Toward a functional characterization of polygenic modifiers of BRCA2 associated prostate cancer risk

Introduction: Male carriers of BRCA2 pathogenic variants are at elevated risk of prostate cancer (PC), with wide variation in disease frequency observed (88% vs. 34%) for men in the top versus bottom 5th percentile of a 147-SNP prostate cancer polygenic risk score (PRS) (PMID: 34320204). Causal variants and genes at most of these PRS loci remain uncharacterized, limiting our understanding of the molecular mediators and biological mechanisms underlying putative modifiers of BRCA2 risk. Methods: We adopted our published informatics pipeline to prioritize candidate functional variants at PC risk loci using Functional Potential Scores (FPS), assembled from disease-relevant annotations of chromatin accessibility, histone marks, and transcriptional factor (TF) binding. We identified 74 loci with a high-scoring lead SNP or strongly correlated variant ( r2 >0.80), typically mapping to a predicted enhancer region, and selected several such loci for experimental interrogation: 10q25, 11q13, 12q14, 19p13. Genomic fragments spanning prioritized SNPs were cloned in forward and reverse orientations upstream of a minimal promoter driving Nanoluc luciferase (pNL3.1), and luciferase reporter assays were conducted to evaluate allele-specific enhancer activity in normal prostate (RWPE-1) and PC cell lines (LnCAP, VCAP and 22PC). CRISPR genome editing studies are underway to assess the impact of enhancers and risk alleles on regional gene expression profiles. Results: Three of the four candidate enhancer fragments tested exhibited enhancer activity in at least two cell lines -- 10q25/SNP5, 11q13/SNP10, 19p13/SNP17. Among these, the enhancer at 10q25 exhibited allele-specific activity, with up to ~two-fold stronger signal observed for allele C versus T in both normal and cancer cell lines. Initial studies also suggested allelic specificity for enhancers at 11q13 (SNP10 G>T) and 19p13 (SNP17 G>A). At 10q25, the functional variant identified maps to the third intron of TCF7L2 , a critical gene in the Wnt signaling pathway, in a region bound by several key TFs in prostate including androgen receptor and FOXA1. Functional studies of validated target genes are being conducted with and without genotoxic stressors using human organoid cultures we have established from prostate biopsies of men with BRCA2 mutations. Conclusions: Our findings support the utility of the FPS informatics framework for prioritizing likely functional/causal variants at inherited cancer susceptibility loci and provide a foundation for downstream functional assays using physiologic model systems to investigate causal mechanisms of genetic modifiers of BRCA2 -associated prostate cancer risk. (Supported by the Breast Cancer Research Foundation, CureBRCA, the MSK Niehaus Center and the Sabin Foundation)