Eugen Buehler

High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy

An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson’s disease. Recent studies of the Parkinson’s disease associated genes PINK1 (ref. 2) and parkin (PARK2, ref. 3) indicate that they may act in a quality control pathway preventing the accumulation of dysfunctional mitochondria. Here we elucidate regulators that have an impact on parkin translocation to damaged mitochondria with genome-wide small interfering RNA (siRNA) screens coupled to high-content microscopy. Screening yielded gene candidates involved in diverse cellular processes that were subsequently validated in low-throughput assays. This led to characterization of TOMM7 as essential for stabilizing PINK1 on the outer mitochondrial membrane following mitochondrial damage. We also discovered that HSPA1L (HSP70 family member) and BAG4 have mutually opposing roles in the regulation of parkin translocation. The screens revealed that SIAH3, found to localize to mitochondria, inhibits PINK1 accumulation after mitochondrial insult, reducing parkin translocation. Overall, our screens provide a rich resource to understand mitochondrial quality control.

siRNA off-target effects in genome-wide screens identify signaling pathway members

We introduce a method for analyzing small interfering RNA (siRNA) genetic screens based entirely on off-target effects. Using a screen for members of the Wnt pathway, we demonstrate that this method identifies known pathway components, some of which are not present in the screening library. This technique can be applied to siRNA screen results retroactively to confirm positives and identify genes missed using conventional methods for on-target gene selection.

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.

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 Brca2cko/ko mouse embryonic stem cells (mESCs), carrying a null (ko) and a conditional (cko) allele of Brca2, results in viable Brca2ko/ko cells. PARP1 deficiency does not restore HR in Brca2ko/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 Brca2cko/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.

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.

The CRASSS plug-in for integrating annotation data with hierarchical clustering results

We describe an algorithm for finding the most statistically significant non-overlapping subtrees of a hierarchical clustering of gene expression data with respect to a set of secondary data labels on genes. The method is implemented as a Java plug-in for a commercial gene expression analysis program (GeneSpring).

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.

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.

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

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

Abstract C222: The identification of kinase targets in Ewing sarcoma cell lines using RNAi and high-throughput investigational agents screens.

Ewing sarcoma (EWS), the second most frequent bone cancer, is an aggressive primitive neuroectodermal tumor that occurs primarily in children, adolescents and young adults. A pathognomonic translocation involving the EWS gene on chromosome 22 and the ETS-type gene FLI1 on chromosome 11 occurs in about 85% of cases. The EWS/FLI1 fusion protein product of this translocation is a potent transcription factor that functions as an oncoprotein. The current study uses complementary approaches of high-throughput siRNA mediated RNAi and Investigational Agents screening to identify new therapeutic strategies for Ewing sarcoma. For RNAi screening the TC32 cell line, a well-characterized EWS cell line that expresses the EWS-FLI1 fusion transcript, was assayed for cell viability (CellTiterGlo, Promega) 72 hrs post siRNA transfection using a library of siRNAs corresponding to the genes encoding proteins with kinase function, three siRNA per gene (Ambion). Fifteen kinase genes were identified as inducing a significant decrease in the survival of TC32 cells when silenced by RNAi. These genes were chosen for validation in further EWS lines and two non-EWS cell lines. Using a cut off of a 40% or greater decrease in cell viability induced by two or more siRNAs per gene in at least at one EWS cell line eleven genes of interest were identified: PLK1, AURKB, AKT1, CDC2L1, CRKRS, ATP6VOB, CDC2L5, AURKA, CHEK1, NRBP2 and CDK4. The importance of several of these kinases in the survival of EWS cell lines was confirmed by an independent high-throughput screen of 63 human pediatric and adult sarcoma lines, including 19 EWS lines, with the Approved Oncology Drugs (AOD) library of compounds and an Investigational Agents library (350 agents) using inhibition of proliferation as an endpoint. Cells were exposed to compounds at varying concentrations (10μM to 1.5nM) for 96 h and the effect of compound on cell viability was monitored using Alamar Blue. The IC50 (50% growth inhibitory) concentrations of each agent on the cell lines were determined using curve fitting algorithms. Compared to the other sarcoma lines, the EWS lines were: more sensitive to eight Aurora kinase inhibitors (IC50 ∼0.05 µM) (IC50 ∼2.5 µM);more sensitive (IC50 ∼0.1 µM) to five checkpoint kinase (CHK) inhibitors (IC50 about 1.3 µM); and more sensitive to eight polo-like kinase (PLK1) inhibitors. These results demonstrate the power of integrating RNAi and compound inhibition studies to identify molecular targets for specific cancer types. We are currently further evaluating the inhibition of the mitosis-associated proteins identified by these complementary screening approaches as a strategy for the treatment EWS. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C222. Citation Format: Amy McCalla, Patrick Grohar, Scott Martin, Eugen Buehler, Suntae Kim, Natasha Caplen, Joel Morris, Mark Kunkel, David Evans, Anne Monks, Beverly Teicher, Lee Helman. The identification of kinase targets in Ewing sarcoma cell lines using RNAi and high-throughput investigational agents screens. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C222.