Fig 1: MAPK activity regulates cellular identity in NKX2-1-negative tumors.(A) Transcriptome analysis comparing control BPN cells with MAPKi-treated BPN tumor cells. Heatmap depicts gastric surface mucous cell markers whose expression diminished versus gastric chief cell or tuft cell markers whose expression increased in MAPK-inhibitor-treated BPN cells relative to untreated. adjP <0.05 for each comparison. RNA-seq data was derived from tdTomato-expressing tumor cells FACS-sorted from BPN mice. Six weeks after tumor initiation, mice were treated for 1 week with control chow (n = 4) or chow containing BRAFV600E inhibitor plus MEK inhibitor (n = 5). (B) GSEA against chief and tuft cell signatures. (C) Violin plots of single-cell sequencing data showing expression levels of Gkn1, Tff1, Tff2, Cym, Pgc, Pga5, and Pou2f3 transcripts in BPN tumor clusters 1–3 and highlighting key drug-mediated cell-identity changes. (D) IHC for a gastric chief cell marker (Pepsinogen C) and a tuft cell marker (POU2F3) on LUAD sections derived 7 weeks following tumor initiation with PGK-Cre lentivirus (5 × 103 pfu/mouse). At 6 weeks, BrafLSL-V600E/+;Trp53f/f;Nkx2-1f/+;Rosa26LSL-tdTomato/LSL-tdTomato and BrafLSL-V600E/+;Trp53f/f;Nkx2-1f/f;Rosa26LSL-tdTomato/LSL-tdTomato mice were given control or MAPK-inhibitor chow for 1 week. Also shown are sections of normal stomach epithelium as positive controls. Note that in hyperplasia that arise from Nkx2-1 deletion in distal lung without concomitant oncogenic activation also upregulate these lineage-restricted markers. Nkx2-1f/f mice were infected with Ad5-Spc-Cre (109 pfu/mouse). Lungs were harvested 16 weeks later. Scale bar: 100 µm. (E) Chief cell markers are rapidly induced in tumor organoids treated with 50 nM Cobimetinib. qRT-PCR analysis of chief cell markers Cym and Pgc on RNA isolated from drug/DMSO-treated organoids for the indicated times. Data are mean ± S.D. ****p<0.0001, ***p<0.001, **p<0.01, ns = not significant by Student’s t-test. A representative experiment of three is shown. Bottom plot: Measurement of viability in organoid cultures treated with vehicle or Cobimetinib using 3D CellTiter-Glo Luminescent cell viability assay at the indicated timepoints. Data are mean ± S.D. A representative experiment of three is shown.