Patel, Minal

Abstract Chromosome 5p15.33 harbors several independent association signals which demonstrate antagonistic pleiotropy across cancer types, with causal mechanisms largely unresolved. To identify functional variants and enhancer elements at this locus, we performed statistical fine-mapping followed by massively parallel reporter assays (MPRA) and proliferation based CRISPRi screens. This approach identified eight multi-cancer functional variants (MCFVs) across three GWAS signals. Targeting rs421629 (part of the CLPTM1L signal marked by rs465498) with CRISPRi revealed opposing effects on TERT expression in pancreatic versus lung cancer cells, consistent with the antagonistic pleiotropy observed for this signal. Furthermore, CRISPRi nominated an intronic CLPTM1L variable number tandem repeat (VNTR) as a potent enhancer. Long-read sequencing established VNTR polymorphisms as potential causal variants for the rs465498 signal. We showed that Hippo-pathway transcription factors mediate VNTR enhancer activity in lung and pancreatic cancer cells. Together, these findings indicate that cancer susceptibility at 5p15.33 may be mediated by both SNPs and VNTRs and provide an integrated framework for resolving complex pleiotropic loci.

Abstract Background: Genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs) to pancreatic ductal adenocarcinoma (PDAC) risk at over 20 genomic loci. The Pancreatic Cancer Cohort Consortium and Pancreatic Cancer Case-Control Consortium are currently expanding on previous GWAS studies for PDAC from ∼9,000 cases and ∼12,000 controls to ∼36,000 cases and ∼800,000 controls in individuals of European and Asian ancestry. This ongoing GWAS phase is estimated to detect up to 50 new risk signals. Post-GWAS studies aim to fine-map and functionally assess risk variants in disease-relevant cell types and contexts to identify target genes and underlying mechanisms of risk. Massively parallel reporter assays (MPRAs) are a powerful method of assessing allele-specific gene regulatory effects. Integrating sequences into the genome using lentiviruses (Lenti-MPRA) enables the capture of variant function in the context of the native cellular chromatin. An added benefit is that Lenti-MPRAs can be used for difficult to transfect cells such as primary cells, that often reflect the disease relevant cell types. Methods: We have used a lentiviral MPRA to test 228 fine-mapped SNPs at the chr5p15.33 multi-cancer risk locus in PDAC cell lines (MIA PaCa-2, PANC-1) and the normal-derived ductal pancreas (HPDE) cell line. Additionally, we have optimized the transduction of large MPRA libraries into primary pancreas cells. Tunicamycin was used to induce ER stress in cell lines and primary cells, assessing downstream gene expression using RT-qPCR. Results: Our pilot MPRA demonstrated high reproducibility in PDAC cell lines (r2=0.93-0.97, MIA PaCa-2, PANC-1 cells) and normal-derived pancreatic ductal cells (r2=0.68-0.86, HPDE cells). We identified 33 SNPs out of 228 with significant allele-specific transcriptional activity in PDAC cell lines and 5 in HPDE cells (FDR ≤0.05). An additional 6-15% of variants exhibited significant allele-specific transcriptional activity when in the frames of the SNP-centered tested oligos were shifted left or right. We demonstrate the inducibility of ER-stress with tunicamycin treatment over 24 hours in cells lines and primary pancreatic cells. Additionally, we observed increased expression of CLPTM1IL at the chr5p15.33 locus after tunicamycin treatment over 24 hours in cell lines and primary pancreatic cells, suggesting ER-stress and inflammation may influence gene expression at this locus. Conclusions: Lentiviral MPRA assays in commonly used PDAC cell lines have demonstrated good reproducibility. The optimization studies and preliminary work in primary pancreatic cells provide a solid foundation for the next steps of assessing all GWAS loci in ER stress, an important disease relevant condition. Citation Format: Minal B Patel, Aidan O'Brien, Jun Zhong, Irene Collins, Jason W Hoskins, Stephen J Chanock, Samuel O Antwi, Rachel Z Stolzenberg-Solomon, Alison P Klein, Brian M Wolpin, Sara Lindström, H. Efsun Arda, Kevin M Brown, Katelyn E Connelly, Laufey T Amundadottir, Meagan Jezek. The functional characterization of pancreatic ductal adenocarcinoma GWAS risk variants in primary pancreatic cells – A pilot study [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A018.