Genomics of Acute Myeloid Leukemia: The Next Generation

Acute myeloid leukemia (AML) is, as other types of cancer, a genetic disorder of somatic cells. The detection of somatic molecular abnormalities that may cause and maintain AML is crucial for patient stratification. The development of mutation-specific therapeutic interventions will hopefully increase cure rates and improve patients’ quality of life. This review illustrates how next generation sequencing technologies are changing the study of cancer genomics of adult AML patients

Time makes histone H3 modifications drift in mouse liver

To detect the epigenetic drift of time passing, we determined the genome-wide distributions of mono- and tri-methylated lysine 4 and acetylated and tri-methylated lysine 27 of histone H3 in the livers of healthy 3, 6 and 12 months old C57BL/6 mice. The comparison of different age profiles of histone H3 marks revealed global redistribution of histone H3 modifications with time, in particular in intergenic regions and near transcription start sites, as well as altered correlation between the profiles of different histone modifications. Moreover, feeding mice with caloric restriction diet, a treatment known to retard aging, reduced the extent of changes occurring during the first year of life in these genomic regions

DG-CST (Disease Gene Conserved Sequence Tags), a database of human–mouse conserved elements associated to disease genes

The identification and study of evolutionarily conserved genomic sequences that surround disease-related genes is a valuable tool to gain insight into the functional role of these genes and to better elucidate the pathogenetic mechanisms of disease. We created the DG-CST (Disease Gene Conserved Sequence Tags) database for the identification and detailed annotation of human–mouse conserved genomic sequences that are localized within or in the vicinity of human disease-related genes. CSTs are defined as sequences that show at least 70% identity between human and mouse over a length of at least 100 bp. The database contains CST data relative to over 1088 genes responsible for monogenetic human genetic diseases or involved in the susceptibility to multifactorial/polygenic diseases. DG-CST is accessible via the internet at http://dgcst.ceinge.unina.it/ and may be searched using both simple and complex queries. A graphic browser allows direct visualization of the CSTs and related annotations within the context of the relative gene and its transcripts

Complex Loci in Human and Mouse Genomes

Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis–antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis–antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis–antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis–antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis–antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis–antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes

AML1/ETO Oncoprotein Is Directed to AML1 Binding Regions and Co-Localizes with AML1 and HEB on Its Targets

A reciprocal translocation involving chromosomes 8 and 21 generates the AML1/ETO oncogenic transcription factor that initiates acute myeloid leukemia by recruiting co-repressor complexes to DNA. AML1/ETO interferes with the function of its wild-type counterpart, AML1, by directly targeting AML1 binding sites. However, transcriptional regulation determined by AML1/ETO probably relies on a more complex network, since the fusion protein has been shown to interact with a number of other transcription factors, in particular E-proteins, and may therefore target other sites on DNA. Genome-wide chromatin immunoprecipitation and expression profiling were exploited to identify AML1/ETO-dependent transcriptional regulation. AML1/ETO was found to co-localize with AML1, demonstrating that the fusion protein follows the binding pattern of the wild-type protein but does not function primarily by displacing it. The DNA binding profile of the E-protein HEB was grossly rearranged upon expression of AML1/ETO, and the fusion protein was found to co-localize with both AML1 and HEB on many of its regulated targets. Furthermore, the level of HEB protein was increased in both primary cells and cell lines expressing AML1/ETO. Our results suggest a major role for the functional interaction of AML1/ETO with AML1 and HEB in transcriptional regulation determined by the fusion protein

Single-cell imaging and transcriptomic analyses of firm adhesion between patient-derived cancer and endothelial cells under shear stress

Adhesion between cancer cells and endothelial cells, lining the blood vessels, is a key event during tumour progression and metastasis formation. However, the analysis of its underlying cellular and molecular mechanisms is largely limited by the intrinsic difficulties to study the interactions between circulating cancer cells and endothelial cells in vivo, and in vitro under conditions that mimic the in vivo blood flow. Here, we developed a method to study cell:cell firm adhesion under shear-stress conditions coupled to high-content live-cell imaging, and single-cell RNAseq analysis. As the model system, we used cancer cells freshly isolated from patient-derived xenografts (PDXs) and human primary endothelial cells. Breast cancer is the most common cancer in women worldwide and the leading cause of cancer-related deaths among women. Therefore, we set up protocols for breast cancer PDX tumour dissociation, isolation and purification to obtain freshly isolated PDX-derived human cancer single cell suspension. We then implemented an in vitro assay to study cancer to endothelial cells firm adhesion under shear-stress, using an all–human microfluidic model coupled to time-lapse and live-cell imaging. Finally, we developed a method to successfully retrieve, separate and enrich alive endothelial and cancer cells from the flow-based firm adhesion assay. Most notably, we used retrieved cells for single-cell RNAseq analysis and showed that samples quality, number of cells and transcripts per cell were consistent and optimal for downstream discovery analyses. In conclusion, we developed a workflow method that can provide insights into the mechanisms of cancer adhesion to endothelial cells, and identify new targets for personalized treatments development for the clinic to prevent and/or treat breast cancer metastasis formation

El Diario de Pontevedra : periódico liberal: Ano XL Número 11752 - 1923 maio 12

Abstract Background The landscape of cancer-predisposing genes has been extensively investigated in the last 30 years with various methodologies ranging from candidate gene to genome-wide association studies. However, sequencing data are still poorly exploited in cancer predisposition studies due to the lack of statistical power when comparing millions of variants at once. Method To overcome these power limitations, we propose a knowledge-based framework founded on the characteristics of known cancer-predisposing variants and genes. Under our framework, we took advantage of a combination of previously generated datasets of sequencing experiments to identify novel breast cancer-predisposing variants, comparing the normal genomes of 673 breast cancer patients of European origin against 27,173 controls matched by ethnicity. Results We detected several expected variants on known breast cancer-predisposing genes, like BRCA1 and BRCA2, and 11 variants on genes associated with other cancer types, like RET and AKT1. Furthermore, we detected 183 variants that overlap with somatic mutations in cancer and 41 variants associated with 38 possible loss-of-function genes, including PIK3CB and KMT2C. Finally, we found a set of 19 variants that are potentially pathogenic, negatively correlate with age at onset, and have never been associated with breast cancer. Conclusions In this study, we demonstrate the usefulness of a genomic-driven approach nested in a classic case-control study to prioritize cancer-predisposing variants. In addition, we provide a resource containing variants that may affect susceptibility to breast cancer

Epigenomic profiling of archived FFPE tissues by enhanced PAT-ChIP (EPAT-ChIP) technology

BACKGROUND: The introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation (ChIP) from formalin-fixed paraffin-embedded (FFPE) tissues, has extended the application of chromatin studies to clinical patient samples. However, extensive crosslinking introduced during routine tissue fixation of clinical specimens may hamper the application of PAT-ChIP to genome-wide studies (PAT-ChIP-Seq) from archived tissue samples. The reduced efficiency in chromatin extraction from over-fixed formalin archival samples is the main hurdle to overcome, especially when low abundant epigenetic marks (e.g., H3K4me3) are investigated. RESULTS: We evaluated different modifications of the original PAT-ChIP protocol to improve chromatin isolation from FFPE tissues. With this aim, we first made extensive usage of a normal human colon specimen fixed at controlled conditions (24\ua0h, 48\ua0h, and 72\ua0h) to mimic the variability of tissue fixation that is most frequently found in archived samples. Different conditions of chromatin extraction were tested applying either diverse sonication protocols or heat-mediated limited reversal of crosslinking (LRC). We found that, if compared with canonical PAT-ChIP protocol, LRC strongly increases chromatin extraction efficiency, especially when 72-h fixed FFPE samples are used. The new procedure, that we named enhanced PAT-ChIP (EPAT-ChIP), was then applied at genome-wide level using an archival sample of invasive breast carcinoma to investigate H3K4me3, a lowly abundant histone modification, and H3K27me3 and H3K27ac, two additional well-known histone marks. CONCLUSIONS: EPAT-ChIP procedure improves the efficiency of chromatin isolation from FFPE samples allowing the study of long time-fixed specimens (72\ua0h), as well as the investigation of low distributed epigenetic marks (e.g., H3K4me3) and the analysis of multiple histone marks from low amounts of starting material. We believe that EPAT-ChIP will facilitate the application of chromatin studies to archived pathology samples, thus contributing to extend the current understanding of cancer epigenomes and enabling the identification of clinically useful tumor biomarkers