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.

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.

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.

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.

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.

Whole-genome RNAi screen highlights components of the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity.

Immunotoxins (antibody-toxin fusion proteins) target surface antigens on cancer cells and kill these cells via toxin-mediated inhibition of protein synthesis. To identify genes controlling this process, an RNAi whole-genome screen (∼ 22,000 genes at three siRNAs per gene) was conducted via monitoring the cytotoxicity of the mesothelin-directed immunotoxin SS1P. SS1P, a Pseudomonas exotoxin-based immunotoxin, was chosen because it is now in clinical trials and has produced objective tumor regressions in patients. High and low concentrations of SS1P were chosen to allow for the identification of both mitigators and sensitizers. As expected, silencing known essential genes in the immunotoxin pathway, such as mesothelin, furin, KDEL receptor 2, or members of the diphthamide pathway, protected cells. Of greater interest was the observation that many RNAi targets increased immunotoxin sensitivity, indicating that these gene products normally contribute to inefficiencies in the killing pathway. Of the top sensitizers, many genes encode proteins that locate to either the endoplasmic reticulum (ER) or Golgi and are annotated as part of the secretory system. Genes related to the ER-associated degradation system were not among high-ranking mitigator or sensitizer candidates. However, the p97 inhibitor eeyarestatin 1 enhanced immunotoxin killing. Our results highlight potential targets for chemical intervention that could increase immunotoxin killing of cancer cells and enhance our understanding of toxin trafficking.

Seed sequence-matched controls reveal limitations of small interfering RNA knockdown in functional and structural studies of hepatitis C virus NS5A-MOBKL1B interaction.

UNLABELLED: Hepatitis C virus (HCV) is a widespread human pathogen causing liver cirrhosis and cancer. Similar to the case for other viruses, HCV depends on host and viral factors to complete its life cycle. We used proteomic and yeast two-hybrid approaches to elucidate host factors involved in HCV nonstructural protein NS5A function and found that MOBKL1B interacts with NS5A. Initial experiments with small interfering RNA (siRNA) knockdown suggesting a role in HCV replication led us to examine the interaction using biochemical and structural approaches. As revealed by a cocrystal structure of a core MOBKL1B-NS5A peptide complex at 1.95 Å, NS5A binds to a hydrophobic patch on the MOBKL1B surface. Biosensor binding assays identified a highly conserved, 18-amino-acid binding site in domain II of NS5A, which encompasses residues implicated in cyclophilin A (CypA)-dependent HCV RNA replication. However, a CypA-independent HCV variant had reduced replication in MOBKL1B knockdown cells, even though its NS5A does not interact with MOBKL1B. These discordant results prompted more extensive studies of MOBKL1B gene knockdowns, which included additional siRNAs and specifically matched seed sequence siRNA controls. We found that reduced virus replication after treating cells with MOBKL1B siRNA was actually due to off-target inhibition, which indicated that the initial finding of virus replication dependence on the MOBKL1B-NS5A interaction was incorrect. Ultimately, using several approaches, we found no relationship of the MOBKL1B-NS5A interaction to virus replication. These findings collectively serve as a reminder to investigators and scientific reviewers of the pervasive impact of siRNA off-target effects on interpretation of biological data. IMPORTANCE: Our study illustrates an underappreciated shortcoming of siRNA gene knockdown technology. We initially identified a cellular protein, MOBKL1B, as a binding partner with the NS5A protein of hepatitis C virus (HCV). MOBKL1B siRNA, but not irrelevant RNA, treatment was associated with both reduced virus replication and the absence of MOBKL1B. Believing that HCV replication depended on the MOBKL1B-NS5A interaction, we carried out structural and biochemical analyses. Unexpectedly, an HCV variant lacking the MOBKL1B-NS5A interaction could not replicate after cells were treated with MOBKL1B siRNA. By repeating the MOBKL1B siRNA knockdowns and including seed sequence-matched siRNA instead of irrelevant siRNA as a control, we found that the MOBKL1B siRNAs utilized had off-target inhibitory effects on virus replication. Collectively, our results suggest that stricter controls must be utilized in all RNA interference (RNAi)-mediated gene knockdown experiments to ensure sound conclusions and a reliable scientific knowledge database.

Common seed analysis to identify off-target effects in siRNA screens.

Genome-scale small interfering RNA (siRNA) screens have become an increasingly popular approach to new target identification and pathway elucidation. However, the large data sets generated from siRNA screens have demonstrated high false-positive rates and the requirement for extensive experimental triage to distinguish true hits. A number of groups have independently reported the presence of siRNAs with identical seed sequences among their top screening hits. Based on these observations, we have developed a comprehensive technique for detecting and visualizing seed-based off-target effects in siRNA screening data. This is accomplished by analyzing the behavior of siRNAs that share identical seed sequences, which we refer to as common seed analysis (CSA). By applying these techniques to primary screening data of the Wnt pathway, we identify 158 distinct seed sequences that have a statistically significant effect on the assay. The promiscuous seed sequences identified in this manner can then be discounted in the analysis of follow-up experiments using single siRNAs. The ability to detect off-target effects when sufficient numbers of siRNAs share a common seed has significant implications for the design of siRNA screening experiments, data analysis, hit selection, and library design.