Molecular Integrative Clustering of Asian Gastric Cell Lines Revealed Two Distinct Chemosensitivity Clusters

<div><p>Cell lines recapitulate cancer heterogeneity without the presence of interfering tissue found in primary tumor. Their heterogeneous characteristics are reflected in their multiple genetic abnormalities and variable responsiveness to drug treatments. In order to understand the heterogeneity observed in Asian gastric cancers, we have performed array comparative genomic hybridization (aCGH) on 18 Asian gastric cell lines. Hierarchical clustering and single-sample Gene Set Enrichment Analysis were performed on the aCGH data together with public gene expression data of the same cell lines obtained from the Cancer Cell Line Encyclopedia. We found a large amount of genetic aberrations, with some cell lines having 13 fold more aberrations than others. Frequently mutated genes and cellular pathways are identified in these Asian gastric cell lines. The combined analyses of aCGH and expression data demonstrate correlation of gene copy number variations and expression profiles in human gastric cancer cells. The gastric cell lines can be grouped into 2 integrative clusters (ICs). Gastric cells in IC1 are enriched with gene associated with mitochondrial activities and oxidative phosphorylation while cells in IC2 are enriched with genes associated with cell signaling and transcription regulations. The two clusters of cell lines were shown to have distinct responsiveness towards several chemotherapeutics agents such as PI3 K and proteosome inhibitors. Our molecular integrative clustering provides insight into critical genes and pathways that may be responsible for the differences in survival in response to chemotherapy.</p></div

Molecular clustering of the Asian gastric cancer cell lines.

<p>(A) Hierarchical clustering using cell lines with both DNA copy number and mRNA expression data. (B) mRNA expression (mean-centered, normalized) heatmap (upper panel) and copy number (lower panel) of 1,762 putative driver genes from 14 gastric cell lines. (C) ssGSEA pathway enrichment score (mean-centered) heatmap for 380 subtype-specific pathways using 27 gastric cell lines from CCLE. Only selected pathway/genesets are labeled. Color code for mRNA expression: red = high expression, green = low expression. Color code for copy number: green = copy number loss, red = copy number gain, black = normal copy number. Color code for pathway enrichment: red = high enrichment, green = low enrichment.</p

Dot plots of IC₅₀ values for targeted inhibitors that have significant differences in toxicity to the Asian gastric cancer cells between the two integrated clusters.

<p>(A) Targeted inhibitors from the CCLE database. (B) Targeted inhibitors from the Sanger COSMIC database. (C) Selected targeted inhibitors in our lab showing significant differences in sensitivity (except XAV939) towards the two clusters of cell lines. Y-axis is the IC₅₀ values in log10 scale. P-value is computed by Mann Whitney U-test. Horizontal bars are medians for sample distributions.</p

Total and kinome genetics aberrations (consisting of gain, loss and LOH) in the 18 Asian gastric cell lines.

<p>Total and kinome genetics aberrations (consisting of gain, loss and LOH) in the 18 Asian gastric cell lines.</p

Top 10 cellular pathways having the most genetic aberrations in the Asian gastric cell lines.

<p>The numerics in red boxes are the number of aberrations. Increased intensity of red corresponds to increased number of aberrations.</p

Pharmacophore Model for Wnt/Porcupine Inhibitors and Its Use in Drug Design

Porcupine is a component of the Wnt pathway which regulates cell proliferation, migration, stem cell self-renewal, and differentiation. The Wnt pathway has been shown to be dysregulated in a variety of cancers. Porcupine is a membrane bound <i>O</i>-acyltransferase that palmitoylates Wnt. Inhibiting porcupine blocks the secretion of Wnt and effectively inhibits the Wnt pathway. Using high throughput screening, we have identified a number of novel porcupine inhibitors with diverse scaffolds. The pharmacophore requirements for our porcupine inhibitors were elucidated, and a pharmacophore model is proposed. Our compounds as well as all currently published porcupine inhibitors may be fitted to this model in low energy conformations with good superimposition of the pharmacophore elements. The model also explains the stereochemical requirements of our chiral porcupine inhibitors. The pharmacophore model was successfully used for designing 3 new series of porcupine inhibitors having a tricyclic xantine, a phtalimide, or a piperidine–maleimide scaffold

Discovery and Optimization of a Porcupine Inhibitor

Wnt proteins regulate various cellular functions and serve distinct roles in normal development throughout life. Wnt signaling is dysregulated in various diseases including cancers. Porcupine (PORCN) is a membrane-bound <i>O</i>-acyltransferase that palmitoleates the Wnts and hence is essential for their secretion and function. The inhibition of PORCN could serve as a therapeutic approach for the treatment of a number of Wnt-dependent cancers. Herein, we describe the identification of a Wnt secretion inhibitor from cellular high throughput screening. Classical SAR based cellular optimization provided us with a PORCN inhibitor with nanomolar activity and excellent bioavailability that demonstrated efficacy in a Wnt-driven murine tumor model. Finally, we also discovered that enantiomeric PORCN inhibitors show very different activity in our reporter assay, suggesting that such compounds may be useful for mode of action studies on the PORCN <i>O</i>-acyltransferase

Scaffold Hopping and Optimization of Maleimide Based Porcupine Inhibitors

Porcupine is an <i>O</i>-acyltransferase that regulates Wnt secretion. Inhibiting porcupine may block the Wnt pathway which is often dysregulated in various cancers. Consequently porcupine inhibitors are thought to be promising oncology therapeutics. A high throughput screen against porcupine revealed several potent hits that were confirmed to be Wnt pathway inhibitors in secondary assays. We developed a pharmacophore model and used the putative bioactive conformation of a xanthine inhibitor for scaffold hopping. The resulting maleimide scaffold was optimized to subnanomolar potency while retaining good physical druglike properties. A preclinical development candidate was selected for which extensive in vitro and in vivo profiling is reported

Fragment-Based Drug Discovery of Potent Protein Kinase C Iota Inhibitors

Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure–activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι

Fragment-Based Drug Discovery of Potent Protein Kinase C Iota Inhibitors

Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure–activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι