Anti-metastatic Inhibitors of Lysyl Oxidase (LOX): Design and Structure-Activity Relationships

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase that crosslinks collagens and elastin in the extracellular matrix (ECM) and is a critical mediator of tumor growth and metastatic spread. LOX is a target for cancer therapy and thus the search for therapeutic agents against LOX has been widely sought. We report herein the medicinal chemistry discovery of a series of LOX inhibitors bearing an aminomethylenethiophene (AMT) scaffold. High throughput screening (HTS) provided the initial hits. Structure-activity relationship (SAR) studies led to the discovery of AMT inhibitors with sub-micromolar half maximal inhibitory concentrations (IC50) in a LOX enzyme activity assay. Further SAR optimisation yielded the orally bioavailable LOX inhibitor CCT365623 with good anti-LOX potency, selectivity, pharmacokinetic properties, as well as anti-metastatic efficacy

Dynamic Gene Regulatory Networks Drive Hematopoietic Specification and Differentiation.

Metazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development.This work was funded by a Longer Larger (LoLa) consortium grant from the Biotechnology and Biological Sciences Research Council, UK, to the senior authors and the corresponding author, computing infrastructure grants from the Wellcome Trust and National Institute for Health Research to B.G., grants from Cancer Research UK to G.L. and V.K., and funding from the Bloodwise charity to C.B.This is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.devcel.2016.01.02

EINCR1 is an EGF inducible lincRNA overexpressed in lung adenocarcinomas

Long non-coding RNAs are being increasingly recognised as important molecules involved in regulating a diverse array of biological functions. For example, many long non-coding RNAs have been associated with tumourigenesis and in this context their molecular functions often involves impacting on chromatin and transcriptional control processes. One important cellular control system that is often deregulated in cancer cells is the ERK MAP kinase pathway. Here we have investigated whether ERK pathway signaling in response to EGF stimulation, leads to changes in the production of long non-coding RNAs. We identify several different classes of EGF pathway-regulated lncRNAs. We focus on one of the inducible lincRNAs, EGF inducible long intergenic non-coding RNA 1 (EINCR1). EINCR1 is predominantly nuclear and shows delayed activation kinetics compared to other immediate-early EGF-inducible genes. In humans it is expressed in a tissue-specific manner and is mainly confined to the heart but it exhibits little evolutionary conservation. Importantly, in several cancers EINCR1 shows elevated expression levels which correlate with poor survival in lung adenocarcinoma patients. In the context of lung adenocarcinomas, EINCR1 expression is anti-correlated with the expression of several protein coding EGF-regulated genes. A potential functional connection is demonstrated as EINCR1 overexpression is shown to reduce the expression of EGF-regulated protein coding genes including FOS and FOSB

Regulation of RUNX1 dosage is crucial for efficient blood formation from Hemogenic Endothelium

ABSTRACT During ontogeny, hematopoietic stem and progenitor cells arise from hemogenic endothelium through an endothelial-to-hematopoietic transition that is strictly dependent on the transcription factor RUNX1. Although it is well established that RUNX1 is essential for the onset of hematopoiesis, little is known about the role of RUNX1 dosage specifically in hemogenic endothelium and during the endothelial-to-hematopoietic transition. Here, we used the mouse embryonic stem cell differentiation system to determine if and how RUNX1 dosage affects hemogenic endothelium differentiation. The use of inducible Runx1 expression combined with alterations in the expression of the RUNX1 co-factor CBFβ allowed us to evaluate a wide range of RUNX1 levels. We demonstrate that low RUNX1 levels are sufficient and necessary to initiate an effective endothelial-to-hematopoietic transition. Subsequently, RUNX1 is also required to complete the endothelial-to-hematopoietic transition and to generate functional hematopoietic precursors. In contrast, elevated levels of RUNX1 are able to drive an accelerated endothelial-to-hematopoietic transition, but the resulting cells are unable to generate mature hematopoietic cells. Together, our results suggest that RUNX1 dosage plays a pivotal role in hemogenic endothelium maturation and the establishment of the hematopoietic system.</jats:p

<i>EINCR1</i> is specifically expressed in normal human heart tissues and is up-regulated in cancer.

<p>(A) Violin plots of the expression of RP11-7F17.7 in normal tissues [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181902#pone.0181902.ref037" target="_blank">37</a>]. (B) Heatmap showing the expression of <i>EINCR1</i> and the indicated protein coding genes across different organs in human embryos [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181902#pone.0181902.ref022" target="_blank">22</a>]. Data are row-normalised to the maximum observed expression (blue scale). The maximum absolute expression of each gene is indicated by the green scale. (C) Boxplots of <i>EINCR1</i> and <i>MALAT1</i> expression in the indicated cancer categories. Expression in both normal (N; blue) and tumour (T; red) samples are shown. Numbers of each type of sample are provided below each cancer subtype. Median values are shown by horizontal lines. Statistically significant differences are indicated: ** = P-value <0.01; *** = P-value<0.001. (D) Kaplan-Meier plot of overall survival in patients with lung adenocarcinoma (n = 206, from the TCGA-LUAD dataset)), using sample groups with either the top or bottom 20% expression of <i>EINCR1</i>. Log rank probabilities between low and high expression are shown. (E) RT-qPCR analysis of <i>EINCR1</i> and <i>FOS</i> expression after EGF stimulation of A549 cells for the indicated times. Data are shown relative to the zero timepoint (taken as 1) and represent mean ± SD (n = 3). * = P-value = 0.05.</p

EINCR1 transcription is regulated via the ERK MAPK pathway.

<p>(A) A screenshot of the genomic features observed near the <i>EINCR1</i> genomic locus compiled from UCSC genome browser data. Data for the histone modifications (H3K3me3, H3K9me3, H3K27ac, H3K27me3, H3K36me3 ChIP-seq are derived from ENCODE data from the MCF7 cell line [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181902#pone.0181902.ref038" target="_blank">38</a>]). RNA polII ChIP-seq data from HeLa cells (before and after EGF induction) are derived from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181902#pone.0181902.ref039" target="_blank">39</a>]. The RNA-seq data are from this study. The promoter sequence of human RP11-7F17.7 (TSS±1000bp) is aligned with five primates, mouse and rat reference sequence using multiz aligner [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181902#pone.0181902.ref040" target="_blank">40</a>] (middle panel). The sequence immediately surrounding the TSS is shown at in the top panel and a putative TATA-like sequence unique to humans is indicated. The TSS is indicated by an arrow. Genomic locations are shown above each panel. (B) RT-qPCR analysis of <i>EINCR1</i> expression following EGF treatment of MCF10A cells for 15 mins in the presence and absence of the MEK inhibitor U0126. n = 2, * = P-value < 0.05. (C) RT-qPCR analysis of <i>FOS</i> and <i>EINCR1</i> after EGF stimulation of MCF10A cells for indicated times in the presence or absence of cycloheximide (CHX). Data are shown relative to the zero timepoint (taken as 1) and represent mean ± SEM from two independent repeats. (D) RT-qPCR analysis of <i>EINCR1</i> after EGF stimulation of MCF10A cells for the indicated times in the presence or absence of dominant negative FOS (A-FOS). Data are shown relative to the zero timepoint (taken as 1) and represent mean ± SEM from two independent repeats.</p

High levels of <i>EINCR1</i> transcription leads to changes in the expression profiles of EGF-regulated genes.

<p>(A) Boxplots of <i>EINCR1</i>, <i>FOSB</i> and <i>FOS</i> expression in lung adenocarcinomas (n = 339 cancer samples from lung adenocarcinomas “not otherwise specified” [NOS], and 108 normal lung samples from TCGA data). Expression in both normal (N; blue) and tumour (T; red) samples are shown. Median values are shown by horizontal lines. Statistically significant differences are indicated: *** = P-value<0.001. (B and C) Scatterplots showing the expression of <i>EINCR1</i> and either <i>FOSB</i> (B) or <i>FOS</i> (C) in lung adenocarcinomas NOS (n = 339). Samples containing either high <i>EINCR1</i> but low protein coding gene expression (or vice versa) are circled. The units on each axis are the RPK10M (reads per 1 kb per 10 M reads) normalised by the maximum value of the corresponding gene and then multiplied by 10. (D) Model of the CRISPR-based upregulation system [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181902#pone.0181902.ref026" target="_blank">26</a>]. dCas9 –mutated Cas9, VP64—4x viral protein 16 transactivation domain, MBP—MS2 binding protein, p65 –transcriptional activation domain of p65. (E-I) RT-qPCR analysis of expression of <i>EINCR1</i> (E) or the indicated protein coding genes (F-I) following EGF stimulation of MCF10A cells for the indicated times either in the presence of guide RNA targeting the <i>EINCR1</i> promoter 218 bp upstream of the TSS (red line) or control non-targeting (NT) guides (black line). Data are shown relative to each sample in the absence of EGF (taken as 1) and are from three independent replicates (except <i>IER3</i> where n = 2). * = P-value <0.05; ** = P-value <0.01; *** = P-value<0.001.</p