Journal: Clinical Cancer Research
Article Title: Overcoming Adaptive Resistance to KRAS G12D Blockade in Pancreatic Cancer through Vertical Pathway Inhibition
doi: 10.1158/1078-0432.CCR-25-1788
Figure Lengend Snippet: FGFR and EGFR mediate distinct adaptive mechanisms of mutant-selective KRASi-mediated signaling rebound in different PDAC subtypes. A, A panel of PDAC cells with KRAS G12C ( n = 1 cell line) or KRAS G12D ( n = 11 cell lines) mutations was treated with MRTX849 or MRTX1133, respectively (M; 10, 30, 100, and 300 nmol/L), alone or combined with CTX (10 μg/mL) at indicated concentrations for 72 hours. Cell viability was quantified by the CellTiter-Glo assay. B, Western blot analysis of EMT markers in untreated KRAS G12C -mutant ( n = 1 cell line) and KRAS G12D -mutant ( n = 14 cell lines) PDAC cell lines. Cell subtypes, mesenchymal or epithelial, were characterized. A subset of PDAC lines ( n = 4) exclusively expressed mesenchymal markers (vimentin and Zeb1) while not expressing the epithelial marker E-cadherin and were identified as mesenchymal lines, including MIAPaCa-2, KP-4, PANC-1, and SW1990. The rest of the cell lines were characterized as epithelial, expressing E-cadherin. β-Actin was used as a loading control. C, Comparison of maximal inhibition indices (I max ) in 3 mesenchymal and 10 epithelial KRAS G12D -mutant PDAC lines. D, Heatmap of DUSP6 expression across KRAS-mutant PDAC cell lines. A library of KRAS-mutant PDAC cell lines ( n = 13) was treated with KRASi (MRTX849 for KRAS G12C -mutant MIAPaCa-2, MRTX1133 for other KRAS G12D -mutant lines) combined with pan-FGFRi (BGJ, 1 μmol/L), an EGFR monoclonal antibody (CTX, 10 μg/mL), or other RTK inhibitors (Supplementary Table S2) for 72 hours. DUSP6 was probed to represent RAS activity at the mRNA level using quantitative real-time PCR analysis and β-actin as a loading control. In each line, the expression quantification of DUSP6 for each treatment was normalized to that of KRASi treatment at 72 hours. Mesenchymal and epithelial PDAC cell lines were characterized above. E, Phospho-ERK was probed to represent RAS protein activity in mesenchymal KRAS-mutant PDAC cell lines after indicated drug treatment, including one KRAS G12C -mutant line MIAPaCa-2 and two KRAS G12D -mutant PDAC lines KP-4 and PANC-1. The other treatments included DMSO control (CTL), KRASi (MRTX, MRTX849, or MRTX1133, 100 nmol/L), inhibitors of RTKs (1 μmol/L), and paired combinations, for 72 hours (except for the “4-hour” MRTX849 treatment in a MIAPaCa-2 sample). The inhibitors of RTK included afatinib (AFA, EGFR/HER2/HER4 inhibitor), BGJ (FGFR1-3 inhibitor), crizotinib (CRI, c-Met/ALK inhibitor), CTX (EGFR antibody), lapatinib (LAP, EGFR/HER2 inhibitor), and linsitinib (LIN, IGF-1R inhibitor). Expression quantification was normalized to that of KRASi treatment at 72 hours (normalized quantification is shown below each band). Data shown are mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.
Article Snippet: Human primers of TaqMan Gene Expression Assays were purchased from Thermo Fisher Scientific, including DUSP6 (Hs04329643_s1), ETV4 (Hs00383361_g1), ETV5 (Hs00927578_g1), SPRY2 (Hs01921749_s1), SPRY4 (Hs01935412_s1), VIMENTIN (Hs00958111_m1), SNAI1 (Hs00195591_m1), and CDH1 (Hs01023895_m1). β - Actin (4326315E, Thermo Fisher Scientific) was used as an endogenous control.
Techniques: Mutagenesis, Glo Assay, Western Blot, Expressing, Marker, Control, Comparison, Inhibition, Activity Assay, Real-time Polymerase Chain Reaction
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