Abstract A016: Functional characterization of the 1p36.33 pancreatic cancer GWAS locus
Authors: Connelly Katelyn E , Hullin Katherine , Abdolalizadeh Ehssan , Zhong Jun , Eiser Daina , Hoskins Jason W. , O'Brien Aidan , Collins Irene , Chanock Stephen J , Stolzenberg-Solomon Rachael Z , Klein Alison P , Wolpin Brian M , Smith Jill P , Amundadottir Laufey 
Published: September 2024

Abstract
Common low effect size germline variants are known to contribute to pancreatic ductal adenocarcinoma (PDAC) susceptibility. Genome-wide association studies (GWAS) for PDAC have identified over 20 loci where common germline variants influence PDAC risk. Understanding the underlying mechanisms of risk could improve detection, prevention, and potentially treatment strategies for this deadly disease. We recently performed a meta-analysis of PanScan I-III, PanC4, and UK Biobank GWAS and identified 1p36.33 (rs13303010, OR=1.24, 95% CI = 1.16-1.32, P=2.09x10-10) as a significant PDAC risk locus. Using fine-mapping strategies, we identified seven candidate functional variants for further follow-up. Electrophoretic mobility shift assays (EMSA) and luciferase reporter assays identified two variants (rs13303327 and rs13303160) with allele-specific protein binding and gene regulatory activity, respectively. In-silico transcription factor (TF) binding analysis indicated that the risk alleles for rs13303327 and rs13303160 disrupt ELF and AP1 TF motifs, respectively. We confirmed preferential binding of ELF2 to the rs13303327 protective allele in vitro but did not observe an allelic preference in cell lines using chromatin immunoprecipitation (ChIP)-qPCR. For rs13303160, JunB and JunD preferentially bound the protective allele in vitro and in vivo, implicating this polymorphism as a functional variant underlying risk at 1p36.33. The GWAS signal strongly colocalized (Posterior Probability=0.99) with a significant KLHL17 expression quantitatitve trait locus (eQTL) signal in the GTEx pancreas (P=2.3x10-10) where the risk allele is associated with lower KLHL17 expression. Additionally, the eQTL effect direction corresponded with our in vitro regulatory results. The KLHL family of proteins, which have been implicated in cancer, are substrate recognition proteins for the Cullin3 E3 ubiquitin ligase complex and are critical for maintaining cellular protein homeostasis. We confirmed through proteomics that KLHL17 is associated with Cullin3 complex members in the PDAC-derived PANC-1 cell line. In PDAC-derived cell lines, knockdown/overexpression of KLHL17 did not exhibit a growth phenotype. Thus, we performed an in silico differential gene expression analysis between the top and bottom quartiles of KLHL17 expression in GTEx Pancreas and TCGA PAAD samples. Differentially expressed genes in both datasets were enriched for inflammation-related pathways, suggesting lower KLHL17 is associated with a pro-inflammatory environment. Persistent inflammation in the pancreas contributes to tumorigenesis. We hypothesize that KLHL17 may mitigate inflammation by recruiting pro-inflammatory proteins for ubiquitination and degradation. As such, lower expression of KLHL17, which associates with PDAC risk, may therefore result in decreased degradation of key inflammatory proteins and unresolved inflammation, a precursor in PDAC pathogenesis.
Citation Format: Katelyn E Connelly, Katherine Hullin, Ehssan Abdolalizadeh, Jun Zhong, Daina Eiser, Jason W. Hoskins, Aidan O'Brien, Irene Collins, Stephen J Chanock, Rachael Z Stolzenberg-Solomon, Alison P Klein, Brian M Wolpin, Jill P Smith, Laufey Amundadottir. Functional characterization of the 1p36.33 pancreatic cancer GWAS locus [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 A016.