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Reinstating wild-type tumor suppressor p53 activity could be a valuable option for the treatment of cancer. To contribute to development of new treatment options for non-small cell lung cancer ( NSCLC), we performed genome-wide si RNA screens for determinants of p53 activity in NSCLC cells. We identified many genes not previously known to be involved in regulating p53 activity. Silencing p53 pathway inhibitor genes was associated with loss of cell viability. The largest functional gene cluster influencing p53 activity was mRNA splicing. Prominent p53 activation was observed upon silencing of specific spliceosome components, rather than by general inhibition of the spliceosome. Ten genes were validated as inhibitors of p53 activity in multiple NSCLC cell lines: genes encoding the Ras pathway activator SOS1, the zinc finger protein TSHZ3, the mitochondrial membrane protein COX16, and the spliceosome components SNRPD3, SF3A3, SF3B1, SF3B6, XAB2, CWC22, and HNRNPL. Silencing these genes generally increased p53 levels, with distinct effects on CDKN1A expression, induction of cell cycle arrest and cell death. Silencing spliceosome components was associated with alternative splicing of MDM4 mRNA, which could contribute to activation of p53. In addition, silencing splice factors was particularly effective in killing NSCLC cells, albeit in a p53-independent manner. Interestingly, silencing SNRPD3 and SF3A3 exerted much stronger cytotoxicity to NSCLC cells than to lung fibroblasts, suggesting that these genes could represent useful therapeutic targets. [ABSTRACT FROM AUTHOR]

Viruses and their hosts can reach balanced states of evolution ensuring mutual survival, which makes it difficult to appreciate the underlying dynamics. To uncover hidden interactions, virus mutants that have lost defense genes may be used. Deletion of the gene that encodes serine protease inhibitor 1 (SPI-1) of rabbit-pox virus and vaccinia virus, two closely related orthopoxviruses, prevents their efficient replication in human cells, whereas certain other mammalian cells remain fully permissive. Our high-throughput genome-wide siRNA screen identified host factors that prevent reproduction and spread of the mutant viruses in human cells. More than 20,000 genes were interrogated with individual siRNAs and those that prominently increased replication of the SPI- 1 deletion mutant were subjected to a secondary screen. The top hits based on the combined data--replication factor C3 (RFC3), FAM111A, and interferon regulatory factor 2 (IRF2)--were confirmed by custom assays. The siRNAs to RFC1, RFC2, RFC4, and RFC5 mRNAs also enhanced spread of the mutant virus, strengthening the biological significance of the RFC complex as a host restriction factor for poxviruses. Whereas association with proliferating cell nuclear antigen and participation in processive genome replication are common features of FAM111A and RFC, IRF2 is a transcriptional regulator. Microarray analysis, quantitative RT-PCR, and immunoblotting revealed that IRF2 regulated the basal level expression of FAM111A, suggesting that the enhancing effect of depleting IRF2 on replication of the SPI-1 mutant was indirect. Thus, the viral SPI-1 protein and the host IRF2, FAM111A, and RFC complex likely form an interaction network that influences the ability of poxviruses to replicate in human cells. [ABSTRACT FROM AUTHOR]

The overwhelming majority of pancreatic ductal adenocarcinoma (PDAC) is not diagnosed until the cancer has metastasized, leading to an abysmal average life expectancy (3-6 months post-diagnosis). Earlier detection and more effective treatments have been hampered by inadequate understanding of the underlying molecular mechanisms controlling metastasis. We hypothesized that metastasis suppressors are involved in controlling metastasis in pancreatic cancer. Using an unbiased genome-wide shRNA screen, an shRNA library was transduced into the non-metastatic PDAC line S2-028 followed by intrasplenic injection. Resulting liver metastases were individually isolated from these mice. One liver metastatic nodule contained shRNA for ITIH5 (Inter-alpha-trypsin inhibitor heavy chain 5), suggesting that ITIH5 may act as a metastasis suppressor. Consistent with this notion, metastatic PDAC cell lines had significantly lower protein expression of ITIH5 compared to immortalized pancreatic ductal epithelial cells and non-/poorly-metastatic PDAC cell lines. By manipulating expression of ITIH5 in different PDAC cell lines (over-expression in metastatic, knockdown in non-metastatic) functional and selective regulation of metastasis was observed for ITIH5. Orthotopic tumor growth of PDAC cells was not blocked following orthotopic injection. In vitro ITIH5 over-expression inhibited motility and invasion. Immunohistochemical analysis of a human PDAC tissue microarray revealed that ITIH5 expression inversely correlated with both survival and invasion/metastasis. ITIH5 is, therefore, functionally validated as a PDAC metastasis suppressor and shows promise as a prognostic biomarker. [ABSTRACT FROM AUTHOR]

Summary The degree of genetic aberrations characteristic of high-grade serous ovarian cancer (HGSC) makes identification of the molecular features that drive tumor progression difficult. Here, we perform genome-wide RNAi screens and comprehensive expression analysis of cell-surface markers in a panel of HGSC cell lines to identify genes that are critical to their survival. We report that the tetraspanin CD151 contributes to survival of a subset of HGSC cell lines associated with a ZEB transcriptional program and supports the growth of HGSC tumors. Moreover, we show that high CD151 expression is prognostic of poor clinical outcome. This study reveals cell-surface vulnerabilities associated with HGSC, provides a framework for identifying therapeutic targets, and reports a role for CD151 in HGSC. [ABSTRACT FROM AUTHOR]

Proteostasis is of vital importance for cellular function and it is challenged upon exposure to acute or chronic insults during neurodegeneration and aging. The proteostasis network is relevant for the maintenance of proteome integrity and mainly comprises molecular chaperones and two degradation pathways, namely, autophagy and the ubiquitin proteasome system. This network is characterized by an impressive functional interrelation and complexity, and occasionally novel factors are discovered that modulate proteostasis. Here, we present an RNAi screen in C. elegans, which aimed to identify modulators of proteostasis in a heat stress paradigm. The screen comprised genes that are located on chromosome I of the nematode and has identified 185 genetic modifiers, whose knockdown has enhanced the misfolding of a reporter protein upon temperature increase. Subsequently, we evaluated the effect of a distinct number of the identified candidates in an additional C. elegans model strain, which expresses the aggregation-prone PolyQ35::YFP protein. Moreover, we annotated the human orthologs of the identified proteins and analyzed their enrichment in functional clusters and, as appropriate, their association with human neuropathologies. The achieved data collection includes several factors that have already been functionally associated with the proteostasis network, which highlights the potential of this heat stress-based proteostasis screen in order to detect novel modulators of proteome integrity.

The development of targeted therapy for patients with Multiple Myeloma (MM) is hampered by the low frequency of actionable genetic abnormalities. Gain or amplification of chr1q (Amp1q) is the most frequent arm-level copy number gain in patients with MM, and it is associated with higher risk of progression and death despite recent advances in therapeutics. Thus, developing targeted therapy for patients with MM and Amp1q stands to benefit a large portion of patients in need of more effective management. Here, we employed large-scale dependency screens and drug screens to systematically characterize the therapeutic vulnerabilities of MM with Amp1q and showed increased sensitivity to the combination of MCL1 and PI3K inhibitors. Using single-cell RNA sequencing, we compared subclones with and without Amp1q within the same patient tumors and showed that Amp1q is associated with higher levels of MCL1 and the PI3K pathway. Furthermore, by isolating isogenic clones with different copy number for part of the chr1q arm, we showed increased sensitivity to MCL1 and PI3K inhibitors with arm-level gain. Lastly, we demonstrated synergy between MCL1 and PI3K inhibitors and dissected their mechanism of action in MM with Amp1q., Competing Interests: DECLARATION OF INTEREST E.D.L., M.R., J.T., M.P.A., D.H.M., D.H., S.J.F.C., L.H., N.J.H., T.W., N.K.S., B.B., J.A., and M.P. declare no competing interests. R.S.P. is a co-founder and equity holder in PreDICTA Biosciences. M.S.D. has received research funding from AbbVie, AstraZeneca, Ascentage Pharma, Genentech, MEI Pharma, Novartis, Surface Oncology, TG Therapeutics and personal consulting income from AbbVie, Adaptive Biosciences, Ascentage Pharma, AstraZeneca, BeiGene, BMS, Eli Lilly, Genentech, Genmab, Janssen, Merck, Mingsight Pharmaceuticals, Nuvalent, Secura Bio, TG Therapeutics, and Takeda. G.G. receives research funds from IBM, Pharmacyclics, and Ultima Genomics and is an inventor on patent applications filed by the Broad Institute related to MSMuTect, MSMutSig, POLYSOLVER, SignatureAnalyzer-GPU, MSIDetect, and MinumuMM-seq. He is also a founder, consultant, and holds privately held equity in Scorpion Therapeutics and PreDICTA Biosciences. I.M.G. has a consulting or advisory role with AbbVie, Adaptive, Amgen, Aptitude Health, Bristol Myers Squibb, GlaxoSmithKline, Huron Consulting, Janssen, Menarini Silicon Biosystems, Oncopeptides, Pfizer, Sanofi, Sognef, Takeda, The Binding Site, and Window Therapeutics; has received speaker fees from Vor Biopharma and Veeva Systems, Inc.; is a co-founder and equity holder in PreDICTA Biosciences; and her spouse is the CMO and equity holder of Disc Medicine. S.M. has a consulting role with Abbvie, Adaptive Biotechnology, Amgen, Celgene/BMS, GlaxoSmithKline, Janssen, Novartis, Oncopeptides, Regeneron, Roche, Takeda, and has received research funding from Abbvie, Adaptive Biotechnology, Amgen, Celgene/BMS, GlaxoSmithKline, Janssen, Novartis, Oncopeptides, Regeneron, Roche, Takeda.

The Hippo pathway is crucial for not only normal growth and apoptosis but also cell fate specification during development. What controls Hippo pathway activity during cell fate specification is incompletely understood. In this article, we identify the insulator protein BEAF-32 as a regulator of Hippo pathway activity in Drosophila photoreceptor differentiation. Though morphologically uniform, the fly eye is composed of two subtypes of R8 photoreceptor neurons defined by expression of light-detecting Rhodopsin proteins. In one R8 subtype, active Hippo signaling induces Rhodopsin 6 (Rh6) and represses Rhodopsin 5 (Rh5), whereas in the other subtype, inactive Hippo signaling induces Rh5 and represses Rh6. The activity state of the Hippo pathway in R8 cells is determined by the expression of warts, a core pathway kinase, which interacts with the growth regulator melted in a doublenegative feedback loop. We show that BEAF-32 is required for expression of warts and repression of melted. Furthermore, BEAF- 32 plays a second role downstream of Warts to induce Rh6 and prevent Rh5 fate. BEAF-32 is dispensable for Warts feedback, indicating that BEAF-32 differentially regulates warts and Rhodopsins. Loss of BEAF-32 does not noticeably impair the functions of the Hippo pathway in eye growth regulation. Our study identifies a context-specific regulator of Hippo pathway activity in post-mitotic neuronal fate, and reveals a developmentally specific role for a broadly expressed insulator protein. [ABSTRACT FROM AUTHOR]

Summary The flaviviruses dengue virus (DENV) and Zika virus (ZIKV) are severe health threats with rapidly expanding ranges. To identify the host cell dependencies of DENV and ZIKV, we completed orthologous functional genomic screens using RNAi and CRISPR/Cas9 approaches. The screens recovered the ZIKV entry factor AXL as well as multiple host factors involved in endocytosis (RAB5C and RABGEF), heparin sulfation (NDST1 and EXT1), and transmembrane protein processing and maturation, including the endoplasmic reticulum membrane complex (EMC). We find that both flaviviruses require the EMC for their early stages of infection. Together, these studies generate a high-confidence, systems-wide view of human-flavivirus interactions and provide insights into the role of the EMC in flavivirus replication. [ABSTRACT FROM AUTHOR]

Proper DNA replication is critical to maintain genome stability. When the DNA replication machinery encounters obstacles to replication, replication forks stall and the replication stress response is activated. This response includes activation of cell cycle checkpoints, stabilization of the replication fork, and DNA damage repair and tolerance mechanisms. Defects in the replication stress response can result in alterations to the DNA sequence causing changes in protein function and expression, ultimately leading to disease states such as cancer. To identify additional genes that control the replication stress response, we performed a three-parameter, high content, whole genome siRNA screen measuring DNA replication before and after a challenge with replication stress as well as a marker of checkpoint kinase signalling. We identified over 200 replication stress response genes and subsequently analyzed how they influence cellular viability in response to replication stress. These data will serve as a useful resource for understanding the replication stress response. [ABSTRACT FROM AUTHOR]

Genetic screens in Drosophila melanogaster and other organisms have been pursued to filter the genome for genetic functions important for memory formation. Such screens have employed primarily chemical or transposon-mediated mutagenesis and have identified numerous mutants including classical memory mutants, dunce and rutabaga. Here, we report the results of a large screen using panneuronal RNAi expression to identify additional genes critical for memory formation. We identified .500 genes that compromise memory when inhibited (low hits), either by disrupting the development and normal function of the adult animal or by participating in the neurophysiological mechanisms underlying memory formation. We also identified .40 genes that enhance memory when inhibited (high hits). The dunce gene was identified as one of the low hits and further experiments were performed to map the effects of the dunce RNAi to the a/b and g mushroom body neurons. Additional behavioral experiments suggest that dunce knockdown in the mushroom body neurons impairs memory without significantly affecting acquisition. We also characterized one high hit, sickie, to show that RNAi knockdown of this gene enhances memory through effects in dopaminergic neurons without apparent effects on acquisition. These studies further our understanding of two genes involved in memory formation, provide a valuable list of genes that impair memory that may be important for understanding the neurophysiology of memory or neurodevelopmental disorders, and offer a new resource of memory suppressor genes that will aid in understanding restraint mechanisms employed by the brain to optimize resources. [ABSTRACT FROM AUTHOR]

The success of T cell-based cancer immunotherapy is limited by tumor's resistance against killing by cytotoxic T lymphocytes (CTLs). Tumor-immune resistance is mediated by cell surface ligands that engage immune-inhibitory receptors on T cells. These ligands represent potent targets for therapeutic inhibition. So far, only few immune-suppressive ligands have been identified. We here describe a rapid high-throughput si RNA-based screening approach that allows a comprehensive identification of ligands on human cancer cells that inhibit CTL-mediated tumor cell killing. We exemplarily demonstrate that CCR9, which is expressed in many cancers, exerts strong immune-regulatory effects on T cell responses in multiple tumors. Unlike PDL1, which inhibits TCR signaling, CCR9 regulates STAT signaling in T cells, resulting in reduced T-helper-1 cytokine secretion and reduced cytotoxic capacity. Moreover, inhibition of CCR9 expression on tumor cells facilitated immunotherapy of human tumors by tumor-specific T cells in vivo. Taken together, this method allows a rapid and comprehensive determination of immune-modulatory genes in human tumors which, as an entity, represent the 'immune modulatome' of cancer. [ABSTRACT FROM AUTHOR]

Aging is associated with a decline in visual function and increased prevalence of ocular disease, correlating with changes in the transcriptome and epigenome of cells in the eye. Here, we sought to identify the transcriptional mechanisms that are necessary to maintain photoreceptor viability and function during aging. To do this, we performed a targeted photoreceptor-specific RNAi screen in Drosophila to identify transcriptional regulators whose knockdown results in premature, age-dependent retinal degeneration. From an initial set of 155 RNAi lines each targeting a unique gene and spanning a diverse set of transcription factors, chromatin remodelers, and histone modifiers, we identified 18 high-confidence target genes whose decreased expression in adult photoreceptors leads to premature and progressive retinal degeneration. These 18 target genes were enriched for factors involved in the regulation of transcription initiation, pausing, and elongation, suggesting that these processes are essential for maintaining the health of aging photoreceptors. To identify the genes regulated by these factors, we profiled the photoreceptor transcriptome in a subset of lines. Strikingly, two of the 18 target genes, Spt5 and domino , show similar changes in gene expression to those observed in photoreceptors with advanced age. Together, our data suggest that dysregulation of factors involved in transcription initiation and elongation plays a key role in shaping the transcriptome of aging photoreceptors. Further, our findings indicate that the age-dependent changes in gene expression not only correlate but might also contribute to an increased risk of retinal degeneration., Competing Interests: Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs harboring premature termination codons (PTCs). We have conducted a genome-wide RNAi screen in Caenorhabditis elegans that resulted in the identification of five novel NMD genes that are conserved throughout evolution. Two of their human homologs, GNL2 ( ngp-1) and SEC13 ( npp-20), are also required for NMD in human cells. We also show that the C. elegans gene noah-2, which is present in Drosophila melanogaster but absent in humans, is an NMD factor in fruit flies. Altogether, these data identify novel NMD factors that are conserved throughout evolution, highlighting the complexity of the NMD pathway and suggesting that yet uncovered novel factors may act to regulate this process. [ABSTRACT FROM AUTHOR]

Background MYC family members are among the most frequently deregulated oncogenes in human cancers, yet direct therapeutic targeting of MYC in cancer has been challenging thus far. Synthetic lethality provides an opportunity for therapeutic intervention of MYC-driven cancers. Methods A pooled kinase shRNA library screen was performed and next-generation deep sequencing efforts identified PRKDC as synthetically lethal in cells overexpressing MYC. Genes and proteins of interest were knockdown or inhibited using RNAi technology and small molecule inhibitors, respectively. Quantitative RT-PCR using TaqMan probes examined total RNA levels and viability was assessed using CellTiter-Glo (Promega). Western blotting was performed to monitor different protein levels in the presence or absence of RNAi or compound treatment. Significant differences among data sets were determined using unpaired t test (Mann--Whitney test) or ANOVA. Results Inhibition of PRKDC using RNAi (RNA interference) or small molecular inhibitors preferentially killed MYC-overexpressing human lung fibroblasts. Moreover, inducible PRKDC knockdown decreased cell viability selectively in high MYC-expressing human small cell lung cancer cell lines. At the molecular level, we found that inhibition of PRKDC downregulated MYC mRNA and protein expression in multiple cancer cell lines. In addition, we confirmed that overexpression of MYC family proteins induced DNA double-strand breaks; our results also revealed that PRKDC inhibition in these cells led to an increase in DNA damage levels. Conclusions Our data suggest that the synthetic lethality between PRKDC and MYC may in part be due to PRKDC dependent modulation of MYC expression, as well as MYC-induced DNA damage where PRKDC plays a key role in DNA damage repair. [ABSTRACT FROM AUTHOR]

The use of molecularly targeted drugs as single agents has shown limited utility in many tumor types, largely due to the complex and redundant nature of oncogenic signaling networks. Targeting of the PI3K/AKT/mTOR pathway through inhibition of mTOR in combination with aromatase inhibitors has seen success in particular sub-types of breast cancer and there is a need to identify additional synergistic combinations to maximize the clinical potential of mTOR inhibitors. We have used loss-of-function RNAi screens of the mTOR inhibitor rapamycin to identify sensitizers of mTOR inhibition. RNAi screens conducted in combination with rapamycin in multiple breast cancer cell lines identified six genes, AURKB, PLK1, PIK3R1, MAPK12, PRKD2 , and PTK6 that when silenced, each enhanced the sensitivity of multiple breast cancer lines to rapamycin. Using selective pharmacological agents we confirmed that inhibition of AURKB or PLK1 synergizes with rapamycin. Compound-associated gene expression data suggested histone deacetylation (HDAC) inhibition as a strategy for reducing the expression of several of the rapamycin-sensitizing genes, and we tested and validated this using the HDAC inhibitor entinostat in vitro and in vivo . Our findings indicate new approaches for enhancing the efficacy of rapamycin including the use of combining its application with HDAC inhibition. [ABSTRACT FROM AUTHOR]

One-year survival rates for newly diagnosed hepatocellular carcinoma (HCC) are <50%, and unresectable HCC carries a dismal prognosis owing to its aggressiveness and the undruggable nature of its main genetic drivers. By screening a custom library of shRNAs directed toward known drug targets in a genetically defined Myc-driven HCC model, we identified cyclin-dependent kinase 9 (Cdk9) as required for disease maintenance. Pharmacological or shRNA-mediated CDK9 inhibition led to robust anti-tumor effects that correlated with MYC expression levels and depended on the role that both CDK9 and MYC exert in transcription elongation. Our results establish CDK9 inhibition as a therapeutic strategy for MYC-overexpressing liver tumors and highlight the relevance of transcription elongation in the addiction of cancer cells to MYC. [ABSTRACT FROM AUTHOR]

Rio kinases are atypical serine/threonine kinases that emerge as potential cooperation partners in Ras-driven tumors. In the current study, we performed an RNAi screen in Caenorhabditis elegans to identify suppressors of oncogenic Ras signaling. Aberrant Ras/Raf signaling in C. elegans leads to the formation of a multi-vulva (Muv) phenotype. We found that depletion of riok-1, the C. elegans orthologue of the mammalian RioK1, suppressed the Muv phenotype. By using a promoter GFP construct, we could show that riok-1 is expressed in neuronal cells, the somatic gonad, the vulva, the uterus and the spermatheca. Furthermore, we observed developmental defects in the gonad upon riok-1 knockdown in a wildtype background. Our data suggest that riok-1 is a modulator of the Ras signaling pathway, suggesting implications for novel interventions in the context of Ras-driven tumors. [ABSTRACT FROM AUTHOR]

Background: Pancreatic cancer is a deadly disease with a five-year survival of less than 5%. A better understanding of the underlying biology may suggest novel therapeutic targets. Recent surveys of the pancreatic cancer genome have uncovered numerous new alterations; yet systematic functional characterization of candidate cancer genes has lagged behind. To address this challenge, here we have devised a highly-parallel RNA interference-based functional screen to evaluate many genomically-nominated candidate pancreatic cancer genes simultaneously. Results: For 185 candidate pancreatic cancer genes, selected from recurrently altered genomic loci, we performed a pooled shRNA library screen of cell growth/viability across 10 different cell lines. Knockdown-associated effects on cell growth were assessed by enrichment or depletion of shRNA hairpins, by hybridization to barcode microarrays. A novel analytical approach (COrrelated Phenotypes for On-Target Effects; COPOTE) was used to discern probable on-target knockdown, based on identifying different shRNAs targeting the same gene and displaying concordant phenotypes across cell lines. Knockdown data were integrated with genomic architecture and gene-expression profiles, and selected findings validated using individual shRNAs and/or independent siRNAs. The pooled shRNA library design delivered reproducible data. In all, COPOTE analysis identified 52 probable on-target gene-knockdowns. Knockdown of known oncogenes (KRAS, MYC, SMURF1 and CCNE1) and a tumor suppressor (CDKN2A) showed the expected contrasting effects on cell growth. In addition, the screen corroborated purported roles of PLEKHG2 and MED29 as 19q13 amplicon drivers. Most notably, the analysis also revealed novel possible oncogenic functions of nucleoporin NUP153 (ostensibly by modulating TGFβ signaling) and Kruppel-like transcription factor KLF5 in pancreatic cancer. Conclusions: By integrating physical and functional genomic data, we were able to simultaneously evaluate many candidate pancreatic cancer genes. Our findings uncover new facets of pancreatic cancer biology, with possible therapeutic implications. More broadly, our study provides a general strategy for the efficient characterization of candidate genes emerging from cancer genome studies. [ABSTRACT FROM AUTHOR]

The large chromosomal deletions frequently observed in cancer genomes are often thought to arise as a "two-hit" mechanism in the process of tumor-suppressor gene (TSG) inactivation. Using a murine model system of hepatocellular carcinoma (HCC) and in vivo RNA1, we test an alternative hypothesis, that such deletions can arise from selective pressure to attenuate the activity of multiple genes. By targeting the mouse orthologs of genes frequently deleted on human 8p22 and adjacent regions, which are lost in approximately half of several other major epithelial cancers, we provide evidence suggesting that multiple genes on chromosome 8p can cooperatively inhibit tumorigenesis in mice, and that their cosuppression can synergistically promote tumor growth. In addition, in human HCC patients, the combined down-regulation of functionally validated 8p TSG5 is associated with poor survival, in contrast to the down-regulation of any individual gene. Our data imply that large cancer-associated deletions can produce phenotypes distinct from those arising through loss of a single TSG, and as such `should be considered and studied as distinct mutational events. [ABSTRACT FROM AUTHOR]

The discovery of RNA interference (RNAi) and the development of technologies exploiting its biology have enabled scientists to rapidly examine the consequences of depleting a particular gene product in a cell or an animal. The availability of genome-wide RNAi libraries targeting the mouse and human genomes has made it possible to carry out large scale, phenotype-based screens, which have yielded seminal information on diverse cellular processes ranging from virology to cancer biology. Today, several strategies are available to perform RNAi screens, each with their own technical and monetary considerations. Special care and budgeting must be taken into account during the design of these screens in order to obtain reliable results. In this review, we discuss a number of critical aspects to consider when planning an effective RNAi screening strategy, including selecting the right biological system, designing an appropriate selection scheme, optimizing technical aspects of the screen, and validating and verifying the hits. Similar to an artistic production, what happens behind the screen has a direct impact on its success. [ABSTRACT FROM AUTHOR]

We present two novel bioassays to be used in the examination of plant-parasitic nematode host-finding ability. The hostfinding 'pipette-bulb assay' was constructed from modelled Pasteur pipette bulbs and connecting barrels using parafilm fastenings. This assay examines the direction of second-stage juvenile (J2) migration in response to a host seedling, through a moistened sand substrate, which underlies terminal upward-facing 'seedling bulbs', one containing a host seedling in potting compost, the other with only potting compost. An equal watering regime through both upward-facing seedling bulbs creates a directional concentration gradient of host diffusate chemotactic factors. Positive chemotactic stimuli cause the J2 to orientate and migrate towards the host plant. We present validation data collected from assays of the root-knot nematode, Meloidogyne incognita, and the potato cyst nematode, Globodera pallida, which indicate a highly significant positive attraction of J2 of both species to respective host plants. This represents a simple, quick and inexpensive method of assessing host-finding behaviour in the laboratory. We consider that the pipette-bulb assay improves on previous host-finding/chemo-attraction assays through creating a more biologically relevant environment for experimental J2; analysis is quick and easy, allowing the straightforward interpretation of results. In addition, we have developed an 'agar trough' sensory assay variant which we believe can be used rapidly to ratify nematode responses to chemical gustatory or olfactory cues. This was constructed from a water agar substrate such that two counting wells were connected by a raised central trough, all flooded with water. Two small water agar plugs were dehydrated briefly in an oven and then hydrated in either an attractant, repellent or water control; these plugs were then placed in the terminal counting wells and subsequently leached the attractant or repellent to form a concentration gradient along the central trough, which contained the initial J2 innoculum. Our data show that both M. incognita and G. pallida J2 are positively attracted to host diffusates. In addition, they displayed a strong repulsion in response to 1 M NaCl2. J2 of M. incognita displayed a mild aversion to a non-host oak root diffusate, whereas G. pallida J2 displayed a strong aversion to the same non-host diffusate; neither species responded to a compost leachate. We believe that the agar trough assay improves on previous methods by facilitating rapid diffusion of attractant or repellents. Both of the aforementioned assays were designed as tools to assess the impact of RNAi-based reverse genetics screens for gene targets involved in chemosensory orientation. [ABSTRACT FROM AUTHOR]

JAK/STAT signaling pathway is evolutionarily conserved and tightly regulated. We carried out a reporter-based genome-wide RNAi in vitro screen to identify genes that regulate Drosophila JAK/STAT pathway and found 5 novel regulators. Of these, CG14225 is a negative regulator structurally related to the Drosophila JAK/STAT pathway receptor Domeless, especially in the extracellular domain, and to the mammalian IL-6 receptor and the signal transducer gp130. CG14225 coimmunoprecipitates with Domeless and its associated kinase hopscotch in S2 cells. CG14225 RNAi caused hyperphosphorylation of the transcription factor Stat92E in S2 cells on stimulation with the Drosophila JAK/STAT pathway ligand unpaired. CGI4225 RNAi in vivo hyperactivated JAK/STAT target genes on septic injury and enhanced unpaired-induced eye overgrowth, and was thus named the eye transformer (ET). In the gastrointestinal infection model, where JAK/STAT signaling is important for stem cell renewal, CG14225/ET RNAi was protective in vivo. In conclusion, we have identified ET as a novel negative regulator of die Drosophila JAK/STAT pathway both in vitro and in vivo, and it functions in regulating Stat92E phosphorylation. [ABSTRACT FROM AUTHOR]

Breast cancers can be classified into those that express the estrogen (ER) and progesterone (PR) receptors, those with ERBB2 ( HER-2/ Neu) amplification, and those without expression of ER, PR, or amplification of ERBB2 (referred to as triple-negative or basal-like breast cancer). In order to identify potential molecular targets in breast cancer, we performed a synthetic siRNA-mediated RNAi screen of the human tyrosine kinome. A primary RNAi screen conducted in the triple-negative/basal-like breast cancer cell line MDA-MB231 followed by secondary RNAi screens and further studies in this cell line and two additional triple-negative/basal-like breast cancer cell lines, BT20 and HCC1937, identified the G2/M checkpoint protein, WEE1, as a potential therapeutic target. Similar sensitivity to WEE1 inhibition was observed in cell lines from all subtypes of breast cancer. RNAi-mediated silencing or small compound inhibition of WEE1 in breast cancer cell lines resulted in an increase in γH2AX levels, arrest in the S-phase of the cell cycle, and a significant decrease in cell proliferation. WEE1-inhibited cells underwent apoptosis as demonstrated by positive Annexin V staining, increased sub-G1 DNA content, apoptotic morphology, caspase activation, and rescue by the pan-caspase inhibitor, Z-VAD-FMK. In contrast, the non-transformed mammary epithelial cell line, MCF10A, did not exhibit any of these downstream effects following WEE1 silencing or inhibition. These results identify WEE1 as a potential molecular target in breast cancer. [ABSTRACT FROM AUTHOR]

Germ cells serve as intriguing examples of differentiated cells that retain the capacity to generate all cell types of an organism. Here we used functional genomic approaches in planarians to identify genes required for proper germ cell development. We conducted microarray analyses and in situ hybridization to discover and validate germ cell-enriched transcripts, and then used RNAi to screen for genes required for discrete stages of germ cell development. The majority of genes we identified encode conserved RNA-binding proteins, several of which have not been implicated previously in germ cell development. We also show that a germ cell-specific subunit of the conserved transcription factor CCAAT-binding protein/nuclear factor-Y is required for maintaining spermatogonial stem cells. Our results demonstrate that conserved transcriptional and post-transcriptional mechanisms regulate germ cell development in planarians. These findings suggest that studies of planarians will inform our understanding of germ cell biology in higher organisms. [ABSTRACT FROM AUTHOR]

Chromosomal instability and the subsequent genetic mutations are considered to be critical factors in the development of the majority of solid tumors, but the mechanisms by which a stable diploid cell loses the ability to maintain genomic integrity are not well characterized. We have approached this critical issue through the use of high-throughput screens in untransformed diploid epithelial cells. In a screen of a cDNA library, we identified 13 kinases whose overexpression leads to increased ploidy. In a series of shRNA screens, we identified 16 kinases whose loss leads to increased ploidy. In both cDNA and shRNA screens, the majority of hits have not been linked previously to genomic stability. We further show that sustained loss of the shRNA screening hits leads to multipolar spindles and heterogeneous chromosome content, two characteristics of chromosomal instability. Loss of several of the kinases leads to loss of contact inhibition and to anchorage-independent growth, vital traits acquired during tumor development. We anticipate that this work will serve as a template for the comprehensive identification of pathways whose dysregulation can drive tumorigenesis through impaired karyotypic maintenance. [ABSTRACT FROM AUTHOR]

Our understanding of the genetic mechanisms of organismal aging has advanced dramatically during the past two decades. With the development of large-scale RNAi screens, the last few years saw the remarkable identifications of hundreds of new longevity genes in the roundworm Caenorhabditis elegans. The various RNAi screens revealed many biological pathways previously unknown to be related to aging. In this review, we focus on findings from the recent large-scale RNAi longevity screens, and discuss insights they have provided into the complex biological process of aging and considerations of the RNAi technology will continue to have on the future development of the aging field. [ABSTRACT FROM PUBLISHER]

RNA interference (RNAi) is a powerful method to unravel the role of a given gene in eukaryotic cells. The development of high throughput assay platforms such as fluorescence plate readers and high throughput microscopy has allowed the design of genome wide RNAi screens to systemically discern members of regulatory networks around various cellular processes. Here we summarize the different strategies employed in RNAi screens to reveal regulators of transcriptional networks. We focus our discussion in experimental approaches designed to uncover regulatory interactions modulating transcription factor activity. [ABSTRACT FROM PUBLISHER]

A major impediment to the effective treatment of cancer is the molecular heterogeneity of the disease, which is also reflected in an equally diverse pattern of clinical responses to therapy. Currently, only few drugs are available that can be used safely and effectively to treat cancer. To improve this situation, the development of novel and highly specific targets for therapy is of utmost importance. Possibly even more importantly, we need better tools to predict which patients will respond to specific therapies. Such drug response biomarkers will be instrumental to individualize the therapy of patients having seemingly similar cancers. In this study, we discuss how RNA interference-based genetic screens can be used to address these two pressing needs in the care for cancer patients. [ABSTRACT FROM AUTHOR]

Intracellular signal transduction networks involving protein kinases are important modulators of cell survival and cell death in multicellular organisms. Functional compromise of these networks has been linked to aberrant apoptosis in diseases such as cancer. To identify novel kinase regulators of cell death, we conducted an RNAi-based screen to identify modulators of the intrinsic apoptosis pathway. Using this approach, we identified MAP4K3 as a novel apoptosis inducer. Here, we present evidence that this pro-apoptotic kinase orchestrates activation of BAX via the concerted posttranscriptional modulation of PUMA, BAD, and BIM. Additionally, we found decreased levels of this kinase in pancreatic cancer samples, suggesting a tumor suppressor role for MAP4K3 in pancreatic tumorigenesis. [ABSTRACT FROM AUTHOR]

RAS-extracellular signal regulated kinase (ERK) signaling governs multiple aspects of cell fate specification, cellular transitions, and growth by regulating downstream substrates through phosphorylation. Understanding how perturbations to the ERK signaling pathway lead to developmental disorders and cancer hinges critically on identification of the substrates. Yet, only a limited number of substrates have been identified that function in vivo to execute ERK-regulated processes. The Caenorhabditis elegans germ line utilizes the well-conserved RAS-ERK signaling pathway in multiple different contexts. Here, we present an integrated functional genomic approach that identified 30 ERK substrates, each of which functions to regulate one or more of seven distinct biological processes during C. elegans germ-line development. Our results provide evidence for three themes that underlie the robustness and specificity of biological outcomes controlled by ERK signaling in C. elegans that are likely relevant to ERK signaling in other organisms: (i) multiple diverse ERK substrates function to control each individual biological process; (ii) different combinations of substrates function to control distinct biological processes; and (iii) regulatory feedback loops between ERK and its substrates help reinforce or attenuate ERK activation. Substrates identified here have conserved orthologs in humans, suggesting that insights from these studies will contribute to our understanding of human diseases involving deregulated ERK activity. [ABSTRACT FROM AUTHOR]

Posttranscriptional silencing by RNAi is initiated by dsRNAs that are processed into siRNAs that ultimately target homologous mRNAs for degradation. We used luciferase reporter constructs and a cultured cell-based assay to perform a genomewide screen for components of the RNAi pathway in Drosophila melanogaster. The screen identified seven genes that affect the RNAi response, five with previously described function (AGO2, Tis11, Hsc70-3, Hsc70-4, and hdc) and two annotated genes (CG17265 and CG10883). [ABSTRACT FROM AUTHOR]