Abstract B39: Characterization of the interaction between KRAS and Argonaute 2
Authors: Waninger Jessica , Shankar Sunita , Siebenaler Ronald , Mody Malay , Tien Jean , Dommeti Vijaya , Beyett Tyler , Smith Richard , Kumar-Sinha Chandan , Carlson Heather , Tesmer John , Chinnaiyan Arul 
Published: June 2021

Abstract
KRAS is one of three members of the RAS family of small GTPases, and nearly one third of all human cancers harbor mutations in the KRAS gene. These mutations lead to a constitutively active GTP-bound form of KRAS that results in aberrant activation of MAPK and PI3K pathways that control cell differentiation, growth, and survival. While much has been learned about the effectors of KRAS and its complex signaling network, little progress has been made in successfully targeting it in the treatment of cancer. Recently, we identified a functional protein-protein interaction between KRAS and Argonaute 2 (AGO2), a core component of the RNA-induced silencing complex (RISC). Investigation of this interaction revealed that the switch II domain of KRAS (both wild-type [WT] and mutant) binds to the N-terminal (Nt) domain of AGO2. Mutant KRAS-AGO2 binding results in attenuation of the RNAi function of AGO2 by preventing microRNA unwinding, a necessary step in the formation of the mature RISC complex (model 1). Analysis of this interaction revealed that KRAS-AGO2 binding is direct through Tyr64 of KRAS and Lys112/Glu114 of AGO2. Recently, using genetically engineered mouse models (GEMM), we showed that mutant KRAS is dependent on AGO2 for the development of pancreatic ductal adenocarcinoma (PDAC) from early precursor lesions in the pancreas, suggesting a critical role for the KRAS-AGO2 interaction in vivo. To further characterize KRAS-AGO2 binding, we purified recombinant proteins individually, isolated complex via size exclusion chromatography (SEC), performed differential scanning fluorimetry (DSF), isothermal titration calorimetry (ITC), and small angle x-ray scattering (SAXS). Additionally, in order to evaluate endogenous regulators of the KRAS-AGO2 interaction we investigated the role of the epidermal growth factor receptor (EGFR), which is known to regulate the RNAi function of AGO2 via phosphorylation at Tyr393. Interestingly, we observed that phosphorylation at this residue disrupts the interaction of AGO2 with WT but not mutant KRAS, providing a basis for differential regulation of the KRAS-AGO2 interaction through growth factor activation. Finally, to examine structural changes induced by EGFR phosphorylation of AGO2 at Tyr393 that may affect KRAS binding, we performed parallel molecular dynamics simulations (MDS) of nonphosphorylated and phospho-Tyr393 AGO2. These studies revealed immediate structural reorganization of the RAS binding site and an outward rotation of the entire Nt domain of AGO2. Taken together, these data shed light on a potential mechanism underlying the differential regulation of mutant vs. WT KRAS.
Citation Format: Jessica Waninger, Sunita Shankar, Ronald Siebenaler, Malay Mody, Jean Tien, Vijaya Dommeti, Tyler Beyett, Richard Smith, Chandan Kumar-Sinha, Heather Carlson, John Tesmer, Arul Chinnaiyan. Characterization of the interaction between KRAS and Argonaute 2 [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 B39.