Søren Jensby Nielsen

Assessing sample and miRNA profile quality in serum and plasma or other biofluids.

MicroRNAs (miRNAs) constitute a class of small cellular RNAs (typically 21-23nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, although they are relatively few in number (less than 2000 human miRNAs). The high relative stability of miRNA in common clinical tissues and biofluids (e.g. plasma, serum, urine, saliva, etc.) and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNA quantification as a promising new tool for a wide range of diagnostic applications. Furthermore miRNAs have been shown to be rapidly released from tissues into the circulation with the development of pathology. To facilitate discovery and clinical development of miRNA-based biomarkers, we developed a genome-wide Locked Nucleic Acid (LNA™)-based miRNA qPCR platform with unparalleled sensitivity and robustness. The platform allows high-throughput profiling of miRNAs from important clinical sources without the need for pre-amplification. Using this system, we have profiled thousands of biofluid samples including blood derived plasma and serum. An extensive quality control (QC) system has been implemented in order to secure technical excellence and reveal any unwanted bias coming from pre-analytical or analytical variables. We present our approaches to sample and RNA QC as well as data QC and normalization. Specifically we have developed normal reference ranges for circulating miRNAs in serum and plasma as well as a hemolysis indicator based on microRNA expression.

Nuclear export inhibitors and kinase inhibitors identified using a MAPK-activated protein kinase 2 redistribution screen.

Redistribution (BioImage) A/S, Søborg, Denmark) is a novel high-throughput screening technology that monitors translocation of specific protein components of intracellular signaling pathways within intact mammalian cells, using green fluorescent protein as a tag. A single Redistribution assay can be used to identify multiple classes of compounds that act at, or upstream of, the level of the protein target used in the primary screening assay. Such compounds may include both conventional and allosteric enzyme inhibitors, as well as protein-protein interaction modulators. We have developed a series of Redistribution assays to discover and characterize compounds that inhibit tumor necrosis factor-alpha biosynthesis via modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. A primary assay was designed to identify low-molecular-weight compounds that inhibit the activation-dependent nuclear export of the p38 kinase substrate MAPK-activated protein kinase 2 (MK2). Hits from the primary screen were categorized, using secondary assays, either as direct inhibitors of MK2 nuclear export, or as inhibitors of the upstream p38 MAPK pathway. Activity profiles are presented for a nuclear export inhibitor, and a compound that structurally and functionally resembles a known p38 kinase inhibitor. These results demonstrate the utility of Redistribution technology as a pathway screening method for the identification of diverse and novel compounds that are active within therapeutically important signaling pathways.

Identification of akt pathway inhibitors using redistribution screening on the FLIPR and the IN cell 3000 analyzer

The PI3-kinase/Akt pathway is an important cell survival pathway that is deregulated in the majority of human cancers. Despite the apparent druggability of several kinases in the pathway, no specific catalytic inhibitors have been reported in the literature. The authors describe the development of a fluorometric imaging plate reader (FLIPR)-based Akt1 translocation assay to discover inhibitors of Akt1 activation. Screening of a diverse chemical library of 45,000 compounds resulted in identification of several classes of Akt1 translocation inhibitors. Using a combination of classical in vitro assays and translocation assays directed at different steps of the Akt pathway, the mechanisms of action of 2 selected chemical classes were further defined. Protein translocation assays emerge as powerful tools for hit identification and characterization. (Journal of Biomolecular Screening 2005:20-29)

Estradiol acutely inhibits whole body lipid oxidation and attenuates lipolysis in subcutaneous adipose tissue: a randomized, placebo-controlled study in postmenopausal women.

CONTEXT Estradiol (E(2)) promotes and maintains the female phenotype characterized by subcutaneous fat accumulation. There is evidence to suggest that this effect is due to increased anti-lipolytic α2A-adrenergic receptors, but whether this requires long-term exposure to E(2) or is an immediate effect is not clear. OBJECTIVE To study acute effects of a single dose (4 mg) of 17β-E(2) on regional and systemic lipolysis. METHODS Sixteen postmenopausal women (age, 595 years; weight, 6710 kg; and BMI, 24.82.9) were studied in a crossover design: i) placebo and ii) 4 mg E(2). Basal and adrenaline-stimulated regional lipolysis was assessed by microdialysis and substrate oxidation rates by indirect calorimetry. Tissue biopsies were obtained to assess lipoprotein lipase activity and mRNA expression of adrenergic, estrogen, cytokine, and vascular reactivity receptors. RESULTS Acute E(2) stimulation significantly attenuated catecholamine-stimulated lipolysis in femoral subcutaneous adipose tissue (interstitial glycerol concentration (micromole/liter) ANOVA time vs treatment interaction, P=0.01) and lipolysis in general in abdominal adipose tissue (ANOVA treatment alone, P<0.05). E(2) also reduced basal lipid oxidation ((mg/kg per min) placebo, 0.58 ± 0.06 vs E(2), 0.45 ± 0.03; P=0.03) and induced a significantly higher expression of anti-lipolytic α2A-adrenergic receptor mRNA (P=0.02) in skeletal muscle tissue as well as an upregulation of eNOS (NOS3) mRNA (P=0.02). CONCLUSION E(2) acutely attenuates the lipolytic response to catecholamines in subcutaneous adipose tissue, shifts muscular adrenergic receptor mRNA toward anti-lipolytic α2A-receptors, decreases whole body lipid oxidation, and enhances expression of markers of vascular reactivity.

Identification of RAS-Mitogen-Activated Protein Kinase Signaling Pathway Modulators in an ERF1 Redistribution® Screen

The RAS-mitogen-activated protein kinase (MAPK) signaling pathway has a central role in regulating the proliferation and survival of both normal and tumor cells. This pathway has been 1 focus area for the development of anticancer drugs, resulting in several compounds, primarily kinase inhibitors, in clinical testing. The authors have undertaken a cell-based, high-throughput screen using a novel ERF1 Redistribution® assay to identify compounds that modulate the signaling pathway. The hit compounds were subsequently tested for activity in a functional cell proliferation assay designed to selectively detect compounds inhibiting the proliferation of MAPK pathway-dependent cancer cells. The authors report the identification of 2 cell membrane-permeable compounds that exhibit activity in the ERF1 Redistribution® assay and selectively inhibit proliferation of MAPK pathway-dependent malignant melanoma cells at similar potencies (IC50 =< 5 μM). These compounds have drug-like structures and are negative in RAF, MEK, and ERK in vitro kinase assays. Drugs belonging to these compound classes may prove useful for treating cancers caused by excessive MAPK pathway signaling. The results also show that cell-based, high-content Redistribution® screens can detect compounds with different modes of action and reveal novel targets in a pathway known to be disease relevant.

Abstract A45: GAS5-encoded intronic snoRNAs produce specific sdRNAs overexpressed in aggressive prostate cancer

Small non-coding RNAs, such as miRNAs, are implicated in carcinogenesis. To investigate changes in the entire small RNA transcriptome in prostate cancer (PCa) we analyzed 11 clinical sample pools representing different stages of PCa by deep sequencing. We found that most C/D-box small nucleolar RNAs (snoRNAs) are specifically processed to smaller snoRNA-derived RNAs (sdRNAs) highly expressed in PCa. In particular, SNORD78 produces sdRNAs strongly up-regulated in PCa. Together with 9 other snoRNAs, SNORD78 is encoded in the introns of the Growth Arrest Specific 5 gene (GAS5). Examination of SNORD78 and the positioned in a neighboring intron SNORD44 showed that both snoRNAs produce predominantly one sdRNA fragment each, specifically originating from the 39-arm (SNORD78) or the 59-arm (SNORD44) of the precursor sequence. Inspection of the secondary structures of SNORD44 and SNORD78 revealed that they have a degenerated C9/D9 box and can fold in a tight hairpin similarly to miRNAs. In contrast, SNORD74 and SNORD81 that also encoded in introns of GAS5, contain canonical C9/D9 boxes and each produce three equally expressed sdRNAs. Quantitative real time PCR analysis in an independent patient cohort of 106 fresh-frozen clinical samples confirmed the significant up-regulation of all four snoRNAs and their derivative sdRNAs in PCa samples compared to normal tissue. Interestingly, the increased expression of snoRNAs and sdRNAs was not associated with elevated levels of GAS5 transcript. Based on these results, we conclude that (i) separate regulatory mechanisms control the posttranscriptional processing of the spliced GAS5 transcript and the encoded in its introns snoRNAs; (ii) SNORD44, SNORD78, SNORD74 and SNORD81 function as precursors of different sdRNAs; (iii) SNORD44, SNORD78, SNORD74 and SNORD81 and their derivative sdRNAs are significantly up-regulated in PCa and carry biomarker potential for this disease. Citation Format: Elena S. Martens-Uzunova, Anton Kalsbeek, Youri Hoogstrate, Adam Baker, Soren Jensby Nielsen, Tapio Visakorpi, Chris Bangma, Guido Jenster. GAS5-encoded intronic snoRNAs produce specific sdRNAs overexpressed in aggressive prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A45.

Abstract LB-135: Selective small molecule inhibition of the N-terminal BET bromodomain

Introduction: The bromodomain acetyl-lysine ‘readers’ is an emerging target class with particular interest centered around the BET family proteins BRD2, 3, and 4. Pre-clinical target validation experiments of BET proteins using genetic tools and/or small molecule bromodomain inhibitors have demonstrated strong disease linkage in several cancers, and a number of BET inhibitors are currently in early clinical trials within oncology. BET proteins contain a unit of tandem bromodomains (BD1 and BD2), each capable of binding to acetylated lysines within partner proteins. However, the contribution of individual bromodomains and of specific BET isoforms to biological activity is currently unclear, and domain-specific inhibition (intra- and/or inter-BET) of BET proteins has been insufficiently explored. Here we report and characterize the first BD1-selective BET inhibitors. Methods: Chemetics DNA-encoding small molecule libraries was used to identify small molecule BET inhibitors. Binding of hit compounds to bromodomains was investigated using AlphaScreen and FRET assays. Compounds were then assayed for AML cell growth inhibition using MTS and CellTiter-Glo assays, and for inhibition of cytokine release from human PBMCs following LPS stimulation. Results: To discover novel inhibitors of BET bromodomains, we screened ∼300 million small molecule compounds for binding towards BD1 of human BRD4. Representative hits from several chemical series were resynthesized and confirmed for binding to BRD4 in the high nM range. Chemical optimization of one series yielded compounds with low nM affinity against BRD4 BD1 with comparable affinity against the BD1s of other BET proteins. Interestingly, the compounds display marked intra-BET BD selectivity, with a strong preference (10-40 fold) for binding to BD1. In cellular growth inhibition assays, the compounds selectively target MLL-translocated AML cell lines. LPS-induced cytokine release from human PBMCs is strongly inhibited by both the pan-BET inhibitors JQ-1/I-BET762 and our BD1-selective compounds. In contrast, the BD2-selective compound RVX-208 show limited activity in the cell models tested here. Conclusions: Using DNA-encoding technology, we discovered low nM small molecule compounds that selectively inhibit the first bromodomain of BET proteins. The compounds strongly inhibit BET-dependent cellular processes. Furthermore, our data suggest that a number of BET biological activities are mediated pimarily by BD1, and that BD1 blockade is sufficient to elicit BET inhibition. Efforts to understand the mechanism and potential therapeutic utility of BD1-selectivity are ongoing. Citation Format: Jimmi G. Seitzberg, Margit H. Hansen, Tine T. Akinleminu Kronborg, Christina R. Underwood, Gitte Friberg, Berit Tonnesen, Lene Teuber, Thomas Franch, Soren J. Nielsen. Selective small molecule inhibition of the N-terminal BET bromodomain. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-135. doi:10.1158/1538-7445.AM2014-LB-135

MicroRNA in biofluids—Robust biomarkers for disease, toxicology, or injury studies: The case of minimally invasive colorectal cancer detection.

20 Background: MicroRNAs function as post-transcriptional regulators of gene expression. Their high relative stability in common clinical source materials (FFPE blocks, plasma, serum, urine, saliva, etc.) and the ability of microRNA expression profiles to accurately classify discrete tissue types and specific disease states have positioned microRNAs as promising new biomarkers for diagnostic application. Furthermore microRNAs have been shown to be rapidly released from tissues into the circulation with the development of pathology. METHODS Thousands of biofluid samples were profiled including blood derived plasma/serum and urine using a genome-wide LNA-based microRNA qPCR platform, which has unparalleled sensitivity and robustness even in biofluids with extremely low microRNA levels. Only a single RT reaction is required to conduct full miRNome profiling thereby facilitating high-throughput profiling without the need for pre-amplification. RESULTS Normal reference ranges for circulating microRNAs were determined in several biofluids, allowing development of qPCR arrays containing only relevant microRNA subsets present in various biofluids together with tissue specific microRNA markers. Procedures were developed to control pre-analytical variables, for quality checking and qualifying biofluid samples in particular serum and plasma but also urine and other biofluids. An extensive QC system was implemented in order to secure technical excellence and reveal any unwanted bias in the dataset. We currently screen and validate microRNAs biomarkers for cancer with the aim of developing minimal invasive tests to be applied in early detection population screens. CONCLUSIONS The qPCR panels support development of robust biomarkers in disease, toxicology, and injury studies. We will demonstrate how panels may be quickly and robustly applied in biomarker discovery/validation projects using the specific case early detection of colorectal cancer in blood. Close attention is required on pre-analytical parameters. Hemolysis and cellular contamination affect miRNA profiles in biofluids and control is required.

Abstract LB-476: A universal method for elimination of haemolyzed plasma samples that improves miRNA signature performance for early detection of colorectal cancer

microRNAs (miRNAs) are short (∼21 bp) non-coding RNAs that regulate gene expression through post-transcriptional interactions with target messenger RNAs. miRNAs are exported to extracellular fluids by both malignant cells and cells of the immune system. Blood plasma and serum miRNAs are stable under standard clinical sampling protocols and have therefore attracted considerable interest for their potential as minimally invasive biomarkers for diverse pathological conditions, including various cancers. Although blood plasma contains as little as 1-20 ng of RNA per mL, plasma miRNAs are easily and reliably quantified using the highly sensitive and specific miRCURY LNA™ Universal RT microRNA PCR platform. However, the relative scarcity of miRNA in plasma creates a potential for contamination of the plasma miRNA expression profile by miRNAs from the cellular constituents in blood. One common cause of plasma sample rejection in clinical chemistry is haemolysis. If not identified, haemolysis can lead to erroneous results in a number of standard clinical laboratory tests, including blood potassium and lactate dehydrogenase (LDH) levels. Here we investigate the effect of sample haemolysis on plasma miRNA profiles. Using isolated red blood cells (RBCs) and genome-wide miRNA PCR profiling, we define the miRNome of RBCs. By spiking RBC extract into a non-haemolyzed plasma sample, we demonstrate that as little as 0.05% contamination of plasma can be detected as an increased expression level of a subset of miRNAs. We also define a set of plasma miRNAs that are not affected by haemolysis. Finally, we discover a miRNA qPCR QC signature that can be used to eliminate haemolyzed plasma samples. In a project aimed at defining a signature for the early detection of colorectal cancer (CRC) from a plasma sample, we screened 325 plasma samples from CRC patients and colonoscopy-verified CRC-negative controls. The samples were part of a clinical trial conducted in 7 different Danish hospitals, and were examined for the expression of 378 miRNAs previously detected in plasma. We show that haemolysis in this sample set correlates with hospital ID, and with the utilization of specific blood sample collection vials. Using our haemolysis signature, we eliminated haemolyzed samples and demonstrated that this step leads to a major improvement of CRC detection (ROC AUC increase from 0.67 to >0.80). We conclude that haemolysis can be a cause of plasma miRNA profile contamination, and that elimination of haemolyzed samples using our miRNA haemolysis QC signature overcomes this clinical problem and leads to an increase in plasma miRNA biomarker performance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-476. doi:1538-7445.AM2012-LB-476

Abstract 2816: Discovery of a miRNA-based RT-qPCR signature able to detect early stage colorectal cancer in blood plasma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Colorectal cancer (CRC) ranks 4th in terms of prevalence and 2nd in number of deaths among cancers of the western world. Although early detection results in improved survival, and regular screening has been proven clinically to lower mortality from CRC, screening rates among the 50-75 year old population are unsatisfactory. There is therefore a clear unmet need for a quick, sensitive, specific, and convenient screening assay to select at risk individuals for definitive diagnosis by colonoscopy. Cellular miRNA profiles vary according to cell type and state, and cellular miRNAs are exported to extracellular fluids by both malignant cells and cells of the immune system. Blood plasma and serum miRNAs are stable under standard clinical sampling protocols and are therefore promising candidates for minimally invasive biomarkers for diverse pathological conditions. To screen for miRNAs in plasma, we developed an LNA-enhanced miRNA RT-qPCR platform with unprecedented sensitivity, selectivity and ease-of-use. An extensive informatics infrastructure has been put in place to allow semi-automated sample- and data-QC, and data analysis of large datasets. A reference melting curve database has been implemented to ensure the integrity of each data point, and appropriate controls monitor plate-to-plate and day-to-day variation. Different normalization protocols are routinely evaluated to ensure correct normalization of the dataset prior to data analysis. Using this platform, we have performed a two-phased discovery program in plasma samples from stage II/III CRC patients and age- and gender-matched colonoscopy-verified healthy controls. A genome wide screen in blood plasma profiled 730 individual miRNAs from 50 stage II cancers and 50 matched controls. This allowed us to develop a candidate panel of miRNAs detectable in plasma and to calculate an appropriate design for the full discovery study. In the second phase of the program we profiled the candidate 378 individual miRNAs in 120 stage II CRC, 50 stage III, and 170 matched controls. The results of these screens have given us a list of miRNAs that are statistically significantly altered between cancers and healthy volunteers. Some of the differentially expressed miRNAs include miRNAs with known roles in cancer and/or inflammatory processes. From this list we have developed a diagnostic miRNA signature that we are moving into assay development. We will present our diagnostic miRNA signature and plans for our first validation study where we will test the signature in samples from a 5,000 patient retrospective clinical trial as well as future plans to test prospectively in an ongoing clinical trial recruiting ∼5,000 symptomatic individuals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2816. doi:10.1158/1538-7445.AM2011-2816