Figure 4 from E2F1-Associated Purine Synthesis Pathway Is a Major Component of the MET-DNA Damage Response Network

Transcriptional effectors involved in regulation of purine synthesis pathway. A, Complementary transcriptomic analysis of purine synthesis pathway regulation effectors in EBC-1 and GTL-16 cells. MYC, E2F1, E2F2, and both large (RRM1) and small (RRM2) ribonucleotide reductase subunits were downregulated upon METi in MET-driven EBC-1 and GTL-16 cell lines (N = 2). Differences between the gene’s expression (METi vs. control) are shown. B, Quantitative real-time PCR assessment of mRNA levels of E2F-1 and E2F-2 upon METi in MET-driven (EBC-1 and GTL-16) and non-MET-driven (HCC821 and H820) cell lines (N = 3, unpaired Student t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). C, Whole-cell lysates of MET-driven cell lines EBC-1 and GTL-16 were subjected to Western blotting using pMET (Tyr1234/5) and E2F1 antibodies following their treatment with vehicle or 50 nmol/L tepotinib for 24, 48, and 72 hours (left). Protein levels of GART and E2F1 were assessed by Western blotting in MET-driven MKN45, KATOII, and SNU-638 cells (right). ß-Actin was used as a loading control. Representative images of N = 3 independent experiments are shown. D, E2F1 expression in EBC-1 (left) and HCC827 (right) tumor xenografts treated by vehicle or METi. Representative images (left) and quantification of E2F1-positive area (right) are provided [EBC-1: N = 5 (control), N = 5 (tepotinib); HCC827: N = 6 (control), N = 4 (tepotinib); unpaired Student t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001]. E, mRNA levels of RRM1 and RRM2 were assessed by real-time PCR following METi (24 hours, 50 nmol/L), irradiation (10 Gy, 1-hour time-point post IR), and upon combinatory treatment in GTL-16 and EBC-1 cells (N = 3, unpaired Student t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). F, Comparisons of E2F1, RRM1, and RRM2 transcript distribution in normal tissue vs. primary tumor in lung, colorectal, stomach, and head and neck cancer TCGA datasets. G, dNTP levels in MET-driven cell lines GTL-16 and EBC-1 and non-MET-driven cell lines HCC827 and H1648 in response to METi by tepotinib (24 hours, 50 nmol/L), N = 3, unpaired Student t test, (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).