register interest

Professor Sebastian Nijman

Research Area: Genetics and Genomics
Technology Exchange: Bioinformatics, Computational biology, Drug discovery and Transcript profiling
Scientific Themes: Cancer Biology and Genetics & Genomics
Keywords: cancer, gene-drug interactions and haploid genetic screens
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Cancer is one of the biggest causes of death in the Western world. Our group develops new approaches to study signaling networks in cancer cells and to uncover specific weaknesses, particularly in breast and lung cancer, that can be used for developing more effective targeted drugs and for guiding treatment decisions. We use high-throughput chemical and genetic screening approaches and haploid genetics to engineer isogenic human cancer cells and tease out chemical and genetic interactions that could be therapeutically exploited. These interactions are then studied in more detail using cell culture, mouse models, and patient samples.

Name Department Institution Country
Professor Paul Brennan Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Professor Jens Rittscher Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Daniel Ebner Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Prof Yvonne Jones (RDM) STRUBI, Nuffield Department of Clinical Medicine University of Oxford United Kingdom
Gerken PA, Wolstenhulme JR, Tumber A, Hatch SB, Zhang Y, Müller S, Chandler SA, Mair B, Li F, Nijman SMB et al. 2017. Discovery of a Highly Selective Cell-Active Inhibitor of the Histone Lysine Demethylases KDM2/7. Angew Chem Int Ed Engl, 56 (49), pp. 15555-15559. | Show Abstract | Read more

Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell-permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure-activity relationships, leading to the development of a small molecule with around 75-fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.

Forment JV, Herzog M, Coates J, Konopka T, Gapp BV, Nijman SM, Adams DJ, Keane TM, Jackson SP. 2017. Genome-wide genetic screening with chemically mutagenized haploid embryonic stem cells. Nat Chem Biol, 13 (1), pp. 12-14. | Show Abstract | Read more

In model organisms, classical genetic screening via random mutagenesis provides key insights into the molecular bases of genetic interactions, helping to define synthetic lethality, synthetic viability and drug-resistance mechanisms. The limited genetic tractability of diploid mammalian cells, however, precludes this approach. Here, we demonstrate the feasibility of classical genetic screening in mammalian systems by using haploid cells, chemical mutagenesis and next-generation sequencing, providing a new tool to explore mammalian genetic interactions.

Smida M, Fece de la Cruz F, Kerzendorfer C, Uras IZ, Mair B, Mazouzi A, Suchankova T, Konopka T, Katz AM, Paz K et al. 2016. MEK inhibitors block growth of lung tumours with mutations in ataxia-telangiectasia mutated. Nat Commun, 7 pp. 13701. | Show Abstract | Read more

Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.

Mair B, Konopka T, Kerzendorfer C, Sleiman K, Salic S, Serra V, Muellner MK, Theodorou V, Nijman SMB. 2016. Gain- and Loss-of-Function Mutations in the Breast Cancer Gene GATA3 Result in Differential Drug Sensitivity. PLoS Genet, 12 (9), pp. e1006279. | Show Abstract | Read more

Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.

Gapp BV, Konopka T, Penz T, Dalal V, Bürckstümmer T, Bock C, Nijman SM. 2016. Parallel reverse genetic screening in mutant human cells using transcriptomics. Mol Syst Biol, 12 (8), pp. 879. | Show Abstract | Read more

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease-relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large-scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.

Lackner DH, Carré A, Guzzardo PM, Banning C, Mangena R, Henley T, Oberndorfer S, Gapp BV, Nijman SMB, Brummelkamp TR, Bürckstümmer T. 2015. A generic strategy for CRISPR-Cas9-mediated gene tagging. Nat Commun, 6 pp. 10237. | Show Abstract | Read more

Genome engineering has been greatly enhanced by the availability of Cas9 endonuclease that can be targeted to almost any genomic locus using so called guide RNAs (gRNAs). However, the introduction of foreign DNA sequences to tag an endogenous gene is still cumbersome as it requires the synthesis or cloning of homology templates. Here we present a strategy that enables the tagging of endogenous loci using one generic donor plasmid. It contains the tag of interest flanked by two gRNA recognition sites that allow excision of the tag from the plasmid. Co-transfection of cells with Cas9, a gRNA specifying the genomic locus of interest, the donor plasmid and a cassette-specific gRNA triggers the insertion of the tag by a homology-independent mechanism. The strategy is efficient and delivers clones that display a predictable integration pattern. As showcases we generated NanoLuc luciferase- and TurboGFP-tagged reporter cell lines.

Inglés-Prieto Á, Reichhart E, Muellner MK, Nowak M, Nijman SMB, Grusch M, Janovjak H. 2015. Light-assisted small-molecule screening against protein kinases. Nat Chem Biol, 11 (12), pp. 952-954. | Show Abstract | Read more

High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.

Nijman SMB. 2015. Functional genomics to uncover drug mechanism of action. Nat Chem Biol, 11 (12), pp. 942-948. | Show Abstract | Read more

The upswing in US Food and Drug Administration and European Medicines Agency drug approvals in 2014 may have marked an end to the dry spell that has troubled the pharmaceutical industry over the past decade. Regardless, the attrition rate of drugs in late clinical phases remains high, and a lack of target validation has been highlighted as an explanation. This has led to a resurgence in appreciation of phenotypic drug screens, as these may be more likely to yield compounds with relevant modes of action. However, cell-based screening approaches do not directly reveal cellular targets, and hence target deconvolution and a detailed understanding of drug action are needed for efficient lead optimization and biomarker development. Here, recently developed functional genomics technologies that address this need are reviewed. The approaches pioneered in model organisms, particularly in yeast, and more recently adapted to mammalian systems are discussed. Finally, areas of particular interest and directions for future tool development are highlighted.

Konopka T, Nijman SMB. 2016. Comparison of genetic variants in matched samples using thesaurus annotation. Bioinformatics, 32 (5), pp. 657-663. | Show Abstract | Read more

MOTIVATION: Calling changes in DNA, e.g. as a result of somatic events in cancer, requires analysis of multiple matched sequenced samples. Events in low-mappability regions of the human genome are difficult to encode in variant call files and have been under-reported as a result. However, they can be described accurately through thesaurus annotation-a technique that links multiple genomic loci together to explicate a single variant. RESULTS: We here describe software and benchmarks for using thesaurus annotation to detect point changes in DNA from matched samples. In benchmarks on matched normal/tumor samples we show that the technique can recover between five and ten percent more true events than conventional approaches, while strictly limiting false discovery and being fully consistent with popular variant analysis workflows. We also demonstrate the utility of the approach for analysis of de novo mutations in parents/child families. AVAILABILITY AND IMPLEMENTATION: Software performing thesaurus annotation is implemented in java; available in source code on github at GeneticThesaurus (https://github.com/tkonopka/GeneticThesaurus) and as an executable on sourceforge at geneticthesaurus (https://sourceforge.net/projects/geneticthesaurus). Mutation calling is implemented in an R package available on github at RGeneticThesaurus (https://github.com/tkonopka/RGeneticThesaurus). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: tomasz.konopka@ludwig.ox.ac.uk.

Kerzendorfer C, Konopka T, Nijman SMB. 2015. A thesaurus of genetic variation for interrogation of repetitive genomic regions. Nucleic Acids Res, 43 (10), pp. e68. | Show Abstract | Read more

Detecting genetic variation is one of the main applications of high-throughput sequencing, but is still challenging wherever aligning short reads poses ambiguities. Current state-of-the-art variant calling approaches avoid such regions, arguing that it is necessary to sacrifice detection sensitivity to limit false discovery. We developed a method that links candidate variant positions within repetitive genomic regions into clusters. The technique relies on a resource, a thesaurus of genetic variation, that enumerates genomic regions with similar sequence. The resource is computationally intensive to generate, but once compiled can be applied efficiently to annotate and prioritize variants in repetitive regions. We show that thesaurus annotation can reduce the rate of false variant calls due to mappability by up to three orders of magnitude. We apply the technique to whole genome datasets and establish that called variants in low mappability regions annotated using the thesaurus can be experimentally validated. We then extend the analysis to a large panel of exomes to show that the annotation technique opens possibilities to study variation in hereto hidden and under-studied parts of the genome.

Muellner MK, Mair B, Ibrahim Y, Kerzendorfer C, Lechtermann H, Trefzer C, Klepsch F, Müller AC, Leitner E, Macho-Maschler S et al. 2015. Targeting a cell state common to triple-negative breast cancers. Mol Syst Biol, 11 (1), pp. 789. | Show Abstract | Read more

Some mutations in cancer cells can be exploited for therapeutic intervention. However, for many cancer subtypes, including triple-negative breast cancer (TNBC), no frequently recurring aberrations could be identified to make such an approach clinically feasible. Characterized by a highly heterogeneous mutational landscape with few common features, many TNBCs cluster together based on their 'basal-like' transcriptional profiles. We therefore hypothesized that targeting TNBC cells on a systems level by exploiting the transcriptional cell state might be a viable strategy to find novel therapies for this highly aggressive disease. We performed a large-scale chemical genetic screen and identified a group of compounds related to the drug PKC412 (midostaurin). PKC412 induced apoptosis in a subset of TNBC cells enriched for the basal-like subtype and inhibited tumor growth in vivo. We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC. Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells. This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies.

Essletzbichler P, Konopka T, Santoro F, Chen D, Gapp BV, Kralovics R, Brummelkamp TR, Nijman SMB, Bürckstümmer T. 2014. Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res, 24 (12), pp. 2059-2065. | Show Abstract | Read more

Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second gene copy. However, no completely haploid human cell line has been described, hampering the genetic accessibility of a subset of genes. The near-haploid human cell line HAP1 contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15. This large fragment encompasses 330 genes and is integrated on the long arm of Chromosome 19. Here, we employ a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state. Importantly, spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping revealed that engineered-HAPloid (eHAP) cells are fully haploid with no gross chromosomal aberrations induced by Cas9. Furthermore, whole-genome sequence and transcriptome analysis of the parental HAP1 and an eHAP cell line showed that transcriptional changes are limited to the excised Chromosome 15 fragment. Together, we demonstrate the feasibility of efficiently engineering megabase deletions with the CRISPR/Cas9 technology and report the first fully haploid human cell line.

Fece de la Cruz F, Gapp BV, Nijman SMB. 2015. Synthetic lethal vulnerabilities of cancer. Annu Rev Pharmacol Toxicol, 55 (1), pp. 513-531. | Show Abstract | Read more

The great majority of targeted anticancer drugs inhibit mutated oncogenes that display increased activity. Yet many tumors do not contain such actionable aberrations, such as those harboring loss-of-function mutations. The notion of targeting synthetic lethal vulnerabilities in cancer cells has provided an alternative approach to exploiting more of the genetic and epigenetic changes acquired during tumorigenesis. Here, we review synthetic lethality as a therapeutic concept that exploits the inherent differences between normal cells and cancer cells. Furthermore, we provide an overview of the screening approaches that can be used to identify synthetic lethal interactions in human cells and present several recently identified interactions that may be pharmacologically exploited. Finally, we indicate some of the challenges of translating synthetic lethal interactions into the clinic and how these may be overcome.

Licciardello MP, Müllner MK, Dürnberger G, Kerzendorfer C, Boidol B, Trefzer C, Sdelci S, Berg T, Penz T, Schuster M et al. 2015. NOTCH1 activation in breast cancer confers sensitivity to inhibition of SUMOylation. Oncogene, 34 (29), pp. 3780-3790. | Show Abstract | Read more

Breast cancer is genetically heterogeneous, and recent studies have underlined a prominent contribution of epigenetics to the development of this disease. To uncover new synthetic lethalities with known breast cancer oncogenes, we screened an epigenome-focused short hairpin RNA library on a panel of engineered breast epithelial cell lines. Here we report a selective interaction between the NOTCH1 signaling pathway and the SUMOylation cascade. Knockdown of the E2-conjugating enzyme UBC9 (UBE2I) as well as inhibition of the E1-activating complex SAE1/UBA2 using ginkgolic acid impairs the growth of NOTCH1-activated breast epithelial cells. We show that upon inhibition of SUMOylation NOTCH1-activated cells proceed slower through the cell cycle and ultimately enter apoptosis. Mechanistically, activation of NOTCH1 signaling depletes the pool of unconjugated small ubiquitin-like modifier 1 (SUMO1) and SUMO2/3 leading to increased sensitivity to perturbation of the SUMOylation cascade. Depletion of unconjugated SUMO correlates with sensitivity to inhibition of SUMOylation also in patient-derived breast cancer cell lines with constitutive NOTCH pathway activation. Our investigation suggests that SUMOylation cascade inhibitors should be further explored as targeted treatment for NOTCH-driven breast cancer.

Muellner MK, Duernberger G, Ganglberger F, Kerzendorfer C, Uras IZ, Schoenegger A, Bagienski K, Colinge J, Nijman SMB. 2014. TOPS: a versatile software tool for statistical analysis and visualization of combinatorial gene-gene and gene-drug interaction screens. BMC Bioinformatics, 15 (1), pp. 98. | Show Abstract | Read more

BACKGROUND: Measuring the impact of combinations of genetic or chemical perturbations on cellular fitness, sometimes referred to as synthetic lethal screening, is a powerful method for obtaining novel insights into gene function and drug action. Especially when performed at large scales, gene-gene or gene-drug interaction screens can reveal complex genetic interactions or drug mechanism of action or even identify novel therapeutics for the treatment of diseases.The result of such large-scale screen results can be represented as a matrix with a numeric score indicating the cellular fitness (e.g. viability or doubling time) for each double perturbation. In a typical screen, the majority of combinations do not impact the cellular fitness. Thus, it is critical to first discern true "hits" from noise. Subsequent data exploration and visualization methods can assist to extract meaningful biological information from the data. However, despite the increasing interest in combination perturbation screens, no user friendly open-source program exists that combines statistical analysis, data exploration tools and visualization. RESULTS: We developed TOPS (Tool for Combination Perturbation Screen Analysis), a Java and R-based software tool with a simple graphical user interface that allows the user to import, analyze, filter and plot data from double perturbation screens as well as other compatible data. TOPS was designed in a modular fashion to allow the user to add alternative importers for data formats or custom analysis scripts not covered by the original release.We demonstrate the utility of TOPS on two datasets derived from functional genetic screens using different methods. Dataset 1 is a gene-drug interaction screen and is based on Luminex xMAP technology. Dataset 2 is a gene-gene short hairpin (sh)RNAi screen exploring the interactions between deubiquitinating enzymes and a number of prominent oncogenes using massive parallel sequencing (MPS). CONCLUSIONS: TOPS provides the benchtop scientist with a free toolset to analyze, filter and visualize data from functional genomic gene-gene and gene-drug interaction screens with a flexible interface to accommodate different technologies and analysis algorithms in addition to those already provided here. TOPS is freely available for academic and non-academic users and is released as open source.

Mair B, Kubicek S, Nijman SMB. 2014. Exploiting epigenetic vulnerabilities for cancer therapeutics. Trends Pharmacol Sci, 35 (3), pp. 136-145. | Show Abstract | Read more

Epigenetic deregulation is a hallmark of cancer, and there has been increasing interest in therapeutics that target chromatin-modifying enzymes and other epigenetic regulators. The rationale for applying epigenetic drugs to treat cancer is twofold. First, epigenetic changes are reversible, and drugs could therefore be used to restore the normal (healthy) epigenetic landscape. However, it is unclear whether drugs can faithfully restore the precancerous epigenetic state. Second, chromatin regulators are often mutated in cancer, making them attractive drug targets. However, in most instances it is unknown whether cancer cells are addicted to these mutated chromatin proteins, or whether their mutation merely results in epigenetic instability conducive to the selection of secondary aberrations. An alternative incentive for targeting chromatin regulators is the exploitation of cancer-specific vulnerabilities, including synthetic lethality, caused by epigenetic deregulation. We review evidence for the hypothesis that mechanisms other than oncogene addiction are a basis for the application of epigenetic drugs, and propose future research directions.

Bürckstümmer T, Banning C, Hainzl P, Schobesberger R, Kerzendorfer C, Pauler FM, Chen D, Them N, Schischlik F, Rebsamen M et al. 2013. A reversible gene trap collection empowers haploid genetics in human cells. Nat Methods, 10 (10), pp. 965-971. | Show Abstract | Read more

Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-β, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.

Kretschy N, Teichmann M, Kopf S, Atanasov AG, Saiko P, Vonach C, Viola K, Giessrigl B, Huttary N, Raab I et al. 2013. In vitro inhibition of breast cancer spheroid-induced lymphendothelial defects resembling intravasation into the lymphatic vasculature by acetohexamide, isoxsuprine, nifedipin and proadifen. Br J Cancer, 108 (3), pp. 570-578. | Show Abstract | Read more

BACKGROUND: As metastasis is the prime cause of death from malignancies, there is vibrant interest to discover options for the management of the different mechanistic steps of tumour spreading. Some approved pharmaceuticals exhibit activities against diseases they have not been developed for. In order to discover such activities that might attenuate lymph node metastasis, we investigated 225 drugs, which are approved by the US Food and Drug Administration. METHODS: A three-dimensional cell co-culture assay was utilised measuring tumour cell-induced disintegrations of the lymphendothelial wall through which tumour emboli can intravasate as a limiting step in lymph node metastasis of ductal breast cancer. The disintegrated areas in the lymphendothelial cell (LEC) monolayers were induced by 12(S)-HETE, which is secreted by MCF-7 tumour cell spheroids, and are called 'circular chemorepellent induced defects' (CCIDs). The putative mechanisms by which active drugs prevented the formation of entry gates were investigated by western blotting, NF-κB activity assay and by the determination of 12(S)-HETE synthesis. RESULTS: Acetohexamide, nifedipin, isoxsuprine and proadifen dose dependently inhibited the formation of CCIDs in LEC monolayers and inhibited markers of epithelial-to-mesenchymal-transition and migration. The migration of LECs is a prerequisite of CCID formation, and these drugs either repressed paxillin levels or the activities of myosin light chain 2, or myosin-binding subunit of myosin phosphatase. Isoxsuprine inhibited all three migration markers, and isoxsuprine and acetohexamide suppressed the synthesis of 12(S)-HETE, whereas proadifen and nifedipin inhibited NF-κB activation. Both the signalling pathways independently cause CCID formation. CONCLUSION: The targeting of different mechanisms was most likely the reason for synergistic effects of different drug combinations on the inhibition of CCID formation. Furthermore, the treatment with drug combinations allowed also a several-fold reduction in drug concentrations. These results encourage further screening of approved drugs and their in vivo testing.

Nijman SMB, Friend SH. 2013. Cancer. Potential of the synthetic lethality principle. Science, 342 (6160), pp. 809-811. | Show Abstract | Read more

Elucidating the first principles of synthetic lethality in cancer, including biological context, will assist clinical translation.

Smida M, Nijman SMB. 2012. Functional drug-gene interactions in lung cancer. Expert Rev Mol Diagn, 12 (3), pp. 291-302. | Show Abstract | Read more

Despite the dawn of the genomic information era, the challenges of cancer treatment remain formidable. Particularly for the most prevalent cancer types, including lung cancer, successful treatment of metastatic disease is rare and escalating costs for modern targeted drugs place an increasing strain on healthcare systems. Although powerful diagnostic tools to characterize individual tumor samples in great molecular detail are becoming rapidly available, the transformation of this information into therapy provides a major challenge. A fundamental difficulty is the molecular complexity of cancer cells that often causes drug resistance, but can also render tumors exquisitely sensitive to targeted agents. By using lung cancer as an example, we outline the principles that govern drug sensitivity and resistance from a genetic perspective and discuss how in vitro chemical-genetic screens can impact on patient stratification in the clinic.

Uras IZ, List T, Nijman SMB. 2012. Ubiquitin-specific protease 4 inhibits mono-ubiquitination of the master growth factor signaling kinase PDK1. PLoS One, 7 (2), pp. e31003. | Show Abstract | Read more

BACKGROUND: Phosphorylation by the phospho-inositide-dependent kinase 1 (PDK1) is essential for many growth factor-activated kinases and thus plays a critical role in various processes such as cell proliferation and metabolism. However, the mechanisms that control PDK1 have not been fully explored and this is of great importance as interfering with PDK1 signaling may be useful to treat diseases, including cancer and diabetes. METHODOLOGY/PRINCIPAL FINDINGS: In human cells, few mono-ubiquitinated proteins have been described but in all cases this post-translational modification has a key regulatory function. Unexpectedly, we find that PDK1 is mono-ubiquitinated in a variety of human cell lines, indicating that PDK1 ubiquitination is a common and regulated process. Ubiquitination occurs in the kinase domain of PDK1 yet is independent of its kinase activity. By screening a library of ubiquitin proteases, we further identify the Ubiquitin-Specific Protease 4 (USP4) as an enzyme that removes ubiquitin from PDK1 in vivo and in vitro and co-localizes with PDK1 at the plasma membrane when the two proteins are overexpressed, indicating direct deubiquitination. CONCLUSIONS: The regulated mono-ubiquitination of PDK1 provides an unanticipated layer of complexity in this central signaling network and offers potential novel avenues for drug discovery.

Muellner MK, Uras IZ, Gapp BV, Kerzendorfer C, Smida M, Lechtermann H, Craig-Mueller N, Colinge J, Duernberger G, Nijman SMB. 2011. A chemical-genetic screen reveals a mechanism of resistance to PI3K inhibitors in cancer. Nat Chem Biol, 7 (11), pp. 787-793. | Show Abstract | Read more

Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. However, there has been no systematic approach for analyzing gene-drug interactions in human cells. Here we establish a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, enabling the systematic analysis of thousands of gene-drug interactions. Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug-resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to phosphoinositide 3-kinase (PI3K) inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC over-rode the dependency of cells on the PI3K-mTOR pathway for proliferation. These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.

Winter GE, Rix U, Lissat A, Stukalov A, Müllner MK, Bennett KL, Colinge J, Nijman SM, Kubicek S, Kovar H et al. 2011. An integrated chemical biology approach identifies specific vulnerability of Ewing's sarcoma to combined inhibition of Aurora kinases A and B. Mol Cancer Ther, 10 (10), pp. 1846-1856. | Show Abstract | Read more

Ewing's sarcoma is a pediatric cancer of the bone that is characterized by the expression of the chimeric transcription factor EWS-FLI1 that confers a highly malignant phenotype and results from the chromosomal translocation t(11;22)(q24;q12). Poor overall survival and pronounced long-term side effects associated with traditional chemotherapy necessitate the development of novel, targeted, therapeutic strategies. We therefore conducted a focused viability screen with 200 small molecule kinase inhibitors in 2 different Ewing's sarcoma cell lines. This resulted in the identification of several potential molecular intervention points. Most notably, tozasertib (VX-680, MK-0457) displayed unique nanomolar efficacy, which extended to other cell lines, but was specific for Ewing's sarcoma. Furthermore, tozasertib showed strong synergies with the chemotherapeutic drugs etoposide and doxorubicin, the current standard agents for Ewing's sarcoma. To identify the relevant targets underlying the specific vulnerability toward tozasertib, we determined its cellular target profile by chemical proteomics. We identified 20 known and unknown serine/threonine and tyrosine protein kinase targets. Additional target deconvolution and functional validation by RNAi showed simultaneous inhibition of Aurora kinases A and B to be responsible for the observed tozasertib sensitivity, thereby revealing a new mechanism for targeting Ewing's sarcoma. We further corroborated our cellular observations with xenograft mouse models. In summary, the multilayered chemical biology approach presented here identified a specific vulnerability of Ewing's sarcoma to concomitant inhibition of Aurora kinases A and B by tozasertib and danusertib, which has the potential to become a new therapeutic option.

Schneller D, Machat G, Sousek A, Proell V, van Zijl F, Zulehner G, Huber H, Mair M, Muellner MK, Nijman SMB et al. 2011. p19(ARF) /p14(ARF) controls oncogenic functions of signal transducer and activator of transcription 3 in hepatocellular carcinoma. Hepatology, 54 (1), pp. 164-172. | Show Abstract | Read more

UNLABELLED: Signal transducer and activator of transcription 3 (Stat3) is activated in a variety of malignancies, including hepatocellular carcinoma (HCC). Activation of Ras occurs frequently at advanced stages of HCC by aberrant signaling through growth factor receptors or inactivation of effectors negatively regulating Ras signaling. Here, we addressed the role of Stat3 in Ras-dependent HCC progression in the presence and absence of p19(ARF) /p14(ARF) . We show that constitutive active (ca) Stat3 is tumor suppressive in Ras-transformed p19(ARF-/-) hepatocytes, whereas the expression of Stat3 lacking Tyr(705) phosphorylation (U-Stat3) enhances tumor formation. Accordingly, Ras-transformed Stat3(Δhc) /p19(ARF-/-) hepatocytes (lacking Stat3 and p19(ARF) ) showed increased tumor growth, compared to those expressing Stat3, demonstrating a tumor-suppressor activity of Stat3 in cells lacking p19(ARF) . Notably, endogenous expression of p19(ARF) in Ras-transformed hepatocytes conveyed oncogenic Stat3 functions, resulting in augmented or reduced HCC progression after the expression of caStat3 or U-Stat3, respectively. In accord with these data, the knockdown of p14(ARF) (the human homolog of p19(ARF) ) in Hep3B cells was associated with reduced pY-Stat3 levels during tumor growth to circumvent the tumor-suppressive effect of Stat3. Inhibition of Janus kinases (Jaks) revealed that Jak causes pY-Stat3 activation independently of p14(ARF) levels, indicating that p14(ARF) controls the oncogenic function of pY-Stat3 downstream of Jak. CONCLUSION: These data show evidence that p19(ARF) /p14(ARF) determines the pro- or anti-oncogenic activity of U-Stat3 and pY-Stat3 in Ras-dependent HCC progression.

Carette JE, Guimaraes CP, Wuethrich I, Blomen VA, Varadarajan M, Sun C, Bell G, Yuan B, Muellner MK, Nijman SM et al. 2011. Global gene disruption in human cells to assign genes to phenotypes by deep sequencing. Nat Biotechnol, 29 (6), pp. 542-546. | Show Abstract | Read more

Insertional mutagenesis in a haploid background can disrupt gene function. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT.

Hoermann G, Cerny-Reiterer S, Perné A, Klauser M, Hoetzenecker K, Klein K, Müllauer L, Gröger M, Nijman SMB, Klepetko W et al. 2011. Identification of oncostatin M as a STAT5-dependent mediator of bone marrow remodeling in KIT D816V-positive systemic mastocytosis. Am J Pathol, 178 (5), pp. 2344-2356. | Show Abstract | Read more

Systemic mastocytosis is a neoplastic disease of mast cells harboring the activating KIT mutation D816V. In most patients, mast cell infiltration in the bone marrow is accompanied by marked microenvironment alterations, including increased angiogenesis, osteosclerosis, and sometimes fibrosis. Little is known about the mast cell-derived molecules contributing to these bone marrow alterations. We show here that neoplastic mast cells in patients with systemic mastocytosis express oncostatin M (OSM), a profibrogenic and angiogenic modulator. To study the regulation of OSM expression, KIT D816V was inducibly expressed in Ba/F3 cells and was found to up-regulate OSM mRNA and protein levels, suggesting that OSM is a KIT D816V-dependent mediator. Correspondingly, KIT D816V(+) HMC-1.2 cells expressed significantly higher amounts of OSM than the KIT D816V(-) HMC-1.1 subclone. RNA interference-induced knockdown of STAT5, a key transcription factor in KIT D816V(+) mast cells, inhibited OSM expression in HMC-1 cells, whereas a constitutively activated STAT5 mutant induced OSM expression. Finally, OSM secreted from KIT D816V(+) mast cells stimulated growth of endothelial cells, fibroblasts, and osteoblasts, suggesting that mast cell-derived OSM may serve as a key modulator of the marrow microenvironment and thus contribute to the pathology of systemic mastocytosis.

Nijman SMB. 2011. Synthetic lethality: general principles, utility and detection using genetic screens in human cells. FEBS Lett, 585 (1), pp. 1-6. | Show Abstract | Read more

Synthetic lethality occurs when the simultaneous perturbation of two genes results in cellular or organismal death. Synthetic lethality also occurs between genes and small molecules, and can be used to elucidate the mechanism of action of drugs. This area has recently attracted attention because of the prospect of a new generation of anti-cancer drugs. Based on studies ranging from yeast to human cells, this review provides an overview of the general principles that underlie synthetic lethality and relates them to its utility for identifying gene function, drug action and cancer therapy. It also identifies the latest strategies for the large-scale mapping of synthetic lethalities in human cells which bring us closer to the generation of comprehensive human genetic interaction maps.

Muellner MK, Nijman SMB. 2010. Giving Rho(d) directions. Nat Chem Biol, 6 (6), pp. 397-398. | Read more

Hoshida Y, Nijman SMB, Kobayashi M, Chan JA, Brunet J-P, Chiang DY, Villanueva A, Newell P, Ikeda K, Hashimoto M et al. 2009. Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma. Cancer Res, 69 (18), pp. 7385-7392. | Show Abstract | Read more

Hepatocellular carcinoma (HCC) is a highly heterogeneous disease, and prior attempts to develop genomic-based classification for HCC have yielded highly divergent results, indicating difficulty in identifying unified molecular anatomy. We performed a meta-analysis of gene expression profiles in data sets from eight independent patient cohorts across the world. In addition, aiming to establish the real world applicability of a classification system, we profiled 118 formalin-fixed, paraffin-embedded tissues from an additional patient cohort. A total of 603 patients were analyzed, representing the major etiologies of HCC (hepatitis B and C) collected from Western and Eastern countries. We observed three robust HCC subclasses (termed S1, S2, and S3), each correlated with clinical parameters such as tumor size, extent of cellular differentiation, and serum alpha-fetoprotein levels. An analysis of the components of the signatures indicated that S1 reflected aberrant activation of the WNT signaling pathway, S2 was characterized by proliferation as well as MYC and AKT activation, and S3 was associated with hepatocyte differentiation. Functional studies indicated that the WNT pathway activation signature characteristic of S1 tumors was not simply the result of beta-catenin mutation but rather was the result of transforming growth factor-beta activation, thus representing a new mechanism of WNT pathway activation in HCC. These experiments establish the first consensus classification framework for HCC based on gene expression profiles and highlight the power of integrating multiple data sets to define a robust molecular taxonomy of the disease.

Perne A, Muellner MK, Steinrueck M, Craig-Mueller N, Mayerhofer J, Schwarzinger I, Sloane M, Uras IZ, Hoermann G, Nijman SMB, Mayerhofer M. 2009. Cardiac glycosides induce cell death in human cells by inhibiting general protein synthesis. PLoS One, 4 (12), pp. e8292. | Show Abstract | Read more

BACKGROUND: Cardiac glycosides are Na(+)/K(+)-pump inhibitors widely used to treat heart failure. They are also highly cytotoxic, and studies have suggested specific anti-tumor activity leading to current clinical trials in cancer patients. However, a definitive demonstration of this putative anti-cancer activity and the underlying molecular mechanism has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: Using an unbiased transcriptomics approach, we found that cardiac glycosides inhibit general protein synthesis. Protein synthesis inhibition and cytotoxicity were not specific for cancer cells as they were observed in both primary and cancer cell lines. These effects were dependent on the Na(+)/K(+)-pump as they were rescued by expression of a cardiac glycoside-resistant Na(+)/K(+)-pump. Unlike human cells, rodent cells are largely resistant to cardiac glycosides in vitro and mice were found to tolerate extremely high levels. CONCLUSIONS/SIGNIFICANCE: The physiological difference between human and mouse explains the previously observed sensitivity of human cancer cells in mouse xenograft experiments. Thus, published mouse xenograft models used to support anti-tumor activity for these drugs require reevaluation. Our finding that cardiac glycosides inhibit protein synthesis provides a mechanism for the cytotoxicity of CGs and raises concerns about ongoing clinical trials to test CGs as anti-cancer agents in humans.

Nijman SMB, Hijmans EM, El Messaoudi S, van Dongen MMW, Sardet C, Bernards R. 2006. A functional genetic screen identifies TFE3 as a gene that confers resistance to the anti-proliferative effects of the retinoblastoma protein and transforming growth factor-beta. J Biol Chem, 281 (31), pp. 21582-21587. | Show Abstract | Read more

The helix-loop-helix transcription factor TFE3 has been suggested to play a role in the control of cell growth by acting as a binding partner of transcriptional regulators such as E2F3, SMAD3, and LEF-1. Furthermore, translocations/TFE3 fusions have been directly implicated in tumorigenesis. Surprisingly, however, a direct functional role for TFE3 in the regulation of proliferation has not been reported. By screening retroviral cDNA expression libraries to identify cDNAs that confer resistance to a pRB-induced proliferation arrest, we have found that TFE3 overrides a growth arrest in Rat1 cells induced by pRB and its upstream regulator p16(INK4A). In addition, TFE3 expression blocks the anti-mitogenic effects of TGF-beta in rodent and human cells. We provide data supporting a role for endogenous TFE3 in the direct regulation of CYCLIN E expression in an E2F3-dependent manner. These observations establish TFE3 as a functional regulator of proliferation and offer a potential mechanism for its involvement in cancer.

Oosterkamp HM, Neering H, Nijman SMB, Dirac AMG, Mooi WJ, Bernards R, Brummelkamp TR. 2006. An evaluation of the efficacy of topical application of salicylic acid for the treatment of familial cylindromatosis. Br J Dermatol, 155 (1), pp. 182-185. | Show Abstract | Read more

BACKGROUND: Familial cylindromatosis is a rare genetic disorder, giving rise to neoplasms of the skin appendages. We have recently shown that loss of the cylindromatosis tumour suppressor gene leads to activation of NF-kappaB, a transcription factor having antiapoptotic activity. This provides a possible explanation for the deregulated growth of cylindromas. In cell-based assays, salicylate can prevent NF-kappaB activation caused by loss of the cylindromatosis gene, suggesting that salicylic acid application might be a potential treatment for cylindromatosis. OBJECTIVES: To assess the effectiveness of topical application of salicylic acid on familial cylindromas. METHODS: Cylindromas in five patients from four different cylindromatosis families were treated with twice daily and then once daily topical salicylic acid. Clinical response was determined by serial tumour measurements. RESULTS: In total 17 cylindromas in five patients were studied: 12 target lesions and five control lesions. The median size of the cylindromas was 1.0 cm (range, 0.6-2.8 cm). Two of the 12 cylindromas showed a complete remission. Another eight lesions showed some response, but not sufficient to qualify as partial remission. The control lesions remained stable or increased in size. CONCLUSIONS: Salicylic acid is a well-tolerated and potential new treatment for cylindromatosis.

Huang TT, Nijman SMB, Mirchandani KD, Galardy PJ, Cohn MA, Haas W, Gygi SP, Ploegh HL, Bernards R, D'Andrea AD. 2006. Regulation of monoubiquitinated PCNA by DUB autocleavage. Nat Cell Biol, 8 (4), pp. 339-347. | Show Abstract | Read more

Monoubiquitination is a reversible post-translational protein modification that has an important regulatory function in many biological processes, including DNA repair. Deubiquitinating enzymes (DUBs) are proteases that are negative regulators of monoubiquitination, but little is known about their regulation and contribution to the control of conjugated-substrate levels. Here, we show that the DUB ubiquitin specific protease 1 (USP1) deubiquitinates the DNA replication processivity factor, PCNA, as a safeguard against error-prone translesion synthesis (TLS) of DNA. Ultraviolet (UV) irradiation inactivates USP1 through an autocleavage event, thus enabling monoubiquitinated PCNA to accumulate and to activate TLS. Significantly, the site of USP1 cleavage is immediately after a conserved internal ubiquitin-like diglycine (Gly-Gly) motif. This mechanism is reminiscent of the processing of precursors of ubiquitin and ubiquitin-like modifiers by DUBs. Our results define a regulatory mechanism for protein ubiquitination that involves the signal-induced degradation of an inhibitory DUB.

Nijman SMB, Luna-Vargas MPA, Velds A, Brummelkamp TR, Dirac AMG, Sixma TK, Bernards R. 2005. A genomic and functional inventory of deubiquitinating enzymes. Cell, 123 (5), pp. 773-786. | Show Abstract | Read more

Posttranslational modification of proteins by the small molecule ubiquitin is a key regulatory event, and the enzymes catalyzing these modifications have been the focus of many studies. Deubiquitinating enzymes, which mediate the removal and processing of ubiquitin, may be functionally as important but are less well understood. Here, we present an inventory of the deubiquitinating enzymes encoded in the human genome. In addition, we review the literature concerning these enzymes, with particular emphasis on their function, specificity, and the regulation of their activity.

Nijman SMB, Huang TT, Dirac AMG, Brummelkamp TR, Kerkhoven RM, D'Andrea AD, Bernards R. 2005. The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway. Mol Cell, 17 (3), pp. 331-339. | Show Abstract | Read more

Protein ubiquitination and deubiquitination are dynamic processes implicated in the regulation of numerous cellular pathways. Monoubiquitination of the Fanconi anemia (FA) protein FANCD2 appears to be critical in the repair of DNA damage because many of the proteins that are mutated in FA are required for FANCD2 ubiquitination. By screening a gene family RNAi library, we identify the deubiquitinating enzyme USP1 as a novel component of the Fanconi anemia pathway. Inhibition of USP1 leads to hyperaccumulation of monoubiquitinated FANCD2. Furthermore, USP1 physically associates with FANCD2, and the proteins colocalize in chromatin after DNA damage. Finally, analysis of crosslinker-induced chromosomal aberrations in USP1 knockdown cells suggests a role in DNA repair. We propose that USP1 deubiquitinates FANCD2 when cells exit S phase or recommence cycling after a DNA damage insult and may play a critical role in the FA pathway by recycling FANCD2.

Dirac AMG, Nijman SMB, Brummelkamp TR, Bernards R. 2005. Functional annotation of deubiquitinating enzymes using RNA interference. Methods Enzymol, 398 pp. 554-567. | Show Abstract | Read more

Protein ubiquitination is a dynamic process, depending on a tightly regulated balance between the activity of ubiquitin ligases and their antagonists, the ubiquitin-specific proteases or deubiquitinating enzymes. The family of ubiquitin ligases has been studied intensively and it is well established that their deregulation contributes to diverse disease processes, including cancer. Much less is known about the function and regulation of the large group of deubiquitinating enzymes. This chapter describes how RNA interference against deubiquitinating enzymes can be used to elucidate their function. The application of this technology will greatly improve the functional annotation of this family of proteases.

Brummelkamp TR, Nijman SMB, Dirac AMG, Bernards R. 2003. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB. Nature, 424 (6950), pp. 797-801. | Show Abstract | Read more

Protein modification by the conjugation of ubiquitin moieties--ubiquitination--plays a major part in many biological processes, including cell cycle and apoptosis. The enzymes that mediate ubiquitin-conjugation have been well-studied, but much less is known about the ubiquitin-specific proteases that mediate de-ubiquitination of cellular substrates. To study this gene family, we designed a collection of RNA interference vectors to suppress 50 human de-ubiquitinating enzymes, and used these vectors to identify de-ubiquitinating enzymes in cancer-relevant pathways. We report here that inhibition of one of these enzymes, the familial cylindromatosis tumour suppressor gene (CYLD), having no known function, enhances activation of the transcription factor NF-kappaB. We show that CYLD binds to the NEMO (also known as IKKgamma) component of the IkappaB kinase (IKK) complex, and appears to regulate its activity through de-ubiquitination of TRAF2, as TRAF2 ubiquitination can be modulated by CYLD. Inhibition of CYLD increases resistance to apoptosis, suggesting a mechanism through which loss of CYLD contributes to oncogenesis. We show that this effect can be relieved by aspirin derivatives that inhibit NF-kappaB activity, which suggests a therapeutic intervention strategy to restore growth control in patients suffering from familial cylindromatosis.

de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL. 1998. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature, 396 (6710), pp. 474-477. | Show Abstract | Read more

Rap1 is a small, Ras-like GTPase that was first identified as a protein that could suppress the oncogenic transformation of cells by Ras. Rap1 is activated by several extracellular stimuli and may be involved in cellular processes such as cell proliferation, cell differentiation, T-cell anergy and platelet activation. At least three different second messengers, namely diacylglycerol, calcium and cyclic AMP, are able to activate Rap1 by promoting its release of the guanine nucleotide GDP and its binding to GTP. Here we report that activation of Rap1 by forskolin and cAMP occurs independently of protein kinase A (also known as cAMP-activated protein kinase). We have cloned the gene encoding a guanine-nucleotide-exchange factor (GEF) which we have named Epac (exchange protein directly activated by cAMP). This protein contains a cAMP-binding site and a domain that is homologous to domains of known GEFs for Ras and Rap1. Epac binds cAMP in vitro and exhibits in vivo and in vitro GEF activity towards Rap1. cAMP strongly induces the GEF activity of Epac towards Rap1 both in vivo and in vitro. We conclude that Epac is a GEF for Rap1 that is regulated directly by cAMP and that Epac is a new target protein for cAMP.

Mair B, Konopka T, Kerzendorfer C, Sleiman K, Salic S, Serra V, Muellner MK, Theodorou V, Nijman SMB. 2016. Gain- and Loss-of-Function Mutations in the Breast Cancer Gene GATA3 Result in Differential Drug Sensitivity. PLoS Genet, 12 (9), pp. e1006279. | Show Abstract | Read more

Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.

Gapp BV, Konopka T, Penz T, Dalal V, Bürckstümmer T, Bock C, Nijman SM. 2016. Parallel reverse genetic screening in mutant human cells using transcriptomics. Mol Syst Biol, 12 (8), pp. 879. | Show Abstract | Read more

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease-relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large-scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.

Nijman SMB. 2015. Functional genomics to uncover drug mechanism of action. Nat Chem Biol, 11 (12), pp. 942-948. | Show Abstract | Read more

The upswing in US Food and Drug Administration and European Medicines Agency drug approvals in 2014 may have marked an end to the dry spell that has troubled the pharmaceutical industry over the past decade. Regardless, the attrition rate of drugs in late clinical phases remains high, and a lack of target validation has been highlighted as an explanation. This has led to a resurgence in appreciation of phenotypic drug screens, as these may be more likely to yield compounds with relevant modes of action. However, cell-based screening approaches do not directly reveal cellular targets, and hence target deconvolution and a detailed understanding of drug action are needed for efficient lead optimization and biomarker development. Here, recently developed functional genomics technologies that address this need are reviewed. The approaches pioneered in model organisms, particularly in yeast, and more recently adapted to mammalian systems are discussed. Finally, areas of particular interest and directions for future tool development are highlighted.

Muellner MK, Mair B, Ibrahim Y, Kerzendorfer C, Lechtermann H, Trefzer C, Klepsch F, Müller AC, Leitner E, Macho-Maschler S et al. 2015. Targeting a cell state common to triple-negative breast cancers. Mol Syst Biol, 11 (1), pp. 789. | Show Abstract | Read more

Some mutations in cancer cells can be exploited for therapeutic intervention. However, for many cancer subtypes, including triple-negative breast cancer (TNBC), no frequently recurring aberrations could be identified to make such an approach clinically feasible. Characterized by a highly heterogeneous mutational landscape with few common features, many TNBCs cluster together based on their 'basal-like' transcriptional profiles. We therefore hypothesized that targeting TNBC cells on a systems level by exploiting the transcriptional cell state might be a viable strategy to find novel therapies for this highly aggressive disease. We performed a large-scale chemical genetic screen and identified a group of compounds related to the drug PKC412 (midostaurin). PKC412 induced apoptosis in a subset of TNBC cells enriched for the basal-like subtype and inhibited tumor growth in vivo. We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC. Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells. This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies.

Essletzbichler P, Konopka T, Santoro F, Chen D, Gapp BV, Kralovics R, Brummelkamp TR, Nijman SMB, Bürckstümmer T. 2014. Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res, 24 (12), pp. 2059-2065. | Show Abstract | Read more

Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second gene copy. However, no completely haploid human cell line has been described, hampering the genetic accessibility of a subset of genes. The near-haploid human cell line HAP1 contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15. This large fragment encompasses 330 genes and is integrated on the long arm of Chromosome 19. Here, we employ a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state. Importantly, spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping revealed that engineered-HAPloid (eHAP) cells are fully haploid with no gross chromosomal aberrations induced by Cas9. Furthermore, whole-genome sequence and transcriptome analysis of the parental HAP1 and an eHAP cell line showed that transcriptional changes are limited to the excised Chromosome 15 fragment. Together, we demonstrate the feasibility of efficiently engineering megabase deletions with the CRISPR/Cas9 technology and report the first fully haploid human cell line.

Fece de la Cruz F, Gapp BV, Nijman SMB. 2015. Synthetic lethal vulnerabilities of cancer. Annu Rev Pharmacol Toxicol, 55 (1), pp. 513-531. | Show Abstract | Read more

The great majority of targeted anticancer drugs inhibit mutated oncogenes that display increased activity. Yet many tumors do not contain such actionable aberrations, such as those harboring loss-of-function mutations. The notion of targeting synthetic lethal vulnerabilities in cancer cells has provided an alternative approach to exploiting more of the genetic and epigenetic changes acquired during tumorigenesis. Here, we review synthetic lethality as a therapeutic concept that exploits the inherent differences between normal cells and cancer cells. Furthermore, we provide an overview of the screening approaches that can be used to identify synthetic lethal interactions in human cells and present several recently identified interactions that may be pharmacologically exploited. Finally, we indicate some of the challenges of translating synthetic lethal interactions into the clinic and how these may be overcome.

Mair B, Kubicek S, Nijman SMB. 2014. Exploiting epigenetic vulnerabilities for cancer therapeutics. Trends Pharmacol Sci, 35 (3), pp. 136-145. | Show Abstract | Read more

Epigenetic deregulation is a hallmark of cancer, and there has been increasing interest in therapeutics that target chromatin-modifying enzymes and other epigenetic regulators. The rationale for applying epigenetic drugs to treat cancer is twofold. First, epigenetic changes are reversible, and drugs could therefore be used to restore the normal (healthy) epigenetic landscape. However, it is unclear whether drugs can faithfully restore the precancerous epigenetic state. Second, chromatin regulators are often mutated in cancer, making them attractive drug targets. However, in most instances it is unknown whether cancer cells are addicted to these mutated chromatin proteins, or whether their mutation merely results in epigenetic instability conducive to the selection of secondary aberrations. An alternative incentive for targeting chromatin regulators is the exploitation of cancer-specific vulnerabilities, including synthetic lethality, caused by epigenetic deregulation. We review evidence for the hypothesis that mechanisms other than oncogene addiction are a basis for the application of epigenetic drugs, and propose future research directions.

Bürckstümmer T, Banning C, Hainzl P, Schobesberger R, Kerzendorfer C, Pauler FM, Chen D, Them N, Schischlik F, Rebsamen M et al. 2013. A reversible gene trap collection empowers haploid genetics in human cells. Nat Methods, 10 (10), pp. 965-971. | Show Abstract | Read more

Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-β, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.

Nijman SMB, Friend SH. 2013. Cancer. Potential of the synthetic lethality principle. Science, 342 (6160), pp. 809-811. | Show Abstract | Read more

Elucidating the first principles of synthetic lethality in cancer, including biological context, will assist clinical translation.

Muellner MK, Uras IZ, Gapp BV, Kerzendorfer C, Smida M, Lechtermann H, Craig-Mueller N, Colinge J, Duernberger G, Nijman SMB. 2011. A chemical-genetic screen reveals a mechanism of resistance to PI3K inhibitors in cancer. Nat Chem Biol, 7 (11), pp. 787-793. | Show Abstract | Read more

Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. However, there has been no systematic approach for analyzing gene-drug interactions in human cells. Here we establish a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, enabling the systematic analysis of thousands of gene-drug interactions. Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug-resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to phosphoinositide 3-kinase (PI3K) inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC over-rode the dependency of cells on the PI3K-mTOR pathway for proliferation. These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.

Nijman SMB, Luna-Vargas MPA, Velds A, Brummelkamp TR, Dirac AMG, Sixma TK, Bernards R. 2005. A genomic and functional inventory of deubiquitinating enzymes. Cell, 123 (5), pp. 773-786. | Show Abstract | Read more

Posttranslational modification of proteins by the small molecule ubiquitin is a key regulatory event, and the enzymes catalyzing these modifications have been the focus of many studies. Deubiquitinating enzymes, which mediate the removal and processing of ubiquitin, may be functionally as important but are less well understood. Here, we present an inventory of the deubiquitinating enzymes encoded in the human genome. In addition, we review the literature concerning these enzymes, with particular emphasis on their function, specificity, and the regulation of their activity.

Brummelkamp TR, Nijman SMB, Dirac AMG, Bernards R. 2003. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB. Nature, 424 (6950), pp. 797-801. | Show Abstract | Read more

Protein modification by the conjugation of ubiquitin moieties--ubiquitination--plays a major part in many biological processes, including cell cycle and apoptosis. The enzymes that mediate ubiquitin-conjugation have been well-studied, but much less is known about the ubiquitin-specific proteases that mediate de-ubiquitination of cellular substrates. To study this gene family, we designed a collection of RNA interference vectors to suppress 50 human de-ubiquitinating enzymes, and used these vectors to identify de-ubiquitinating enzymes in cancer-relevant pathways. We report here that inhibition of one of these enzymes, the familial cylindromatosis tumour suppressor gene (CYLD), having no known function, enhances activation of the transcription factor NF-kappaB. We show that CYLD binds to the NEMO (also known as IKKgamma) component of the IkappaB kinase (IKK) complex, and appears to regulate its activity through de-ubiquitination of TRAF2, as TRAF2 ubiquitination can be modulated by CYLD. Inhibition of CYLD increases resistance to apoptosis, suggesting a mechanism through which loss of CYLD contributes to oncogenesis. We show that this effect can be relieved by aspirin derivatives that inhibit NF-kappaB activity, which suggests a therapeutic intervention strategy to restore growth control in patients suffering from familial cylindromatosis.

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