Spatial intratumoral heterogeneity and temporal clonal evolution in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is among the most common malignancies, but little is known about its spatial intratumoral heterogeneity (ITH) and temporal clonal evolutionary processes. To address this, we performed multiregion whole-exome sequencing on 51 tumor regions from 13 ESCC cases and multiregion global methylation profiling for 3 of these 13 cases. We found an average of 35.8% heterogeneous somatic mutations with strong evidence of ITH. Half of the driver mutations located on the branches of tumor phylogenetic trees targeted oncogenes, including PIK3CA, NFE2L2 and MTOR, among others. By contrast, the majority of truncal and clonal driver mutations occurred in tumor-suppressor genes, including TP53, KMT2D and ZNF750, among others. Interestingly, phyloepigenetic trees robustly recapitulated the topological structures of the phylogenetic trees, indicating a possible relationship between genetic and epigenetic alterations. Our integrated investigations of spatial ITH and clonal evolution provide an important molecular foundation for enhanced understanding of tumorigenesis and progression in ESCC

Topography of transcriptionally active chromatin in glioblastoma

Molecular profiling of the most aggressive brain tumor glioblastoma (GBM) on the basis of gene expression, DNA methylation, and genomic variations advances both cancer research and clinical diagnosis. The enhancer architectures and regulatory circuitries governing tumor-intrinsic transcriptional diversity and subtype identity are still elusive. Here, by mapping H3K27ac deposition, we analyze the active regulatory landscapes across 95 GBM biopsies, 12 normal brain tissues, and 38 cell line counterparts. Analyses of differentially regulated enhancers and super-enhancers uncovered previously unrecognized layers of intertumor heterogeneity. Integrative analysis of variant enhancer loci and transcriptome identified topographies of transcriptional enhancers and core regulatory circuitries in four molecular subtypes of primary tumors: AC1-mesenchymal, AC1-classical, AC2-proneural, and AC3-proneural. Moreover, this study reveals core oncogenic dependency on super-enhancer–driven transcriptional factors, long noncoding RNAs, and druggable targets in GBM. Through profiling of transcriptional enhancers, we provide clinically relevant insights into molecular classification, pathogenesis, and therapeutic intervention of GBM

Epigenetic modulation of radiation-induced diacylglycerol kinase alpha expression prevents pro-fibrotic fibroblast response

Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis

Epigenetic blueprint of human thymopoiesis and adult T-cell Acute Lymphoblastic Leukemia

Thymopoiesis is a process by which bone-marrow-derived lymphoid progenitor cells migrate to the thymus and undergo multi-step differentiation into mature CD4+ or CD8+ Tlymphocytes. The entire process is tightly regulated and governed by the transcriptional/epigenetic changes necessary for lineage commitment and cellular identity. Genetic lesions such as somatically acquired point mutations or chromosomal rearrangements lead to differentiation blockade resulting in hematological malignancy known as T-cell acute lymphoblastic leukemia (T-ALL). While the thymopoiesis and T-ALL are well characterized by transcriptional studies, high-resolution mapping of the epigenetic changes is still lacking. DNA methylation (DNAm) changes involving the addition of de-novo, or the erasure of existing methyl groups from the cytosine nucleotide, are dynamic during cellular differentiation and form the cell-type-specific signatures. In this doctoral thesis, DNAm dynamics during the human thymopoiesis is studied by whole-genome bisulfite sequencing of seven distinct intra-thymic cell types. DNAm changes during the thymopoiesis are characterized by the uni-directional and irreversible loss methylation primarily occurring at the transcription factor binding sites critical for T-cell lineage commitment (e.g., NOTCH1 and MYB) and T-cell receptor rearrangements. A DNAm atlas of thymopoiesis is established by identifying 381 de-novo differentially methylated regions (tDMRs) that are highly conserved across cell-types originating from the thymic lineage. The tDMRs can recapitulate the in-silico ontogeny of T-cell differentiation and are validated in an independent dataset. Remarkably, combined analysis with bone-marrow-derived hematopoietic progenitors and peripheral derived mature blood cells shows the hypermethylation of tDMRs among non-lymphoid cell types suggesting the epigenetic silencing of pathways necessary for thymic lineage commitment. To further highlight the role of tDMRs in disease development, a combined array-centric analysis of intra-thymic cell types and a well-defined cohort of 143 primary adult T-ALLs was performed. Interestingly, DNAm classified the T-ALL cohort into five distinct subtypes (C1-C5) with characteristic levels of DNAm (C1 lowest level and C5 the highest). Moreover, each II subtype is correlated with a specific somatic event, including a novel adult T-ALL specific subtype with co-occurring DNMT3A/IDH2 mutations (C1), and well known transcriptionally deregulated subtypes resulting from TAL1 (C2), TLX3 (C3), TLX1/in cis-HOXA9 (C4), or in trans- HOXA9 (C5) overexpression. Utilizing tDMRs as the blueprint, maturation arrest stages of TALL subtypes are established, revealing a hierarchical ordering, with C1 and C5 arising earlier during the T-cell development followed by TLX3/1 overexpression (C3, C4) and TAL1 deregulation (C5). Although tDMRs highlight the ontogeny of T-ALL subtypes, global DNAm levels did not correlate with the maturation arrest stages suggesting a non-linear association of DNAm and differentiation blockade. Subsequent integrative analysis with epigenetic marks associated with active transcription (H3K27ac and H3K4me1) revealed the hypomethylation of pathogenic enhancer elements. Importantly, careful survival analysis identified an unexpected, clinically aggressive hypermethylated subtype (C5) that can be targeted with DNA hypomethylating agents. Finally, using machine learning models, a 79 CpG classifier was developed for de-novo classification of newly diagnosed adult T-ALLs. In summary, results from the comprehensive analysis of DNAm changes during human thymopoiesis and the subsequent modifications leading to T-ALL provide meaningful insights into the role of DNAm in maintaining the cellular identity and disease development. Furthermore, the identification of clinically actionable hypermethylated T-ALL subtype paves the way for targeted epigenetic therapies

A Top-Down Methodology for Global Urban Air Mobility Demand Estimation

International audienceThe convergence of several key technologies during the past decade are enabling the ideation of new Urban Air Mobility (UAM) concepts of operations. UAM systems have the potential to bring significant improvements to the way people move and commute within cities and these include a reduction in commuting time, a reduction of roadway congestion, and a reduction of emissions. Understanding the potential demand for UAM services is crucial for the various stakeholders in order to ensure that the air traffic management systems, the regulations, and the supporting infrastructure are ready and do not slow down the introduction of these services. This paper presents a top-down methodology to estimate the demand for UAM transportation worldwide by estimating the travelers' willingness to pay for UAM services and by estimating the potential volume of UAM traffic. The exercise is implemented as a case study for a set of 31 cities distributed all across the world in 2035