Role of Argonaute 2 in oncogene induced senescence in a pancreatic cancer mouse model

Pancreatic cancer is a highly devastating malignancy with a very poor survival rate of 7%. Mutations in KRAS have been identified in more than 90% of PDAC patients. Previous work from our lab has shown that KRAS directly interacts with Argonaute 2 (AGO2) to promote cellular transformation. To study the involvement of AGO2 in KRASG12D-driven cancers, AGO2 expression was ablated in the KrasG12D/+; p48 Cre model (KC) model of pancreatic cancer.AGO2 floxed mice (AGO2loxP/loxP) were crossed with KC mice, resulting in oncogenic KRAS expression along with knockout of AGO2 in pancreatic acinar cells. Survival and disease progression were compared between wild-type (AGO2+/+; KRASG12D; p48Cre), heterozygous (AGO2fl/+; KRASG12D; p48Cre), and homozygous (AGO2fl/fl; KRASG12D; p48Cre) experimental mice groups. Homozygous knockout of AGO2 in KC mice resulted in significantly increased survival as compared to wild type and heterozygous mice. Pancreatic ductal adenocarcinoma (PDAC) and metastases were restricted to the wild-type and heterozygous mice. Pancreas from AGO2fl/fl; KRASG12D; p48Cre mice develop only early pancreatic intraepithelial lesions (PanINs), which fail to progress to PDAC. Senescence-associated β-galactosidase staining showed strong and significant increase in senescence in PanIN lesions mice lacking AGO2 expression as compared to AGO2+/+; KRASG12D; p48Cre mice. This suggests that AGO2 prevents oncogene induced senescence (OIS) as a result of KRASG12D expression and allows PanIN to PDAC progression. To gain mechanistic insights of OIS due to AGO2 loss, we evaluated markers for OIS including p16, p53, p21, gamma γH2AX, and RAS-associated signaling (pERK and pAkt). Analysis of PanIN lesions lacking AGO2 showed increased p16 levels and high levels of phospho-ERK, compared to PDAC from pancreas with AGO2 expression. In order to extend these observations in cell line models, we performed AGO2 knockdown in T24 cells harboring HRASG12V. Surprisingly, cells with low AGO2 levels underwent OIS, which was similar to the pancreatic mouse model and was accompanied with increased phospho-ERK signaling and p16 expression. Further studies are underway to determine the contribution of the RAS-AGO2 interaction in the development of OIS. Additionally, we are using CRISPR/Cas9 technology to screen pancreatic cancer cell lines with AGO2 knockout for their dependence on AGO2 and their ability to undergo OIS in the absence of AGO2 expression. We will present findings from our ongoing studies involving the role of AGO2 loss in the KPC (KRASG12D; p53fl/+; Cre) model, wherein OIS will be assessed in the absence of p53, a canonical inducer of cellular senescence. Citation Format: Seema Chugh, Jean C. Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Z. Wang, Sanjana Eyunni, Kristin M. Juckette, Lisha Wang, Sunita Shankar, Arul M. Chinnaiyan. Role of Argonaute 2 in oncogene induced senescence in a pancreatic cancer mouse model [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 94.