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.

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During embryonic development, a large number of cells die naturally to shape the new organism. Members of the caspase family of proteases are essential intracellular death effectors. Herein, we generated caspase-2-deficient mice to evaluate the requirement for this enzyme in various paradigms of apoptosis. Excess numbers of germ cells were endowed in ovaries of mutant mice and the oocytes were found to be resistant to cell death following exposure to chemotherapeutic drugs. Apoptosis mediated by granzyme B and perforin was defective in caspase-2-deficient B lymphoblasts. In contrast, cell death of motor neurons during development was accelerated in caspase-2-deficient mice. In addition, caspase-2-deficient sympathetic neurons underwent apoptosis more effectively than wild-type neurons when deprived of NGF. Thus, caspase-2 acts both as a positive and negative cell death effector, depending upon cell lineage and stage of development.

The mammalian DAF-16-like transcription factors, FKHR, FKHRL1, and AFX, function as key regulators of insulin signaling, cell cycle progression, and apoptosis downstream of phosphoinositide 3-kinase. Gene activation through binding to insulin response sequences (IRS) has been thought to be essential for mediating these functions. However, using transcriptional profiling, chromatin immunoprecipitation, and functional experiments, we demonstrate that rather than activation of IRS regulated genes (Class I transcripts), transcriptional repression of D-type cyclins (in Class III) is required for FKHR mediated inhibition of cell cycle progression and transformation. These data suggest that a novel mechanism of FKHR-mediated gene regulation is linked to its activity as a suppressor of tumor growth.

Interleukin-1␤-converting enzyme (ICE)/Ced-3 proteases play a critical role in apoptosis. One well characterized substrate of these proteases is the DNA repair enzyme poly(ADP-ribose) polymerase. We report here that ␣-fodrin, an abundant membrane-associated cytoskeletal protein, is cleaved rapidly and specifically during Fas-and tumor necrosis factor-induced apoptosis; this cleavage is mediated by an ICE/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. Studies in cells treated with these apoptotic stimuli reveal that both fodrin and poly(ADP-ribose) polymerase proteolysis are inhibited by acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and CrmA, specific inhibitors of ICE/ Ced-3 proteases. However, fodrin proteolysis can be distinguished from poly(ADP-ribose) polymerase proteolysis by its relative insensitivity to acetyl-Asp-Glu-ValAsp aldehyde (DEVD-CHO), a selective inhibitor of a subset of ICE/Ced-3 proteases that includes CPP32. DEVD-CHO protects cells from Fas-induced apoptosis but does not prevent fodrin proteolysis, indicating that cleavage of this protein can be uncoupled from apoptotic cell death. Moreover, purified fodrin is cleaved in vitro by CPP32 (but not by ICE) into fragments of the same size observed in vivo during apoptosis. These findings suggest that fodrin proteolysis in vivo may reflect the activity of multiple ICE/Ced-3 proteases whose partial sensitivity to DEVD-CHO reflects a limited contribution from CPP32, or an ICE/Ced-3 protease less sensitive than CPP32 to DEVD-CHO inhibition.

Members of the CED-3/interleukin-1␤-converting enzyme (ICE) protease (caspase) family are synthesized as proforms, which are proteolytically cleaved and activated during apoptosis. We report here that caspase-2 (ICH-1/NEDD-2), a member of the ICE family, is activated during apoptosis by another ICE member, a caspase-3 (CPP32)-like protease(s). When cells are induced to undergo apoptosis, endogenous caspase-2 is first cleaved into three fragments of 32-33 kDa and 14 kDa, which are then further processed into 18-and 12-kDa active subunits. Up to 50 M N-acetyl-Asp-Glu-ValAsp-aldehyde (DEVD-CHO), a caspase-3-preferred peptide inhibitor, inhibits caspase-2 activation and DNA fragmentation in vivo, but does not prevent loss of mitochondrial function, while higher concentrations of DEVD-CHO (>50 M) inhibit both. In comparison, although the activity of caspase-3 is very sensitive to the inhibition of DEVD-CHO (<50 nM), inhibition of caspase-3 activation as marked by processing of the proform requires more than 100 M DEVD-CHO. Our results suggest that the first cleavage of caspase-2 is accomplished by a caspase-3-like activity, and other ICE-like proteases less sensitive to DEVD-CHO may be responsible for activation of caspase-3 and loss of mitochondrial function. Interleukin-1␤-converting enzyme (ICE)1 caspase-1 (1, 2) was identified as the first mammalian homolog of the Caenorhabditis elegans cell death gene product CED-3 (3, 4). Subsequently, a growing number of ICE-like cysteine proteases have been isolated and characterized, including caspase-2 (NEDD-2/ICH-1) (5, 6), caspase-3 (CPP32/YAMA/Apopain) (7,8,39), caspase-6 (Mch-2) (9), caspase-4 (TX/Ich-2/ICE rel II) (10 -12), caspase-5 (ICE rel III) (12), caspase-7 (Mch-3/CMH-1/ICE-LAP3) (13-15), caspase-8 (FLICE/MACH/Mch-5) (16 -18), caspase-10 (Mch-4) (18), and caspase-9 (ICE-LAP6/Mch-6) (19,20). Increasing evidence suggests that caspases play critical roles in the control of programmed cell death (for review, see Refs. 21-23). Microinjection of an expression vector encoding CrmA, a serpin encoded by cowpox virus, inhibits the death of dorsal root ganglia neurons induced by nerve growth factor deprivation (24). Viral inhibitors of caspases, p35 and CrmA, inhibit serum withdrawal-, tumor necrosis factor-, and Fas-induced apoptosis, as well as cytotoxic T lymphocyte (CTL)-mediated apoptosis (6,(25)(26)(27)(28)(29). Ice Ϫ/Ϫ thymocytes undergo apoptosis normally when treated with dexamethasome and ␥-irradiation but are partially resistant to Fas-induced apoptosis (30). Peptide inhibitors of caspases prevent programmed cell death when administered to tissue culture cells and animals (31). These results indicate that the ICE family plays important roles in mammalian apoptosis. The roles played by individual members of the caspase family in controlling apoptosis are the subjects of intensive debates and investigations.Nedd-2, the murine caspase-2, was identified by Kumar et al. (32) as a mRNA expressed mostly during early embryonic brain development and down-regulated in ad...

Changes were introduced into conserved amino acids within the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp4l transmembrane envelope glycoprotein. The effect of these changes on the structure and function of the HIV-1 envelope glycoproteins was examined. The gp4l glycoprotein contains an amino-terminal fusion peptide (residues 512 to 527) and a disulfide loop near the middle of the extracellular domain (residues 598 to 604). Mutations affecting the hydrophobic sequences between these two regions resulted in two phenotypes. Some changes in amino acids 528 to 562 resulted in a loss of the noncovalent association between gp4l and the gpl20 exterior glycoprotein. Amino acid changes in other parts of the gp4l glycoprotein (residues 608 and 628) also resulted in subunit dissociation. Some changes affecting amino acids 568 to 596 resulted in envelope glycoproteins partially or completely defective in mediating membrane fusion. Syncytium formation was more sensitive than virus entry to these changes. Changes in several amino acids from 647 to 675 resulted in higher-than-wild-type syncytium-forming ability. One of these amino acid changes affecting tryptophan 666 resulted in escape from neutralization by an anti-gp4l human monoclonal antibody, 2F5. These results contribute to an understanding of the functional regions of the HIV-1 gp4l ectodomain. Human immunodeficiency virus type 1 (HIV-1) is the cause of AIDS, which is characterized by the depletion of CD4-positive lymphocytes (6, 26, 33). HIV-1 exhibits a tropism for CD4-bearing cells that is mediated by a specific interaction between the viral envelope glycoproteins and the CD4 glycoprotein (18, 44, 53, 55). The HIV-1 envelope glycoproteins are synthesized as a 160-kDa precursor (gp160) that is cleaved into the exterior envelope glycoprotein (gpl20), which binds to the CD4 molecule, and a transmembrane glycoprotein (gp4l) (1, 70, 82). The HIV-1 gpl20 and gp41 glycoproteins remain associated through noncovalent interactions (38, 46, 56). Following CD4 binding, the envelope glycoproteins mediate a pH-independent process that results in the fusion of the viral and target cell membranes and that is necessary for virus entry (37, 77). HIV-1 envelope glycoproteins expressed on the surface of infected cells initiate receptor-binding and membrane fusion events with adjacent CD4-positive cells, resulting in the formation of multinucleated syncytia (52, 75). Structural and mutagenic studies have provided some insight into the molecular events underlying HIV-1 entry and syncytium formation. The CD4-binding region of gpl20 consists of a conformation-dependent structure with elements derived from discontinuous sequences in the third and fourth conserved regions of the glycoprotein (16, 17, 46, 50, 62). The gpl20 glycoprotein binds to the CD4 region bearing similarity to the second complementarity-determining (CDR2) region of immunoglobulins (3, 4, 7, 11, 12, 14, 15, 42, 49, 57, 66). The CDR2-like region of CD4, located in the aminoterminal domain of the glycopro...

The histone methyltransferase enhancer of Zeste homolog 2 (EZH2) is a candidate oncogene due to its prevalent overexpression in malignant diseases, including late stage prostate and breast cancers. The dependency of cancer cells on EZH2 activity is also predicated by recurrent missense mutations residing in the catalytic domain of EZH2 that have been identified in subtypes of diffuse large B cell lymphoma, follicular lymphoma and melanoma. Herein, we report the identification of a highly selective small molecule inhibitor series of EZH2 and EZH1. These compounds inhibit wild-type and mutant versions of EZH2 with nanomolar potency, suppress global histone H3-lysine 27 methylation, affect gene expression, and cause selective proliferation defects. These compounds represent a structurally distinct EZH2 inhibitor chemotype for the exploration of the role of Polycomb Repressive Complex 2-mediated H3K27 methylation in various biological contexts.