Publications by authors named "Huy Q Dinh"

25 Publications

  • Page 1 of 1

Single-cell transcriptomics identifies gene expression networks driving differentiation and tumorigenesis in the human fallopian tube.

Cell Rep 2021 Apr;35(2):108978

Women's Cancer Research Program at the Samuel Oschin Comprehensive Cancer Center, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Center for Bioinformatics and Functional Genomics, Cedars-Sinai Medical Center, Los Angeles, CA, USA. Electronic address:

The human fallopian tube harbors the cell of origin for the majority of high-grade serous "ovarian" cancers (HGSCs), but its cellular composition, particularly the epithelial component, is poorly characterized. We perform single-cell transcriptomic profiling of around 53,000 individual cells from 12 primary fallopian specimens to map their major cell types. We identify 10 epithelial subpopulations with diverse transcriptional programs. Based on transcriptional signatures, we reconstruct a trajectory whereby secretory cells differentiate into ciliated cells via a RUNX3 intermediate. Computational deconvolution of advanced HGSCs identifies the "early secretory" population as a likely precursor state for the majority of HGSCs. Its signature comprises both epithelial and mesenchymal features and is enriched in mesenchymal-type HGSCs (p = 6.7 × 10), a group known to have particularly poor prognoses. This cellular and molecular compendium of the human fallopian tube in cancer-free women is expected to advance our understanding of the earliest stages of fallopian epithelial neoplasia.
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http://dx.doi.org/10.1016/j.celrep.2021.108978DOI Listing
April 2021

Naive CD8 T Cells Expressing CD95 Increase Human Cardiovascular Disease Severity.

Arterioscler Thromb Vasc Biol 2020 12 15;40(12):2845-2859. Epub 2020 Oct 15.

Division of Inflammation Biology, La Jolla Institute for Immunology, CA (L.E.P., H.Q.D., R.W., D.E.G., D.J.A., H.W., C.C.H.).

Objective: Cardiovascular disease (CVD) remains a significant global health concern with a high degree of mortality. While CD4 T cells have been extensively studied in CVD, the importance of CD8 T cells in this disease, despite their abundance and increased activation in human atherosclerotic plaques, remains largely unknown. Thus, the objective of this study was to compare peripheral T-cell signatures between humans with a high (severe) risk of CVD (including myocardial infarction or stroke) and those with a low risk of CVD. Approach and Results: Using mass cytometry, we uncovered a naive CD8 T (T) cell population expressing CD95 (termed CD95CD8 stem cell memory T [CD8 T] cells) that was enriched in patients with high compared with low CVD. This T-cell subset enrichment within individuals with high CVD was a relative increase and resulted from the loss of CD95 cells within the T compartment. We found that CD8 T cells positively correlated with CVD risk in humans, while CD8 T cells were inversely correlated. Atherosclerotic apolipoprotein E-deficient (ApoE) mice also displayed respective 7- and 2-fold increases in CD8 T frequencies within the peripheral blood and aorta-draining paraaortic lymph nodes compared with C57BL/6J mice. CD8 T cells were 1.7-fold increased in aortas from western diet fed ApoE mice compared with normal laboratory diet-fed ApoE mice. Importantly, transfer of T cells into immune-deficient recipient mice that lacked T cells increased atherosclerosis, illustrating the importance of these cells in atherogenesis.

Conclusions: CD8 T cells are increased in humans with high CVD. As these T cells promote atherosclerosis, targeting them may attenuate atherosclerotic plaque progression.
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http://dx.doi.org/10.1161/ATVBAHA.120.315106DOI Listing
December 2020

Coexpression of CD71 and CD117 Identifies an Early Unipotent Neutrophil Progenitor Population in Human Bone Marrow.

Immunity 2020 08;53(2):319-334.e6

Center for Cancer Immunotherapy & Center for Autoimmunity and Inflammation, La Jolla Institute for Immunology, La Jolla, CA 92037, USA. Electronic address:

Neutrophils are the most abundant peripheral immune cells and thus, are continually replenished by bone marrow-derived progenitors. Still, how newly identified neutrophil subsets fit into the bone marrow neutrophil lineage remains unclear. Here, we use mass cytometry to show that two recently defined human neutrophil progenitor populations contain a homogeneous progenitor subset we term "early neutrophil progenitors" (eNePs) (LinCD66bCD117CD71). Surface marker- and RNA-expression analyses, together with in vitro colony formation and in vivo adoptive humanized mouse transfers, indicate that eNePs are the earliest human neutrophil progenitors. Furthermore, we identified CD71 as a marker associated with the earliest neutrophil developmental stages. Expression of CD71 marks proliferating neutrophils, which were expanded in the blood of melanoma patients and detectable in blood and tumors from lung cancer patients. In summary, we establish CD117CD71 eNeP as the inceptive human neutrophil progenitor and propose a refined model of the neutrophil developmental lineage in bone marrow.
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http://dx.doi.org/10.1016/j.immuni.2020.07.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7942809PMC
August 2020

Meta-Analysis of Leukocyte Diversity in Atherosclerotic Mouse Aortas.

Circ Res 2020 Jul 16;127(3):402-426. Epub 2020 Jul 16.

La Jolla Institute for Immunology, CA (C.C.H., Y.G., H.Q.D., K.L.).

The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single-cell RNA sequencing and 2 mass cytometry studies. In a comprehensive meta-analysis, we confirm 4 known macrophage subsets-resident, inflammatory, interferon-inducible cell, and Trem2 (triggering receptor expressed on myeloid cells-2) foamy macrophages-and identify a new macrophage subset resembling cavity macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2, and CD (cluster of differentiation)-8 T cells form prominent and separate immune cell populations in atherosclerotic aortas. Many CD4 T cells express IL (interleukin)-17 and the chemokine receptor CXCR (C-X-C chemokine receptor)-6. A small number of regulatory T cells and T helper 1 cells is also identified. Immature and naive T cells are present in both healthy and atherosclerotic aortas. Our meta-analysis overcomes limitations of individual studies that, because of their experimental approach, over- or underrepresent certain cell populations. Mass cytometry studies demonstrate that cell surface phenotype provides valuable information beyond the cell transcriptomes. The present analysis helps resolve some long-standing controversies in the field. First, Trem2 foamy macrophages are not proinflammatory but interferon-inducible cell and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, , which had been considered specific for platelets and megakaryocytes, is also prominently expressed in the main population of resident vascular macrophages. Fourth, a new type of resident macrophage shares transcripts with cavity macrophages. Finally, the discovery of a prominent innate lymphoid cell-2 cluster links the single-cell RNA sequencing work to recent flow cytometry data suggesting a strong atheroprotective role of innate lymphoid cells-2. This resolves apparent discrepancies regarding the role of T helper 2 cells in atherosclerosis based on studies that predated the discovery of innate lymphoid cells-2 cells.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.316903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371244PMC
July 2020

Frontline Science: Kindlin-3 is essential for patrolling and phagocytosis functions of nonclassical monocytes during metastatic cancer surveillance.

J Leukoc Biol 2020 06 9;107(6):883-892. Epub 2020 May 9.

Center for Cancer Immunotherapy, La Jolla Institute for Immunology, La Jolla, California, USA.

Nonclassical monocytes maintain vascular homeostasis by patrolling the vascular endothelium, responding to inflammatory signals, and scavenging cellular debris. Nonclassical monocytes also prevent metastatic tumor cells from seeding new tissues, but whether the patrolling function of nonclassical monocytes is required for this process is unknown. To answer this question, we utilized an inducible-knockout mouse that exhibits loss of the integrin-adaptor protein Kindlin-3 specifically in nonclassical monocytes. We show that Kindlin-3-deficient nonclassical monocytes are unable to patrol the vascular endothelium in either the lungs or periphery. We also find that Kindlin-3-deficient nonclassical monocytes cannot firmly adhere to, and instead "slip" along, the vascular endothelium. Loss of patrolling activity by nonclassical monocytes was phenocopied by ablation of LFA-1, an integrin-binding partner of Kindlin-3. When B16F10 murine melanoma tumor cells were introduced into Kindlin-3-deficient mice, nonclassical monocytes showed defective patrolling towards tumor cells and failure to ingest tumor particles in vivo. Consequently, we observed a significant, 4-fold increase in lung tumor metastases in mice possessing Kindlin-3-deficient nonclassical monocytes. Thus, we conclude that the patrolling function of nonclassical monocytes is mediated by Kindlin-3 and essential for these cells to maintain vascular endothelial homeostasis and prevent tumor metastasis to the lung.
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http://dx.doi.org/10.1002/JLB.4HI0420-098RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986576PMC
June 2020

Macrophage Syk-PI3Kγ Inhibits Antitumor Immunity: SRX3207, a Novel Dual Syk-PI3K Inhibitory Chemotype Relieves Tumor Immunosuppression.

Mol Cancer Ther 2020 03 23;19(3):755-764. Epub 2020 Jan 23.

UCSD Department of Pediatrics, University of California, San Diego, San Diego, California.

Macrophages (MΦ) play a critical role in tumor growth, immunosuppression, and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that an MΦ Syk-PI3K axis drives polarization of immunosuppressive MΦs that establish an immunosuppressive tumor microenvironment in syngeneic tumor models. Genetic or pharmacologic inhibition of Syk and/or PI3Kγ in MΦs promotes a proinflammatory MΦ phenotype, restores CD8 T-cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow-derived macrophages (BMDM) show that inhibition of Syk kinase promotes activation and binding of NF-κB motif in Syk BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs to suppress tumor growth. Finally, we have developed the "first-in-class" dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450492PMC
March 2020

A Study of High-Grade Serous Ovarian Cancer Origins Implicates the SOX18 Transcription Factor in Tumor Development.

Cell Rep 2019 12;29(11):3726-3735.e4

Department of Neurosurgery, Henry Ford Hospital, Detroit, MI, USA; Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, São Paulo, Brazil. Electronic address:

Fallopian tube secretory epithelial cells (FTSECs) are likely the main precursor cell type of high-grade serous ovarian cancers (HGSOCs), but these tumors may also arise from ovarian surface epithelial cells (OSECs). We profiled global landscapes of gene expression and active chromatin to characterize molecular similarities between OSECs (n = 114), FTSECs (n = 74), and HGSOCs (n = 394). A one-class machine learning algorithm predicts that most HGSOCs derive from FTSECs, with particularly high FTSEC scores in mesenchymal-type HGSOCs (p < 8 × 10). However, a subset of HGSOCs likely derive from OSECs, particularly HGSOCs of the proliferative type (p < 2 × 10), suggesting a dualistic model for HGSOC origins. Super-enhancer (SE) landscapes were also more similar between FTSECs and HGSOCs than between OSECs and HGSOCs (p < 2.2 × 10). The SOX18 transcription factor (TF) coincided with a HGSOC-specific SE, and ectopic overexpression of SOX18 in FTSECs caused epithelial-to-mesenchymal transition, indicating that SOX18 plays a role in establishing the mesenchymal signature of fallopian-derived HGSOCs.
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http://dx.doi.org/10.1016/j.celrep.2019.10.122DOI Listing
December 2019

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells.

J Vis Exp 2019 06 19(148). Epub 2019 Jun 19.

Division of Inflammation Biology, La Jolla Institute for Immunology;

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.
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http://dx.doi.org/10.3791/59617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726111PMC
June 2019

Monocyte heterogeneity and functions in cancer.

J Leukoc Biol 2019 08 18;106(2):309-322. Epub 2019 Feb 18.

La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.

Monocytes are innate immune cells of the mononuclear phagocyte system that have emerged as important regulators of cancer development and progression. Our understanding of monocytes has advanced from viewing these cells as a homogenous population to a heterogeneous system of cells that display diverse responses to different stimuli. During cancer, different monocyte subsets perform functions that contribute to both pro- and antitumoral immunity, including phagocytosis, secretion of tumoricidal mediators, promotion of angiogenesis, remodeling of the extracellular matrix, recruitment of lymphocytes, and differentiation into tumor-associated macrophages and dendritic cells. The ability of cancer to evade immune recognition and clearance requires protumoral signals to outweigh ongoing attempts by the host immune system to prevent tumor growth. This review discusses current understanding of monocyte heterogeneity during homeostasis, highlights monocyte functions in cancer progression, and describes monocyte-targeted therapeutic strategies for cancer treatment.
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http://dx.doi.org/10.1002/JLB.4RI0818-311RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658332PMC
August 2019

Human Monocyte Heterogeneity as Revealed by High-Dimensional Mass Cytometry.

Arterioscler Thromb Vasc Biol 2019 01;39(1):25-36

From the Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA (A.A.J.H., H.Q.D., G.D.T., P.M., A.B., C.S.N., C.C.H.).

Objective- Three distinct human monocyte subsets have been identified based on the surface marker expression of CD14 and CD16. We hypothesized that monocytes were likely more heterogeneous in composition. Approach and Results- We used the high dimensionality of mass cytometry together with the FlowSOM clustering algorithm to accurately identify and define monocyte subsets in blood of healthy human subjects and those with coronary artery disease (CAD). To study the behavior and functionality of the newly defined monocyte subsets, we performed RNA sequencing, transwell migration, and efferocytosis assays. Here, we identify 8 human monocyte subsets based on their surface marker phenotype. We found that 3 of these subsets fall within the CD16 nonclassical monocyte population and 4 subsets belong to the CD14 classical monocytes, illustrating significant monocyte heterogeneity in humans. As nonclassical monocytes are important in modulating atherosclerosis in mice, we studied the functions of our 3 newly identified nonclassical monocytes in subjects with CAD. We found a marked expansion of a SlanCXCR6 nonclassical monocyte subset in CAD subjects, which was positively correlated with CAD severity. This nonclassical subset can migrate towards CXCL16 and shows an increased efferocytosis capacity, indicating it may play an atheroprotective role. Conclusions- Our data demonstrate that human nonclassical monocytes are a heterogeneous population, existing of several subsets with functional differences. These subsets have changed frequencies in the setting of severe CAD. Understanding how these newly identified subsets modulate CAD will be important for CAD-based therapies that target myeloid cells.
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http://dx.doi.org/10.1161/ATVBAHA.118.311022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697379PMC
January 2019

Identification of an Early Unipotent Neutrophil Progenitor with Pro-tumoral Activity in Mouse and Human Bone Marrow.

Cell Rep 2018 08;24(9):2329-2341.e8

Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. Electronic address:

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however, unipotent neutrophil progenitors are not well defined. Here, we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly, we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models, including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases, including cancer.
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http://dx.doi.org/10.1016/j.celrep.2018.07.097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542273PMC
August 2018

Targetable BET proteins- and E2F1-dependent transcriptional program maintains the malignancy of glioblastoma.

Proc Natl Acad Sci U S A 2018 05 15;115(22):E5086-E5095. Epub 2018 May 15.

Cancer Science Institute of Singapore, National University of Singapore, 117599 Singapore.

Competitive BET bromodomain inhibitors (BBIs) targeting BET proteins (BRD2, BRD3, BRD4, and BRDT) show promising preclinical activities against brain cancers. However, the BET protein-dependent glioblastoma (GBM)-promoting transcriptional network remains elusive. Here, with mechanistic exploration of a next-generation chemical degrader of BET proteins (dBET6), we reveal a profound and consistent impact of BET proteins on E2F1- dependent transcriptional program in both differentiated GBM cells and brain tumor-initiating cells. dBET6 treatment drastically reduces BET protein genomic occupancy, RNA-Pol2 activity, and permissive chromatin marks. Subsequently, dBET6 represses the proliferation, self-renewal, and tumorigenic ability of GBM cells. Moreover, dBET6-induced degradation of BET proteins exerts superior antiproliferation effects compared to conventional BBIs and overcomes both intrinsic and acquired resistance to BBIs in GBM cells. Our study reveals crucial functions of BET proteins and provides the rationale and therapeutic merits of targeted degradation of BET proteins in GBM.
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http://dx.doi.org/10.1073/pnas.1712363115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984485PMC
May 2018

DNA methylation loss in late-replicating domains is linked to mitotic cell division.

Nat Genet 2018 04 2;50(4):591-602. Epub 2018 Apr 2.

Center for Bioinformatics and Functional Genomics, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

DNA methylation loss occurs frequently in cancer genomes, primarily within lamina-associated, late-replicating regions termed partially methylated domains (PMDs). We profiled 39 diverse primary tumors and 8 matched adjacent tissues using whole-genome bisulfite sequencing (WGBS) and analyzed them alongside 343 additional human and 206 mouse WGBS datasets. We identified a local CpG sequence context associated with preferential hypomethylation in PMDs. Analysis of CpGs in this context ('solo-WCGWs') identified previously undetected PMD hypomethylation in almost all healthy tissue types. PMD hypomethylation increased with age, beginning during fetal development, and appeared to track the accumulation of cell divisions. In cancer, PMD hypomethylation depth correlated with somatic mutation density and cell cycle gene expression, consistent with its reflection of mitotic history and suggesting its application as a mitotic clock. We propose that late replication leads to lifelong progressive methylation loss, which acts as a biomarker for cellular aging and which may contribute to oncogenesis.
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http://dx.doi.org/10.1038/s41588-018-0073-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893360PMC
April 2018

Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry.

Circ Res 2018 06 15;122(12):1675-1688. Epub 2018 Mar 15.

Institute of Experimental Biomedicine, University Hospital Würzburg, Germany (C.C., A.Z.)

Rationale: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood.

Objective: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis.

Methods And Results: Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed and mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic and mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients.

Conclusions: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.312513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993603PMC
June 2018

Identification of distinct mutational patterns and new driver genes in oesophageal squamous cell carcinomas and adenocarcinomas.

Gut 2018 10 31;67(10):1769-1779. Epub 2017 Aug 31.

Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

Objectives: Oesophageal squamous cell carcinoma (OSCC) and adenocarcinoma (OAC) are distinct cancers in terms of a number of clinical and epidemiological characteristics, complicating the design of clinical trials and biomarker developments. We analysed 1048 oesophageal tumour-germline pairs from both subtypes, to characterise their genomic features, and biological and clinical significance.

Design: Previously exome-sequenced samples were re-analysed to identify significantly mutated genes (SMGs) and mutational signatures. The biological functions of novel SMGs were investigated using cell line and xenograft models. We further performed whole-genome bisulfite sequencing and chromatin immunoprecipitation (ChIP)-seq to characterise epigenetic alterations.

Results: OSCC and OAC displayed nearly mutually exclusive sets of driver genes, indicating that they follow independent developmental paths. The combined sample size allowed the statistical identification of a number of novel subtype-specific SMGs, mutational signatures and prognostic biomarkers. Particularly, we identified a novel mutational signature similar to Catalogue Of Somatic Mutations In Cancer (COSMIC)signature 16, which has prognostic value in OSCC. Two newly discovered SMGs, and , were validated as important tumour-suppressors specific to the OSCC subtype. We further identified their additional loss-of-function mechanisms. was homozygously deleted specifically in OSCC and other squamous cell cancers (SCCs). Notably, is associated with super-enhancer in healthy oesophageal mucosa; DNA hypermethylation in its super-enhancer reduced active histone markers in squamous cancer cells, suggesting an epigenetic inactivation of a super-enhancer-associated SCC suppressor.

Conclusions: These data comprehensively contrast differences between OSCC and OAC at both genomic and epigenomic levels, and reveal novel molecular features for further delineating the pathophysiological mechanisms and treatment strategies for these cancers.
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http://dx.doi.org/10.1136/gutjnl-2017-314607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980794PMC
October 2018

Genomic and Epigenomic Heterogeneity of Hepatocellular Carcinoma.

Cancer Res 2017 05 20;77(9):2255-2265. Epub 2017 Feb 20.

Division of Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California.

Understanding the intratumoral heterogeneity of hepatocellular carcinoma is instructive for developing personalized therapy and identifying molecular biomarkers. Here we applied whole-exome sequencing to 69 samples from 11 patients to resolve the genetic architecture of subclonal diversification. Spatial genomic diversity was found in all 11 hepatocellular carcinoma cases, with 29% of driver mutations being heterogeneous, including TERT, ARID1A, NOTCH2, and STAG2. Similar with other cancer types, TP53 mutations were always shared between all tumor regions, that is, located on the "trunk" of the evolutionary tree. In addition, we found that variants within several drug targets such as KIT, SYK, and PIK3CA were mutated in a fully clonal manner, indicating their therapeutic potentials for hepatocellular carcinoma. Temporal dissection of mutational signatures suggested that mutagenic processes associated with exposure to aristolochic acid and aflatoxin might play a more important role in early, as opposed to late, stages of hepatocellular carcinoma development. Moreover, we observed extensive intratumoral epigenetic heterogeneity in hepatocellular carcinoma based on multiple independent analytical methods and showed that intratumoral methylation heterogeneity might play important roles in the biology of hepatocellular carcinoma cells. Our results also demonstrated prominent heterogeneity of intratumoral methylation even in a stable hepatocellular carcinoma genome. Together, these findings highlight widespread intratumoral heterogeneity at both the genomic and epigenomic levels in hepatocellular carcinoma and provide an important molecular foundation for better understanding the pathogenesis of this malignancy. .
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http://dx.doi.org/10.1158/0008-5472.CAN-16-2822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413372PMC
May 2017

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

Nat Genet 2016 12 17;48(12):1500-1507. Epub 2016 Oct 17.

Division of Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California, USA.

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.
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http://dx.doi.org/10.1038/ng.3683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127772PMC
December 2016

The RdDM pathway is required for basal heat tolerance in Arabidopsis.

Mol Plant 2013 Mar 1;6(2):396-410. Epub 2013 Feb 1.

GMI-Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030 Vienna, Austria.

Heat stress affects epigenetic gene silencing in Arabidopsis. To test for a mechanistic involvement of epigenetic regulation in heat-stress responses, we analyzed the heat tolerance of mutants defective in DNA methylation, histone modifications, chromatin-remodeling, or siRNA-based silencing pathways. Plants deficient in NRPD2, the common second-largest subunit of RNA polymerases IV and V, and in the Rpd3-type histone deacetylase HDA6 were hypersensitive to heat exposure. Microarray analysis demonstrated that NRPD2 and HDA6 have independent roles in transcriptional reprogramming in response to temperature stress. The misexpression of protein-coding genes in nrpd2 mutants recovering from heat correlated with defective epigenetic regulation of adjacent transposon remnants which involved the loss of control of heat-stress-induced read-through transcription. We provide evidence that the transcriptional response to temperature stress, at least partially, relies on the integrity of the RNA-dependent DNA methylation pathway.
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http://dx.doi.org/10.1093/mp/sst023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3603006PMC
March 2013

Advanced methylome analysis after bisulfite deep sequencing: an example in Arabidopsis.

PLoS One 2012 20;7(7):e41528. Epub 2012 Jul 20.

Max F. Perutz Laboratories, Center for Integrative Bioinformatics Vienna, University of Vienna, Vienna, Austria.

Deep sequencing after bisulfite conversion (BS-Seq) is the method of choice to generate whole genome maps of cytosine methylation at single base-pair resolution. Its application to genomic DNA of Arabidopsis flower bud tissue resulted in the first complete methylome, determining a methylation rate of 6.7% in this tissue. BS-Seq reads were mapped onto an in silico converted reference genome, applying the so-called 3-letter genome method. Here, we present BiSS (Bisufite Sequencing Scorer), a new method applying Smith-Waterman alignment to map bisulfite-converted reads to a reference genome. In addition, we introduce a comprehensive adaptive error estimate that accounts for sequencing errors, erroneous bisulfite conversion and also wrongly mapped reads. The re-analysis of the Arabidopsis methylome data with BiSS mapped substantially more reads to the genome. As a result, it determines the methylation status of an extra 10% of cytosines and estimates the methylation rate to be 7.7%. We validated the results by individual traditional bisulfite sequencing for selected genomic regions. In addition to predicting the methylation status of each cytosine, BiSS also provides an estimate of the methylation degree at each genomic site. Thus, BiSS explores BS-Seq data more extensively and provides more information for downstream analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041528PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401099PMC
February 2013

Uncovering cis-regulatory sequence requirements for context-specific transcription factor binding.

Genome Res 2012 Oct 25;22(10):2018-30. Epub 2012 Apr 25.

Research Institute of Molecular Pathology, 1030 Vienna, Austria.

The regulation of gene expression is mediated at the transcriptional level by enhancer regions that are bound by sequence-specific transcription factors (TFs). Recent studies have shown that the in vivo binding sites of single TFs differ between developmental or cellular contexts. How this context-specific binding is encoded in the cis-regulatory DNA sequence has, however, remained unclear. We computationally dissect context-specific TF binding sites in Drosophila, Caenorhabditis elegans, mouse, and human and find distinct combinations of sequence motifs for partner factors, which are predictive and reveal specific motif requirements of individual binding sites. We predict that TF binding in the early Drosophila embryo depends on motifs for the early zygotic TFs Vielfaltig (also known as Zelda) and Tramtrack. We validate experimentally that the activity of Twist-bound enhancers and Twist binding itself depend on Vielfaltig motifs, suggesting that Vielfaltig is more generally important for early transcription. Our finding that the motif content can predict context-specific binding and that the predictions work across different Drosophila species suggests that characteristic motif combinations are shared between sites, revealing context-specific motif codes (cis-regulatory signatures), which appear to be conserved during evolution. Taken together, this study establishes a novel approach to derive predictive cis-regulatory motif requirements for individual TF binding sites and enhancers. Importantly, the method is generally applicable across different cell types and organisms to elucidate cis-regulatory sequence determinants and the corresponding trans-acting factors from the increasing number of tissue- and cell-type-specific TF binding studies.
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http://dx.doi.org/10.1101/gr.132811.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460196PMC
October 2012

Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis.

Nucleic Acids Res 2012 Mar 29;40(6):2454-69. Epub 2011 Nov 29.

Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria.

Alternative splicing (AS) coupled to nonsense-mediated decay (NMD) is a post-transcriptional mechanism for regulating gene expression. We have used a high-resolution AS RT-PCR panel to identify endogenous AS isoforms which increase in abundance when NMD is impaired in the Arabidopsis NMD factor mutants, upf1-5 and upf3-1. Of 270 AS genes (950 transcripts) on the panel, 102 transcripts from 97 genes (32%) were identified as NMD targets. Extrapolating from these data around 13% of intron-containing genes in the Arabidopsis genome are potentially regulated by AS/NMD. This cohort of naturally occurring NMD-sensitive AS transcripts also allowed the analysis of the signals for NMD in plants. We show the importance of AS in introns in 5' or 3'UTRs in modulating NMD-sensitivity of mRNA transcripts. In particular, we identified upstream open reading frames overlapping the main start codon as a new trigger for NMD in plants and determined that NMD is induced if 3'-UTRs were >350 nt. Unexpectedly, although many intron retention transcripts possess NMD features, they are not sensitive to NMD. Finally, we have shown that AS/NMD regulates the abundance of transcripts of many genes important for plant development and adaptation including transcription factors, RNA processing factors and stress response genes.
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http://dx.doi.org/10.1093/nar/gkr932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3315328PMC
March 2012

Genetic rearrangements can modify chromatin features at epialleles.

PLoS Genet 2011 Oct 20;7(10):e1002331. Epub 2011 Oct 20.

Gregor Mendel Institute of Molecular Plant Biology (GMI), Austrian Academy of Sciences, Vienna, Austria.

Analogous to genetically distinct alleles, epialleles represent heritable states of different gene expression from sequence-identical genes. Alleles and epialleles both contribute to phenotypic heterogeneity. While alleles originate from mutation and recombination, the source of epialleles is less well understood. We analyze active and inactive epialleles that were found at a transgenic insert with a selectable marker gene in Arabidopsis. Both converse expression states are stably transmitted to progeny. The silent epiallele was previously shown to change its state upon loss-of-function of trans-acting regulators and drug treatments. We analyzed the composition of the epialleles, their chromatin features, their nuclear localization, transcripts, and homologous small RNA. After mutagenesis by T-DNA transformation of plants carrying the silent epiallele, we found new active alleles. These switches were associated with different, larger or smaller, and non-overlapping deletions or rearrangements in the 3' regions of the epiallele. These cis-mutations caused different degrees of gene expression stability depending on the nature of the sequence alteration, the consequences for transcription and transcripts, and the resulting chromatin organization upstream. This illustrates a tight dependence of epigenetic regulation on local structures and indicates that sequence alterations can cause epigenetic changes at some distance in regions not directly affected by the mutation. Similar effects may also be involved in gene expression and chromatin changes in the vicinity of transposon insertions or excisions, recombination events, or DNA repair processes and could contribute to the origin of new epialleles.
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http://dx.doi.org/10.1371/journal.pgen.1002331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197671PMC
October 2011

Pleiotropic effects of the wheat dehydrin DHN-5 on stress responses in Arabidopsis.

Plant Cell Physiol 2011 Apr 18;52(4):676-88. Epub 2011 Mar 18.

Laboratory of Plant Protection and Improvement, Centre of Biotechnology of Sfax/University of Sfax, BP '1177' 3018 Sfax-Tunisia.

We have previously reported that transgenic Arabidopsis plants overexpressing the wheat dehydrin DHN-5 show enhanced tolerance to osmotic stresses. In order to understand the mechanisms through which DHN-5 exerts this effect, we performed transcriptome profiling using the Affymetrix ATH1 microarray. Our data show an altered expression of 77 genes involved mainly in transcriptional regulation, cellular metabolism, stress tolerance and signaling. Among the up-regulated genes, we identified those which are known to be stress-related genes. Several late embryogenesis abundant (LEA) genes, ABA/stress-related genes (such as RD29B) and those involved in pathogen responses (PR genes) are among the most up-regulated genes. In addition, the MDHAR gene involved in the ascorbate biosynthetic pathway was also up-regulated. This up-regulation was correlated with higher ascorbate content in two dehydrin transgenic lines. In agreement with this result and as ascorbate is known to be an antioxidant, we found that both transgenic lines show enhanced tolerance to oxidative stress caused by H₂O₂. On the other hand, multiple types of transcription factors constitute the largest group of the down-regulated genes. Moreover, three members of the jasmonate-ZIM domain (JAZ) proteins which are negative regulators of jasmonate signaling were severely down-regulated. Interestingly, the dehydrin-overexpressing lines exhibit less sensitivity to jasmonate than wild-type plants and changes in regulation of jasmonate-responsive genes, in a manner similar to that in the jasmonate-insensitive jai3-1 mutant. Altogether, our data unravel the potential pleiotropic effects of DHN-5 on both abiotic and biotic stress responses in Arabidopsis.
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http://dx.doi.org/10.1093/pcp/pcr030DOI Listing
April 2011

Epigenetic regulation of repetitive elements is attenuated by prolonged heat stress in Arabidopsis.

Plant Cell 2010 Sep 28;22(9):3118-29. Epub 2010 Sep 28.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, 1030 Viena, Austria.

Epigenetic factors determine responses to internal and external stimuli in eukaryotic organisms. Whether and how environmental conditions feed back to the epigenetic landscape is more a matter of suggestion than of substantiation. Plants are suitable organisms with which to address this question due to their sessile lifestyle and diversification of epigenetic regulators. We show that several repetitive elements of Arabidopsis thaliana that are under epigenetic regulation by transcriptional gene silencing at ambient temperatures and upon short term heat exposure become activated by prolonged heat stress. Activation can occur without loss of DNA methylation and with only minor changes to histone modifications but is accompanied by loss of nucleosomes and by heterochromatin decondensation. Whereas decondensation persists, nucleosome loading and transcriptional silencing are restored upon recovery from heat stress but are delayed in mutants with impaired chromatin assembly functions. The results provide evidence that environmental conditions can override epigenetic regulation, at least transiently, which might open a window for more permanent epigenetic changes.
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http://dx.doi.org/10.1105/tpc.110.078493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965555PMC
September 2010

Cooperation of multiple chromatin modifications can generate unanticipated stability of epigenetic States in Arabidopsis.

Plant Cell 2010 Jan 22;22(1):34-47. Epub 2010 Jan 22.

Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, 1030 Viena, Austria.

Epigenetic changes of gene expression can potentially be reversed by developmental programs, genetic manipulation, or pharmacological interference. However, a case of transcriptional gene silencing, originally observed in tetraploid Arabidopsis thaliana plants, created an epiallele resistant to many mutations or inhibitor treatments that activate many other suppressed genes. This raised the question about the molecular basis of this extreme stability. A combination of forward and reverse genetics and drug application provides evidence for an epigenetic double lock that is only alleviated upon the simultaneous removal of both DNA methylation and histone methylation. Therefore, the cooperation of multiple chromatin modifications can generate unanticipated stability of epigenetic states and contributes to heritable diversity of gene expression patterns.
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http://dx.doi.org/10.1105/tpc.109.072819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2828703PMC
January 2010