Journal: Molecular Cancer

Article Title: Overexpression of wild type RRAS2 , without oncogenic mutations, drives chronic lymphocytic leukemia

doi: 10.1186/s12943-022-01496-x

Figure Lengend Snippet: Overexpression of the unmutated RRAS2 leads to a B cell leukemia in mice. a & b Genomic alterations in the KRAS and RRAS2 genes according to cBioportal for cancer genomics ( http://www.cbioportal.org ). Data represent a combined study of 78,278 patients/81072 samples. The X-axis represents alteration frequency of the gene (colors represent the alteration type). The Y-axis represents different cancer types. c Relative mRNA expression of RRAS2 in different cancers. Data are represented in log2 scale and were obtained from the Pan-Cancer Analysis of Whole Genomes (PCAWG). d Representative images of the relative sizes of spleens from 12 month-old control and Sox2-Cre + mice. e Quantification of the total number of B cells per spleen of 6 month-old control and Sox2-Cre + mice. Data shown correspond to triplicate measurements of one control and three Sox2-Cre mice. Unpaired t-test with Welch’s correction. f Hematoxylin and eosin images of the spleen structure in an 8-month old control mouse and in 4-, 8- and 12-month-old Sox2-Cre + mice. g Two-parameter flow cytometry of the expression of CD21 and CD23, and IgD and IgM, respectively, in B cells in spleens of 8 month-old control and Sox2Cre + mice h Left, two-parameter flow cytometry of the expression of CD5 and IgM in B cells of the blood of 8 month-old control and Sox2-Cre + mice. i, quantification of the number of CD5 + IgM+ B cells in the blood of 40 wk-old control and Sox2-Cre + mice. Data shown correspond to duplicate measurements of four control and six Sox2-Cre mice. Unpaired t-test with Welch’s correction. In all figures, control mice refer to Rosa26- RRAS2 fl/fl without Cre recombinase

Article Snippet: Ten-week Rag2 −/− γc −/− male mice were subcutaneously injected in their left flank with 10 × 10 6 MEC-1 control or MEC-1 RRAS2 knockdown cells as described in [ ], in a mixture with PBS/Matrigel (1:1) (Becton Dickinson).

Techniques: Over Expression, Expressing, Control, Flow Cytometry

Journal: Molecular Cancer

Article Title: Overexpression of wild type RRAS2 , without oncogenic mutations, drives chronic lymphocytic leukemia

doi: 10.1186/s12943-022-01496-x

Figure Lengend Snippet: CD5 + IgM+ B cells are oligoclonal and can be transferred to wild type mice. a Two-parameter flow cytometry of the expression of CD19 and CD5 in blood cells of 35 wk-old control and Rosa26- RRAS2 fl/fl xmb1-Cre (mb1-Cre+) mice. b Quantification of the number of CD5 + IgM+ B cells (CD19+) in the blood of 30–35 wk-old control ( n = 10) and mb1-Cre ( n = 47) mice. Unpaired t-test with Welch’s correction. c Dot plot representation of the quantification of CD5 + IgM+ B cells (CD19+) in the blood of mb1-Cre mice ( n = 14) and its evolution over time. Data points were adjusted to an exponential growth. d Kaplan-Meier survival plot of Rosa26- RRAS2 fl/fl xmb1-Cre (mb1-Cre+) mice ( n = 22) and WT control C57BL/6 J mice ( n = 40) allowed to age in the same housing conditions. Median survival for mb1-Cre = 13.57 months; median survival for WT controls = 32.39 months. P < 0.0001, long-rank Mantel-Cox test and Gehan-Breslow-Wilcoxon test. e Experimental setup for the adoptive transfer experiment. Total B cells from mouse donors bearing the hematopoietic cell marker allele CD45.2 were purified and inoculated i.v. in the tail vein of sublethaly irradiated wild type recipient of the same strain (C57BL/6) but bearing the CD45.1 allele. Donor control mice refer to Rosa26- RRAS2 fl/fl without Cre recombinase 11 wk-old and the donor problem mice corresponded to 16 wk-old Rosa26- RRAS2 fl/fl x mb1-Cre. f Quantification by flow cytometry of the expression of CD45.2 and CD5 within the CD19+ population of inoculated CD45.1+ mice ( n = 8) and bled at the indicated time points. In this figure, control mice refer to Rosa26- RRAS2 fl/fl without Cre recombinase. Two-way ANOVA test. g PCR results of using specific forward oligonucleotides for different V H families and a constant reverse oligonucleotide against the J region of the immunoglobulin heavy chain gene. The specific V H families analyzed in each case are indicated on top of each section of the agarose gel. H 2 O is used as a negative control. In each V H family, BM corresponds to bone marrow cells of a wild-type 12 wk-old mouse, T corresponds to sorted naïve thymocytes (no recombination) from a wild type 6 wk-old mouse, and each column corresponds to the spleen of an individual mb1-Cre mouse of ages between 35 and 40 weeks. h Representative flow cytometry plot showing the presence of GFP low and GFP high populations in the spleen of a 30 wk-old Rosa26- RRAS2 fl/fl xmb1-Cre mouse. i RT-qPCR for the differentially expressed RRAS2 gene in the CD19 + CD5+ GFP high and GFP low populations sorted from spleens of n = 7 30–35 wk-old Rosa26- RRAS2 fl/fl xmb1-Cre mice compared to follicular B cells of n = 6 30 wk-old WT mouse controls. One-way ANOVA test. j Dot plot representation of GFP high CD5+ leukemic B cell evolution in blood from mb1-Cre mice over time, showing each mouse individually ( n = 14). Data points were adjusted to a linear fit. k Paraffin-embedded lung tissue section stained with hematoxylin-eosin, showing representative lymphocytic infiltration in one out of three 35 wk-old Rosa26- RRAS2 fl/fl xmb1-Cre mice. l Percentage of IgM + CD5+ B cells within the lymphoid population infiltrating the lungs of n = 2 35 wk-old Rosa26- RRAS2 fl/fl xmb1-Cre mice compared to n = 2 32 wk-old Rosa26- RRAS2 fl/fl control ones. m Bar plot of the distribution of lymphoid cells between the GFP low and GFP high populations in the lungs and lymph nodes (LN) of 20 wk-old Rosa26- RRAS2 fl/fl xmb1-Cre mice ( n = 2)

Article Snippet: Ten-week Rag2 −/− γc −/− male mice were subcutaneously injected in their left flank with 10 × 10 6 MEC-1 control or MEC-1 RRAS2 knockdown cells as described in [ ], in a mixture with PBS/Matrigel (1:1) (Becton Dickinson).

Techniques: Flow Cytometry, Expressing, Control, Adoptive Transfer Assay, Marker, Purification, Irradiation, Agarose Gel Electrophoresis, Negative Control, Quantitative RT-PCR, Staining

Journal: Molecular Cancer

Article Title: Overexpression of wild type RRAS2 , without oncogenic mutations, drives chronic lymphocytic leukemia

doi: 10.1186/s12943-022-01496-x

Figure Lengend Snippet: RRAS2 -driven CLL harbors a highly conserved pattern of somatic mutations. a Pie chart representation of the somatic mutation distribution from CD19 + CD5+ leukemic cells by chromosome. Spleens from seven Rosa26- RRAS2 fl/fl xmb1-Cre mice were included in this analysis (ages in panel e ). b Ingenuity Pathway Analysis (IPA) diagram of the immunological and hematological neoplastic signature generated from the 270 mutations present in all seven different Rosa26- RRAS2 fl/fl xmb1-Cre mice. c Pie chart representation of the co-occurrence of gene mutations in murine CLL vs human CLL. d Summary of most frequently mutated genes in 1094 cases of human CLL also identified in Rosa26-RRAS2 murine CLL. The panel also shows each gene’s condition in MBL (monoclonal B lymphocytosis) and other human leukemias (BL:Burkitt Lymphoma; MCL: Mantle Cell Lymphoma; DLBCL: Diffuse Large B Cell Lymphoma; CTL:Cutaneous T cell lymphoma; FL: Follicular Lymphoma; MM: Multiple Myeloma; MZL; Marginal Zone Lymphoma; SS: Sezary Syndrome). e Heatmap of mutation rates of 107 mutated genes detected in CD19 + CD5+ leukemic cells from seven Rosa26- RRAS2 fl/fl xmb1-Cre independent mice (age of mice: Ctrl 1–3, 12 wk.; Ctrl 4–6 25 wk.; BCLL-9, 104 wk.; BCLL10–11 and BCLL16–17, 54 wk.; BCLL13–14, 32 wk). Black: homozygous; grey: heterozygous; white: unmutated. f Schematic representation of registered missense mutations in SPEN , ARID1A and AKAP13 in human CLL patients (green dots: missense mutation; black dots: truncating mutations; orange dots: splicing site mutation). Red text indicates mutations found in Rosa26- RRAS2 fl/fl xmb1-Cre mice which are also present in human genes

Article Snippet: Ten-week Rag2 −/− γc −/− male mice were subcutaneously injected in their left flank with 10 × 10 6 MEC-1 control or MEC-1 RRAS2 knockdown cells as described in [ ], in a mixture with PBS/Matrigel (1:1) (Becton Dickinson).

Techniques: Mutagenesis, Generated

Journal: Molecular Cancer

Article Title: Overexpression of wild type RRAS2 , without oncogenic mutations, drives chronic lymphocytic leukemia

doi: 10.1186/s12943-022-01496-x

Figure Lengend Snippet: 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- RRAS2 fl/fl xmb1-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 × 10 6 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

Article Snippet: Ten-week Rag2 −/− γc −/− male mice were subcutaneously injected in their left flank with 10 × 10 6 MEC-1 control or MEC-1 RRAS2 knockdown cells as described in [ ], in a mixture with PBS/Matrigel (1:1) (Becton Dickinson).

Techniques: Activation Assay, Binding Assay, Expressing, Inhibition, Modification, Phospho-proteomics, De-Phosphorylation Assay, Protein Binding, RNA Binding Assay, Translocation Assay, Ubiquitin Proteomics, Western Blot, Two-Dimensional Gel Electrophoresis, Electrophoresis, Purification, Immunoprecipitation, Control, Incubation, Molecular Weight, Membrane, Quantitative RT-PCR, Transduction, shRNA, In Vitro, Proliferation Assay, Knockdown, Injection, Flow Cytometry

Journal: Molecular Cancer

Article Title: Overexpression of wild type RRAS2 , without oncogenic mutations, drives chronic lymphocytic leukemia

doi: 10.1186/s12943-022-01496-x

Figure Lengend Snippet: RRAS2 expression is increased in CLL patients and associated to more aggressive disease. a Box and whiskers plots showing all points and median value of RT-qPCR data of RRAS2 mRNA expression in PBMCs from healthy individuals and from MBL and CLL patients. Expression data is normalized to the mean value of healthy blood donor values ( n = 17). Two-tailed unpaired t-test with Welch’s correction. b RT-qPCR analysis of RRAS2 mRNA expression compared to the lymphocyte count in the blood of CLL patients. Patients are classified according the number of total lymphocytes in blood (in ranges, in the x-axis). Values represent the mean ± SEM. All datapoints are represented. One-way ANOVA test. c RT-qPCR analysis of RRAS2 expression compared to the percentage of CD19 + CD5+ cells (in ranges in the x-axis) in the blood of CLL patients. All datapoints are represented. One-way ANOVA test. d Box and whiskers plots showing all points and median value of RT-qPCR data of RRAS2 mRNA expression in blood from MBL and CLL patients. Expression data is normalized to the mean value of healthy blood donor values. Two-tailed unpaired t-test with Welch’s correction. e Box and whiskers plots showing all points and median values of RT-qPCR data of RRAS2 mRNA expression versus age of the patient at diagnosis (in 10-year intervals) and divided according to the diagnosis as CLL or MBL. One-way ANOVA test. f Box and whiskers plot showing all points and median value of RT-qPCR data of RRAS2 mRNA expression in CLL patients classified according to the expression of a mutated or an unmutated IgH gene. ns, not significant (two-tailed unpaired t-test with Welch’s correction). g Contigency test of the distribution of mutated and unmutated IgHV gene within MBL and CLL patients in our study cohort. h Box and whiskers plot showing all points and median value of RT-qPCR data of RRAS2 mRNA expression in our study cohort classified according to MBL vs CLL diagnosis and having mutated or unmutated IgHV. i, Box and whiskers plots showing all points and median value of RT-qPCR data of RRAS2 mRNA expression in blood from all patients in our study cohort classified according to male or female sex. Two-tailed unpaired t-test with Welch’s correction. j Pie chart diagram of the relative fold variations in the expression of RRAS2 mRNA in male and female CLL patients, classified in fold intervals. 1 is the mean of expression in healthy blood donors. k Box and whiskers plot showing all points and median value of RT-qPCR RRAS2 mRNA expression in blood from healthy individuals and CLL patients, and in spleen B cells from control mice, mb1-Cre mice, and Sox2-Cre mice. One-way ANOVA test). In all panels, when there is no distinction between MBL and CLL patients, CLL refers to both groups

Article Snippet: Ten-week Rag2 −/− γc −/− male mice were subcutaneously injected in their left flank with 10 × 10 6 MEC-1 control or MEC-1 RRAS2 knockdown cells as described in [ ], in a mixture with PBS/Matrigel (1:1) (Becton Dickinson).

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Biomarker Discovery, Control

Journal: Molecular Cancer

Article Title: Overexpression of wild type RRAS2 , without oncogenic mutations, drives chronic lymphocytic leukemia

doi: 10.1186/s12943-022-01496-x

Figure Lengend Snippet: The C allele at SNP rs8570 is associated with several parameters of more aggressive disease in CLL patients. a Schematic representation of the rs8570 location in the 3’UTR of RRAS2 mRNA. b Representative examples of the Sanger sequencing chromatograms showing the 3 possibilities of allele dosage at the position of rs8570. The rs8570 position is highlighted by a red rectangle. c Classification of patient samples according to the genotype at SNP rs8570 detected by a dedicated RT-qPCR strategy using specific primers for each of the 2 alleles at that position. The patients sequenced as GG, GC or CC with Sanger sequencing are plotted with dark blue, purple and orange dots, respectively. d Pie chart representation of the Observed versus the Expected distribution of GG, GC and CC genotypes at position of the SNP rs8570 in our study cohort of CLL patients. e Box and whiskers plots showing all points and median value of RT-qPCR RRAS2 mRNA expression in blood from all patients in our study cohort classified according to GG, GC or CC genotype at position of the SNP rs8570. Ttwo-tailed unpaired t-test with Welch’s correction. f Box and whiskers plots showing all points and median value of RT-qPCR RRAS2 mRNA expression in blood from all patients in our study cohort harboring none (GG) or at least one (GC and CC) 124C at position of the SNP rs8570. Two-tailed unpaired t-test with Welch’s correction. g Box and whiskers plots showing all points and median value of total lymphocyte count in the blood of patients harboring two G alleles or at least one C. Two-tailed unpaired t-test with Welch’s correction. h Box and whiskers plots showing all points and median value of the percentage of CD19+ B cells in the blood of patients harboring two G alleles or at least one C. Two-tailed unpaired t-test with Welch’s correction. i Box and whiskers plots showing all points and median value of the percentage of CD19 + CD5+ leukemic B cells in the blood of patients harboring two G alleles or at least one C. Two-tailed unpaired t-test with Welch’s correction. j Box and whiskers plots showing all points and median value of the platelet count in the blood of patients harboring two G alleles or at least one C. Two-tailed unpaired t-test with Welch’s correction. k Contigency test of the distribution of patients diagnosed as having MBL or CLL according to a GG or GC + CC genotype. l Contigency test of the distribution of all samples in our study cohort according to presenting or not chromosomal alterations by FISH and a GG or GC + CC genotype. m Contigency test of the distribution of samples in our study cohort according to presenting normal caryotype or presenting deletions in chromosome 11q or in chromosome 17p (or both) and a GG or GC + CC genotype. n Contingency test of the distribution of all samples in our study cohort according to presenting or not mutated IgHV and a GG or GC genotype. o Contingency test of the distribution of all samples in our study cohort according to male or female sex and a GG or GC + CC genotype

Article Snippet: Ten-week Rag2 −/− γc −/− male mice were subcutaneously injected in their left flank with 10 × 10 6 MEC-1 control or MEC-1 RRAS2 knockdown cells as described in [ ], in a mixture with PBS/Matrigel (1:1) (Becton Dickinson).

Techniques: Sequencing, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: 3D Bioprinting Allows the Establishment of Long-Term 3D Culture Model for Chronic Lymphocytic Leukemia Cells

doi: 10.3389/fimmu.2021.639572

Figure Lengend Snippet: 3D bioprinting strategy. Schematic representation of our 3D bioprinting strategy: CLL cell line MEC1 or CLL primary B cells were used. The cells are pre-mixed with hydrogels specifically designed to support cellular adhesion and functions, as well as high printability and biocompatibility. Once the geometry of the 3D bioprinted scaffolds is defined, the cells are printed, encapsulated in the hydrogel matrix, crosslinked with CaCl 2 and placed in a culture plate. The constructs are then processed to extract the RNA, perform histological analyses, assess cell surface markers and cell viability.

Article Snippet: Indeed, we also tested MEC1 cells printability in the laminin hydrogel series but we didn’t observe an improvement in cell survival in comparison with CELLINK Bioink (data not shown).

Techniques: Construct

Journal: Frontiers in Immunology

Article Title: 3D Bioprinting Allows the Establishment of Long-Term 3D Culture Model for Chronic Lymphocytic Leukemia Cells

doi: 10.3389/fimmu.2021.639572

Figure Lengend Snippet: CLL cells are viable and homogeneously distributed in the hydrogel matrix. MEC1 and/or MEC-GFP cell lines were used to define the optimal number of cells and hydrogel quantity to be printed. (A) Representative images of Live/Dead assay of 3D bioprinted MEC1 cell line acquired with Axio Observer Zeiss fluorescent microscope. Green cells are alive cells; red cells are dead cells. An example of a grid scheme used to quantify alive and dead cells is shown on the top right of each image. (B) The graph shows quantification of alive (green column portion) and dead (red column portion) 3D bioprinted MEC1 cells. (C) Representative z-stack images of 3D bioprinted MEC-GFP cells in the scaffold overtime showing their distribution. Images were obtained with Axio Observer Zeiss fluorescent microscope. (D) The graph shows quantification by cell count, at different time points (day 0-7-14-21), of viable MEC-GFP cells found outside the 3D bioprinted scaffold. Data are represented as mean ± SEM, n=3 (B) and n=5 (D) .

Article Snippet: Indeed, we also tested MEC1 cells printability in the laminin hydrogel series but we didn’t observe an improvement in cell survival in comparison with CELLINK Bioink (data not shown).

Techniques: Live Dead Assay, Microscopy, Cell Counting

Journal: Frontiers in Immunology

Article Title: 3D Bioprinting Allows the Establishment of Long-Term 3D Culture Model for Chronic Lymphocytic Leukemia Cells

doi: 10.3389/fimmu.2021.639572

Figure Lengend Snippet: Differences in gene expression between 3D bioprinted and 2D cultured MEC1 cells: clustering analysis and top genes validation. (A) PCA plot built using the 500 most variable genes (in RPKM). (B) Heatmap of the 500 most variable genes (in RPKM), clustering row (genes) and columns (samples). Expression is scaled. (C) Intersection between up-regulated (left) and down-regulated (right) genes between the comparisons 3D_7d_vs_2D and 3D_14d_vs_2D. (D) Vulcano plot for the comparison 3D_7d_vs_2D highlighting the 20 most significantly up-regulated (red) and down-regulated (blue) features for the FDR filter. (E) Heatmap summarizing genes among the first 100 most modulated in the comparison 3D_7d_vs_2D. (F) The graphs show mRNA levels of n=8 genes selected among the first 100 most modulated in the comparison 3D_7d_vs_2D which have been validated by RT-qPCR ( AICDA, SELL, CXCR3, CCL22, HCLS1, PIM3, MYC, BCL2 ). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are represented as mean ± SEM, n=3 MEC1 cell line samples. Student’s t-test was performed for statistical analysis.

Article Snippet: Indeed, we also tested MEC1 cells printability in the laminin hydrogel series but we didn’t observe an improvement in cell survival in comparison with CELLINK Bioink (data not shown).

Techniques: Gene Expression, Cell Culture, Biomarker Discovery, Expressing, Comparison, Quantitative RT-PCR