Elucidation and therapeutic strategies to overcome intrinsic resistance mechanisms in KRAS G12D-mutant tumors

[Background and Aim] KRAS, a small GTPase transmitting signals from receptor tyrosine kinases to MAPK and PI3K pathways, plays a pivotal role in cell proliferation and survival. Oncogenic KRAS mutations cause constitutive activation of downstream signaling and are common in pancreatic, colorectal, and lung cancers. The G12D variant is particularly prevalent in pancreatic and colorectal cancers and also occurs in non-small cell lung cancer, especially among never-smokers. While the success of covalent KRAS G12C inhibitors has validated KRAS as a druggable target, KRAS G12D remained challenging. Recently, selective non-covalent inhibitors such as MRTX1133 have shown potent preclinical efficacy. However, the efficacy of KRAS G12D inhibitor monotherapy remains limited, underscoring the need to clarify and overcome resistance mechanisms. [Method] KRAS G12D-mutant lung and pancreatic cancer cell lines (A427, SK-LU1, AsPC1, Panc1, T3M10, and HPAF-II) were used to evaluate the antitumor effects of MRTX1133 monotherapy and combination therapies. In vitro, three combinations were assessed: MRTX1133 plus an AXL inhibitor, MRTX1133 plus both AXL and FGFR1 inhibitors, and MRTX1133 plus a SHP2 inhibitor. Cell viability was determined by MTT assay, signaling alterations were analyzed by Western blotting, and apoptosis was evaluated by flow cytometry. In vivo, the efficacy of MRTX1133 combined with a SHP2 inhibitor was validated using cell-derived xenograft (CDX) mouse models. [Results] MRTX1133 monotherapy exhibited variable efficacy across cell lines. Knockdown or pharmacological inhibition of AXL (dual therapy) significantly enhanced the antiproliferative effects of MRTX1133. However, some cell lines showed ERK reactivation, indicating incomplete pathway suppression. Further addition of the FGFR1 inhibition (triple therapy) effectively suppressed ERK reactivation and markedly improved antitumor activity. Moreover, targeting adaptor proteins such as SHP2 amplified the therapeutic efficacy, suggesting that convergence nodes downstream of RTKs play a crucial role in resistance mechanisms. While SHP2 inhibition alone showed limited activity, its combination with MRTX1133 prevented ERK reactivation and achieved substantial growth suppression. [Discussion and Conclusion] Resistance to MRTX1133 in KRAS G12D-mutant tumors is driven by AXL and FGFR1 activation, which can be suppressed through dual or triple combination therapies. Inhibition of adaptor proteins such as SHP2 further provides a strategy to overcome heterogeneous RTK-mediated resistance, supporting combination approaches targeting adaptor signaling.