High-throughput RNA Interference Research Articles

Identification of factors regulating MET receptor endocytosis by high-throughput siRNA screening.

The tyrosine kinase MET, a receptor for hepatocyte growth factor, is a key regulator for normal development and organ renewal via stem cell maintenance. Dysregulated MET signaling contributes to tumor progression and metastasis and is considered a potent therapeutic target for a growing number of malignancies. Toward that goal it is critical to develop high-throughput assays to identify candidate regulators for the termination of MET signaling. We describe here a rapid and efficient method for identifying cellular factors required for MET ubiquitination, which utilizes high-throughput RNA interference screening (HT-siRNA) with a receptor internalization assay and an In-Cell ELISA in a 96-well format. The assay is amenable to a large array of cell surface proteins as well as genome-wide siRNA libraries, with high signal-to-background ratio and low well-to-well variability.

A high-throughput siRNA screen identifies genes that regulate mannose 6-phosphate receptor trafficking.

The delivery of newly synthesized soluble lysosomal hydrolases to the endosomal system is essential for lysosome function and cell homeostasis. This process relies on the proper trafficking of the mannose 6-phosphate receptors (MPRs) between the trans-Golgi network (TGN), endosomes and the plasma membrane. Many transmembrane proteins regulating diverse biological processes ranging from virus production to the development of multicellular organisms also use these pathways. To explore how cell signaling modulates MPR trafficking, we used high-throughput RNA interference (RNAi) to target the human kinome and phosphatome. Using high-content image analysis, we identified 127 kinases and phosphatases belonging to different signaling networks that regulate MPR trafficking and/or the dynamic states of the subcellular compartments encountered by the MPRs. Our analysis maps the MPR trafficking pathways based on enzymes regulating phosphatidylinositol phosphate metabolism. Furthermore, it reveals how cell signaling controls the biogenesis of post-Golgi tubular carriers destined to enter the endosomal system through a SRC-dependent pathway regulating ARF1 and RAC1 signaling and myosin II activity.

High-content functional genomic screening to identify novel regulators of the PINK1-Parkin pathway.

PINK1/PARK6 and Parkin/PARK2 are amongst the most commonly mutated genes associated with recessive forms of familial Parkinson's disease. Recent evidence indicates that the proteins they encode, PINK1 and Parkin, function in the same pathway to mediate the selective autophagic clearance of dysfunctional mitochondria. Upon mitochondrial damage, PINK1 is stabilized on the outer mitochondrial membrane where it phosphorylates ubiquitin, generating a signal for the recruitment and activation of Parkin. However, key mechanistic questions still exist regarding Parkin recruitment, including whether or not other factors are required for the PINK1 and Parkin pathway. We describe a method below using high-throughput RNA interference technology to interrogate the genome for novel components of the PINK1 and Parkin pathway.

A novel phenotypic dissimilarity method for image-based high-throughput screens

BackgroundDiscovering functional relationships of genes through cell-based phenotyping has become an important approach in functional genomics. High-throughput imaging offers the ability to quantitatively assess complex phenotypes after perturbation by RNA interference (RNAi). Such image-based high-throughput RNAi screening studies have facilitated the discovery of novel components of gene networks and their interactions. Images generated by automated microscopy are typically analyzed by extracting quantitative features of individual cells, resulting in large multidimensional data sets. Robust and sensitive methods to interpret these data sets and to derive biologically relevant information in a high-throughput and unbiased manner remain to be developed.ResultsHere we propose a new analysis method, PhenoDissim, which computes the phenotypic dissimilarity between cell populations via Support Vector Machine classification and cross validation. Applying this method to a kinome RNAi screening data set, we demonstrate that the proposed method shows a good replicate reproducibility, separation of controls and clustering quality, and we are able to identify siRNA phenotypes and discover potential functional links between genes.ConclusionsPhenoDissim is a novel analysis method for image-based high-throughput screen, relying on two parameters which can be automatically optimized without a priori knowledge. PhenoDissim is freely available as an R package.

PubChem BioAssay: 2014 update

PubChem’s BioAssay database (http://pubchem.ncbi.nlm.nih.gov) is a public repository for archiving biological tests of small molecules generated through high-throughput screening experiments, medicinal chemistry studies, chemical biology research and drug discovery programs. In addition, the BioAssay database contains data from high-throughput RNA interference screening aimed at identifying critical genes responsible for a biological process or disease condition. The mission of PubChem is to serve the community by providing free and easy access to all deposited data. To this end, PubChem BioAssay is integrated into the National Center for Biotechnology Information retrieval system, making them searchable by Entrez queries and cross-linked to other biomedical information archived at National Center for Biotechnology Information. Moreover, PubChem BioAssay provides web-based and programmatic tools allowing users to search, access and analyze bioassay test results and metadata. In this work, we provide an update for the PubChem BioAssay resource, such as information content growth, new developments supporting data integration and search, and the recently deployed PubChem Upload to streamline chemical structure and bioassay submissions.

Metabolic and Functional Genomic Studies Identify Deoxythymidylate Kinase as a Target in LKB1-Mutant Lung Cancer

The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase, which coordinates cell growth, polarity, motility, and metabolism. In non-small cell lung carcinoma, LKB1 is somatically inactivated in 25% to 30% of cases, often concurrently with activating KRAS mutations. Here, we used an integrative approach to define novel therapeutic targets in KRAS-driven LKB1-mutant lung cancers. High-throughput RNA interference screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase (DTYMK), which catalyzes dTTP biosynthesis, as synthetically lethal with Lkb1 deficiency in mouse and human lung cancer lines. Global metabolite profiling showed that Lkb1-null cells had a striking decrease in multiple nucleotide metabolites as compared with the Lkb1-wild-type cells. Thus, LKB1-mutant lung cancers have deficits in nucleotide metabolism that confer hypersensitivity to DTYMK inhibition, suggesting that DTYMK is a potential therapeutic target in this aggressive subset of tumors.

PPP2R2C Loss Promotes Castration-Resistance and Is Associated with Increased Prostate Cancer-Specific Mortality

Metastatic prostate cancers generally rely on androgen receptor (AR) signaling for growth and survival, even following systemic androgen-deprivation therapy (ADT). However, recent evidence suggests that some advanced prostate cancers escape ADT by using signaling programs and growth factors that bypass canonical AR ligand-mediated mechanisms. We used an in vitro high-throughput RNA interference (RNAi) screen to identify pathways in androgen-dependent prostate cancer cell lines whose loss-of-function promotes androgen ligand-independent growth. We identified 40 genes where knockdown promoted proliferation of both LNCaP and VCaP prostate cancer cells in the absence of androgen. Of these, 14 were downregulated in primary and metastatic prostate cancer, including two subunits of the protein phosphatase 2 (PP2A) holoenzyme complex: PPP2R1A, a structural subunit with known tumor-suppressor properties in several tumor types; and PPP2R2C, a PP2A substrate-binding regulatory subunit that has not been previously identified as a tumor suppressor. We show that loss of PPP2R2C promotes androgen ligand depletion-resistant prostate cancer growth without altering AR expression or canonical AR-regulated gene expression. Furthermore, cell proliferation induced by PPP2R2C loss was not inhibited by the AR antagonist MDV3100, indicating that PPP2R2C loss may promote growth independently of known AR-mediated transcriptional programs. Immunohistochemical analysis of PPP2R2C protein levels in primary prostate tumors determined that low PPP2R2C expression significantly associated with an increased likelihood of cancer recurrence and cancer-specific mortality. These findings provide insights into mechanisms by which prostate cancers resist AR-pathway suppression and support inhibiting PPP2R2C complexes or the growth pathway(s) activated by PPP2R2C as a therapeutic strategy.

Highlights of This Issue

Cancer cells undergo a well-described metabolic reprogramming that is important for tumorigenesis. However, metabolic reprogramming in non-epithelial cells residing in the tumor microenvironment has not been examined in detail. Chaudhri and colleagues performed comprehensive mass-spectrometry–based metabolic profiling to uncover several metabolic pathways that distinguish cancer-associated fibroblasts (CAF) from adjacent “normal” fibroblasts as well as distinguishing CAFs from high- versus low-glycolytic tumors. One such change was an increase in dipeptides from CAFs, which was positively correlated with the glycolytic activity of their parent tumor. This increase in dipeptides was associated with upregulated basal macroautophagy, indicating that augmented catabolism was responsible for the increase in dipeptides. These results demonstrate that metabolic reprogramming and increased macroautophagy are not limited to cancer cells of the tumor, thereby establishing the importance of understanding the role of this metabolic reprogramming in the protumorigenic activities of CAFs.Prostate cancer, including metastatic disease, is largely dependent on androgen receptor (AR) signaling. Despite initially effective androgen deprivation therapies (ADT), disease recurrence is common and may involve circumventing AR ligand-mediated pathways. To test this hypothesis, Bluemn and colleagues used a high-throughput RNA interference screening approach to identify 40 high-confidence targets that, when lost, drive growth under conditions that mimic ADT. Two components (PPP2R: 1A and 2C) of the protein phosphatase 2 (PP2A) complex were shown to be downregulated as a function of disease progression. Additional investigation revealed that PPP2R2C loss was sufficient to promote bypass of enzalutamide-based ADT, and immunohistochemical stratification of PPP2R2C levels in patient specimens indicated a significant association between low PPP2R2C levels and increased recurrence and mortality. The results of this study not only define PPP2R2C as a new player in the mechanisms of ADT resistance but also suggest prognostic and therapeutic potential.The cytokine mda-7/interleukin (IL)-24 has been suggested to function as a tumor suppressor in a number of solid tumor types and to promote cancer-specific cell death in a context-dependent manner. Li and colleagues reveal that mammary tumorigenesis in rats was suppressed by single administration of an adenovirus-expressing mda-7/IL-24. Mechanistically, mda-7/IL-24 induced the expression of growth arrest–specific gene 3 (GAS3/PMP22) to inhibit the attachment and proliferation of tumor cells in part by blocking the interaction of β1-integrin with fibronectin. Using primary human breast cancer specimens, they found a positive correlation between the expression of the IL-24 receptor (IL20RA) and GAS3. Moreover, IL20RA expression was correlated with metastasis-free survival in HER2+ patient samples. Thus, a novel GAS3-integrin/fibronectin inhibitory pathway has been identified downstream of mda-7/IL-24 that can be targeted for therapy.To understand the pathogenesis of lung cancer and identify new therapeutic targets, it is crucial to develop new preclinical model systems. Sato and colleagues demonstrate that nonmalignant CDK4/hTERT–immortalized human bronchial epithelial cells (HBEC) can be progressed to fully malignant cells capable of in vivo tumor formation, representing the gamut of non–small cell lung cancer (NSCLC) histologies, following the introduction of defined oncogenic alterations (oncogenic KRAS, p53 knockdown, and c-MYC overexpression) that mimic genetic alterations commonly found in NSCLC. Genomic data show that human lung cancers exhibit a large number of sequence-altering mutations that require sorting out of “passenger” and “driver” changes. Therefore, the HBEC model provides a mechanism to test these candidates and identify the minimal, most crucial set of alterations required for full malignant transformation of lung epithelial cells.

Abstract A37: Cross-cancer evaluation of MYC synthetic lethal targets in The Cancer Genome Atlas

Abstract Overview: Synthetic lethality in cancer has been investigated for the last decade, but only recently do we have the convergence of high throughput RNA interference screening capabilities with large genomic and molecular data sets derived from primary tumor samples that allows for more comprehensive characterization of putative therapeutic targets against multiple cancers. Accurately relating cancer cell line RNA interference synthetic lethal screening data to primary human tumors has proven to be challenging, yet critically important, for improving validation rates and therapeutic efficacy. To this end, we have combined siRNA synthetic lethal screening hits in MYC amplified cell lines [Toyoshima et al. PNAS 2012] with genomic and molecular data from twelve tumor types in The Cancer Genome Atlas (TCGA) to identify and prioritize cross-cancer therapeutic targets with direct evidence for synthetic lethal relationships in both cell line and primary tumor samples. Approach: In order to relate TCGA data to synthetic lethal siRNA screening in cell lines, we began by integrating all data types produced by TCGA, including: clinical data, sample characteristics, somatic mutations, gene expression, DNA methylation, miRNA expression, reverse-phase protein array data, and somatic copy number variation. From these data, feature matrices have been constructed which combine all available information regarding patients and samples for each given tumor type. For each such heterogeneous feature matrix, we have performed statistical analysis to infer associations in the data between MYC amplification and all other molecular features. Hits from MYC synthetic lethal cell line screening were evaluated for statistical significance in pairwise analysis of twelve TCGA tumor types, classes of pairwise data-derived associations as well as properties of network-level topologies were characterized, and relationships between genomic data and clinical features were investigated where possible. Results: Evidence for synthetic lethal relationships between MYC amplification and molecular features (e.g., gene expression and DNA methylation) for numerous experimentally validates hits was present across multiple TCGA tumor types. For example, statistically significant associations between MYC amplification and decreased CSNK1E methylation, indicative of increased expression, were present in head and neck squamous cell carcinoma, cutaneous melanoma, and stomach adenocarcinoma, corroborating the experimental findings of Toyoshima et al. Similarly, PES1 gene expression is increased in breast carcinoma, colon adenocarcinoma, and head and neck squamous cell carcinoma samples with MYC amplification. Synthetic lethal hits had differential expression profiles reflective of both increases and decreases in correlation with MYC amplification. Based on these preliminary findings, the intersection of siRNA cell line screening hits with accompanying statistical associations in primary tumor sample data may enable prioritization of preclinical models of MYC driven cancers. While the present approach has been applied to MYC amplification, synthetic lethal relationships are present for a multitude of significantly mutated, copy number aberrated, or epigenetically silenced genes in TCGA. Citation Format: Brady Bernard, Silvia Cermelli, Carla Grandori, Ilya Shmulevich. Cross-cancer evaluation of MYC synthetic lethal targets in The Cancer Genome Atlas. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A37.

High-Throughput RNA Interference Screening: Tricks of the Trade

The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater. HTS assays are usually evaluated using Z' factor, which incorporates both standard deviation and signal window. A Z' factor value of 0.5 or higher is acceptable for HTS. We used a standard HTS validation procedure in developing small interfering RNA (siRNA) screening technology at the HTS center at Southern Research. Initially, our assay performance was similar to published screens, with CV values greater than 10% and Z' factor values of 0.51 ± 0.16 (average ± standard deviation). After optimizing the siRNA assay, we got CV values averaging 7.2% and a robust Z' factor value of 0.78 ± 0.06 (average ± standard deviation). We present an overview of the problems encountered in developing this whole-genome siRNA screening program at Southern Research and how equipment optimization led to improved data quality.

Akt-Mediated Phosphorylation of Argonaute 2 Downregulates Cleavage and Upregulates Translational Repression of MicroRNA Targets

A high-throughput RNA interference (RNAi) screen targeting 542 genes of the human kinome was usedto discover regulators of RNAi. Here we report that the proto-oncogene Akt-3/PKBγ (Akt3) phosphorylates Argonaute 2 (Ago2) at S387, which downregulates cleavage and upregulates translational repression of endogenous microRNA (miRNA)-targeted messenger RNAs (mRNAs). We further demonstrate that Akt3 coimmunoprecipitates with Ago2 and phosphorylation of Ago2 at S387 facilitates its interaction with GW182 and localization to cytoplasmic processing bodies (P bodies), where miRNA-targeted mRNAs are thought to be stored and degraded. Therefore, Akt3-mediated phosphorylation of Ago2 is a molecular switch between target mRNA cleavage and translational repression activities of Ago2.

Abstract 3765: High-throughput RNAi screening identifies genes controlling glioblastoma stem cell migration and survival.

Abstract Glioblastoma is incurable. Glioblastoma stem cells (GSC) became a model for explaining tumor recurrence due to their tumorigenic capacity, migratory nature, and radio-chemoresistant phenotype. To prevent tumor recurrence, a strategy targeting of GSC must be identified and added into the treatment. To identify vital genes that promote GSC migration and sustain self-renewal and anti-apoptotic features we have established tumorigenic CD133+ GSC lines (n=3 patients), which are capable of clonal self-renewal and asymmetric division for producing fast-growing CD133- daughter cells that form tumor spheres. CD133+ GSC can migrate outward from primary spheres and form spheres again, which provide an visible, functional model for identifying essential genes of CD133+GSC via loss-of-function RNA interference (RNAi) screen. To identify genes and pathways that confer the migration nature and super longevity of GSC, we constructed a siRNA library consisting of siRNA clones targeting 2079 genes overexpressed in tumorigenic CD133+ GSC when compared to non-tumorigenic, autologous tumor cell lines, normal brain tissue, human embryonic stem cells, normal neural stem cells, fetal neural progenitor cells and glioblastoma tumors, respectively, as determined by the comparative expression microarray analysis (E/B>2X, E-B>100, p value<0.05). An automated high-throughput siRNA screen was performed in 384-well plates using a reverse transfection procedures and the functional consequence were scored in 3 categories: score 1- degree of cell migration and size of tumor spheres were slightly decreased, score 2- cell migration was interrupted, and score 3-cell apoptosis was detected thereby no tumor spheres were formed. The siRNA targeting endothelin 3 served as positive siRNA control, which suppress GSC migration and induction of GSC apoptosis. Our primary screen has identified 193 genes whose inhibition by RNAi had led to impaired cell migration while knockdown of 128 genes led to the cell apoptosis and loss of self-repopulating capacity. Overlapped gene functions in regulation of both GSC migration and survival were detected, and genes which have never been reported to be associated with cell migration and cell survival were identified. Genes that maintain GSC survival and self-renewal capacity (score 3) mainly include those with roles in stemness maintenance, tumor suppressor/antigrowth/antidifferentiation, DNA repair, cell cycle checkpoint, While genes involved in GSC migration mainly include those with a role in cell adhesion, cell spreading, chemotaxis, axon guidance, neural crest cell migration. A high confidence gene list will be presented in the meeting. The development of an innovative therapy targeting essential genes of GSC that promote cell migration and/or cell survival could attenuate the infiltrating nature of GBM tumor, prolong treatment sensitivity, and prevent tumor recurrence. Citation Format: Yue Liu, Yibei Zhang, Jonathan L. Tso, Jimmy C. Menjivar, Jane Y. Tian, Linda Liau, William McBride, Cho-Lea Tso. High-throughput RNAi screening identifies genes controlling glioblastoma stem cell migration and survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3765. doi:10.1158/1538-7445.AM2013-3765

Abstract 3151: High-throughput RNAi screening identifies a novel metastatic melanoma drug target in the context of PTEN mutation.

Abstract It has become increasingly important to identify targeted therapy in a defined molecular context. Here we will present our data which suggests TERT, a telomerase reverse transcriptase, serves as a novel drug target in melanoma in the context of PTEN mutation. PTEN is mutated in ∼15% of melanoma patients. Genomic RNA-interference (RNAi) technology was applied for synthetic lethality screens in an effort to identify the targets which upon knock-down significantly decrease cell viability specifically in the context of somatic mutations of the PTEN gene. We carried out such screen in a panel of 15 melanoma cell lines, including four PTEN mutated cell lines, against an siRNA library covering the kinome (∼700 genes with 4 siRNA per target) using CellTiter Glo as the endpoint to measure cell viability. Lethal genes, which decreased cell viability significantly upon knockdown, were initially identified for each cell line. Then the hit lists were compared between wild-type PTEN and mutant PTEN using a chi-square test, a statistical analysis. 29 gene candidates were selected for hit confirmation using the same set of siRNA, among which 24 genes were confirmed as potential PTEN synthetic lethal candidates, this results in a high confirmation rate of 83%. Based upon its association with melanoma risk (Law et al, J Invest Dermatol, 2012), We further validated TERT using a commercially available small molecule inhibitor. This inhibitor has significantly higherpotency in the PTEN mutant context (average IC50 of 0.34uM) compared to PTEN wild-type background (average IC50 of 0.75uM). This result both validates our RNAi screening results, but more importantly, this compound can serve as an initial structure scaffold for further optimization towards development as a novel drug for metastatic melanoma patients who carry PTEN mutations. Evaluation of more TERT inhibitors in a diverse panel of melanoma cell lines is presently underway. Citation Format: Holly Yin, Donald Chow, Chris Sereduk, Meraj Aziz, Seungchan Kim, Kevin M. Brown, Jeffrey M. Trent. High-throughput RNAi screening identifies a novel metastatic melanoma drug target in the context of PTEN mutation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3151. doi:10.1158/1538-7445.AM2013-3151

Expanding the Diversity of Imaging-Based RNAi Screen Applications Using Cell Spot Microarrays.

Over the past decade, great strides have been made in identifying gene aberrations and deregulated pathways that are associated with specific disease states. These association studies guide experimental studies aimed at identifying the aberrant genes and networks that cause the disease states. This requires functional manipulation of these genes and networks in laboratory models of normal and diseased cells. One approach is to assess molecular and biological responses to high-throughput RNA interference (RNAi)-induced gene knockdown. These responses can be revealed by immunofluorescent staining for a molecular or cellular process of interest and quantified using fluorescence image analysis. These applications are typically performed in multiwell format, but are limited by high reagent costs and long plate processing times. These limitations can be mitigated by analyzing cells grown in cell spot microarray (CSMA) format. CSMAs are produced by growing cells on small (~200 μm diameter) spots with each spot carrying an siRNA with transfection reagent. The spacing between spots is only a few hundred micrometers, thus thousands of cell spots can be arranged on a single cell culture surface. These high-density cell cultures can be immunofluorescently stained with minimal reagent consumption and analyzed quickly using automated fluorescence microscopy platforms. This review covers basic aspects of imaging-based CSMA technology, describes a wide range of immunofluorescence assays that have already been implemented successfully for CSMA screening and suggests future directions for advanced RNAi screening experiments.

Combining integrated genomics and functional genomics to dissect the biology of a cancer‐associated, aberrant transcription factor, the ASPSCR1–TFE3 fusion oncoprotein

Oncogenic rearrangements of the TFE3 transcription factor gene are found in two distinct human cancers. These include ASPSCR1-TFE3 in all cases of alveolar soft part sarcoma (ASPS) and ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3 and others in a subset of paediatric and adult RCCs. Here we examined the functional properties of the ASPSCR1-TFE3 fusion oncoprotein, defined its target promoters on a genome-wide basis and performed a high-throughput RNA interference screen to identify which of its transcriptional targets contribute to cancer cell proliferation. We first confirmed that ASPSCR1-TFE3 has a predominantly nuclear localization and functions as a stronger transactivator than native TFE3. Genome-wide location analysis performed on the FU-UR-1 cell line, which expresses endogenous ASPSCR1-TFE3, identified 2193 genes bound by ASPSCR1-TFE3. Integration of these data with expression profiles of ASPS tumour samples and inducible cell lines expressing ASPSCR1-TFE3 defined a subset of 332 genes as putative up-regulated direct targets of ASPSCR1-TFE3, including MET (a previously known target gene) and 64 genes as down-regulated targets of ASPSCR1-TFE3. As validation of this approach to identify genuine ASPSCR1-TFE3 target genes, two up-regulated genes bound by ASPSCR1-TFE3, CYP17A1 and UPP1, were shown by multiple lines of evidence to be direct, endogenous targets of transactivation by ASPSCR1-TFE3. As the results indicated that ASPSCR1-TFE3 functions predominantly as a strong transcriptional activator, we hypothesized that a subset of its up-regulated direct targets mediate its oncogenic properties. We therefore chose 130 of these up-regulated direct target genes to study in high-throughput RNAi screens, using FU-UR-1 cells. In addition to MET, we provide evidence that 11 other ASPSCR1-TFE3 target genes contribute to the growth of ASPSCR1-TFE3-positive cells. Our data suggest new therapeutic possibilities for cancers driven by TFE3 fusions. More generally, this work establishes a combined integrated genomics/functional genomics strategy to dissect the biology of oncogenic, chimeric transcription factors.

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.

High-throughput RNA interference screens integrative analysis: Towards a comprehensive understanding of the virus-host interplay

Viruses are extremely heterogeneous entities; the size and the nature of their genetic information, as well as the strategies employed to amplify and propagate their genomes, are highly variable. However, as obligatory intracellular parasites, replication of all viruses relies on the host cell. Having co-evolved with their host for several million years, viruses have developed very sophisticated strategies to hijack cellular factors that promote virus uptake, replication, and spread. Identification of host cell factors (HCFs) required for these processes is a major challenge for researchers, but it enables the identification of new, highly selective targets for anti viral therapeutics. To this end, the establishment of platforms enabling genome-wide high-throughput RNA interference (HT-RNAi) screens has led to the identification of several key factors involved in the viral life cycle. A number of genome-wide HT-RNAi screens have been performed for major human pathogens. These studies enable first inter-viral comparisons related to HCF requirements. Although several cellular functions appear to be uniformly required for the life cycle of most viruses tested (such as the proteasome and the Golgi-mediated secretory pathways), some factors, like the lipid kinase Phosphatidylinositol 4-kinase IIIα in the case of hepatitis C virus, are selectively required for individual viruses. However, despite the amount of data available, we are still far away from a comprehensive understanding of the interplay between viruses and host factors. Major limitations towards this goal are the low sensitivity and specificity of such screens, resulting in limited overlap between different screens performed with the same virus. This review focuses on how statistical and bioinformatic analysis methods applied to HT-RNAi screens can help overcoming these issues thus increasing the reliability and impact of such studies.

Analysis of small-sample clinical genomics studies using multi-parameter shrinkage: application to high-throughput RNA interference screening

High-throughput (HT) RNA interference (RNAi) screens are increasingly used for reverse genetics and drug discovery. These experiments are laborious and costly, hence sample sizes are often very small. Powerful statistical techniques to detect siRNAs that potentially enhance treatment are currently lacking, because they do not optimally use the amount of data in the other dimension, the feature dimension.We introduce ShrinkHT, a Bayesian method for shrinking multiple parameters in a statistical model, where 'shrinkage' refers to borrowing information across features. ShrinkHT is very flexible in fitting the effect size distribution for the main parameter of interest, thereby accommodating skewness that naturally occurs when siRNAs are compared with controls. In addition, it naturally down-weights the impact of nuisance parameters (e.g. assay-specific effects) when these tend to have little effects across siRNAs. We show that these properties lead to better ROC-curves than with the popular limma software. Moreover, in a 3 + 3 treatment vs control experiment with 'assay' as an additional nuisance factor, ShrinkHT is able to detect three (out of 960) significant siRNAs with stronger enhancement effects than the positive control. These were not detected by limma. In the context of gene-targeted (conjugate) treatment, these are interesting candidates for further research.

Antiviral Stratagems against HIV-1 Using RNA Interference (RNAi) Technology

The versatility of human immunodeficiency virus (HIV)-1 and its evolutionary potential to elude antiretroviral agents by mutating may be its most invincible weapon. Viruses, including HIV, in order to adapt and survive in their environment evolve at extremely fast rates. Given that conventional approaches which have been applied against HIV have failed, novel and more promising approaches must be employed. Recent studies advocate RNA interference (RNAi) as a promising therapeutic tool against HIV. In this regard, targeting multiple HIV sites in the context of a combinatorial RNAi-based approach may efficiently stop viral propagation at an early stage. Moreover, large high-throughput RNAi screens are widely used in the fields of drug development and reverse genetics. Computer-based algorithms, bioinformatics, and biostatistical approaches have been employed in traditional medicinal chemistry discovery protocols for low molecular weight compounds. However, the diversity and complexity of RNAi screens cannot be efficiently addressed by these outdated approaches. Herein, a series of novel workflows for both wet- and dry-lab strategies are presented in an effort to provide an updated review of state-of-the-art RNAi technologies, which may enable adequate progress in the fight against the HIV-1 virus.

Microfluidic platforms for RNA interference screening of virus–host interactions

RNA interference (RNAi) is a powerful tool for functional genomics with the capacity to comprehensively analyze host-pathogen interactions. High-throughput RNAi screening is used to systematically perturb cellular pathways and discover therapeutic targets, but the method can be tedious and requires extensive capital equipment and expensive reagents. To aid in the development of an inexpensive miniaturized RNAi screening platform, we have developed a two part microfluidic system for patterning and screening gene targets on-chip to examine cellular pathways involved in virus entry and infection. First, a multilayer polydimethylsiloxane (PDMS)-based spotting device was used to array siRNA molecules into 96 microwells targeting markers of endocytosis, along with siRNA controls. By using a PDMS-based spotting device, we remove the need for a microarray printer necessary to perform previously described small scale (e.g. cellular microarrays) and microchip-based RNAi screening, while still minimizing reagent usage tenfold compared to conventional screening. Second, the siRNA spotted array was transferred to a reversibly sealed PDMS-based screening platform containing microchannels designed to enable efficient cell loading and transfection of mammalian cells while preventing cross-contamination between experimental conditions. Validation of the screening platform was examined using Vesicular stomatitis virus and emerging pathogen Rift Valley fever virus, which demonstrated virus entry pathways of clathrin-mediated endocytosis and caveolae-mediated endocytosis, respectively. The techniques here are adaptable to other well-characterized infection pathways with a potential for large scale screening in high containment biosafety laboratories.