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

[Background]: Chronic lymphocytic leukemia (CLL) is the most frequent, and still incurable, form of leukemia in the Western World. It is widely accepted that cancer results from an evolutionary process shaped by the acquisition of driver mutations which confer selective growth advantage to cells that harbor them. Clear examples are missense mutations in classic RAS genes (KRAS, HRAS and NRAS) that underlie the development of approximately 13% of human cancers. Although autonomous B cell antigen receptor (BCR) signaling is involved and mutations in many tumor suppressor genes and oncogenes have been identified, an oncogenic driver gene has not still been identified for CLL. [Methods]: Conditional knock-in mice were generated to overexpress wild type RRAS2 and prove its driver role. RT-qPCR analysis of a human CLL sample cohort was carried out to measure RRAS2 transcriptional expression. Sanger DNA sequencing was used to identify a SNP in the 3’UTR region of RRAS2 in human CLL samples. RNAseq of murine CLL was carried out to identify activated pathways, molecular mechanisms and to pinpoint somatic mutations accompanying RRAS2 overexpression. Flow cytometry was used for phenotypic characterization and shRNA techniques to knockdown RRAS2 expression in human CLL. [Results]: RRAS2 mRNA is found overexpressed in its wild type form in 82% of the human CLL samples analyzed (n = 178, mean and median = 5-fold) as well as in the explored metadata. A single nucleotide polymorphism (rs8570) in the 3’UTR of the RRAS2 mRNA has been identified in CLL patients, linking higher expression of RRAS2 with more aggressive disease. Deliberate overexpression of wild type RRAS2 in mice, but not an oncogenic Q72L mutation in the coding sequence, provokes the development of CLL. Overexpression of wild type RRAS2 in mice is accompanied by a strong convergent selection of somatic mutations in genes that have been identified in human CLL. R-RAS2 protein is physically bound to the BCR and mediates BCR signals in CLL. [Conclusions]: The results indicate that overexpression of wild type RRAS2 is behind the development of CLL.This work was supported by grants from the Spanish Association against Cancer (GC16173472GARC), PID2019-104935RB-I00 from the ‘Comision Interministerial de Ciencia y Tecnología’, the ‘Fundación Ramón Areces’, and by the European Research Council ERC 2013-Advanced Grant 334763 “NOVARIPP”, Instituto de Salud Carlos III (ISCIII) (CIBERONC – groups CB16/12/00233, CB16/12/00351), the Health Council of the Junta de Castilla y León (GRS 2036/A/19) and private Gilead (GLD15/00348). Juan de la Cierva (FJCI-2016-28756)

Additional file 8 of Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia [Dataset]

Fundación Científica Asociación Española Contra el Cáncer Ministerio de Ciencia, Innovación y Universidades H2020 European Research Council Instituto de Salud Carlos III Consejería de Educación, Junta de Castilla y LeónPeer reviewe

Additional file 2 of Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia [Dataset]

Additional file 2: Figure S2. a, Flow cytometry analysis of GFP populations in 23 wk-old Rosa26-RRAS2fl/flxSox2-Cre mouse spleen. Representative two-color contour plots of GFPhigh and GFPlow populations in total B cells (CD19+), CD5+ leukemic and CD23+ follicular B cells. Bottom, representation of GFP populations in T lymphocytes (CD3+). b, Percentage of GFPhigh cells in the indicated populations determined by flow cytometry. Data show means ± SEM from n = 8 mice (23 wk-old mice). ****p < 0.0001 (one-way ANOVA test). c, Western blot analysis of R-RAS2 expression of sorted GFPlow and GFPhigh leukemic cells from the spleen of a 25 wk-old Rosa26-RRAS2fl/flxSox2-Cre mouse (β-actin as loading control). d, Dot plot representation of GFPlow CD5+ leukemic B cell evolution in mb1-Cre mice over time, showing each mouse individually (n = 14). Data points were adjusted to a linear fit. These data were retrieved from the same mice as in Fig. 2i. e, Percentage of CD5+ cells in the indicated populations comparing GFPhigh and GFPlow distribution. Data show means ± SEM from n = 4 30 wk-old mice. Two-way ANOVA test. f, Heatmap of RNAseq expression data showing the genes differentially regulated in wild-type, follicular B cells (n = 6, 12wk-old), leukemic CD19 + CD5+ B cells (n = 6, 54wk-old), CD19+ GFPhigh (n = 2, 54wk-old) and CD19+ GFPlow (n = 2, 54wk-old) populations. Only genes significantly different between GFPhigh GFPlow populations (p < 0.05) and with a difference of 2-fold or more were used. Gene expression is shown in normalized log2 fold change.Peer reviewe

Additional file 1 of Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia [Dataset]

Additional file 1: Figure S1. a, Relative mRNA expression of RRAS2 in different types of leukemia. Data comes from (Haferlach et al., 2010) and has been retrieved from www.oncomine.org . b, Schematic representation of the overexpression cassette inserted into the Rosa26 locus. c, Relative expression of RRAS2 measured by RT-qPCR in different organs of Rosa26-RRAS2fl/flxSox2-Cre (Sox2-Cre+) mice compared to that of WT C57BL/6 J Control mice using 18S as the reference gene. All expression numbers were normalized to those of liver from WT Control mice (mean = 1). Data show relative expression of RRAS2 in the indicated organs in n = 3–4 8 month-old independent mice. d, Quantification of spleen weight from control and 6 month-old Sox2-Cre + mice. Data shown correspond to four control mice and eleven Sox2-Cre mice. Two-tailed unpaired t-test with Welch’s correction. e, Two-parameter flow cytometry of the expression of CD5 and IgM in B cells in the spleen of 6 month-old control and Sox2-Cre + mice. f, Quantification of the number of CD5 + IgM+ B cells in the spleens and bone marrow of 6 month-old control and Sox2-Cre + mice. Data correspond to triplicate measurements of one control and three Sox2-Cre mice. Unpaired t-test with Welch’s correction. g, Quantification of the serum IgM concentration in the blood of 35–40 wk-old control (n = 3) and mb1-Cre (n = 8) mice by ELISA. Unpaired t-test with Welch’s correction. h, Representative images from Giemsa stainings of blood smears of 36 wk-old control and mb1-Cre mice. i, Two-parameter flow cytometry of the forward scatter and CD5 expression in CD19+ cells in the blood of 16 wk-old mb1-Cre mice. The gated population represents large cells. j, Two-parameter flow cytometry of CD5 expression and BrdU incorporation in CD19+ cells in the blood of 16 wk-old mb1-Cre mice. k, Quantification of the percentage of CD19+ cells that are CD5+ blasts and of the CD19+ CD5+ cells that have incorporated BrdU.Peer reviewe

Additional file 10 of Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia [Dataset]

Fundación Científica Asociación Española Contra el Cáncer Ministerio de Ciencia, Innovación y Universidades H2020 European Research Council Instituto de Salud Carlos III Consejería de Educación, Junta de Castilla y LeónPeer reviewe

Additional file 9 of Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia [Dataset]

Fundación Científica Asociación Española Contra el Cáncer Ministerio de Ciencia, Innovación y Universidades H2020 European Research Council Instituto de Salud Carlos III Consejería de Educación, Junta de Castilla y LeónPeer reviewe

Additional file 3 of Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia [Dataset]

Additional file 3: Figure S3. a, Representative two-color contour plots of B cell populations in a peritoneal wash and the spleen of 12 wk-old mice according to the expression of the CD11b and CD5 markers in the CD19+ population. The blue square indicates CD11b + CD5- B1b cells in the peritoneum. Red square, the presence of CD11b + CD5+ B1a cells in control mice and leukemic cells. Quantification of CD11b + CD5+ cells is shown to the right in box and whiskers plots showing all points and median value. **p < 0.01; *** p < 0.001, two-tailed unpaired t-test with Welch’s correction. b, Representative two-color contour plots of IgM and GFP expression within the CD11b + CD5+ populations shown in a. Quantification of IgMbright cells within the CD11b + CD5+ B cell population is shown to the right in box and whiskers plots showing all points and median value. **** p < 0.0001, two-tailed unpaired t-test with Welch’s correction.Peer reviewe

Additional file 6 of Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia [Dataset]

Additional file 6: Figure S6. a, Mutations found in human cancer involving the RRAS2 gene. Data obtained from cBioPortal (97,250 patients/100669 samples). Refseq: NM_012250. Ensembl: ENST00000256196. CCDS: CCDS7814. Uniprot: RRAS2_HUMAN. Missense mutations (green dots): 36. Truncating mutations (black dots): 6. Splice mutations (orange dots): 5. b, Quantification by RT-qPCR or total mouse (Rras2) and human (RRAS2) mRNA expression in purified splenic CD19+ B cells from Rras2(Q72L)fl/fl xmb1-Cre (Q72L) mice compared to purified B CD19+ B cells from control WT C57BL/6 mice and to CD19 + CD5+ leukemic B cells from Rosa26-RRAS2fl/flxmb1-Cre mice. Results show data obtained in triplicate normalized to the C57BL/6 control for n = 3 mice per group. All mice were 14 month-old. Data show means ± SEM for three mice per group. *p < 0.05; ns. Not significant (one-way ANOVA test). c, Left, quantification by flow cytometry of total B-cell number in spleens of 14 month-old control and Rras2(Q72L)fl/fl xmb1-Cre mice. Right, two-parameter flow cytometry plot showing frequency of IgM + CD5+ cells within CD19+ splenic B cells of control and Rras2(Q72L)fl/fl xmb1-Cre mice. d, Left, concentration of B-cells per microliter in blood of control and Rras2(Q72L)fl/fl xmb1-Cre mice. Right, two-parameter flow cytometry plot showing frequency of CD19 + CD5+ cells within blood B cells of control and Rras2(Q72L)fl/fl xmb1-Cre mice. e, Frequency of marginal zone (MZ) phenotype (CD21high, CD23low), and follicular (CD21low, CD23high) B cells within CD19+ splenic B cells of control and Rras2(Q72L)fl/fl xmb1-Cre mice. f, Phosflow cytometry analysis of different elements from PI3K-Akt-mTOR, Raf-Erk and proximal BCR signaling pathways. Wild-type CD19+ follicular B cells, CD19 + CD5+ leukemic cells from spleens of Rosa26-RRAS2fl/flxmb1-Cre mice and CD19+ non-leukemic B cells from Rras2(Q72L)fl/fl xmb1-Cre are shown. In grey, background fluorescence of the secondary antibodies. All mice were 23 wk-old. Data show means ± SEM from three mice per group. *p < 0.05; **p < 0.01; ****p < 0.0001 (one-way ANOVA test). g, Phosflow cytometry analysis of different elements from PI3K-Akt-mTOR, Raf-Erk and proximal BCR signaling pathways. CD19 + CD5+ leukemic cells from 30 wk-old Rosa26-RRAS2fl/flxmb1-Cre mice are compared with WT Control follicular (CD23highCD21−), marginal zone (MZ, CD23−CD21high), B1a (CD11b + CD5+) and B1b (CD11b + CD5−) spleen B cell populations. Data show means ± SEM from n = 3 mice per group. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant (one-way ANOVA test).Fundación Científica Asociación Española Contra el Cáncer Ministerio de Ciencia, Innovación y Universidades H2020 European Research Council Instituto de Salud Carlos III Consejería de Educación, Junta de Castilla y LeónPeer reviewe

Additional file 4 of Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia [Dataset]

Additional file 4: Figure S4. a, Representative two-color contour plots of lymphoid populations in liver and spleen from 2 wk-old mice according to the expression of CD19 and CD5 and within the CD19 + CD5+ population according to the expression of CD21, B220, CD24, CD23 and CD38 markers. b, Column plots show the quantification of the percentage of CD19 + CD5+ B cells in liver and spleen bearing the markers shown in a. n = 4 mice per group. ** p < 0.01 ****p < 0.0001, ns, not significant (one-way ANOVA test).Peer reviewe

Additional file 5 of Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia [Dataset]

Additional file 5: Figure S5. a, Principal component analysis of CD19 + CD21-CD23+ follicular B cells from Rosa26-RRAS2xmb1-Cre mice, CD19 + CD21-CD23+ follicular B cells from WT C57BL/6 J mice and of leukemic CD19 + CD5+, GFPlow and GFPhigh cells from Rosa26-RRAS2xmb1-Cre mice. b, Ingenuity Pathway Analysis (IPA) of differentially expressed genes associated with molecular mechanisms of cancer 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; vertical rectangle: G protein-coupled receptor; circle: other. Black arrows: direct interactions; grey/white arrows: indirect interactions. 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.Peer reviewe