Publications

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors.

Supplementary Table 1 from Discovery of Epigenetically Silenced Genes by Methylated DNA Immunoprecipitation in Colon Cancer Cells

Abstract Triple-negative breast cancer (TNBC) represents the most heterogeneous and aggressive subtype of breast carcinomas, characterized by the absence of clinical biomarkers (ERα, PR, and HER2) and the lack of targeted therapies. In this regard, several clinical trials have consistently failed to effectively stratify patients and identify specific treatments that elicit substantial responses. This study aims to pinpoint biomarkers expressed exclusively in the basal mammary epithelial compartment, facilitating a refined subclassification of this breast cancer subtype. Using computational analyses of single-cell RNA sequencing data, we have identified a list of genes associated with basal identity (BC-markers). Histological validation in 137 human samples has enabled us to categorize TNBC patients into BC-positive and BC-negative TNBC subtypes. Significantly, the presence of these markers correlates with a poorer prognosis in TNBC patients. Functional analyses have revealed a pivotal role for TAGLN in cell migration, likely influencing tumor aggressiveness. Further, we discovered that BC-marker expression is associated with the mesenchymal phenotype and increased sensitivity to the tyrosine kinase inhibitor dasatinib, particularly in BC-positive TNBC, suggesting novel therapeutic avenues. In our study, TAGLN emerged as a potential predictive biomarker for dasatinib responsiveness, offering new directions for personalized therapy for TNBC patients.

Univariate and multivariate logistic regression of the second CRC patient cohort. (XLSX 9 kb)

Rett syndrome (RTT) is a complex neurological disorder that is one of the most frequent causes of mental retardation in women. A great landmark in research in this field was the discovery of a relationship between the disease and the presence of mutations in the gene that codes for the methyl-CpG binding protein 2 (MeCP2). Currently, MeCP2 is thought to act as a transcriptional repressor that couples DNA methylation and transcriptional silencing. The present study aimed to identify new target genes regulated by Mecp2 in a mouse model of RTT.We have compared the gene expression profiles of wild type (WT) and Mecp2-null (KO) mice in three regions of the brain (cortex, midbrain, and cerebellum) by using cDNA microarrays. The results obtained were confirmed by quantitative real-time PCR. Subsequent chromatin immunoprecipitation assays revealed seven direct target genes of Mecp2 bound in vivo (Fkbp5, Mobp, Plagl1, Ddc, Mllt2h, Eya2, and S100a9), and three overexpressed genes due to an indirect effect of a lack of Mecp2 (Irak1, Prodh and Dlk1). The regions bound by Mecp2 were always methylated, suggesting the involvement of the methyl-CpG binding domain of the protein in the mechanism of interaction.We identified new genes that are overexpressed in Mecp2-KO mice and are excellent candidate genes for involvement in various features of the neurological disease. Our results demonstrate new targets of MeCP2 and provide us with a better understanding of the underlying mechanisms of RTT.

The approval of histone deacetylase inhibitors for treatment of lymphoma subtypes has positioned histone modifications as potential targets for the development of new classes of anticancer drugs. Histones also undergo phosphorylation events, and Haspin is a protein kinase the only known target of which is phosphorylation of histone H3 at Thr3 residue (H3T3ph), which is necessary for mitosis progression. Mitotic kinases can be blocked by small drugs and several clinical trials are underway with these agents. As occurs with Aurora kinase inhibitors, Haspin might be an optimal candidate for the pharmacological development of these compounds. A high-throughput screening for Haspin inhibitors identified the CHR-6494 compound as being one promising such agent. We demonstrate that CHR-6494 reduces H3T3ph levels in a dose-dependent manner and causes a mitotic catastrophe characterized by metaphase misalignment, spindle abnormalities and centrosome amplification. From the cellular standpoint, the identified small-molecule Haspin inhibitor causes arrest in G2/M and subsequently apoptosis. Importantly, ex vivo assays also demonstrate its anti-angiogenetic features; in vivo, it shows antitumor potential in xenografted nude mice without any observed toxicity. Thus, CHR-6494 is a first-in-class Haspin inhibitor with a wide spectrum of anticancer effects that merits further preclinical research as a new member of the family of mitotic kinase inhibitors.

Aberrant DNA methylation often occurs in colorectal cancer (CRC). In our study we applied a genome-wide DNA methylation analysis approach, MethylCap-seq, to map the differentially methylated regions (DMRs) in 24 tumors and matched normal colon samples. In total, 2687 frequently hypermethylated and 468 frequently hypomethylated regions were identified, which include potential biomarkers for CRC diagnosis. Hypermethylation in the tumor samples was enriched at CpG islands and gene promoters, while hypomethylation was distributed throughout the genome. Using epigenetic data from human embryonic stem cells, we show that frequently hypermethylated regions coincide with bivalent loci in human embryonic stem cells. DNA methylation is commonly thought to lead to gene silencing; however, integration of publically available gene expression data indicates that 75% of the frequently hypermethylated genes were most likely already lowly or not expressed in normal tissue. Collectively, our study provides genome-wide DNA methylation maps of CRC, comprehensive lists of DMRs, and gives insights into the role of aberrant DNA methylation in CRC formation.

<p>Captions for supplementary Figures and Tables</p>

Abstract Genetic analyses of chromatin remodelers in cancer Laia S. Riudalbas1, Sónia A. Melo, Anna Portela & Manel Esteller. 1Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), 08907 L'Hospitalet, Barcelona, Catalonia, Spain Chromatin remodelers are ATP dependent proteins that enable dynamic access to packaged DNA and tailor nucleosome composition in chromosomal regions. These protein complexes have been shown to regulate a wide range of cellular processes, including transcription regulation, DNA-damage response and DNA replication. Being that some components of this remodeler machinery were already characterized as tumor suppressor genes and/or oncogenic, we can hypothesize that the dysregulation of any of these molecules in tumor cells with microsatellite instability (MSI+), that show a higher rate of insertions and deletions, can lead to neoplastic transformation. For that we decided to screen all the exonic mononucleotide repeats of some of the most important chromatin remodelers with ATPase activity in colorectal, gastric and endometrial cancer cell lines, on the search for frameshift mutations that could alter protein function. We have mainly analyzed the catalytic subunits of remodeling complexes because any alteration of these crucial proteins could drive into a tumorigenic process. Until now no alteration was found in the cancer cell lines studied. This could be underscoring the importance of these genes to normal cellular functioning being that their disruption could impair cell viability. This work was supported by Grants SAF2007-00027-65134, FIS PI08-0517, Consolider CSD2006-49, and CANCERDIP FP7-200620. L.S.R. is a research fellow of FIS - Instituto de Salud Carlos III, FI09/00218. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4887.

Some but not all neonates are affected by prenatal exposure to serotonin reuptake inhibitor antidepressants (SRI) and maternal mood disturbances. Distinguishing the impact of these two exposures is challenging and raises critical questions about whether pharmacological, genetic, or epigenetic factors can explain the spectrum of reported outcomes. Using unbiased DNA methylation array measurements followed by a detailed candidate gene approach, we examined whether prenatal SRI exposure was associated with neonatal DNA methylation changes and whether such changes were associated with differences in birth outcomes. Prenatal SRI exposure was first associated with increased DNA methylation status primarily at <i>CYP2E1</i>(β_Non-exposed = 0.06, β_SRI-exposed = 0.30, FDR = 0); however, this finding could not be distinguished from the potential impact of prenatal maternal depressed mood. Then, using pyrosequencing of <i>CYP2E1</i> regulatory regions in an expanded cohort, higher DNA methylation status—both the mean across 16 CpG sites (<i>P</i> &lt; 0.01) and at each specific CpG site (<i>P</i> &lt; 0.05)—was associated with exposure to lower 3^rd trimester maternal depressed mood symptoms only in the SRI-exposed neonates, indicating a maternal mood × SRI exposure interaction. In addition, higher DNA methylation levels at CpG2 (<i>P</i> = 0.04), CpG9 (<i>P</i> = 0.04) and CpG10 (<i>P</i> = 0.02), in the interrogated <i>CYP2E1</i> region, were associated with increased birth weight independently of prenatal maternal mood, SRI drug exposure, or gestational age at birth. Prenatal SRI antidepressant exposure and maternal depressed mood were associated with altered neonatal <i>CYP2E1</i> methylation status, which, in turn, appeared to be associated with birth weight.

Background: Colorectal cancer (CRC) is characterized by marked inter-tumor heterogeneity, both molecularly and clinically. Patient stratification using transcriptional subtyping show promise to resolve the heterogeneity and guide precision medicine. This requires high quality RNA, which can be purified from fresh frozen (FF) tissue, but not from clinically collected formalin-fixed paraffin-embedded (FFPE) tissue. Consequently, transcriptional subtyping is not applicable to most CRC patients.Aim: To establish a DNA methylation-based approach for molecular characterization and subtyping, which is compatible with both FF and FFPE tissue, and use this to improve patient prognostication as compared to histopathological TNM staging.Methods: Using paired RNA expression and DNA methylation profiles (450K array) from 394 CRCs, we identified 200 CpG sites genome-wide, whose methylation levels correlated best with expression for each gene. With this information, we 1) devised an approach, methCORR, that can infer RNA expression in both FF and FFPE samples using only DNA methylation profiles, 2) established a methCORR network that clusters genes according to overlap in the CpG sites associated with their expression level and 3) used the network to determine and characterize biological traits associated with tumor aggressiveness.Results: The methCORR-inferred RNA expression profiles in FF tissue consistently exhibited high correlation to matched RNA-seq. profiles (median Pearson r=0.97; range 0.91-0.98). We found a higher correlation between FF RNA-seq. and FFPE inferred RNA profiles (median r= 0.97; range 0.96-0.98) than between FF and FFPE RNA-seq. profiles (median r= 0.78; range 0.51-0.92). Gene expression profiles were inferred in two FF (n=231, n=203), and two FFPE cohorts (n=113, n=56). Clustering of these profiles identified the same two CRC subtypes independently in all cohorts. Characterization using the methCORR network showed that the two subtypes resembled conventional and serrated CRC. Moreover, methCORR network analysis identified subtype-specific traits that associated strongly with tumor aggressiveness, such as T-cell, fibroblast, and epithelial-mesenchymal transition activity, and thus allowed identification of subtype-specific prognostic biomarkers. These better predicted relapse-free survival (HR=3.22, 95%CI 2.00-5.17) than TNM staging (HR=1.99, 95%CI 1.28-3.08). Finally, we derived four simple and clinically applicable DNA methylation-specific PCR assays for subtyping and prognostication of CRC FFPE samples.Conclusion: We have developed a novel method for characterization, subtyping, and prognostication of CRC, which is compatible with FF and FFPE samples. We envision that application of the methCORR approach to other cancer types will generate similar fruitful results.Citation Format: Trine B. Mattesen, Mads H. Rasmussen, Juan Sandoval, Halit Ongen, Sigrid S. Árnadóttir, Anders H. Madsen, Søren Laurberg, Emmanouil T. Dermitzakis, Manel Esteller, Claus L. Andersen, Jesper B. Bramsen. A novel DNA methylation-based approach for molecular subtyping and improved prognostication of colorectal cancer using formalin-fixed and paraffin-embedded tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 466.

&lt;div&gt;Abstract&lt;p&gt;The tumor-promoting fibrotic stroma rich in tumor-associated fibroblasts (TAF) is drawing increased therapeutic attention. Intriguingly, a trial with the antifibrotic drug nintedanib in non–small cell lung cancer reported clinical benefits in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC), even though the stroma is fibrotic in both histotypes. Likewise, we reported that nintedanib inhibited the tumor-promoting fibrotic phenotype of TAFs selectively in ADC. Here we show that tumor fibrosis is actually higher in ADC-TAFs than SCC-TAFs &lt;i&gt;in vitro&lt;/i&gt; and patient samples. Mechanistically, the reduced fibrosis and nintedanib response of SCC-TAFs was associated with increased promoter methylation of the profibrotic TGFβ transcription factor &lt;i&gt;SMAD3&lt;/i&gt; compared with ADC-TAFs, which elicited a compensatory increase in TGFβ1/SMAD2 activation. Consistently, forcing global DNA demethylation of SCC-TAFs with 5-AZA rescued TGFβ1/SMAD3 activation, whereas genetic downregulation of &lt;i&gt;SMAD3&lt;/i&gt; in ADC-TAFs and control fibroblasts increased TGFβ1/SMAD2 activation, and reduced their fibrotic phenotype and antitumor responses to nintedanib &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;. Our results also support that smoking and/or the anatomic location of SCC in the proximal airways, which are more exposed to cigarette smoke particles, may prime SCC-TAFs to stronger &lt;i&gt;SMAD3&lt;/i&gt; epigenetic repression, because cigarette smoke condensate selectively increased &lt;i&gt;SMAD3&lt;/i&gt; promoter methylation. Our results unveil that the histotype-specific regulation of tumor fibrosis in lung cancer is mediated through differential &lt;i&gt;SMAD3&lt;/i&gt; promoter methylation in TAFs and provide new mechanistic insights on the selective poor response of SCC-TAFs to nintedanib. Moreover, our findings support that patients with ADC may be more responsive to antifibrotic drugs targeting their stromal TGFβ1/SMAD3 activation.&lt;/p&gt;Significance:&lt;p&gt;This study implicates the selective epigenetic repression of &lt;i&gt;SMAD3&lt;/i&gt; in SCC-TAFs in the clinical failure of nintedanib in SCC and supports that patients with ADC may benefit from antifibrotic drugs targeting stromal TGFβ1/SMAD3.&lt;/p&gt;&lt;/div&gt;