Publications

Supplementary Table 1 from Epigenetic Inactivation of the Circadian Clock Gene <i>BMAL1</i> in Hematologic Malignancies

Summary Maintenance of pluripotency is a multifactorial process in which NF-κB is a negative regulator. Our previous work identified a chromatin role for IκBα, the master regulator of NF-κB signaling, that is critical for the proper regulation of various tissue stem cells. Here, we found that IκBα accumulates specifically in the chromatin fraction of pluripotent embryonic stem cells. IκBα depletion does not affect NF-kB-dependent transcription, but causes a profound epigenetic rewiring in pluripotent stem cells, including alterations in H3K27me3, a histone mark catalyzed by Polycomb repression complex 2. Chromatin changes induced by IκBα depletion affect a subset of pluripotency genes and are associated with altered gene transcription. At the cellular level, IκBα-deficient embryonic stem cells are arrested in a naive pluripotency state when cultured in serum/LIF conditions and fail to exit pluripotency under differentiation conditions. By constructing separation-of-function mutants, we show that the effects of IκBα in regulating stem cell pluripotency are NF-κB-independent, but mainly rely on its chromatin-related function. Taken together, our results reveal a novel mechanism by which IκBα participates in the regulation of the pluripotent state of embryonic stem cells and shed light on the interplay between inflammatory signals and the regulation of pluripotency.

Abstract Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by systemic inflammation involving various immune cell types. Monocytes, pivotal in promoting and regulating inflammation in SLE, differentiate from classical monocytes into intermediate monocytes and non-classical monocytes, assuming diverse roles. In this study, we investigated the epigenetic and transcriptomic profiles of these three monocyte subsets in an SLE cohort. In addition to common DNA methylation and transcriptomic alterations, we identified monocyte subset-specific alterations, especially in DNA methylation, which reflect an impact of SLE on the monocyte differentiation process. SLE classical monocytes exhibited a stronger proinflammatory profile, with an interferon signature and were primed for macrophage differentiation. SLE non-classical monocytes displayed a phenotype related to T cell differentiation regulation, and a Th17-promoting phenotype. Changes in monocyte proportions, DNA methylation and expression occurred in relation to disease activity and involved the STAT1 pathway. Integrating bulk datasets with single-cell RNA-seq data of SLE patients further supported the interferon signature in classical monocytes, associating intermediate and non-classical populations with exacerbated complement activation pathways. Our results indicate a subversion of the epigenome and transcriptome in monocyte differentiation toward non-classical subsets in SLE, impacting function, in relation to disease activity and progression.

Rett syndrome (RTT) is the second leading cause of mental impairment in girls and is currently untreatable. RTT is caused, in more than 95% of cases, by loss-of-function mutations in the methyl CpG-binding protein 2 gene (MeCP2). We propose here a molecular target involved in RTT: the glycogen synthase kinase-3b (Gsk3b) pathway. Gsk3b activity is deregulated in Mecp2-knockout (KO) mice models, and SB216763, a specific inhibitor, is able to alleviate the clinical symptoms with consequences at the molecular and cellular levels. In vivo, inhibition of Gsk3b prolongs the lifespan of Mecp2-KO mice and reduces motor deficits. At the molecular level, SB216763 rescues dendritic networks and spine density, while inducing changes in the properties of excitatory synapses. Gsk3b inhibition can also decrease the nuclear activity of the Nfkb1 pathway and neuroinflammation. Altogether, our findings indicate that Mecp2 deficiency in the RTT mouse model is partially rescued following treatment with SB216763.

e14609 Background: Resistance acquisition to chemotherapeutic agents is one of the main problems that come up during cancer treatment. hSRBC is a tumor suppressor gene whose inactivation has been associated with malignant tumor progression. In a previous work, we investigated the influence of hSRBC promoter methylation alterations in oxaliplatin (OXA) resistance acquisition by using a CRC “in vitro” model, detecting an hSRBC promoter hypermethylation in OXA–resistant derived cells when compared with the sensitive counterpart. These results were validated by functional analyses in the same model. Taking this into account, our aim in the present work is to determine the role of hSRBC methylation status as a potential biomarker of OXA resistance, in metastatic CRC patients, treated at first line with fluouropirimidines plus OXA based chemotherapy. Methods: hSRBC promoter hypermethylation was analyzed in DNA extracted from paraffin embebbed tissue of 111 metastatic CRC tumors by using Methylation Specific PCR. Methylation data was correlated to overall response (OR) and progression free survival (PFS) by using F-Fisher test and Kaplan-Meyer Survival curves respectively. A multivariate analysis was carried out by Cox regression. p-values under 0.05 were considered statistic significant. Results: Two independent cohorts of stage IV CRC tumors were included. Twenty-two out of 111 patients received radical surgery for metastasis, that became to be a positive prognostic factor (p-value = 0.04). Gene hypermethylation was detected in a 33% of cases. Although OR was not associated with hSRBC methylation, we observed a significant correlation between hypermethylation of gene and a worse PFS in patients without metastasis surgery (Log Rank; p-value = 0.04). Conclusions: Remarkably, hSRBC promoter hypermethylation is associated with worse PFS in metastatic CRC patients. We suggest hSRBC methylation status as a predictive biomarker of OXA-based treatment outcome in metastatic CRC patients. However, further studies are warranted in order to elucidate the clinical application of these findings.

<p>TAGL as a predictive marker for dasatinib sensitivity. <b>A,</b> Western blot of control and <i>TAGLN</i>-KO cells in Hs578T (top) and BT-549 (bottom) cells. <b>B,</b> Representative immunofluorescence of Hs578T and BT549 control and <i>TAGLN</i>-KO cells, stained with αTAGL (green) and DAPI (blue). Scale bar, 50 μm. <b>C,</b> Cell proliferation in control (black) and <i>TAGLN</i>-KO (purple) cells measured by MTT assays over 72 hours. <b>D,</b> Relative percentage of migrated cells in control and <i>TAGLN</i>-KO cells, assessed by transwell assays. In <b>C</b> and <b>D</b>, values represent the mean ± SEM of at least three experimental replicates, and statistical significance was determined using one-way ANOVA. <b>E,</b> Drug–response curves for cell viability of control and <i>TAGLN</i>-KO cells treated with dasatinib at increasing concentrations. <b>F,</b> Western blot of known dasatinib targets in control and <i>TAGLN</i>-KO cells. <b>G,</b> Data from the <i>DepMap</i> portal showing the correlation between <i>TAGLN</i> and <i>PDGFRB</i> in breast cancer cell lines. <b>H,</b> Western blot of PDGFRβ in <i>TAGLN</i>-KO and cells and clones constitutively expressing <i>PDGFRB</i>. <b>I,</b> Drug–response curves for cell viability of <i>TAGLN</i>-KO and <i>TAGLN</i>-KO/<i>PBGFRB</i> cells treated with dasatinib at increasing concentrations. In <b>E</b> and <b>I</b>, solid lines represent the mean of three biological replicates performed in technical replicates. The dashed line indicates their IC<sub>50</sub> value. **, <i>P</i> < 0.01; ****, <i>P</i> < 0.0001; ns, not significant.</p>

// Juan Huertas-Martínez 1 , Santiago Rello-Varona 1 , David Herrero-Martín 1 , Ignasi Barrau 1 , Silvia García-Monclús 1 , Miguel Sáinz-Jaspeado 1 , Laura Lagares-Tena 1 , Yaiza Núñez-Álvarez 5 , Silvia Mateo-Lozano 2 , Jaume Mora 2 , Josep Roma 3 , Nuria Toran 3 , Sebastian Moran 4 , Roser López-Alemany 1 , Soledad Gallego 3 , Manel Esteller 4 , Miguel A. Peinado 5 , Xavier García del Muro 1 and Oscar M. Tirado 1 1 Sarcoma research group, Molecular Oncology Lab, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain 2 Developmental Tumor Biology Laboratory, Hospital Sant Joan de Deu, Barcelona, Spain 3 Biomedical Research Unit, Hospital Universitari Vall d'Hebron, Barcelona, Spain 4 Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L' Hospitalet de Llobregat, Barcelona, Spain 5 Institut de Medicina Predictiva i Personalitzada del Càncer, Badalona, Barcelona, Spain Correspondence: Oscar M. Tirado, email: // Keywords : alveolar rhabdomyosarcoma, Caveolin-1, muscular differentiation, 5-AZA-2'-deoxycytidine, epigenetics, cell death Received : June 26, 2014 Accepted : August 26, 2014 Published : August 27, 2014 Abstract Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with histological variant of rhabdomyosarcoma known as alveolar rhabdomyosarcoma (ARMS) have a 5-year survival of less than 30%. Caveolin-1 (CAV1), encoding the structural component of cellular caveolae, is a suggested tumor suppressor gene involved in cell signaling. In the present study we report that compared to other forms of rhabdomyosarcoma (RMS) CAV1 expression is either undetectable or very low in ARMS cell lines and tumor samples. DNA methylation analysis of the promoter region and azacytidine-induced re-expression suggest the involvement of epigenetic mechanisms in the silencing of CAV1. Reintroduction of CAV1 in three of these cell lines impairs their clonogenic capacity and promotes features of muscular differentiation. In vitro , CAV1-expressing cells show high expression of Caveolin-3 (CAV3), a muscular differentiation marker. Blockade of MAPK signaling is also observed. In vivo , CAV1-expressing xenografts show growth delay, features of muscular differentiation and increased cell death. In summary, our results suggest that CAV1 could function as a potent tumor suppressor in ARMS tumors. Inhibition of CAV1 function therefore, could contribute to aberrant cell proliferation, leading to ARMS development.

Abstract Alterations in epigenetic marks, such as DNA methylation, represent a hallmark of cancer that has been successfully exploited for therapy in myeloid malignancies. Hypomethylating agents (HMA), such as azacitidine, have become standard-of-care therapy to treat myelodysplastic syndromes (MDS), myeloid neoplasms that can evolve into acute myeloid leukemia. However, our capacity to identify who will respond to HMAs, and the duration of response, remains limited. To shed light on this question, we have leveraged the unprecedented analytic power of single-cell technologies to simultaneously map the genome and immunoproteome of MDS samples throughout clinical evolution. We were able to chart the architecture and evolution of molecular clones in precious paired bone marrow MDS samples at diagnosis and posttreatment to show that a combined imbalance of specific cell lineages with diverse mutational profiles is associated with the clinical response of patients with MDS to hypomethylating therapy. Significance: MDS are myeloid clonal hemopathies with a low 5-year survival rate, and approximately half of the cases do not respond to standard HMA therapy. Our innovative single-cell multiomics approach offers valuable biological insights and potential biomarkers associated with the demethylating agent efficacy. It also identifies vulnerabilities that can be targeted using personalized combinations of small drugs and antibodies.

<p>Supplementary Figure 8. Effect of KAT6B shRNA-mediated depletion in ATM expression and H2AX phosphorylation</p>

Human tumors show altered patterns of protein isoforms that can be related to the dysregulation of messenger RNA alternative splicing also observed in transformed cells. Although somatic mutations in core spliceosome components and their associated factors have been described in some cases, almost nothing is known about the contribution of distorted epigenetic patterns to aberrant splicing. Herein, we show that the splicing RNA-binding protein CELF2 is targeted by promoter hypermethylation-associated transcriptional silencing in human cancer. Focusing on the context of breast cancer, we also demonstrate that CELF2 restoration has growth-inhibitory effects and that its epigenetic loss induces an aberrant downstream pattern of alternative splicing, affecting key genes in breast cancer biology such as the autophagy factor ULK1 and the apoptotic protein CARD10. Furthermore, the presence of CELF2 hypermethylation in the clinical setting is associated with shorter overall survival of the breast cancer patients carrying this epigenetic lesion.

Abstract Anaplastic meningioma is a rare and aggressive brain tumor characterised by intractable recurrences and dismal outcomes. Here, we present an integrated analysis of the whole genome, transcriptome and methylation profiles of primary and recurrent anaplastic meningioma. A key finding was the delineation of two distinct molecular subgroups that were associated with diametrically opposed survival outcomes. Relative to lower grade meningiomas, anaplastic tumors harbored frequent driver mutations in SWI/SNF complex genes, which were confined to the poor prognosis subgroup. Our analyses discern two biologically distinct variants of anaplastic meningioma with potential prognostic and therapeutic significance.

Abstract Natural antisense transcripts (NATs) are the most abundant class of long noncoding RNAs (lncRNAs). Importantly, NATs are one of the most unknown ncRNAs. However, due to their highly gene locus-specific effects, NATs may provide a unique entry point for therapeutic intervention in targeted gene regulation. For this reason we are interested in studying the regulation of sense/antisense transcription in cancer related locus. In our previous work we found that, at the important cancer-related locus vimentin (VIM) there is a previously uncharacterized antisense transcript (VIM-AS1), which is transcribed head-to-head with VIM gene. This antisense transcript enhances R loop formation; it allows the binding of transcription factors as NF-kB, low nucleosome occupancy and the transcription of VIM gene. Our results are the first example of R loop mediated enhancement of gene expression involving head-to-head antisense transcription at cancer related locus. We also observed that in colon and breast cancer there was a positive expression correlation between VIM and VIM-AS1 and we have observed that hypermethylation silences both transcripts in colon primary tumors. In the present work we have seen that this positive correlation between sense and antisense transcripts is present in most of the tumor types from patients that we have obtained from Genomic Data Commons Data Portal (GDC Portal). Importantly this positive correlation correlates better in the cases in which the antisense transcript is downregulated or maintains the expression than in tumors where VIM and VIM-AS1 are up-regulated. Furthermore, we wanted to see if the cellular function is also coordinated. VIM is a component of the intermediate filament family of proteins that has an important role in epithelial mesenchymal transition. We found that specific depletion of VIM-AS1 results in a decrease of VIM expression, and now we have found that this depletion produces low migration rate and more invasion capability, as expected. On the other hand, the mechanisms used by antisense transcripts to regulate their corresponding sense mRNAs are not fully understood and in view of these results we are investigating from a genome-wide perspective the regulation of sense/antisense transcription, the involvement of R loop structures and DNA methylation in cancer. For this, we have applied a number of techniques: RNAseq strand specific, Illumina’s HumanMethylation450 Bead Chip array, RNAseq strand specific with overexpression of RNaseH (which specifically degrades the RNA in RNA/DNA hybrids), and DRIPseq (which immunoprecipitates the hybrids DNA-RNAs). The analysis of these data sheds light on the study of the role of sense/antisense transcripts in cancer-related locus. Citation Format: Raquel Boque-Sastre, Manuel Castro de Moura, Antonio Gomez, Sònia Guil, Manel Esteller. Genome-wide analysis of the antisense transcriptome in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3483. doi:10.1158/1538-7445.AM2017-3483