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  • 1
    Publication Date: 2014-12-24
    Description: Models derived from human pluripotent stem cells that accurately recapitulate neural development in vitro and allow for the generation of specific neuronal subtypes are of major interest to the stem cell and biomedical community. Notch signalling, particularly through the Notch effector HES5, is a major pathway critical for the onset and maintenance of neural progenitor cells in the embryonic and adult nervous system. Here we report the transcriptional and epigenomic analysis of six consecutive neural progenitor cell stages derived from a HES5::eGFP reporter human embryonic stem cell line. Using this system, we aimed to model cell-fate decisions including specification, expansion and patterning during the ontogeny of cortical neural stem and progenitor cells. In order to dissect regulatory mechanisms that orchestrate the stage-specific differentiation process, we developed a computational framework to infer key regulators of each cell-state transition based on the progressive remodelling of the epigenetic landscape and then validated these through a pooled short hairpin RNA screen. We were also able to refine our previous observations on epigenetic priming at transcription factor binding sites and suggest here that they are mediated by combinations of core and stage-specific factors. Taken together, we demonstrate the utility of our system and outline a general framework, not limited to the context of the neural lineage, to dissect regulatory circuits of differentiation.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336237/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336237/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Ziller, Michael J -- Edri, Reuven -- Yaffe, Yakey -- Donaghey, Julie -- Pop, Ramona -- Mallard, William -- Issner, Robbyn -- Gifford, Casey A -- Goren, Alon -- Xing, Jeffrey -- Gu, Hongcang -- Cacchiarelli, Davide -- Tsankov, Alexander M -- Epstein, Charles -- Rinn, John L -- Mikkelsen, Tarjei S -- Kohlbacher, Oliver -- Gnirke, Andreas -- Bernstein, Bradley E -- Elkabetz, Yechiel -- Meissner, Alexander -- F32 DK095537/DK/NIDDK NIH HHS/ -- HG006911/HG/NHGRI NIH HHS/ -- P01 GM099117/GM/NIGMS NIH HHS/ -- P01GM099117/GM/NIGMS NIH HHS/ -- U01 ES017155/ES/NIEHS NIH HHS/ -- U01ES017155/ES/NIEHS NIH HHS/ -- U54 HG006991/HG/NHGRI NIH HHS/ -- England -- Nature. 2015 Feb 19;518(7539):355-9. doi: 10.1038/nature13990. Epub 2014 Dec 24.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA [2] Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA. ; Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 6997801, Israel. ; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA [2] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA. ; Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA. ; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA [2] Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA [3] Center for Systems Biology and Center for Cancer Research, Massachusetts General Hospital, Boston, Massachusetts 02114, USA. ; Applied Bioinformatics, Center for Bioinformatics and Quantitative Biology Center, University of Tubingen, Tubingen 72076, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25533951" target="_blank"〉PubMed〈/a〉
    Keywords: Binding Sites ; Cell Differentiation/*genetics ; Cell Lineage/genetics ; Embryonic Stem Cells/*cytology/metabolism ; Epigenesis, Genetic/*genetics ; Epigenomics/*methods ; Humans ; Neural Stem Cells/*cytology/*metabolism ; RNA, Small Interfering/analysis/genetics ; Reproducibility of Results ; Transcription Factors/metabolism ; Transcription, Genetic/genetics
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 2
    Publication Date: 2013-08-09
    Description: DNA methylation is a defining feature of mammalian cellular identity and is essential for normal development. Most cell types, except germ cells and pre-implantation embryos, display relatively stable DNA methylation patterns, with 70-80% of all CpGs being methylated. Despite recent advances, we still have a limited understanding of when, where and how many CpGs participate in genomic regulation. Here we report the in-depth analysis of 42 whole-genome bisulphite sequencing data sets across 30 diverse human cell and tissue types. We observe dynamic regulation for only 21.8% of autosomal CpGs within a normal developmental context, most of which are distal to transcription start sites. These dynamic CpGs co-localize with gene regulatory elements, particularly enhancers and transcription-factor-binding sites, which allow identification of key lineage-specific regulators. In addition, differentially methylated regions (DMRs) often contain single nucleotide polymorphisms associated with cell-type-related diseases as determined by genome-wide association studies. The results also highlight the general inefficiency of whole-genome bisulphite sequencing, as 70-80% of the sequencing reads across these data sets provided little or no relevant information about CpG methylation. To demonstrate further the utility of our DMR set, we use it to classify unknown samples and identify representative signature regions that recapitulate major DNA methylation dynamics. In summary, although in theory every CpG can change its methylation state, our results suggest that only a fraction does so as part of coordinated regulatory programs. Therefore, our selected DMRs can serve as a starting point to guide new, more effective reduced representation approaches to capture the most informative fraction of CpGs, as well as further pinpoint putative regulatory elements.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3821869/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3821869/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Ziller, Michael J -- Gu, Hongcang -- Muller, Fabian -- Donaghey, Julie -- Tsai, Linus T-Y -- Kohlbacher, Oliver -- De Jager, Philip L -- Rosen, Evan D -- Bennett, David A -- Bernstein, Bradley E -- Gnirke, Andreas -- Meissner, Alexander -- ES017690/ES/NIEHS NIH HHS/ -- P01 GM099117/GM/NIGMS NIH HHS/ -- P01GM099117/GM/NIGMS NIH HHS/ -- P30AG10161/AG/NIA NIH HHS/ -- R01 AG017917/AG/NIA NIH HHS/ -- R01AG15819/AG/NIA NIH HHS/ -- R01AG17917/AG/NIA NIH HHS/ -- R01AG36042/AG/NIA NIH HHS/ -- U01 ES017155/ES/NIEHS NIH HHS/ -- U01ES017155/ES/NIEHS NIH HHS/ -- England -- Nature. 2013 Aug 22;500(7463):477-81. doi: 10.1038/nature12433. Epub 2013 Aug 7.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/23925113" target="_blank"〉PubMed〈/a〉
    Keywords: Binding Sites ; CpG Islands/genetics ; *DNA Methylation ; Enhancer Elements, Genetic/genetics ; Genome, Human/*genetics ; Genome-Wide Association Study ; Humans ; Organ Specificity ; Polymorphism, Single Nucleotide/genetics ; Sequence Analysis, DNA ; Sulfites/metabolism ; Transcription Factors/metabolism
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 3
    Keywords: GRAFT ; TRANSPLANTATION
    Abstract: Allogenic stem cell transplantation has shown considerable success in a number of hematological malignancies, in particular in leukemia. The beneficial effect is mediated by donor T cells recognizing patient-specific HLA-binding peptides. These peptides are called minor histocompatibility antigens (miHAs) and are typically caused by single nucleotide polymorphisms. Tissue-specific miHAs have successfully been used in anti-tumor therapy without causing unspecific graft-versus-host reactions. However, only a small number of miHAs have been identified to date, limiting the clinical use. Here we present an immunoinformatics pipeline for the identification of miHAs. The pipeline can be applied to large-scale miHA screening, for example, in the development of diagnostic tests. Another interesting application is the design of personalized miHA-based cancer therapies based on patient-donor pair-specific miHAs detected by this pipeline. The suggested method covers various aspects of genetic variant detection, effects of alternative transcripts, and HLA-peptide binding. A comparison of our computational pipeline and experimentally derived datasets shows excellent agreement and coverage of the computationally predicted miHAs.
    Type of Publication: Journal article published
    PubMed ID: 22985828
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  • 4
    Abstract: Direct analysis of HLA-presented antigens by mass spectrometry provides a comprehensive view on the antigenic landscape of different tissues/malignancies and enables the identification of novel, pathophysiologically relevant T-cell epitopes. Here, we present a systematic and comparative study of the HLA class I and II presented, nonmutant antigenome of multiple myeloma (MM). Quantification of HLA surface expression revealed elevated HLA molecule counts on malignant plasma cells compared with normal B cells, excluding relevant HLA downregulation in MM. Analyzing the presentation of established myeloma-associated T-cell antigens on the HLA ligandome level, we found a substantial proportion of antigens to be only infrequently presented on primary myelomas or to display suboptimal degrees of myeloma specificity. However, unsupervised analysis of our extensive HLA ligand data set delineated a panel of 58 highly specific myeloma-associated antigens (including multiple myeloma SET domain containing protein) which are characterized by frequent and exclusive presentation on myeloma samples. Functional characterization of these target antigens revealed peptide-specific, preexisting CD8(+) T-cell responses exclusively in myeloma patients, which is indicative of pathophysiological relevance. Furthermore, in vitro priming experiments revealed that peptide-specific T-cell responses can be induced in response-naive myeloma patients. Together, our results serve to guide antigen selection for T-cell-based immunotherapy of MM.
    Type of Publication: Journal article published
    PubMed ID: 26138685
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  • 5
    Abstract: Recent studies suggest that multiple myeloma is an immunogenic disease, which might be effectively targeted by antigen-specific T-cell immunotherapy. As standard of care in myeloma includes proteasome inhibitor therapy, it is of great importance to characterize the effects of this treatment on HLA-restricted antigen presentation and implement only robustly presented targets for immunotherapeutic intervention. Here, we present a study that longitudinally and semi-quantitatively maps the effects of the proteasome inhibitor carfilzomib on HLA-restricted antigen presentation. The relative presentation levels of 4780 different HLA ligands were quantified in an in vitro model employing carfilzomib treatment of MM.1S and U266 myeloma cells, which revealed significant modulation of a substantial fraction of the HLA-presented peptidome. Strikingly, we detected selective down-modulation of HLA ligands with aromatic C-terminal anchor amino acids. This particularly manifested as a marked reduction in the presentation of HLA ligands through the HLA allotypes A*23:01 and A*24:02 on MM.1S cells. These findings implicate that carfilzomib mediates a direct, peptide motif-specific inhibitory effect on HLA ligand processing and presentation. As a substantial proportion of HLA allotypes present peptides with aromatic C-termini, our results may have broad implications for the implementation of antigen-specific treatment approaches in patients undergoing carfilzomib treatment.
    Type of Publication: Journal article published
    PubMed ID: 27058226
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  • 6
    Abstract: BACKGROUND & AIMS: We report a novel experimental immunotherapeutic approach in a patient with metastatic intrahepatic cholangiocarcinoma. In the 5year course of the disease, the initial tumor mass, two local recurrences and a lung metastasis were surgically removed. Lacking alternative treatment options, aiming at the induction of anti-tumor T cells responses, we initiated a personalized multi-peptide vaccination, based on in-depth analysis of tumor antigens (immunopeptidome) and sequencing. METHODS: Tumors were characterized by immunohistochemistry, next-generation sequencing and mass spectrometry of HLA ligands. RESULTS: Although several tumor-specific neo-epitopes were predicted in silico, none could be validated by mass spectrometry. Instead, a personalized multi-peptide vaccine containing non-mutated tumor-associated epitopes was designed and applied. Immunomonitoring showed vaccine-induced T cell responses to three out of seven peptides administered. The pulmonary metastasis resected after start of vaccination showed strong immune cell infiltration and perforin positivity, in contrast to the previous lesions. The patient remains clinically healthy, without any radiologically detectable tumors since March 2013 and the vaccination is continued. CONCLUSIONS: This remarkable clinical course encourages formal clinical studies on adjuvant personalized peptide vaccination in cholangiocarcinoma. LAY SUMMARY: Metastatic cholangiocarcinomas, cancers that originate from the liver bile ducts, have very limited treatment options and a fatal prognosis. We describe a novel therapeutic approach in such a patient using a personalized multi-peptide vaccine. This vaccine, developed based on the characterization of the patient's tumor, evoked detectable anti-tumor immune responses, associating with long-term tumor-free survival.
    Type of Publication: Journal article published
    PubMed ID: 27397612
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  • 7
  • 8
    Abstract: Hematological malignancies (HM) are highly amenable targets for immunotherapeutic intervention and may be effectively treated by antigen-specific T-cell based treatment. Recent studies demonstrate that physiologically occurring anti-cancer T-cell responses in certain HM entities target broadly presented non-mutated epitopes. HLA ligands are thus implied as prime targets for broadly applicable and antigen-specific off-the-shelf compounds. With the aim of assessing the presence of common targets shared among different HM which may enable addressing a larger patient collective we conducted a meta-analysis of 83 mass spectrometry-based HLA peptidome datasets (comprising 40,361 unique peptide identifications) across four major HM (19 AML, 16 CML, 35 CLL, and 13 MM/MCL samples) and investigated similarities and differences within the HLA presented antigenic landscape. We found the cancer HLA peptidome datasets to cluster specifically along entity and lineage lines, suggesting that the immunopeptidome directly reflects the differences in the underlying (tumor-)biology. In line with these findings, we only detected a small set of entity-spanning antigens, which were predominantly characterized by low presentation frequencies within the different patient cohorts. These findings suggest that design of T-cell immunotherapies for the treatment of HM should ideally be conducted in an entity-specific fashion.
    Type of Publication: Journal article published
    PubMed ID: 28159928
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  • 9
    Abstract: Immunotherapies, particularly checkpoint inhibitors, have set off a revolution in cancer therapy by releasing the power of the immune system. However, only little is known about the antigens that are essentially presented on cancer cells, capable of exposing them to immune cells. Large-scale HLA ligandome analysis has enabled us to exhaustively characterize the immunopeptidomic landscape of epithelial ovarian cancers (EOCs). Additional comparative profiling with the immunopeptidome of a variety of benign sources has unveiled a multitude of ovarian cancer antigens (MUC16, MSLN, LGALS1, IDO1, KLK10) to be presented by HLA class I and class II molecules exclusively on ovarian cancer cells. Most strikingly, ligands derived from mucin 16 and mesothelin, a molecular axis of prognostic importance in EOC, are prominent in a majority of patients. Differential gene-expression analysis has allowed us to confirm the relevance of these targets for EOC and further provided important insights into the relationship between gene transcript levels and HLA ligand presentation.
    Type of Publication: Journal article published
    PubMed ID: 29093164
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  • 10
    Abstract: BACKGROUND: Variability in drug efficacy and adverse effects are observed in clinical practice. While the extent of genetic variability in classic pharmacokinetic genes is rather well understood, the role of genetic variation in drug targets is typically less studied. METHODS: Based on 60,706 human exomes from the ExAC dataset, we performed an in-depth computational analysis of the prevalence of functional variants in 806 drug-related genes, including 628 known drug targets. We further computed the likelihood of 1236 FDA-approved drugs to be affected by functional variants in their targets in the whole ExAC population as well as different geographic sub-populations. RESULTS: We find that most genetic variants in drug-related genes are very rare (f 〈 0.1%) and thus will likely not be observed in clinical trials. Furthermore, we show that patient risk varies for many drugs and with respect to geographic ancestry. A focused analysis of oncological drug targets indicates that the probability of a patient carrying germline variants in oncological drug targets is, at 44%, high enough to suggest that not only somatic alterations but also germline variants carried over into the tumor genome could affect the response to antineoplastic agents. CONCLUSIONS: This study indicates that even though many variants are very rare and thus likely not observed in clinical trials, four in five patients are likely to carry a variant with possibly functional effects in a target for commonly prescribed drugs. Such variants could potentially alter drug efficacy.
    Type of Publication: Journal article published
    PubMed ID: 29273096
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