGene expression and immunoblot analysis of six plasma cell cancer models showed upregulation of various BCL2 family members, notably MCL1 and BCL-xL. In a dose-dependent manner, AT-101 was able to downregulate BCL2 and MCL1 and induced apoptotic cell death in MM and WM cells in a dose dependent manner. Tumor cell death was associated with caspase and PARP-1 cleavage accompanied by an increase in MOMP. This cytotoxic effect and BCL2 downregulation were further potentiated when AT-101 was combined with lenalidomide/dexamethasone (LDA). Nanostring nCounter mRNA quantification and IPA analysis revealed differential changes in the CCNA, FRZB, FYN, IRF1, PTPN11 genes in LDA treated cells. IPA canonical pathway analysis demonstrated the p53 signaling and the cyclins and cell cycle regulation pathways to be the most significantly activated by LDA therapy. SummaryIn summary, we describe for the first time the cellular and molecular events associated with the use of AT-101 in combination with lenalidomide/dexamethasone in preclinical models of plasma cell malignancy. This study lays the rationale for engaging and handicapping the intrinsic apoptotic system through the pan-BCL2 targeting capabilities of AT-101; thus enhancing the anti-MM effects of lenalidomide/dexamethasone. Disclosures:Foran:Celgene: Research Funding.

Cardiac progenitor cells from the left atrial appendage may originate from a resident non-hematopoietic myeloid progenitor population

Purpose: We recently identified three distinct cardiac progenitor cell (CPC) populations from the left atrial appendages (LAAs) of adult murine hearts. These populations have distinct phenotypes and characteristics. Type A CPCs are c-kit+/Sca-1+, Type B CPCs are c-kit+/CD45+, and more mature Type C CPCs can be derived from Type B CPCs in vitro. The purpose of this study was to analyze the embryologic origin and the connection between the different CPC populations. Methods: We performed a whole-genome expression array to the cell sorted Type A, Type B and Type C CPC populations. The LAAs were analyzed using immunohistochemistry. Tissue culture derived cells were analyzed using immunostaining and FACS. Neural crest lineage tracing was performed with Pax3-cre eYFP reporter mice. We investigated the spontaneous cardiac differentiation process from Type B to Type C using live microscopy and FACS. Results: Transcriptome comparison demonstrated a significant up-regulation of the neural crest differentiation –pathway genes in Type A and C CPCs. Type B CPCs expressed a gene profile, which matches that of non-hematopoietic, resident macrophage –like cells found in other tissues. Pax3 lineage tracing demonstrated traced cells in the subepicardial zone of the LAA, co-stained with F4/80 macrophage marker and c-kit. Closely attached were epicardially lined F4/80+ cells with no eYFP signal. In tissue culture, eYFP signal was strongest in the Type B CPCs and lower in the Type A CPCs. Fibroblasts in the culture were eYFP negative. The CD45 expression in the Type B CPCs was stable (∼90% of the cells) up to five passages folllowed by a rapid decline of CD45, c-kit and Flk-1 expression. In live microscopy, formation of giant Sca-1+ and Gata-4+ cells by cell fusion was followed by asymmetric divisions creating Type C CPCs, which expressed cardiac myosin heavy chain and atrial natriuretic factor. The transcriptome was significantly skewed toward a cardiomyocyte phenotype with the spontaneous differentiation, although this differentiation was not complete, as seen by electron microscopy. The partially differentiated Type C CPC population could be grown for >60 passages. Conclusion: Our results suggest that rather than having distinct embryonic origins, the different types of CPCs in the LAA originate from a resident, non-hematopoietic myeloid progenitor cell population. The neural crest differentiation pathway can be activated in these cells during adulthood, possibly in cardiac stress situations, enabling differentiation to cardiac lineage with a novel mechanism consisting of cell fusion and asymmetric division.

Endoscopically Acquired Pancreatic Cyst Fluid MicroRNA 21 and 221 Are Associated With Invasive Cancer

Pancreatic cysts are a group of lesions with heterogeneous malignant potential. Currently, there are no reliable biomarkers to aid in cyst diagnosis and classification. The objective of this study was to identify potential microRNA (miR) biomarkers in endoscopically acquired pancreatic cyst fluid that could be used to distinguish between benign, premalignant, and malignant cysts. A list of candidate miRs was developed using a whole-genome expression array analysis of pancreatic cancer (pancreatic ductal adenocarcinoma) and nonmalignant samples overlapped with existing literature and predicted gene targets. Endoscopically acquired pancreatic cyst fluid samples were obtained from a group of 38 patients who underwent cyst fluid aspiration and surgical resection. Selected miR expression levels in cyst fluid samples were assessed by quantitative real-time-PCR. Additionally, in situ hybridization (ISH) on corresponding cyst tissue samples was performed to identify the source and validate the expression level of fluid miRs. Of the six miRs that were profiled in the study, two showed differential expression in malignant cysts. miR-221 was expressed at significantly higher levels in malignant cysts compared with benign or premalignant cysts (P=0.05). miR-21 was also expressed at significantly higher levels in malignant cysts (P<0.01). Additionally, the expression of miR-21 was significantly higher in premalignant cysts than benign cysts (P=0.03). The differential expression of miR-21 among cyst categories was confirmed by ISH. In this small single-center study, miRs are potential pancreatic cyst fluid diagnostic biomarkers. In particular, miR-21 is identified as a candidate biomarker to distinguish between benign, premalignant, and malignant cysts. Additionally miR-221 may be of use in the identification of more advanced malignant disease.

Combining Small-Volume Metabolomic and Transcriptomic Approaches for Assessing Brain Chemistry

The integration of disparate data types provides a more complete picture of complex biological systems. Here we combine small-volume metabolomic and transcriptomic platforms to determine subtle chemical changes and to link metabolites and genes to biochemical pathways. Capillary electrophoresis–mass spectrometry (CE–MS) and whole-genome gene expression arrays, aided by integrative pathway analysis, were utilized to survey metabolomic/transcriptomic hippocampal neurochemistry. We measured changes in individual hippocampi from the mast cell mutant mouse strain, C57BL/6 KitW-sh/W-sh. These mice have a naturally occurring mutation in the white spotting locus that causes reduced c-Kit receptor expression and an inability of mast cells to differentiate from their hematopoietic progenitors. Compared with their littermates, the mast cell-deficient mice have profound deficits in spatial learning, memory, and neurogenesis. A total of 18 distinct metabolites were identified in the hippocampus that discriminated between the C57BL/6 KitW-sh/W-sh and control mice. The combined analysis of metabolite and gene expression changes revealed a number of altered pathways. Importantly, results from both platforms indicated that multiple pathways are impacted, including amino acid metabolism, increasing the confidence in each approach. Because the CE–MS and expression profiling are both amenable to small-volume analysis, this integrated analysis is applicable to a range of volume-limited biological systems.

A cross-platform analysis of 14,177 expression quantitative trait loci derived from lymphoblastoid cell lines

Gene expression levels can be an important link DNA between variation and phenotypic manifestations. Our previous map of global gene expression, based on ∼400K single nucleotide polymorphisms (SNPs) and 50K transcripts in 400 sib pairs from the MRCA family panel, has been widely used to interpret the results of genome-wide association studies (GWASs). Here, we more than double the size of our initial data set with expression data on 550 additional individuals from the MRCE family panel using the Illumina whole-genome expression array. We have used new statistical methods for dimension reduction to account for nongenetic effects in estimates of expression levels, and we have also included SNPs imputed from the 1000 Genomes Project. Our methods reduced false-discovery rates and increased the number of expression quantitative trait loci (eQTLs) mapped either locally or at a distance (i.e., in cis or trans) from 1534 in the MRCA data set to 4452 (with <5% FDR). Imputation of 1000 Genomes SNPs further increased the number of eQTLs to 7302. Using the same methods and imputed SNPs in the newly acquired MRCE data set, we identified eQTLs for 9000 genes. The combined results identify strong local and distant effects for transcripts from 14,177 genes. Our eQTL database based on these results is freely available to help define the function of disease-associated variants.

Epigenetic Regulation of Cell Adhesion and Communication by Enhancer of Zeste Homolog 2 in Human Endothelial Cells

The histone methyltransferase enhancer of zeste homolog 2 (Ezh2) mediates trimethylation of lysine 27 in histone 3, which acts as a repressive epigenetic mark. Ezh2 is essential for maintaining pluripotency of stem cells, but information on its role in differentiated cells is sparse. Whole-genome mRNA expression arrays identified 964 genes that were regulated by >2-fold 72 hours after small interfering RNA-mediated silencing of Ezh2 in human umbilical vein endothelial cells. Among them, genes associated with the gene ontology terms cell communication and cell adhesion were significantly overrepresented, suggesting a functional role for Ezh2 in the regulation of angiogenesis. Indeed, adhesion, migration, and tube formation assays revealed significantly altered angiogenic properties of human umbilical vein endothelial cells after silencing of Ezh2. To identify direct target genes of Ezh2, we performed chromatin immunoprecipitation experiments followed by whole-genome promoter arrays (chromatin immunoprecipitation-on-chip) and identified 5585 genes associated with trimethylation of lysine 27 in histone 3. Comparative analysis with our mRNA expression data identified 276 genes that met our criteria for putative Ezh2 target genes, upregulation by >2-fold after Ezh2 silencing and association with trimethylation of lysine 27 in histone 3. Notably, we observed a striking overrepresentation of genes involved in wingless-type mouse mammary tumor virus integration site (WNT) signaling pathways. Epigenetic regulation of several of these genes by Ezh2 was specifically confirmed by polymerase chain reaction analysis of DNA enrichment after chromatin immunoprecipitation using an antibody specific for trimethylation of lysine 27 in histone 3. Combining mRNA expression arrays and chromatin immunoprecipitation-on-chip analysis, we identified 276 Ezh2 target genes in endothelial cells. Ezh2-dependent repression of genes involved in cell adhesion and communication contributes to the regulation of angiogenesis.