Targeted resequencing of cis-regulatory elements in human leukemia

Mutations in protein-coding genes are well established as the basis for human cancer, yet it remains elusive how alterations within non-coding genome, a substantial fraction of which contain cis-regulatory elements, contribute to cancer pathophysiology largely due to lack of high throughput assays to assess their functional effects. Here we developed an integrative approach to systematically identify and characterize non-coding regulatory variants in human hematopoietic malignancies by combining targeted resequencing, mutation discovery, CRISPR-based enhancer-selective epigenome editing, and enhancer reporter assays. We identify 4,629 recurrent non-coding alterations and 939 mutation-associated pathogenic enhancers controlling proto-oncogenes or tumor suppressors. Enhancer variants at KRAS and PER2 co-localize with nuclear receptor (NR) binding sites and modulate transcriptional activities in response to NR signaling in leukemia cells. NR binding sites frequently associate with non-coding variants across cancer types. Hence, recurrent non-coding somatic variants connect enhancer dysregulation with nuclear receptor signaling in hematopoietic malignancies. Overall design: Targeted resequencing of enhancer elements was performed using Ovation® Target Enrichment System (NuGEN) following the manufacture's protocols. Target enriched library were purified, quantified, and sequenced on an Illumina NextSeq500 system using the 150bp high output sequencing kit