Abstract PR002: Identification of a RAS-GTP Threshold for Malignant Transformation

KRAS G12, G13, and Q61 mutations perturb GTPase-activating protein (GAP) and, in some cases, guanine nucleotide exchange factor (GEF) activity, resulting in a change in total KRAS-GTP levels and upregulation of MAPK signaling. Despite G12P altering GAP sensitivity, it is the only G12 mutation that is non-transforming. We used immunoprecipitation coupled with high performance liquid chromatography (IP-HPLC) to quantify KRAS GTP and KRAS GDP levels for a panel of FLAG-tagged KRAS mutants expressed in HeLa cells. KRASG12P was found to be significantly less GTP loaded at ∼25% when compared to other oncogenic mutants at >50%, but higher than KRASWT at ∼5%. This result suggests a percentage of GTP-bound KRAS exists between 25% and 50% under which cells are non-transforming. We further sought to understand how the biochemical parameters of different KRAS mutants contribute to elevated GTP levels and malignant transformation. GAP and GEF catalysis rates were measured for a panel of G12, G13, and Q61 mutants via phosphate sensor and MANT-GDP assays respectively. As expected, mutations at G12 and Q61 significantly impaired GAP activity. However, while reduced compared to KRASWT, KRASG12P and KRASG13D showed GAP sensitivity, and KRASG13D showed elevated GEF activity. Affinity of KRAS for the RAS Binding Domain (RBD) of RAF1 was also measured for the panel of mutants by SPR and found to be similar, demonstrating that the GTP-bound KRAS level and not the affinity for substrate is the main driver of oncogenicity. Given that G13D is oncogenic and G12P is non-transforming, yet both show GAP sensitivity, a detailed analysis of their rate constants was performed using stopped-flow. A 50-fold decrease in GAP hydrolysis rate and a 5-fold increase in GEF exchange rate for G13D was measured compared to WT. A 100-fold decrease in GAP hydrolysis rate and a 3-fold decrease in GEF exchange rate for G12P was measured compared to WT. Despite a similar loss in GAP sensitivity for G12P and G13D, the increase in GEF exchange for G13D contributes to its higher KRAS-GTP levels and oncogenic potential. The measured rate constants for GAP and GEF were used to calculate the theoretical KRAS-GTP percentage and compared to the experimentally measured KRAS-GTP percentage in cells. The measured biochemical parameters predict KRAS-GTP percentages that were comparable to those experimentally determined. Taken together, this work provides a quantitative target of GTP-bound KRAS for which therapeutics can aim to achieve by modulating GAP and GEF activity. Citation Format: Sophie Krahnke, Erik K. Larsen, Dana Rabara, Nicole Fer, Timothy Waybright, William Burgan, Frank McCormick, Andrew G. Stephen. Identification of a RAS-GTP Threshold for Malignant Transformation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr PR002.