RESUMO
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.
Assuntos
Carcinogênese/genética , Aberrações Cromossômicas , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Oncogenes/genética , Succinato Desidrogenase/deficiência , Animais , Sistemas CRISPR-Cas/genética , Metilação de DNA , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Fator 4 de Crescimento de Fibroblastos/genética , Tumores do Estroma Gastrointestinal/enzimologia , Humanos , Camundongos , Mutação , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Succinato Desidrogenase/genéticaRESUMO
The human genome is folded into regulatory units termed 'topologically-associated domains' (TADs). Genome-wide studies support a global role for the insulator protein CTCF in mediating chromosomal looping and the topological constraint of TAD boundaries. However, the impact of individual insulators on enhancer-gene interactions and transcription remains poorly understood. Here, we investigate epigenome editing strategies for perturbing individual CTCF insulators and evaluating consequent effects on genome topology and transcription. We show that fusions of catalytically-inactive Cas9 (dCas9) to transcriptional repressors (dCas9-KRAB) and DNA methyltransferases (dCas9-DNMT3A, dCas9-DNMT3A3L) can selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif. We further demonstrate that stable, partially-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing. Finally, we apply these strategies to simulate an insulator loss mechanism implicated in brain tumorigenesis. Our study provides strategies for stably modifying genome organization and gene activity without altering the underlying DNA sequence.
Assuntos
Fator de Ligação a CCCTC/genética , Proteína 9 Associada à CRISPR/genética , Carcinogênese/genética , DNA (Citosina-5-)-Metiltransferases/genética , Edição de Genes/métodos , Proteínas Recombinantes de Fusão/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Sistemas CRISPR-Cas/genética , Carcinogênese/patologia , Linhagem Celular , Metilação de DNA , DNA Metiltransferase 3A , Epigênese Genética/genética , Genoma Humano/genética , Células HEK293 , Humanos , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/metabolismoRESUMO
Gain-of-function Notch mutations are recurrent in mature small B cell lymphomas such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), but the Notch target genes that contribute to B cell oncogenesis are largely unknown. We performed integrative analysis of Notch-regulated transcripts, genomic binding of Notch transcription complexes, and genome conformation data to identify direct Notch target genes in MCL cell lines. This B cell Notch regulome is largely controlled through Notch-bound distal enhancers and includes genes involved in B cell receptor and cytokine signaling and the oncogene MYC, which sustains proliferation of Notch-dependent MCL cell lines via a Notch-regulated lineage-restricted enhancer complex. Expression of direct Notch target genes is associated with Notch activity in an MCL xenograft model and in CLL lymph node biopsies. Our findings provide key insights into the role of Notch in MCL and other B cell malignancies and have important implications for therapeutic targeting of Notch-dependent oncogenic pathways.