Abstract 6154: Targeted CRISPRi screen identifies functional variants and novel target genes at multiple renal cell carcinoma (RCC) susceptibility loci

Abstract Genome-wide association studies (GWAS) have identified 13 renal cell carcinoma (RCC) risk regions, as well as 7 regions with nominal significance, but detailed investigation of how these regions function is required to reveal the underlying biological bases of disease susceptibility. While detailed study of four loci have implicated critical pathways in RCC, causal genes and pathways for most loci remain unidentified. To identify such causative variants and target genes, we evaluated a set of variants based on Massively Parallel Reporter Assays (MPRA), eQTL analysis, capture Hi-C, and an arrayed CRISPR inhibition (CRISPRi) screen. The MPRA identified 196 variants across 19 regions with significant allele-specific effects, indicating cis-regulatory activity. Of these, 39 variants across 10 RCC loci displayed chromatin loops allowing physical interaction with the promoter of 24 nearby putative gene targets, as well as a significant cis-eQTL with the same gene. To confirm the presence of a cis-regulatory relationship between these 39 candidate variants and 24 putative target genes, we performed an arrayed CRISPRi screen in ACHN (RCC) and HEK293T (embryonic renal) cells covering each of the 10 nominated regions. Cells were stably transduced with inactive Cas9 fused to the ZIM3 transcriptional repressor domain and cis-regulatory relationships were assessed by TaqMan qPCR for the target genes. The screen identified multiple novel relevant target genes of RCC predisposition variants, including UCK2 at 1q24, ABL2 and TDRD5 at 1q25, GTDC1 at 2q22, GPR37 at 7q31, AGBL3 and CALD1 at 7q33, and MAP2K1 at 15q22. At some regions, evidence indicates cis-regulatory relationships occur between a target gene and all variants predicted to be ‘causative’ at the region (e.g., UCK2 at 1q24 and ABL2 at 1q25). For other regions, cis-regulatory relationships occur between a gene and only one variant tested (e.g., GPR37 at 7q31 and TDRD5 at 1q25).Previously, target genes of only four RCC susceptibility regions had been identified and published. This study identifies eight novel target genes across six additional RCC susceptibility regions, representing a significant advance in understanding of the underlying biology of RCC susceptibility variants. Furthermore, ongoing screening using the orthologous method, CRISPR activation (CRISPRa), and integration of both normoxic and hypoxic conditions continues to provide further insight into the underlying biology at these regions. Citation Format: Timothy Winter, Timothy Myers, Leandro Colli, Lea Jessop, Jiyeon Choi, Mitchell J. Machiela, Mark P. Purdue, Kevin Brown, Stephen Chanock. Targeted CRISPRi screen identifies functional variants and novel target genes at multiple renal cell carcinoma (RCC) susceptibility loci [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6154.