Fig 2: Screen results reveal cooccurring KRASG12C mutations resistant/sensitizing to G12C inhibitors. (A and B) Z-score analysis demonstrating the enrichment (red) and depletion (blue) of all single amino acid substitution mutants of KRASG12C during treatment using sotarasib (A) or adagrasib (B). The green bars above the heatmaps indicate the positions where the KRASG12C protein has direct contact with the inhibitor. (C) Combined number of all KRAS missense mutations that have been reported in noncancer records of the gnomAD and UK Biobank, along with predicted germline mutations from the FMI. The color key indicates the number of individuals combined from these three databases. (D) Number of all secondary mutations that co-occurred with KRASG12C identified in the FMI solid tumor database. The color key indicates the number of individuals. (E and F) Cell viability validation of the resistant/sensitizing phenotypes in H358 by generating 18 cell lines individually expressing KRAS (G12C + secondary mutation). Cells were treated with either sotarasib or adagrasib at indicated concentrations for 7 d after doxycycline induction. (G) Summary of IC50 shift (LFC before and after induction) and screen Z-score for sotarasib or adagrasib treatments in 18 mutant H358 cell lines as well as the parental line. Green boxes indicate mutations also identified in human genetic databases or FMI inquiries.

Fig 3: miR-202 overexpression inhibits ESC invasion and migration by targeting K-Ras. ESCs were co-transfected with miR-202 mimics or mimics NC and pcDNA-empty vector or pcDNA-K-Ras for 24 h, following which cells were harvested for subsequent experiments. (A) Transfection efficiency of pcDNA-K-Ras was measured using western blotting in ESCs. (B) Protein levels of K-Ras were detected by western blot analysis. (C) Cell invasion and (D) wound healing assay were used to measure the invasion and migration of ESCs. Representative images of (E) cell invasion and (F) wound healing assays. Magnification, ×200. Data represent the mean ± SD from three independent experiments. *P<0.05 and **P<0.01 vs. Control ESCs. ##P<0.01 vs. miR-202 mimics + pcDNA-empty vector. ESCs, endometrial stromal cells isolated from ectopic endometriosis tissues; miR, microRNA; NC, negative control.

Fig 4: Rnf43 -/-; KrasG12D mice have shortened lifespan. A, Cross-mating schedule for generating pancreatic Kras G12D and Rnf43 deficient mice. B, PCR genotyping of mouse tail genomic DNA. #2, 4, 6 and 9 are Rnf43 -/-; Kras G12D; #161 and 162 are Kras G12D; #3 is Rnf43 -/-; #1, 5 and 160 are Rnf43 f/f. C, Immunoblotting of pancreatic lysates from 6-month-old Rnf43-/-; Kras G12D and control mice. D, Kaplan-Meier curves of Rnf43 -/-; Kras G12D mice (red) (n = 42) comparing with Rnf43 f/f (black) (n = 46), Rnf43 -/- (purple) (n = 42) and Kras G12D (blue) (n = 42) mice. Log-rank p < 0.0001

Fig 5: Anti-tumor effects of the KRAS(G12D) inhibitors alone and in combination with anti-PD-L1 antibody.a Nude mice injected with Panc 04.03 cells at Day 0 were later intraperitoneally injected with TH-Z827 (at 10 mg/kg or 30 mg/kg) according to the indicated dosage schedule (blue arrow). The tumor volumes (means ± SEM, n = 10) were measured with digital calipers and assessed using one-way ANOVA followed by Dunnett’s test, **** Padj < 0.0001. b Mice were injected with KPC cells at Day 0 and TH-Z835 at Day 10. The tumor volumes (means ± SEM, n = 10) were assessed using one-way ANOVA followed by Dunnett’s test, ** Padj < 0.01, **** Padj < 0.0001. c Left panel: Immunofluorescence (IF) analysis of pERK and cleaved Caspase-3 in tumor sections from C57BL/6 mice (b) treated with TH-Z835 or vehicle. Scale bar, 20 µm. Right panel: quantification of IF-positive staining. Data are shown as means ± SEM. n = 9, two-tailed Student’s t-test, ** P < 0.01. d Flow cytometry analysis of the immunogenic cell death (ICD) markers CRT and ERp57 and the immune checkpoint protein PD-L1 on the surface of KPC cells after 24 h treatment with TH-Z835. Data are shown as means ± SEM (n = 3), two-tailed Student’s t-test, * P < 0.05, ** P < 0.01, *** P < 0.001. e mRNA expression level of PD-L1 in PANC-1 cells after 24 h treatment with TH-Z835. Data are shown as means ± SEM (n = 3), two-tailed Student’s t-test, **** P < 0.0001. f C57BL/6 mice were injected with KPC cells at Day 0, after which TH-Z835, anti-PD-1 antibody, or a combination therapy (10 mg/kg TH-Z835 and 100 µg per dose anti-PD-1 antibody) was intraperitoneally administered. The tumor volumes (means ± SEM, n = 5) were assessed using one-way ANOVA followed by Dunnett’s test, * Padj < 0.05, **** Padj < 0.0001.