Abstract 394: Elucidating tissue-specific effects of the 8q24 multicancer risk locus via CRISPR/Cas9 scarless genome editing

Abstract

Abstract In the past decade, genome-wide association studies have identified a large number of genomic variants, specifically single-nucleotide polymorphisms (SNPs), associated with increased cancer risk. The molecular mechanisms underlying the vast majority of these associations remain largely unknown. Precise cellular models containing the variant of interest could be a crucial tool for establishing disease-relevant function in order to translate these discoveries into clinical application. CRISPR/Cas9 gene editing has provided a convenient and flexible method for creating isogenic cell lines via the homology-directed repair (HDR) pathway; however, methods for efficient generation and subsequent isolation of precisely edited cells have proven both expensive and time consuming. In response to this need, we have combined CRISPR/Cas9 HDR gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) technology to create scarless isogenic cell models of cancer risk variants in ~1 month without selectable markers or specialized methods such as digital droplet PCR or NGS. Utilizing this technology, we have been able to create cell lines differing by only a single base to model risk-associated SNPs located in the 8q24 risk locus in multiple cell types. The 8q24 risk locus is associated with increased risk for colorectal, breast, prostate, and, more recently, thyroid cancer. In this study, we use engineered isogenic cell lines modeling functional risk SNP rs6983267 in each of these cancer types to identify tissue-specific mechanisms of this multicancer risk locus. Rs6983267 is located within a known c-Myc enhancer and has been shown to exert risk-allele specific increase in enhancer activity. Oncogenic c-Myc is known to play a significant role in the pathogenesis of many cancers. However, how or if this mechanism varies between the different cancers this locus has been associated with remains uncertain. From preliminary studies, we do observe cell type variation in the regulatory effect of rs6983267. Not only does there seem to be a varying effect of the risk allele on c-Myc expression across the different cell types, but also risk allele enhancement of binding of the TCF7L2 transcription factor to the c-Myc enhancer rs6983267 is located within. Interestingly, rs6983267's regulatory effect seems most significant between homozygous reference cells and heterozygous cells within the same tissue type. We plan to delve further into these differences using RNA-seq and 4C-seq to assess whole transcriptomic and chromosomal interaction changes with changes in risk status. This study is one of first to compare risk mechanisms across different cancer tissue types; using precisely edited cellular models, we are able to detect cell-type specific variations in functional risk SNP effects on cancer development. Citation Format: Nicole B. Coggins, Henriette O'Geen, David J. Segal, Luis G. Carvajal-Carmona. Elucidating tissue-specific effects of the 8q24 multicancer risk locus via CRISPR/Cas9 scarless genome editing [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 394.