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Shareef, Sarah

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Shareef

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Sarah

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Shareef, Sarah
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    Altered Chromosomal Topology Drives Oncogenic Programs in SDH-Deficient GISTs
    (Springer Science and Business Media LLC, 2019-10-16) Flavahan, William A.; Drier, Yotam; Johnstone, Sarah E.; Hemming, Matthew; Tarjan, Daniel R.; Hegazi, Esmat; Shareef, Sarah; Javed, Nauman; Eschle, Benjamin K.; Gokhale, Prafulla C.; Hornick, Jason; Sicinska, Ewa; Demetri, George; Bernstein, Bradley
    Epigenetic aberrations are widespread in cancer, yet the underlying mechanisms and causality remain poorly understood1-3. A subset of gastrointestinal stromal tumors (GISTs) lack canonical kinase mutations but instead have succinate dehydrogenase (SDH)-deficiency and global DNA hyper-methylation4,5. Here we associate this hyper-methylation with changes in genome topology that activate oncogenic programs. To investigate epigenetic alterations systematically, we mapped DNA methylation, CTCF insulators, enhancers, and chromosome topology in KIT-mutant, PDGFRA-mutant, and SDH-deficient GISTs. Although these respective subtypes shared similar enhancer landscapes, we identified hundreds of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. We focused on a disrupted insulator that normally partitions a core GIST super-enhancer from the FGF4 oncogene. Recurrent loss of this insulator alters locus topology in SDH-deficient GISTs, allowing aberrant physical interaction between enhancer and oncogene. CRISPR-mediated excision of the corresponding CTCF motifs in an SDH-intact GIST model disrupted the boundary and strongly up-regulated FGF4 expression. We also identified a second recurrent insulator loss event near the KIT oncogene, which is also highly expressed across SDH-deficient GISTs. Finally, we established a patient-derived xenograft (PDX) from an SDH-deficient GIST that faithfully maintains the epigenetics of the parental tumor, including hyper-methylation and insulator defects. This PDX model is highly sensitive to FGF receptor (FGFR) inhibitor, and more so to combined FGFR and KIT inhibition, validating the functional significance of the underlying epigenetic lesions. Our study reveals how epigenetic alterations can drive oncogenic programs in the absence of canonical kinase mutations, with implications for mechanistic targeting of aberrant pathways in cancers.
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    Extended-representation bisulfite sequencing of gene regulatory elements in multiplexed samples and single cells
    (Springer Science and Business Media LLC, 2021-05-06) Shareef, Sarah; Bevill, Samantha; Raman, Ayush; Aryee, Martin; van Galen, Peter; Hovestadt, Volker; Bernstein, Bradley
    The biological roles of DNA methylation have been elucidated by profiling methods based on whole-genome or reduced-representation bisulfite sequencing, but these approaches do not efficiently survey the vast numbers of noncoding regulatory elements in mammalian genomes. Here we present a novel extended representation bisulfite sequencing (XRBS) method for targeted profiling of DNA methylation. Our design strikes a balance between expanding coverage of regulatory elements and reproducibly enriching informative CpG dinucleotides in promoters, enhancers, and CTCF binding sites. Barcoded DNA fragments are pooled prior to bisulfite conversion, allowing multiplex processing and technical consistency in low input samples. Application of XRBS to single leukemia cells enabled us to evaluate genetic copy-number variations and methylation variability across individual cells. Our analysis highlights heterochromatic H3K9me3 regions as having the highest cell-to-cell variability in their methylation, likely reflecting inherent epigenetic instability of these late replicating regions, compounded by differences in cell cycle stages among sampled cells.