Increased synthesis of Apolipoprotein A-I (ApoA-I) and HDL is believed to provide a new approach to treating atherosclerosis through the stimulation of reverse cholesterol transport. RVX-208 increases the production of ApoA-I in hepatocytes in vitro, and in vivo in monkeys and humans, which results in increased HDL-C, but the molecular target was not previously reported. Using binding assays and X-ray crystallography, we now show that RVX-208 selectively binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a site bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological activity. siRNA experiments further suggest that induction of ApoA-I mRNA is mediated by BET family member BRD4. These data indicate that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggests that BET inhibition may be a promising new approach to the treatment of atherosclerosis.

In bacteria and archaea, small RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci are involved in an adaptable and heritable gene-silencing pathway. Resistance to phage infection is conferred by the incorporation of short invading DNA sequences into the genome as CRISPR spacer elements separated by short repeat sequences. Processing of long primary transcripts (pre-crRNAs) containing these repeats by an RNA endonuclease generates the mature effector RNAs that interfere with phage gene expression. Here we describe structural and functional analyses of the Thermus thermophilus CRISPR Cse3 endonuclease. High-resolution X-ray structures of Cse3 bound to repeat RNAs model both the pre- and post-cleavage complexes associated with processing the pre-crRNA. These structures establish the molecular basis of a specific CRISPR RNA recognition and suggest the mechanism for generation of effector RNAs responsible for gene silencing.

The bromodomain and extraterminal (BET) domain family of proteins binds to acetylated lysines on histones and regulates gene transcription. Recently, BET inhibitors (BETi) have been developed that show promise as potent anticancer drugs against various solid and hematological malignancies. Here we show that the structurally novel and orally bioavailable BET inhibitor RVX2135 inhibits proliferation and induces apoptosis of lymphoma cells arising in Myctransgenic mice in vitro and in vivo. We find that BET inhibition exhibits broad transcriptional effects in Myc-transgenic lymphoma cells affecting many transcription factor networks. By examining the genes induced by BETi, which have largely been ignored to date, we discovered that these were similar to those induced by histone deacetylase inhibitors (HDACi). HDACi also induced cell-cycle arrest and cell death of Myc-induced murine lymphoma cells and synergized with BETi. Our data suggest that BETi sensitize Myc-overexpressing lymphoma cells partly by inducing HDAC-silenced genes, and suggest synergistic and therapeutic combinations by targeting the genetic link between BETi and HDACi.T he bromodomain and extraterminal (BET) domain family of proteins Brd2, Brd3, Brd4, and BrdT bind via their tandem bromodomains (BD1 and BD2) to acetylated lysines in histones and other proteins (1). On binding, they regulate the transcription of genes critical for cell-cycle progression and apoptosis. Therefore, BET proteins have emerged as interesting proteins for targeted intervention of cancer.Recently, the small-molecule BET inhibitor (+)-JQ-1 (hereafter JQ1) was found to be a potent and specific suppressor of B cell-lineage malignancies (2, 3). In acute myelogenous leukemia, BRD4 is essential for tumor maintenance, and JQ1 recapitulates the effects of RNA interference of BRD4 (4, 5). JQ1 was subsequently shown to have an antiproliferative effect in other hematological malignancies and solid organ tumors including glioblastoma, prostate cancer, and neuroblastoma (6-10). The current model of how BET inhibitors (BETi) inhibit tumor cell proliferation places inhibition of MYC as mediating activity in lymphoid tumors, with Myc-independent activity in some solid tumor types such as lung adenocarcinoma (11). However, it has not been clear in hematopoietic tumor types whether the antiproliferative effects of BETi are mediated by suppression of MYC expression or whether effects on MYC are a correlative bystander of the mechanism, perhaps useful as a biomarker but not necessarily mechanistic (12).We have assessed the effect of RVX2135, a novel and orally bioavailable selective inhibitor of Brd2, Brd3, Brd4, and BrdT, in in vitro and in vivo models of Myc-induced lymphoma. We find that the effects are mediated by broad transcriptional changes and that these are genetically and functionally linked to histone deacetylase inhibitors. Results RVX2135 Blocks Proliferation of Myc-Induced Mouse Lymphoma Cellsand Induces Caspase-Dependent Apoptosis. RVX2135 is a novel small-molecule BET bromodoma...

MicroRNAs (miRNAs) regulate gene expression in a variety of biological pathways such as development and tumourigenesis. miRNAs are initially expressed as long primary transcripts (pri-miRNAs) that undergo sequential processing by Drosha and then Dicer to yield mature miRNAs. miR-17~92 is a miRNA cluster that encodes 6 miRNAs and while it is essential for development it also has reported oncogenic activity. To date, the role of RNA structure in miRNA biogenesis has only been considered in terms of the secondary structural elements required for processing of pri-miRNAs by Drosha. Here we report that the miR-17~92 cluster has a compact globular tertiary structure where miRNAs internalized within the core of the folded structure are processed less efficiently than miRNAs on the surface of the structure. Increased miR-92 expression resulting from disruption of the compact miR-17~92 structure results in increased repression of integrin α5 mRNA, a known target of miR-92a. In summary, we describe the first example of pri-miRNA structure modulating differential expression of constituent miRNAs.

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