The General Hospital Corporation

Massachusetts Institute of Technology

The Broad Institute, Inc.

Harvard College

Dana-farber-cancer Institute Inc.

The Brigham and Women's Hospital, Inc.

Seqll LLC

Tarjan, Daniel

Bernstein, Bradley

Drier, Yotam

Flavahan, William

Bradley Bernstein

Assaf Rotem

Alon Goren

Oren Ram

Volker Hovestadt

Peter Van Galen

David A Weitz

James Elliott Bradner

Aviv Regev

J Christopher Love

Todd Michael Gierahn

Alexander K Shalek

Jon C Aster

Tarjei Mikkelsen

Travis K Hughes

David Root

Marc H Wadsworth, Ii

Jae Keith Joung

Itay Tirosh

Christopher J Ott

Deepak Reyon

Mario Suva

Eric M Mendenhall

Birgit Knoechel

Anoop P Patel

William Flavahan

Cem Sievers

Mariella Filbin

Brian B Liau

Mazhar Adli

Efrat Shema-Yaacoby

Daniel Tarjan

Daniel Jones

Ang A Tu

Yotam Drier

Charles B Epstein

The subject matter disclosed herein is generally directed to compositions and methods for treating diffuse gliomas with histone H3 lysine27-to-methionine mutations (H3K27M-gliomas). Disclosed herein are gene signatures specific for tumor cell types and compositions for treatment of H3K27M gliomas. In one embodiment, PRC1 is targeted in a treatment regimen for H3K27M-gliomas.

Methods and compositions for targeting developmental and oncogenic programs in H3K27M gliomas

The present invention provides compositions and methods for the treatment of drug-tolerant glioblastoma, and in particular glioblastoma stem cells. Specifically, the methods comprise contacting a glioblastoma stem cell with a platelet-derived growth factor receptor alpha inhibitor and one or more of a histone lysine demethylase inhibitor and a Notch inhibitor.

Compositions and methods for treating drug-tolerant glioblastoma

Fusion proteins comprising a DNA binding domain, e.g., a TAL effector repeat array (TALE) or zinc finger array, and a catalytic domain comprising a sequence that catalyzes histone demethylation, and methods of use thereof.

Transcription activator-like effector (tale) - lysine-specific demethylase 1 (LSD1) fusion proteins

The present invention relates to methods of detecting region(s) of interest in a gene comprising a polyA tail. The region(s) of interest can include gene(s), region(s), mutation(s), deletion(s), insertion(s), indel(s), and/or translocation(s). The region(s) can be greater than or less than 1 kilobases from the polyA tail. Methods can include forming a library of single cell transcripts comprising the region(s) in close proximity to a cell barcode and a unique molecular identifier (UMI). Methods for distinguishing cells by genotype can include amplifying the transcripts using PCR methods and detecting the cell barcode and UMI using single cell sequencing methods. Transcripts can be enriched using tagged region-specific PCR primers. Cell barcodes can be brought into close proximity to the region(s) by circularizing the transcripts. Sequencing of the transcripts can include using primer binding sites added during PCR amplification and library indexes for multiplexed sequencing.

Single cell sequencing libraries of genomic transcript regions of interest in proximity to barcodes, and genotyping of said libraries

The present invention provides for single-molecule profiling of combinatorial protein modifications and single-molecule profiling of combinatorial protein modifications combined with single-molecule sequencing of protein/nucleic acids complexes. High-throughput single-molecule imaging was applied to decode combinatorial modifications on millions of individual nucleosomes from pluripotent stem cells and lineage-committed cells. Applicants identified bivalent nucleosomes with concomitant repressive and activating marks, as well as other combinatorial modification states whose prevalence varies with developmental potency. Applying genetic and chemical perturbations of chromatin enzymes show a preferential affect on nucleosomes harboring specific modification states. The present invention also combines this proteomic platform with single-molecule DNA sequencing technology to simultaneously determine the modification states and genomic positions of individual nucleosomes. This novel single-molecule technology can be used to address fundamental questions in chromatin biology and epigenetic regulation leading to novel therapeutics and diagnostics.

Combinatorial single molecule analysis of chromatin

The present application generally to the diagnosis and treatment of diseases resulting from the alteration of chromatin boundaries between topologically-associated domains. In particular, the present application relates to detection of mutations causing DNA hypermethylation phenotypes, CpG methylation within CTCF binding motifs, and aberrant gene expression caused by altered chromatin topology. Applicants show that IDH mutant gliomas exhibit hyper-methylation at CTCF binding sites, compromising binding of this methylation-sensitive insulator protein. Applicants also demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to aberrantly interact with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Thus, Applicants have uncovered that IDH mutations may promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.

Methods of detecting insulator dysfunction and oncogene activation for screening, diagnosis and treatment of patients in need thereof

The present invention generally relates to microfluidics and/or epigenetic sequencing. In one set of embodiments, cells contained within a plurality of microfluidic droplets are lysed and the DNA (e.g., from nucleosomes) within the droplets are labeled, e.g., with adapters containing an identification sequence. The adapters may also contain other sequences, e.g., restriction sites, primer sites, etc., to assist with later analysis. After labeling with adapters, the DNA from the different cells may be combined and analyzed, e.g., to determine epigenetic information about the cells. For example, the DNA may be separated on the basis of certain modifications (e.g., methylation), and the DNA from the separated nucleosomes may be sequenced using techniques such as chromatin immunoprecipitation ('ChIP'). In some cases, the DNA sequences may also be aligned with genomes, e.g., to determine which portions of the genome were epigenetically modified, e.g., via methylation.

Systems and methods for epigenetic sequencing

Embodiments disclosed herein provide methods for identifying cell-type-specific nucleosomal DNA modifications. The methods leverage nucleosomal DNA barcoding and pool-and-split multiplexing to provide high-throughout, quantitative profiling of nucleosomal DNA states. The methods enable the profiling of multiple nucleosomal DNA marks across different cell types and/or conditions thereby linking quantitative changes in chromatin landscapes to different genotypes and chemical and physical perturbations.

Nucleic acid constructs and methods for labeling and detecting nucleosomal DNA modifications

The invention relates to methods of treatment and diagnosis of subjects with cancer. In some aspects, the invention relates to methods of treatment and diagnosis of subjects with cancer, wherein the cancer is characterized by a Notch pathway activation mutation or by resistance to a Notch pathway inhibitor.

Diagnostic and treatment methods in subjects having or at risk of developing resistance to cancer therapy

Control agents for immunoprecipitation assays, methods of using the control agents and kits comprising the control agents are provided.

Agents providing controls and standards for immunoprecipitation assays

Control agents for immuno-precipitation assays, methods of using the control agents and kits comprising the control agents are provided.

Agents providing controls and standards for immuno-precipitation assays

Improvements in chromatin immunoprecipitation-high throughput sequencing techniques has allowed the creation of chromatin maps from limited biological sample sizes that cannot be evaluated using conventional chromatin immunoprecipitation-sequencing protocols. For example, a modified universal primer is utilized that incorporates restriction enzymes into chromatin immunoprecipitation fragments before amplification. The improved method allows the sample sizes to be several orders of magnitude less than that required for standard ChIP-Seq techniques.

Methods for preparing sequencing libraries

Growing community of inventors

Cambridge, MA, United States of America

Bradley Bernstein