Abstract 4441: Functional and clinical implications of an INDEL within the HuR regulatory region of the mitotic kinase inhibitor WEE1

Abstract

Abstract The RNA-binding protein Human Antigen R (HuR) is upregulated in pancreatic ductal adenocarcinoma (PDA), where it promotes tumorigenesis via its mRNA pro-survival targets. PDA cells exposed to DNA damage upregulate the mitotic inhibitor kinase, WEE1, in a HuR-dependent manner to induce cell cycle arrest and facilitate drug-resistance (1). Herein, we further evaluate a 56 base-pair (bp) region within WEE1's 3'UTR (labeled WEE1.3UTR) where HuR binds and stabilizes expression. Within this regulatory site, we observed that a 10-thymidine (T) track contains frequent polymorphisms (mean allele frequency 8.67%) of thymidine insertions (i.e, an INDEL). Using a combined approach of Sanger sequencing and a more sensitive capillary-electrophoresis (CE) assay, we screened this region in various cancer cell lines and patient samples. Results revealed three distinct alleles between individual cohorts: the wild-type (10-T, 56 bps), a 1-T insertion (11-T, 57 bps), and a 2-T insertion (12-T, 58 bps). Luciferase reporter constructs were subcloned with the HuR regulatory region embedded in the WEE1.3'UTR. In response to stress, constructs with the wild-type allele reported a higher level of expression compared to the 11-T and 12-T alleles (p<0.01). Complementary RNA-binding protein immunoprecipitation (RNP-IP) assays validated the enhanced binding of HuR to the wild-type construct as compared to the others. Collectively, these data suggest that HuR's regulation of WEE1 is impaired when 11-Ts or 12-Ts are present. Electrophoretic mobility shift assay (EMSA) experiments will investigate HuR's physical interaction by quantifying the relative affinity of the protein to each variant transcript, by itself and in direct competition with each other. To investigate the clinical implication of these findings, we sequenced (via Sanger and CE) a cohort of resected patient tumor samples (n=99), and found a significant enrichment for individuals homozygous for 12-T among those with a unique, Lynch-like familial history of cancer (odds ratio 2.4-6.9, p<0.05). We postulate that the addition of the INDEL in the WEE1.3UTR disrupts the association of HuR, and therefore, the functional upregulation of the WEE1 transcript in response to the stressful PDA microenvironment. Thus, a dysregulated G2/M checkpoint could result in accumulated DNA-damage and eventually promote PDA tumorigenesis. Paradoxically, the disruption of the HuR-WEE1 axis may render PDA cells more sensitive to genotoxic agents, thus providing a potential therapeutic window for patients screened for the polymorphism. Reference: 1. Lal et al., Cancer Res 2014;74(4):1128-40. Citation Format: Samantha Z. Brown, Avinoam Nevler, Henry T. Thomsett, Shruti Lal, Mahsa Zarei, Fernando Blanco, Joseph A. Cozzitorto, Alexis L. Norris-Kirby, Charles J. Yeo, Jordan M. Winter, James R. Eshleman, Jonathan R. Brody. Functional and clinical implications of an INDEL within the HuR regulatory region of the mitotic kinase inhibitor WEE1 [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 4441.