Abstract A001: RAS:RAF proximity ligation assay may predict response to KRASG12C inhibitors in NSCLC
Authors: Kato Ryoji , Solanki Hitendra S. , Imbody Denis , Majumder Anurima , Stern Yaakov , Bridenstine Liznair , Johnson Joseph , Haura Eric B. 
Published: May 2023

Abstract
Sotorasib demonstrated efficacy in patients with non-small cell lung cancer (NSCLC). However, a substantial proportion of patients do not respond. There is therefore a clear need for a predictive biomarker to guide treatment decisions. Proximity ligation assay (PLA) is an immunofluorescent-based approach for analysis of endogenous protein-protein interactions, in which the signal can be detected only when two target proteins are within 40 nm. This method measures drug-targetable signaling-associated protein complexes in cells and tissues. KRAS activity has recently been shown to be related to the sensitivity to sotorasib. We therefore conducted a preclinical study to evaluate whether RAS activation inferred by PLA might predict response to KRASG12C inhibitors. We first examined panRAS:CRAF PLA reaction for a KRASG12C-mutant H358 NSCLC cell line, by comparing a complete PLA reaction with both panRAS and CRAF antibodies and an incomplete PLA reaction in the absence of either panRAS or CRAF antibodies. We observed high panRAS:CRAF PLA intensity in H358 with both antibodies, which was abrogated in the absence of either panRAS or CRAF antibodies. We next evaluated the specificity of the PLA reaction by RNA interference. Transfection with sipanRAS or siCRAF resulted in a reduction in panRAS:CRAF PLA signal. We then performed panRAS:CRAF PLA with a panel of KRAS G12C-mutant NSCLC cell lines. The intensity of panRAS:CRAF PLA signal varied between these cell lines, with H358 and LU65 having higher and H1792 and LU99 having lower intensity of panRAS:CRAF PLA signal. To further understand the RAS-RAF interaction, we evaluated a CRAF-RAS binding domain pulldown assay for the cell lines. Different levels of CRAF-bound panRAS were observed in the cell lines, with H358 and LU65 having higher and H1792 and LU99 having lower level of CRAF-bound panRAS. The intensity of panRAS:CRAF PLA strongly correlated with CRAF-bound panRAS (r = 0.70, P = 0.037), suggesting that panRAS:CRAF PLA works sufficiently well to detect the RAS-RAF complex. We investigated the antitumor efficacy of AMG510 in vitro and found that a heterogeneous response to AMG510 was observed in the cell lines, with the 50% inhibitory concentration values of AMG510 for H358 and LU65 below 30 nmol/L and those for H1792, and LU99 above 10 μmol/L. The intensity of panRAS:CRAF PLA was strongly associated with the sensitivity of the G12C-mutated cells to AMG510 (r = 0.74, P = 0.01). We next examined sections of FFPE tumors from NSCLC cell line-derived xenograft models. The panRAS:CRAF PLA signal was higher in mice with sotorasib-sensitive H358 than with resistant H1792 (P = 0.02). Our results suggest that RAS:RAF PLA may have values as a predictive marker to identify which patients can obtain the greatest benefit from sotorasib.
Citation Format: Ryoji Kato, Hitendra S. Solanki, Denis Imbody, Anurima Majumder, Yaakov Stern, Liznair Bridenstine, Joseph Johnson, Eric B. Haura. RAS:RAF proximity ligation assay may predict response to KRASG12C inhibitors in NSCLC [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A001.