Abstract 2577: AGO2 interaction limits wild type RAS activation yet essential for disease progression in oncogenic KRAS driven cancers
Authors: Shankar Sunita , Tien Jean Ching-Yi , Siebenaler Ronald F. , Chugh Seema , Dommeti Vijaya L. , Zelenka-Wang Sylvia , Waninger Jessica , Juckette Kristin M. , Wang Xiao-Ming , Eyunni Sanjana , Goodrum Andrew , Zhang Yuping , Apel Ingrid J. , Siddiqui Javed , Cao Xuhong , Shi Jiaqi , Pitchiaya Sethuramsundaram , Kumar-Sinha Chandan , Crawford Howard C. , Chinnaiyan Arul M. 
Published: September 2020

Abstract
KRAS mutations drive over 30% of all cancers and 90% of pancreatic cancer. To investigate additional potential modulators of RAS-mediated oncogenesis, we previously performed a screen for direct interactors of RAS in a panel of cancer cell lines and identified a direct interaction between KRAS and Argonaute 2 (AGO2), independent of KRAS mutation status.
To define the effects of conditional loss of AGO2 in KRASG12D-driven cancer, we employed a genetically engineered mouse model of 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 prevented progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastatic progression. This resulted in increased 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 RASWT-phospho ERK signaling including reduced baseline autophagy in mice with AGO2 loss. Interestingly, in the KPC model simultaneous expression of oncogenic KRAS and loss of a p53 allele abrogates this phenotype, suggesting that p53 deficiency overcomes the OIS check by AGO2. However, in both mouse and human PDAC, AGO2 expression increased with disease progression and, interestingly, was predominant at the plasma membrane, where it co-localized with RAS. These in vivo analyses support a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR and WT RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression. In the KRASG12D driven lung cancer mouse model, we see a similar requirement for AGO2 for diseases progression–but not for initiation–despite p53 loss indicating a context dependent requirement for AGO2.
Given that AGO2 binds both the mutant and the WT forms of KRAS, we also investigated the role of RASWT-AGO2 interaction. Analysis using purified proteins showed that AGO2 competes with SOS1 to limit RASWT activation. AGO2 had no effect on the intrinsic GTPase activity of RAS or NF1-mediated GAP activity. In line with this data, AGO2 null mouse embryonic fibroblasts showed increased RAS activity and activation of MAPK/PI3K pathways. Further, using cell line models, we also showed that phosphorylation of AGO2Y393 by EGFR disrupted the interaction of RASWT with AGO2 at the membrane, but did not affect the interaction of mutant KRAS with AGO2. On the other hand, ARS-1620, a G12C-specific inhibitor, disrupted the KRASG12C-AGO2 interaction specifically in cells harboring this mutant, demonstrating that the oncogenic KRAS-AGO2 interaction can be pharmacologically targeted.
Altogether, our findings reveal that the AGO2 interaction regulates RASWT and is essential for mutant KRAS driven oncogenesis.
Citation Format: Sunita Shankar, Jean Ching-Yi Tien, Ronald F. Siebenaler, Seema Chugh, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Kristin M. Juckette, Xiao-Ming Wang, Sanjana Eyunni, Andrew Goodrum, Yuping Zhang, Ingrid J. Apel, Javed Siddiqui, Xuhong Cao, Jiaqi Shi, Sethuramsundaram Pitchiaya, Chandan Kumar-Sinha, Howard C. Crawford, Arul M. Chinnaiyan. AGO2 interaction limits wild type RAS activation yet essential for disease progression in oncogenic KRAS driven cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2577.