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Epigenetic aberrations are widespread in cancer, yet the underlying mechanisms and causality remain poorly understood1–3. A subset of gastrointestinal stromal tumours (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 between enhancer and oncogene, and strongly upregulated 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 tumour, including hypermethylation and insulator defects. This PDX model is highly sensitive to FGF receptor (FGFR) inhibition, 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. Gastrointestinal stromal tumours can be initiated by gain-of-function mutations of the KIT or PDGFRA oncogenes but also by loss of the metabolic complex succinate dehydrogenase (SDH), which leads to DNA hypermethylation; this study shows that in SDH-deficient tumours, displacement of CTCF insulators by DNA methylation activates oncogene expression, illustrating how epigenetic alterations can drive oncogenic signalling in the absence of kinase mutations.

Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.

Metabolic lesions with profound effects on epigenetic regulation are widely implicated in cancer, yet the mechanistic links between this epigenetic dysregulation and tumorigenesis remain unclear. Succinate dehydrogenase (SDH) deficiency, responsible for a subset of gastrointestinal stromal tumors (GISTs), causes accumulation of the metabolite succinate and DNA hypermethylation. We identified convergent mechanisms involving altered chromosomal conformation and pseudo-hypoxia that mediate the tumorigenic effects of SDH deficiency in GIST. To investigate epigenetic alterations in this disease, we created epigenetic maps of 14 clinical GIST specimens; including KIT and PDGFRA mutant, and SDH-deficient tumors. We characterized the landscapes of enhancers, genetic regulatory elements which can drive gene expression, through histone H3 lysine 27 acetylation chromatin immunoprecipitation sequencing (ChIP-seq). We characterized both the DNA methylation and CTCF occupancy of insulators, elements which help control chromatin conformation and restrict enhancer-gene interactions, through hybrid selection bisulfite sequencing and CTCF ChIP-seq, respectively. Analyzing these data, we uncovered thousands of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. One of the strongest disrupted insulators protected the receptor tyrosine kinase and known driver of GIST, c-KIT, from a nearby superenhancer. Chromatin conformation studies confirmed an SDH-deficient-specific interaction of this superenhancer with the KIT gene. CRISPR-mediated excision of the insulator in an SDH-intact GIST model resulted in enhancer interaction and KIT upregulation. Immunohistochemical studies confirm strong expression of c-KIT in SDH-deficient GIST clinical samples. SDH deficiency has also been reported to cause pseudohypoxia in tumors. We confirmed that the enhancer landscape of SDH-deficient tumors had a signature of pseudohypoxia. Additionally, following pseudohypoxia induction in a SDH-intact GIST model, the c-KIT ligand Stem Cell Factor (SCF/KITLG) was upregulated 12-fold. While activating KIT mutations drive the majority (~75%) of GIST tumors and are mutually exclusive with SDH deficiency, we show that a primary consequence of SDH loss is in fact induction of KIT signaling. Our findings demonstrate how metabolic lesions can provide alternate epigenetic mechanisms to activate classic tumorigenic pathways in the absence of canonical genetic mutations. Citation Format: William A. Flavahan, Yotam Drier, Sarah E. Johnstone, Daniel R. Tarjan, Esmat Hegazi, Ewa T. Sicinska, Matthew L. Hemming, Chandrajit P. Raut, Jason L. Hornick, George D. Demetri, Bradley E. Bernstein. Insulator dysfunction and epigenetic oncogene activation in SDH-deficient gastrointestinal stromal tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2996.