Publications

The Epigenomic Revolution in Breast Cancer

Circadian Cycle-Dependent MeCP2 and Brain Chromatin Changes

Methyl CpG binding protein 2 (MeCP2) is a chromosomal protein of the brain, very abundant especially in neurons, where it plays an important role in the regulation of gene expression. Hence it has the potential to be affected by the mammalian circadian cycle. We performed expression analyses of mice brain frontal cortices obtained at different time points and we found that the levels of MeCP2 are altered circadianly, affecting overall organization of brain chromatin and resulting in a circadian-dependent regulation of well-stablished MeCP2 target genes. Furthermore, this data suggests that alterations of MeCP2 can be responsible for the sleeping disorders arising from pathological stages, such as in autism and Rett syndrome.

Infinium DNA Methylation Microarrays on Formalin-Fixed, Paraffin-Embedded Samples

Capillary electrophoresis-based method to quantitate DNA–protein interactions

Hypermethylation of a Small CpGuanine-Rich Region Correlates with Loss of Activator Protein-2α Expression during Progression of Breast Cancer

Abstract The transcription factor activator protein-2α (AP-2α) has recently been implicated as a tumor suppressor protein that can be lost during tumor progression and that exhibits growth-inhibitory properties when overexpressed in cancer cell lines. We now demonstrate that hypermethylation of a discrete 5′ region within a promoter CpG island of the gene is associated in breast cancer with the loss of AP-2α expression. Multiple CpG sites within the island become hypermethylated during breast cancer evolution. However, only hypermethylation of the most CpG-rich region, a small, ∼300-bp area at the 3′ end of exon 1, fully distinguishes neoplastic from normal breast tissue and correlates with transcriptional silencing. In cell culture, silenced AP-2α, associated with exon 1 hypermethylation, is re-expressed by 5-aza-2′deoxycytidine resulting in the restoration of a functional DNA sequence-specific binding protein. In vivo, as detected by a very sensitive nested PCR approach, methylation of the discrete AP-2α exon 1 region does not occur in normal breast epithelium and occurs in only 3 (16%) of 19 ductal carcinoma in situ (DCIS) lesions, but is present in 12 (75%) of 16 invasive breast tumors (P < 0.001; DCIS versus invasive cancers). Tumors unmethylated for this region expressed AP-2α protein throughout, whereas tumors with hypermethylation showed large areas of loss. Our studies then determine that hypermethylation of a small region of a CpG island correlates with silencing of AP-2α in breast cancer and suggest that inactivation of this gene could be a factor in, and a useful marker for, the progression of DCIS lesions.

Genome-wide DNA methylation pattern in visceral adipose tissue differentiates insulin-resistant from insulin-sensitive obese subjects

SirT7 auto-ADP-ribosylation regulates glucose starvation response through macroH2A1.1

Abstract Sirtuins are key players in the response to oxidative, metabolic and genotoxic stress, and are involved in genome stability, metabolic homeostasis and aging. Originally described as NAD + -dependent deacetylases, some sirtuins are also characterized by a poorly understood mono-ADP-ribosyltransferase (mADPRT) activity. Here we report that the deacetylase SirT7 is a dual sirtuin as it also features auto-mADPRT activity. Molecular and structural evidence suggests that this novel activity occurs at a second previously undefined active site that is physically separated in another domain. Specific abrogation of this activity alters SirT7 chromatin distribution, suggesting a role for this modification in SirT7 chromatin binding specificity. We uncover an epigenetic pathway by which ADP-ribosyl-SirT7 is recognized by the ADP-ribose reader macroH2A1.1, a histone variant involved in chromatin organization, metabolism and differentiation. Glucose starvation (GS) boosts this interaction and promotes SirT7 relocalization to intergenic regions in a macroH2A1-dependent manner. Both SirT7 activities are in turn required to promote GS-dependent enrichment of macroH2A1 in a subset of nearby genes, which results in their specific up- or downregulation. Consistently, the expression changes of these genes associated to calorie restriction (CR) or aging are abrogated in SirT7 -/- mice, reinforcing the link between Sirtuins, CR and aging. Our work provides a novel perspective about sirtuin duality and suggests a key role for SirT7/macroH2A1.1 axis in mammalian glucose homeostasis, calorie restriction signaling and aging.

Identification of a mononucleotide repeat as a major target for mitochondrial DNA alterations in human tumors.

Mitochondrial DNA (mtDNA) mutations scattered through coding and noncoding regions have been reported in cancer. The mechanisms that generate such mutations and the importance of mtDNA mutations in tumor development are still not clear. Here we present the identification of a specific and highly polymorphic homopolymeric C stretch (D310), located within the displacement (D) loop, as a mutational hotspot in primary tumors. Twenty-two % of the 247 primary tumors analyzed harbored somatic deletions/insertions at this mononucleotide repeat. Moreover, these alterations were also present in head and neck preneoplastic lesions. We further characterized the D310 variants that appeared in the lung and head and neck tumors. Most of the somatic alterations found in tumors showed deletion/insertions of 1- or 2-bp generating D310 variants identical to constitutive polymorphisms described previously. Sequencing analysis of individual clones from lymphocytes revealed that patients with D310 mutations in the tumors had statistically significant higher levels of D310 heteroplasmy (more than one length variant) in the lymphocyte mtDNA as compared with the patients without D310 mutations in the tumor mtDNA. On the basis of our observations, we propose a model in which D310 alterations are already present in normal cells and achieve homoplasmy in the tumor through a restriction/amplification event attributable to random genetic drift and clonal expansion.

PRC2 Loss and DNMT Inhibition Boost Viral Mimicry in Cancer

Summary: In this issue of Cancer Discovery, Patel and colleagues explore the synergistic lethality of PRC2 inactivation and DNMT inhibition in malignant peripheral nerve sheath tumor cells. Reactivation of retrotransposons under this dual control suggests that the viral mimicry response contributes to enhanced cytotoxicity with potential clinical implications. See related article by Patel et al., p. 2120 (5).

Supplemental Table S3 from The NCI-60 Methylome and Its Integration into CellMiner

<p>Linear regression analysis of 15,798 genes with with transcript expression, DNA methylation, and DNA copy number data available.</p>

Validation of the new EPIC DNA methylation microarray (900K EPIC v2) for high-throughput profiling of the human DNA methylome

DNA methylation, one of the best characterized epigenetic marks in the human genome, plays a pivotal role in gene transcription regulation and other biological processes in humans. On top of that, the DNA methylome undergoes profound changes in cancer and other disorders. However, large-scale and population-based studies are limited by high costs and the need for considerable expertise in data analysis for whole-genome bisulphite-sequencing methodologies. Following the success of the EPIC DNA methylation microarray, the newly developed Infinium HumanMethylationEPIC version 2.0 (900K EPIC v2) is now available. This new array contains more than 900,000 CpG probes covering the human genome and excluding masked probes from the previous version. The 900K EPIC v2 microarray adds more than 200,000 probes covering extra DNA cis-regulatory regions such as enhancers, super-enhancers and CTCF binding regions. Herein, we have technically and biologically validated the new methylation array to show its high reproducibility and consistency among technical replicates and with DNA extracted from FFPE tissue. In addition, we have hybridized primary normal and tumoural tissues and cancer cell lines from different sources and tested the robustness of the 900K EPIC v2 microarray when analysing the different DNA methylation profiles. The validation highlights the improvements offered by the new array and demonstrates the versatility of this updated tool for characterizing the DNA methylome in human health and disease.

MAX mutant small-cell lung cancers exhibit impaired activities of MGA-dependent noncanonical polycomb repressive complex

The MYC axis is disrupted in cancer, predominantly through activation of the MYC family oncogenes but also through inactivation of the MYC partner MAX or of the MAX partner MGA. MGA and MAX are also members of the polycomb repressive complex, ncPRC1.6. Here, we use genetically modified MAX-deficient small-cell lung cancer (SCLC) cells and carry out genome-wide and proteomics analyses to study the tumor suppressor function of MAX. We find that MAX mutant SCLCs have ASCL1 or NEUROD1 or combined ASCL1/NEUROD1 characteristics and lack MYC transcriptional activity. MAX restitution triggers prodifferentiation expression profiles that shift when MAX and oncogenic MYC are coexpressed. Although ncPRC1.6 can be formed, the lack of MAX restricts global MGA occupancy, selectively driving its recruitment toward E2F6-binding motifs. Conversely, MAX restitution enhances MGA occupancy to repress genes involved in different functions, including stem cell and DNA repair/replication. Collectively, these findings reveal that MAX mutant SCLCs have either ASCL1 or NEUROD1 or combined characteristics and are MYC independent and exhibit deficient ncPRC1.6-mediated gene repression.

MeCP2-E1 isoform is a dynamically expressed, weakly DNA-bound protein with different protein and DNA interactions compared to MeCP2-E2

Abstract MeCP2 – a chromatin-binding protein associated with Rett syndrome – has two main isoforms, MeCP2-E1 and MeCP2-E2, with 96% amino acid identity differing in a few N-terminal amino acid residues. Previous studies have shown brain region-specific expression of these isoforms which, in addition to their different cellular localization and differential expression during brain development, suggest they may also have non-overlapping molecular mechanisms. However, differential functions of MeCP2-E1 and E2 remain largely unexplored. Here, we show that the N-terminal domains (NTD) of MeCP2-E1 and E2 modulate the ability of the methyl binding domain (MBD) to interact with DNA as well as influencing the turnover rates, binding dynamics, response to nuclear depolarization, and circadian oscillations of the two isoforms. Our proteomics data indicate that both isoforms exhibit unique interacting protein partners. Moreover, genome-wide analysis using ChIP-seq provide evidence for a shared as well as a specific regulation of different sets of genes. Our findings provide insight into the functional complexity of MeCP2 by dissecting differential aspects of its two isoforms. Significance Whether the two E1 and E2 isoforms of MeCP2 have different structural and/or functional implications has been highly controversial and is not well known. Here we show that the relatively short N-terminal sequence variation between the two isoforms impinges them with an important DNA binding difference. Moreover, MeCP2-E1 and E2 exhibit a different cellular dynamic behavior and have some distinctive interacting partners. In addition, while sharing genome occupancy they specifically bind to several distinctive genes.

Abstract 1819: Non-coding RNAs as key players for understanding oncogenic pathways in ovarian cancer: Studying new therapeutic strategies

Abstract The largest part of the human genome is transcribed into non-coding RNAs (ncRNAs) which are functional transcripts displaying relevant roles in many biological processes, and their dysregulation has been shown to be involved in the development of diseases like cancer. The group of long non-coding RNAs (lncRNAs) has been elucidated to be key regulators of gene expression by acting at different levels, as regulators of epigenetic modifications and chromatin remodelling by acting as structural scaffolds, decoys or guides in the nucleus; as well as post-transcriptional regulators of messenger RNA stability and translation in the cytoplasm. Hence, the knowledge about their mechanisms of action may provide a better understanding of cell biology and likewise, lncRNAs may be used as biomarkers or therapeutical tools. The aim of the current project has been to describe the mechanism of action of a lncRNA called RPSAP52 (Ribosomal Protein SA Pseudogene 52) in ovarian cancer, as well as to study the potential use of RPSAP52 as a tool for therapeutical strategies. Our previous results indicate that RPSAP52, which is an antisense transcript to the well described oncogene HMGA2 (High Mobility Group A2), positively regulates HMGA2 expression through the formation of an R loop structure, and it has been shown that RPSAP52 enhances the oncofetal pathway HMGA2-IGF2BP2-RAS axis through LIN28B-dependent and independent inhibition of let-7 miRNAs in breast cancer and sarcomas. Since the dysregulation of this pathway has been described to be involved in the development of ovarian cancer; which is among the most common types of cancer with poor prognosis, the goal of this project has been to reveal the relevance of RPSAP52 in this system. The oncogenic potential of RPSAP52 has been confirmed by the analysis of a battery of xenograft samples from ovarian cancer patients, in which significant higher levels of RPSAP52 in those samples versus normal ovary have been observed, and it is positively correlated with higher levels of the sense gene HMGA2. Preliminary in vitro studies have shown that the depletion of RPSAP52 with shRNAs is related with a down-regulation of the oncogenic protein LIN28A in SKOV3 cells. Our findings suggest that RPSAP52 may be involved in the pathogenesis of ovarian cancer, and a further analysis of the link between RPSAP52 and LIN28A needs to be performed, together with the study of other candidate protein partners of RPSAP52 revealed by RNA pull down and RAP-MS techniques. Interestingly, the use of the LNA Gapmers as a strategy for the depletion of RPSAP52 also allows us to use them in vivo to confirm in vitro results as well as to study its potential role as a therapeutic target in ovarian cancer. Citation Format: Anaís Sánchez Castillo, Cristina Oliveira Mateos, Marta Soler Riera, Manel Esteller, Sonia Guil. Non-coding RNAs as key players for understanding oncogenic pathways in ovarian cancer: Studying new therapeutic strategies [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 1819.

Point mutation and homozygous deletion of PTEN/MMAC1 in primary bladder cancers

Predictive value of integrative epigenetic profiling in metastatic melanoma (MM) patients (p) treated with BRAF/MEK inhibitors (BMI) and chemotherapy (CT).

e20059 Background: The treatment of MM has evolved over the last four years to include novel treatments in addition to CT, requiring predictive markers of response. Treatment with BMI provides benefit in overall survival, although with early resistance development. The value of CT in these p is limited. Hence, the aim of this work is to perform an integrative epigenetic profiling as predictive tool of resistance to treatment in MM p treated, in one hand with BRAF/MEK inhibitors, and in the other hand, CT-based treatment. The final goal is obtain epigenetic biomarkers to optimize treatment selection and p's outcome. Additionally, we pretend to identify epigenetic biomarkers related to metastatic process. Methods: It was analyzed 35 paraffin-embebbed primary tumor tissue samples from 35 MM p, 20 of them had been treated with BMI and 15 with CT. Additionally, it was analyzed the paired metastasis tissue for a half of them to study the effect of epigenetics in metastatic process. Epigenetic screening was performed using DNA methylation array technology Infinium 450K from Illumina, following the manufacturer's instructions. Data analysis was performed using R statistics software. Results: Comparing responders and non-responders p, we obtained an epigenetic profile associated with drug resistance prediction for each treatment strategy (target therapy and CT). Regarding metastatic process, we compared primary tumor with paired metastasis identifying a set of epigenetic biomarkers candidates. In all cases, it was considered as significant those methylation changes with beta-values differences over 10% and ANOVA with p-value under 0.01. Conclusions: According our high-throughput epigenetic screening, we have been capable to discriminate in a cohort of MM p between responders and refractory tumors focusing in DNA methylation changes. Moreover, epigenetic changes are also presented as a strategy to understand better the metastasis process in this neoplasia. Hence, integrated epigenetic screening could be a useful tool to optimize treatment selection. Nevertheless, further studies are warranted to elucidate their potential role in the clinical setting.

Supplementary Figure 2 from KAT6B Is a Tumor Suppressor Histone H3 Lysine 23 Acetyltransferase Undergoing Genomic Loss in Small Cell Lung Cancer

<p>Supplementary Figure 2. Copy number and expression levels of six KATs in SCLC cell lines</p>

IL16 PRODUCTION IS A MECHANISM OF RESISTANCE TO BTK INHIBITORS AND TO R‐CHOP

Introduction: Covalent and non-covalent BTK inhibitors (BTKi) are used or being explored for the treatment of patients with B-cell malignancies, such as marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) of the activated B-cell type (ABC). The identification of mechanisms of resistance can lead to improvements in the current therapeutic approaches. We had reported IL16 production in a MZL model of secondary resistance to ibrutinib (Arribas ENA2019). Here, we expand the initial observation to additional lymphoma types and treatments modalities, performing therapeutic interventions to block it and providing clinical validation data on its expression and role in patients. Methods: VL51 (marginal zone lymphoma, MZL) parental (PAR) and Ibrutinib-resistant (RES) cells underwent RNA (mRNA & miRNA by RNA-Seq), DNA (promoter methylation, WES) and secretome (Cytokine array) profiling. RES cells exhibited resistance to covalent and non-covalent BTK inhibitors. No DNA copy number variations or mutations were detected by WES in RES compared to PAR cells, including those affecting BTK or PLCG2 genes. Results: Multi-omics (DNA, mRNA, miRNA, secretome) characterization of RES and PAR cells identified hypomethylation, up-regulation and secretion of IL16 in RES but not in PAR cells. Elevated levels of p-AKT and p-ERK was observed in RES compared to PAR, suggesting a signaling activation in RES cells. Stimulation with recombinant IL16 reduced sensitivity to BTKi not only in VL51 PAR cells, but also in models derived from MCL (REC1) and CLL (MEC1). Conversely, the use of IL16 blocking antibody recovered sensitivity to BTKi in RES cells, along with other lines with high expression of IL16 and low sensitivity to ibrutinib derived from MCL (MAVER1) and MZL (SSK41). IL16 is part of the ABC-DLBCL signature. We observed that high IL16 expression was associated with shorter overall survival in ABC but not in germinal center like DLBCL patients (n = 233, log-rank test p = 0.013, multivariate Cox: HR (95% CI) 0.43 (0.22–0.85), p = 0.016). IL16 stimulation also decreased sensitivity to RCHOP in ABC DBCL cells (OCILy10, HBL1). Finally, secreted levels of IL16 in the serum of CLL patients (n = 17) with resistance to ibrutinib (in absence of BTK or PLCG2 mutations) were significantly higher compared to patients responding to the drug and paired for clinical features. Conclusions: Starting from a MZL model of secondary resistance to ibrutinib, we showed that IL16 can sustain resistance to BTK inhibitors also in CLL and MCL. Furthermore, increased expression of IL16 associated with shorter outcome in ABC DLBCL patients and the interleukin induced in vitro resistance to RCHOP. The demonstration that IL16 blocking therapies recovered sensitivity to therapies can lead to novel therapeutic approaches to overcome resistance in lymphoma patients. The research was funded by: Swiss National Science Foundation (SNSF 31003A_163232/1) Keyword: molecular targeted therapies Conflicts of interests pertinent to the abstract A. Arribas Educational grants: Astra Zeneca