Fig 2: Spectrum of MAP2K1 variants, the structure of the MEK1 protein, and a schematic overview of the MANO method. A, Patterns of MAP2K1 mutations in various cancers. The bar charts illustrate the number of tumor samples with MAP2K1 mutations in the COSMIC database for each cancer type. Mutation types are coded in green (missense mutation), black (nonsense mutation), light green (inframe deletion), and pink (frameshift). B, Circle chart showing the percentage of MAP2K1 mutation types selected in this study. C, Categorization of MEK1 mutations in the crystal structure location. Helix A (blue), Helix C (orange) as well as β3-αC loop and αC-β4 loop (pink) are highlighted. D, Distribution of MAP2K1 mutations detected in the COSMIC database. E, Schematic representation of the MANO method. 3T3 cells and A375 cells were infected with recombination retrovirus-expressing MAP2K1 variants with individual 10 bp barcodes. The transforming activity of MAP2K1 variants in 3T3 cells was evaluated using a focus formation assay. Equal numbers of stabilized, transduced cells were mixed and cultured with two types of medium in 3T3 cells or treated with drugs in A375 cells. Genomic DNA was extracted from the cells at the end of each experiment. The barcode sequences were PCR-amplified and subjected to deep sequencing to quantitate the relative abundance. F, Construction of MEK-dependent cells and evaluation of MEKi sensitivity against MEK1 variants. Both cell lines were transfected with MAP2K1 variants by retrovirus using pCX6 plasmid to generate MEK-dependent cell lines. MEK1-dependent 3T3 cells were assessed for drug sensitivity and RAF dependency, and sensitivity to MEKis was evaluated in A375 cells transfected with MAP2K1 variants, with or without RAF inhibitors.

Fig 3: The FGFR-1 receptor regulates HGF secretion, and it is targeted by miR-16. a CAF154-hTERT fibroblasts (3 × 105 cells/well) were serum-starved for 48 h and then stimulated with increasing doses of FGF-2. CM was collected after 24 h and analyzed by ELISA to evaluate the levels of secreted HGF. b CAF154-hTERT fibroblasts were transfected with the non-targeting miR-C, miR-16, a control siRNA, or a siRNA targeting FGFR-1 or HGF. After 72 h, the CM was collected and, together with a non-conditioned medium, used to stimulate the A549 cells. Western blot was performed on transfected CAF154-hTERT fibroblasts and on stimulated A549 cells to detect the activated state of cMet and its total levels, the levels of FGFR-1, and the activated form of ERK1/2. Actin is shown as a loading control. c Luciferase assay performed as in Fig. 2e, by using 293 T cells transfected with the pMirTarget FGFR-1 3'UTR and a Renilla-expressing plasmid. The FGFR-1 3'UTR displays two putative miR-16 binding sites (Additional file 3: Figure S3). d Levels of HGF in CM of CAF154-hTERT fibroblasts transfected with the control miR-C and miR-16 or a control siRNA and siRNA specific for FGFR-1 or HGF. Non-conditioned medium is shown as a negative control. e CAF154-hTERT fibroblasts were treated with 10 µM FGFR-1 inhibitor SU5402 and CM collected at the indicated times to evaluate HGF concentration by ELISA. The graph is representative of two independent experiments. f Correlation between the expression of HGF and FGFR-1 in primary fibroblasts derived from lung cancer patients and expressing high levels of HGF (R2 = 0.3607, slope = 0.3797 ± 0.1054). g CAF154-hTERT fibroblasts were transduced with lentiviral particles to stably express FGFR-1 and transfected with control miR-C and miR-16. Western blot was performed 72 h after transfection to detect MEK1/2 and FGFR-1 levels and the activated form of ERK1/2. Actin is shown as a loading control. h ELISA performed to evaluate the levels of HGF in the CM of cells transfected as described in g

Fig 4: NUPR1 promotes angiogenesis through a PDGFA/MEK/ERK signaling cascade. (A, B) The expression levels of total and phosphorylated forms of PDGFRa, MEK1/2, and ERK1/2 proteins in HUVECs cultured with CM from NUPR1-overexpressing, -knockdown, or control Mahlavu and Huh7 cells for 48 h were analyzed by western blotting. GAPDH was used as a loading control. (C) HUVECs were pretreated with DMSO (vehicle control) or the ERK inhibitor U0126 (10 µM) for 1 h, then seeded on matrigel together with CM from NUPR1-overexpressing and control Mahlavu cells for 16 h. (D) Chick embryo were treated with DMSO or U0126 for 1 h, after which a mixture of matrigel and CM from NUPR1-overexpressing or control Mahlavu cells was implanted on the chick embryo. After a 4-day incubation, the number of vessel branching points were quantified by image analysis. (E) Serially deleted pA3TK-PDGFA promoter-report constructs and pcDNA3-NUPR1 expression plasmid were co-transfected in 293TN cells, after 24 h, cells were lysed and promoter activity was determined (left panel). Wild type and mutant forms of PDGFA promoter constructs were co-transfected with pcDNA3-NUPR1 plasmid in 293TN cells, after 24 h, cells were lysed and promoter activity was determined (right panel). (F) NUPR1-overexpression Mahlavu cell lysates were immunoprecipitated with nonspecific rabbit IgG or antibodies against NUPR1. GAPDH promoter region was used as negative control, respectively. Bar plot represents means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA).

Fig 5: RREB1-BioID reveals a mechanism of epigenetic histone regulation.a RREB1 interactors identified by BioID and classified according to information compiled at NCBI-Gene, Gene Cards, and literature searches. Dot color intensity represents spectral sum counts (SpecSum) as indicated in the scale. b Western blot analysis of histone modifications in HEK293 cells expressing control shRNA (Con) or sh1-RREB1 (sh1)and c histone modifications in WT and Rreb1 + /− MEFs and 6 month LV total lysates (Hearts). H3 and ERK are loading controls. All blots representative of n = 3 independent experiments. d ChIP analysis of H3K4me2 modification at the indicated promoters. Upper panels show the gene track view of H3K4me2 ChIP-seq (ENCODE, Broad Institute, K562 cells) at the FGFR4, HRAS and MAP2K2 loci. Red arrows point to the RRE. Lower graphs show ChIP-PCR of the indicated histone marks at promoters from HEK293 cells expressing sh-control (Con) or sh1-RREB1 (sh1, n = 3). e ChIP analysis of histone modifications at the indicated promoters in WT and Rreb1 + /− MEFs and (f) WT and Rreb1 + /− 6 month hearts (n = 3; *p < 0.01, **p < 0.001, ***p < 0.0001 two-tailed student’s t-test). For panels d, e, f error bars presented as mean values ± SD. Source data are provided as a Source Data file.