Functional characterizacion of RRAS2 mutations and role of RRAS2Q72L in ovarian cancer

[EN] R-RAS2 is a small GTPase with high structural proximity to classical RAS proteins. RRAS2 gain-of-function mutations have been identified at low frecuency in recent PanCancer studies. However, the cancer driver and pathobiological roles of this GTPase remain poorly characterized. In this thesis we have used in vitro and in vivo models to tackle those issues. We have demonstrated that tumor-found RRAS2 mutations targeting residues involved in the GTP-binding are able to induce cell transformation in vitro. Analyses of R-RAS2Q72L-expressing cancer cell lines have shown that this protein in required for the tumoral fitness of the cells. In these cell models, R-RAS2Q72L modulates pathways involved in cell proliferation and survival and regulates basic processes such as polysomal translation and cell metabolism. However, these effects are not driven by R-RAS2 expression. This work also exposes a driver role for R-Ras2Q72L in tumorigenesis. The R-Ras 5$62Q72L-driven tumors exhibit diffential sensitivity to mTORCI and/or P13K phamarcological inhibition, with some tumors showing resistance to all the inhibitors tested. The characterization of the R-Ras2Q72L-triggered ovarian cystadenomas has revealed a rete ovarii origin and a sexreversal phenotype in these tumors. Beyond tumorigenesis, R-Ras2Q72L also triggers follicular atresia- and absent spermatogenesis-induced infertility in mice. Altogether, oir findings unveil novel phatological functions of R-RAS2 and expand the current knowledge of cellular functions of the wild-type and the oncogenic R-RAS2 versions

Elaboración e implementación de un nuevo programa de prácticas de laboratorio para una asignatura de grado de Biología Molecular

Memoria ID-191. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2019-2020

A hotspot mutation targeting the R-RAS2 GTPase acts as a potent oncogenic driver in a wide spectrum of tumors

A missense change in RRAS2 (Gln to Leu), analogous to the Gln-to-Leu mutation of RAS oncoproteins, has been identified as a long-tail hotspot mutation in cancer and Noonan syndrome. However, the relevance of this mutation for in vivo tumorigenesis remains understudied. Here we show, using an inducible knockin mouse model, that R-Ras2 triggers rapid development of a wide spectrum of tumors when somatically expressed in adult tissues. These tumors show limited overlap with those originated by classical Ras oncogenes. R-Ras2-driven tumors can be classified into different subtypes according to therapeutic susceptibility. Importantly, the most relevant R-Ras2-driven tumors are dependent on mTORC1 but independent of phosphatidylinositol 3-kinase-, MEK-, and Ral guanosine diphosphate (GDP) dissociation stimulator. This pharmacological vulnerability is due to the extensive rewiring by R-Ras2 of pathways that orthogonally stimulate mTORC1 signaling. These findings demonstrate that RRAS2 is a bona fide oncogenic driver and unveil therapeutic strategies for patients with cancer and Noonan syndrome bearing RRAS2 mutations.We thank M. Blázquez and the personnel of the CIC Flow Cytometry, Microscopy, Pathology, and Genomics Units for expert technical work. X.R.B.’s project leading to these results has received funding from the Spanish Association against Cancer (GC16173472GARC), the Castilla-León government (CSI252P18, CSI145P20, and CLC-2017-01), the RTI2018-096481-B-100 grant cofounded by MCIN/AEI/10.13039/501100011033 and the European Research Development Fund “A way of making Europe” of the European Union, and “la Caixa” Banking Foundation (HR20-00164). X.R.B.’s institution is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia of the Castilla-León government (CLC-2017-01). J.R.-V. received funding from the Carlos III Health Institute (PI20/01724). J.R.-V.’s contract is supported by a senior postdoctoral contract of the Spanish Association against Cancer. L.C.’s contract was supported by contracts from the Spanish Association against Cancer and the CLC-2017-01 grant. L.F.L.-M.’s contract was mostly supported by funding from the Spanish Ministry of Education, Culture and Sports (FPU13/02923) and, subsequently, by the CLC-2017-01 grant. R.C. was supported by a predoctoral contract from the MSI (BES-2016-0077909) and the CLC-2017-01 grant

Characterization of mutant versions of the R-RAS2/TC21 GTPase found in tumors

The R-RAS2 GTP hydrolase (GTPase) (also known as TC21) has been traditionally considered quite similar to classical RAS proteins at the regulatory and signaling levels. Recently, a long-tail hotspot mutation targeting the R-RAS2/TC21 Gln72 residue (Q72L) was identified as a potent oncogenic driver. Additional point mutations were also found in other tumors at low frequencies. Despite this, little information is available regarding the transforming role of these mutant versions and their relevance for the tumorigenic properties of already-transformed cancer cells. Here, we report that many of the RRAS2 mutations found in human cancers are highly transforming when expressed in immortalized cell lines. Moreover, the expression of endogenous R-RAS2Q72L is important for maintaining optimal levels of PI3K and ERK activities as well as for the adhesion, invasiveness, proliferation, and mitochondrial respiration of ovarian and breast cancer cell lines. Endogenous R-RAS2Q72L also regulates gene expression programs linked to both cell adhesion and inflammatory/immune-related responses. Endogenous R-RAS2Q72L is also quite relevant for the in vivo tumorigenic activity of these cells. This dependency is observed even though these cancer cell lines bear concurrent gain-of-function mutations in genes encoding RAS signaling elements. Finally, we show that endogenous R-RAS2, unlike the case of classical RAS proteins, specifically localizes in focal adhesions. Collectively, these results indicate that gain-of-function mutations of R-RAS2/TC21 play roles in tumor initiation and maintenance that are not fully redundant with those regulated by classical RAS oncoproteins

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)

Efficient fractionation and analysis of ribosome assembly intermediates in human cells

© 2021 The Author(s).Biochemical studies of the human ribosome synthesis pathway have been hindered by technical difficulties in obtaining intact preribosomal complexes from internal regions of the nucleolus. Here we provide a detailed description of an extraction method that enables efficient detection, isolation, and characterization of nucleolar preribosomes containing large pre-rRNA species. The three-step Preribosome Sequential Extraction (PSE) protocol preserves the integrity of early preribosomal complexes and yields preparations amenable to biochemical analyses from low amounts of starting material. We validate this procedure through the detection of specific trans-acting factors and pre-rRNAs in the extracted preribosomes using affinity matrix pull-downs and sedimentation assays. In addition, we describe the application of the PSE method for monitoring cellular levels of ribosome-free 5S RNP complexes as an indicator of ribosome biogenesis stress. Our optimized experimental procedures will facilitate studies of human ribosome biogenesis in normal, mutant and stressed-cell scenarios, including the characterization of candidate ribosome biogenesis factors, preribosome interactors under specific physiological conditions or effects of drugs on ribosome maturation.This work was supported by the Spanish Ministry of Science and Innovation [BFU2017-88192-P to MD][RTI2018-096481-B-100 to XRB]; the National Institutes of Health [R03CA246009 to DGP]; the Castilla-León autonomous government [CSI252P18 and CSI145P2 to XRB]; the Spanish Association against Cancer [GC16173472GARC to XRB]; “la Caixa” Banking Foundation [HR20-00164 to XRB]; and the Samuel Solórzano Foundation [FS/36-2017 to MD]. BN has been supported by a predoctoral contract sponsored by the University of Salamanca and Santander Bank, SGG by a predoctoral FPU contract of the Spanish Ministry of Education, Culture and Sports, and LC by a predoctoral contract from the Spanish Association against Cancer. The Centro de Investigación del Cáncer is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia of the Ministry of Education of the Castilla-León Government (CLC-2017-01). Both the Spanish and Castilla-León government-associated funding is partially supported by the European Regional Development Fund

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 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 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