Active Motif Epigenetic Services Grant Program

November 16, 2022

Warren Fiskus, PhD
University of Texas
MD Anderson Cancer Center

"Collectively with the single-cell multiome analyses, data generated will allow us to determine the effect of a chromatin remodeling inhibitor (CRI) on the distinct chromatin states (open vs closed chromatin) and transcriptome profiles in clusters of AML cells, immune-effector cells and stromal cells, which could potentially be correlated in the future with the therapeutic response or resistance to CRI treatment.”

Winning Abstract

Acute Myeloid Leukemia (AML) afflicts approximately 20,000 adults per year and is lethal in the majority, especially in elderly patients. AML stem/progenitor cells (LSCs) resist differentiation, retain leukemia-initiating potential and mediate relapse of AML. Driver genetic alterations dysregulate enhancers (Es) and transcriptome that orchestrates the phenotype and malignant behavior of AML LSCs. Chromatin remodelers (CRs) control chromatin accessibility, at the enhancers/promoters and activity of the key transcription factors (TFs) and co-factors that induce the dysregulated transcriptome in AML. A chromatin remodeling inhibitor (CRI) that induces lethality in AML cells is currently being evaluated for efficacy in early phase clinical trials in AML at MD Anderson Cancer Center. However, CRI-induced perturbations in the activity of Es and their targeted gene-expressions have not been characterized in AML cells. It is important to determine and correlate this activity of CRI with its anti-AML efficacy. Single-cell Multiome ATAC-Seq and RNA-Seq analysis (utilizing the 10X chromium controller) is a powerful technique to determine the chromatin accessibility, active Es and transcriptome at the single cell level, which can be bioinformatically linked to delineate drug-induced gene-expression signature not only in the AML bone marrow aspirate samples but also in the immune-effector and stromal cells present in the AML micro-environment. The CRI-induced signature of transcriptional perturbations in AML LSCs and microenvironment can be utilized as a biomarker signature for correlation with the clinical efficacy/resistance of CRI. To accomplish this goal, we first determined the median LD50 (50% lethal dose) of CRI in PD AML cells, by evaluating the dose-response lethal effect of CRI in patient-derived (PD), poor prognosis AML cells (with MLL1 rearrangement) to different doses and exposure intervals of CRI. We next treated two samples of BMA-derived mononuclear cells from AML with MLL1r to the LD50 concentration of CRI for 16 hours and cryopreserved the cells (total of 4 samples). We anticipate that we will have available two additional samples (untreated and treated with CRI), making a total of six AML samples for conducing the single-cell multiome ATAC-Seq and RNA-Seq analysis. We will also apply GSVA to identify effects of CRI on functional gene-sets highlighting alterations in signaling pathways in the cellular subpopulations. We will concomitantly perform ChIP-Seq analyses on the AML samples, utilizing H3K27Ac and BRD4 antibodies to determine the effects of CRI treatment on the active chromatin. Collectively with the single-cell multiome analyses, data generated will allow us to determine the effect of CRI on the distinct chromatin states (open vs closed chromatin) and transcriptome profiles in clusters of AML cells, immune-effector cells and stromal cells, which could potentially be correlated in the future with the therapeutic response or resistance to CRI treatment.

May 23, 2019

We would like to thank all of the researchers that took the time to submit abstracts for this year’s competition. There were many outstanding abstracts that we selected two winners this year! We would like to congratulate our winners, Dr. Enrico Glaab and Dr. Lucy Stead. We were excited about Dr. Glaab’s research, which aims to understand how epigenetics may account for the “missing heritability” and mediate environmental influences in Parkinsonian disorders. We were equally as excited about Dr. Stead’s research. The aim of the study is to understand the role of histone modifications at bivalent promoters that induce transcriptional reprogramming of cells derived from Glioblastoma to survive radiation and chemotherapy. Both of these studies hope to shed light on the importance of understanding the role of epigenetics in disease and how understanding the mechanism of such will one day lead to better diagnosis and outcomes for many devastating diseases. We look forward to participating in these exciting projects!

Services Grant Competition Winners

Enrico Glaab, PhD
Assistant Professor, Luxembourg Centre for Systems Biomedicine
University of Luxembourg

Biomedical Data Science group and Epigenetics group

Front row (left to right): Yujuan Gui, Borja Gomez Ramos, Sergio Helgueta, Muhammad Ali.
Back row (left to right): Enrico Glaab (PI of the Biomedical Data Science group), Julia Becker, Armin Rauschenberger, Jochen Ohnmacht, Marios Gavriil, Lasse Sinkkonen (PI of the Epigenetics group), Léon-Charles Tranchevent, Diana Hendrickx

Winning Abstract

Our main objective is to improve the understanding of epigenetic alterations in Parkinsonian disorders and their utility in differential diagnosis.

Parkinson's disease (PD) and Progressive Supranuclear Palsy (PSP) are two neurodegenerative movement disorders with many similar symptoms. Due to their phenotypic resemblance, they can be easily misdiagnosed, and objective and reliable diagnostic biomarker signatures are needed for personalized therapies.

Disease-associated genetic variants have been identified in recent years for both disorders. Still, a large fraction of the heritability of PD and PSP, and the underlying molecular disease causes, remain largely unknown. Epigenetic mechanisms influencing chromatin accessibility, such as DNA and histone modifications, may account for a significant part of this ‘missing heritability’ and mediate environmental influences on disease susceptibility. Previous studies revealed that genes containing PD-associated genetic risk variants display altered DNA methylation in various brain tissues in PD, suggesting that their chromatin accessibility is also altered. These PD-linked methylation changes in brain tissue show a high concordance with methylome alterations in blood. Thus, chromatin accessibility changes in blood cells, such as monocytes, could serve as an easy-to-access surrogate biomarker with major diagnostic value. However, epigenetic changes in PD have not yet been compared against those in other forms of parkinsonism, such as PSP, and no chromatin accessibility studies have been performed in the context of PD.

We hypothesize that:
(1) Significant chromatin accessibility changes occur in monocytes from PD and PSP patients as compared to unaffected controls.
(2) Multivariate signatures of these changes provide significant discriminative information for differential diagnostic model building using machine learning methods.

To test these hypotheses we aim to integrate data from genome-wide ATAC-seq chromatin accessibility profiles of monocytes from our local patient cohort for PD and atypical parkinsonism syndromes (LuxPARK), with state-of-the-art molecular network perturbation analyses exploiting prior transcriptomic and genomic data we previously collected for these disorders. By comparing ATAC-seq data from monocytes of idiopathic PD patients, PSP patients and healthy controls, we will determine genomic regions and target genes with altered chromatin accessibility. After mapping this data onto a genome-scale gene and protein regulatory network, we will apply our machine learning algorithm to determine the most affected sub-networks. The derived perturbed sub-networks will enable a network-based interpretation of disease-associated epigenetic changes and their interrelations with transcriptomic and genomic changes. Finally, we will assess the potential of network-based multi-omics signatures to provide more robust diagnostic models than conventional methods focused on single biomarker molecules.

Lucy Stead, PhD
University Academic Fellow, Leeds Institute of Medical Research at St James's
University of Leeds

Winning Abstract

Glioblastoma (GBM) is the most common and most deadly form of adult brain cancer. It has a median survival of just 15 months and kills more people in their 40s than any other cancer. This is because GBM tumours are incurable; cells break away from the main tumour and invade into the surrounding brain tissue making complete surgical removal impossible. The cells that remain are treated with radiation and chemotherapy but some of them inevitably survive, leading to tumour regrowth. To address the unmet clinical need for more effective treatment of GBM, we must specifically characterise the cells that currently resist treatment and find ways to kills them. To this end, we have been doing large-scale profiling and comparison of matched primary and post-treatment recurrent GBM tumours from the same patient. Our work so far has indicated that treatment resistance is not conferred by tumour specific mutations as is the case of some other cancers. This partially explains why drugs that have been developed to target genetic abnormalities within GBM tumours have failed to yield clinical impact. Furthermore, we have identified evidence for transcriptional reprogramming of GBM cells in response to treatment, which may facilitate their survival and consequently provide a novel therapeutic opportunity. This reprogramming is indicated to occur via chromatin remodelling, especially around bivalent promoters, implicating two specific histone marks: H3K27me3 and H3K4me3. We have formalin-fixed pre-and post-treatment GBM tumour samples from the same patient, from which we have already acquired the expression profiles via RNAseq. We propose to now additionally characterise and compare the locations of both trimethylated H3K27 and H3K4 within these samples, using the Active Motif FFPE ChIPseq service, to acquire preliminary data to address our hypothesis: that remodelling of bivalent promoters enables transcriptional reprogramming in response to treatment in GBM. We will acquire raw data from Active Motif for this single pair and integrate it with our RNAseq and exome sequencing data, in house, to determine a) which promoters are repressed (H3K7me3), active (H3K4me3), or primed for activity (bivalent i.e. both marks are present) in the primary GBM, b) how the status of each promoter is changed after treatment by comparing the presence of these marks in isolation and combination in the recurrent tumour, c) how well gene expression from each promoter correlates with its histone-mark status in each tumour independently, and d) how expression changes after treatment correlates with therapy-driven changes in promoter status. This will confirm whether the transcriptional reprogramming we observe in recurrent versus primary GBM tumours is driven by epigenetic changes. If so, this highlights the deposition or removal of these histone marks as potentially therapeutically targetable mechanisms to more effectively treat GBM.

May 16, 2018

We would like to thank all of the researchers that took the time to submit abstracts and we'd also like to congratulate Maha Abdellatif on being selected to receive $20,000 in free services. We were especially excited about Dr. Abdellatif’s research, which connects metabolism and metabolic enzymes directly with chromatin bound complexes, which results in a local supply of cofactors that are required for histone modifying enzyme function. The potential role of this epigenetic and metabolic connection to cardiovascular disease makes this research all the more important and we look forward to participating in this exciting project.

Services Grant Competition Winner

Maha Abdellatif, PhD
Professor, Cell Biology & Molecular Medicine
Rutgers University

Abdellatif Lab

From left to right: Zhi Yang (Surgeon), Sujung Choi (Graduate Student), Maha Abdellatif (PI), Kathy He (Lab Manager), Yong Heui Jeon (Graduate Student).

Winning Abstract

Our overall goal is to understand the mechanisms that govern transcription in the heart during health and disease.

Transcription is a highly dynamic process that requires metabolic intermediates for its activation or deactivation, these include: acetyl-CoA for histone acetylation, alpha-ketoglutarate as a cofactor for histone and DNA demethylases, and succinyl-CoA for histone succinylation. Since none of the CoA-linked metabolites could be exported out of the mitochondria, the nucleus must acquire its acetyl-CoA (Ac-CoA), mainly, via export of citrate from the mitochondria during substrate abundance, which is then converted to acetyl-CoA in the nucleus via ATP citrate lyase. On the other hand, the nucleus’s source of alpha-ketoglutarate (alpha-KG), succinyl-CoA (Suc-CoA), or other short-chain acyl-CoA is not established. The other unanswered question, is how are histones selectively modified at promoters and how does this influence an organ’s homeostasis? In a recent unbiased screen for discovery of proteins that associated with chromatin-bound histone variant H2A.Z in the heart, we uncovered mitochondrial enzymes of the TCA cycle, beta-oxidation, and branched-chain amino acid catabolism in the nucleus, uniquely localized to the transcription start sites (TSS) of genes. The data have been confirmed by immunostaining and Western blots in mouse heart tissue, isolated adult and neonatal myocytes, human iPSC-derived myocytes, and mouse embryos, and metabolomics that identified the metabolites in the nucleus after inhibiting the respective metabolizing enzyme. Importantly, we also uniquely show, using chromatin immunoprecipitation-sequencing (ChIP-Seq) with anti-acetyl-CoA acyltransferase (ACAA2), that this enzyme localizes selectively to the TSS of genes that have H2A.Z in the heart. Knockdown of ACAA2 in cardiac myocytes reduced histone modifications in those promoters. We are currently focusing our investigation on the nuclear role of 4 enzymes, representatives of the pathways that catabolize glucose and fatty acids. These include isocitrate dehydrogenase 2 (IDH2), which converts isocitrate into alpha-ketoglutarate; OGDH, which converts alpha-KG into Suc-CoA; pyruvate dehydrogenase A1 (PDHA1), which converts pyruvate into Ac-CoA; ACAA2, which converts 3-ketoacyl-CoA into Ac-CoA and acyl-CoA.

We hypothesize that:
1) The nucleus harbors mitochondrial enzymes of the TCA cycle and beta-oxidation of fatty acids, that are specifically localized to H2A.Z-bound chromatin at the TSS of select genes.
2) This allows for the local production of Ac-CoA, Suc-CoA, and the production/consumption of alphaKG, which are required for histone modifications necessary for transcriptional activation or repression. Disruption of the nuclear localization of these genes results in the reduction of histone acetylation and succinylation, or enhances methylation at select gene promoters, dysregulating gene expression, and promoting or inhibiting cardiomyopathy, depending on the genes that are selectively regulated.

Using ChIP-Seq, our aim is to identify the chromatin association sites of the metabolic enzymes PDHA1, IDH2, OGDH, and ACAA2 in the normal and hypertrophied hearts, and the effect of their knockdown on histone modifications.