Fig 1: Prediction and design of amino acid substitutions in each amino acid position of KRpep-2d. The complex of ribbon diagram of K-Ras(G12D)GDP and stick model of KRpep-2d (PDB ID: 5XCO) are shown in the center. Purple amino acids are Cys5 and Cys15. Orange amino acids indicate pharmacophore sequence (Pro6–Val14). Cyan amino acids present Arg1–4,16–19 residues. Chemical structures of representative amino acid examples that were predicted to improve biochemical functions of KRpep-2d in Leu7, Ile9, and Tyr11 are listed. In the upper left side box, Arg residues at both termini of the KRpep-2d were excluded for clarity. The figure was made using PyMOL.
Fig 2: Biochemical characters of KS-58. (A) Binding activity of biotinylated peptides to recombinant K-Ras(G12D) protein in ELISA (n = 4, ± SEM). (B) Binding activity of different concentrations (from the highest 3 µM and subsequent threefold dilution) of Biotin-KS-58 to recombinant Ras proteins as measured by ELISA (n = 4, ± SEM). (C) Inhibition activity of peptides to intracellular Ras–effector proteins interactions. Intracellular PPI in the presence or absence of peptides are detected as luminescence, and the PPI is showed as % value (30 µM, n = 4, ± SEM). (D) Proposed K-Ras(G12D)-inhibition mechanism of KS-58.
Fig 3: Screening of KRpep-2d derivatives. (A) K-Ras(G12D)-binding activity of Biotin-KRpep-2d in ELISA (n = 4, ± SEM). (B) Competitive inhibition activities of KRpep-2d and KS-36 against the interaction of plate-coated K-Ras(G12D) and Biotin-KRpep-2d (100 nM) (n = 4, ± SEM). (C) Amino acid structures introduced into KRpep-2d derivatives and their K-Ras(G12D)-binding activities. Positions of the substituted amino acids in KRpep-2d and introduced amino acids are listed. K-Ras(G12D)-binding activities are listed as fold-value compared to the parental KRpep-2d set as 1.0 (n = 4, ± SEM). 2d-amide is an amide bond (Dap5–Asp15) cyclized KRpep-2d, and 2d-nc is main chain cyclized KRpep-2d.
Fig 4: MD simulations of KS-58, KS-58(monocyclic), and KS-58(Ser6Dap). (A) Structural comparison of K-Ras(G12D)GDP-binding mode and K-Ras(G12D)GTP-binding mode of KS-58 (stable snap shots) with K-Ras(G12D)GDP (PDB ID: 4EPR), K-Ras(G12D)GDP/KRpep-2d (PDB ID: 5XCO), K-Ras(G12D)GppNHp (PDB ID: 5USJ), and K-Ras(G12D)GppNHp/KD2 (PDB ID: 6WGN). The magnesium ions are presented as large spheres. Dash lines indicate hydrogen bonds. Arg residues at both termini of the KRpep-2d were excluded for clarity. (B) Lipid membrane accessibility of peptides in MD simulations. Line graph shows moving positions of underlined atoms of Asp8 (Cα–CH2–COOH, red) and Nle3/Anon5 (Cα–CH2–CH2–CH2–CH3, Cα–CH2–CH2–CH2–CH2–CH2–CH2–CH3, green), respectively. KS-58: left panel, KS-58(monocyclic): center panel, KS-58(Ser6Dap): right panel.
Fig 5: Rearrangements at PVT1 stabilize MYC in human tumors:(A) Schematic representation of the role of RAS/MAPK signaling in increased phospho-MYCSer62 (p-MYCSer62) (B) Western blot and quantitative analysis of p-MYCSer62 in D458 stably transduced with sh.Cntrl, shRNA1-PVT1ts and shRNA2-PVT1ts. β ACTIN was used as loading control (p-values obtained by ANOVA). (C, D) Cycloheximide chase assay and quantification of c-MYC in D458 cells transduced with sh.Cntrl (as control) and shRNA1PVT1ts. β-ACTIN was used as a control (p-value obtained by simple linear regression, n=3, for each treatment condition). (E, F) GSEA analysis of MYC target genes upregulated due to activation of c-MYC in D458 stably transduced with shRNA1-PVT1ts (n=3). (G) PVT1 fusion enrichment analysis from RNASeq data from the MB tumors (N= 545). (H) Pan-cancer analysis of unbalanced PVT1 rearrangement with a genomic breakpoint between FFX and HNB. Bar charts on the left column show copy number changes. The vertical axis indicates the segmented copy number ratio, and the dotted line indicates the number of two. The red triangles correspond to the breakpoints. The chromosomal coordinates from the human genome reference (GRCh38) are shown at the bottom. The red and green lines represent the position of the 3′ end of FFX and the 5′ end of HNB, respectively. Representative isoforms of MYC and PVT1 are also shown from GENCODE v.38. The right column shows the screenshots of the IGV at the breakpoint of each case. (I) q-RT-PCR of FFX (red) and HNB (Blue) transcripts from RNA samples derived from G3 (n=30) and G4 (n=20) MB patients (P value determined by unpaired t-test). (J) Proposed model for how AKT and RAS/MAPK signaling pathways converge, enhancing MYC activation due to rearrangements at the PVT1 locus in MYC-driven cancers.
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