In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fung et al., 2015), that described how we intended to replicate selected experiments from the paper "Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia" (Dawson et al.

The development of selective histone deacetylase (HDAC) inhibitors with anti-cancer and anti-inflammatory properties remains challenging in large part owing to the difficulty of probing the interaction of small molecules with megadalton protein complexes. A combination of affinity capture and quantitative mass spectrometry revealed the selectivity with which 16 HDAC inhibitors target multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex. We identified several non-HDAC targets for hydroxamate inhibitors. HDAC inhibitors with distinct profiles have correspondingly different effects on downstream targets. We also identified the anti-inflammatory drug bufexamac as a class IIb (HDAC6, HDAC10) HDAC inhibitor. Our approach enables the discovery of novel targets and inhibitors and suggests that the selectivity of HDAC inhibitors should be evaluated in the context of HDAC complexes and not purified catalytic subunits.

55 Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which confer a poor prognosis and are often refractory to conventional therapies. Recent efforts have begun to unravel the molecular pathogenesis of these malignancies. Several groups have demonstrated that MLL-fusions associate with two macromolecular chromatin complexes; the polymerase associated factor (PAFc) complex, which interacts with the N-terminal domain of MLL, a portion of the protein that is retained in all the described fusions, or the super elongation complex (SEC), via interaction with the C-terminal fusion partner. These complexes play an integral role in regulating transcriptional elongation and this function appears to be aberrantly co-opted by the MLL-fusions to initiate and perpetuate transcriptional programmes that culminate in leukaemia. In this study we used a systematic global proteomic survey incorporating quantitative mass spectrometry to demonstrate that MLL-fusions, as part of SEC and PAFc complexes, are associated with the BET family of acetyl lysine recognition chromatin “adaptor” proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. Targeting the BET proteins to alter aberrant transcriptional elongation has recently been demonstrated to be possible using small molecule inhibitors that selectively bind the tandem bromodomain at the amino-terminus of the ubiquitously expressed BET proteins (BRD2/BRD3/BRD4). We developed a novel class of potent small molecule inhibitors to the BET family, which is chemically distinct to previously published BET-inhibitors. We then used this new compound (I-BET151) to demonstrate its profound and selective efficacy against human MLL-fusion leukaemic cell lines in liquid culture as well as clonogenic assays in methylcellulose. We also establish that primary murine progenitors retrovirally transformed with MLL-ENL and MLL-AF9 are equally susceptible to treatment with I-BET151. We show that the main phenotypic consequence of BET inhibition in MLL fusion leukaemia is a dramatic early induction of cell cycle arrest and apoptosis. Global gene-expression profiling, following I-BET151 treatment in two different human MLL-fusion leukaemia cell lines (expressing MLL-AF4 and MLL-AF9), highlights a common differentially expressed gene signature that accounts for this phenotype. Importantly, chromatin immunoprecipitation analyses at direct MLL target genes including BCL2, C-MYC and CDK6, indicate that I-BET151 selectively inhibits the recruitment of BET family members BRD3/BRD4, and SEC and PAFc components. These events result in the inefficient phosphorylation and release of paused POL-II from the TSS of these genes providing mechanistic insight into the mode of action of I-BET151 in MLL-fusion leukaemia. We subsequently established the therapeutic efficacy of I-BET151 in vivo by demonstrating dramatic disease control in murine models of MLL-AF4 and MLL-AF9 leukaemia. Finally, we also demonstrate that I-BET151 accelerates apoptosis in primary leukaemic cells from a large number of patients with various MLL-fusion leukaemias, by affecting a similar transcription programme to that identified in the human leukaemic cell lines. Importantly, we also demonstrate that I-BET151 significantly reduces the clonogenic potential of isolated primary leukaemic stem cells, suggesting that disease eradication may be possible. These data highlight a new paradigm for drug discovery targeting the protein-protein interactions of chromatin-associated proteins. We demonstrate that small molecules that perturb the interaction of BRD3/4 with chromatin have therapeutic potential in MLL fusion leukaemias and moreover, we provide the molecular mechanism to account for this therapeutic efficacy. Finally, our results emphasize an emerging role for targeting aberrant transcriptional elongation in oncogenesis. Disclosures: Prinjha: GSK: Employment. Chung:GSK: Employment. Lugo:GSK: Employment. Beinke:GSK: Employment. Soden:GSK: Employment. Mirguet:GSK: Employment. Jeffrey:GSK: Employment. Lee:GSK: Employment. Kouzarides:GSK: Consultancy.

Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has commonly been suggested, and we demonstrate that many of the potential alternative gene products will have markedly different structure and function from their constitutively spliced counterparts. For the vast majority of these alternative isoforms, little evidence exists to suggest they have a role as functional proteins, and it seems unlikely that the spectrum of conventional enzymatic or structural functions can be substantially extended through alternative splicing.function ͉ human ͉ isoforms ͉ splice ͉ structure A lternative mRNA splicing, the generation of a diverse range of mature RNAs, has considerable potential to expand the cellular protein repertoire (1-3), and recent studies have estimated that 40-80% of multiexon human genes can produce differently spliced mRNAs (4, 5). The importance of alternative splicing in processes such as development (6) has long been recognized, and proteins coded by alternatively spliced transcripts have been implicated in a number of cellular pathways (7-9). The extent of alternative splicing in eukaryotic genomes has lead to suggestions that alternative splicing is key to understanding how human complexity can be encoded by so few genes (10).The pilot project of the Encyclopedia of DNA Elements (ENCODE) (11), which aims to identify all the functional elements in the human genome, has undertaken a comprehensive analysis of 44 selected regions that make up 1% of the human genome. One valuable element of the project has been the detailing of a reference set of manually annotated splice variants by the GENCODE consortium (12). The annotation by the GENCODE consortium is an extension of the manually curated annotation by the Havana team at The Sanger Institute.Although a full understanding of the functional implications of alternative splicing is still a long way off, the GENCODE set has provided us with the material to make an in-depth assessment of a systematically collected reference set of splice variants. ResultsAlternative Splicing Frequency. The GENCODE set is made up of 2,608 annotated transcripts for 487 distinct loci. A total of 1,097 transcripts from 434 loci are predicted to be protein coding. There are on average 2.53 protein coding variants per locus; 182 loci have only one variant, whereas one locus, RP1-309K20.2 (CPNE1) has 17 coding variants.A total of 57.8% of the loci are annotated with alternatively spliced transcripts, although there are differences between target re...

BackgroundDespite enormous efforts to combat malaria the disease still afflicts up to half a billion people each year of which more than one million die. Currently no approved vaccine is available and resistances to antimalarials are widely spread. Hence, new antimalarial drugs are urgently needed.ResultsHere, we present a computational analysis of the metabolism of Plasmodium falciparum, the deadliest malaria pathogen. We assembled a compartmentalized metabolic model and predicted life cycle stage specific metabolism with the help of a flux balance approach that integrates gene expression data. Predicted metabolite exchanges between parasite and host were found to be in good accordance with experimental findings when the parasite's metabolic network was embedded into that of its host (erythrocyte). Knock-out simulations identified 307 indispensable metabolic reactions within the parasite. 35 out of 57 experimentally demonstrated essential enzymes were recovered and another 16 enzymes, if additionally the assumption was made that nutrient uptake from the host cell is limited and all reactions catalyzed by the inhibited enzyme are blocked. This predicted set of putative drug targets, shown to be enriched with true targets by a factor of at least 2.75, was further analyzed with respect to homology to human enzymes, functional similarity to therapeutic targets in other organisms and their predicted potency for prophylaxis and disease treatment.ConclusionsThe results suggest that the set of essential enzymes predicted by our flux balance approach represents a promising starting point for further drug development.

Kinetic modelling of complex metabolic networks - a central goal of computational systems biology - is currently hampered by the lack of reliable rate equations for the majority of the underlying biochemical reactions and membrane transporters. On the basis of biochemically substantiated evidence that metabolic control is exerted by a narrow set of key regulatory enzymes, we propose here a hybrid modelling approach in which only the central regulatory enzymes are described by detailed mechanistic rate equations, and the majority of enzymes are approximated by simplified(non mechanistic) rate equations (e.g. mass action, LinLog, Michaelis-Menten and power law) capturing only a few basic kinetic features and hence containing only a small number of parameters to be experimentally determined. To check the reliability of this approach, we have applied it to two different metabolic networks, the energy and redox metabolism of red blood cells, and the purine metabolism of hepatocytes, using in both cases available comprehensive mechanistic models as reference standards. Identification of the central regulatory enzymes was performed by employing only information on network topology and the metabolic data for a single reference state of the network [Grimbs S, Selbig J, Bulik S, Holzhutter HG & Steuer R (2007) Mol Syst Biol 3, 146, doi:10.1038/msb4100186].Calculations of stationary and temporary states under various physiological challenges demonstrate the good performance of the hybrid models. We propose the hybrid modelling approach as a means to speed up the development of reliable kinetic models for complex metabolic networks.

No abstract

Supplementary data are available at Bioinformatics online.