Abstract 5476: A novel PARP inhibitor resistance mechanism mediated by the RNA-binding protein HuR

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Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer-related deaths in the United States, and the 3rd most common cancer associated with BRCA mutations. Frontline therapies have significant toxicities and only minimally extend overall survival, highlighting the need to optimize targeted therapies. Poly-ADP ribose polymerase (PARP) inhibitors (PARPi), a ‘poster child’ for personalized medicine, depend on the concept of synthetic lethality where the combined perturbation of DNA repair genes, via genetic mutations within the tumor cells, and pharmacological PARP inhibition effectively targets BRCA-deficient tumors. Although PARPi have delivered promising preclinical and clinical results, initially- responsive patients ultimately develop resistance. A unique mechanism elucidated by our lab demonstrates that the mRNA-binding protein HuR mediates resistance to DNA damaging agents through post-transcriptional regulation of select mRNA cargo. Predominantly expressed in the nucleus, HuR translocates to the cytoplasm upon tumor-associated stress. Cytoplasmic HuR binds and stabilizes unique pro-survival transcripts, resulting in resistance to a harsh tumor microenvironment. Here, we sought to evaluate the role of HuR in regulating PARPi response. Methods and Results: Through immunofluorescence and western blot of fractionated lysates, we demonstrate that the PARP inhibitors Veliparib, Olaparib, and Rucaparib induced cytoplasmic HuR localization. Conversely, pre-treatment with MS-444 (Novartis), an established small molecule inhibitor of HuR, abrogated its nuclear export induced by PARPi treatment. Consistent with these findings, the growth-inhibitory effects of PARPi treatment were significantly potentiated upon HuR silencing whereas ectopic HuR overexpression promoted resistance, as observed in short term cell survival and long-term anchorage-independent growth assays. Additionally, silencing of HuR enhanced PARPi-induced cytotoxicity, assessed by increased accumulation of DNA damage (γH2Ax) foci and Poly ADP-ribose (PAR) polymers. Ribonucleotide protein immunoprecipitation (RNP-IP) assays demonstrated that HuR binds and upregulates Poly-ADP Ribose Glycohydrolase (PARG) mRNA, the major enzyme responsible for catabolism of PAR. Taken together, when PDA cells are exposed to PARPi, HuR mediates upregulation of PARG, thereby decreasing PARylation and facilitating DNA repair. Conversely, HuR inhibition results in detrimental accumulation of PAR and enhanced DNA damage, which ultimately leads to increased PARPi-conferred cytotoxicity. Discussion: These results demonstrate that HuR imposes a significant barrier to PARPi therapy by orchestrating a strong chemoresistance mechanism. Thus, we provide evidence that HuR (and/or its target) inhibition via an HuR inhibitor (MS-444) can optimize PARPi-based therapies for better patient outcomes. Citation Format: Saswati N. Chand, Akshay R. Kamath, Nicole Meisner-Kober, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. A novel PARP inhibitor resistance mechanism mediated by the RNA-binding protein HuR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5476. doi:10.1158/1538-7445.AM2015-5476