Fig 2: Phospho-ERK activation and WT Kras mRNA levels in syngeneic allograft tumor model.(A) Quantification of phospho-ERK1/2 (normalized to total ERK1/2 and β-actin) as assessed via Western blot. Exo-Fect + KrasG12D synthetic guide RNA1, n = 7 mice; n = 8 mice for all other groups. Data are presented as mean ± SEM. Kruskall–Wallis with Dunn’s multiple comparison test performed. (B) Quantitative PCR for WT Kras mRNA. Data are normalized to 18s and Exosomes + Exo-Fect control and presented as mean ± SEM. n = 5 mice per group. One-way ANOVA with Tukey’s multiple comparison test performed. (C) Full-length blots for phospho-ERK1/2, total ERK1/2, and β-actin. *P < 0.05, **P < 0.01.Source data are available for this figure.

Fig 3: Wild-type Kras deletion alters progression and metastasis of KPN tumours.a Kaplan–Meier survival curve of Kras+/G12D Trp53fl/fl Rosa26N1icd/+ (KPN) and Krasfl/G12D Trp53fl/fl Rosa26N1icd/+ (KPN KF) mice aged until clinical endpoint (KPN, n = 10, 5M, 5F, KPN KF, n = 12, 6M, 6F) ***P = 2 × 10-4, log-rank (Mantel-Cox) test. b Representative H&E images of primary tumour and metastasis (liver) of mice from (a). Scale, 200 μm. c Incidence of metastasis (%) in KPN and KPN KF mice aged until clinical endpoint (KPN, n = 11, 6M, 5F; KPN KF, n = 11, 5M, 6F) ****P = 1 × 10-15, two-tailed chi-square test. d GSEA of hallmark WNT-β-Catenin signalling in KPN KF tumours compared to KPN. X-axis shows normalised enrichment score (NES), and the P value (computed and corrected for multiple testing using the Benjamini–Hochberg procedure). e Representative Notum in situ hybridisation (ISH) in KPN and KPN KF tumours. Scale, 100 μm. f Boxplots showing Notum (ISH) positive cells per tumour (%) of KPN and KPN KF tumours (KPN, n = 7, 2M, 5F; KPN KF, n = 5, 2M, 3F). **P = 0.0013. g Dotplot showing neutrophils percentage in systemic blood of KPN and KPN KF mice aged to clinical endpoint (KPN, n = 8, 6M, 2F; KPN KF, n = 7, 5M, 2F). ***P = 0.0006. h Representative Cxcl5 ISH in KPN and KPN KF tumours. Representative of five mice per genotype. Scale, 100 μm. i Boxplots showing Cxcl5 positive cells per tumour (%) of KPN and KPN KF tumours (KPN, n = 6, 2M, 4F; KPN KF, n = 5, 2M, 3F). *P = 0.0411. j Volcano plot of differentially expressed genes in organoids derived from KPN KF tumours. k Relative expression of Tgfβ ligands and chemokines from organoids derived from KPN and KPN KF tumours. Data are mean ± s.e.m. KPN, n = 3, 3M; KPN KF, n = 4, 1M, 3F. l Representative αSMA (alpha-smooth muscle actin) and Sirius Red staining of KPN and KPN KF tumours. Scale, 200 µm. m Boxplots showing Sirius Red positivity (%) and αSMA positive cells (%) of ageing or vehicle-treated KPN and KPN KF tumours (KPN n = 7, 2M, 5F; KPN KF n = 8, 4M, 4F). *P = 0.0103. f, i and m boxes depict interquartile range, central line indicates median and whiskers indicate minimum/maximum values. f, g, i and m are one-way Mann–Whitney U tests. Source data are provided as a Source Data file.

Fig 4: REDD1 deficiency cooperates with KRAS to drive tumor progression and metastatic dissemination. (A) Kaplan-Meier analysis of p48-Cre;KrasG12D; Redd1−/− (p48KR) mice (n = 6, median survival = 364 d) and p48-Cre;KrasG12D;Redd1+/+ (p48K) mice (n = 12, median survival = 610.5 d). All animals euthanized exhibited PanIN and/or PDAC. P = 0.0005 by log-rank test. (B, top) representative gross photographs depicting enlarged, nodule-studded pancreas and enlarged spleen (left) and liver with metastatic foci (right) from 17-mo-old p48KR mice. (Bottom) Hematoxylin and eosin (H&E)-stained tissue sections from the indicated genotypes. The right panels represent higher magnification of the respective left panels. p48KR mice show PDAC and liver metastasis (indicated by an asterisk) compared with p48K mice that show early PanIN histology. Scale bars: left, 50 µm; right 20 µm. (C) Summary of histological findings of PDAC and metastasis including those reported in A and additional mice sacrificed at 11 mo. Total n = 20 mice analyzed per genotype. (D) Kaplan-Meier analysis of KrasG12D; Redd1+/+ and KrasG12D; Redd1−/−mice after intratracheal adenoviral Cre (AdK and AdKR mice, respectively). AdK mice (median survival = 229 d, n = 7); AdKR mice, median survival = 196 d, n = 13. Survival times are after infection. P = 0.011 by log-rank test. (E, left) Representative H&E-stained sections of whole-mount lung tissues from a pair of age- and sex-matched AdK and AdKR mice 20 wk after intratracheal delivery of Cre. Tumors are indicated by arrows. Scale bars, 2 mm. (Right) Quantification of lung tumor burden per lung lobe from mice of the denoted genotypes. Each dot indicates one lobe (n ≥ 2 mice for each time point per genotype). Horizontal lines indicate mean ± SEM. (*) P = 0.01, by two-tailed t-test. (F) Necropsy of AdKR mice euthanized 28 wk after intratracheal Cre treatment showing metastasis to kidney (K), diaphragm (D), and pancreas (P). H&E-stained sections of primary lung tumor and metastatic foci (indicated by an asterisk) are shown. Scale bars, 100 µm. See also Supplemental Figure S1.

Fig 5: Development of a KRAS saturation mutagenesis screen for KRAS G12C inhibitors in NCI-H358 (NSCLC) cell model. (A) Schematic diagram of the saturation mutagenesis screen. (B) Western blot analysis of engineered H358 cell lines harboring doxycycline-induced KRASWT, KRASG12D, and KRASG12C/Q61H variants. KRAS relative protein level was quantified from the gel image. (C) Cell viability assays for parental H358 and engineered H358 cell lines harboring KRASG12D or KRASG12C/Q61H in culturing conditions with or without doxycycline induction treated with sotarasib and adagrasib for 7 d (shown as mean ± SD, n = 3). (D) Cell growth curves of H358 parental and engineered H358 cells expressing KRASG12D or KRASG12C/Q61H under continuous treatment using sotarasib (750 nM) or adagrasib (750 nM) for 16 d. Cell confluency images were acquired every 6 h, n = 2.