Abstract 2578: An essential role for Argonaute 2 in HRAS and NRAS driven oncogenesis
Authors: Siebenaler Ronald F. , Waninger Jessica , Shankar Sunita , Chugh Seema , Chu Shih-Chun , Tien Jean , Dommeti Vijaya L. , Mody Malay , Gautam Anudeeta , Kenum Carson , Kumar-Sinha Chandan , Chinnaiyan Arul M. 
Published: September 2020

Abstract
The RAS gene family is among the most commonly mutated genes in cancer, and targeting the potential binding partners of mutated RAS may present a promising alternative therapeutic strategy. We recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as a novel partner of KRAS through its Switch II domain. Further, we demonstrated a role for AGO2 through the development of a KRASG12D driven mouse model of pancreatic cancer. Specifically, our findings revealed that AGO2 ablation drove early precursor pancreatic intraepithelial (PanIN) lesions to enter oncogene-induced senescence in lieu of progressing to late stage PanINs and pancreatic ductal adenocarcinoma. These studies suggested that AGO2 plays a key role in the development of mutant KRAS driven cancers.
In order to assess the role of AGO2 in other mutant RAS driven cancers, we performed co-IP of purified AGO2 and purified NRAS/HRAS proteins using isoform specific antibodies. Both HRAS and NRAS bound AGO2, regardless of GDP or GTP nucleotide loading. Using a panel of cell lines (including T24 cells (HRASG12V) and Mel-Juso cells (NRASQ61L)), we observed that AGO2 interacted with both wild-type (WT) and mutant forms of HRAS and NRAS. Additionally, using a variety of cell line models, we observed that EGFR-mediated phosphorylation of AGO2Y393 disrupted the interaction between WT RAS and AGO2. However, the mutant NRAS-, HRAS-, and KRAS-AGO2 interactions were resistant to EGFR regulation.
Considering the strong role of AGO2 in mutant KRAS driven cancer, we next asked if loss of AGO2 could induce a similar inhibition of RAS driven disease in human cell line models. In a panel of mutant NRAS and HRAS driven cell lines, AGO2 knockdown led to a substantial decrease in cell proliferation; however, WT RAS expressing cells were unperturbed following loss of AGO2. Similar to the loss of AGO2 in the KRASG12D driven mouse model, knockdown of AGO2 led to increased beta-galactosidase staining in a panel of oncogenic NRAS/HRAS driven cells. Furthermore, knockdown of AGO2 was associated with an induction of multiple senescence pathways, including increased p53, p21, and p16 expression. These results suggest that decreased AGO2 expression is sufficient to induce senescence in mutant but not WT RAS driven cells. In these cell line models, AGO2 loss reduced not only the mutant RAS isoform expression but also the WT RAS isoforms. Finally, knockdown of AGO2 led to an inhibition of T24 and Mel-Juso cell migration and metastasis in a zebrafish xenograft model. Taken together, these results suggest that 1) AGO2 interactions with mutant HRAS and NRAS play a key role in mutant RAS driven oncogenesis and 2) both mutant HRAS and NRAS depend on AGO2 to overcome senescence.
Citation Format: Ronald F. Siebenaler, Jessica Waninger, Sunita Shankar, Seema Chugh, Shih-Chun Chu, Jean Tien, Vijaya L. Dommeti, Malay Mody, Anudeeta Gautam, Carson Kenum, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in HRAS and NRAS driven oncogenesis [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 2578.