The RASopathies: from pathogenetics to therapeutics

Abstract

The RASopathies are a group of disorders caused by a germline mutation in one of the genes encoding a component of the RAS/MAPK pathway. These disorders, including neurofibromatosis type 1, Noonan syndrome, cardiofaciocutaneous syndrome, Costello syndrome and Legius syndrome, among others, have overlapping clinical features due to RAS/MAPK dysfunction. Although several of the RASopathies are very rare, collectively, these disorders are relatively common. In this Review, we discuss the pathogenesis of the RASopathy-associated genetic variants and the knowledge gained about RAS/MAPK signaling that resulted from studying RASopathies. We also describe the cell and animal models of the RASopathies and explore emerging RASopathy genes. Preclinical and clinical experiences with targeted agents as therapeutics for RASopathies are also discussed. Finally, we review how the recently developed drugs targeting RAS/MAPK-driven malignancies, such as inhibitors of RAS activation, direct RAS inhibitors and RAS/MAPK pathway inhibitors, might be leveraged for patients with RASopathies.

Keywords: Cardiofaciocutaneous syndrome; Costello syndrome; Legius syndrome; Noonan syndrome; RAS; RASopathies.

Fig. 1.

Modeling the RASopathies. (A) Biochemical assays with purified proteins. These assays can compare the impacts of RASopathy-associated mutations on the enzymatic activities of the chosen proteins. Some examples include kinase, phosphatase, GEF-stimulated exchange, GEF-independent exchange, GAP-stimulated hydrolysis and GAP-independent hydrolysis activities. In addition, purified protein can be used to determine the impact of RASopathy variants on protein–protein interactions. Bmax, maximum specific binding; Kd, dissociation constant. (B) Transfection of RASopathy variants into mammalian cells. These assays assess the ability of the variants to stimulate the RAS/MAPK pathway, as well as define the subcellular localization of the variants in comparison to the wild-type protein. (C) Expression of RASopathy variants in model organisms. These models facilitate the study of the impact of RAS/MAPK pathway activation at the organismal level. In Drosophila melanogaster, aberrant RAS/MAPK signaling in the eye leads to small, rough eyes, while aberrant signaling in the wing leads to ectopic wing development. Similarly, aberrant RAS/MAPK signaling in the zebrafish (Danio rerio) embryo leads to elongated embryos with an increased major:minor axis ratio, and aberrant RAS/MAPK signaling in developing Caenorhabditis elegans embryos leads to various vulvar phenotypes. These phenotypes can be quantified, elucidating the genotype/phenotype correlations when comparing RASopathy-associated variants in these systems. The D. melanogaster, D. rerio, C. elegans and Mus musculus orthologs of each of the human RASopathy-associated genes are listed in Table S2. (D) Patient-derived cell lines. Fibroblast lines, lymphoblastoid lines and induced pluripotent stem cells can be used in a variety of cell signaling and phenotypic assays, provided an appropriate control line is available for comparison. Model organisms and patient-derived cell lines can both be used for preclinical drug screening. (E) Knock-in/knockout mouse models. These can be used to determine the developmental stage at which a variant can trigger the RASopathy phenotype, as well as identify the cell of origin for the RASopathy phenotypes. Germline expression of RASopathy variants in mouse models can identify genetic modifiers of the phenotype through backcrossing onto different genetic backgrounds. Moreover, these mouse models can be used in preclinical studies of potential therapeutics. Knockout models of RASopathy genes are summarized in Table S1, while knock-in models are described in Tables S3-S7, which are organized by RASopathy.

Fig. 2.

Pathogenesis of Noonan syndrome with loose anagen hair (NS-LAH) and related disorders. (A) In the basal state, MRAS and HRAS/KRAS/NRAS (depicted as RAS) are GDP bound, and RAF1 is held in the autoinhibited confirmation by 14-3-3 binding to phospho-serines 259 and 621. (B) The NS-LAH-associated SHOC2^S2G causes aberrant myristylation and membrane localization of SHOC2. Activation of MRAS, which can occur via guanine nucleotide exchange factor activity or via mutation, myristylation of SHOC2, or mutation of the PPP1CB phosphatase, facilitates the formation of a complex between MRAS-GTP and PPP1CB, SHOC2 and SCRIB. RAF1 membrane localization via binding to RAS-GTP puts RAF1 in proximity to the MRAS/SHOC2/PPP1CB complex, which allows PPP1CB to dephosphorylate S259 on RAF1. (C) In the active state, S259-dephosphorylated RAF1 is now able to homo- or heterodimerize with other RAF proteins, activating its kinase activity and triggering the signaling through MEK to ERK. 14-3-3 binding to S621 stabilizes the dimerization.

Fig. 3.

Pathogenesis of LZTR1-associated Noonan syndrome (NS). LZTR1 facilitates the CUL3-mediated polyubiquitination of several RAS isoforms, which leads to their degradation. Loss-of-function alterations in LZTR1, then, increase RAS stability and signaling through the RAS/MAPK pathway, driving NS phenotype.

Fig. 4.

Treating RASopathies. Researchers have developed several classes of drugs that target RAS activation and components of the MAPK or the PI3K/AKT pathway. Drugs that inhibit RAS activation include those that target RAS directly (e.g. sotorasib), inhibit RAS membrane localization (e.g. tipifarnib), prevent the activity of SHP2 (e.g. RMC-4550), inhibit the interaction of RAS with its exchange factor SOS1 (e.g. BI 1701963), or block the kinase activity of receptor tyrosine kinases (e.g. dasatinib). Please see the text for more details. Blue font indicates preclinical tool compounds; black font indicates a drug in clinical development. Several have been FDA approved for the treatment of cancer, including sotorasib, trametinib and copanlisib, among others, and could potentially be used for the treatment of RASopathies. TTM, tetrathiomolybate.