Funding for developing the data integration workflow was obtained from European Community&#8217;s Seventh Framework Programme under grant agreement nu. 279113 (OCTIPS). Adaption of data within this publication was kindly approved by Public Library of Sciences Publications and Impact Journals, LLC.

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AH and PP were employees of emergentec biodevelopment GmbH at the time of performing the analyses leading to the results presented in the representative results section. MM and MK have nothing to disclose.

We have outlined a workflow to identify drug combinations impacting synthetic lethal interactions. This workflow makes use of (i) data on synthetic lethal interactions from model organisms, (ii) information of human orthologs, (iii) information on drug-target associations, (iv) drug information on clinical trials in the context of cancer, as well as (v) on information of drug-disease and gene-disease associations extracted from scientific literature. The consolidated information can be used to evaluate the impact of a gi...

Synthetic lethality defines an association of two genes, where loss of one gene does not affect viability, but loss of both genes leads to cell death. It was first described in 1946 by Dobzhansky while analyzing various phenotypes of drosophila by breeding homozygous mutants1. Mutants that did not produce viable offspring, although viable themselves, exhibited lethal phenotypes when crossed with certain other mutants, setting ground for the establishment of the theory of synthetic lethality. Hartwell and colleagues suggested that this concept might be applicable for cancer therapy in humans2. Pharmacologically provoked synthetic lethality could rely on just one mutation, given that the mutated gene&#8217;s synthetic lethal partner is targetable by a pharmacological compound. The first gene pair to enable pharmacological induction of synthetic lethality was BRCA(1/2) and PARP1. PARP1 functions as a sensor for DNA damage, and is tied to sites of double and single DNA strand-breaks, supercoils and crossovers3. BRCA1 and 2 play major roles in repair of DNA double-strand breaks through homologous recombination4. Farmer and colleagues published findings that cells deficient for BRCA1/2 were susceptible to PARP inhibition, while no cytotoxicity was observed in BRCA wild-type cells5. Ultimately, PARP inhibitors were approved for the treatment of BRCA deficient breast and ovarian cancer6,7. Further, synthetic lethality gene pairs leading to clinical approval of pharmacological compounds are much anticipated and a major area of recent cancer research efforts8.
Synthetic lethal gene interactions were modelled in multiple organisms including fruit flies, C. elegans and yeast2. Using various approaches including RNA-interference- and CRISPR/CAS-library knockouts, novel synthetic lethal gene pairs were discovered in recent years9,10,11. A protocol on the experimental procedures of RNAi in combination with CRISPR/CAS was recently published by Housden and colleagues12. Meanwhile, researchers also conducted large screens in haploid human cells to identify synthetic lethal interactions13,14. In silico methods like biological network analysis and machine learning have also shown promise in the discovery of synthetic lethal interactions15,16.
Conceptionally, one approach to make use of synthetic lethal interactions in the context of anti-tumor therapy is to identify mutated or non-functional proteins in tumor cells, making their synthetic lethal interaction partners promising drug targets for therapeutic intervention. Due to the heterogeneity of most tumor types, researchers have started the search for so-called synthetic lethal hub proteins. These synthetic lethal hubs have a number of synthetic lethal interaction partners that are either mutated and therefore non-functional or significantly downregulated in tumor samples. Addressing such synthetic lethal hubs holds promise in increasing drug efficacy or overcoming drug resistance as could be shown for instance in the context of vincristine resistant neuroblastoma17. A second approach to enhance drug treatment making use of the concept of synthetic lethal interactions is to identify drug combinations targeting synthetic lethal interactions. This could lead to new combinations of already approved single anti-tumor therapies and to the repositioning of drugs from other disease areas to the field of oncology.
In this article, we present a step-by-step procedure to yield a list of drug combinations that target synthetic lethal interaction pairs. In this workflow, we (i) use data on synthetic lethal interactions from BioGRID and (ii) information on homologous genes from Ensembl, (iii) retrieve drug-target pairs from DrugBank, (iv) build disease-drug associations from ClinicalTrials.gov, and (v) hence generate a set of drug combinations addressing synthetic lethal interactions. Lastly, we provide drug combinations in the context of ovarian and breast cancer in the representative results section.

<table>
	<tbody>
		<tr>
			<td>BioGRID</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>thebiogrid.org</td>
		</tr>
		<tr>
			<td>ClinicalTrials.gov</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>ClinicalTrials.gov</td>
		</tr>
		<tr>
			<td>DrugBank</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>drugbank.ca</td>
		</tr>
		<tr>
			<td>Ensembl BioMart</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>ensembl.org</td>
		</tr>
		<tr>
			<td colspan="4">for alternative computational databases please refer to the manuscript</td>
		</tr>
		<tr>
			<td>7-AAD</td>
			<td>ebioscience</td>
			<td>00-6993-50</td>
			<td></td>
		</tr>
		<tr>
			<td>AnnexinV-APC</td>
			<td>BD Bioscience</td>
			<td>550474</td>
			<td></td>
		</tr>
		<tr>
			<td>celecoxib</td>
			<td>Sigma-Aldrich</td>
			<td>PZ0008-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Blue Viability Assay</td>
			<td>Promega</td>
			<td>G8080</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Canto II</td>
			<td>BD Bioscience</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Fisher Scientific/Gibco</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>FloJo Software</td>
			<td>FloJo LLC</td>
			<td>V10</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5a Medium Modified</td>
			<td>Fisher Scientific/Gibco</td>
			<td>16600082</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin G/streptomycin sulfate</td>
			<td>Fisher Scientific/Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>SKBR-3 cells</td>
			<td>American Type Culture Collection (ATCC)</td>
			<td>ATCC HTB-30</td>
			<td></td>
		</tr>
		<tr>
			<td>zoledronic acid</td>
			<td>Sigma-Aldrich</td>
			<td>SML0223-50MG</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">further materials or equipment will be made available upon request</td>
		</tr>
	</tbody>
</table>

a data integration workflow to identify drug combinations targeting synthetic lethal interactions

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of both synthetic lethal interactors leads to loss of cell viability or cell death. The best studied synthetic lethal interaction is between BRCA1/2 and PARP1, with PARP1 inhibitors being used in clinical practice to treat patients with BRCA1/2 mutated tumors. Large genetic screens in model organisms but also in haploid human cell lines have led to the identification of numerous additional synthetic lethal interaction pairs, all being potential targets of interest in the development of novel tumor therapies. One approach is to therapeutically target genes with a synthetic lethal interactor that is mutated or significantly downregulated in the tumor of interest. A second approach is to formulate drug combinations addressing synthetic lethal interactions. In this article, we outline a data integration workflow to evaluate and identify drug combinations targeting synthetic lethal interactions. We make use of available datasets on synthetic lethal interaction pairs, homology mapping resources, drug-target links from dedicated databases, as well as information on drugs being investigated in clinical trials in the disease area of interest. We further highlight key findings of two recent studies of our group on drug combination assessment in the context of ovarian and breast cancer.

Our group has recently published two studies applying the workflow depicted in this manuscript to identify drug combinations targeting synthetic lethal interactions in the context of ovarian and breast cancer24,25. In the first study, we evaluated drug combinations that are currently tested in late stage clinical trials (phase III and IV) or already being used in clinical practice to treat ovarian cancer patients regarding their impact on synthetic lethal interac...

Watch this Scientific Journal Video about A Data Integration Workflow to Identify Drug Combinations Targeting Synthetic Lethal Interactions at JoVE.com

A Data Integration Workflow to Identify Drug Combinations Targeting Synthetic Lethal Interactions

Large genetic screens in model organisms have led to the identification of negative genetic interactions. Here, we describe a data integration workflow using data from genetic screens in model organisms to delineate drug combinations targeting synthetic lethal interactions in cancer.

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of ...

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4.1K Views.  Medical University of Vienna. Large genetic screens in model organisms have led to the identification of negative genetic interactions. Here, we describe a data integration workflow using data from genetic screens in model organisms to delineate drug combinations targeting synthetic lethal interactions in cancer.

Video: A Data Integration Workflow to Identify Drug Combinations Targeting Synthetic Lethal Interactions

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH receptors. However, the quantitative analysis of GnRH analogues&#39; cellular uptake and the investigated cell types in GnRH-based drug delivery systems are currently limited. Previously introduced, selectively labeled fluorescent GnRH I, -II and -III derivatives provide great detectability, and they have suitable chemical properties for reproducible and robust experiments. We also found that the appropriate up-to-date methods with these labeled GnRH analogues could offer novel information about the GnRH-based drug delivery systems. This manuscript introduces some simple and fast experiments regarding the cellular uptake of [D-Lys6(FITC)]-GnRH-I, [D-Lys6(FITC)]-GnRH-II and [Lys8(FITC)]-GnRH-III on the EBC-1 (lung), the BxPC-3 (pancreas) and on the Detroit-562- (pharynx) malignant tumor cells. In parallel with these GnRH-FITC conjugates, the cell surface level of GnRH-I receptors was also examined on these cell lines before and after the GnRH treatment by confocal laser scanning microscopy. The cellular uptake of GnRH-FITC conjugates was quantified by fluorescence-activated cell sorting. In these experiments minor differences among GnRH analogues and major differences among cell types was observed. The significant differences among cell lines are correlated with their distinct level of cell surface GnRH-I receptors. The introduced experiments contain practical methods to visualize, quantify and compare the uptake efficiency of GnRH-FITC conjugates in a time- and concentration-dependent manner on various adherent cell cultures. These results could predict the drug targeting efficiency of GnRH conjugates on the given cell culture, and offer a good basis for further experiments in the examination of GnRH-based drug delivery systems.

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

Selectively labeled fluorescent GnRH-I, -II and -III derivatives are reliable tools for tracking and quantifying their cellular uptake. This manuscript introduces experiments to visualize, quantify and compare the uptake efficiency of these GnRH conjugates in various cell lines.

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH ...

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal models are required for bridging the nanotechnology to its biomedical application. The mouse represents the traditional animal model for preclinical testing; however, mice are relatively expensive to keep and have long experimental cycles due to the limited progeny from each mother. The zebrafish has emerged as a powerful model system for developmental and biomedical research, including cancer research. In particular, due to its optical transparency and rapid development, zebrafish embryos are well suited for real-time in vivo monitoring of the behavior of cancer cells and their interactions with their microenvironment. This method was developed to sequentially introduce human cancer cells and functionalized nanoparticles in transparent Casper zebrafish embryos and monitor in vivo recognition and targeting of the cancer cells by nanoparticles in real time. This optimized protocol shows that fluorescently labeled nanoparticles, which are functionalized with folate groups, can specifically recognize and target metastatic human cervical epithelial cancer cells labeled with a different fluorochrome. The recognition and targeting process can occur as early as 30 min postinjection of the nanoparticles tested. The whole experiment only requires the breeding of a few pairs of adult fish and takes less than 4 days to complete. Moreover, zebrafish embryos lack a functional adaptive immune system, allowing the engraftment of a wide range of human cancer cells. Hence, the utility of the protocol described here enables the testing of nanoparticles on various types of human cancer cells, facilitating the selection of optimal nanoparticles in each specific cancer context for future testing in mammals and the clinic.

Described here is a method for utilizing zebrafish embryos to study the ability of functionalized nanoparticles to target human cancer cells in vivo. This method allows for the evaluation and selection of optimal nanoparticles for future testing in large animals and in clinical trials.

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...