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Both KRAS and EGFR are essential mediators of pancreatic cancer development and interact with Argonaute 2 (AGO2) to perturb its function. Here, in a mouse model of mutant KRAS-driven pancreatic cancer, loss of AGO2 allows precursor lesion (PanIN) formation yet prevents progression to pancreatic ductal adenocarcinoma (PDAC). Precursor lesions with AGO2 ablation undergo oncogene-induced senescence with altered microRNA expression and EGFR/RAS signaling, bypassed by loss of p53. In mouse and human pancreatic tissues, PDAC progression is associated with increased plasma membrane localization of RAS/AGO2. Furthermore, phosphorylation of AGO2Y393 disrupts both the wild-type and oncogenic KRAS-AGO2 interaction, albeit under different conditions. ARS-1620 (G12C-specific inhibitor) disrupts the KRASG12C-AGO2 interaction, suggesting that the interaction is targetable. Altogether, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR-RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression., Argonaute 2 (AGO2) binds RAS and is required for cellular transformation. Here, the authors establish a KRAS-driven mouse model of pancreatic cancer with conditional loss of AGO2 and show that the early phase of neoplastic lesion initiation is dependent on EGFR/RAS but not AGO2, while AGO2 is required for pancreatic ductal adenocarcinoma progression and metastasis.

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, however, presents 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 Switch II domain of KRAS. In order to assess the role of AGO2 in KRAS-G12D 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, loss of AGO2 prevented progression to late-stage PanINs, pancreactic ductal adenocarcinoma (PDAC), and metastatic disease. AGO2 null lesions displayed increased activation of the EGFR-RAS signaling axis during PanIN development. This signaling resulted in an increase in WT RAS-GTP activation, pEGFR-Y1068, and pERK levels leading to the development of oncogene-induced senescence in these PanIN lesions. Furthermore, we observed that EGFR-mediated phosphorylation of AGO2-Y393 disrupted the interaction between WT RAS and AGO2. This regulation by EGFR, however, was blocked in cells expressing mutant KRAS. These results suggested that the interaction of mutant RAS and AGO2 was vital to tumor development. To better assess the role of AGO2 loss in mutant RAS driven cancer, we performed AGO2 knockdown in multiple cell lines expressing mutations in either NRAS or HRAS isoforms. In each cell line, AGO2 directly interacted with KRAS, NRAS, and KRAS. In addition to suppressing growth in mutant RAS-driven cells (T24: HRAS-G12V, SK-MEL-2: NRAS-Q61H), loss of AGO2 produced marked increases in beta-galactosidase and p16 expression, as well as a decrease in cyclin D1, suggesting development of oncogene-induced senescence. Interestingly, upon AGO2 loss, cells displayed induction of pEGFR and pERK similar to what was observed in our pancreatic mouse model, and despite decreased expression of mutant RAS, WT RAS-GTP loading upon AGO2 loss was strongly induced. Together these results suggest a unique EGFR-AGO2-RAS signaling axis that requires AGO2-RAS interaction to prevent induction of oncogene-induced senescence in mutant RAS-driven cancers. Citation Format: Ronald F. Siebenaler, Sunita Shankar, Jean C. Tien, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Malay Mody, Seema Chugh, Chandan Kumar-Sinha, Arul M. Chinnaiyan. Loss of Argonaute 2 leads to oncogene-induced senescence in mutant RAS-driven cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A21.

In 2016, we identified a direct interaction between RAS and Argonaute 2 (AGO2), a key mediator of RNA-mediated gene silencing that is required for KRAS-driven oncogenesis using pancreatic and lung cancer cell line models. Recently, we employed the genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss of AGO2 in KRASG12D driven pancreatic cancer. 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 survival of KRASG12D mutant mice deficient in AGO2 expression. 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. AGO2 ablation permits PanIN initiation driven by the EGFR-RAS axis; however rather than progressing to PDAC, these lesions undergo profound oncogene-induced senescence (OIS). Since PanIN development requires EGFR and is not AGO2 dependent, we probed the effects of EGF stimulation in cell lines expressing wild-type and mutant forms of KRAS (using co-IP and PLA analyses). In wild-type RAS expressing cells, grown in media containing serum, RAS-AGO2 co-localization was limited to the intracellular regions of the cells, which 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 basal levels. Using phosphorylation-deficient AGO2 mutants, we demonstrate that the disruption of wild type-RAS-AGO2 interaction is due to AGO2Y393 phosphorylation, a target of EGFR. Interestingly, the 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. Taken together, our study 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. In the lung cancer mouse model, we also observed a similar dependence of AGO2 in KRAS-driven lung adenocarcinoma. Along with related abstracts detailing the mechanisms of OIS mediated by AGO2 (Ronald Siebenaler) and evidence of direct interaction between oncogenic KRAS and AGO2 with an affinity of 200nM (Jessica Waninger), we present our latest studies related to the KRAS-AGO2 interaction. Citation Format: Sunita Shankar, Jean Ching-Yi Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Xiao-Ming Wang, Kristin M. Juckette, Alice Xu, Seema Chugh, Malay Mody, Sanjana Eyunni, Andrew Goodrum, Grace Tsaloff, Yuping Zhang, Ingrid J. Apel, Lisha Wang, 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 AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A20.

KRAS and EGFR are known essential mediators of pancreatic cancer development. In addition, KRAS and EGFR have both been shown to interact with and perturb the function of Argonaute 2 (AGO2), a key regulator of RNA-mediated gene silencing. Here, we employed a genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss ofAGO2inKRASG12D-driven pancreatic cancer. Genetic ablation ofAGO2does not interfere with development of the normal pancreas orKRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. Remarkably, however,AGO2is required for progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastasis.AGO2ablation permits PanIN initiation driven by the EGFR-RAS axis, but rather than progressing to PDAC, these lesions undergo profound oncogene-induced senescence (OIS). Loss ofTrp53(p53) in this model obviates the requirement ofAGO2for PDAC development. In mouse and human pancreatic tissues, increased expression of AGO2 and elevated co-localization with RAS at the plasma membrane is associated with PDAC progression. Furthermore, phosphorylation of AGO2Y393by EGFR disrupts the interaction of wild-type RAS with AGO2 at the membrane, but does not affect the interaction of mutant KRAS with AGO2. ARS-1620, a G12C-specific inhibitor, disrupts the KRASG12C-AGO2 interaction specifically in pancreatic cancer cells harboring this mutant, demonstrating that the oncogenic KRAS-AGO2 interaction can be pharmacologically targeted. Taken together, our study supports a biphasic model of pancreatic cancer development: anAGO2-independent early phase of PanIN formation reliant on EGFR-RAS signaling, and anAGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical to prevent OIS in PanINs and allow progression to PDAC.

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.

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.