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

Abstract

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.

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

“…These results suggest that other gain-of-function mutations of RRAS2 that are found at low frequency in human tumors and Noonan syndrome patients could also be oncogenic. Consistent with this idea, we have found that R-RAS2 proteins with mutations at residues Gly 23 , Gly 24 , Ala 70 or Gln 72 can transform NIH3T3 cells. Our signaling experiments also indicate that the oncogenic versions of R-RAS2 can potently activate the RAF-ERK and PI3K-AKT pathways in the transformed NIH3T3 cells.…”

“…1A, B). These data indicate that, like mutations of classical RAS proteins [22], the mutations targeting the Gly 23 , Gly 24 , Ala 70 or Gln 72 residues of R-RAS2 are oncogenic.…”

“…Based on this, we tested the transforming activity of a collection of 21 additional RRAS2 mutations that are found in human tumors at very low frequency, according to data from the cBioPortal database. Of note, all RRAS2 mutations targeting the residues Gly 23 (G23A/C/S/V), Gly 24 (G24C/D/V), Ala 70 (A70T) or Gln 72 (Q72L/H) display transforming activity in focus formation assays (Fig. 1A, B).…”

“…Thus, we have shown that the elimination of endogenous R-Ras2 leads the abrogation of Her2-driven mammary tumorigenesis as well as of Notch1-driven T cell acute lymphoblastic leukemia [ 18 , 23 ]. Conversely, the overexpression of R-Ras2 per se promotes the development of a B-cell chronic lymphocytic leukemia-like condition in mice [ 24 ]. R-RAS2 also plays roles in normal physiological conditions, including in mammary gland development, the negative selection of T lymphocytes, B-cell–dependent germinal responses, T cell phagocytosis and T cell receptor homeostatic signaling [ 25 – 30 ].…”

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

“…Beyond the limited number of tumors bearing oncogenic RRAS2 mutations, it is possible that the WT version of R-RAS2 could play more general roles in human tumors because of gene amplification, upregulated expression, enhanced activity, or presence of mutations in upstream and negative regulatory elements in cancer cells. Genetic evidence in favor of the importance of endogenous R-Ras2 WT in Her2-driven mammary tumorigenesis (Larive et al, 2014), B cell chronic lymphocytic leukemia (Hortal et al, 2022), and Notch1-driven leukemogenesis (this work) is already available for mice. Future studies are needed to better understand the effect of the WT and oncogenic versions of this GTPase in human tumors.…”