In situ RAS:RAF binding correlates with response to KRASG12C inhibitors in KRASG12C­-mutant non–small cell lung cancer

Abstract Purpose: Therapeutic efficacy of KRASG12C(OFF) inhibitors (KRASG12Ci) in KRASG12C-mutant non–small cell lung cancer (NSCLC) varies widely. The activation status of RAS signaling in tumors with KRASG12C mutation remains unclear, as its ability to cycle between the active GTP-bound and inactive GDP-bound states may influence downstream pathway activation and therapeutic responses. We hypothesized that the interaction between RAS and its downstream effector RAF in tumors may serve as indicators of RAS activity, rendering NSCLC tumors with a high degree of RAS engagement and downstream effects more responsive to KRASG12Ci compared to tumors with lower RAS­­–RAF interaction. Experimental Design: We developed a method for measuring in situ RAS binding to RAF in cancer samples using proximity ligation assays (PLAs) designed to detect panRAS–CRAF interactions. Results: The panRAS–CRAF PLA signal correlated with levels of both RAS-GTP and phosphorylated ERK protein, suggesting that this assay can effectively assess active RAS signaling. We found that elevated panRAS–CRAF PLA signals were associated with increased sensitivity to KRASG12Ci in KRASG12C-mutant NSCLC cell lines, xenograft models, and patient samples. Applying a similar PLA approach to measure the interactions between EGFR and its adaptor protein GRB2 as a surrogate for EGFR activity, we found no relationship between EGFR activity and response to KRASG12Ci in the same samples. Conclusions: Our study highlights the importance of evaluating in situ RAS–RAF interactions as a potential predictive biomarker for identifying NSCLC patients most likely to benefit from KRASG12Ci. The PLA developed for quantifying these interactions represents a valuable tool for guiding treatment strategies.