Augustine Chen

Transterm: a database to aid the analysis of regulatory sequences in mRNAs

Messenger RNAs, in addition to coding for proteins, may contain regulatory elements that affect how the protein is translated. These include protein and microRNA-binding sites. Transterm (http://mRNA.otago.ac.nz/Transterm.html) is a database of regions and elements that affect translation with two major unique components. The first is integrated results of analysis of general features that affect translation (initiation, elongation, termination) for species or strains in Genbank, processed through a standard pipeline. The second is curated descriptions of experimentally determined regulatory elements that function as translational control elements in mRNAs. Transterm focuses on protein binding sites, particularly those in 3′-untranslated regions (3′-UTR). For this release the interface has been extensively updated based on user feedback. The data is now accessible by strain rather than species, for example there are 10 Escherichia coli strains (genomes) analysed separately. In addition to providing a repository of data, the database also provides tools for users to query their own mRNA sequences. Users can search sequences for Transterm or user defined regulatory elements, including protein or miRNA targets. Transterm also provides a central core of links to related resources for complementary analyses.

Translation of the first upstream ORF in the hepatitis B virus pregenomic RNA modulates translation at the core and polymerase initiation codons

The human hepatitis B virus (HBV) has a compact genome encoding four major overlapping coding regions: the core, polymerase, surface and X. The polymerase initiation codon is preceded by the partially overlapping core and four or more upstream initiation codons. There is evidence that several mechanisms are used to enable the synthesis of the polymerase protein, including leaky scanning and ribosome reinitiation. We have examined the first AUG in the pregenomic RNA, it precedes that of the core. It initiates an uncharacterized short upstream open reading frame (uORF), highly conserved in all HBV subtypes, we designated the C0 ORF. This arrangement suggested that expression of the core and polymerase may be affected by this uORF. Initiation at the C0 ORF was confirmed in reporter constructs in transfected cells. The C0 ORF had an inhibitory role in downstream expression from the core initiation site in HepG2 cells and in vitro, but also stimulated reinitiation at the polymerase start when in an optimal context. Our results indicate that the C0 ORF is a determinant in balancing the synthesis of the core and polymerase proteins.

Characterization of the chemokine response of RAW264.7 cells to infection by murine norovirus

Noroviruses are an emerging threat to public health, causing large health and economic costs, including at least 200,000 deaths annually. The inability to replicate in cell culture or small animal models has limited the understanding of the interaction between human noroviruses and their hosts. However, an alternative strategy to gain insights into norovirus pathogenesis is to study murine norovirus (MNV-1) that replicates in cultured macrophages. While the innate immune response is central to the resolution of norovirus disease, the adaptive immune response is required for viral clearance. The specific responses of macrophages and dendritic cells to infection drive the adaptive immune response, with chemokines playing an important role. In this study, we have conducted microarray analysis of RAW264.7 macrophages infected with MNV-1 and examined the changes in chemokine transcriptional expression during infection. While the majority of chemokines showed no change, there was specific up-regulation in chemokines reflective of a bias toward a Th1 response, specifically CCL2, CCL3, CCL4, CCL5, CXCL2, CXCL10 and CXCL11. These changes in gene expression were reflected in protein levels as determined by ELISA assay. This virus-induced chemokine response will affect the resolution of infection and may limit the humoral response to norovirus infection.

Synthetic Lethal Screens Identify Vulnerabilities in GPCR Signaling and Cytoskeletal Organization in E-Cadherin–Deficient Cells

The CDH1 gene, which encodes the cell-to-cell adhesion protein E-cadherin, is frequently mutated in lobular breast cancer (LBC) and diffuse gastric cancer (DGC). However, because E-cadherin is a tumor suppressor protein and lost from the cancer cell, it is not a conventional drug target. To overcome this, we have taken a synthetic lethal approach to determine whether the loss of E-cadherin creates druggable vulnerabilities. We first conducted a genome-wide siRNA screen of isogenic MCF10A cells with and without CDH1 expression. Gene ontology analysis demonstrated that G-protein–coupled receptor (GPCR) signaling proteins were highly enriched among the synthetic lethal candidates. Diverse families of cytoskeletal proteins were also frequently represented. These broad classes of E-cadherin synthetic lethal hits were validated using both lentiviral-mediated shRNA knockdown and specific antagonists, including the JAK inhibitor LY2784544, Pertussis toxin, and the aurora kinase inhibitors alisertib and danusertib. Next, we conducted a 4,057 known drug screen and time course studies on the CDH1 isogenic MCF10A cell lines and identified additional drug classes with linkages to GPCR signaling and cytoskeletal function that showed evidence of E-cadherin synthetic lethality. These included multiple histone deacetylase inhibitors, including vorinostat and entinostat, PI3K inhibitors, and the tyrosine kinase inhibitors crizotinib and saracatinib. Together, these results demonstrate that E-cadherin loss creates druggable vulnerabilities that have the potential to improve the management of both sporadic and familial LBC and DGC. Mol Cancer Ther; 14(5); 1213–23. ©2015 AACR.

A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation:

Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines.

Distinct families of cis-acting RNA replication elements epsilon from hepatitis B viruses

The hepadnavirus encapsidation signal, epsilon (ε), is an RNA structure located at the 5′ end of the viral pregenomic RNA. It is essential for viral replication and functions in polymerase protein binding and priming. This structure could also have potential regulatory roles in controlling the expression of viral replicative proteins. In addition to its structure, the primary sequence of this RNA element has crucial functional roles in the viral lifecycle. Although the ε elements in hepadnaviruses share common critical functions, there are some significant differences in mammalian and avian hepadnaviruses, which include both sequence and structural variations. Here we present several covariance models for ε elements from the Hepadnaviridae. The model building included experimentally determined data from previous studies using chemical probing and NMR analysis. These models have sufficient similarity to comprise a clan. The clan has in common a highly conserved overall structure consisting of a lower-stem, bulge, upper-stem and apical-loop. The models differ in functionally critical regions—notably the two types of avian ε elements have a tetra-loop (UGUU) including a non-canonical UU base pair, while the hepatitis B virus (HBV) epsilon has a tri-loop (UGU). The avian epsilon elements have a less stable dynamic structure in the upper stem. Comparisons between these models and all other Rfam models, and searches of genomes, showed these structures are specific to the Hepadnaviridae. Two family models and the clan are available from the Rfam database.

E-cadherin-deficient cells have synthetic lethal vulnerabilities in plasma membrane organisation, dynamics and function

BackgroundThe E-cadherin gene (CDH1) is frequently mutated in diffuse gastric cancer and lobular breast cancer, and germline mutations predispose to the cancer syndrome Hereditary Diffuse Gastric Cancer. We are taking a synthetic lethal approach to identify druggable vulnerabilities in CDH1-mutant cancers.MethodsDensity distributions of cell viability data from a genome-wide RNAi screen of isogenic MCF10A and MCF10A-CDH1−/− cells were used to identify protein classes affected by CDH1 mutation. The synthetic lethal relationship between selected protein classes and E-cadherin was characterised by drug sensitivity assays in both the isogenic breast MCF10A cells and CDH1-isogenic gastric NCI-N87. Endocytosis efficiency was quantified using cholera toxin B uptake. Pathway metagene expression of 415 TCGA gastric tumours was statistically correlated with CDH1 expression.ResultsMCF10A-CDH1−/− cells showed significantly altered sensitivity to RNAi inhibition of groups of genes including the PI3K/AKT pathway, GPCRs, ion channels, proteosomal subunit proteins and ubiquitinylation enzymes. Both MCF10A-CDH1−/− and NCI-N87-CDH1−/− cells were more sensitive than wild-type cells to compounds that disrupted plasma membrane composition and trafficking, but showed contrasting sensitivities to inhibitors of actin polymerisation and the chloride channel inhibitor NS3728. The MCF10A-CDH1−/− cell lines showed reduced capacity to endocytose cholera toxin B. Pathway metagene analysis identified 20 Reactome pathways that were potentially synthetic lethal in tumours. Genes involved in GPCR signalling, vesicle transport and the metabolism of PI3K and membrane lipids were strongly represented amongst the candidate synthetic lethal genes.ConclusionsE-cadherin loss leads to disturbances in receptor signalling and plasma membrane trafficking and organisation, creating druggable vulnerabilities.

Abstract B41: Statins show synthetic lethality in E-cadherin-deficient cells and are synergistic with SRC and HDAC inhibitors

E-cadherin (CDH1) is a cell-cell adhesion protein implicated in the epithelial-mesenchymal transition and frequently dysregulated in diffuse gastric cancer (DGC) and invasive lobular breast cancer (ILBC). Germline CDH1 mutations define hereditary diffuse gastric cancer (HDGC), a rare cancer syndrome characterised by highly penetrant DGC and an elevated rate of ILBC in females. There is a strong need to develop specific chemopreventative strategies for CDH1 germline mutation carriers which are capable of reducing the viability of early stage CDH1-deficient cancers whilst minimizing side effects. We have previously applied a synthetic lethal approach to an isogenic pair of MCF10A breast cell lines, one with and one without functional CDH1 expression (MCF10A CDH1-/-), to screen for drugs which preferentially reduce the viability of CDH1-deficient cells. This led to our finding that histone deacetylase inhibitors (HDACi; entinostat and vorinostat) and SRC inhibitors (SRCi; saracatinib) are involved in synthetic lethal interactions with MCF10A CDH1-/- cells. Single agent therapy, however, only produces a modest synthetic lethal effect, leading to our interest in developing synergistic drug combinations. We have now shown that statins, inhibitors of the HMG-CoA reductase enzyme, also show a synthetic lethal phenotype in the MCF10A isogenic cell line pair. This result suggests that survival of E-cadherin-deficient cells may be enhanced by the mevalonate pathway, a metabolic pathway responsible for synthesizing prenyl groups and subsequently activating the small GTPase proteins Rho, Rac, and Cdc42. SRC is also a known activator of GTPase proteins and combined statin and saracatinib treatment caused an enhanced reduction in cell viability in the isogenic MCF10A cells whilst maintaining the synthetic lethal phenotype. This drug combination was shown to be synergistic in both cell lines using the Chou-Talalay median effect analysis and is the first known example of synergy between a statin and SRC inhibitor. Combined statin and HDACi treatment also enhanced cell viability inhibition and was synthetic lethal in the MCF10A pair. This effect was observed using both class-I specific (entinostat) and pan- (vorinostat) HDACi and was synergistic in both cell lines using median effect analysis. These are the first known examples of synergistic drug combinations being used in the field of synthetic lethality and serve as a foundation for novel DGC and ILBC treatment options. Citation Format: Andrew Single, Augustine Chen, Bryony Telford, Henry Beetham, Parry Guilford. Statins show synthetic lethality in E-cadherin-deficient cells and are synergistic with SRC and HDAC inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B41.

Abstract B04: Synthetic lethal targeting of E-cadherin-deficient cancers

Germline mutation of the E-cadherin gene (CDH1) genetically defines the inherited cancer syndrome hereditary diffuse gastric cancer (HDGC). HDGC is characterized by highly penetrant diffuse gastric cancer (DGC) and an elevated rate of lobular breast cancer (LBC). Somatic CDH1 mutations also occur frequently in the sporadic forms of these cancers. We propose that the loss of E-cadherin, a tumor suppressor gene, creates vulnerabilities in the cancer cell that can be exploited with drugs (“synthetic lethal” interactions). We are particularly interested in identifying drugs that can be used for the chemoprevention of advanced disease in HDGC family members by targeting the multifocal precursor lesions that occur in stomach and breast tissue. In the stomach, these early lesions are mucosally-confined stage T1a signet ring cell carcinomas. Up to 400 independent foci have been identified in the stomachs of CDH1 mutation carriers; these foci are genetically homogenous and are likely to only require the 2nd CDH1 hit for their initiation. To identify the vulnerabilities created by E-cadherin loss, we have conducted a genome-wide siRNA knockdown screen, a 4000 compound known drug screen and a 114,000 compound library screen in isogenic MCF10a breast cell lines with and without E-cadherin expression. The functional screen has shown that GPCR signaling proteins are highly enriched amongst the candidate synthetic lethal proteins, as well as many protein classes associated cell signaling and cytoskeletal function. Drugs that show increased activity against the E-cadherin-deficient cells include the JAK inhibitor LY2784544, the c-SRC inhibitor saracatanib, the beta-2 adrenoreceptor (GPCR) agonist formoterol, several HDAC inhibitors and statins. Detail on synergistic combinations involving many of these drugs is provided in the abstract entitled “Statins show synthetic lethality in E-cadherin-deficient cells and are synergistic with SRC and HDAC inhibitors”. To identify mechanisms associated with E-cadherin synthetic lethality, we used the cell viability data from the siRNA screen to examine the distributions of cell viabilities for the genes that make up the major KEGG signaling pathways. Using the Kolomogorov-Smirnof method to test for significance, we identified the PI3K-AKT survival pathway as being central to E-cadherin9s synthetic lethal associations. These findings pave the way for the development of rationally designed drug combinations for the chemoprevention and treatment of E-cadherin-negative cancers. Citation Format: Parry J. Guilford, Augustine Chen, Bryony Telford, Andrew Single, Henry Beetham, Tanis Godwin. Synthetic lethal targeting of E-cadherin-deficient cancers. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B04.

Abstract 3814: Synthetic lethal approaches targeting E-cadherin-deficient cancers

E-cadherin is a cellular adhesion protein that is frequently mutated in lobular breast cancer and diffuse gastric cancer. The E-cadherin protein which is encoded by the CDH1 gene has key roles in establishing and maintaining cell polarity and differentiation, the organization of cell migration and architecture and the mediation of signaling through various proliferation and survival pathways. E-cadherin also has a tumor suppressor role and its loss in cancer cells would preclude drug targeting by conventional therapy. To circumvent this, we have taken a synthetic lethal approach to exploit any vulnerability created by the loss of E-cadherin. In the therapeutic setting, synthetic lethality refers to a combination of a mutated gene and a drug targeted at a second gene or protein causing cell death (specifically in cancer cells). To identify the vulnerabilities created by E-cadherin loss, we performed a genome-wide siRNA knockdown screen in isogenic MCF10A cell lines with and without E-cadherin expression. From the functional screen, we identified broad classes of G-protein-coupled receptor (GPCR) signaling proteins and families of cytoskeletal proteins which were highly enriched among the synthetic lethal candidates. Indeed, we identified drug classes with linkages to several of the GPCR and cytoskeletal targets that showed evidence of E-cadherin synthetic lethality when we performed a 4,057 known drug screen. These included certain PI3K inhibitors (PI-103), anti-glucocorticoid (mifepristone), tyrosine kinase inhibitor (saracatinib) and multiple histone deacetylase inhibitors (vorinostat and entinostat). Interestingly, the combination of saracatinib and mifepristone gave a synergistic effect (combination index Citation Format: Augustine Chen, Bryony J. Telford, Andrew Single, Henry Beetham, Kaylene J. Simpson, Parry Guilford. Synthetic lethal approaches targeting E-cadherin-deficient cancers. [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 3814.