Unmutated RRAS2 emerges as a key oncogene in post-partum-associated triple negative breast cancer.

BACKGROUND: Breast cancer (BC) is the most common cancer in women, with triple negative BC (TNBC) accounting for 20% of cases. While early detection and targeted therapies have improved overall life expectancy, TNBC remains resistant to current treatments. Although parity reduces the lifetime risk of developing BC, pregnancy increases the risk of developing TNBC for years after childbirth. Although numerous gene mutations have been associated with BC, no single gene alteration has been identified as a universal driver. RRAS2 is a RAS-related GTPase rarely found mutated in cancer. METHODS: Conditional knock-in mice were generated to overexpress wild type human RRAS2 in mammary epithelial cells. A human sample cohort was analyzed by RT-qPCR to measure RRAS2 transcriptional expression and to determine the frequency of both a single-nucleotide polymorphism (SNP rs8570) in the 3'UTR region of RRAS2 and of genomic DNA amplification in tumoral and non-tumoral human BC samples. RESULTS: Here we show that overexpression of wild-type RRAS2 in mice is sufficient to develop TNBC in 100% of females in a pregnancy-dependent manner. In human BC, wild-type RRAS2 is overexpressed in 68% of tumors across grade, location, and molecular type, surpassing the prevalence of any previously implicated alteration. Still, RRAS2 overexpression is notably higher and more frequent in TNBC and young parous patients. The increased prevalence of the alternate C allele at the SNP position in tumor samples, along with frequent RRAS2 gene amplification in both tumors and blood of BC patients, suggests a cause-and-effect relationship between RRAS2 overexpression and breast cancer. CONCLUSIONS: Higher than normal expression of RRAS2 not bearing activating mutations is a key driver in the majority of breast cancers, especially those of the triple-negative type and those linked to pregnancy.

Transfection of Sponge Cells and Intracellular Localization of Cancer-Related MYC, RRAS2, and DRG1 Proteins.

The determination of the protein's intracellular localization is essential for understanding its biological function. Protein localization studies are mainly performed on primary and secondary vertebrate cell lines for which most protocols have been optimized. In spite of experimental difficulties, studies on invertebrate cells, including basal Metazoa, have greatly advanced. In recent years, the interest in studying human diseases from an evolutionary perspective has significantly increased. Sponges, placed at the base of the animal tree, are simple animals without true tissues and organs but with a complex genome containing many genes whose human homologs have been implicated in human diseases, including cancer. Therefore, sponges are an innovative model for elucidating the fundamental role of the proteins involved in cancer. In this study, we overexpressed human cancer-related proteins and their sponge homologs in human cancer cells, human fibroblasts, and sponge cells. We demonstrated that human and sponge MYC proteins localize in the nucleus, the RRAS2 in the plasma membrane, the membranes of the endolysosomal vesicles, and the DRG1 in the cell's cytosol. Despite the very low transfection efficiency of sponge cells, we observed an identical localization of human proteins and their sponge homologs, indicating their similar cellular functions.

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.

The molecular genetics of RASopathies: An update on novel disease genes and new disorders.

Enhanced signaling through RAS and the mitogen-associated protein kinase (MAPK) cascade underlies the RASopathies, a family of clinically related disorders affecting development and growth. In RASopathies, increased RAS-MAPK signaling can result from the upregulated activity of various RAS GTPases, enhanced function of proteins positively controlling RAS function or favoring the efficient transmission of RAS signaling to downstream transducers, functional upregulation of RAS effectors belonging to the MAPK cascade, or inefficient signaling switch-off operated by feedback mechanisms acting at different levels. The massive effort in RASopathy gene discovery performed in the last 20 years has identified more than 20 genes implicated in these disorders. It has also facilitated the characterization of several molecular activating mechanisms that had remained unappreciated due to their minor impact in oncogenesis. Here, we provide an overview on the discoveries collected during the last 5 years that have delivered unexpected insights (e.g., Noonan syndrome as a recessive disease) and allowed to profile new RASopathies, novel disease genes and new molecular circuits contributing to the control of RAS-MAPK signaling.

Activating Mutations of RRAS2 Are a Rare Cause of Noonan Syndrome.

Aberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%-20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations.

Germline-Activating RRAS2 Mutations Cause Noonan Syndrome.

Noonan syndrome (NS) is characterized by distinctive craniofacial appearance, short stature, and congenital heart disease. Approximately 80% of individuals with NS harbor mutations in genes whose products are involved in the RAS/mitogen-activating protein kinase (MAPK) pathway. However, the underlying genetic causes in nearly 20% of individuals with NS phenotype remain unexplained. Here, we report four de novo RRAS2 variants in three individuals with NS. RRAS2 is a member of the RAS subfamily and is ubiquitously expressed. Three variants, c.70_78dup (p.Gly24_Gly26dup), c.216A>T (p.Gln72His), and c.215A>T (p.Gln72Leu), have been found in cancers; our functional analyses showed that these three changes induced elevated association of RAF1 and that they activated ERK1/2 and ELK1. Notably, prominent activation of ERK1/2 and ELK1 by p.Gln72Leu associates with the severe phenotype of the individual harboring this change. To examine variant pathogenicity in vivo, we generated zebrafish models. Larvae overexpressing c.70_78dup (p.Gly24_Gly26dup) or c.216A>T (p.Gln72His) variants, but not wild-type RRAS2 RNAs, showed craniofacial defects and macrocephaly. The same dose injection of mRNA encoding c.215A>T (p.Gln72Leu) caused severe developmental impairments and low dose overexpression of this variant induced craniofacial defects. In contrast, the RRAS2 c.224T>G (p.Phe75Cys) change, located on the same allele with p.Gln72His in an individual with NS, resulted in no aberrant in vitro or in vivo phenotypes by itself. Together, our findings suggest that activating RRAS2 mutations can cause NS and expand the involvement of RRAS2 proto-oncogene to rare germline disorders.

The RRAS2 pathogenic variant p.Q72L produces severe Noonan syndrome with hydrocephalus: A case report.

Noonan syndrome (NS) is the most common disease among RASopathies, characterized by short stature, distinctive facial features, congenital cardiac defects, and variable developmental delay. NS rarely presents with overt neurologic manifestations, in particular hydrocephalus. Recent evidence suggests that pathogenic variants in the gene RRAS2 are a rare cause of NS. Specifically, an RRAS2 pathogenic variant, p.Q72L, may be particularly severe, manifesting with lethal neurologic findings. Here, we report a NS patient with documented p.Q72L variant in RRAS2. The patient was identified in utero to have hydrocephalus and a Dandy Walker malformation. Postnatal examination revealed multiple dysmorphic features, some reminiscent of NS including low-set posteriorly rotated ears, redundant nuchal skin, widely spaced nipples, and cryptorchidism. Despite suspicion of NS, results of a 14-gene Noonan syndrome panel (Invitae) were negative. Follow-up rapid whole exome sequencing revealed a de novo p.Q72L variant in RRAS2, a poorly studied gene recently identified as a cause of NS. The patient herein reported brings to three the total number of cases reported with the RRAS2 p.Q72L pathogenic variant. All three documented patients presented with a particularly fulminant course of NS, which included hydrocephalus. RRAS2, specifically p.Q72L, should be considered in severe NS cases with neurologic manifestations.

The RRAS2 pathogenic variant (c.67G>T; p. Gly23Cys) produces Noonan syndrome with embryonal rhabdomyosarcoma.

BACKGROUND: Noonan syndrome (NS) due to the RRAS2 gene, the pathogenic variant is an extremely rare RASopathies. Our objective was to identify the potential site of RRAS2, combined with the literature review, to find the correlation between clinical phenotype and genotype. De novo missense mutations affect different aspects of the RRAS2 function, leading to hyperactivation of the RAS-MAPK signaling cascade. METHODS: Conventional G-banding was used to analyze the chromosome karyotype of the patient. Copy number variation sequencing (CNV-seq) was used to detect the chromosomal gene microstructure of the patient and her parents. The exomes of the patient and her parents were sequenced using trio-based whole exome sequencing (trio-WES) technology. The candidate variant was verified by Sanger sequencing. The pathogenicity of the variant was predicted with a variety of bioinformatics tools. RESULTS: Chromosome analysis of the proband revealed 46, XX, and no abnormality was found by CNV-seq. After sequencing and bioinformatics filtering, the variant of RRAS2(c.67G>T; p. Gly23Cys) was found in the proband, while the mutation was absent in her parents. To the best of our knowledge, our patient was with the typical Noonan syndrome, such as short stature, facial dysmorphism, and developmental delay. Furthermore, our study is the first case of NS with embryonal rhabdomyosarcoma (ERMS) caused by the RRAS2 gene mutation reported in China. CONCLUSIONS: Our investigations suggested that the heterozygous missense of RRAS2 may be a potential causal variant in a rare cause of Noonan syndrome, expanding our understanding of the causally relevant mutations for this disorder.

Functional analysis of RRAS2 pathogenic variants with a Noonan-like phenotype.

Introduction: RRAS2, a member of the R-Ras subfamily of Ras-like low-molecular-weight GTPases, is considered to regulate cell proliferation and differentiation via the RAS/MAPK signaling pathway. Seven RRAS2 pathogenic variants have been reported in patients with Noonan syndrome; however, few functional analyses have been conducted. Herein, we report two patients who presented with a Noonan-like phenotype with recurrent and novel RRAS2 pathogenic variants (p.Gly23Val and p.Gly24Glu, respectively) and the results of their functional analysis. Materials and methods: Wild-type (WT) and mutant RRAS2 genes were transiently expressed in Human Embryonic Kidney293 cells. Expression of RRAS2 and phosphorylation of ERK1/2 were confirmed by Western blotting, and the RAS signaling pathway activity was measured using a reporter assay system with the serum response element-luciferase construct. WT and p.Gly23Val RRAS2 were expressed in Drosophila eye using the glass multiple reporter-Gal4 driver. Mutant mRNA microinjection into zebrafish embryos was performed, and the embryo jaws were observed. Results: No obvious differences in the expression of proteins WT, p.Gly23Val, and p.Gly24Glu were observed. The luciferase reporter assay showed that the activity of p.Gly23Val was 2.45 ± 0.95-fold higher than WT, and p.Gly24Glu was 3.06 ± 1.35-fold higher than WT. For transgenic flies, the p.Gly23Val expression resulted in no adults flies emerging, indicating lethality. For mutant mRNA-injected zebrafish embryos, an oval shape and delayed jaw development were observed compared with WT mRNA-injected embryos. These indicated hyperactivity of the RAS signaling pathway. Discussion: Recurrent and novel RRAS2 variants that we reported showed increased in vitro or in vivo RAS signaling pathway activity because of gain-of-function RRAS2 variants. Clinical features are similar to those previously reported, suggesting that RRAS2 gain-of-function variants cause this disease in patients.

Clinical analysis of Noonan syndrome caused by RRAS2 mutations and literature review.

Noonan syndrome is a common developmental disorder characterized by distinctive facial dysmorphism, short stature, congenital heart defects, pectus deformity, and developmental delay. It is related to the abnormal activation of genes involved in the RAS-MAPK signaling pathway, more than a dozen of which can be affected. However, mutations of the RRAS2 gene are rare, with only 6 different RRAS2 variants in 13 patients reported to date. In this case report, whole-exome sequencing revealed a novel heterozygous variant in the RRAS2 gene NM_012250: c.212G > A, p.(Gly71Glu). Phenotypically, our patient had typical Noonan syndrome-related clinical manifestations consistent with published reports, such as short stature, facial dysmorphism, short neck, patent foramen ovale, moderate global developmental delay, and hearing impairment. In addition, our patient also had a distal middle finger deformity and hair defect, which have not been reported in previous cases. We analyzed the clinical characteristics of all patients with Noonan syndrome caused by RRAS2 variants and reviewed the literature. This discovery expands the genetic and phenotypic spectrum of Noonan syndrome.

Characterization of Three Somatic Mutations in the 3'UTR of RRAS2 and Their Inverse Correlation with Lymphocytosis in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a hematologic malignancy characterized by progressive accumulation of a rare population of CD5+ B-lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. CLL exhibits remarkable clinical heterogeneity, with some patients presenting with indolent disease and others progressing rapidly to aggressive CLL. The significant heterogeneity of CLL underscores the importance of identifying novel prognostic markers. Recently, the RAS-related gene RRAS2 has emerged as both a driver oncogene and a potential marker for CLL progression, with higher RRAS2 expression associated with poorer disease prognosis. Although missense somatic mutations in the coding sequence of RRAS2 have not been described in CLL, this study reports the frequent detection of three somatic mutations in the 3' untranslated region (3'UTR) affecting positions +26, +53, and +180 downstream of the stop codon in the mRNA. An inverse relationship was observed between these three somatic mutations and RRAS2 mRNA expression, which correlated with lower blood lymphocytosis. These findings highlight the importance of RRAS2 overexpression in CLL development and prognosis and point to somatic mutations in its 3'UTR as novel mechanistic clues. Our results may contribute to the development of targeted therapeutic strategies and improved risk stratification for CLL patients.

An Optimized Single Nucleotide Polymorphism-Based Detection Method Suggests That Allelic Variants in the 3' Untranslated Region of RRAS2 Correlate with Treatment Response in Chronic Lymphocytic Leukemia Patients.

Unlike classical RAS genes, oncogenic mutations on RRAS2 are seldomly found in human cancer. By contrast, RRAS2 is frequently found overexpressed in a number of human tumors, including B and T cell lymphomas, breast, gastric, head and neck cancers. In this regard, we have recently shown that overexpression of wild-type RRAS2 drives the formation of both chronic lymphocytic leukemia (CLL) and breast cancer in mice. In support for the relevance of overexpression of wild type RRAS2 in human cancer, we have found that RRAS2 expression is influenced by the presence of a specific single nucleotide polymorphism (SNP) located in the 3'-untranslated region (UTR) of the RRAS2 mRNA. Perhaps more importantly, the presence of the alternate C, rather than the G allele, at the RRAS2 SNP designated as rs8570 is also associated with worse patient prognosis in CLL. This indicates that the detection of this SNP allelic variants can be informative to predict RRAS2 expression levels and disease long-term evolution in patients. Here, we describe a polymerase chain reaction (PCR)-based method that facilitates the rapid and easy determination of G and C allelic variants of the SNP. Using this approach, we confirm that the C allelic variant is associated with higher expression levels of RRAS2 transcripts and poor patient prognosis. However, we have also found that expression of the C allelic variants correlates with better response to ibrutinib, a Bruton kinase inhibitor commonly used in CLL treatments. This suggests that this method for detecting the RRAS2 rs8570 SNP might be a useful as a tool to predict both patient prognosis and response to targeted therapy in CLL.