Targeting the KRAS G12D mutant suppresses tumor growth in a syngeneic mouse cholangiocarcinoma model through activation of immune cells 2258895

Abstract Introduction Cholangiocarcinoma (CCA), is an aggressive malignancy often diagnosed at an advanced, inoperable stage, with 5-year survival rate of 3%. KRAS mutations are frequent driver alterations linked to poorer prognosis in this cancer. The KRAS G12C mutation inhibitor Adagrasib has been approved by the FDA for the treatment of advanced non-small-cell lung cancer, indicating that the KRAS mutation is a druggable target. However, KRAS mutant inhibitors have yet to be employed in CCA treatment. Methods A mouse CCA model, designated AKP, was generated by crossing genetically engineered mouse strains harboring Alb-Cre, LSL-KrasG12D, and p53L/L. Subsequently, the AKP-M4 cell line was derived by serially passaging tumor cells from AKP mice across three generations. Sanger sequencing and immunohistochemical (IHC) staining were used to determine the KRAS G12D mutation and cytokeratin 19, respectively. MTT assay was used to assess the cell proliferation inhibition by MRTX-1133. Immune cell profiles in AKP-M4-bearing mice treated with MRTX-1133 were determined by flow cytometry. Results Sanger sequencing confirmed G12D mutation in the KRAS gene in AKP-M4 cells. IHC analysis demonstrated that AKP-M4 tumors expressed cytokeratin 19, a key marker for CCA. The in vitro proliferation of AKP-M4 cells, but not KRAS wild-type SNU-1079 and SSP-25 cells, was reduced by the KRAS G12D mutation inhibitor MRTX-1133. We further assessed the anti-cancer efficacy of MRTX-1133 in AKP-M4-bearing C57BL/6 mice, observing a significant dose-dependent reduction in tumor volume and weight. Flow cytometry analysis showed that MRTX-1133 elevated CD4+ T cells, CD8+ T cells, and M1 macrophages, while decreasing MDSCs and M2 macrophages. Notably, PD-L1 expression on tumor cells was diminished. Conclusion KRAS G12D mutant inhibition reduces cancer cell proliferation and stimulates immune cells. This supports the development of KRAS mutation inhibitor for CCA treatment. Funding Source National Science and Technology Council, Taiwan Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)