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)

Predictive Power of the "Trigger Tool" for the detection of adverse events in general surgery: a multicenter observational validation study

Background In spite of the global implementation of standardized surgical safety checklists and evidence-based practices, general surgery remains associated with a high residual risk of preventable perioperative complications and adverse events. This study was designed to validate the hypothesis that a new “Trigger Tool” represents a sensitive predictor of adverse events in general surgery. Methods An observational multicenter validation study was performed among 31 hospitals in Spain. The previously described “Trigger Tool” based on 40 specific triggers was applied to validate the predictive power of predicting adverse events in the perioperative care of surgical patients. A prediction model was used by means of a binary logistic regression analysis. Results The prevalence of adverse events among a total of 1,132 surgical cases included in this study was 31.53%. The “Trigger Tool” had a sensitivity and specificity of 86.27% and 79.55% respectively for predicting these adverse events. A total of 12 selected triggers of overall 40 triggers were identified for optimizing the predictive power of the “Trigger Tool”. Conclusions The “Trigger Tool” has a high predictive capacity for predicting adverse events in surgical procedures. We recommend a revision of the original 40 triggers to 12 selected triggers to optimize the predictive power of this tool, which will have to be validated in future studies

Receptores tipo Toll y de Esfingosina1 fosfato y su implicación en la fisiopatología cardiovascular

Dado que los receptores tipo Toll (TLR) y esfingosina-1-fosfato (S1P) juegan un papel muy importante en inflamación, en esta tesis se estudió la potencial interacción entre los receptores de S1P y TLRs en las vías de adhesión/ inflamación en células endoteliales de origen humano. Determinamos por Western Blot y por citometría de Flujo, que las células tratadas con LPS (ligando de TLR4) y S1P, expresaban una mayor cantidad de ICAM-1 y E-selectina comparado con las células estimuladas con cada ligando por separado, observando diferencias entre las endoteliales de origen venoso y aórtico en cuanto a la expresión de E-selectina. Sin embargo, no observamos ningún efecto cooperativo cuando estimulamos las células con un ligando de TLR2/TLR1. En consecuencia con el aumento de la expresión de las moléculas de adhesión, los experimentos de adhesión bajo flujo laminar mostraron que LPS y S1P cooperan para aumentar el número de leucocitos adheridos a la monocapa endotelial y la fuerza de adhesión en comparación con las células estimuladas con los ligandos por separado. Además la fuerza de adhesión resultó ser similar a la inducida por TNF-¿. Además, la coestimulación con LPS y S1P coopera en el aumento de la expresión de moléculas proinflamatorias como IL-6, COX-2 y prostaciclina. El análisis de las rutas de señalización implicadas reveló una fosforilación sinergística de la MAPK ERK tras el tratamiento con LPS y S1P y la implicación del factor de tanscripción NF-kB, además en el caso de las endoteliales de origen venoso, también participa la MAPK p38. Además, dilucidamos los receptores de S1P implicados en el efecto cooperativo, así como la posible implicación de la enzima esfingosina Kinasa en este efecto. Por otro lado, debido a la prominente acumulación lipídica y los cambios inflamatorios en la estenosis aórtica, y la implicación de S1P en la patofisiología cardiovascular, estudiamos el papel de S1P en las vías proinflamatorias/proosteogénicas en células intersticiales procedentes de válvulas aórticas (AVIC) tanto sanas como estenóticas y de válvulas pulmonares (PVIC). Los resultados fueron que la coestimulación con LPS y S1P tenía un efecto sinérgico en el aumento de la expresión de ICAM-1, COX-2 y cooperan en la expresión de ALP (un marcador inicial de calcificación), en la de VEGF (un factor angiogénico) y en la secreción de sICAM-1 (un marcador cardiovascular) y prostaglandinas (un mediador inflamatorio derivado de la expresión de COX-2). Además, estos efectos, son mayores en las células AVIC provenientes de válvulas estenóticas que de las sanas, y más aún que en las PVIC. El efecto sinergístico en este caso, fue inhibido por inhibidores de los receptores de S1P1/3 en cuanto a las moléculasDepartamento de Bioquímica y Biología Molecular y Fisiologí

RAS GTPase-dependent pathways in developmental diseases: old guys, new lads, and current challenges

Deregulated RAS signaling is associated with increasing numbers of congenital diseases usually referred to as RASopathies. The spectrum of genes and mutant alleles causing these diseases has been significantly expanded in recent years. This progress has triggered new challenges, including the origin and subsequent selection of the mutations driving these diseases, the specific pathobiological programs triggered by those mutations, the type of correlations that exist between the genotype and the clinical features of patients, and the ancillary genetic factors that influence the severity of the disease in patients. These issues also directly impinge on the feasibility of using RAS pathway drugs to treat RASopathy patients. Here, we will review the main developments and pending challenges in this research topic.The authors wish to apologize to scientists who have not been cited in this work due to space constraints. XRB work is supported by grants from the Castilla-León Government (CSI049U16), Spanish Ministry of Economy and Competitiveness (SAF2015-64556-R), Worldwide Cancer Research (14-1248), Ramón Areces Foundation, and Spanish Society against Cancer (GC16173472GARC). PC work is supported by grants from both the Spanish Ministry of Economy and Competitiveness (SAF-2015 63638R) and the Spanish Society against Cancer (GCB141423113). Funding from Spanish national and regional governments to both XRB and PC is partially contributed by the European Regional Development Fund

An unexpected tumor suppressor role for VAV1a

RHO GDP/GTP exchange factors, including VAV1, are considered key protumorigenic factors. Against this paradigm, we have found that VAV1 plays tumor suppressor roles by buffering NOTCH1 signals in thymocytes. The silencing of this pathway contributes to the pathogenesis of T cell acute lymphoblastic leukemia of the early cortical, TLX+ subtype

Distinct roles of vav family members in adaptive and innate immune models of arthritis

© 2021 by the authors.Genetic evidence suggests that three members of the VAV family (VAV1, VAV2 and VAV3) of signal transduction proteins could play important roles in rheumatoid arthritis. However, it is not known currently whether the inhibition of these proteins protects against this disease and, if so, the number of family members that must be eliminated to get a therapeutic impact. To address this issue, we have used a collection of single and compound Vav family knockout mice in experimental models for antigen-dependent (methylated bovine serum albumin injections) and neutrophil-dependent (Zymosan A injections) rheumatoid arthritis in mice. We show here that the specific elimination of Vav1 is sufficient to block the development of antigen-induced arthritis. This protection is likely associated with the roles of this Vav family member in the development and selection of immature T cells within the thymus as well as in the subsequent proliferation and differentiation of effector T cells. By contrast, we have found that depletion of Vav2 reduces the number of neutrophils present in the joints of Zymosan A-treated mice. Despite this, the elimination of Vav2 does not protect against the joint degeneration triggered by this experimental model. These findings indicate that Vav1 is the most important pharmacological target within this family, although its main role is limited to the protection against antigen-induced rheumatoid arthritis. They also indicate that the three Vav family proteins do not play redundant roles in these pathobiological processes.This research was funded by the Castilla-León autonomous government (CSI252P18, CSI145P20, CLC-2017-01), the Spanish Ministry of Science and Innovation (MSI) (RTI2018-096481-B-100), the Spanish Association against Cancer (GC16173472GARC) and the “la Caixa” Banking Foundation (HR20-00164). J.C. has been supported by the Sara Borrell contract from the Spanish Carlos III Health Institute (CD15/00113). 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 autonomous government (CLC-2017-01). Both the Spanish and Castilla-León government-associated funding is partially supported by the European Regional Development Fun

An unexpected tumor suppressor role for VAV1a

RHO GDP/GTP exchange factors, including VAV1, are considered key protumorigenic factors. Against this paradigm, we have found that VAV1 plays tumor suppressor roles by buffering NOTCH1 signals in thymocytes. The silencing of this pathway contributes to the pathogenesis of T cell acute lymphoblastic leukemia of the early cortical, TLX+ subtype.Castilla-Leon Government (BIO/SA01/15, CSI049U16), Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2015-64556-R, RD12/0036/0002), Worldwide Cancer Research (14-1248), Ramon Areces Foundation, and Spanish Society against Cancer (GC16173472GARC). Spanish governmental funding is partially supported by the European Regional Development Fund

Sphingosine 1-phosphate induces inflammation and osteogenesis and increases the activity of the LPS/TLR4 route in human aortic valve interstitial cells

Resumen del póster presentado al 22nd IUBMB & 37th FEBS Congress: "From Single Molecules to Systems Biology" celebrado en Sevilla (España) del 4 al 9 de septiembre de 2012.-- et al.[Aims]: Lipid accumulation in the aortic valve is a characteristic of aortic stenosis and sphingosine 1-phosphate (S1P) plays a relevant functional role in the cardiovascular system. On this basis, we investigated the possible role of S1P on the induction of proinflammatory and pro-osteogenic changes in human interstitial cells from aortic valve (AVIC) and pulmonary valves (PVIC). [Methods and Results]: As regards pro-inflammatory routes, S1P up-regulated IL-6, IL-8, and cyclooxygenase (COX)-2 in AVICs, as determined by Western blot and ELISA experiments. AVIC exposure to a combination of S1P and bacterial lipopolysaccharide (LPS) a Toll-like receptor (TLR)-4 ligand known to promote pro-inflammatory and pro-osteogenic phenotypes in AVICs, resulted in the synergistic induction of COX-2, PGE2, and intercellular adhesion molecule (ICAM)-1. Strikingly, the cooperative effect was stronger in stenotic than in control cells, and more prominent in cells from the aortic valves than from the pulmonary valves, which rarely undergo stenosis. Pharmacological and gene silencing experiments revealed the involvement of several S1P receptors in the synergistic effect. As regards proosteogenic processes, S1P induced the expression/activity of the calcification marker alkaline phosphatase (ALP) in in vitro calcification experiments. In addition, S1P cooperated with LPS to enhance significantly ALP activity and the cooperative effect was partially blocked by S1P receptor antagonists. [Conclusions]: S1P induces pro-inflammatory and pro-osteogenic changes and increases the effect of TLR4 ligands in AVICs, what might be relevant for the pathogenesis of aortic stenosis and could open the way for new therapeutic approaches for this disease.Peer Reviewe