RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia

Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.     

Tissue-specific and reversible RNA interference in transgenic mice

Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice. Transgenic mice harboring a tet-responsive RNA polymerase II promoter driving a microRNA-based short hairpin RNA targeting the tumor suppressor Trp53 reversibly express short hairpin RNA when crossed with existing mouse strains expressing general or tissue-specific 'tet-on' or 'tet-off' transactivators. Reversible Trp53 knockdown can be achieved in several tissues, and restoring Trp53 expression in lymphomas whose development is promoted by Trp53 knockdown leads to tumor regression. By leaving the target gene unaltered, this approach permits tissue-specific, reversible regulation of endogenous gene expression in vivo, with potential broad application in basic biology and drug target validation.     

Photosynthetic microbial mats in the 3,416-Myr-old ocean

Recent re-evaluations of the geological record of the earliest life on Earth have led to the suggestion that some of the oldest putative microfossils and carbonaceous matter were formed through abiotic hydrothermal processes. Similarly, many early Archaean (more than 3,400-Myr-old) cherts have been reinterpreted as hydrothermal deposits rather than products of normal marine sedimentary processes. Here we present the results of a field, petrographic and geochemical study testing these hypotheses for the 3,416-Myr-old Buck Reef Chert, South Africa. From sedimentary structures and distributions of sand and mud, we infer that deposition occurred in normal open shallow to deep marine environments. The siderite enrichment that we observe in deep-water sediments is consistent with a stratified early ocean. We show that most carbonaceous matter was formed by photosynthetic mats within the euphotic zone and distributed as detrital matter by waves and currents to surrounding environments. We find no evidence that hydrothermal processes had any direct role in the deposition of either the carbonaceous matter or the enclosing sediments. Instead, we conclude that photosynthetic organisms had evolved and were living in a stratified ocean supersaturated in dissolved silica 3,416 Myr ago.     

An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo

The application of RNA interference (RNAi) to mammalian systems has the potential to revolutionize genetics and produce novel therapies. Here we investigate whether RNAi applied to a well-characterized gene can stably suppress gene expression in hematopoietic stem cells and produce detectable phenotypes in mice. Deletion of the Trp53 tumor suppressor gene greatly accelerates Myc-induced lymphomagenesis, resulting in highly disseminated disease. To determine whether RNAi suppression of Trp53 could produce a similar phenotype, we introduced several Trp53 short hairpin RNAs (shRNAs) into hematopoietic stem cells derived from E(mu)-Myc transgenic mice, and monitored tumor onset and overall pathology in lethally irradiated recipients. Different Trp53 shRNAs produced distinct phenotypes in vivo, ranging from benign lymphoid hyperplasias to highly disseminated lymphomas that paralleled Trp53-/- lymphomagenesis in the E(mu)-Myc mouse. In all cases, the severity and type of disease correlated with the extent to which specific shRNAs inhibited p53 activity. Therefore, RNAi can stably suppress gene expression in stem cells and reconstituted organs derived from those cells. In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an 'epi-allelic series' for dissecting gene function in vivo.     

Acute physiological responses during blood substitution with colloidal gelatin in conscious rats

The effects of exchange-transfusion with a proprietary gelatin-based plasma volume expander, Haemaccel, have been investigated in conscious, chronically-catheterized rats. No adverse changes in basic cardiovascular or respiratory functions occurred during the procedure although an increase in intravascular fluid sodium but not potassium concentration was observed.     

The stereochemical course of amino acid activation by methionyl- and tyrosyl-tRNA synthetases.

Stereochemical analysis has long been recognised as a powerful tool for elucidating the mechanisms of chemical and enzyme-catalysed reactions. Although much is known about the stereochemical course of reactions at saturated carbon, phosphate and thiophosphate esters whose ligands to phosphorus are also tetrahedrally disposed, are capable in principle of revealing sterochemical information about events at the active site of enzymes that transform such substrates. Nucleotidyl transferases are a group of enzymes which in general selectively use one of the diastereoisomers of a nucleoside 5'(1-thiotriphosphate), such as isomers A and B of adenosine 5'(1-thiotriphosphate), designated ATP alpha S-A and ATP alpha S-B, and allow investigation of the stereochemical course of nucleotidyl transfer. We have developed a simple method based on 31P nuclear magnetic resonance spectroscopy for determining the stereochemical course of these reactions, and using this method show here that the nucleotidyl transfer step in two aminoacyl-tRNA synthetases from Escherichia coli occurs with inversion of configuration at phosphorus. These observations greatly constrain the mechanistic possibilities for these enzymes, and are interpreted most simply as a direct 'in line' transfer from ATP to the amino acid.     

The role of crustecdysone in the moulting crayfish

Résumé La crustecdysone injectée dans l'écrevisse,Procambarus sinulans ne cause pas la mue chez l'animal intact mais l'accélère après ablation des pédoncules oculaires.     

A microRNA polycistron as a potential human oncogene

To date, more than 200 microRNAs have been described in humans; however, the precise functions of these regulatory, non-coding RNAs remains largely obscure. One cluster of microRNAs, the mir-17-92 polycistron, is located in a region of DNA that is amplified in human B-cell lymphomas. Here we compared B-cell lymphoma samples and cell lines to normal tissues, and found that the levels of the primary or mature microRNAs derived from the mir-17-92 locus are often substantially increased in these cancers. Enforced expression of the mir-17-92 cluster acted with c-myc expression to accelerate tumour development in a mouse B-cell lymphoma model. Tumours derived from haematopoietic stem cells expressing a subset of the mir-17-92 cluster and c-myc could be distinguished by an absence of apoptosis that was otherwise prevalent in c-myc-induced lymphomas. Together, these studies indicate that non-coding RNAs, specifically microRNAs, can modulate tumour formation, and implicate the mir-17-92 cluster as a potential human oncogene.