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TRICKING CANCER, THE CDK WAY
this occurs by counteracting the positive feedback loop of MYCN-driven transcriptional amplification and that superenhancers, like the one associated with MYCN’s expression itself, are especially sensitive to the drug. This study demonstrates how careful inhibition of a ubiquitous and important regulator like a CDK can prove fruitful and may someday yield better therapy options for MYC-driven cancers. Jason G. Underwood, Ph.D.
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HARNESSING THE POWER OF THE SUN... TAG
Reprinted from Cell 159, Chipumuro, E., et al., CDK7 Inhibition Suppresses Super-Enhancer-Linked Oncogenic Transcription in MYCN-Driven Cancer, DOI: 10.1016/j.cell.2014.10.024. Copyright 2014, with permission from Elsevier. Reprinted from Cell 159, Tanenbaum, M. E., et al., A ProteinTagging System for Signal Amplification in Gene Expression and Fluorescence Imaging, DOI: 10.1016/j.cell.2014.09.039. Copyright 2014, with permission from Elsevier.
The MYC family of transcription factors participates in the expression of most nuclear genes including those involved in essential cellular processes such as cell cycle progression and apoptosis. Due to its many functions, perhaps it is not surprising that mutations, translocations, or genomic amplifications involving MYC lead to a variety of cancers in multiple tissues. Disregulated MYC can drive a cascade of proliferative events as many transcript levels are ramped up, including that of the driver itself. Other important players in orchestrating transcription are the cyclin-dependent kinases (CDKs), some which phosphorylate Pol II to modulate its initiation and elongation activities. Now, a group (Chipumuro et al. Cell 2014, DOI: 10.1016/j.cell.2014.10.024) has taken lessons from both MYC and the CDKs to turn down the runaway transcription associated with many MYC-driven cancers. The researchers chose to target neuroblastoma, a cancer type that usually affects the very young, and a subset is characterized by high genomic amplification of the neural MYC family member, MYCN, conferring a poor prognosis. Neuroblastoma cell lines with and without amplified MYCN were used as a test set and controls, respectively, for the studies. After screening a variety of transcription-associated CDK inhibitors, a first-inclass covalent inhibitor of CDK7, THZ1, was shown to be far more potent in killing MYCN-amplified cell lines than those lacking amplification. The drug was tested in a tumor assay in mice and found to be a potent inhibitor of tumor growth with low toxicity. Gene expression studies demonstrated that THZ1 acts to turn down transcription on a nearly global scale in the MYCN-amplified cell lines. Further experiments showed that © 2014 American Chemical Society
Comparing the inner workings of single cells has improved significantly with advancements in microscopy and high throughput sequencing technologies. Enabling such fine grain observations are improvements in signal amplification, and in the case of microscopy, multimers of chemical or protein fluorophores are often the tool of choice. Prior studies have demonstrated single molecule sensitivity in detecting mRNA by delivering a fluorescently tagged RNA-binding protein to the cell along with an expression construct expressing multimers of its cognate binding sequence. Delivering many fluorescent proteins to one place on the RNA affords the necessary sensitivity of detection. Similarly, functional assays can benefit from fusing a candidate RNA processing or decay factor to that RNA binding protein since the multimer target can amplify the effect that the candidate has on the construct’s gene expression. Now, a new study (Tanenbaum et al. Cell 2014, DOI: 10.1016/ j.cell.2014.09.039) takes the multimer concept back to the protein side, with SunTag, a tandem array of identical peptides that can recruit many copies of an antipeptide antibody. The key to sensitive detection was identifying the ideal combination of a soluble peptide that could be expressed in tandem and a single-chain antibody to recognize that peptide. Published: December 19, 2014 2699
dx.doi.org/10.1021/cb501003y | ACS Chem. Biol. 2014, 9, 2699−2701
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reducing end or an azido group at the nonreducing end could stimulate the production of GH-, SSEA3-, and SSEA4-targeting antibodies, with the latter vaccine eliciting an especially favorable high ratio of IgG/IgM antibodies not usually achieved in anticancer vaccines. Encouragingly, the antibodies produced in response to these vaccines mediated complement-dependent cytotoxicity toward cultured GH-positive human breast cancer cells. Heidi A. Dahlmann, Ph.D.
After a good pair was identified, further optimization led to a peptide array that could be expressed at even higher levels in vivo. By expressing a protein fused to SunTag, the researchers could visualize single protein molecules by virtue of a bound array of 24 antibody-GFP fusions, a tag so large that it actually affected the rate of protein diffusion. The researchers demonstrate that the SunTag can be used to light up organelles or to watch cytoskeletal motors in action. Targetting a SunTag to a specific DNA location by fusing it to a catalytically dead Cas9 and a crRNA showed yet another way to amplify the signal, but in this case it was gene expression as the cognate antibody was fused to a transcriptional activation domain instead of GFP. This study shows an exciting direction for localizing tagged proteins in the cell and combining that tag with a cleverly engineered antibody fusion protein delivers a variety of high utility opportunities. Jason G. Underwood, Ph.D.
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POTENT AND STABLE INHIBITORS OF ACID CERAMIDASE
MODIFIED CARBOHYDRATES SHARPEN IMMUNE RESPONSE
Reprinted with permission from Lee, H.-Y., et al., J. Am. Chem. Soc., DOI: 10.1021/ja508040d. Copyright 2014 American Chemical Society.
Adapted with permission from Angew. Chem., Int. Ed. from Wiley-VCH, Pizzirani, D., et al., 2014, DOI: 10.1002/ anie.201409042.
Over the past century, vaccines that prime the immune system to recognize nonself antigens such as viral and bacterial invaders have become ubiquitous. In a new twist, researchers have begun to develop therapeutic cancer vaccines, taking on the unique challenge of sharpening the immune response toward endogenously produced molecules. To make these vaccines work, researchers typically target molecules that are differentially expressed on cancer cells versus normal cell surfaces. Among the plethora of molecules in extracellular matrix is Globo H (GH), a carbohydrate overexpressed in many types of human cancer cells. Chi-Huey Wong and co-workers originally reported an anticancer vaccine consisting of GH cross-linked to diphtheria toxoid cross-reactive material (CRM197) as a carrier and the glycolipid C34 as an adjuvant (Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 2517). In their recent attempts to improve the immunogenicity of the vaccine, the research team synthesized a variety of GH derivatives containing modifications at either the reducing end or the nonreducing end of the carbohydrate and prepared next-generation vaccines using the previously optimized DT carrier and C34 adjuvant (J. Am. Chem. Soc., 2014, DOI: 10.1021/ja508040d). To determine if the GH modifications affected the immunogenicity of the vaccines, the research team administered them to mice and after 10 days collected the mice serum, which was subsequently screened for antibodies active against GH and the structurally similar stage-specific embryonic antigens 3 and 4 (SSEA3 and SSEA4). The team found that vaccines containing GH modified with a fluoro, azido, or phenyl group at the
Ceramides, N-acetylated amino alcohols adorned with long-chain fatty acids, are believed to play a role in cellular senescence, inflammation, and apoptosis. These multipurpose biomolecules can be broken down by an enzyme called acid ceramidase (AC) to form sphingosine, a precursor of sphingosine-1-phosphate (S1P). S1P activates G protein-coupled receptors to enhance cell survival and proliferation, two processes which are critical for tumor formation. The intracellular ceramide-to-sphingosine ratio is controlled by AC activity; inhibition of AC activity would allow a higher concentration of ceramide to persist in cells, enabling cells to undergo apoptosis upon sustaining high levels of damage and thus suppressing tumor formation. Therefore, AC inhibitors have been pursued as potential anticancer therapeutics and as tools for investigating the pathophysiological roles of ceramide. However, most reported AC inhibitors have either low potency or are chemically or metabolically unstable under physiologically relevant conditions, rendering them ineffective in vivo. A newly reported class of AC inhibitors, benzoxazolone carboxamides, appears to circumvent these shortcomings; according to Daniele Piomelli and co-workers, these AC inhibitors are the first to have high potency (with IC50 values below 100 nM) as well as pronounced systemic activity (Angew. Chem., Int. Ed. 2014, DOI: 10.1002/anie.201409042). The urea moieties of the benzoxazalone carboxamides undergo nucleophilic attack by the active cysteine of AC, causing the inhibitors to covalently bind to and inactivate the enzyme. The reported inhibitors strike a balance between potency and 2700
dx.doi.org/10.1021/cb501003y | ACS Chem. Biol. 2014, 9, 2699−2701
ACS Chemical Biology
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structures, may affect many aspects of mRNA metabolism, and they suggest that misregulation of mRNA modification may be involved in the pathology of diseases associated with PUS mutations. Heidi A. Dahlmann, Ph.D.
chemical stability; modifications to enhance their potency by making the inhibitors more electrophilic rendered the molecules more susceptible to decomposition in a buffer. Further testing showed that the most stable reported inhibitor effectively reduced AC activity and significantly increased the ceramide-to-sphingosine ratio in both cultured cells as well as in the multiple organs of treated mice. The authors expect that benzoxazolone carboxamides will serve as leads for novel therapeutic agents and as probes for basic research. Heidi A. Dahlmann, Ph.D.
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DETECTING THE UNEXPECTED: PSEUDOURIDINE IN MRNA
Adapted by permission from Macmillan Publishers Ltd.: Nature Gilbert, W. V., et al., 515, 143−146, copyright 2014.
Uridine, one of the canonical nucleotides that makes up RNA, can be isomerized by an enzyme called pseudouridine synthase (PUS) to form the C-glycoside pseudouridine in a process called pseudouridylation. Although pseudouridine is the most abundant modified nucleotide in noncoding RNAs such as tRNA and rRNA, it had until recently never been detected in mRNA. A new report by Wendy V. Gilbert and co-workers indicates that not only does pseudouridine exist in mRNA but pseudouridylation also appears to be regulated in response to environmental signals (Nature 2014, 515, 143−146). The research team developed a high-throughput method, “Pseudo-seq,” to identify pseudouridine in mRNA. As a first step, the team applied the known method of exposing RNA to N-cyclohexyl-N′-(2-morpholinoethyl)-carbodiimide metho-ptoluenesulfonate (CMC), which alkylates pseudouridine to create a bulky adduct that blocks reverse transcription. Subsequent next-generation sequencing maps the 3′ ends of cDNAs to reveal the site at which pseudouridine adducts blocked further transcription. Applying Pseudoseq to study yeast mRNA, the team discovered at least 260 pseudouridylated sites in mRNAs and 74 new sites in noncoding RNAs, many of which were differentially pseudouridylated during different phases of growth. To determine which enzymes were involved, the team examined a series of yeast strains with specific PUS deletions; 52% of the mRNA and 31% of the novel noncoding RNA pseudouridylation sites could be assigned to particular PUS enzymes known to modify tRNA targets. Because these PUS enzymes are conserved in all eukaryotes, Gilbert and co-workers also used Pseudoseq to determine whether mRNA pseudouridylation occurred in mammalian cells, specifically in human cervical carcinoma (HeLA) cells. Over 90 sites were found, many of which were differentially pseudouridylated during different states of cell growth. The authors note that pseudouridylation, which stabilizes RNA 2701
dx.doi.org/10.1021/cb501003y | ACS Chem. Biol. 2014, 9, 2699−2701
