Tamara Maes

Down-regulation of the maize and Arabidopsis thaliana caffeic acid O -methyl-transferase genes by two new maize R2R3-MYB transcription factors

The maize (Zea mays L.) caffeic acid O-methyl-transferase (COMT) is a key enzyme in the biosynthesis of lignin. In this work we have characterized the involvement of COMT in the lignification process through the study of the molecular mechanisms involved in its regulation. The examination of the maize COMT gene promoter revealed a putative ACIII box, typically recognized by R2R3-MYB transcription factors. We used the sequence of known R2R3-MYB factors to isolate five maize R2R3-MYB factors (ZmMYB2, ZmMYB8, ZmMYB31, ZmMYB39, and ZmMYB42) and study their possible roles as regulators of the maize COMT gene. The factors ZmMYB8, ZmMY31, and ZmMYB42 belong to the subgroup 4 of the R2R3-MYB family along with other factors associated with lignin biosynthesis repression. In addition, the induction pattern of ZmMYB31 and ZmMYB42 gene expression on wounding is that expected for repressors of the maize COMT gene. Arabidopsisthaliana plants over-expressing ZmMYB31 and ZmMYB42 down-regulate both the A. thaliana and the maize COMT genes. Furthermore, the over-expression of ZmMYB31 and ZmMYB42 also affect the expression of other genes of the lignin pathway and produces a decrease in lignin content of the transgenic plants.

Mouse neuron navigator 1, a novel microtubule-associated protein involved in neuronal migration.

The development of the nervous system (NS) requires the coordinated migration of multiple waves of neurons and subsequent processes of neurite maturation, both involving selective guidance mechanisms. In Caenorhabditis elegans, unc-53 codes for a new multidomain protein involved in the directional migration of a subset of cells. We describe here the first functional characterization of the mouse homologue, mouse Neuron navigator 1 (mNAV1), whose expression is largely restricted to the NS during development. EGFP-mNAV1 associates with microtubules (MTs) plus ends present in the growth cone through a new microtubule-binding (MTB) domain. Moreover, its overexpression in transfected cells leads to MT bundling. The abolition of mNAV1 causes loss of directionality in the leading processes of pontine-migrating cells, providing evidence for a role of mNAV1 in mediating Netrin-1-induced directional migration.

Molecular markers of endometrial carcinoma detected in uterine aspirates.

Endometrial cancer (EC) is the most frequent of the invasive tumors of the female genital tract. Although usually detected in its initial stages, a 20% of the patients present with advanced disease. To date, no characterized molecular marker has been validated for the diagnosis of EC. In addition, new methods for prognosis and classification of EC are needed to combat this deadly disease. We thus aimed to identify new molecular markers of EC and to evaluate their validity on endometrial aspirates. Gene expression screening on 52 carcinoma samples and series of real‐time quantitative PCR validation on 19 paired carcinomas and normal tissue samples and on 50 carcinoma and noncarcinoma uterine aspirates were performed to identify and validate potential biomarkers of EC. Candidate markers were further confirmed at the protein level by immunohistochemistry and Western blot. We identified ACAA1, AP1M2, CGN, DDR1, EPS8L2, FASTKD1, GMIP, IKBKE, P2RX4, P4HB, PHKG2, PPFIBP2, PPP1R16A, RASSF7, RNF183, SIRT6, TJP3, EFEMP2, SOCS2 and DCN as differentially expressed in ECs. Furthermore, the differential expression of these biomarkers in primary endometrial tumors is correlated to their expression level in corresponding uterine fluid samples. Finally, these biomarkers significantly identified EC with area under the receiver‐operating‐characteristic values ranging from 0.74 to 0.95 in uterine aspirates. Interestingly, analogous values were found among initial stages. We present the discovery of molecular biomarkers of EC and describe their utility in uterine aspirates. These findings represent the basis for the development of a highly sensitive and specific minimally invasive method for screening ECs.

Reduced ubiquitin C-terminal hydrolase-1 expression levels in dementia with Lewy bodies.

Parkinson disease (PD) and dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal alpha-synuclein and ubiquitin in protein aggregates conforming Lewy bodies and Lewy neurites. Ubiquitin C-terminal hydrolase-1 (UCHL-1) disassembles polyubiquitin chains to increase the availability of free monomeric ubiquitin to the ubiquitin proteasome system (UPS) thus favoring protein degradation. Since mutations in the UCHL-1 gene, reducing UPS activity by 50%, have been reported in autosomal dominant PD, and UCHL-1 inhibition results in the formation of alpha-synuclein aggregates in mesencephalic cultured neurons, the present study was initiated to test UCHL-1 mRNA and protein levels in post-mortem frontal cortex (area 8) of PD and DLB cases, compared with age-matched controls. TaqMan PCR assays, and Western blots demonstrated down-regulation of UCHL-1 mRNA and UCHL-1 protein in the cerebral cortex in DLB (either in pure forms, not associated with Alzheimer disease: AD, and in common forms, with accompanying AD changes), but not in PD, when compared with age-matched controls. Interestingly, UCHL-1 mRNA and protein expressions were reduced in the medulla oblongata in the same PD cases. Moreover, UCHL-1 protein was decreased in the substantia nigra in cases with Lewy body pathology. UCHL-1 down-regulation was not associated with reduced protein levels of several proteasomal subunits, including 20SX, 20SY, 19S and 11Salpha. Yet UCHL-3 expression was reduced in the cerebral cortex of PD and DLB patients. Together, these observations show reduced UCHL-1 expression as a contributory factor in the abnormal protein aggregation in DLB, and points UCHL-1 as a putative therapeutic target in the treatment of DLB.

KDM1 histone lysine demethylases as targets for treatments of oncological and neurodegenerative disease

Histone methylation and demethylation are important processes associated with the regulation of gene transcription, and alterations in histone methylation status have been linked to a large number of human diseases. Initially thought to be an irreversible process, histone methylation is now known to be reversed by two families of proteins containing over 30 members that act to remove methyl groups from specific lysine residues present in the tails of histone H3 and histone H4. A rapidly growing number of reports have implicated the FAD-dependent lysine specific demethylase (KDM1) family in cancer, and several small-molecule inhibitors are in development for the treatment of cancer. An additional role has emerged for KDM1 in brain function, offering additional opportunities for the development of novel therapeutic strategies in neurodegenerative disease. A decade after the identification of KDM1A as a histone demethylase, the first selective inhibitors have now reached the clinic.

Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

BackgroundMicroarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events.ResultsThe array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer.ConclusionsThis methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies.

Molecular diagnosis of endometrial cancer from uterine aspirates

Rapid and reliable diagnosis of endometrial cancer (EC) in uterine aspirates is highly desirable. Current sensitivity and failure rate of histological diagnosis limit the success of this method and subsequent hysteroscopy is often necessary. Using quantitative reverse transcriptase‐polymerase chain reaction on RNA from uterine aspirates samples, we measured the expression level of 20 previously identified genes involved in EC pathology, created five algorithms based on combinations of five genes and evaluated their ability to diagnose EC. The algorithms were tested in a prospective, double‐blind, multicenter study. We enlisted 514 patients who presented with abnormal uterine bleeding. EC was diagnosed in 60 of the 514 patients (12%). Molecular analysis was performed on the remnants of aspirates and results were compared to the final histological diagnoses obtained through biopsies acquired by aspiration or guided by hysteroscopy, or from the specimens resected by hysterectomy. Algorithm 5 was the best performing molecular diagnostic classifier in the case–control and validation study. The molecular test had a sensitivity of 81%, specificity of 96%, positive predictive value (PPV) of 75% and negative predictive value (NPV) of 97%. A combination of the molecular and histological diagnosis had a sensitivity of 91%, specificity of 97%, PPV of 79% and NPV of 99% and the cases that could be diagnosed on uterine aspirate rose from 76 to 93% when combined with the molecular test. Incorporation of the molecular diagnosis increases the reliability of a negative diagnosis, reduces the need for hysteroscopies and helps to identify additional cases.

The dual lsd1/maob inhibitor ory2001 prevents the development of the memory deficit in samp8 mice through induction of neuronal plasticity and reduction of neuroinflammation

P4-315 EVALUATING AMYLOID-BETA PROCESSING USING NOVEL GAMMA-SECRETASE MODULATORS IN PRECLINICAL ANIMAL MODELS Kathleen M. Wood, Cathleen Gonzales, Feng Pan, Nikolay Pozdnyakov, Michael Marconi, Ashley Robshaw, David Riddell, Dmitri Volfson, Martin Pettersson, Eva Hajos-Korcsok, Kelly R. Bales, Pfizer Inc., Cambridge, MA, USA; Merck, Cambridge, MA, USA; Merck, Indianapolis, IN, USA. Contact e-mail: kathleen.m. wood@pfizer.com

Abstract 4708: Neuroendocrine gene transcript expression is associated with efficacy to lysine-specific demethylase-1 inhibitor RG6016 in small cell lung cancer-derived cell lines

Small cell lung cancer (SCLC), which comprises 15% of lung neoplasms, is an aggressive malignancy with limited treatment options. Survival in refractory settings is typically less than one year, exemplifying the need for more effective therapeutics. Recent published results suggest that epigenetic modulation mediated by Lysine Specific Demethylase-1 (LSD1) inhibition can impact this disease setting (Mohammed et al., Cell, 2015 28(1):57-69). RG6016 is a potent and selective inhibitor of LSD1 that promotes growth inhibition in vitro and in vivo SCLC xenograft models. Here we describe the identification of a gene expression profile associated with neuroendocrine lineages that predicts responses to RG6016 in SCLC cell lines. RG6016 was screened on a panel of 19 SCLC cell lines; potent sub-nanomolar to nanomolar activity was exhibited on a subset of these cell lines. Growth inhibitory responses were greater in classic compared to variant SCLC cell lines (p-value 0.0055). 9 of the 11 classic SCLC cell lines tested were responsive to growth inhibitory effects of RG6016, while 7 of the 8 variant cell lines were insensitive to RG6016 treatment. Differential gene expression analysis comparing classic to variant cell lines ranked neuroendocrine lineage-associated markers, such as ASCL1, amongst the highest differentially expressed genes (adj. p-value = 2.6 × 10-23). ASCL1 is a transcription factor required for proper development of pulmonary neuroendocrine cells, and was recently identified as essential for the survival of a majority of small cell lung cancers (Augustyn et al., Proc Natl Acad Sci USA 2014 111(41):14788-93). Two other established neuroendocrine markers, DDC and GRP, ranked within the top ten differentially expressed genes. We also found that a subset of insensitive SCLC cell lines harbor c-MYC amplifications, suggesting a potential pathway for resistance to LSD1 inhibition. An RG6016 response gene expression pattern that highly correlates with growth inhibition was defined using baseline expression levels of neuroendocrine lineage transcripts and c-MYC amplification. In vitro activity also correlated with in vivo responses; RG60106 treatment inhibited xenograft growth of response signature positive cell line NCI-H510A, while the response signature negative NCI-H526 xenografts were refractory to RG6016 exposure. Similar gene expression patterns were observed within SCLC cell lines and a panel of SCLC patient samples, suggesting that use of the RG6016 response gene signature may increase the likelihood of identifying patients who will clinically benefit from LSD1- based therapies. Citation Format: Francesca Milletti, Wei-Yi Cheng, Tamara Maes, Serena Lunardi, Mark DeMario, William E. Pierceall, Fiona Mack. Neuroendocrine gene transcript expression is associated with efficacy to lysine-specific demethylase-1 inhibitor RG6016 in small cell lung cancer-derived cell lines. [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 4708.

Orymold: ontology based gene expression data integration and analysis tool applied to rice

BackgroundIntegration and exploration of data obtained from genome wide monitoring technologies has become a major challenge for many bioinformaticists and biologists due to its heterogeneity and high dimensionality. A widely accepted approach to solve these issues has been the creation and use of controlled vocabularies (ontologies). Ontologies allow for the formalization of domain knowledge, which in turn enables generalization in the creation of querying interfaces as well as in the integration of heterogeneous data, providing both human and machine readable interfaces.ResultsWe designed and implemented a software tool that allows investigators to create their own semantic model of an organism and to use it to dynamically integrate expression data obtained from DNA microarrays and other probe based technologies. The software provides tools to use the semantic model to postulate and validate of hypotheses on the spatial and temporal expression and function of genes. In order to illustrate the softwares use and features, we used it to build a semantic model of rice (Oryza sativa) and integrated experimental data into it.ConclusionIn this paper we describe the development and features of a flexible software application for dynamic gene expression data annotation, integration, and exploration called Orymold. Orymold is freely available for non-commercial users from http://www.oryzon.com/media/orymold.html