Quantitative Assessment of the Target Engagement of a KRAS G12C Inhibitor in Formalin-Fixed Paraffin-Embedded Tumor...

Understanding the target engagement of covalent inhibitors, such as KRAS G12C inhibitors, is essential for evaluating their mechanisms of action and efficacy, particularly given the KRAS G12C mutation's significant role in cancer progression. We previously developed a method to directly evaluate KRAS G12C target engagement by measuring both free and drug-bound proteins in frozen tissues without the need for control samples. Since frozen tissues may not be readily available, we aimed to extend our method to formalin-fixed, paraffin-embedded (FFPE) tissues, which are the standard clinical specimens due to their superior preservation and long-term storage capabilities. This adaptation required addressing the analytical challenges associated with fixation-induced protein cross-linking and structural complexity. Here, we report an optimized workflow for quantification of both free and drug-bound KRAS G12C in FFPE tissues. The workflow integrates a specialized pretreatment process including deparaffinization, heat-induced antigen retrieval, and protein-level immunoaffinity enrichment with a targeted 2D-LC-MS/MS to enable a precise and reproducible determination of KRAS G12C target engagement in complex FFPE matrices. The method typically requires a minimum of 5 μg of total protein input, making it suitable for clinical applications with limited sample quantities such as core needle biopsies. A direct comparison of KRAS G12C target engagement measured in both FFPE and corresponding frozen tissues from a xenograft mouse study demonstrated a strong correlation between the two tissue formats. Overall, the reported workflow may serve as a sensitive and reliable tool for assessing the KRAS G12C engagement and potentially that of other covalent inhibitors.