The MYC transcription factor is a master regulator of diverse cellular functions and has been long considered a compelling therapeutic target because of its role in a range of human malignancies. However, pharmacologic inhibition of MYC function has proven challenging because of both the diverse mechanisms driving its aberrant expression and the challenge of disrupting protein-DNA interactions. Here, we demonstrate the rapid and potent abrogation of MYC gene transcription by representative small molecule inhibitors of the BET family of chromatin adaptors. MYC transcriptional suppression was observed in the context of the natural, chromosomally translocated, and amplified gene locus. Inhibition of BET bromodomain-promoter interactions and subsequent reduction of MYC transcript and protein levels resulted in G 1 arrest and extensive apoptosis in a variety of leukemia and lymphoma cell lines. Exogenous expression of MYC from an artificial promoter that is resistant to BET regulation significantly protected cells from cell cycle arrest and growth suppression by BET inhibitors. MYC suppression was accompanied by deregulation of the MYC transcriptome, including potent reactivation of the p21 tumor suppressor. Treatment with a BET inhibitor resulted in significant antitumor activity in xenograft models of Burkitt's lymphoma and acute myeloid leukemia. These findings demonstrate that pharmacologic inhibition of MYC is achievable through targeting BET bromodomains. Such inhibitors may have clinical utility given the widespread pathogenetic role of MYC in cancer.

Mouse mammary tumor virus (MMTV) has been classified as a simple retrovirus with two accessory genes, dut and sag. Cloned MMTV proviruses carrying a trimethoprim (trim) cassette in the envelope gene were defective for Gag protein production and the nuclear export of unspliced gag-pol RNA. Complementation experiments indicated that a trans-acting product was responsible for the Gag defect of such mutants. Analysis of MMTV-infected cells revealed the presence of a novel, doubly spliced RNA that encodes a putative product of 301 amino acids. Overexpression of cDNA from this RNA increased Gag levels from env mutant proviruses or reporter gene expression from unspliced mRNAs and allowed detection of a 33-kDa protein product, which has been named regulator of export of MMTV mRNA, or Rem. The Rem N terminus has motifs similar to the Rev-like export proteins of complex retroviruses, and mutation of the nuclear localization signal (NLS) abolished RNA export and detection within the nucleus. The Rem C terminus has few identifiable features, but removal of this domain increased Rem-mediated export, suggesting an autoregulatory function. A reporter vector developed from the 3 end of the MMTV provirus was Rem responsive and required both the presence of the MMTV env-U3 junction and a functional Crm1 pathway. The identification of a third accessory protein from a doubly spliced transcript suggests that MMTV is the first murine complex retrovirus to be documented. Manipulation of the MMTV genome may provide mouse models for human retroviral diseases, such as AIDS.

In recent years, inhibition of the interaction between the bromodomain and extra-terminal domain (BET) family of chromatin adaptors and acetyl-lysine residues on chromatin has emerged as a promising approach to regulate the expression of important disease-relevant genes, including MYC, BCL-2, and NF-κB. Here we describe the identification and characterization of a potent and selective benzoisoxazoloazepine BET bromodomain inhibitor that attenuates BET-dependent gene expression in vivo, demonstrates antitumor efficacy in an MV-4-11 mouse xenograft model, and is currently undergoing human clinical trials for hematological malignancies (CPI-0610).

Mouse mammary tumor virus (MMTV) is a complex murine retrovirus that encodes an HIV Rev-like export protein, Rem, from a doubly spliced version of envelope (Env) mRNA. Previously, the Nterminal 98-amino acid sequence of Rem, which is identical to Env signal peptide (SP), and full-length Rem were shown to be functional in a reporter assay that measures a postexport function. Here we show that MMTV-infected cells or cells transfected with rem or env cDNAs express SP, which is the active component in the reporter assay. Uncleaved Rem was partially glycosylated, but mutations in both glycosylation sites within the C terminus prevented Rem function. Mutations that reduced Rem or Env cleavage by signal peptidase greatly reduced SP levels and functional activity in the reporter assay and allowed accumulation of the uncleaved protein. Fluorescence microscopy revealed that GFP-tagged cleavagesite mutants are unstable and lack fluorescence compared with wild-type Rem, suggesting improper folding. Proteasome inhibitors allowed accumulation of uncleaved Rem relative to SP and increased reporter activity, consistent with SP retrotranslocation and proteasome escape before nuclear entry. Expression of a dominant-negative p97 ATPase did not alter levels of unprocessed Rem and SP but decreased reporter activity, suggesting p97-facilitated retrotranslocation of SP. Our results provide an example of a SP that is processed by signal peptidase and retrotranslocated to allow nuclear localization and function. mouse mammary tumor virus | retrotranslocation | retrovirus | signal peptide | ERAD M ouse mammary tumor virus (MMTV) has provided many insights into cell and cancer biology. MMTV is a complex mouse retrovirus with regulatory features similar to those found in human complex retroviruses, such as HIV and human T-cell leukemia virus (HTLV) (1). These features include the ability to manipulate the immune system and to produce a doubly spliced mRNA encoding a protein (Rem) functionally similar to HIV Rev (1, 2). Rem mRNA is translated in the same reading frame as the MMTV envelope (env) gene but has a deletion of internal sequences encoding the surface (SU) and transmembrane (TM) proteins (1, 2) (Fig. 1A).MMTV Rem is a 301-amino acid protein that regulates expression and export of full-length viral RNA from the nucleus to the cytoplasm using the Crm1 export pathway (1, 2). The Rem protein contains several Rev-like motifs, including a nuclear localization signal (NLS), a nucleolar localization sequence, an arginine-rich RNA-binding motif, and a leucine-rich nuclear export sequence (Fig. 1A). These motifs map to the N-terminal 98 amino acids, which are identical in sequence to the signal peptide (SP) of the MMTV Env protein (1, 2). Similar to HIV Rev or HTLV-1 Rex (3, 4), GFP fusions to Rem or the SP are localized to nucleoli. Both GFPRem and GFP-SP fusions are functional in a reporter assay that measures a postexport function (e.g., translation) (5). Rem-dependent reporter activity requires the NLS as well as the presence of a Rem-re...

Pharmacological inhibition of chromatin co-regulatory factors represents a clinically validated strategy to modulate oncogenic signaling through selective attenuation of gene expression. Here, we demonstrate that CBP/EP300 bromodomain inhibition preferentially abrogates the viability of multiple myeloma cell lines. Selective targeting of multiple myeloma cell lines through CBP/EP300 bromodomain inhibition is the result of direct transcriptional suppression of the lymphocyte-specific transcription factor IRF4, which is essential for the viability of myeloma cells, and the concomitant repression of the IRF4 target gene c-MYC. Ectopic expression of either IRF4 or MYC antagonizes the phenotypic and transcriptional effects of CBP/EP300 bromodomain inhibition, highlighting the IRF4/MYC axis as a key component of its mechanism of action. These findings suggest that CBP/EP300 bromodomain inhibition represents a viable therapeutic strategy for targeting multiple myeloma and other lymphoid malignancies dependent on the IRF4 network.DOI: http://dx.doi.org/10.7554/eLife.10483.001

The identification of a novel series of small molecule BET inhibitors is described. Using crystallographic binding modes of an amino-isoxazole fragment and known BET inhibitors, a structure-based drug design effort lead to a novel isoxazole azepine scaffold. This scaffold showed good potency in biochemical and cellular assays and oral activity in an in vivo model of BET inhibition.

No abstract

No abstract