TDI Publications 2013

Ferguson FM, Fedorov O, Chaikuad A, Philpott M, Muniz JRC, Felletar I, von Delft F, Heightman T, Knapp S, Abell C, Ciulli A. 2013. Targeting low-druggability bromodomains: fragment based screening and inhibitor design against the BAZ2B bromodomain. J Med Chem, 56 (24), pp. 10183-10187. | Citations: 61 (Scopus) | Show Abstract | Read more

Bromodomains are epigenetic reader domains that have recently become popular targets. In contrast to BET bromodomains, which have proven druggable, bromodomains from other regions of the phylogenetic tree have shallower pockets. We describe successful targeting of the challenging BAZ2B bromodomain using biophysical fragment screening and structure-based optimization of high ligand-efficiency fragments into a novel series of low-micromolar inhibitors. Our results provide attractive leads for development of BAZ2B chemical probes and indicate the whole family may be tractable.

Morley AD, Pugliese A, Birchall K, Bower J, Brennan P, Brown N, Chapman T, Drysdale M, Gilbert IH, Hoelder S et al. 2013. Fragment-based hit identification: thinking in 3D. Drug Discov Today, 18 (23-24), pp. 1221-1227. | Citations: 63 (Web of Science Lite) | Show Abstract | Read more

The identification of high-quality hits during the early phases of drug discovery is essential if projects are to have a realistic chance of progressing into clinical development and delivering marketed drugs. As the pharmaceutical industry goes through unprecedented change, there are increasing opportunities to collaborate via pre-competitive networks to marshal multifunctional resources and knowledge to drive impactful, innovative science. The 3D Fragment Consortium is developing fragment-screening libraries with enhanced 3D characteristics and evaluating their effect on the quality of fragment-based hit identification (FBHI) projects.

McGouran JF, Gaertner SR, Altun M, Kramer HB, Kessler BM. 2013. Deubiquitinating enzyme specificity for ubiquitin chain topology profiled by di-ubiquitin activity probes. Chem Biol, 20 (12), pp. 1447-1455. | Citations: 45 (Scopus) | Show Abstract | Read more

Posttranslational modification with ubiquitin (Ub) controls many cellular processes, and aberrant ubiquitination can contribute to cancer, immunopathology, and neurodegeneration. The versatility arises from the ability of Ub to form polymer chains with eight distinct linkages via lysine side chains and the N terminus. In this study, we engineered Di-Ub probes mimicking all eight different poly-Ub linkages and profiled the deubiquitinating enzyme (DUB) selectivity for recognizing Di-Ub moieties in cellular extracts. Mass spectrometric profiling revealed that most DUBs examined have broad selectivity, whereas a subset displays a clear preference for recognizing noncanonical over K48/K63 Ub linkages. Our results expand knowledge of Ub processing enzyme functions in cellular contexts that currently depends largely on using recombinant enzymes and substrates.

Lee R, Adlam D, Fischer R, Di Gleria K, Valli A, Charles P, McGouran J, Ruparelia N, Dawkins S, Kharbanda RK et al. 2013. Integrated Plaque and Plasma Proteo-Lipidomics Reveal Novel Signatures of Coronary Atherosclerotic Plaque Disruption CIRCULATION, 128 (22),

Picaud S, Wells C, Felletar I, Brotherton D, Martin S, Savitsky P, Diez-Dacal B, Philpott M, Bountra C, Lingard H et al. 2013. RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain. Proc Natl Acad Sci U S A, 110 (49), pp. 19754-19759. | Citations: 175 (Scopus) | Show Abstract | Read more

Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo and extraterminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here, we report that RVX-208, a compound currently in phase II clinical trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2s). Cocrystal structures revealed binding modes of RVX-208 and its synthetic precursor, and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene-expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation.

Schäfer G, Milić J, Eldahshan A, Götz F, Zühlke K, Schillinger C, Kreuchwig A, Elkins JM, Abdul Azeez KR, Oder A et al. 2013. Highly functionalized terpyridines as competitive inhibitors of AKAP-PKA interactions. Angew Chem Int Ed Engl, 52 (46), pp. 12187-12191. | Citations: 33 (Scopus) | Show Abstract | Read more

A good fit: Interactions between A-kinase anchoring proteins (AKAPs) and protein kinaseA (PKA) play key roles in a plethora of physiologically relevant processes whose dysregulation causes or is associated with diseases such as heart failure. Terpyridines have been developed as α-helix mimetics for the inhibition of such interactions and are the first biologically active, nonpeptidic compounds that block the AKAP binding site of PKA. © 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Fisher K, Gee F, Wang S, Xue F, Knapp S, Philpott M, Wells C, Rodriguez M, Snoek LB, Kammenga J, Poulin GB. 2013. Maintenance of muscle myosin levels in adult C. elegans requires both the double bromodomain protein BET-1 and sumoylation. Biol Open, 2 (12), pp. 1354-1363. | Citations: 4 (European Pubmed Central) | Show Abstract | Read more

Attenuation of RAS-mediated signalling is a conserved process essential to control cell proliferation, differentiation, and apoptosis. Cooperative interactions between histone modifications such as acetylation, methylation and sumoylation are crucial for proper attenuation in C. elegans, implying that the proteins recognising these histone modifications could also play an important role in attenuation of RAS-mediated signalling. We sought to systematically identify these proteins and found BET-1. BET-1 is a conserved double bromodomain protein that recognises acetyl-lysines on histone tails and maintains the stable fate of various lineages. Unexpectedly, adults lacking both BET-1 and SUMO-1 are depleted of muscle myosin, an essential component of myofibrils. We also show that this muscle myosin depletion does not occur in all animals at a specific time, but rather that the penetrance of the phenotype increases with age. To gain mechanistic insights into this process, we sought to delay the occurrence of the muscle myosin depletion phenotype and found that it requires caspase activity and MEK-dependent signalling. We also performed transcription profiling on these mutants and found an up-regulation of the FGF receptor, egl-15, a tyrosine kinase receptor acting upstream of MEK. Consistent with a MEK requirement, we could delay the muscle phenotype by systemic or hypodermal knock down of egl-15. Thus, this work uncovered a caspase- and MEK-dependent mechanism that acts specifically on ageing adults to maintain the appropriate net level of muscle myosin.

Harrington L, Cheley S, Alexander LT, Knapp S, Bayley H. 2013. Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate. Proc Natl Acad Sci U S A, 110 (47), pp. E4417-E4426. | Citations: 17 (Web of Science Lite) | Show Abstract | Read more

In stochastic sensing, the association and dissociation of analyte molecules is observed as the modulation of an ionic current flowing through a single engineered protein pore, enabling the label-free determination of rate and equilibrium constants with respect to a specific binding site. We engineered sensors based on the staphylococcal α-hemolysin pore to allow the single-molecule detection and characterization of protein kinase-peptide interactions. We enhanced this approach by using site-specific proteolysis to generate pores bearing a single peptide sensor element attached by an N-terminal peptide bond to the trans mouth of the pore. Kinetics and affinities for the Pim protein kinases (Pim-1, Pim-2, and Pim-3) and cAMP-dependent protein kinase were measured and found to be independent of membrane potential and in good agreement with previously reported data. Kinase binding exhibited a distinct current noise behavior that forms a basis for analyte discrimination. Finally, we observed unusually high association rate constants for the interaction of Pim kinases with their consensus substrate Pimtide (~10(7) to 10(8) M(-1) · s(-1)), the result of electrostatic enhancement, and propose a cellular role for this phenomenon.

van Ameijde J, Overvoorde J, Knapp S, den Hertog J, Ruijtenbeek R, Liskamp RMJ. 2013. Real-Time Monitoring of the Dephosphorylating Activity of Protein Tyrosine Phosphatases Using Microarrays with 3-Nitrophosphotyrosine Substrates CHEMPLUSCHEM, 78 (11), pp. 1349-1357. | Citations: 2 (Web of Science Lite) | Show Abstract | Read more

Phosphatases and kinases regulate the crucial phosphorylation post-translational modification. In spite of their similarly important role in many diseases and therapeutic potential, phosphatases have received arguably less attention. One reason for this is a scarcity of high-throughput phosphatase assays. Herein, a new real-time, dynamic protein tyrosine phosphatase (PTP) substrate microarray assay measuring product formation is described. PTP substrates comprising a novel 3-nitrophosphotyrosine residue are immobilized in discrete spots. After reaction catalyzed by a PTP a 3-nitrotyrosine residue is formed that can be detected by specific, sequence-independent antibodies. The resulting microarray was successfully evaluated with a panel of recombinant PTPs and cell lysates, which afforded results comparable to data from other assays. Its parallel nature, convenience, and low sample requirements facilitate investigation of the therapeutically relevant PTP enzyme family. Keeping it real: The activity of important protein tyrosine phosphatases has been monitored in real time in parallel with a novel substrate microarray through formation of 3-nitrotyrosine (see figure). Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Knapp S, Weinmann H. 2013. Small-molecule modulators for epigenetics targets ChemMedChem, 8 (11), pp. 1885-1891. | Citations: 17 (Scopus) | Read more

Nijman SMB, Friend SH. 2013. Cancer. Potential of the synthetic lethality principle. Science, 342 (6160), pp. 809-811. | Citations: 35 (Scopus) | Show Abstract | Read more

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

Fischer R, Bowness P, Kessler BM. 2013. Two birds with one stone: doing metabolomics with your proteomics kit. Proteomics, 13 (23-24), pp. 3371-3386. | Citations: 11 (Scopus) | Show Abstract | Read more

Proteomic research facilities and laboratories are facing increasing demands for the integration of biological data from multiple '-OMICS' approaches. The aim to fully understand biological processes requires the integrated study of genomes, proteomes and metabolomes. While genomic and proteomic workflows are different, the study of the metabolome overlaps significantly with the latter, both in instrumentation and methodology. However, chemical diversity complicates an easy and direct access to the metabolome by mass spectrometry (MS). The present review provides an introduction into metabolomics workflows from the viewpoint of proteomic researchers. We compare the physicochemical properties of proteins and peptides with metabolites/small molecules to establish principle differences between these analyte classes based on human data. We highlight the implications this may have on sample preparation, separation, ionisation, detection and data analysis. We argue that a typical proteomic workflow (nLC-MS) can be exploited for the detection of a number of aliphatic and aromatic metabolites, including fatty acids, lipids, prostaglandins, di/tripeptides, steroids and vitamins, thereby providing a straightforward entry point for metabolomics-based studies. Limitations and requirements are discussed as well as extensions to the LC-MS workflow to expand the range of detectable molecular classes without investing in dedicated instrumentation such as GC-MS, CE-MS or NMR.

Hipp MM, Shepherd D, Gileadi U, Aichinger MC, Kessler BM, Edelmann MJ, Essalmani R, Seidah NG, Reis e Sousa C, Cerundolo V. 2013. Processing of human toll-like receptor 7 by furin-like proprotein convertases is required for its accumulation and activity in endosomes. Immunity, 39 (4), pp. 711-721. | Citations: 37 (Web of Science Lite) | Show Abstract | Read more

Toll-like receptor 7 (TLR7) triggers antiviral immune responses by recognizing viral single-stranded RNA in endosomes, but the biosynthetic pathway of human TLR7 (hTLR7) remains unclear. Here, we show that hTLR7 is proteolytically processed and that the C-terminal fragment selectively accumulates in endocytic compartments. hTLR7 processing occurred at neutral pH and was dependent on furin-like proprotein convertases (PCs). Furthermore, TLR7 processing was required for its functional response to TLR7 agonists such as R837 or influenza virus. Notably, proinflammatory and differentiation stimuli increased the expression of furin-like PCs in immune cells, suggesting a positive feedback mechanism for TLR7 processing during infection. Because self-RNA can under certain conditions activate TLR7 and trigger autoimmunity, our results identify furin-like PCs as a possible target to attenuate TLR7-dependent autoimmunity and other immune pathologies.

Knapp S, Weinmann H. 2013. Small-molecule modulators for epigenetics targets. ChemMedChem, 8 (11), pp. 1885-1891. | Citations: 14 (Web of Science Lite) | Show Abstract | Read more

A capital conference: Influencing epigenetic mechanisms may be highly relevant for future therapies of various diseases such as cancer, inflammation, and metabolic disorders. Leading experts in the field gathered in Berlin on June 5-6, 2013 at a Bayer HealthCare Life Science Workshop to share recent success stories and to discuss future trends.

Vidler LR, Filippakopoulos P, Fedorov O, Picaud S, Martin S, Tomsett M, Woodward H, Brown N, Knapp S, Hoelder S. 2013. Discovery of novel small-molecule inhibitors of BRD4 using structure-based virtual screening. J Med Chem, 56 (20), pp. 8073-8088. | Citations: 77 (Scopus) | Show Abstract | Read more

Bromodomains (BRDs) are epigenetic readers that recognize acetylated-lysine (KAc) on proteins and are implicated in a number of diseases. We describe a virtual screening approach to identify BRD inhibitors. Key elements of this approach are the extensive design and use of substructure queries to compile a set of commercially available compounds featuring novel putative KAc mimetics and docking this set for final compound selection. We describe the validation of this approach by applying it to the first BRD of BRD4. The selection and testing of 143 compounds lead to the discovery of six novel hits, including four unprecedented KAc mimetics. We solved the crystal structure of four hits, determined their binding mode, and improved their potency through synthesis and the purchase of derivatives. This work provides a validated virtual screening approach that is applicable to other BRDs and describes novel KAc mimetics that can be further explored to design more potent inhibitors.

De Nicola GF, Martin ED, Chaikuad A, Bassi R, Clark J, Martino L, Verma S, Sicard P, Tata R, Atkinson RA et al. 2013. Mechanism and consequence of the autoactivation of p38α mitogen-activated protein kinase promoted by TAB1 Nature Structural and Molecular Biology, 20 (10), pp. 1182-1192. | Citations: 40 (Scopus) | Show Abstract | Read more

p38α mitogen-activated protein kinase (p38α) is activated by a variety of mechanisms, including autophosphorylation initiated by TGFβ-activated kinase 1 binding protein 1 (TAB1) during myocardial ischemia and other stresses. Chemical-genetic approaches and coexpression in mammalian, bacterial and cell-free systems revealed that mouse p38α autophosphorylation occurs in cis by direct interaction with TAB1(371-416). In isolated rat cardiac myocytes and perfused mouse hearts, TAT-TAB1(371-416) rapidly activates p38 and profoundly perturbs function. Crystal structures and characterization in solution revealed a bipartite docking site for TAB1 in the p38α C-terminal kinase lobe. TAB1 binding stabilizes active p38α and induces rearrangements within the activation segment by helical extension of the Thr-Gly-Tyr motif, allowing autophosphorylation in cis. Interference with p38α recognition by TAB1 abolishes its cardiac toxicity. Such intervention could potentially circumvent the drawbacks of clinical pharmacological inhibitors of p38 catalytic activity. © 2013 Nature America, Inc. All rights reserved.

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. | Citations: 50 (Scopus) | 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.

Canning P, Cooper CD, Krojer T, Murray JW, Pike AC, Chaikuad A, Keates T, Thangaratnarajah C, Hojzan V, Ayinampudi V et al. 2013. Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases. J Biol Chem, 288 (39), pp. 28304. | Citations: 1 (Scopus) | Read more

Zheng S, Moehlenbrink J, Lu Y-C, Zalmas L-P, Sagum CA, Carr S, McGouran JF, Alexander L, Fedorov O, Munro S et al. 2013. Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1. Mol Cell, 52 (1), pp. 37-51. | Citations: 54 (Scopus) | Show Abstract | Read more

The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase 1 (PRMT1) and symmetric dimethylating PRMT5 and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favors proliferation by antagonizing methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell-cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN downregulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity.

Gammons MV, Fedorov O, Ivison D, Du C, Clark T, Hopkins C, Hagiwara M, Dick AD, Cox R, Harper SJ et al. 2013. Topical antiangiogenic SRPK1 inhibitors reduce choroidal neovascularization in rodent models of exudative AMD. Invest Ophthalmol Vis Sci, 54 (9), pp. 6052-6062. | Citations: 28 (Scopus) | Show Abstract | Read more

PURPOSE: Exudative AMD (wet AMD) is treated by monthly injection into the eye of anti-VEGF proteins. VEGF is alternatively spliced to produce numerous isoforms that differ in angiogenic activity. Serine-rich protein kinase-1 (SRPK1) has been identified as a regulator of pro-angiogenic VEGF splicing by phosphorylating serine-rich splicing factor-1 (SRSF1), which binds to VEGF pre-mRNA. We tested the hypothesis that topical (eye drop) SRPK1-selective inhibitors could be generated that reduce pro-angiogenic isoforms, and prevent choroidal neovascularization in vivo. METHODS: Novel inhibitors were tested for SRPK inhibition in vitro, pro-angiogenic VEGF production in RPE cells by PCR and ELISA, and for inhibition of choroidal neovascularisation in mice and rats. RESULTS: A novel disubstituted furan inhibitor was selective for the SRPK family of kinases and reduced expression of pro-angiogenic but not antiangiogenic VEGF isoforms. This inhibitor and previously identified SRPK inhibitors significantly reduced choroidal neovascularisation in vivo. Topical administration of SRPK inhibitors dose-dependently blocked CNV with an EC50 of 9 μM. CONCLUSIONS: These results indicate that novel SRPK1 selective inhibitors could be a potentially novel topical (eye drop) therapeutic for wet AMD.

Lercher L, McGouran JF, Kessler BM, Schofield CJ, Davis BG. 2013. DNA modification under mild conditions by Suzuki-Miyaura cross-coupling for the generation of functional probes. Angew Chem Int Ed Engl, 52 (40), pp. 10553-10558. | Citations: 56 (Scopus) | Show Abstract | Read more

Quick and clean: A method for Pd-catalyzed Suzuki-Miyaura cross-coupling to iododeoxyuridine (IdU) in DNA is described. Key to the reactivity is the choice of the ligand and the buffer. A covalent [Pd]-DNA intermediate was isolated and characterized. Photocrosslinking probes were generated to trap proteins that bind to epigenetic DNA modifications.

Bogani D, Morgan MAJ, Nelson AC, Costello I, McGouran JF, Kessler BM, Robertson EJ, Bikoff EK. 2013. The PR/SET domain zinc finger protein Prdm4 regulates gene expression in embryonic stem cells but plays a nonessential role in the developing mouse embryo. Mol Cell Biol, 33 (19), pp. 3936-3950. | Citations: 14 (Scopus) | Show Abstract | Read more

Prdm4 is a highly conserved member of the Prdm family of PR/SET domain zinc finger proteins. Many well-studied Prdm family members play critical roles in development and display striking loss-of-function phenotypes. Prdm4 functional contributions have yet to be characterized. Here, we describe its widespread expression in the early embryo and adult tissues. We demonstrate that DNA binding is exclusively mediated by the Prdm4 zinc finger domain, and we characterize its tripartite consensus sequence via SELEX (systematic evolution of ligands by exponential enrichment) and ChIP-seq (chromatin immunoprecipitation-sequencing) experiments. In embryonic stem cells (ESCs), Prdm4 regulates key pluripotency and differentiation pathways. Two independent strategies, namely, targeted deletion of the zinc finger domain and generation of a EUCOMM LacZ reporter allele, resulted in functional null alleles. However, homozygous mutant embryos develop normally and adults are healthy and fertile. Collectively, these results strongly suggest that Prdm4 functions redundantly with other transcriptional partners to cooperatively regulate gene expression in the embryo and adult animal.

Hopkinson RJ, Tumber A, Yapp C, Chowdhury R, Aik W, Che KH, Li XS, Kristensen JBL, King ONF, Chan MC et al. 2013. 5-Carboxy-8-hydroxyquinoline is a Broad Spectrum 2-Oxoglutarate Oxygenase Inhibitor which Causes Iron Translocation. Chem Sci, 4 (8), pp. 3110-3117. | Citations: 72 (Scopus) | Show Abstract | Read more

2-Oxoglutarate and iron dependent oxygenases are therapeutic targets for human diseases. Using a representative 2OG oxygenase panel, we compare the inhibitory activities of 5-carboxy-8-hydroxyquinoline (IOX1) and 4-carboxy-8-hydroxyquinoline (4C8HQ) with that of two other commonly used 2OG oxygenase inhibitors, N-oxalylglycine (NOG) and 2,4-pyridinedicarboxylic acid (2,4-PDCA). The results reveal that IOX1 has a broad spectrum of activity, as demonstrated by the inhibition of transcription factor hydroxylases, representatives of all 2OG dependent histone demethylase subfamilies, nucleic acid demethylases and γ-butyrobetaine hydroxylase. Cellular assays show that, unlike NOG and 2,4-PDCA, IOX1 is active against both cytosolic and nuclear 2OG oxygenases without ester derivatisation. Unexpectedly, crystallographic studies on these oxygenases demonstrate that IOX1, but not 4C8HQ, can cause translocation of the active site metal, revealing a rare example of protein ligand-induced metal movement.

Knapp S. 2013. Testis specific gene expression drives disease progression and Rituximab resistance in lymphoma. EMBO Mol Med, 5 (8), pp. 1149-1150. | Citations: 1 (Scopus) | Read more

Dürnberger G, Bürckstümmer T, Huber K, Giambruno R, Doerks T, Karayel E, Burkard TR, Kaupe I, Müller AC, Schönegger A et al. 2013. Experimental characterization of the human non-sequence-specific nucleic acid interactome. Genome Biol, 14 (7), pp. R81. | Citations: 3 (European Pubmed Central) | Show Abstract | Read more

BACKGROUND: The interactions between proteins and nucleic acids have a fundamental function in many biological processes, including gene transcription, RNA homeostasis, protein translation and pathogen sensing for innate immunity. While our knowledge of the ensemble of proteins that bind individual mRNAs in mammalian cells has been greatly augmented by recent surveys, no systematic study on the non-sequence-specific engagement of native human proteins with various types of nucleic acids has been reported. RESULTS: We designed an experimental approach to achieve broad coverage of the non-sequence-specific RNA and DNA binding space, including methylated cytosine, and tested for interaction potential with the human proteome. We used 25 rationally designed nucleic acid probes in an affinity purification mass spectrometry and bioinformatics workflow to identify proteins from whole cell extracts of three different human cell lines. The proteins were profiled for their binding preferences to the different general types of nucleic acids. The study identified 746 high-confidence direct binders, 139 of which were novel and 237 devoid of previous experimental evidence. We could assign specific affinities for sub-types of nucleic acid probes to 219 distinct proteins and individual domains. The evolutionarily conserved protein YB-1, previously associated with cancer and drug resistance, was shown to bind methylated cytosine preferentially, potentially conferring upon YB-1 an epigenetics-related function. CONCLUSIONS: The dataset described here represents a rich resource of experimentally determined nucleic acid-binding proteins, and our methodology has great potential for further exploration of the interface between the protein and nucleic acid realms.

Guitart AV, Subramani C, Armesilla-Diaz A, Smith G, Sepulveda C, Gezer D, Vukovic M, Dunn K, Pollard P, Holyoake TL et al. 2013. Hif-2α is not essential for cell-autonomous hematopoietic stem cell maintenance. Blood, 122 (10), pp. 1741-1745. | Citations: 35 (Scopus) | Show Abstract | Read more

Local hypoxia in hematopoietic stem cell (HSC) niches is thought to regulate HSC functions. Hypoxia-inducible factor-1 (Hif-1) and Hif-2 are key mediators of cellular responses to hypoxia. Although oxygen-regulated α-subunits of Hifs, namely Hif-1α and Hif-2α, are closely related, they play overlapping and also distinct functions in nonhematopoietic tissues. Although Hif-1α-deficient HSCs lose their activity on serial transplantation, the role for Hif-2α in cell-autonomous HSC maintenance remains unknown. Here, we demonstrate that constitutive or inducible hematopoiesis-specific Hif-2α deletion does not affect HSC numbers and steady-state hematopoiesis. Furthermore, using serial transplantations and 5-fluorouracil treatment, we demonstrate that HSCs do not require Hif-2α to self-renew and recover after hematopoietic injury. Finally, we show that Hif-1α deletion has no major impact on steady-state maintenance of Hif-2α-deficient HSCs and their ability to repopulate primary recipients, indicating that Hif-1α expression does not account for normal behavior of Hif-2α-deficient HSCs.

Da Costa D, Agathanggelou A, Perry T, Weston V, Petermann E, Zlatanou A, Oldreive C, Wei W, Stewart G, Longman J et al. 2013. BET inhibition as a single or combined therapeutic approach in primary paediatric B-precursor acute lymphoblastic leukaemia. Blood Cancer J, 3 (7), pp. e126. | Citations: 40 (Web of Science Lite) | Show Abstract | Read more

Paediatric B-precursor ALL is a highly curable disease, however, treatment resistance in some patients and the long-term toxic effects of current therapies pose the need for more targeted therapeutic approaches. We addressed the cytotoxic effect of JQ1, a highly selective inhibitor against the transcriptional regulators, bromodomain and extra-terminal (BET) family of proteins, in paediatric ALL. We showed a potent in vitro cytotoxic response of a panel of primary ALL to JQ1, independent of their prognostic features but dependent on high MYC expression and coupled with transcriptional downregulation of multiple pro-survival pathways. In agreement with earlier studies, JQ1 induced cell cycle arrest. Here we show that BET inhibition also reduced c-Myc protein stability and suppressed progression of DNA replication forks in ALL cells. Consistent with c-Myc depletion and downregulation of pro-survival pathways JQ1 sensitised primary ALL samples to the classic ALL therapeutic agent dexamethasone. Finally, we demonstrated that JQ1 reduces ALL growth in ALL xenograft models, both as a single agent and in combination with dexamethasone. We conclude that targeting BET proteins should be considered as a new therapeutic strategy for the treatment of paediatric ALL and particularly those cases that exhibit suboptimal responses to standard treatment.

Weirauch U, Beckmann N, Thomas M, Grünweller A, Huber K, Bracher F, Hartmann RK, Aigner A. 2013. Functional role and therapeutic potential of the pim-1 kinase in colon carcinoma. Neoplasia, 15 (7), pp. 783-794. | Citations: 37 (Scopus) | Show Abstract | Read more

PURPOSE: The provirus integration site for Moloney murine leukemia virus 1 (Pim-1) kinase is overexpressed in various tumors and has been linked to poor prognosis. Its role as proto-oncogene is based on several Pim-1 target proteins involved in pivotal cellular processes. Here, we explore the functional relevance of Pim-1 in colon carcinoma. EXPERIMENTAL DESIGN: RNAi-based knockdown approaches, as well as a specific small molecule inhibitor, were used to inhibit Pim-1 in colon carcinoma cells. The effects were analyzed regarding proliferation, apoptosis, sensitization toward cytostatic treatment, and overall antitumor effect in vitro and in mouse tumor models in vivo. RESULTS: We demonstrate antiproliferative, proapoptotic, and overall antitumor effects of Pim-1 inhibition. The sensitization to 5-fluorouracil (5-FU) treatment upon Pim-1 knockdown offers new possibilities for combinatorial treatment approaches. Importantly, this also antagonizes a 5-FU-triggered Pim-1 up-regulation, which is mediated by decreased levels of miR-15b, a microRNA we newly identify to regulate Pim-1. The analysis of the molecular effects of Pim-1 inhibition reveals a complex regulatory network, with therapeutic Pim-1 repression leading to major changes in oncogenic signal transduction with regard to p21(Cip1/WAF1), STAT3, c-jun-N-terminal kinase (JNK), c-Myc, and survivin and in the levels of apoptosis-related proteins Puma, Bax, and Bcl-xL. CONCLUSIONS: We demonstrate that Pim-1 plays a pivotal role in several tumor-relevant signaling pathways and establish the functional relevance of Pim-1 in colon carcinoma. Our results also substantiate the RNAi-mediated Pim-1 knockdown based on polymeric polyethylenimine/small interfering RNA nanoparticles as a promising therapeutic approach.

Eisenreich A, Zakrzewicz A, Huber K, Thierbach H, Pepke W, Goldin-Lang P, Schultheiss H-P, Pries A, Rauch U. 2013. Regulation of pro-angiogenic tissue factor expression in hypoxia-induced human lung cancer cells. Oncol Rep, 30 (1), pp. 462-470. | Citations: 27 (Scopus) | Show Abstract | Read more

Alternative splicing is a key regulatory mechanism for cellular metabolism controlling cell proliferation and angiogenesis, both of which are crucial processes for tumorigenesis under hypoxia. Human cells express two tissue factor (TF) isoforms, alternatively spliced TF (asTF) and 'full length' TF (flTF). flTF is the major source of thrombogenicity whereas, the function of soluble asTF, particularly in cancer, is widely unknown. In the present study, we examined the impact of alternative splicing on the pro-angiogenic potential and the TF expression pattern of A549 cells under hypoxia. We focused our efforts toward alternative splicing factors, such as Clk1, and pro-angiogenic proliferation-regulating factors, such as Cyr61. We further examined the influence of asTF overexpression on the expression of MCP-1, Cyr61 and VEGF, as well as on cell number and pro-angiogenic properties of A549 cells. Notably, we found hypoxia to induce the expression of alternative splicing factors (Clk1 and Clk4) as well as proliferation- and angiogenesis-promoting factors (Cyr61 and flTF). asTF overexpression in A549 cells also increased both cell number and tube formation. These effects were mediated by the induction of Cyr61, MCP-1 and VEGF, as well as by integrin α(v)β(3). Taken together, our results suggest that the pro-angiogenic potential of A549 lung cancer cells is modulated under hypoxic conditions via modulation of TF isoform expression which in turn is controlled by alternative splicing.

McKee CM, Xu D, Kessler BM, Muschel RJ. 2013. Proteomic analysis reveals a proteolytic feedback loop in murine seminal fluid. Prostate, 73 (13), pp. 1427-1440. | Citations: 5 (Scopus) | Show Abstract | Read more

BACKGROUND: Matrix metalloproteinase 9 (MMP9) has been implicated in extracellular matrix (ECM) remodelling, angiogenesis and inflammation. However, the targets for proteolysis that lead to these physiological consequences are often undefined as is the regulation of MMP9 itself. Therefore, identification of both the potential direct and indirect targets of MMP9 is critical for further understanding the effects of its proteolytic cascades. METHODS: To study these cascades on a wider scale, transgenic mouse "knock-out" models and ultra-high performance liquid chromatography mass spectroscopy (UPLC-MS(E) ) were used to elucidate the MMP9 targets, inhibitors, and interactors found in mouse seminal vesicle fluid (SVF). RESULTS: Proteomics analysis of SVF from wild type, mmp9-/- or pn1-/- mice detected differences in serine protease inhibitors (serpins), reproductive proteins, developmental regulators, and cancer proto-oncogenes, including Renin 1/2. Protease nexin 1 (PN1), an ECM-based inhibitor of urokinase, was elevated in the SVF of mmp9-/- mice. We observed that MMP9-mediated N-terminal cleavage of PN1 reduces this serpin's functional activity. Our data also suggest a feedback loop in which inhibition of PN1 is a critical step in permitting greater activity of MMP9. CONCLUSION: This study extends the degradome of MMP9 and examines components relevant to seminal fluid physiology. PN1 is proposed to be a novel inhibitor of MMP9 activity and a block to collagen cleavage, a frequent antecedent to cancer cell invasion. The interaction of MMP9 with PN1 and other serpins may lead to a better understanding of seminal vesicle function and possible impacts on fertility, as well as provide novel therapeutic targets.

Ekambaram R, Enkvist E, Vaasa A, Kasari M, Raidaru G, Knapp S, Uri A. 2013. Selective bisubstrate inhibitors with sub-nanomolar affinity for protein kinase Pim-1. ChemMedChem, 8 (6), pp. 909-913. | Citations: 12 (Scopus) | Show Abstract | Read more

Potent and selective: The unique nature of the ATP binding pocket structure of Pim family protein kinases (PKs) was used for the development of bisubstrate inhibitors and a fluorescent probe with sub-nanomolar affinity. Conjugates of arginine-rich peptides with two ATP mimetic scaffolds were synthesized and tested as inhibitors of Pim-1. Against a panel of 124 protein kinases, a novel ARC-PIM conjugate selectively inhibited PKs of the Pim family.

New M, Olzscha H, Liu G, Khan O, Stimson L, McGouran J, Kerr D, Coutts A, Kessler B, Middleton M, La Thangue NB. 2013. A regulatory circuit that involves HR23B and HDAC6 governs the biological response to HDAC inhibitors. Cell Death Differ, 20 (10), pp. 1306-1316. | Citations: 21 (Scopus) | Show Abstract | Read more

Histone deacetylase (HDAC) is an emergent anticancer target, and HR23B is a biomarker for response to HDAC inhibitors. We show here that HR23B has impacts on two documented effects of HDAC inhibitors; HDAC inhibitors cause apoptosis in cells expressing high levels of HR23B, whereas in cells with low level expression, HDAC inhibitor treatment is frequently associated with autophagy. The mechanism responsible involves the interaction of HDAC6 with HR23B, which downregulates HR23B and thereby reduces the level of ubiquitinated substrates targeted to the proteasome, ultimately desensitising cells to apoptosis. Significantly, the ability of HDAC6 to downregulate HR23B occurs independently of its deacetylase activity. An analysis of the HDAC6 interactome identified HSP90 as a key effector of HDAC6 on HR23B levels. Our results define a regulatory mechanism that involves the interplay between HR23B and HDAC6 that influences the biological outcome of HDAC inhibitor treatment.

Wolf A, Mantri M, Heim A, Müller U, Fichter E, Mackeen MM, Schermelleh L, Dadie G, Leonhardt H, Vénien-Bryan C et al. 2013. The polyserine domain of the lysyl-5 hydroxylase Jmjd6 mediates subnuclear localization. Biochem J, 453 (3), pp. 357-370. | Citations: 15 (Web of Science Lite) | Show Abstract | Read more

Jmjd6 (jumonji-domain-containing protein 6) is an Fe(II)- and 2OG (2-oxoglutarate)-dependent oxygenase that catalyses hydroxylation of lysine residues in proteins involved in pre-mRNA splicing. Jmjd6 plays an essential role in vertebrate embryonic development and has been shown to modulate alternative splicing in response to hypoxic stress. In the present study we show that an alternatively spliced version of Jmjd6 lacking the polyS (polyserine) domain localizes to the nucleolus, predominantly in the fibrillar centre. Jmjd6 with the polyS domain deleted also interacts with nucleolar proteins. Furthermore, co-immunoprecipitation experiments and F2H (fluorescent 2-hybrid) assays demonstrate that Jmjd6 homo-oligomerization occurs in cells. In correlation with the observed variations in the subnuclear distribution of Jmjd6, the structure of Jmjd6 oligomers in vitro changes in the absence of the polyS domain, possibly reflecting the role of the polyS domain in nuclear/nucleolar shuttling of Jmjd6.

Bishop T, Talbot NP, Turner PJ, Nicholls LG, Pascual A, Hodson EJ, Douglas G, Fielding JW, Smith TG, Demetriades M et al. 2013. Carotid body hyperplasia and enhanced ventilatory responses to hypoxia in mice with heterozygous deficiency of PHD2. J Physiol, 591 (14), pp. 3565-3577. | Citations: 29 (Web of Science Lite) | Show Abstract | Read more

Oxygen-dependent prolyl hydroxylation of hypoxia-inducible factor (HIF) by a set of closely related prolyl hydroxylase domain enzymes (PHD1, 2 and 3) regulates a range of transcriptional responses to hypoxia. This raises important questions about the role of these oxygen-sensing enzymes in integrative physiology. We investigated the effect of both genetic deficiency and pharmacological inhibition on the change in ventilation in response to acute hypoxic stimulation in mice. Mice exposed to chronic hypoxia for 7 days manifest an exaggerated hypoxic ventilatory response (HVR) (10.8 ± 0.3 versus 4.1 ± 0.7 ml min(-1) g(-1) in controls; P < 0.01). HVR was similarly exaggerated in PHD2(+/-) animals compared to littermate controls (8.4 ± 0.7 versus 5.0 ± 0.8 ml min(-1) g(-1); P < 0.01). Carotid body volume increased (0.0025 ± 0.00017 in PHD2(+/-) animals versus 0.0015 ± 0.00019 mm(3) in controls; P < 0.01). In contrast, HVR in PHD1(-/-) and PHD3(-/-) mice was similar to littermate controls. Acute exposure to a small molecule PHD inhibitor (PHI) (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid) did not mimic the ventilatory response to hypoxia. Further, 7 day administration of the PHI induced only modest increases in HVR and carotid body cell proliferation, despite marked stimulation of erythropoiesis. This was in contrast with chronic hypoxia, which elicited both exaggerated HVR and cellular proliferation. The findings demonstrate that PHD enzymes modulate ventilatory sensitivity to hypoxia and identify PHD2 as the most important enzyme in this response. They also reveal differences between genetic inactivation of PHDs, responses to hypoxia and responses to a pharmacological inhibitor, demonstrating the need for caution in predicting the effects of therapeutic modulation of the HIF hydroxylase system on different physiological responses.

Elkins JM, Wang J, Deng X, Pattison MJ, Arthur JSC, Erazo T, Gomez N, Lizcano JM, Gray NS, Knapp S. 2013. X-ray crystal structure of ERK5 (MAPK7) in complex with a specific inhibitor. J Med Chem, 56 (11), pp. 4413-4421. | Citations: 16 (Scopus) | Show Abstract | Read more

The protein kinase ERK5 (MAPK7) is an emerging drug target for a variety of indications, in particular for cancer where it plays a key role mediating cell proliferation, survival, epithelial-mesenchymal transition, and angiogenesis. To date, no three-dimensional structure has been published that would allow rational design of inhibitors. To address this, we determined the X-ray crystal structure of the human ERK5 kinase domain in complex with a highly specific benzo[e]pyrimido[5,4-b]diazepine-6(11H)-one inhibitor. The structure reveals that specific residue differences in the ATP-binding site, compared to the related ERKs p38s and JNKs, allow for the development of ERK5-specific inhibitors. The selectivity of previously observed ERK5 inhibitors can also be rationalized using this structure, which provides a template for future development of inhibitors with potential for treatment of disease.

Chowdhury R, Candela-Lena JI, Chan MC, Greenald DJ, Yeoh KK, Tian Y-M, McDonough MA, Tumber A, Rose NR, Conejo-Garcia A et al. 2013. Selective small molecule probes for the hypoxia inducible factor (HIF) prolyl hydroxylases. ACS Chem Biol, 8 (7), pp. 1488-1496. | Citations: 51 (Scopus) | Show Abstract | Read more

The hypoxia inducible factor (HIF) system is central to the signaling of low oxygen (hypoxia) in animals. The levels of HIF-α isoforms are regulated in an oxygen-dependent manner by the activity of the HIF prolyl-hydroxylases (PHD or EGLN enzymes), which are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Here, we describe biochemical, crystallographic, cellular profiling, and animal studies on PHD inhibitors including selectivity studies using a representative set of human 2OG oxygenases. We identify suitable probe compounds for use in studies on the functional effects of PHD inhibition in cells and in animals.

Lu M, Breyssens H, Salter V, Zhong S, Hu Y, Baer C, Ratnayaka I, Sullivan A, Brown NR, Endicott J et al. 2013. Restoring p53 function in human melanoma cells by inhibiting MDM2 and cyclin B1/CDK1-phosphorylated nuclear iASPP. Cancer Cell, 23 (5), pp. 618-633. | Citations: 79 (Scopus) | Show Abstract | Read more

Nearly 90% of human melanomas contain inactivated wild-type p53, the underlying mechanisms for which are not fully understood. Here, we identify that cyclin B1/CDK1-phosphorylates iASPP, which leads to the inhibition of iASPP dimerization, promotion of iASPP monomer nuclear entry, and exposure of its p53 binding sites, leading to increased p53 inhibition. Nuclear iASPP is enriched in melanoma metastasis and associates with poor patient survival. Most wild-type p53-expressing melanoma cell lines coexpress high levels of phosphorylated nuclear iASPP, MDM2, and cyclin B1. Inhibition of MDM2 and iASPP phosphorylation with small molecules induced p53-dependent apoptosis and growth suppression. Concurrent p53 reactivation and BRAFV600E inhibition achieved additive suppression in vivo, presenting an alternative for melanoma therapy.

Soundararajan M, Roos AK, Savitsky P, Filippakopoulos P, Kettenbach AN, Olsen JV, Gerber SA, Eswaran J, Knapp S, Elkins JM. 2013. Structures of Down syndrome kinases, DYRKs, reveal mechanisms of kinase activation and substrate recognition. Structure, 21 (6), pp. 986-996. | Citations: 47 (Scopus) | Show Abstract | Read more

Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinases (DYRKs) play key roles in brain development, regulation of splicing, and apoptosis, and are potential drug targets for neurodegenerative diseases and cancer. We present crystal structures of one representative member of each DYRK subfamily: DYRK1A with an ATP-mimetic inhibitor and consensus peptide, and DYRK2 including NAPA and DH (DYRK homology) box regions. The current activation model suggests that DYRKs are Ser/Thr kinases that only autophosphorylate the second tyrosine of the activation loop YxY motif during protein translation. The structures explain the roles of this tyrosine and of the DH box in DYRK activation and provide a structural model for DYRK substrate recognition. Phosphorylation of a library of naturally occurring peptides identified substrate motifs that lack proline in the P+1 position, suggesting that DYRK1A is not a strictly proline-directed kinase. Our data also show that DYRK1A wild-type and Y321F mutant retain tyrosine autophosphorylation activity.

Adam J, Yang M, Bauerschmidt C, Kitagawa M, O'Flaherty L, Maheswaran P, Özkan G, Sahgal N, Baban D, Kato K et al. 2013. A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia. Cell Rep, 3 (5), pp. 1440-1448. | Citations: 38 (Scopus) | Show Abstract | Read more

The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

Hipp MM, Shepherd D, Gileadi U, Aichinger MC, Kessler BM, Edelmann MJ, Essalmani R, Seidah NG, Reis e Sousa C, Cerundolo V. 2013. Processing of human toll-like receptor 7 by furin-like proprotein convertases is required for its accumulation and activity in endosomes. Immunity, 39 (4), pp. 711-721. | Citations: 38 (Scopus) | Show Abstract | Read more

Toll-like receptor 7 (TLR7) triggers antiviral immune responses by recognizing viral single-stranded RNA in endosomes, but the biosynthetic pathway of human TLR7 (hTLR7) remains unclear. Here, we show that hTLR7 is proteolytically processed and that the C-terminal fragment selectively accumulates in endocytic compartments. hTLR7 processing occurred at neutral pH and was dependent on furin-like proprotein convertases (PCs). Furthermore, TLR7 processing was required for its functional response to TLR7 agonists such as R837 or influenza virus. Notably, proinflammatory and differentiation stimuli increased the expression of furin-like PCs in immune cells, suggesting a positive feedback mechanism for TLR7 processing during infection. Because self-RNA can under certain conditions activate TLR7 and trigger autoimmunity, our results identify furin-like PCs as a possible target to attenuate TLR7-dependent autoimmunity and other immune pathologies.

Picaud S, Da Costa D, Thanasopoulou A, Filippakopoulos P, Fish PV, Philpott M, Fedorov O, Brennan P, Bunnage ME, Owen DR et al. 2013. PFI-1, a highly selective protein interaction inhibitor, targeting BET Bromodomains. Cancer Res, 73 (11), pp. 3336-3346. | Citations: 144 (Scopus) | Show Abstract | Read more

Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are transcriptional regulators required for efficient expression of several growth promoting and antiapoptotic genes as well as for cell-cycle progression. BET proteins are recruited on transcriptionally active chromatin via their two N-terminal bromodomains (BRD), a protein interaction module that specifically recognizes acetylated lysine residues in histones H3 and H4. Inhibition of the BET-histone interaction results in transcriptional downregulation of a number of oncogenes, providing a novel pharmacologic strategy for the treatment of cancer. Here, we present a potent and highly selective dihydroquinazoline-2-one inhibitor, PFI-1, which efficiently blocks the interaction of BET BRDs with acetylated histone tails. Cocrystal structures showed that PFI-1 acts as an acetyl-lysine (Kac) mimetic inhibitor efficiently occupying the Kac binding site in BRD4 and BRD2. PFI-1 has antiproliferative effects on leukemic cell lines and efficiently abrogates their clonogenic growth. Exposure of sensitive cell lines with PFI-1 results in G1 cell-cycle arrest, downregulation of MYC expression, as well as induction of apoptosis and induces differentiation of primary leukemic blasts. Intriguingly, cells exposed to PFI-1 showed significant downregulation of Aurora B kinase, thus attenuating phosphorylation of the Aurora substrate H3S10, providing an alternative strategy for the specific inhibition of this well-established oncology target.

Kumar R, Horvath A, Mazumder R, Toi M, Sato F, Pillai MR, Costa L, Carmo-Fonseca M, Knapp S, Dutt A et al. 2013. The Global Cancer Genomics Consortium's Second Annual Symposium: Genomics Medicine in Cancer Research Genes & Cancer, 4 (5-6), pp. 196-200. | Show Abstract | Read more

The Second Annual Symposium of the Global Cancer Genomics Consortium (GCGC) was held at the Tata Memorial Center in Mumbai, India, from November 19 to 20, 2012. Founded in late 2010, the GCGC aims to provide a platform for highly productive, collaborative efforts on next-generation cancer research through bridging the latest scientific and technology developments with clinical oncology challenges. This year's presenters brought together highly innovative interdisciplinary views and strategies to meet major challenges in cancer research. The symposium featured 3 major themes: OMICS approaches toward the identification of cancer molecular drivers, single-cell analysis in cancer, and clinical and translational genomics. Each theme was represented in presentations of new findings, with an obvious implication in cross-disciplinary components of OMICs and an overwhelming participation by students. In summary, the GCGC symposium provided a discussion and congregation of the latest advances in basic and translational cancer research and offered the participants with a highly cooperative network environment for future collaboration. © The Author(s) 2013.

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