3D chromatin connectivity underlies replication origin efficiency in mouse embryonic stem cells.

In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled. Replicative stress triggers additional initiation events from 'dormant' origins whose genomic distribution and regulation are not well understood. In this study, we have analyzed origin activity in mouse embryonic stem cells in the absence or presence of mild replicative stress induced by aphidicolin, a DNA polymerase inhibitor, or by deregulation of origin licensing factor CDC6. In both cases, we observe that the majority of stress-responsive origins are also active in a small fraction of the cell population in a normal S phase, and stress increases their frequency of activation. In a search for the molecular determinants of origin efficiency, we compared the genetic and epigenetic features of origins displaying different levels of activation, and integrated their genomic positions in three-dimensional chromatin interaction networks derived from high-depth Hi-C and promoter-capture Hi-C data. We report that origin efficiency is directly proportional to the proximity to transcriptional start sites and to the number of contacts established between origin-containing chromatin fragments, supporting the organization of origins in higher-level DNA replication factories.

Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment.

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.

CREBBP/EP300 bromodomains are critical to sustain the GATA1/MYC regulatory axis in proliferation.

BACKGROUND: The reported antitumor activity of the BET family bromodomain inhibitors has prompted the development of inhibitors against other bromodomains. However, the human genome encodes more than 60 different bromodomains and most of them remain unexplored. RESULTS: We report that the bromodomains of the histone acetyltransferases CREBBP/EP300 are critical to sustain the proliferation of human leukemia and lymphoma cell lines. EP300 is very abundant at super-enhancers in K562 and is coincident with sites of GATA1 and MYC occupancy. In accordance, CREBBP/EP300 bromodomain inhibitors interfere with GATA1- and MYC-driven transcription, causing the accumulation of cells in the G0/G1 phase of the cell cycle. The CREBBP/CBP30 bromodomain inhibitor CBP30 displaces CREBBP and EP300 from GATA1 and MYC binding sites at enhancers, resulting in a decrease in the levels of histone acetylation at these regulatory regions and consequently reduced gene expression of critical genes controlled by these transcription factors. CONCLUSIONS: Our data shows that inhibition of CREBBP/EP300 bromodomains can interfere with oncogene-driven transcriptional programs in cancer cells and consequently hold therapeutic potential.

Bicycle: a bioinformatics pipeline to analyze bisulfite sequencing data.

Summary: High-throughput sequencing of bisulfite-converted DNA is a technique used to measure DNA methylation levels. Although a considerable number of computational pipelines have been developed to analyze such data, none of them tackles all the peculiarities of the analysis together, revealing limitations that can force the user to manually perform additional steps needed for a complete processing of the data. This article presents bicycle, an integrated, flexible analysis pipeline for bisulfite sequencing data. Bicycle analyzes whole genome bisulfite sequencing data, targeted bisulfite sequencing data and hydroxymethylation data. To show how bicycle overtakes other available pipelines, we compared them on a defined number of features that are summarized in a table. We also tested bicycle with both simulated and real datasets, to show its level of performance, and compared it to different state-of-the-art methylation analysis pipelines. Availability and implementation: Bicycle is publicly available under GNU LGPL v3.0 license at http://www.sing-group.org/bicycle. Users can also download a customized Ubuntu LiveCD including bicycle and other bisulfite sequencing data pipelines compared here. In addition, a docker image with bicycle and its dependencies, which allows a straightforward use of bicycle in any platform (e.g. Linux, OS X or Windows), is also available. Contact: ograna@cnio.es or dgpena@uvigo.es. Supplementary information: Supplementary data are available at Bioinformatics online.

A Braf kinase-inactive mutant induces lung adenocarcinoma.

The initiating oncogenic event in almost half of human lung adenocarcinomas is still unknown, a fact that complicates the development of selective targeted therapies. Yet these tumours harbour a number of alterations without obvious oncogenic function including BRAF-inactivating mutations. Inactivating BRAF mutants in lung predominate over the activating V600E mutant that is frequently observed in other tumour types. Here we demonstrate that the expression of an endogenous Braf(D631A) kinase-inactive isoform in mice (corresponding to the human BRAF(D594A) mutation) triggers lung adenocarcinoma in vivo, indicating that BRAF-inactivating mutations are initiating events in lung oncogenesis. Moreover, inactivating BRAF mutations have also been identified in a subset of KRAS-driven human lung tumours. Co-expression of Kras(G12V) and Braf(D631A) in mouse lung cells markedly enhances tumour initiation, a phenomenon mediated by Craf kinase activity, and effectively accelerates tumour progression when activated in advanced lung adenocarcinomas. We also report a key role for the wild-type Braf kinase in sustaining Kras(G12V)/Braf(D631A)-driven tumours. Ablation of the wild-type Braf allele prevents the development of lung adenocarcinoma by inducing a further increase in MAPK signalling that results in oncogenic toxicity; this effect can be abolished by pharmacological inhibition of Mek to restore tumour growth. However, the loss of wild-type Braf also induces transdifferentiation of club cells, which leads to the rapid development of lethal intrabronchiolar lesions. These observations indicate that the signal intensity of the MAPK pathway is a critical determinant not only in tumour development, but also in dictating the nature of the cancer-initiating cell and ultimately the resulting tumour phenotype.

Genomic and Molecular Screenings Identify Different Mechanisms for Acquired Resistance to MET Inhibitors in Lung Cancer Cells.

The development of resistance to tyrosine kinase inhibitors (TKI) limits the long-term efficacy of cancer treatments involving them. We aimed to understand the mechanisms that underlie acquired resistance (AR) to MET inhibitors in lung cancer. EBC1 cells, which have MET amplification and are sensitive to TKIs against MET, were used to generate multiple clones with AR to a MET-TKI. Whole-exome sequencing, RNA sequencing, and global DNA methylation analysis were used to scrutinize the genetic and molecular characteristics of the resistant cells. AR to the MET-TKI involved changes common to all resistant cells, that is, phenotypic modifications, specific changes in gene expression, and reactivation of AKT, ERK, and mTOR. The gene expression, global DNA methylation, and mutational profiles distinguished at least two groups of resistant cells. In one of these, the cells have acquired sensitivity to erlotinib, concomitantly with mutations of the KIRREL, HDAC11, HIATL1, and MAPK1IP1L genes, among others. In the other group, some cells have acquired inactivation of neurofibromatosis type 2 (NF2) concomitantly with strong overexpression of NRG1 and a mutational profile that includes changes in LMLN and TOMM34 Multiple independent and simultaneous strategies lead to AR to the MET-TKIs in lung cancer cells. The acquired sensitivity to erlotinib supports the known crosstalk between MET and the HER family of receptors. For the first time, we show inactivation of NF2 during acquisition of resistance to MET-TKI that may explain the refractoriness to erlotinib in these cells. Mol Cancer Ther; 16(7); 1366-76. ©2017 AACR.

Prediction of miRNA-mRNA Interactions Using miRGate.

miRGate ( http://mirgate.bioinfo.cnio.es /) is a freely available database that contains predicted and experimentally validated microRNA-messenger RNA (miRNA-mRNA) target pairs. This resource includes novel predictions from five well-established algorithms, but recalculated from a common and comprehensive sequence dataset. It includes all 3'-UTR sequences of all known genes of the three more widely employed genomes (human, mouse, and rat), and all annotated miRNA sequences from those genomes. Besides, it also contains predictions for all genes in human targeted by miRNA viruses such as Epstein-Barr and Kaposi sarcoma-associated herpes virus.The approach intends to circumvent one of the main drawbacks in this area, as diverse sequences and gene database versions cause poor overlap among different target prediction methods even with experimentally confirmed targets. As a result, miRGate predictions have been successfully validated using functional assays in several laboratories.This chapter describes how a user can access target information via miRGate's web interface. It also shows how automatically access the database through the programmatic interface based on representational state transfer services (REST), using the application programming interface (API) available at http://mirgate.bioinfo.cnio.es/API .

Genomic Profiling of Patient-Derived Xenografts for Lung Cancer Identifies B2M Inactivation Impairing Immunorecognition.

Purpose: We aimed to maximize the performance of detecting genetic alterations in lung cancer using high-throughput sequencing for patient-derived xenografts (PDXs).Experimental Design: We undertook an integrated RNA and whole-exome sequencing of 14 PDXs. We focused on the genetic and functional analysis of ß2-microglobulin (B2M), a component of the HLA class-I complex.Results: We identified alterations in genes involved in various functions, such as B2M involved in immunosurveillance. We extended the mutational analysis of B2M to about 230 lung cancers. Five percent of the lung cancers carried somatic mutations, most of which impaired the correct formation of the HLA-I complex. We also report that genes such as CALR, PDIA3, and TAP1, which are involved in the maturation of the HLA-I complex, are altered in lung cancer. By gene expression microarrays, we observed that restitution of B2M in lung cancer cells upregulated targets of IFNα/IFNγ. Furthermore, one third of the lung cancers lacked the HLA-I complex, which was associated with lower cytotoxic CD8+ lymphocyte infiltration. The levels of B2M and HLA-I proteins correlated with those of PD-L1. Finally, a deficiency in HLA-I complex and CD8+ infiltration tended to correlate with reduced survival of patients with lung cancer treated with anti-PD-1/anti-PD-L1.Conclusions: Here, we report recurrent inactivation of B2M in lung cancer. These observations, coupled with the mutations found at CALR, PDIA3, and TAP1, and the downregulation of the HLA-I complex, indicate that an abnormal immunosurveillance axis contributes to lung cancer development. Finally, our observations suggest that an impaired HLA-I complex affects the response to anti-PD-1/anti-PD-L1 therapies. Clin Cancer Res; 23(12); 3203-13. ©2016 AACR.

Vitamin D receptor expression and associated gene signature in tumour stromal fibroblasts predict clinical outcome in colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is a major health concern. Vitamin D deficiency is associated with high CRC incidence and mortality, suggesting a protective effect of vitamin D against this disease. Given the strong influence of tumour stroma on cancer progression, we investigated the potential effects of the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on CRC stroma. DESIGN: Expression of vitamin D receptor (VDR) and two 1,25(OH)2D3 target genes was analysed in 658 patients with CRC with prolonged clinical follow-up. 1,25(OH)2D3 effects on primary cultures of patient-derived colon normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were studied using collagen gel contraction and migration assays and global gene expression analyses. Publicly available data sets (n=877) were used to correlate the 1,25(OH)2D3-associated gene signature in CAFs with CRC outcome. RESULTS: High VDR expression in tumour stromal fibroblasts was associated with better overall survival (OS) and progression-free survival in CRC, independently of its expression in carcinoma cells. 1,25(OH)2D3 inhibited the protumoural activation of NFs and CAFs and imposed in CAFs a 1,25(OH)2D3-associated gene signature that correlated with longer OS and disease-free survival in CRC. Furthermore, expression of two genes from the signature, CD82 and S100A4, correlated with stromal VDR expression and clinical outcome in our cohort of patients with CRC. CONCLUSIONS: 1,25(OH)2D3 has protective effects against CRC through the regulation of stromal fibroblasts. Accordingly, expression of VDR and 1,25(OH)2D3-associated gene signature in stromal fibroblasts predicts a favourable clinical outcome in CRC. Therefore, treatment of patients with CRC with VDR agonists could be explored even in the absence of VDR expression in carcinoma cells.

Deep sequencing reveals microRNAs predictive of antiangiogenic drug response.

The majority of metastatic renal cell carcinoma (RCC) patients are treated with tyrosine kinase inhibitors (TKI) in first-line treatment; however, a fraction are refractory to these antiangiogenic drugs. MicroRNAs (miRNAs) are regulatory molecules proven to be accurate biomarkers in cancer. Here, we identified miRNAs predictive of progressive disease under TKI treatment through deep sequencing of 74 metastatic clear cell RCC cases uniformly treated with these drugs. Twenty-nine miRNAs were differentially expressed in the tumors of patients who progressed under TKI therapy (P values from 6 × 10-9 to 3 × 10-3). Among 6 miRNAs selected for validation in an independent series, the most relevant associations corresponded to miR-1307-3p, miR-155-5p, and miR-221-3p (P = 4.6 × 10-3, 6.5 × 10-3, and 3.4 × 10-2, respectively). Furthermore, a 2 miRNA-based classifier discriminated individuals with progressive disease upon TKI treatment (AUC = 0.75, 95% CI, 0.64-0.85; P = 1.3 × 10-4) with better predictive value than clinicopathological risk factors commonly used. We also identified miRNAs significantly associated with progression-free survival and overall survival (P = 6.8 × 10-8 and 7.8 × 10-7 for top hits, respectively), and 7 overlapped with early progressive disease. In conclusion, this is the first miRNome comprehensive study, to our knowledge, that demonstrates a predictive value of miRNAs for TKI response and provides a new set of relevant markers that can help rationalize metastatic RCC treatment.

NSD2 contributes to oncogenic RAS-driven transcription in lung cancer cells through long-range epigenetic activation.

The histone methyltransferase NSD2/WHSC1/MMSET is overexpressed in a number of solid tumors but its contribution to the biology of these tumors is not well understood. Here, we describe that NSD2 contributes to the proliferation of a subset of lung cancer cell lines by supporting oncogenic RAS transcriptional responses. NSD2 knock down combined with MEK or BRD4 inhibitors causes co-operative inhibitory responses on cell growth. However, while MEK and BRD4 inhibitors converge in the downregulation of genes associated with cancer-acquired super-enhancers, NSD2 inhibition affects the expression of clusters of genes embedded in megabase-scale regions marked with H3K36me2 and that contribute to the RAS transcription program. Thus, combinatorial therapies using MEK or BRD4 inhibitors together with NSD2 inhibition are likely to be needed to ensure a more comprehensive inhibition of oncogenic RAS-driven transcription programs in lung cancers with NSD2 overexpression.

A genetic interaction between RAP1 and telomerase reveals an unanticipated role for RAP1 in telomere maintenance.

RAP1 is one of the components of shelterin, the capping complex at chromosome ends or telomeres, although its role in telomere length maintenance and protection has remained elusive. RAP1 also binds subtelomeric repeats and along chromosome arms, where it regulates gene expression and has been shown to function in metabolism control. Telomerase is the enzyme that elongates telomeres, and its deficiency causes a premature aging in humans and mice. We describe an unanticipated genetic interaction between RAP1 and telomerase. While RAP1 deficiency alone does not impact on mouse survival, mice lacking both RAP1 and telomerase show a progressively decreased survival with increasing mouse generations compared to telomerase single mutants. Telomere shortening is more pronounced in Rap1-/- Terc-/- doubly deficient mice than in the single-mutant Terc-/- counterparts, leading to an earlier onset of telomere-induced DNA damage and degenerative pathologies. Telomerase deficiency abolishes obesity and liver steatohepatitis provoked by RAP1 deficiency. Using genomewide ChIP sequencing, we find that progressive telomere shortening owing to telomerase deficiency leads to re-localization of RAP1 from telomeres and subtelomeric regions to extratelomeric sites in a genomewide manner. These findings suggest that although in the presence of sufficient telomere reserve RAP1 is not a key factor for telomere maintenance and protection, it plays a crucial role in the context of telomerase deficiency, thus in agreement with its evolutionary conservation as a telomere component from yeast to humans.

Analysis of Paired Primary-Metastatic Hormone-Receptor Positive Breast Tumors (HRPBC) Uncovers Potential Novel Drivers of Hormonal Resistance.

We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role in vitro, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only MYC, DNAH5, CSFR1, EPHA7, ARID1B, and KMT2C preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of MYC, KMT2C, and EPHA7 best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25 (p<0.0001), and early relapse during the adjuvant hormonal treatment. The presence of the D348N mutation in KMT2C and/or the T666I mutation in the kinase domain of EPHA7 conferred hormonal resistance in vitro. Novel inactivating mutations in KMT2C and EPHA7, which confer hormonal resistance, are linked to adverse clinical course in HRPBC.

A novel high-content analysis tool reveals Rab8-driven cytoskeletal reorganization through Rho GTPases, calpain and MT1-MMP.

Rab8 is a small Ras-related GTPase that regulates polarized membrane transport to the plasma membrane. Here, we developed a high-content analysis (HCA) tool to dissect Rab8-mediated actin and focal adhesion reorganization that revealed that Rab8 activation significantly induced Rac1 and Tiam1 to mediate cortical actin polymerization and RhoA-dependent stress fibre disassembly. Rab8 activation increased Rac1 activity, whereas its depletion activated RhoA, which led to reorganization of the actin cytoskeleton. Rab8 was also associated with focal adhesions, promoting their disassembly in a microtubule-dependent manner. This Rab8 effect involved calpain, MT1-MMP (also known as MMP14) and Rho GTPases. Moreover, we demonstrate the role of Rab8 in the cell migration process. Indeed, Rab8 is required for EGF-induced cell polarization and chemotaxis, as well as for the directional persistency of intrinsic cell motility. These data reveal that Rab8 drives cell motility by mechanisms both dependent and independent of Rho GTPases, thereby regulating the establishment of cell polarity, turnover of focal adhesions and actin cytoskeleton rearrangements, thus determining the directionality of cell migration.

Profiling of Sox4-dependent transcriptome in skin links tumour suppression and adult stem cell activation.

Adult stem cells (ASCs) reside in specific niches in a quiescent state in adult mammals. Upon specific cues they become activated and respond by self-renewing and differentiating into newly generated specialised cells that ensure appropriate tissue fitness. ASC quiescence also serves as a tumour suppression mechanism by hampering cellular transformation and expansion (White AC et al., 2014). Some genes restricted to early embryonic development and adult stem cell niches are often potent modulators of stem cell quiescence, and derailed expression of these is commonly associated to cancer (Vervoort SJ et al., 2013). Among them, it has been shown that recommissioned Sox4 expression facilitates proliferation, survival and migration of malignant cells. By generating a conditional Knockout mouse model in stratified epithelia (Sox4 (cKO) mice), we demonstrated a delayed plucking-induced Anagen in the absence of Sox4. Skin global transcriptome analysis revealed a prominent defect in the induction of transcriptional networks that control hair follicle stem cell (HFSC) activation such as those regulated by Wnt/Ctnnb1, Shh, Myc or Sox9, cell cycle and DNA damage response-associated pathways. Besides, Sox4 (cKO) mice are resistant to skin carcinogenesis, thus linking Sox4 to both normal and pathological HFSC activation (Foronda M et al., 2014). Here we provide additional details on the analysis of Sox4-regulated transcriptome in Telogen and Anagen skin. The raw and processed microarray data is deposited in GEO under GSE58155.

miRNA expression profiling of formalin-fixed paraffin-embedded (FFPE) hereditary breast tumors.

Hereditary breast cancer constitutes only 5-10% of all breast cancer cases and is characterized by strong family history of breast and/or other associated cancer types. Only ~ 25% of hereditary breast cancer cases carry a mutation in BRCA1 or BRCA2 gene, while mutations in other rare high and moderate-risk genes and common low penetrance variants may account for additional 20% of the cases. Thus the majority of cases are still unaccounted for and designated as BRCAX tumors. MicroRNAs are small non-coding RNAs that play important roles as regulators of gene expression and are deregulated in cancer. To characterize hereditary breast tumors based on their miRNA expression profiles we performed global microarray miRNA expression profiling on a retrospective cohort of 80 FFPE breast tissues, including 66 hereditary breast tumors (13 BRCA1, 10 BRCA2 and 43 BRCAX), 10 sporadic breast carcinomas and 4 normal breast tissues, using Exiqon miRCURY LNA™ microRNA Array v.11.0. Here we describe in detail the miRNA microarray expression data and tumor samples used for the study of BRCAX tumor heterogeneity (Tanic et al., 2013) and biomarkers associated with positive BRCA1/2 mutation status (Tanic et al., 2014). Additionally, we provide the R code for data preprocessing and quality control.

Non-coding recurrent mutations in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3' region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia.