Etoposide-induced DNA damage is increased in p53 mutants: identification of ATR and other genes that influence effects of p53 mutations on Top2-induced cytotoxicity.

The functional status of the tumor suppressor p53 is a critical component in determining the sensitivity of cancer cells to many chemotherapeutic agents. DNA topoisomerase II (Top2) plays essential roles in DNA metabolism and is the target of FDA approved chemotherapeutic agents. Topoisomerase targeting drugs convert the enzyme into a DNA damaging agent and p53 influences cellular responses to these agents. We assessed the impact of the loss of p53 function on the formation of DNA damage induced by the Top2 poison etoposide. Using human HCT116 cells, we found resistance to etoposide in cell growth assays upon the functional loss of p53. Nonetheless, cells lacking fully functional p53 were etoposide hypersensitive in clonogenic survival assays. This complex role of p53 led us to directly examine the effects of p53 status on topoisomerase-induced DNA damage. A deficiency in functional p53 resulted in elevated levels of the Top2 covalent complexes (Top2cc) in multiple cell lines. Employing genome-wide siRNA screens, we identified a set of genes for which reduced expression resulted in enhanced synthetic lethality upon etoposide treatment of p53 defective cells. We focused on one hit from this screen, ATR, and showed that decreased expression sensitized the p53-defective cells to etoposide in all assays and generated elevated levels of Top2cc in both p53 proficient and deficient cells. Our findings suggest that a combination of etoposide treatment with functional inactivation of DNA repair in p53 defective cells could be used to enhance the therapeutic efficacy of Top2 targeting agents.

A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation.

Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes. NDPK-D was required for both mitochondrial DNA synthesis and cardiolipin exposure on the mitochondrial surface in response to inflammasome priming signals mediated by TLRs, and macrophages deficient in NDPK-D had multiple defects in LPS-induced inflammasome activation. In addition, NDPK-D was required for the recruitment of TNF receptor-associated factor 6 (TRAF6) to mitochondria, which was critical for reactive oxygen species (ROS) production and the metabolic reprogramming that supported the TLR-induced gene program. NDPK-D knockout mice were protected from LPS-induced shock, consistent with decreased ROS production and attenuated glycolytic commitment during priming. Our findings suggest that, in response to microbial challenge, NDPK-D-dependent TRAF6 mitochondrial recruitment triggers an energetic fitness checkpoint required to engage and maintain the transcriptional program necessary for inflammasome activation.

A high-throughput genome-wide RNAi screen identifies modifiers of survival motor neuron protein.

Spinal muscular atrophy (SMA) is a debilitating neurological disorder marked by degeneration of spinal motor neurons and muscle atrophy. SMA results from mutations in survival motor neuron 1 (SMN1), leading to deficiency of survival motor neuron (SMN) protein. Current therapies increase SMN protein and improve patient survival but have variable improvements in motor function, making it necessary to identify complementary strategies to further improve disease outcomes. Here, we perform a genome-wide RNAi screen using a luciferase-based activity reporter and identify genes involved in regulating SMN gene expression, RNA processing, and protein stability. We show that reduced expression of Transcription Export complex components increases SMN levels through the regulation of nuclear/cytoplasmic RNA transport. We also show that the E3 ligase, Neurl2, works cooperatively with Mib1 to ubiquitinate and promote SMN degradation. Together, our screen uncovers pathways through which SMN expression is regulated, potentially revealing additional strategies to treat SMA.

Machine learning prediction of methionine and tryptophan photooxidation susceptibility.

Photooxidation of methionine (Met) and tryptophan (Trp) residues is common and includes major degradation pathways that often pose a serious threat to the success of therapeutic proteins. Oxidation impacts all steps of protein production, manufacturing, and shelf life. Prediction of oxidation liability as early as possible in development is important because many more candidate drugs are discovered than can be tested experimentally. Undetected oxidation liabilities necessitate expensive and time-consuming remediation strategies in development and may lead to good drugs reaching patients slowly. Conversely, sites mischaracterized as oxidation liabilities could result in overengineering and lead to good drugs never reaching patients. To our knowledge, no predictive model for photooxidation of Met or Trp is currently available. We applied the random forest machine learning algorithm to in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) datasets (Met, n = 421; Trp, n = 342) of tryptic therapeutic protein peptides to create computational models for Met and Trp photooxidation. We show that our machine learning models predict Met and Trp photooxidation likelihood with 0.926 and 0.860 area under the curve (AUC), respectively, and Met photooxidation rate with a correlation coefficient (Q2) of 0.511 and root-mean-square error (RMSE) of 10.9%. We further identify important physical, chemical, and formulation parameters that influence photooxidation. Improvement of biopharmaceutical liability predictions will result in better, more stable drugs, increasing development throughput, product quality, and likelihood of clinical success.

Degradation of 5hmC-marked stalled replication forks by APE1 causes genomic instability.

Synthetic lethality between poly(ADP-ribose) polymerase (PARP) inhibition and BRCA deficiency is exploited to treat breast and ovarian tumors. However, resistance to PARP inhibitors (PARPis) is common. To identify potential resistance mechanisms, we performed a genome-wide RNAi screen in BRCA2-deficient mouse embryonic stem cells and validation in KB2P1.21 mouse mammary tumor cells. We found that resistance to multiple PARPi emerged with reduced expression of TET2 (ten-eleven translocation), which promotes DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC) and other products. TET2 knockdown in BRCA2-deficient cells protected stalled replication forks (RFs). Increasing 5hmC abundance induced the degradation of stalled RFs in KB2P1.21 and human cancer cells by recruiting the base excision repair-associated apurinic/apyrimidinic endonuclease APE1, independent of the BRCA2 status. TET2 loss did not affect the recruitment of the repair protein RAD51 to sites of double-strand breaks (DSBs) or the abundance of proteins associated with RF integrity. The loss of TET2, of its product 5hmC, and of APE1 recruitment to stalled RFs promoted resistance to the chemotherapeutic cisplatin. Our findings reveal a previously unknown role for the epigenetic mark 5hmC in maintaining the integrity of stalled RFs and a potential resistance mechanism to PARPi and cisplatin.

A genome-wide siRNA screen identifies a druggable host pathway essential for the Ebola virus life cycle.

BACKGROUND: The 2014-2016 Ebola virus (EBOV) outbreak in West Africa highlighted the need for improved therapeutic options against this virus. Approaches targeting host factors/pathways essential for the virus are advantageous because they can potentially target a wide range of viruses, including newly emerging ones and because the development of resistance is less likely than when targeting the virus directly. However, systematic approaches for screening host factors important for EBOV have been hampered by the necessity to work with this virus at biosafety level 4 (BSL4). METHODS: In order to identify host factors involved in the EBOV life cycle, we performed a genome-wide siRNA screen comprising 64,755 individual siRNAs against 21,566 human genes to assess their activity in EBOV genome replication and transcription. As a screening platform, we used reverse genetics-based life cycle modelling systems that recapitulate these processes without the need for a BSL4 laboratory. RESULTS: Among others, we identified the de novo pyrimidine synthesis pathway as an essential host pathway for EBOV genome replication and transcription, and confirmed this using infectious EBOV under BSL4 conditions. An FDA-approved drug targeting this pathway showed antiviral activity against infectious EBOV, as well as other non-segmented negative-sense RNA viruses. CONCLUSIONS: This study provides a minable data set for every human gene regarding its role in EBOV genome replication and transcription, shows that an FDA-approved drug targeting one of the identified pathways is highly efficacious in vitro, and demonstrates the power of life cycle modelling systems for conducting genome-wide host factor screens for BSL4 viruses.

ICE1 promotes the link between splicing and nonsense-mediated mRNA decay.

The nonsense-mediated mRNA decay (NMD) pathway detects aberrant transcripts containing premature termination codons (PTCs) and regulates expression of 5-10% of non-aberrant human mRNAs. To date, most proteins involved in NMD have been identified by genetic screens in model organisms; however, the increased complexity of gene expression regulation in human cells suggests that additional proteins may participate in the human NMD pathway. To identify proteins required for NMD, we performed a genome-wide RNAi screen against >21,000 genes. Canonical members of the NMD pathway were highly enriched as top hits in the siRNA screen, along with numerous candidate NMD factors, including the conserved ICE1/KIAA0947 protein. RNAseq studies reveal that depletion of ICE1 globally enhances accumulation and stability of NMD-target mRNAs. Further, our data suggest that ICE1 uses a putative MIF4G domain to interact with exon junction complex (EJC) proteins and promotes the association of the NMD protein UPF3B with the EJC.

Identifying HIPK1 as Target of miR-22-3p Enhancing Recombinant Protein Production From HEK 293 Cell by Using Microarray and HTP siRNA Screen.

Protein expression from human embryonic kidney cells (HEK 293) is an important tool for structural and clinical studies. It is previously shown that microRNAs (small, noncoding RNAs) are effective means for improved protein expression from these cells, and by conducting a high-throughput screening of the human microRNA library, several microRNAs are identified as potential candidates for improving expression. From these, miR-22-3p is chosen for further study since it increased the expression of luciferase, two membrane proteins and a secreted fusion protein with minimal effect on the cells' growth and viability. Since each microRNA can interact with several gene targets, it is of interest to identify the repressed genes for understanding and exploring the improved expression mechanism for further implementation. Here, the authors describe a novel approach for identification of the target genes by integrating the differential gene expression analysis with information obtained from our previously conducted high-throughput siRNA screening. The identified genes were validated as being involved in improving luciferase expression by using siRNA and qRT-PCR. Repressing the target gene, HIPK1, is found to increase luciferase and GPC3 expression 3.3- and 2.2-fold, respectively.

Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons.

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.

RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions.

The widespread use of two-dimensional (2D) monolayer cultures for high-throughput screening (HTS) to identify targets in drug discovery has led to attrition in the number of drug targets being validated. Solid tumors are complex, aberrantly growing microenvironments that harness structural components from stroma, nutrients fed through vasculature, and immunosuppressive factors. Increasing evidence of stromally-derived signaling broadens the complexity of our understanding of the tumor microenvironment while stressing the importance of developing better models that reflect these interactions. Three-dimensional (3D) models may be more sensitive to certain gene-silencing events than 2D models because of their components of hypoxia, nutrient gradients, and increased dependence on cell-cell interactions and therefore are more representative of in vivo interactions. Colorectal cancer (CRC) and breast cancer (BC) models composed of epithelial cells only, deemed single-cell-type tumor spheroids (SCTS) and multi-cell-type tumor spheroids (MCTS), containing fibroblasts were developed for RNAi HTS in 384-well microplates with flat-bottom wells for 2D screening and round-bottom, ultra-low-attachment wells for 3D screening. We describe the development of a high-throughput assay platform that can assess physiologically relevant phenotypic differences between screening 2D versus 3D SCTS, 3D SCTS, and MCTS in the context of different cancer subtypes. This assay platform represents a paradigm shift in how we approach drug discovery that can reduce the attrition rate of drugs that enter the clinic.

Triad of human cellular proteins, IRF2, FAM111A, and RFC3, restrict replication of orthopoxvirus SPI-1 host-range mutants.

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 rabbitpox 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.

TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral Protein (ZAP).

The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP's antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP's ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity.

Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread.

Human respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral cause of acute pediatric lower respiratory tract illness worldwide, and lacks a vaccine or effective antiviral drug. The involvement of host factors in the RSV replicative cycle remains poorly characterized. A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 (ARP2) as a host factor involved in RSV infection. ARP2 knockdown did not reduce RSV entry, and did not markedly reduce gene expression during the first 24 hr of infection, but decreased viral gene expression thereafter, an effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, there was a 10-fold reduction in the release of infectious progeny virions in ARP2-depleted cells at 72 hr post-infection. In addition, we found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was ARP2 dependent. Filopodia appeared to shuttle RSV to nearby uninfected cells, facilitating virus spread. Expression of the RSV F protein alone from a plasmid or heterologous viral vector in A549 cells induced filopodia, indicating a new role for the RSV F protein, driving filopodia induction and virus spread. Thus, this study identified roles for ARP2 and filopodia in RSV-induced cell motility, RSV production, and RSV cell-to-cell spread.

Synthetic viability by BRCA2 and PARP1/ARTD1 deficiencies.

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib has been approved for treatment of advanced ovarian cancer associated with BRCA1 and BRCA2 mutations. BRCA1- and BRCA2-mutated cells, which are homologous recombination (HR) deficient, are hypersensitive to PARPi through the mechanism of synthetic lethality. Here we examine the effect of PARPi on HR-proficient cells. Olaparib pretreatment, PARP1 knockdown or Parp1 heterozygosity of Brca2(cko/ko) mouse embryonic stem cells (mESCs), carrying a null (ko) and a conditional (cko) allele of Brca2, results in viable Brca2(ko/ko) cells. PARP1 deficiency does not restore HR in Brca2(ko/ko) cells, but protects stalled replication forks from MRE11-mediated degradation through its impaired recruitment. The functional consequence of Parp1 heterozygosity on BRCA2 loss is demonstrated by a significant increase in tumorigenesis in Brca2(cko/cko) mice. Thus, while olaparib efficiently kills BRCA2-deficient cells, we demonstrate that it can also contribute to the synthetic viability if PARP is inhibited before BRCA2 loss.

Genome-scale RNA interference screen identifies antizyme 1 (OAZ1) as a target for improvement of recombinant protein production in mammalian cells.

For the purpose of improving recombinant protein production from mammalian cells, an unbiased, high-throughput whole-genome RNA interference screen was conducted using human embryonic kidney 293 (HEK 293) cells expressing firefly luciferase. A 21,585 human genes were individually silenced with three different siRNAs for each gene. The screen identified 56 genes that led to the greatest improvement in luciferase expression. These genes were found to be included in several pathways involved in spliceosome formation and mRNA processing, transcription, metabolic processes, transport, and protein folding. The 10 genes that most enhanced protein expression when downregulated, were further confirmed by measuring the effect of their silencing on the expression of three additional recombinant proteins. Among the confirmed genes, OAZ1-the gene encoding the ornithine decarboxylase antizyme1-was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing OAZ1 caused an increase of the ornithine decarboxylase enzyme and the cellular levels of putrescine and spermidine; an indication that increased cellular polyamines enhances luciferase expression without affecting its transcription. The study shows that OAZ1 is a novel target for improving expression of recombinant proteins. The genome-scale screening performed in this work can establish the foundation for targeted design of an efficient mammalian cell platform for various biotechnological applications. Biotechnol. Bioeng. 2016;113: 2403-2415. © 2016 Wiley Periodicals, Inc.

An interactive web-based application for Comprehensive Analysis of RNAi-screen Data.

RNAi screens are widely used in functional genomics. Although the screen data can be susceptible to a number of experimental biases, many of these can be corrected by computational analysis. For this purpose, here we have developed a web-based platform for integrated analysis and visualization of RNAi screen data named CARD (for Comprehensive Analysis of RNAi Data; available at https://card.niaid.nih.gov). CARD allows the user to seamlessly carry out sequential steps in a rigorous data analysis workflow, including normalization, off-target analysis, integration of gene expression data, optimal thresholds for hit selection and network/pathway analysis. To evaluate the utility of CARD, we describe analysis of three genome-scale siRNA screens and demonstrate: (i) a significant increase both in selection of subsequently validated hits and in rejection of false positives, (ii) an increased overlap of hits from independent screens of the same biology and (iii) insight to microRNA (miRNA) activity based on siRNA seed enrichment.

Functional Genomic Screening Reveals Splicing of the EWS-FLI1 Fusion Transcript as a Vulnerability in Ewing Sarcoma.

Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.

Aurora B kinase is a potent and selective target in MYCN-driven neuroblastoma.

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials.