Genome-wide Screening Approach Research Articles

134. Gene Editing Approaches for Investigating Therapy-Resistance in Soft-Tissue Sarcoma

Soft-tissue sarcoma (STS) is a rare type of tumor that accounts for approximately 1 % of adult cancers. STS tumors frequently recur or result in distant metastasis after surgical resection. Cytotoxic chemotherapy, the current “gold standard”, is often confronted with development of resistance, leading to patient survival periods below 15 months. Therefore, further insight into new therapies and mechanisms leading to drug resistance in sarcoma is required to improve therapeutic success. In the present study, we analyzed drug resistance in vitro using (i) a genome-wide assay for identification of drug resistance-associated genetic alterations and (ii) generation of isogenic cell lines using gene editing approaches to functionally characterize the genetic alterations. To address drug response and resistance in sarcoma, we have established a genome-wide insertional mutagenesis screening approach. To this end, a panel of STS cell lines is screened for the sensitivity towards drugs currently used for standard therapy of STS. Sensitive cell lines will be treated with transforming lentiviral vectors to identify genes involved in drug resistance. The genetic alterations potentially involved in acquired resistance mechanisms will be functionally characterized using isogenic cell lines created by gene editing approaches. Towards this end, we have successfully employed transcription activator-like effector nucleases and CRISPR/Cas9 system RNA guided endonucleases targeting the BRAF locus to introduce the previously described V600E mutation in 293T cells, as a “proof of concept”. Analysis of the target site showed that 31 % of the sequences were positive for the mutation. The mutation was also confirmed in single cell clones by tetra-primer ARMS-PCR and western blot using an antibody specific for V600E mutated B-Raf. To test the method in a model relevant to STS, we applied gene editing to introduce a previously undescribed KRT8 mutation found in STS patients into a sarcoma cell line. For this mutation, wild type and D10A-mutated Cas9 expressing constructs were generated to allow use of single nucleases and paired nickases. We applied single stranded oligodeoxynucleotides as donor template for KRT8. Using high-throughput sequencing, we detected successful recombination of the mutated KRT8 gene locus in 0.6 % of the treated STS cells. We are using improved guide RNA and donor design as well as compounds affecting non-homologous end-joining and homologous recombination to improve efficiency of successful recombination. To summarize, we successfully edited wild-type BRAF to BRAF V600E in 293T cells and showed that gene editing of KRT8 is possible in sarcoma cells. The methodology will be further optimized to characterize genes involved in resistance to therapy identified in the genome-wide screens. In addition, our gene editing approaches will also be used to model genetic alterations identified in STS patients by next generation sequencing. Our findings will provide interesting insights in nominating drugs for combinatorial therapy to circumvent drug resistance in STS.

Effects of Controlled Cortical Impact on the Mouse Brain Vasculome.

Perturbations in blood vessels play a critical role in the pathophysiology of brain injury and neurodegeneration. Here, we use a systematic genome-wide transcriptome screening approach to investigate the vasculome after brain trauma in mice. Mice were subjected to controlled cortical impact and brains were extracted for analysis at 24 h post-injury. The core of the traumatic lesion was removed and then cortical microvesels were isolated from nondirectly damaged ipsilateral cortex. Compared to contralateral cortex and normal cortex from sham-operated mice, we identified a wide spectrum of responses in the vasculome after trauma. Up-regulated pathways included those involved in regulation of inflammation and extracellular matrix processes. Decreased pathways included those involved in regulation of metabolism, mitochondrial function, and transport systems. These findings suggest that microvascular perturbations can be widespread and not necessarily localized to core areas of direct injury per se and may further provide a broader gene network context for existing knowledge regarding inflammation, metabolism, and blood-brain barrier alterations after brain trauma. Further efforts are warranted to map the vasculome with higher spatial and temporal resolution from acute to delayed phase post-trauma. Investigating the widespread network responses in the vasculome may reveal potential mechanisms, therapeutic targets, and biomarkers for traumatic brain injury.

Up-regulation of Cathepsin G in the Development of Chronic Postsurgical Pain: An Experimental and Clinical Genetic Study.

Proteases have been shown to modulate pain signaling in the spinal cord and may contribute to the development of chronic postsurgical pain. By using peripheral inflammation in rats as a chronic pain model, the authors identified the deregulation of proteases and their inhibitors as a hallmark of chronic pain development using a genome-wide screening approach. A microarray analysis was performed and identified spinal cathepsin G (CTSG) as the most up-regulated gene in rats with persistent hyperalgesia after intraplantar injection of complete Freund's adjuvant (n = 4). Further experiments were performed to elucidate the mechanisms of CTSG-induced hyperalgesia by intrathecally applying specific CTSG inhibitor (n = 10). The authors also evaluated the association between CTSG gene polymorphisms and the risk of chronic postsurgical pain in 1,152 surgical patients. CTSG blockade reduced heat hyperalgesia, accompanied by a reduction in neutrophil infiltration and interleukin 1β levels in the dorsal horns. In the gene association study, 246 patients (21.4%) reported chronic postsurgical pain at 12-month follow-up. Patients with AA genotypes at polymorphisms rs2070697 (AA-15.3%, GA-24.1%, and GG-22.3%) or rs2236742 (AA-6.4%, GA-20.4%, and GG-22.6%) in the CTSG gene had lower risk for chronic postsurgical pain compared with wild-types. The adjusted odds ratios were 0.67 (95% CI, 0.26 to 0.99) and 0.34 (95% CI, 0.21 to 0.98), respectively. This study demonstrated that CTSG is a pronociceptive mediator in both animal model and human study. CTSG represents a new target for pain control and a potential marker to predict patients who are prone to develop chronic pain after surgery.

Identification of Endoglin as an epigenetically regulated tumour-suppressor gene in lung cancer.

Background:The transforming growth factor-beta (TGF- β) pathway has been implicated in proliferation, migration and invasion of various cancers. Endoglin is a TGF-β accessory receptor that modulates signalling. We identified Endoglin as an epigenetically silenced tumour-suppressor gene in lung cancer by means of a genome-wide screening approach, then sought to characterise its effect on lung cancer progression.Methods:Methylation microarray and RNA sequencing were carried out on lung cancer cell lines. Epigenetic silencing of Endoglin was confirmed by methylation and expression analyses. An expression vector and a 20-gene expression panel were used to evaluate Endoglin function. Pyrosequencing was carried out on two independent cohorts comprising 112 and 202 NSCLC cases, respectively, and the impact of Endoglin methylation on overall survival (OS) was evaluated.Results:Methylation in the promoter region resulted in silencing of Endoglin, which could be reactivated by demethylation. Increased invasion coupled with altered EMT marker expression was observed in cell lines with an epithelial-like, but not those with a mesenchymal-like, profile when Endoglin was absent. Methylation was associated with decreased OS in stage I but not in stages II–III disease.Conclusions:We show that Endoglin is a common target of epigenetic silencing in lung cancer. We reveal a link between Endoglin silencing and EMT progression that might be associated with decreased survival in stage I disease.

Complete genome-wide screening and subtractive genomic approach revealed new virulence factors, potential drug targets against bio-war pathogen Brucella melitensis 16M

Brucella melitensis 16M is a Gram-negative coccobacillus that infects both animals and humans. It causes a disease known as brucellosis, which is characterized by acute febrile illness in humans and causes abortions in livestock. To prevent and control brucellosis, identification of putative drug targets is crucial. The present study aimed to identify drug targets in B. melitensis 16M by using a subtractive genomic approach. We used available database repositories (Database of Essential Genes, Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server, and Kyoto Encyclopedia of Genes and Genomes) to identify putative genes that are nonhomologous to humans and essential for pathogen B. melitensis 16M. The results revealed that among 3 Mb genome size of pathogen, 53 putative characterized and 13 uncharacterized hypothetical genes were identified; further, from Basic Local Alignment Search Tool protein analysis, one hypothetical protein showed a close resemblance (50%) to Silicibacter pomeroyi DUF1285 family protein (2RE3). A further homology model of the target was constructed using MODELLER 9.12 and optimized through variable target function method by molecular dynamics optimization with simulating annealing. The stereochemical quality of the restrained model was evaluated by PROCHECK, VERIFY-3D, ERRAT, and WHATIF servers. Furthermore, structure-based virtual screening was carried out against the predicted active site of the respective protein using the glycerol structural analogs from the PubChem database. We identified five best inhibitors with strong affinities, stable interactions, and also with reliable drug-like properties. Hence, these leads might be used as the most effective inhibitors of modeled protein. The outcome of the present work of virtual screening of putative gene targets might facilitate design of potential drugs for better treatment against brucellosis.

Annual research review: The (epi)genetics of neurodevelopmental disorders in the era of whole-genome sequencing--unveiling the dark matter.

Neurodevelopmental disorders (NDDs) are defined by a wide variety of behavioural phenotypes, psychopathology and clinically informed categorical classifications. Diagnostic entities include intellectual disability (ID), the autism spectrum (ASD) and attention-deficit/hyperactivity disorder (ADHD). The aetiopathogenesis of these conditions and disorders involves an interaction between both genetic and environmental risk factors on the developmental trajectory. Despite their remarkable genetic heterogeneity and complexity of pathophysiological mechanisms, NDDs display an overlap in their phenotypic features, a considerable degree of comorbidity as well as sharing of genetic and environmental risk factors. This review aims to provide an overview of the genetics and epigenetic of NDDs. Recent evidence suggests a critical role of defined and tightly regulated neurodevelopmental programs running out of control in NDDs, most notably neuronal proliferation and migration, synapse formation and remodelling, as well as neural network configuration resulting in compromised systems connectivity and function. Moreover, the machinery of epigenetic programming, interacting with genetic liability, impacts many of those processes and pathways, thus modifying vulnerability of, and resilience to, NDDs. Consequently, the categorically defined entities of ID, ADHD and ASD are increasingly viewed as disorders on a multidimensional continuum of molecular and cellular deficiencies in neurodevelopment. As such, this range of NDDs displays a broad phenotypic diversity, which may be explained by a combination and interplay of underlying loss- and potential gain-of-function traits. In this overview, we discuss a backbone continuum concept of NDDs by summarizing pertinent findings in genetics and epigenetics. We also provide an appraisal of the genetic overlap versus differences, with a focus on genome-wide screening approaches for (epi)genetic variation. Finally, we conclude with insights from evolutionary psychobiology suggesting positive selection for discrete NDD-associated traits.

Variations in genome-wide RNAi screens: lessons from influenza research.

Genome-wide RNA interference (RNAi) screening is an emerging and powerful technique for genetic screens, which can be divided into arrayed RNAi screen and pooled RNAi screen/selection based on different screening strategies. To date, several genome-wide RNAi screens have been successfully performed to identify host factors essential for influenza virus replication. However, the host factors identified by different research groups are not always consistent. Taking influenza virus screens as an example, we found that a number of screening parameters may directly or indirectly influence the primary hits identified by the screens. This review highlights the differences among the published genome-wide screening approaches and offers recommendations for performing a good pooled shRNA screen/selection.

Abstract 1363: Epigenetic inactivation of VGF in urothelial cell carcinoma and its potential as a non-invasive biomarker using urine

Abstract Background: Urothelial Cell Carcinoma (UCC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new epigenetic markers for UCC we scrutinized the methylation status of 19 genes that has been shown to be cancer specific methylated genes in other solid tumors in our comprehensive genome-wide discovery approach. Methods: We performed promoter methylation of 19 genes (CAMK4, CKMT1b, FKBP4, GALE, HOXB5, HPN, KRT14, LPAR2, MAL, NBR1, NDP, NF1, PDLIM3, PHKA2, PVRL1, RGS4, SGK1, VGF and ZMYM2) that are related to key molecular pathways involved in urothelial carcinogenesis including MAPK pathway or PI3K/AKT pathway. The methods we used includes bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR(QMSP). All the 19 genes and/or a particular gene were evaluated in urothelial cancer cell lines, primary tumor tissues and urine sediments. Results: By bisulfite sequencing, methylation frequencies for CAMK4, CKMT1b, FKBP4, GALE, HOXB5, HPN, KRT14, LPAR2, MAL, NBR1, NDP, NF1, PDLIM3, PHKA2, PVRL1, RGS4, SGK1, VGF and ZMYM2 were 33.3%, 42.9%, 71.4%, 100.0%, 42.9%, 71.4%, 85.7%, 28.6%, 100.0%, 16.7%, 42.9%, 100.0%, 33.3%, 16.7%, 16.7%, 33.3%, 16.7%, 57.1% and 57.1% respectively in urothelial cancer cell lines. Based on methylation frequencies and known functional evidence, CAMK4, HOXB5, MAL, RGS4, VGF and ZMYM2 were selected for re-expression analysis after 5-Aza-2′-deoxycytidine treatment of UCC cell lines. We also found that promoter methylation of these 6 genes were inversely correlated with gene expression. Since VGF gene was found to be completely methylated in the representative cell lines, we further performed QMSP analysis on 19 primary UCC tissues of different stages and grades with paired normal. Quantitative methylation values were higher in almost all the primary UCC tissues than the paired normal (student's t-test, P=0.005). In an independent cohort of 35 low grade UCC primary tissues, we performed QMSP assay. Twelve out of thirty-five (34.3%) of low grade UCC cases displayed VGF methylation that indicate VGF could be a marker for determining initiation of neoplastic transformation. To explore the potential of methylated VGF as a biomarker for non-invasive detection of UCC, we tested 20 urine samples from UCC cases and 20 urine samples from subjects without any known neoplastic diseases. VGF showed significantly high frequency of QMSP positive status (Fisher's exact test, P=0.020) in urine from UCC cases (8/20) compared to controls (1/20). Conclusion: Our selected genes from genome-wide screening approach successfully identified UCC specific methylated genes that can be determined in primary tissues and urine. Further studies are needed to understand the role of VGF in urothelial carcinogenesis and its potential for the detection of low grade UCC using urine. Citation Format: Masamichi Hayashi, Heike Bernert, Luciane Tsukamoto Kagohara, Mariana Brait, Mark Schoenberg, Trinity Bivalacqua, George Netto, Wayne Koch, David Sidransky, Mohammad O. Hoque. Epigenetic inactivation of VGF in urothelial cell carcinoma and its potential as a non-invasive biomarker using urine. [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 1363. doi:10.1158/1538-7445.AM2014-1363

Genome-wide screening identifies new genes required for stress-induced phase 2 detoxification gene expression in animals.

BackgroundPhase 2 detoxification enzymes provide a vital defence against reactive oxygen species, including xenobiotic metabolites, which cause the oxidative damage involved in drug toxicity and many diseases. Hence, there is great interest in understanding how levels of these enzymes are regulated. CnC transcription factors, such as mammalian Nrf2, drive the expression of phase 2 enzymes and are activated as an important conserved response to oxidative stress and xenobiotics. For instance, the Caenorhabditis elegans Nrf2 orthologue, SKN-1, is activated in response to arsenite by the stress-activated p38-related kinase, PMK-1, leading to increased expression of phase 2 enzymes. Here we have used a genome-wide screening approach to identify other C. elegans genes that are required for stress-induced increases in phase 2 detoxification gene expression.ResultsTaking advantage of the elevated phase 2 gene expression in a mutant lacking the peroxidase PRDX-2, we have identified many new genes that are required for stress-induced expression of gcs-1, a phase 2 enzyme critically required for glutathione synthesis. Significantly, these include genes previously implicated in resistance to ionizing radiation, longevity and responses to pathogenic infection. Many of these new candidate activators of gcs-1 are also required for the stress-induced intestinal expression of other phase 2 genes. However, intriguingly, our data suggest other factors may be specifically required for the stress-induced expression of gcs-1. Notably, we demonstrate that the candidate activator TIR-1(SARM1) and the MAPKKK NSY-1(Ask1) are required for the arsenite-induced activation of PMK-1. However, our data suggest that the majority of candidates participate in novel mechanisms to promote gcs-1 expression. For example, the E4 ubiquitin ligase UFD-2(UBE4B) is dispensable for PMK-1 activation but important for maintaining nuclear levels of SKN-1, the stress-induced expression of multiple SKN-1-target genes and oxidative stress resistance.ConclusionsHere we present the first functional, genome-wide analysis identifying genes that are required for activation of phase 2 detoxification genes in an animal. Our study identifies potential new regulators of Nrf2, reveals that additional mechanisms promote the stress-induced expression of specific phase 2 detoxification genes and provides new insight into the relationships between these universally important stress defences, oxidative stress resistance and aging.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0064-6) contains supplementary material, which is available to authorized users.

Epigenetic inactivation of VGF associated with Urothelial Cell Carcinoma and its potential as a non-invasive biomarker using urine

To identify new epigenetic markers and further characterize Urothelial Cell Carcinoma (UCC), we tested the promoter methylation (PM) status of 19 genes previously identified as cancer specific methylated genes in other solid tumors. We used bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR (QMSP) to test the PM status of 19 genes in urothelial cancer cell lines. Among the 19 genes tested, VGF was found to be completely methylated in several UCC cell lines. VGF QMSP analysis showed that methylation values of almost all the primary 19 UCC tissues were higher than the paired normal tissues (P=0.009). In another cohort, 12/35 (34.3%) of low grade UCC cases displayed VGF methylation. As a biomarker for non-invasive detection of UCC, VGF showed a significantly higher frequency of methylation in urine from UCC cases (8/20) compared to controls (1/20) (P=0.020). After treatment of cell lines with 5-Aza-2'-deoxycytidine, VGF was robustly re-expressed. Forced expression of VGF in bladder cancer cell lines inhibited cell growth. Selection of candidates from genome-wide screening approach in other solid tumors successfully identified UCC specific methylated genes.

Differential DNA Methylation Predicts Response To Combined Treatment Regimens With a DNA Methyltransferase Inhibitor In Acute Myeloid Leukemia (AML)

Deregulated epigenetic mechanisms have been identified as major components of acute myeloid leukemia (AML) pathogenesis. This improved mechanistic understanding has started to translate into clinics and leads to the development of novel therapeutic options as exemplified by the DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-azaC) and decitabine (DAC). However, biomarkers for response prediction to epigenetic therapy are urgently needed. Recently, we and others demonstrated that in-depth characterization of leukemia-associated DNA-methylation patterns contributes to refinement of the molecular classification and of prognostication in AML. Thus, disease associated methylation patterns might also harbor predictive relevance for identification of patients who will profit from DNMT inhibitor therapy and for support of therapeutic decision making.In order to identify a DNA methylation based response predictor, we applied a two-step strategy and generated genome-wide profiles underlying response and resistance to a combination chemotherapy applied within the AMLSG 12-09 Study (ClinicalTrials.gov Identifier: NCT01180322) comprising the drugs idarubicin and etoposide plus the demethylating agent 5-azaC as induction therapy. By methylated-CpG immune-precipitation and next generation sequencing (MCIp-seq), we generated DNA methylation profiles of responders (n=12) and non-responders (n=23). A supervised empirical Bayes approach for the analysis of sequencing read count data (“edgeR”) was applied to identify differentially methylated regions (DMRs) associated with 5-azaC response.We identified 550 genomic regions (based on 500 bp binning) that exposed highly significant read count differences indicating differential DNA methylation between both patient groups. The GC content distribution within the identified differentially methylated regions (DMRs) was comparable to the entire genome. 14% of the DMRs were located in gene promoter regions, 60% in intragenic and 26% in intergenic regions. Overall, the detected DMRs were considerably enriched in the vicinity of transcriptional start sites and preferentially targeted genes acting as transcriptional regulators (including transcription factors involved in hematopoiesis).Within the set of 550 DMRs, we selected the 40 most significantly discriminating regions and validated them with quantitative DNA methylation data from the Illumina Infinium® HumanMethylation450 Bead Chip. 25% of the selected DMRs were covered by only one probe whereas the majority was covered by up to six probes totaling in 107 probes (CpGs). We detected a good correlation between MCIp-seq und 450k-derived methylation data for each patient (median Spearman's rho = 0.69, 95%-CI [0.32, 0.87]) and could validate 90% of DMRs via quantitative 450k array data. Comprising 95 probes, these validated DMRs were used to create a multivariable signature for therapy response prediction.Through a penalized logistic regression model (“elastic-net”-penalty) applied to the 450k M-values in our discovery sample set, we identified a signature containing 17 probes (CpGs) associated with 12 genes which predicted response perfectly. Four of the identified CpGs were located in promoters, 11 in intragenic and two in intergenic regions. Among the genes targeted by differential methylation in our signature, we found WNT10A, a component of the WNT-beta-catenin-TCF signaling pathway, and PKMYT1. The latter one is a membrane-associated serine/threonine protein kinase which is regulated by polo-like kinase 1. Its inhibition has been reported recently to sensitize for cytarabine-mediated toxicity in vitro. Furthermore, two DMRs associated with the promoters of miRNAs (miR-3154, miR-3186) were contained in the signature.In summary, by genome-wide screening approaches, we identified differentially methylated genes and genomic regions that are associated with response to treatment regimens containing the DNMT inhibitor 5-azaC. At the same time, the predictive DMRs also harbor high potential to be functionally linked to molecular mechanisms and pathways involved in therapy response. By variable selection, we created a minimal signature that accurately predicts response in our discovery sample set. Further validation of this response-signature in independent cohorts of AML cases also comprising patients treated with decitabine are underway. Disclosures:Schlenk:Celgene: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Chugai: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Ambit: Honoraria.

Drosophila as a screening tool to study human neurodegenerative diseases

In an aging society, research involving neurodegenerative disorders is of paramount importance. Over the past few years, research on Alzheimer's and Parkinson's diseases has made tremendous progress. Experimental studies, however, rely mostly on transgenic animal models, preferentially using mice. Although experiments on mice have enormous advantages, they also have some inherent limitations, some of which can be overcome by the use of Drosophila melanogaster as an experimental animal. Among the major advantages of using the fly is its small genome, which can also be modified very easily. The fact that its genome lends itself to diverse alterations (e. g. mutagenesis, transposons) has made the fly a useful organism to perform large-scale and genome-wide screening approaches. This has opened up an entirely new field of experimental research aiming to elucidate genetic interactions and screen for modifiers of disease processes in vivo. Here, we provide a brief overview of how flies can be used to analyze molecular mechanisms underlying human neurodegenerative diseases.

Abstract B19: Functional genomics approach links anchorage-independence with tumor dormancy in ovarian cancer

Abstract Although a number of genes are aberrantly regulated in ovarian cancer, little is known about which genes are functionally important. The aim of this study was to identify new genes relevant to anchorage-independent growth of ovarian cancer cells through a genome-wide functional screening approach. We performed lentiviral transduction of a library of 81,000 shRNAs (Cellecta, Inc.) targeting 15,000 genes into an immortalized human ovarian surface epithelium cell line and 7 ovarian cancer cell lines (OVCA420, OVCA433, OVCA429, CAOV3, SKOV8, Tyk-nu and DOV13) which do not generate anchorage-independent colonies in soft agar. Among these, only OVCA420 generated colonies larger than 100 micrometers after shRNA library transduction. We extracted DNA from these colonies, amplified the shRNA constructs with PCR, and inserted the amplicons back into the original lentiviral vector, thus generating a secondary library of 68 shRNAs. Subsequent analysis, performed to examine the reproducible formation of anchorage-independent colonies using the secondary library, identified 5 shRNAs targeting ABHD2, CYB5R3, MR1, ELAC2 and CCL22. Interestingly, population doubling time was increased following transduction of these shRNAs (44.8, 50.1, 43.9, 28.0 and 33.2 hours, respectively) compared to the original or control shRNA-transducted OVCA420 cells (22.4 and 23.7 hours, respectively). Furthermore, side populations detected by efflux of Hoechst 33342 dye were drastically increased (50.5, 46.6, 55.5, 76.5 and 41.1%, respectively) compared to the original or control shRNA-transducted OVCA420 cells (7.3 and 10.4%, respectively). Although these data are not conclusive, they support that a phenotypic change occurs as ovarian cancer cells leave the primary tumor and are suspended in ascites. We found that repression of any one of these particular genes was sufficient to avoid anoikis. This change was accompanied by suppression of cell proliferation and increased efflux of Hoechst 33342 dye, which indicates activation of pumping function, suggesting a change to chemoresistant dormant cells. In summary, our functional genomics approach suggests a molecular mechanism underlying the process of anchorage independence, tumor dormancy and chemoresistance in ovarian cancer. Citation Format: Noriomi Matsumura, Koji Yamanoi, Yasuaki Amano, Junzo Hamanishi, Tsukasa Baba, Susan K. Murphy, Ikuo Konishi. Functional genomics approach links anchorage-independence with tumor dormancy in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B19.

Genes Required for Growth at High Hydrostatic Pressure in Escherichia coli K-12 Identified by Genome-Wide Screening

Despite the fact that much of the global microbial biosphere is believed to exist in high pressure environments, the effects of hydrostatic pressure on microbial physiology remain poorly understood. We use a genome-wide screening approach, combined with a novel high-throughput high-pressure cell culture method, to investigate the effects of hydrostatic pressure on microbial physiology in vivo. The Keio collection of single-gene deletion mutants in Escherichia coli K-12 was screened for growth at a range of pressures from 0.1 MPa to 60 MPa. This led to the identification of 6 genes, rodZ, holC, priA, dnaT, dedD and tatC, whose products were required for growth at 30 MPa and a further 3 genes, tolB, rffT and iscS, whose products were required for growth at 40 MPa. Our results support the view that the effects of pressure on cell physiology are pleiotropic, with DNA replication, cell division, the cytoskeleton and cell envelope physiology all being potential failure points for cell physiology during growth at elevated pressure.

Genome-wide shRNA screening approach towards identification and characterization of therapy resistance determinants in leukemia

Genome-wide shRNA screening approach towards identification and characterization of therapy resistance determinants in leukemia

Identification of cancer gene fusions based on advanced analysis of the human genome or transcriptome

Many gene fusions have been recognized as important diagnostic and/or prognostic markers in human malignancies. In recent years, novel gene fusions have been identified in cases without prior knowledge of the genetic background. Accompanied by a powerful computational data analysis method, new genome-wide screening approaches were used to detect cryptic genomic aberrations. This review focused on advanced genomewide screening approaches in fusion gene identification, such as microarray-based approaches, next-generation sequencing, and NanoString nCounter gene expression system. The fundamental rationale and strategy for fusion gene identification using each biotech platform are also discussed.

Rif2 Promotes a Telomere Fold-Back Structure through Rpd3L Recruitment in Budding Yeast

Using a genome-wide screening approach, we have established the genetic requirements for proper telomere structure in Saccharomyces cerevisiae. We uncovered 112 genes, many of which have not previously been implicated in telomere function, that are required to form a fold-back structure at chromosome ends. Among other biological processes, lysine deacetylation, through the Rpd3L, Rpd3S, and Hda1 complexes, emerged as being a critical regulator of telomere structure. The telomeric-bound protein, Rif2, was also found to promote a telomere fold-back through the recruitment of Rpd3L to telomeres. In the absence of Rpd3 function, telomeres have an increased susceptibility to nucleolytic degradation, telomere loss, and the initiation of premature senescence, suggesting that an Rpd3-mediated structure may have protective functions. Together these data reveal that multiple genetic pathways may directly or indirectly impinge on telomere structure, thus broadening the potential targets available to manipulate telomere function.

Comparative Chemogenomics To Examine the Mechanism of Action of DNA-Targeted Platinum-Acridine Anticancer Agents

Platinum-based drugs have been used to successfully treat diverse cancers for several decades. Cisplatin, the original compound of this class, cross-links DNA, resulting in cell cycle arrest and cell death via apoptosis. Cisplatin is effective against several tumor types, yet it exhibits toxic side effects and tumors often develop resistance. To mitigate these liabilities while maintaining potency, we generated a library of non-classical platinum-acridine hybrid agents and assessed their mechanisms of action using a validated genome-wide screening approach in Saccharomyces cerevisiae and in the distantly related yeast Schizosaccharomyces pombe. Chemogenomic profiles from both S. cerevisiae and S. pombe demonstrate that several of the platinum-acridines damage DNA differently than cisplatin based on their requirement for distinct modules of DNA repair.

Genome-Wide Aberrant Splicing in Patients with Acute Myeloid Leukemia (AML) Indetifies Potential Novel Targets

Abstract 761 Background:AML is characterized by specific chromosomal translocations along with a number of other recurrent mutations. However, it is also becoming clear that AML cells have frequent epigenetic abnormalities that also contribute to transformation. Alternative splicing (AS) is an epigenetic event that is used in normal cells to generate protein diversity from single genes. Using genome-wide screening approaches, we have shown that alternative splicing of numerous genes is aberrant in most cases of AML, and that several common myeloid cell surface proteins may have novel and potentially antigenic epitopes as a result. Methods:Using Affymetrix Human Exon 10ST arrays we performed a genome-wide analysis of AS in bone marrow (BM) aspirates from two cohorts (training cohort including 40 patients and test cohort including 41 patients) of patients with AML (total 81 patients) and CD34+ BM progenitor cells from 8 normal donors (NDs). After principle component analysis, 6 patients were removed from the test group, because they were outliers. In final array analysis for the test group we analysed 35 patients. Aberrant splicing of several selected genes was confirmed through cloning and sequencing analysis followed evaluating expression patterns of cloned novel splice variant transcripts in 193 patients compared to NDs. To evaluate the clinical significance of AS in AML we determined if there were any correlations between novel aberrant splice variant expression and clinical features of patients. Results and conclusions:We found that AS is a common event in AML involving many genes in any given patient. Overall, we identified ∼550 genes that were significantly spliced in patients compared to normal CD34+ cells. Three genes encoding myeloid cell surface proteins, NOTCH2, CD13, and FLT3, were selected for further study. By cloning and sequencing analysis we found that novel splice variants of the selected genes are the results of either exon skipping, usage of cryptic 5’ or 3’ splice sites on exons and/or partial retention of an intron. We also found that these splice variants encode proteins, as assessed by expressing splice variant transcripts in the HEK293 cell line. To identify the most frequently expressed variants of NOTCH2, FLT3 and CD13 in AML, we performed expression profiling of these transcripts and their variants in an independent validation group of 193 AML patients. These analyses identified NOTCH2-Va (74%), FLT3-Va (50%) and CD13-Va (60%) as the most frequently expressed variants in AML. Expression of these novel splice variants, other than NOTCH2-Vb, was undetectable in BM CD34+ cells, normal monocytes or neutrophils. To examine whether expression of novel variants are associated with disease evolution, we performed longitudinal monitoring of expression levels of NOTCH2-FL, FLT3-FL, and CD13-FL and their splice variants in paired samples of 13 patients taken over the course of the disease. Our study suggests the “splicing profile” normalizes in remission and recurs when patients relapse. Correlation analysis among NOTCH2, CD13 variants and FLT3 splice variants, and clinical features of patients with AML, showed that splice variant NOTCH2-Va expression is significantly negatively associated with overall survival (OS) of patients (P=0.008). Our study also showed that deregulated expression of NOTCH2-Va identifies two subgroups of patients within the intermediate-risk profile patients. We also observed significantly inferior OS of the patients that overexpressed NOTCH2-Va within this group (P=0.007). Thus, NOTCH2-Va expression can be used to stratify patients with cytogenetically determined intermediate-risk profiles. Overall, our study demonstrates that genome-wide study of AS can be used to discover new prognostic markers, and potentially creates new surface antigenic epitopes that could be targeted in AML. Disclosures:No relevant conflicts of interest to declare.

Genome-wide screen identifies PVT1 as a regulator of Gemcitabine sensitivity in human pancreatic cancer cells

Gemcitabine has been a first-line chemotherapy agent for advanced pancreatic cancer, which is associated with one of the lowest 5 years survival rates among human cancers. Due to our lack of understanding of the genetic determinants of Gemcitabine sensitivity in pancreatic cancer, the therapeutic effectiveness of Gemcitabine chemotherapy is typically unpredictable. Using a genome-wide and piggyBac transposon-based genetic screening platform, we identified the PVT1 gene as a regulator of Gemcitabine sensitivity and showed that functional inactivation of the PVT1 gene led to enhanced Gemcitabine sensitivity in human pancreatic cancer ASPC-1 cells. The integration of the piggyBac transposon-based vector system into intron 3 of PVT1 was within a common site of oncogenic retroviral insertions and chromosomal translocations. PVT1 is a non-protein encoding gene; the genomic arrangement of PVT1 and its co-amplification with MYC have been implicated in the tumorigenesis of a variety of cancers. The molecular mechanism of PVT1 transcripts in gene regulation remains a puzzle. We demonstrated that overexpression of a full length PVT1 cDNA in the antisense orientation reconstituted enhanced sensitivity to Gemcitabine in naïve ASPC-1 cells, whereas overexpression of a full length PVT1 cDNA in the sense orientation resulted in decreased sensitivity to Gemcitabine. Our results identified PVT1 as a regulator of Gemcitabine sensitivity in pancreatic cancer cells and validated the genome-wide genetic screening approach for the identification of genetic determinants as well as potential biomarkers for the rational design of Gemcitabine chemotherapies for pancreatic cancer.