86 research outputs found
Abstract 4025: New candidate therapy for BAP1-mutant cancer identified using novel screen
No full textAbstract Inactivation of the oncogene BAP1 is associated with poor prognosis in many cancers, including uveal melanoma (UM). Therapies for mediating the detrimental effects of BAP1 mutations have yet to be developed. Previously, we found that BAP1 depletion in Xenopus causes a striking developmental phenotype and a global dysregulation of differentiation markers across the genetic landscape. This was caused by impaired promoter assembly at commitment genes, marked by decreased H3K27ac levels. Inhibition of HDAC4 protein rescues the affected BAP1-depleted phenotype and genetic regulations, further emphasizing the role of H3K27ac in BAP1 function. Pairing these findings with a lab-devised multitiered screening process, we identified an epigenetic compound that successfully reverses the detrimental effects of the BAP1 deficient phenotype back to phenotypically normal conditions. This multitiered screening process tests prospective compounds for the ability to mediate transcriptional aberrations caused by BAP1 loss in UM cells (phase 1), rescue the BAP1 loss phenotype in Xenopus (phase 2), and provide significant inhibition of UM tumor growth in a mouse model (phase 3). These findings reveal a new application for a promising epigenetic compound as a therapeutic and reversing agent for BAP1 mutant UM. Furthermore, our streamlined process for the testing of promising therapeutic agents can accelerate the testing of such agents and getting them to clinical trials in a timely manner. Citation Format: DAWN A. OWENS, Jeffim N. Kuznetsov, Andy Lopez, Stefan Kurtenbach, Daniel Bilbao, Evan R. Roberts, Claude-Henry Volmar, Claes R. Wahlestedt, Shaun P. Brothers, J. William Harbour. New candidate therapy for BAP1-mutant cancer identified using novel screen [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4025
Recommended from our members
DNA Hydroxymethylation in Non-coding Repeat Expansion Disorders
No full textHexanucleotide repeat expansion in C9ORF72 gene has recently been shown to cause familial amyotrophic lateral sclerosis, a neurodegenerative disease caused by global death of motor neurons. The expansion leads to partial heterochromatinization of the locus, yet mutant RNAs and dipeptide repeat proteins are still produced in sufficient quantities to confer neurotoxicity. So far several research groups have identified DNA hypermethylation at C9ORF72 promoter CpG sites in a fraction of patients, but the developmental timing and the reason of its occurrence only in a subset of individuals remains unknown. In order to model the acquisition of C9ORF72 hypermethylation, we generated induced pluripotent stem cells from an ALS patient with C9ORF72 promoter hypermethylation. Our data show that methylation levels are reduced by reprogramming and then re-acquired upon neuronal specification, while hydroxymethylation levels increase following reprogramming and are highest in iPSCs and motor neurons. We confirmed the presence of hydroxymethylation within the C9ORF72 promoter in post-mortem brain tissues of hypermethylated patients. Using iPSC neurons, we found that preventing R-loop formation did not impede heterochromatinization of the expanded locus. Moreover, we show that in C9-BAC mouse model of ALS, partial heterochromatinization of the C9ORF72 occurs during the first weeks of the lifespan, indicating that epigenetic repression is developmentally regulated. Taken together, these observations provide further insight into mechanism and developmental time-course of epigenetic perturbations conferred by the C9ORF72 repeat expansion. The Fragile X Syndrome (FXS) results from a repeat expansion mutation near the FMR1 gene promoter and is the most common form of heritable intellectual disability and autism. Mutations larger than 200 CGG repeats trigger FMR1 heterochromatinization and loss of gene expression, which is primarily responsible for the pathological features of FXS. While the role of 5-methylcytosine (5mC) in FMR1 gene silencing has been studied extensively, the role of 5-hydroxymethylation (5hmC), a newly discovered epigenetic mark produced through active DNA demethylation, has not been previously investigated in FXS neurons. Here, we used two complementary epigenetic assays, hydroxymethylation sensitive restriction digest and TET-assisted bisulfite pyrosequencing, to quantify FMR1 5mC and 5hmC levels. We observed increased levels of 5hmC at the FMR1 promoter in FXS patient brains with full-mutations relative to pre-mutation carriers and unaffected controls. In addition, we found that 5hmC enrichment at the FMR1 locus in FXS cells is specific to neurons by utilizing a nuclei sorting technique to separate neuronal and glial DNA fractions from post-mortem brain tissues. Future studies could investigate the potential to leverage this epigenetic pathway to restore FMR1 expression and discern whether levels of 5hmC correlate with phenotypic severity.</p
RNAi Joins the "Singles Club"
No full textBiotechnology & Applied MicrobiologyGenetics & HeredityMedicine, Research & ExperimentalSCI(E)1EDITORIAL MATERIAL112010-20112
Recommended from our members
Antisense RNA-Mediated Epigenetic Regulation of Brain-Derived Neurotrophic Factor.
No full textLong noncoding RNAs (lncRNAs) regulate chromatin remodeling through their interactions with epigenetic enzymes during development and disease. The inhibition of the natural antisense transcript of Brain-derived neurotrophic factor (BDNF-AS), results in BDNF promoter de-repression and transcriptional upregulation, both in vitro and in vivo. Recently, we showed that BDNF-AS interacts with the histone methyltransferase enhancer of zeste homolog 2 (EZH2) to suppress BDNF mRNA and protein expression. BDNF is an important neurotrophin that is required for neural development and maintenance of the nervous system. Dysregulation of BDNF occurs in a number of neurological disorders, including: Alzheimer’s Disease, Parkinson’s Disease, Rett syndrome, and amyotrophic lateral sclerosis. Previous attempts to upregulate BDNF by administering the recombinant form in various parts of the central nervous system have failed, mostly due to the challenge of delivering BDNF to the correct cells and neural networks. Our approach to upregulating BDNF by modulating its interaction with an epigenetic enzyme is a highly specific target with potential therapeutic value. To achieve this, we developed a novel pharmacological assay to characterize the interaction between long noncoding RNAs and their epigenetic targets using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) technology. With this assay, we are able to quantify lncRNA-protein interactions rapidly for the purpose of high throughput screening, enabling drug discovery efforts for this novel class of drug targets. In this work, we present our assay development and screening findings, including the identification of potential small molecule modulators of lncRNA-protein interactions. Furthermore, we describe the application of this lncRNA-protein interaction assay to detect RNA requirements for EZH2 recruitment, a much debated and important question that lingers in the field. From our work, it is evident that BDNF-AS has several regions of RNA that are required for EZH2 recruitment, potentially due to the importance of this transcript in regulating BDNF. This work describes exploratory drug discovery for a novel class of drug targets as well as applications to understand the basic biochemistry governing lncRNA-protein interactions.</p
Antisense RNA-Mediated Epigenetic Regulation of Brain-Derived Neurotrophic Factor.
No full textLong noncoding RNAs (lncRNAs) regulate chromatin remodeling through their interactions with epigenetic enzymes during development and disease. The inhibition of the natural antisense transcript of Brain-derived neurotrophic factor (BDNF-AS), results in BDNF promoter de-repression and transcriptional upregulation, both in vitro and in vivo. Recently, we showed that BDNF-AS interacts with the histone methyltransferase enhancer of zeste homolog 2 (EZH2) to suppress BDNF mRNA and protein expression. BDNF is an important neurotrophin that is required for neural development and maintenance of the nervous system. Dysregulation of BDNF occurs in a number of neurological disorders, including: Alzheimer’s Disease, Parkinson’s Disease, Rett syndrome, and amyotrophic lateral sclerosis. Previous attempts to upregulate BDNF by administering the recombinant form in various parts of the central nervous system have failed, mostly due to the challenge of delivering BDNF to the correct cells and neural networks. Our approach to upregulating BDNF by modulating its interaction with an epigenetic enzyme is a highly specific target with potential therapeutic value. To achieve this, we developed a novel pharmacological assay to characterize the interaction between long noncoding RNAs and their epigenetic targets using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) technology. With this assay, we are able to quantify lncRNA-protein interactions rapidly for the purpose of high throughput screening, enabling drug discovery efforts for this novel class of drug targets. In this work, we present our assay development and screening findings, including the identification of potential small molecule modulators of lncRNA-protein interactions. Furthermore, we describe the application of this lncRNA-protein interaction assay to detect RNA requirements for EZH2 recruitment, a much debated and important question that lingers in the field. From our work, it is evident that BDNF-AS has several regions of RNA that are required for EZH2 recruitment, potentially due to the importance of this transcript in regulating BDNF. This work describes exploratory drug discovery for a novel class of drug targets as well as applications to understand the basic biochemistry governing lncRNA-protein interactions.</p
Recommended from our members
Novel Signaling Mechanisms Implicated in Epithelial Mesenchymal Transition and Metastasis of Human Cancers
No full textDistant metastases are the leading cause of breast cancer related death. It is important to understand how the metastatic process is regulated in order to develop new therapies that would oppose metastasis and improve cancer outcome. Initiation of metastasis requires cell invasion and escape from the primary tumor into the vasculature followed by colonization of secondary sites. Tumor invasion and intravasation are enabled by the epithelial mesenchymal transition (EMT), a process in which epithelial cells lose polarity and intracellular adhesion and acquire motility and invasiveness. This thesis work has revealed novel mechanisms whereby cancer metastasis is regulated via induction of EMT. First, this thesis work provides a novel mechanism whereby VEGFA promotes metastasis through activation of EMT. VEGFA is best known as an angiogenic agent, but it also promotes cancer invasion and metastasis through mechanisms that are not fully understood. Our prior work showed VEGFA mediates cancer stem cell (CSC) expansion via induction of SOX2, a key stem cell driver in breast cancer models. Here, we showed VEGFA rapidly upregulates SOX2, leading to SNAI2 induction and EMT. Sox2 downregulates miR-452, a novel metastasis suppressor, that we show directly targets the SNAI2 3’ UTR. VEGFA induction of SOX2 leads to loss of miR452 and upregulation of Slug, driving EMT and metastasis in breast cancer models. Second, we also uncovered a new mechanism whereby T157 and T198-phosphorylated, deregulated p27 contributes to cancer metastasis. In normal cells, p27 regulates cell cycle and functions as an atypical tumor suppressor. While p27 is rarely completely lost, it is frequently deregulated through either excess degradation or through key C-terminal phosphorylations in human cancers. We and others have previously identified an oncogenic role for p27 in motility, invasion and metastasis resulting from these C-terminal phosphorylations. Here we showed that p27CK-DD induced EMT and enhanced metastatic potential of breast cancer cell lines. Knockdown of p27 in highly metastatic EMT-transformed cell lines with high levels of p27pT157pT198 (p27pTpT) reverted EMT and impaired metastatic potential. Mechanistically, we provides evidence for p27 as a transcriptional co-regulator of cJun. We showed that C-terminally phophorylated p27 binds and activates cJun, and forms a complex with cJun at an enhancer region upstream of the TGF-β2 gene to induce TGF-β2 and EMT. My Ph.D. data identify novel pathways in which Sox2, upregulated by VEGFA, contributes to activation of EMT and metastasis through Slug. Furthermore, this thesis work reveals an oncogenic function of p27 to promote tumor progression through EMT via cJun-mediated TGF-β2 induction. Since treatment of metastasis is the final therapeutic frontier, it is hoped that mechanistic insights into acquisition of metastatic potential through EMT may ultimately generate new strategies for opposing metastasis and improving outcome of cancer.</p
Recommended from our members
Histone Modification ChIP-seq Algorithm Engineering and High Performance Bioinformatics Graphics and Analysis Software for Computational Epigenetics
No full textNovel algorithm design, implementation, and optimization in histone modification ChIP-seq analysis of broad chromatin mark data is the subject of part I of this dissertation, focusing on data-driven precision medicine computational strategies for mapping ChIP-seq peaks to genomic features (and biological function) as well as coverage island analysis of low-sample size ChIP-seq experiments within individual biological replicates. Part II of this dissertation focuses on novel algorithm design, implementation, and analysis of high performance visualization techniques for histone modification ChIP-seq data using static and interactive biological gene expression heatmaps.</p
Novel Signaling Mechanisms Implicated in Epithelial Mesenchymal Transition and Metastasis of Human Cancers
No full textDistant metastases are the leading cause of breast cancer related death. It is important to understand how the metastatic process is regulated in order to develop new therapies that would oppose metastasis and improve cancer outcome. Initiation of metastasis requires cell invasion and escape from the primary tumor into the vasculature followed by colonization of secondary sites. Tumor invasion and intravasation are enabled by the epithelial mesenchymal transition (EMT), a process in which epithelial cells lose polarity and intracellular adhesion and acquire motility and invasiveness. This thesis work has revealed novel mechanisms whereby cancer metastasis is regulated via induction of EMT. First, this thesis work provides a novel mechanism whereby VEGFA promotes metastasis through activation of EMT. VEGFA is best known as an angiogenic agent, but it also promotes cancer invasion and metastasis through mechanisms that are not fully understood. Our prior work showed VEGFA mediates cancer stem cell (CSC) expansion via induction of SOX2, a key stem cell driver in breast cancer models. Here, we showed VEGFA rapidly upregulates SOX2, leading to SNAI2 induction and EMT. Sox2 downregulates miR-452, a novel metastasis suppressor, that we show directly targets the SNAI2 3’ UTR. VEGFA induction of SOX2 leads to loss of miR452 and upregulation of Slug, driving EMT and metastasis in breast cancer models. Second, we also uncovered a new mechanism whereby T157 and T198-phosphorylated, deregulated p27 contributes to cancer metastasis. In normal cells, p27 regulates cell cycle and functions as an atypical tumor suppressor. While p27 is rarely completely lost, it is frequently deregulated through either excess degradation or through key C-terminal phosphorylations in human cancers. We and others have previously identified an oncogenic role for p27 in motility, invasion and metastasis resulting from these C-terminal phosphorylations. Here we showed that p27CK-DD induced EMT and enhanced metastatic potential of breast cancer cell lines. Knockdown of p27 in highly metastatic EMT-transformed cell lines with high levels of p27pT157pT198 (p27pTpT) reverted EMT and impaired metastatic potential. Mechanistically, we provides evidence for p27 as a transcriptional co-regulator of cJun. We showed that C-terminally phophorylated p27 binds and activates cJun, and forms a complex with cJun at an enhancer region upstream of the TGF-β2 gene to induce TGF-β2 and EMT. My Ph.D. data identify novel pathways in which Sox2, upregulated by VEGFA, contributes to activation of EMT and metastasis through Slug. Furthermore, this thesis work reveals an oncogenic function of p27 to promote tumor progression through EMT via cJun-mediated TGF-β2 induction. Since treatment of metastasis is the final therapeutic frontier, it is hoped that mechanistic insights into acquisition of metastatic potential through EMT may ultimately generate new strategies for opposing metastasis and improving outcome of cancer.</p
HeatmapGenerator: high performance RNAseq and microarray visualization software suite to examine differential gene expression levels using an R and C++ hybrid computational pipeline
No full textMicroScope: ChIP-seq and RNA-seq software analysis suite for gene expression heatmaps
No full textHeatmaps are an indispensible visualization tool for examining large-scale snapshots of genomic activity across various types of next-generation sequencing datasets. However, traditional heatmap software do not typically offer multi-scale insight across multiple layers of genomic analysis (e.g., differential expression analysis, principal component analysis, gene ontology analysis, and network analysis) or multiple types of next-generation sequencing datasets (e.g., ChIP-seq and RNA-seq). As such, it is natural to want to interact with a heatmap's contents using an extensive set of integrated analysis tools applicable to a broad array of genomic data types.
We propose a user-friendly ChIP-seq and RNA-seq software suite for the interactive visualization and analysis of genomic data, including integrated features to support differential expression analysis, interactive heatmap production, principal component analysis, gene ontology analysis, and dynamic network analysis.
MicroScope is hosted online as an R Shiny web application based on the D3 JavaScript library: http://microscopebioinformatics.org/ . The methods are implemented in R, and are available as part of the MicroScope project at: https://github.com/Bohdan-Khomtchouk/Microscope
- …
