In-cell chemical construction of a photoswitchable CENP-E using a photochromic covalent inhibitor.

An arylazopyrazole-based covalent inhibitor targeting the mitotic motor protein of centromere-associated protein E (CENP-E) was developed. Using this photoswitchable inhibitor, a photoswitchable CENP-E was chemically constructed in cells, which enabled to local control of mitotic cell division with light illumination.

Rational Design and Development of Selective BRD7 Bromodomain Inhibitors and Their Activity in Prostate Cancer.

Bromodomain-containing proteins are readers of acetylated lysine and play important roles in cancer. Bromodomain-containing protein 7 (BRD7) is implicated in multiple malignancies; however, there are no selective chemical probes to study its function in disease. Using crystal structures of BRD7 and BRD9 bromodomains (BDs) bound to BRD9-selective ligands, we identified a binding pocket exclusive to BRD7. We synthesized a series of ligands designed to occupy this binding region and identified two inhibitors with increased selectivity toward BRD7, 1-78 and 2-77, which bind with submicromolar affinity to the BRD7 BD. Our binding mode analyses indicate that these ligands occupy a uniquely accessible binding cleft in BRD7 and maintain key interactions with the asparagine and tyrosine residues critical for acetylated lysine binding. Finally, we validated the utility and selectivity of the compounds in cell-based models of prostate cancer.

Tetraploidy-linked sensitization to CENP-E inhibition in human cells.

Tetraploidy is a hallmark of cancer cells, and tetraploidy-selective cell growth suppression is a potential strategy for targeted cancer therapy. However, how tetraploid cells differ from normal diploids in their sensitivity to anti-proliferative treatments remains largely unknown. In this study, we found that tetraploid cells are significantly more susceptible to inhibitors of a mitotic kinesin (CENP-E) than are diploids. Treatment with a CENP-E inhibitor preferentially diminished the tetraploid cell population in a diploid-tetraploid co-culture at optimum conditions. Live imaging revealed that a tetraploidy-linked increase in unsolvable chromosome misalignment caused substantially longer mitotic delay in tetraploids than in diploids upon moderate CENP-E inhibition. This time gap of mitotic arrest resulted in cohesion fatigue and subsequent cell death, specifically in tetraploids, leading to tetraploidy-selective cell growth suppression. In contrast, the microtubule-stabilizing compound paclitaxel caused tetraploidy-selective suppression through the aggravation of spindle multipolarization. We also found that treatment with a CENP-E inhibitor had superior generality to paclitaxel in its tetraploidy selectivity across a broader spectrum of cell lines. Our results highlight the unique properties of CENP-E inhibitors in tetraploidy-selective suppression and their potential use in the development of tetraploidy-targeting interventions in cancer.

Host Poly(A) Polymerases PAPD5 and PAPD7 Provide Two Layers of Protection That Ensure the Integrity and Stability of Hepatitis B Virus RNA.

Noncanonical poly(A) polymerases PAPD5 and PAPD7 (PAPD5/7) stabilize hepatitis B virus (HBV) RNA via the interaction with the viral posttranscriptional regulatory element (PRE), representing new antiviral targets to control HBV RNA metabolism, hepatitis B surface antigen (HBsAg) production, and viral replication. Inhibitors targeting these proteins are being developed as antiviral therapies; therefore, it is important to understand how PAPD5/7 coordinate to stabilize HBV RNA. Here, we utilized a potent small-molecule AB-452 as a chemical probe, along with genetic analyses to dissect the individual roles of PAPD5/7 in HBV RNA stability. AB-452 inhibits PAPD5/7 enzymatic activities and reduces HBsAg both in vitro (50% effective concentration [EC50] ranged from 1.4 to 6.8 nM) and in vivo by 0.94 log10. Our genetic studies demonstrate that the stem-loop alpha sequence within PRE is essential for both maintaining HBV poly(A) tail integrity and determining sensitivity toward the inhibitory effect of AB-452. Although neither single knockout (KO) of PAPD5 nor PAPD7 reduces HBsAg RNA and protein production, PAPD5 KO does impair poly(A) tail integrity and confers partial resistance to AB-452. In contrast, PAPD7 KO did not result in any measurable changes within the HBV poly(A) tails, but cells with both PAPD5 and PAPD7 KO show reduced HBsAg production and conferred complete resistance to AB-452 treatment. Our results indicate that PAPD5 plays a dominant role in stabilizing viral RNA by protecting the integrity of its poly(A) tail, while PAPD7 serves as a second line of protection. These findings inform PAPD5-targeted therapeutic strategies and open avenues for further investigating PAPD5/7 in HBV replication. IMPORTANCE Chronic hepatitis B affects more than 250 million patients and is a major public health concern worldwide. HBsAg plays a central role in maintaining HBV persistence, and as such, therapies that aim at reducing HBsAg through destabilizing or degrading HBV RNA have been extensively investigated. Besides directly degrading HBV transcripts through antisense oligonucleotides or RNA silencing technologies, small-molecule compounds targeting host factors such as the noncanonical poly(A) polymerase PAPD5 and PAPD7 have been reported to interfere with HBV RNA metabolism. Herein, our antiviral and genetic studies using relevant HBV infection and replication models further characterize the interplays between the cis element within the viral sequence and the trans elements from the host factors. PAPD5/7-targeting inhibitors, with oral bioavailability, thus represent an opportunity to reduce HBsAg through destabilizing HBV RNA.

A global chromatin compaction pathway that represses germline gene expression during starvation.

While much is known about how transcription is controlled at individual genes, comparatively little is known about how cells regulate gene expression on a genome-wide level. Here, we identify a molecular pathway in the C. elegans germline that controls transcription globally in response to nutritional stress. We report that when embryos hatch into L1 larvae, they sense the nutritional status of their environment, and if food is unavailable, they repress gene expression via a global chromatin compaction (GCC) pathway. GCC is triggered by the energy-sensing kinase AMPK and is mediated by a novel mechanism that involves the topoisomerase II/condensin II axis acting upstream of heterochromatin assembly. When the GCC pathway is inactivated, then transcription persists during starvation. These results define a new mode of whole-genome control of transcription.

Exposure to the bromodomain inhibitor N-methyl pyrrolidone blocks spermatogenesis in a hormonal and non-hormonal fashion.

N-methyl pyrrolidone (NMP) is an FDA approved molecule used as an excipient in pharmaceutical industry. Besides having a central role in formulation of drugs, the most important function of any excipient is to guarantee the safety of the medicine during and after its administration. Several studies have shown that exposure to NMP and especially in rats produce a gonadotoxic effect leading to infertility. However, the mechanisms underlying the effect of NMP on male reproduction are unknown. The aim of this study was to assess the reproductive toxicity of NMP in male rats and to elucidate the underlying mechanism. Male Sprague Dawley rats were injected intraperitoneally, twice/ week, at a dose of 108 mg/ 100 g of body weight with NMP. Analysis of reproductive parameters revealed testicular atrophy in NMP treated animals compared to control animals. Germ cell composition within the seminiferous tubules was disturbed and manifested in an increase in number of cells with fragmented DNA. A subsequent decrease in number of spermatocytes and spermatids was observed. Alpha screen assay shows that NMP acts at the concentrations we applied in vivo as a low affinity inhibitor for BRDT (testis specific bromodomain protein). BRDT inhibition is mirrored by a significant decrease in the expression of early stage spermatocyte markers (lmna, aurkc and ccna1), during which BRDT expression predominates. A significant decrease in testosterone levels was also observed. Since NMP interferes with spermatogenesis on various levels, its use in humans must be carefully monitored.

Exploiting vulnerabilities of SWI/SNF chromatin remodelling complexes for cancer therapy.

Multi-subunit ATPase-dependent chromatin remodelling complexes SWI/SNF (switch/sucrose non-fermentable) are fundamental epigenetic regulators of gene transcription. Functional genomic studies revealed a remarkable mutation prevalence of SWI/SNF-encoding genes in 20-25% of all human cancers, frequently driving oncogenic programmes. Some SWI/SNF-mutant cancers are hypersensitive to perturbations in other SWI/SNF subunits, regulatory proteins and distinct biological pathways, often resulting in sustained anticancer effects and synthetic lethal interactions. Exploiting these vulnerabilities is a promising therapeutic strategy. Here, we review the importance of SWI/SNF chromatin remodellers in gene regulation as well as mechanisms leading to assembly defects and their role in cancer development. We will focus in particular on emerging strategies for the targeted therapy of SWI/SNF-deficient cancers using chemical probes, including proteolysis targeting chimeras, to induce synthetic lethality.

Phase transition of fibrillarin LC domain regulates localization and protein interaction of fibrillarin.

A key nucleolar protein, fibrillarin, has emerged as an important pharmacological target as its aberrant expression and localization are related to tumorigenesis, chemoresistance and poor survival in breast cancer patients. Fibrillarin contains a N-terminal low complexity sequence (LC) domain with a skewed amino acid distribution, which is known to undergo a phase transition to liquid-like droplets. However, the underlying mechanism of the phase transition of the fibrillarin LC domain and its physiological function are still elusive. In this study, we show that the localization of fibrillarin and its association with RNA binding proteins is regulated by this phase transition. Phenylalanine-to-serine substitutions of the phenylalanine:glycine repeats in the fibrillarin LC domain impede its phase transition into liquid-like droplets, as well as the hydrogel-like state composed of polymers, and also its incorporation into hydrogel or liquid-like droplets composed of wild-type LC domains. When expressed in cultured cells, fibrillarin containing the mutant LC domain fails to localize to the dense fibrillar component of nucleoli in the same way as intact fibrillarin. Moreover, the phase transition of the fibrillarin LC domain is required for the interaction of fibrillarin with other RNA binding proteins, such as FUS, TAF15, DDX5 and DHX9. Taken together, the results suggest that the phenylalanine residues in the LC domain are critical for the phase transition of fibrillarin, which in turn regulates the sub-nucleolar localization of fibrillarin and its interaction with RNA binding proteins, providing a useful framework for regulating the function of fibrillarin.

Discovery of BAZ1A bromodomain inhibitors with the aid of virtual screening and activity evaluation.

BAZ1A is a bromodomain-containing protein, and has been recognized as a potential target for multiple diseases, particularly cancer. However, there is no BAZ1A inhibitor reported so far. In this study, we used a consensus docking/scoring strategy to screen for BAZ1A bromodomain inhibitors from commercial chemical libraries and an in-house chemical database. The retrieved hit compounds were evaluated experimentally and four compounds were found to be active against BAZ1A bromodomain. To the most active compounds, similarity and substructure searches were used to find more BAZ1A bromodomain inhibitors. Among all the obtained active compounds, Cpd-2 is the most potent one, which showed a KD value of 0.52 µM. The interaction model of Cpd-2 with BAZ1A bromodomain was revealed by molecular docking. In a cellular assay, Cpd-2 displayed good anti-viability activity against cancer cell lines expressing a high level of BAZ1A. Overall, we discovered a number of BAZ1A bromodomain inhibitors for the first time, which can be a good starting point for subsequent drug discovery targeting BAZ1A bromodomain.

4-Acyl Pyrroles as Dual BET-BRD7/9 Bromodomain Inhibitors Address BETi Insensitive Human Cancer Cell Lines.

Various malignant human diseases show disturbed signaling pathways due to increased activity of proteins within the epigenetic machinery. Recently, various novel inhibitors for epigenetic regulation have been introduced which promise a great therapeutic benefit. Inhibitors for the bromo- and extra-terminal domain (BET) family were of particular interest after inhibitors had shown a strong antiproliferative effect. More recently, the focus has increasingly shifted to bromodomains (BDs) outside the BET family. Based on previously developed inhibitors, we have optimized a small series of 4-acyl pyrroles, which we further analyzed by ITC, X-ray crystallography, selectivity studies, the NCI60 cell-panel, and GI50 determinations for several cancer cell lines. The inhibitors address both, BET and BRD7/9 BDs, with very high affinity and show a strong antiproliferative effect on various cancer cell lines that could not be observed for BD family selective inhibitors. Furthermore, a synergistic effect on breast cancer (MCF-7) and melanoma (SK-MEL-5) was proven.

Opa-Interacting Protein 5 Expression in Human Glioma Tissues Is Essential to the Biological Function of U251 Human Malignant Glioma Cells.

Opa-interacting protein 5 (OIP5) is a member of the cancer-testis antigen (CTA) family that elicits a spontaneous antitumor immune response. The failure of current immunotherapies for glioma has prompted the search for novel biomarkers that may be utilized as therapeutic targets. This study aimed to investigate whether OIP5 serves as a target for malignant glioma immunotherapy. Glioma specimens from 53 adult patients were evaluated for OIP5 expression by immunohistochemical (IHC) staining, and the correlation of OIP5 expression with World Health Organization (WHO) tumor grade was analyzed. Endogenous expression of OIP5 in glioma cell lines was determined via real-time polymerase chain reaction (RT-PCR). Using lentiviral siOIP5, the effect of OIP5 gene knockdown on proliferation, cell cycle, and apoptosis in U251 glioma cells was studied. The results show that OIP5 is overexpressed in glioma tissues and is correlated with WHO tumor grade (P < 0.001). However, OIP5 protein expression is barely detectable in normal adult brain tissues. MTT assays and analysis using the Celigo Imaging Cytometry System reveal that the silencing of OIP5 inhibits U251 cell growth. Cell cycle assays and Annexin V staining show that OIP5 silencing disrupts the balance of the cell cycle and increases U251 cell death. These results indicate that OIP5 is upregulated in malignant glioma specimens but barely detected in normal brain tissues. OIP5 knockdown inhibits the biological function of glioma cells, reinforcing that OIP5 may serve as an immunotherapeutic target for malignant glioma.

DEK-targeting aptamer DTA-64 attenuates bronchial EMT-mediated airway remodelling by suppressing TGF-ß1/Smad, MAPK and PI3K signalling pathway in asthma.

This study is to investigate the inhibitory effects and mechanisms of DEK-targeting aptamer (DTA-64) on epithelial mesenchymaltransition (EMT)-mediated airway remodelling in mice and human bronchial epithelial cell line BEAS-2B. In the ovalbumin (OVA)-induced asthmatic mice, DTA-64 significantly reduced the infiltration of eosinophils and neutrophils in lung tissue, attenuated the airway resistance and the proliferation of goblet cells. In addition, DTA-64 reduced collagen deposition, transforming growth factor 1 (TGF-ß1) level in BALF and IgE levels in serum, balanced Th1/Th2/Th17 ratio, and decreased mesenchymal proteins (vimentin and α-SMA), as well as weekend matrix metalloproteinases (MMP-2 and MMP-9) and NF-κB p65 activity. In the in vitro experiments, we used TGF-ß1 to induce EMT in the human epithelial cell line BEAS-2B. DEK overexpression (ovDEK) or silencing (shDEK) up-regulated or down-regulated TGF-ß1 expression, respectively, on the contrary, TGF-ß1 exposure had no effect on DEK expression. Furthermore, ovDEK and TGF-ß1 synergistically promoted EMT, whereas shDEK significantly reduced mesenchymal markers and increased epithelial markers, thus inhibiting EMT. Additionally, shDEK inhibited key proteins in TGF-ß1-mediated signalling pathways, including Smad2/3, Smad4, p38 MAPK, ERK1/2, JNK and PI3K/AKT/mTOR. In conclusion, the effects of DTA-64 against EMT of asthmatic mice and BEAS-2B might partially be achieved through suppressing TGF-ß1/Smad, MAPK and PI3K signalling pathways. DTA-64 may be a new therapeutic option for the management of airway remodelling in asthma patients.

Application of Atypical Acetyl-lysine Methyl Mimetics in the Development of Selective Inhibitors of the Bromodomain-Containing Protein 7 (BRD7)/Bromodomain-Containing Protein 9 (BRD9) Bromodomains.

Non-BET bromodomain-containing proteins have become attractive targets for the development of novel therapeutics targeting epigenetic pathways. To help facilitate the target validation of this class of proteins, structurally diverse small-molecule ligands and methodologies to produce selective inhibitors in a predictable fashion are in high demand. Herein, we report the development and application of atypical acetyl-lysine (KAc) methyl mimetics to take advantage of the differential stability of conserved water molecules in the bromodomain binding site. Discovery of the n-butyl group as an atypical KAc methyl mimetic allowed generation of 31 (GSK6776) as a soluble, permeable, and selective BRD7/9 inhibitor from a pyridazinone template. The n-butyl group was then used to enhance the bromodomain selectivity of an existing BRD9 inhibitor and to transform pan-bromodomain inhibitors into BRD7/9 selective compounds. Finally, a solvent-exposed vector was defined from the pyridazinone template to enable bifunctional molecule synthesis, and affinity enrichment chemoproteomic experiments were used to confirm several of the endogenous protein partners of BRD7 and BRD9, which form part of the chromatin remodeling PBAF and BAF complexes, respectively.

Distinct Mechanisms of Resistance to a CENP-E Inhibitor Emerge in Near-Haploid and Diploid Cancer Cells.

Aberrant chromosome numbers in cancer cells may impose distinct constraints on the emergence of drug resistance-a major factor limiting the long-term efficacy of molecularly targeted therapeutics. However, for most anticancer drugs we lack analyses of drug-resistance mechanisms in cells with different karyotypes. Here, we focus on GSK923295, a mitotic kinesin CENP-E inhibitor that was evaluated in clinical trials as a cancer therapeutic. We performed unbiased selections to isolate inhibitor-resistant clones in diploid and near-haploid cancer cell lines. In diploid cells we identified single-point mutations that can suppress inhibitor binding. In contrast,transcriptome analyses revealed that the C-terminus of CENP-E was disrupted in GSK923295-resistant near-haploid cells. While chemical inhibition of CENP-E is toxic to near-haploid cells, knockout of the CENPE gene does not suppress haploid cell proliferation, suggesting that deletion of the CENP-E C-terminus can confer resistance to GSK923295. Together, these findings indicate that different chromosome copy numbers in cells can alter epistatic dependencies and lead to distinct modes of chemotype-specific resistance.

Structural Basis of Inhibitor Selectivity in the BRD7/9 Subfamily of Bromodomains.

Inhibition of the bromodomain containing protein 9 (BRD9) by small molecules is an attractive strategy to target mutated SWI/SNF chromatin-remodeling complexes in cancer. However, reported BRD9 inhibitors also inhibit the closely related bromodomain-containing protein 7 (BRD7), which has different biological functions. The structural basis for differential potency and selectivity of BRD9 inhibitors is largely unknown because of the lack of structural information on BRD7. Here, we biochemically and structurally characterized diverse inhibitors with varying degrees of potency and selectivity for BRD9 over BRD7. Novel cocrystal structures of BRD7 liganded with new and previously reported inhibitors of five different chemical scaffolds were determined alongside BRD9 and BRD4. We also report the discovery of first-in-class dual bromodomain-kinase inhibitors outside the bromodomain and extraterminal family targeting BRD7 and BRD9. Combined, the data provide a new framework for the development of BRD7/9 inhibitors with improved selectivity or additional polypharmacologic properties.

Cordycepin Inhibits Cancer Cell Proliferation and Angiogenesis through a DEK Interaction via ERK Signaling in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a malignant tumor that arises from the epithelial cells of the bile duct and is notorious for its poor prognosis. The clinical outcome remains disappointing, and thus more effective therapeutic options are urgently required. Cordycepin, a traditional Chinese medicine, provides multiple pharmacological strategies in antitumors, but its mechanisms have not been fully elucidated. In this study, we reported that cordycepin inhibited the viability and proliferation capacity of CCA cells in a time- and dose-dependent manner determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and colony formation assay. Flow cytometry and Hoechst dye showed that cordycepin induced cancer cell apoptosis via extracellular signal-regulated kinase (ERK) 1/2 deactivation. Moreover, cordycepin significantly reduced the angiogenetic capabilities of CCA in vitro as examined by tube formation assay. We also discovered that cordycepin inhibited DEK expression by using Western blot assay. DEK serves as an oncogenic protein that is overexpressed in various gastrointestinal tumors. DEK silencing inhibited CCA cell viability and angiogenesis but not apoptosis induction determined by Western blot and flow cytometry. Furthermore, cordycepin significantly inhibited tumor growth and angiogenic capacities in a xenograft model by downregulating the expression of DEK, phosphorylated ERK1/2 CD31 and von Willebrand factor (vWF). Taken together, we demonstrated that cordycepin inhibited CCA cell proliferation and angiogenesis with a DEK interaction via downregulation in ERK signaling. These data indicate that cordycepin may serve as a novel agent for CCA clinical treatment and prognosis improvement. SIGNIFICANCE STATEMENT: Cordycepin provides multiple strategies in antitumors, but its mechanisms are not fully elucidated, especially on cholangiocarcinoma (CCA). We reported that cordycepin inhibited the viability of CCA cells, induced apoptosis via extracellular signal-regulated kinase 1/2 deactivation and DEK inhibition, and reduced the angiogenetic capabilities of CCA both in vivo and in vitro.

Targeting Spt5-Pol II by Small-Molecule Inhibitors Uncouples Distinct Activities and Reveals Additional Regulatory Roles.

Spt5 is a conserved and essential transcription elongation factor that promotes promoter-proximal pausing, promoter escape, elongation, and mRNA processing. Spt5 plays specific roles in the transcription of inflammation and stress-induced genes and tri-nucleotide expanded-repeat genes involved in inherited neurological pathologies. Here, we report the identification of Spt5-Pol II small-molecule inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on promoter-proximal pausing, NF-κB activation, and expanded-repeat huntingtin gene transcription. Using SPIs, we identified Spt5 target genes that responded with profoundly diverse kinetics. SPIs uncovered the regulatory role of Spt5 in metabolism via GDF15, a food intake- and body weight-inhibitory hormone. SPIs further unveiled a role for Spt5 in promoting the 3' end processing of histone genes. While several SPIs affect all Spt5 functions, a few inhibit a single one, implying uncoupling and selective targeting of Spt5 activities. SPIs expand the understanding of Spt5-Pol II functions and are potential drugs against metabolic and neurodegenerative diseases.

Genome-wide RNAi screen in Drosophila reveals Enok as a novel trithorax group regulator.

BACKGROUND: Polycomb group (PcG) and trithorax group (trxG) proteins contribute to the specialization of cell types by maintaining differential gene expression patterns. Initially discovered as positive regulators of HOX genes in forward genetic screens, trxG counteracts PcG-mediated repression of cell type-specific genes. Despite decades of extensive analysis, molecular understanding of trxG action and regulation are still punctuated by many unknowns. This study aimed at discovering novel factors that elicit an anti-silencing effect to facilitate trxG-mediated gene activation. RESULTS: We have developed a cell-based reporter system and performed a genome-wide RNAi screen to discover novel factors involved in trxG-mediated gene regulation in Drosophila. We identified more than 200 genes affecting the reporter in a manner similar to trxG genes. From the list of top candidates, we have characterized Enoki mushroom (Enok), a known histone acetyltransferase, as an important regulator of trxG in Drosophila. Mutants of enok strongly suppressed extra sex comb phenotype of Pc mutants and enhanced homeotic transformations associated with trx mutations. Enok colocalizes with both TRX and PC at chromatin. Moreover, depletion of Enok specifically resulted in an increased enrichment of PC and consequently silencing of trxG targets. This downregulation of trxG targets was also accompanied by a decreased occupancy of RNA-Pol-II in the gene body, correlating with an increased stalling at the transcription start sites of these genes. We propose that Enok facilitates trxG-mediated maintenance of gene activation by specifically counteracting PcG-mediated repression. CONCLUSION: Our ex vivo approach led to identification of new trxG candidate genes that warrant further investigation. Presence of chromatin modifiers as well as known members of trxG and their interactors in the genome-wide RNAi screen validated our reverse genetics approach. Genetic and molecular characterization of Enok revealed a hitherto unknown interplay between Enok and PcG/trxG system. We conclude that histone acetylation by Enok positively impacts the maintenance of trxG-regulated gene activation by inhibiting PRC1-mediated transcriptional repression.

Identification of benzo[d]pyrrolo[2,1-b]thiazole derivatives as CENP-E inhibitors.

Kinesin centromere-associated protein E (CENP-E) has emerged as a potential target for the development of anticancer drugs due to its involvement in the mitotic progression of the cell cycle. Although several CENP-E inhibitors have been reported, more knowledge of chemical structures and inhibitory mechanisms is necessary for developing CENP-E inhibitors. Here, we describe the identification of new CENP-E inhibitors. Screening of a small-molecule chemical library identified benzo[d]pyrrolo[2,1-b]thiazole derivatives, including 1, as compounds with inhibitory activity against the microtubule-stimulated ATPase of the CENP-E motor domain. Among the mitotic kinesins examined, 1 selectively inhibited the kinesin ATPase activity of CENP-E. In a steady-state ATPase assay, 1 exhibited ATP-competitive behavior, which was different from the CENP-E inhibitor GSK923295. Compound 1 inhibited the proliferation of tumor-derived HeLa and HCT116 cells more efficiently than that of non-cancerous WI-38 cells. The inhibition of cell proliferation was attributed to the ability of 1 to induce apoptotic cell death. The compound showed antimitotic activity, which caused cell cycle arrest at mitosis via interference with proper chromosome alignment. We identified 1 and its derivatives as the lead compounds that target CENP-E, thus providing a new opportunity for the development of anticancer agents targeting kinesins.

A Cohesin-Mediated Intrachromosomal Loop Drives Oncogenic ROR lncRNA to Accelerate Tumorigenesis.

Long noncoding RNAs (lncRNAs) are an important class of pervasive noncoding RNA involved in a variety of biological functions. Numerous studies have demonstrated their important regulatory role in human disease, especially cancer. However, the mechanism underlying the transcription of lncRNAs is not fully elucidated. Here, a comparison of local chromatin structure of the ROR lncRNA locus revealed a cohesin-complex-mediated intrachromosomal loop that is juxtaposed with an upstream enhancer to the ROR promoter, enabling activation of endogenous ROR lncRNA in tumor cells. This chromosomal interaction was not observed in normal control cells. Knockdown of SMC1 by RNAi or deletion of the enhancer DNA by CRISPR/Cas9 abolished the intrachromosomal interaction, resulting in ROR lncRNA silencing and inhibition of the tumor progression in animals carrying tumor xenografts. Our results reveal a novel mechanism by which the cohesin-orchestrated intrachromosomal looping may serve as a critical epigenetic driver to activate transcription of ROR lncRNA, subsequently inducing tumorigenesis. Our data represent a novel chromosomal folding pattern of lncRNA regulation, thereby providing a novel alternative concept of chromosomal interaction in lncRNA-triggered tumorigenesis.