EZH2 K63-polyubiquitination affecting migration in extranodal natural killer/T-cell lymphoma.

BACKGROUND: Overexpressed EZH2 is oncogenically involved in the pathogenesis of different cancerous contexts including extranodal natural killer/T cell lymphoma (ENKTL). However, the underlying mechanisms of EZH2 upregulation have not been fully clarified and it is still difficult to target EZH2 in ENKTL. RESULTS: Current study identifies an E3 ligase TRIP12 that triggers K63-linked polyubiquitination of EZH2 in ENKTL and unexpectedly, stabilizes EZH2. As determined by gene expression profiling (GEP), TRIP12 and EZH2 levels correlate with each other in ENKTL patient samples. Aided by quantitative mass spectrometry (MS) and follow-up analysis, we identify K634 as the ubiquitination site of EZH2. Further study confirms that TRIP12-mediated EZH2 K634 ubiquitination enhances the interaction between EZH2 and SUZ12 or CDK1 and increases the level of EZH2 T487 phosphorylation. This study further demonstrates the TRIP12-EZH2 signaling might be regulated by cytoplasmic HSP60. Importantly, the TRIP12-EZH2 axis mediates ENKTL cell migration via accelerating epithelial-mesenchymal transition (EMT). Moreover, our study finds out dexamethasone treatment manipulates TRIP12-EZH2 signaling and may represent a novel therapeutic strategy against ENKTL metastasis. CONCLUSIONS: Altogether, TRIP12 induces K63-linked site-specific polyubiquitination of EZH2 for stabilization, which promotes ENKTL cell migration and could be targeted by dexamethasone treatment.

Immune pathway upregulation and lower genomic instability distinguish EBV-positive nodal T/NK-cell lymphoma from ENKTL and PTCL-NOS.

Primary Epstein-Barr virus (EBV)-positive nodal T/NK-cell lymphoma (PTCL-EBV) is a poorly understood disease which shows features resembling extranodal NK/T-cell lymphoma (ENKTL) and is currently not recognized as a distinct entity but categorized as a variant of primary T-cell lymphoma not otherwise specified (PTCL-NOS). Herein, we analyzed copynumber aberrations (n=77) with a focus on global measures of genomic instability and homologous recombination deficiency and performed gene expression (n=84) and EBV miRNA expression (n=24) profiling as well as targeted mutational analysis (n=16) to further characterize PTCL-EBV in relation to ENKTL and PTCL-NOS. Multivariate analysis revealed that patients with PTCL-EBV had a significantly worse outcome compared to patients with PTCL-NOS (P=0.002) but not to those with ENKTL. Remarkably, PTCL-EBV exhibited significantly lower genomic instability and homologous recombination deficiency scores compared to ENKTL and PTCL-NOS. Gene set enrichment analysis revealed that many immune-related pathways, interferon α/γ response, and IL6_JAK_STAT3 signaling were significantly upregulated in PTCLEBV and correlated with lower genomic instability scores. We also identified that NFκB-associated genes, BIRC3, NFKB1 (P50) and CD27, and their proteins are upregulated in PTCL-EBV. Most PTCL-EBV demonstrated a type 2 EBV latency pattern and, strikingly, exhibited downregulated expression of most EBV miRNA compared to ENKTL and their target genes were also enriched in immune-related pathways. PTCL-EBV also showed frequent mutations of TET2, PIK3CD and STAT3, and are characterized by microsatellite stability. Overall, poor outcome, low genomic instability, upregulation of immune pathways and downregulation of EBV miRNA are distinctive features of PTCL-EBV. Our data support the concept that PTCL-EBV could be considered as a distinct entity, provide novel insights into the pathogenesis of the disease and offer potential new therapeutic targets for this tumor.

T-Cell Lymphoma Clonality by Copy Number Variation Analysis of T-Cell Receptor Genes.

T-cell lymphomas arise from a single neoplastic clone and exhibit identical patterns of deletions in T-cell receptor (TCR) genes. Whole genome sequencing (WGS) data represent a treasure trove of information for the development of novel clinical applications. However, the use of WGS to identify clonal T-cell proliferations has not been systematically studied. In this study, based on WGS data, we identified monoclonal rearrangements (MRs) of T-cell receptors (TCR) genes using a novel segmentation algorithm and copy number computation. We evaluated the feasibility of this technique as a marker of T-cell clonality using T-cell lymphomas (TCL, n = 44) and extranodal NK/T-cell lymphomas (ENKTLs, n = 20), and identified 98% of TCLs with one or more TCR gene MRs, against 91% detected using PCR. TCR MRs were absent in all ENKTLs and NK cell lines. Sensitivity-wise, this platform is sufficiently competent, with MRs detected in the majority of samples with tumor content under 25% and it can also distinguish monoallelic from biallelic MRs. Understanding the copy number landscape of TCR using WGS data may engender new diagnostic applications in hematolymphoid pathology, which can be readily adapted to the analysis of B-cell receptor loci for B-cell clonality determination.

Combining kinase inhibitors for optimally co-targeting cancer and drug escape by exploitation of drug target promiscuities.

Cancers resist targeted therapeutics by drug-escape signaling. Multitarget drugs co-targeting cancer and drug-escape mediators (DEMs) are clinically advantageous. DEM coverage may be expanded by drug combinations. This work evaluated to what extent the kinase DEMs (KDEMs) can be optimally co-targeted by drug combinations based on target promiscuities of individual drugs. We focused on 41 approved and 28 clinical trial small molecule kinase inhibitor drugs with available experimental kinome and clinical pharmacokinetic data. From the kinome inhibitory profiles of these drugs, drug combinations were assembled for optimally co-targeting an established cancer target (EGFR, HER2, ABL1, or MEK1) and 9-16 target-associated KDEMs at comparable potency levels as that against the cancer target. Each set of two-, three-, and four-drug combinations co-target 36-71%, 44-89%, 50-88%, and 27-55% KDEMs of EGFR, HER2, ABL1, and MEK1, respectively, compared with the 36, 33, 38, and 18% KDEMs maximally co-targeted by an existing drug or drug combination approved or clinically tested for the respective cancer. Some co-targeted KDEMs are not covered by any existing drug or drug combination. Our work suggested that novel drug combinations may be constructed for optimally co-targeting cancer and drug escape by the exploitation of drug target promiscuities.

Drug sales confirm clinical advantage of multi-target inhibition of drug escapes by anticancer kinase inhibitors.

The clinical advantage of co-targeting cancer drug escape has been indicated by the percentage of these co-targeting drugs among all multi-target drugs in clinics and clinical trials. This clinical advantage needs to be further interrogated from such perspectives as the clinical impact of multi-target inhibition of drug-escape mediators. This impact may be reflected by drug sales data, that is, multi-target inhibition of higher number of drug-escape mediators favors the expanded coverage of drug-resistant patients leading to higher sales. We investigated whether this expectation is followed by the 25 FDA-approved anticancer kinase inhibitors, which were divided into 11 groups of comparable therapeutic mechanisms and approval years. We found 19 (76%) drugs to follow and 3 (12%) drugs not to follow this expectation. The remaining two (8%) and one (4%) drugs cannot be assessed due to insufficient data and incomparability. Therefore, drug sales strongly indicate the clinical advantage of multi-target inhibition of cancer drug escapes.

Pharmacological relationships and ligand discovery of G protein-coupled receptors revealed by simultaneous ligand and receptor clustering.

Conventional ligand and receptor similarity methods have been extensively used for exposing pharmacological relationships and drug lead discovery. They may in some cases neglect minor relationships useful for target hopping particularly against the remote family members. To complement the conventional methods for capturing these minor relationships, we developed a new method that uses a SLARC (Simultaneous Ligand And Receptor Clustering) 2D map to simultaneously characterize the ligand structural and receptor binding-site sequence relationships of a receptor family. The SLARC maps of the rhodopsin-like GPCR family comprehensively revealed scaffold hopping, target hopping, and multi-target relationships for the ligands of both homologous and remote family members. Their usefulness in new ligand discovery was validated by guiding the prospective discovery of novel indole piperazinylpyrimidine dual-targeting adenosine A2A receptor antagonist and dopamine D2 agonist compounds. The SLARC approach is useful for revealing pharmacological relationships and discovering new ligands at target family levels.

HEROD: a human ethnic and regional specific omics database.

MOTIVATION: Genetic and gene expression variations within and between populations and across geographical regions have substantial effects on the biological phenotypes, diseases, and therapeutic response. The development of precision medicines can be facilitated by the OMICS studies of the patients of specific ethnicity and geographic region. However, there is an inadequate facility for broadly and conveniently accessing the ethnic and regional specific OMICS data. RESULTS: Here, we introduced a new free database, HEROD, a human ethnic and regional specific OMICS database. Its first version contains the gene expression data of 53 070 patients of 169 diseases in seven ethnic populations from 193 cities/regions in 49 nations curated from the Gene Expression Omnibus (GEO), the ArrayExpress Archive of Functional Genomics Data (ArrayExpress), the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). Geographic region information of curated patients was mainly manually extracted from referenced publications of each original study. These data can be accessed and downloaded via keyword search, World map search, and menu-bar search of disease name, the international classification of disease code, geographical region, location of sample collection, ethnic population, gender, age, sample source organ, patient type (patient or healthy), sample type (disease or normal tissue) and assay type on the web interface. AVAILABILITY AND IMPLEMENTATION: The HEROD database is freely accessible at http://bidd2.nus.edu.sg/herod/index.php. The database and web interface are implemented in MySQL, PHP and HTML with all major browsers supported. CONTACT: phacyz@nus.edu.sg.

Discovery of novel dual VEGFR2 and Src inhibitors using a multistep virtual screening approach.

AIM: Simultaneous inhibition of VEGFR2 and Src may enhance the efficacy of VEGFR2-targeted cancer therapeutics. Hence, development of dual inhibitors on VEGFR2 and Src can be a useful strategy for such treatments. MATERIALS & METHODS: A multistep virtual screening protocol, comprising ligand-based support vector machines method, drug-likeness rules filter and structure-based molecular docking, was developed and employed to identify dual inhibitors of VEGFR2 and Src from a large commercial chemical library. Kinase inhibitory assays and cell viability assays were then used for experimental validation. RESULTS: A set of compounds belonging to six different molecular scaffolds was identified and sent for biological evaluation. Compound 3c belonging to the 2-amino-3-cyanopyridine scaffold exhibited good antiproliferative effect and dual-target activities against VEGFR2 and Src. CONCLUSION: This study demonstrated the ability of the multistep virtual screening approach to identify novel multitarget agents.

Applying the designed multiple ligands approach to inhibit dihydrofolate reductase and thioredoxin reductase for anti-proliferative activity.

The development of multi-targeting drugs is currently being explored as an attractive alternative to combination therapy, especially for the treatment of complex diseases such as cancer. Dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR) are enzymes belonging to two unrelated cellular pathways that are known to contribute towards cancer cell growth and survival. In order to evaluate whether simultaneous inhibition of DHFR and TrxR by dihydrotriazines (DHFR-targeting) and chalcones (TrxR-targeting) may be beneficial, breast MCF-7 and colorectal HCT116 carcinoma cells were treated with combinations of selected dihydrotriazines and chalcones at a 1:1 M ratio. Two combinations demonstrated synergy at low-to-moderate concentrations. Based on this result, four merged dihydrotriazine-chalcone compounds were designed and synthesized. Two compounds, 11a [DHFR IC50 = 56.4 µM, TrxR IC50 (60 min) = 12.6 µM] and 11b [DHFR IC50 = 2.4 µM, TrxR IC50 (60 min) = 10.1 µM], demonstrated in vitro inhibition of DHFR and TrxR. The compounds showed growth inhibitory activity against MCF-7 and HCT116 cells, but lacked cytotoxicity. Molecular docking experiments showed 11b to possess rational binding modes to both the enzymes. In conclusion, this study has not only identified the dihydrotriazine and chalcone scaffolds as good starting points for the development of dual inhibitors of DHFR and TrxR, but also demonstrated the synthetic feasibility of producing a chemical entity that could result in simultaneous inhibition of DHFR and TrxR. Future efforts to improve the antiproliferative profiles of such compounds will look at alternative ways of integrating the two pharmacophoric scaffolds.

Design, synthesis, and biological evaluation of curcumin analogues as multifunctional agents for the treatment of Alzheimer's disease.

A series of novel curcumin analogues were designed, synthesized, and evaluated as potential multifunctional agents for the treatment of AD. The in vitro studies showed that these compounds had better inhibitory properties against Aß aggregation than curcumin. Superior anti-oxidant properties (better than the reference compound Trolox) of these compounds were observed by the oxygen radical absorbance capacity (ORAC) method and a cell-based assay using DCFH-DA as a probe. In addition they were able to chelate metals such as iron and copper and decrease metal-induced Aß aggregation. The structure-activity relationships were discussed. The results suggested that our curcumin analogues could be selected as multifunctional agents for further investigation of AD treatment.