Abstract 956: An essential role for Argonaute 2 in EGFR-KRAS signaling in pancreatic cancer development
Authors: Siebenaler Ronald F. , Shankar Sunita , Tien Jean C. , Dommeti Vijaya L. , Zelenka-Wang Sylvia , Chugh Seema , Apel Ingrid J. , Mody Malay , Gautam Anudeeta , Kumar-Sinha Chandan , Chinnaiyan Arul M. 
Published: August 2019

Abstract
The RAS gene family is among the most commonly mutated genes within cancer, but little progress has been made in successfully targeting RAS mutations. Targeting binding partners of mutated RAS presents as a promising alternative therapeutic strategy. With the goal of uncovering novel interactors of RAS, we recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as a novel partner of the KRAS through its Switch II domain. In order to assess the role of AGO2 in KRASG12D driven disease, we developed a mouse model of pancreatic cancer with conditional loss of AGO2. While AGO2 knockout did not prevent development of early precursor pancreatic intraepithelial (PanIN) lesions, AGO2 null lesions displayed increased activation of the EGFR-RAS signaling axis during PanIN development that failed to progress to late stage PanINs, pancreatic ductal adenocarcinoma (PDAC), and metastatic disease. This resulted in a dramatic increase in the survival of mice with AGO2 ablation. Unlike the PanINs in AGO2 sufficient mice, the early PanIN lesions with AGO2 ablation showed staining for the senescence associated beta galactosidase activity, suggesting that AGO2 loss induces oncogene induced senescence. To extend these observations and explore the role of AGO2 interaction with mutant forms of HRAS and NRAS proteins, we performed co-IP of AGO2 with RAS proteins using isoform specific antibodies. Both HRAS and NRAS bound AGO2 in T24 cells (HRASG12V) and SK-MEL-2 cells (NRASQ61H), respectively. In T24 cells, AGO2 knockdown led to the senescent phenotype and was accompanied with changes in the EGFR-RAS signaling axis, similar to that observed in the PanINs of the mice with AGO2 loss. In this cell line model, AGO2 loss reduced mutant HRAS expression and increased wild type RAS activity. These signaling effects were also consistent with our observation that AGO2 loss increased RAS activation in the mouse embryonic fibroblast (MEF) model. Together with our previous work with mutant KRAS dependent cells, these data suggest that 1) AGO2-wild type RAS binding prevents RAS activation and 2) mutant RAS-AGO2 association regulates oncogenic RAS levels in cell line models. Studies on the mouse model and the close proximity of RAS and AGO2 with EGFR also furthered our understanding of the RAS-AGO2 interaction. Using a variety of cell line models, we observed that EGFR-mediated phosphorylation of AGO2Y393 disrupts the interaction between WT RAS and AGO2. However, the mutant KRAS-AGO2 interaction was recalcitrant to EGFR regulation. This provides the first instance of a nucleotide dependent association of RAS and AGO2 and sheds light on the dynamic nature of the RAS-AGO2 interaction.
Citation Format: Ronald F. Siebenaler, Sunita Shankar, Jean C. Tien, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Seema Chugh, Ingrid J. Apel, Malay Mody, Anudeeta Gautam, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in EGFR-KRAS signaling in pancreatic cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 956.