Abstract 4385: Preclinical evaluation of RAS pathway inhibitors in gastroesophageal adenocarcinoma
Authors: Zachary Hammer, Jack Rifkin, Ralph Fleuranvil, Babita Pokharel, Stella Savone, Sandra W. Ryeom, Mariam El-Ashmawy
Journal: Cancer Research (2025)

Abstract Gastric cancer is the 4th leading cause of cancer-related deaths worldwide and up to 25% are driven by alterations in the RAS pathway. RAS activation in cancer is commonly a result of a mutation locking the protein in the ON state. However, KRAS amplification (KRASAMP), defined as &amp;gt;6 copies of wild type KRAS allele, occurs more frequently in gastroesophageal adenocarcinoma (GEA). Western blot analysis revealed differences in MAPK signaling amplitude between KRASAMP and KRASMUT cell lines after synchronization. KRASAMP cells show a sharp spike in signaling upon stimulation, followed by attenuation, we speculate due to negative feedback. Historically KRASAMP has been difficult to target due to adaptive signaling requiring MAPK inhibition at multiple signaling nodes, yet these combinations are clinically limited by toxicity. Here we test novel RAS pathway inhibitors in KRASAMP/MUT GEA cell lines, to investigate toxicity, synergy with drug combinations, and efficacy of MAPK pathway target inhibition by quantifying levels of p-ERK. KRASAMP GEA cells ranged in copy number from 8-68; while all GEA mutant cell lines harbored KRASG12D mutations. GEA cells were seeded in 96 well plates, treated with drug for 5 days then assessed for viability via Cell titer glo. The half-maximal effective drug concentration (EC50) for p-ERK inhibition was determined by Western blot analysis after 24 hours of drug treatment. The pan-KRAS (OFF) Inhibitor, BI-2865 inhibits KRAS altered GEA cell viability with an IC50 ranging from 0.2 - 1.4 μM in KRASAMP and KRASG12D mutant cell lines. The RAF/MEK clamp VS-6766 demonstrated an IC50 range from 0.81 μM in KRASAMP in cell lines, while mutant KRASG12D lines exhibited 40-fold lower sensitivity. The allosteric SHP2 inhibitor RMC-4550 demonstrated an IC50 of 0.08-6.67 μM in KRASAMP and KRASG12D lines. BI-2865 suppressed p-ERK protein expression with an EC50 of 16 and 45 nM in KRASAMP and KRASMUT GEA cells respectively. There was no adaptive signaling through PI3K pathways based on suppressed p-AKT levels, in comparison to trametinib which resulted in increases in pAKT. The RAF/MEK clamp VS-6766 also inhibits signaling through MAPK as evidenced by decreased p-AKT, and an EC50 for p-ERK of 131.83 nM (KRASAMP) and 10.4725 nM (KRASMUT) GEA cells. Our data indicates a positive correlation between KRAS copy number and the IC50 of RAS-MAPK pathway inhibitors, suggesting cells with more KRAS copies require higher doses to achieve cytotoxicity; this is also reflected in the dose required for target engagement. This highlights the differential sensitivity of KRASAMP versus mutant cell lines, emphasizing the complexity of KRASAMP tumors and their reliance on high copy numbers. Our results suggest that combination strategies will be essential to overcome adaptive resistance and maximize therapeutic benefit in KRASAMP cells. Future in vivo studies will validate the potential of these inhibitors and combination approaches. Citation Format: Zachary Hammer, Jack Rifkin, Ralph Fleuranvil, Babita Pokharel, Stella Savone, Sandra W. Ryeom, Mariam El-Ashmawy. Preclinical evaluation of RAS pathway inhibitors in gastroesophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4385.