Abstract 957: An essential role for Argonaute 2 in mouse models of KRAS-driven cancers
Authors: Shankar Sunita , Tien Jean Ching-Yi , Siebenaler Ronald F. , Dommeti Vijaya L. , Zelenka-Wang Sylvia , Waninger Jessica , Juckette Kristin M. , Xu Alice , Wang Xiao-Ming , Chugh Seema , Mody Malay , Eyunni Sanjana , Goodrum Andrew , Tsaloff Grace , Zhang Yuping , Apel Ingrid J. , Siddiqui Javed , Smith Richard D. , Carlson Heather A. , Tesmer John , Cao Xuhong , Shi Jiaqi , Kumar-Sinha Chandan , Chinnaiyan Arul M. 
Published: August 2019

In 2016, we identified a direct interaction between RAS and Argonaute 2 (AGO2), a key mediator of RNA-mediated gene silencing, that is essential for KRAS-driven oncogenesis using pancreatic and lung cancer cell line models. Recently, we employed a genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss of AGO2 in KRASG12D-driven pancreatic cancer (KC model). Genetic ablation of AGO2 did not interfere with development of the normal pancreas or KRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. However, AGO2 loss prevents progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastatic progression. This results in a dramatic increase in the survival of KRASG12D mutant mice deficient in AGO2 expression. Mechanistically, lack of PanIN to PDAC progression was due to oncogene-induced senescence (OIS) through activation of EGFR-wild type RAS-phosphoERK signaling in the absence of AGO2.

Using validated pan-RAS and AGO2 antibodies for immunofluorescence (IF) and proximity ligation assay (PLA), we observed increased RAS and AGO2 co-localization at the plasma membrane in mouse and human pancreatic tissues associated with PDAC progression. While AGO2 ablation permits PanIN initiation driven by the EGFR-RAS axis, these lesions undergo OIS rather than progressing to PDAC. Further, we used co-IP and PLA analyses to probe the effects of EGF stimulation in cell lines expressing wild-type and mutant forms of KRAS. In wild-type RAS expressing cells, RAS-AGO2 co-localization and interaction were limited to the intracellular regions of the cells, and dramatically increased and shifted to the plasma membrane under conditions of stress (serum starvation). Interestingly, EGF stimulation disrupted this membrane RAS-AGO2 interaction and restored it to intracellular levels. Using phosphorylation-deficient AGO2 mutants, we further demonstrate that the disruption of wild-type RAS-AGO2 interaction is due to EGFR-mediated AGO2Y393 phosphorylation. Interestingly, mutant KRAS-AGO2 interaction is not subject to EGFR activation, suggesting that although both the wild type and mutant RAS bind AGO2, they are differentially regulated through growth factor receptor activation.

We will discuss our ongoing studies evaluating the effects of AGO2 ablation in the KRASG12Ddriven lung cancer mouse model and PDAC progression with p53 loss (KPC model). Our recent in vivo work supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR and wild-type RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression.

Citation Format: Sunita Shankar, Jean Ching-Yi Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Kristin M. Juckette, Alice Xu, Xiao-Ming Wang, Seema Chugh, Malay Mody, Sanjana Eyunni, Andrew Goodrum, Grace Tsaloff, Yuping Zhang, Ingrid J. Apel, Javed Siddiqui, Richard D. Smith, Heather A. Carlson, John Tesmer, Xuhong Cao, Jiaqi Shi, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in mouse models of KRAS-driven cancers [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 957.