Abstract 2000: Different oncogenic KRAS mutations produce distinct heterogenous outcomes in signaling pathways of isogenic mouse embryonic fibroblasts
Authors: El Gazzah Emna , Baldelli Elisa , Ruhunsiri Chamodya , Petricoin Emanuel F. , Pierobon Mariaelena 
Published: July 2021

Background: Growing evidence points towards the concept that not all RAS allelic variances are equal. Different RAS mutations produce distinct variations in signal transduction to sustain their proliferative needs accordingly. This is an especially important concept to consider as medicine shifts towards a more personalized approach. In accordance with this notion, through this study, we aim to explore the network dynamics and pinpoint disparities in signaling events associated with seven common KRAS allelic variances in non-small cell lung carcinoma.

Methods: We used Mouse Embryonic Fibroblasts (MEF) provided by the RAS initiative (https://www.cancer.gov/research/key-initiatives/ras/outreach/reference-reagents/cell-lines). MEF were engineered to selectively harbor and express either wild type KRAS (4A and 4B WT) or one of seven following mutations: 4B G12D, G12C, G12R, G12V, G13D, Q61R and Q61L. Using high throughput immunoassay, Reverse Phase Protein Microarray (RPPA), we explored broad changes in signaling dynamics across our models. Specifically, we captured expression and/or activation levels of 68 proteins and phosphoproteins including: Receptor Tyrosine Kinases, MAPK, AKT/mTOR, JNK/STAT. Data were analyzed using unsupervised hierarchical clustering analysis using the Ward methods and the Kruskal Wallis test. P values &lt;0.05 were considered significant.

Results: Based on the unsupervised clustering analysis, Q61R and Q61L mutations showed the least activated signaling when compared to all other models. Activation of Raf was increased in cells harboring mutations of the G12 residues compared to those with mutations of the G13 and Q61 residues. Furthermore, the five G12 mutations clustered together with an overall higher active signaling compared to other cell lines. However, among the group of G12 mutants, unique signal transduction profiles were identified. We report distinct significant differences that divide the clustered G12 mutants into high versus low activation of specific signaling proteins. For example, ERK T202/Y204 (p=0.011) and S6 Ribosomal Protein S235/236 (p=0.001) phosphorylation were significantly higher in G12R compared to G12C and G12D models. On the contrary, STAT1 and STAT3 protein phosphorylation at residues Y701 and Y705 respectively were decreased in the G12R model. G12C has the lowest activation levels of p90RSK S380 (p=0.029), NF-KappaB p65 S536 (p=0.027) and P70 S6 Kinase S371 (p=0.013).

Conclusions: In alignment with other findings, we show that based on RAS mutation, individual signaling components are affected differently, producing extensive heterogeneity in signaling dynamics. Even within grouped mutations presenting an overall similar net effect on downstream signaling, each individual variant produced discrete alterations.

Citation Format: Emna El Gazzah, Elisa Baldelli, Chamodya Ruhunsiri, Emanuel F. Petricoin, Mariaelena Pierobon. Different oncogenic KRAS mutations produce distinct heterogenous outcomes in signaling pathways of isogenic mouse embryonic fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2000.