Fig. 4
From: Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia
R-RAS2 is associated with the BCR in leukemic cells and is required for human CLL cell proliferation. a Ingenuity Pathway Analysis (IPA) of differentially expressed genes in leukemic versus normal follicular B cells. Pink-filled symbols: upregulated genes. Green-filled: downregulated genes. Double circle: protein complex; horizontal ellipse: transcription regulator; vertical ellipse: transmembrane receptor, diamond: enzyme; trapezium: transporter; triangle: phosphatase; inverted triangle: kinase; circle: other. Relationship labels: A: activation; B: binding; C: causation; CO: correlation; E: expression; EC: enzyme catalysis; I: inhibition; L: molecular cleavage; LO: localization; M: biochemical modification; miT: microRNA Targeting; P: phosphorylation/dephosphorylation; PD: protein-DNA binding; PP: protein-protein binding; PR: protein-RNA binding, RB: regulation of binding; RE: reaction; T: transcription; TR: translocation; UB: ubiquitination. b Western Blot of two-dimensional (2D) gel electrophoresis under non-reducing/reducing conditions of purified CD19 + CD5+ cells from the spleen of a 45 wk-old Rosa26-RRAS2fl/flxmb1-Cre mouse. Left: co-immunoprecipitation of R-RAS2-interacting components using an anti-HA antibody. Right: isotype IgG2b control. Membranes were serially incubated with streptavidin-PO, anti-IgM and anti-HA. The positions of molecular weight markers are indicated to the left of each membrane. The blue line represents the mobility of proteins without inter-chain disulfide bridges. c Schematic representation of R-RAS2 interaction with the B-cell receptor and its downstream effects on canonical BCR signaling and the PI3K-Akt-mTOR pathway. d RT-qPCR analysis of RRAS2 expression in MEC-1 cell line transduced with scrambled control lentiviral particles (blue), shRNA for human RRAS2 (red), and expression of RRAS2 in healthy peripheral blood lymphocytes (grey). One-way ANOVA test. e In vitro proliferation assay of RRAS2 knockdown MEC-1 cells (red) and control (blue). Data show means ± SEM from three biological replicates. Two-way ANOVA test, row factor. f Tumor growth in immunodeficient mice. 10 × 106 transduced MEC-1 cells per animal were subcutaneously injected. Data show means ± SEM from a total of eight mice. Two-way ANOVA test, row factor. g & h Flow cytometry analysis of Raf-ERK pathway and proximal BCR signaling components ERK (T202/Y204), VAV (Y174), BTK (Y223) and BLNK (Y96) phosphorylation in MEC-1 cell line transduced with scrambled control lentiviral particles (blue), shRNA for human RRAS2 (red) and treated with Src-kinases inhibitor PP2 20 μM, Btk inhibitor Ibrutinib 10 μM and MEK inhibitor U0126 10 μM