OBJECTIVE: Pathogenic LZTR1 variants cause schwannomatosis and dominant/recessive Noonan syndrome (NS). We aim to establish an association between heterozygous loss-of-function (LoF) LZTR1 alleles and isolated multiple café-au-lait macules (CaLMs).
METHODS: 849 unrelated participants with multiple CaLMs, lacking pathogenic/likely pathogenic NF1 and SPRED1 variants, underwent RASopathy gene panel sequencing. Data on 125 individuals with heterozygous LZTR1 variants were collected for characterizing their clinical features and the associated molecular spectrum. In vitro functional assessment was performed on a representative panel of missense variants and small in-frame deletions.
RESULTS: Analysis revealed heterozygous LZTR1 variants in 6.0% (51/849) of participants, exceeding the general population prevalence. LZTR1-related CaLMs varied in number, displayed sharp or irregular borders, and were generally isolated, but occasionally associated with features recurring in RASopathies. In two families, CaLMs and schwannomas co-occurred. The molecular spectrum mainly consisted of truncating variants, indicating LoF. These variants substantially overlapped with those occurring in schwannomatosis and recessive NS. Functional characterization showed accelerated protein degradation or mislocalization, and failure to downregulate MAPK signaling.
CONCLUSIONS: Our findings expand the phenotypic variability associated with LZTR1 variants, which, in addition to conferring susceptibility to schwannomatosis and causing dominant and recessive NS, occur in individuals with isolated multiple CaLMs.

Pulmonary arterial hypertension (PAH) is a debilitating vascular disorder characterized by abnormal pulmonary artery smooth muscle cell (PASMC) proliferation and collagen synthesis, contributing to vascular remodeling and elevated pulmonary vascular resistance. This study investigated the critical role of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) in cell proliferation and collagen synthesis in PASMCs in PAH. Here we show that ATIC levels are significantly increased in the lungs of MCT-induced PAH rat model and hypoxia-induced PAH mouse model and PDGF-stimulated PASMCs. Inhibition of ATIC attenuated platelet-derived growth factor (PDGF)-induced cell proliferation and collagen I synthesis in PASMCs. Conversely, overexpression or knockdown of ATIC causes a significant promotion or inhibition of Ras and ERK activation, cell proliferation and collagen synthesis in PASMCs. Moreover, ATIC deficiency attenuated Ras activation in the lungs of hypoxia-induced PAH mice. Further, Ras inhibition attenuates ATIC overexpression- and PDGF-induced collagen synthesis and PASMC proliferation. Notably, we identified that transcription factor MYC, EGR1, and SP1 directly binds to promoters of Atic gene and regulate ATIC expression. These results provide the first evidence that ATIC promotes PASMC proliferation in pulmonary vascular remodeling through the Ras signaling pathway.

The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.

Casitas B-lineage lymphoma (CBL) encodes an adaptor protein with E3-ligase activity negatively controlling intracellular signaling downstream of receptor tyrosine kinases. Somatic CBL mutations play a driver role in a variety of cancers, particularly myeloid malignancies, whereas germline defects in the same gene underlie a RASopathy having clinical overlap with Noonan syndrome (NS) and predisposing to juvenile myelomonocytic leukemia and vasculitis. Other features of the disorder include cardiac defects, postnatal growth delay, cryptorchidism, facial dysmorphisms, and predisposition to develop autoimmune disorders. Here we report a novel CBL variant (c.1202G>T; p.Cys401Phe) occurring de novo in a subject with café-au-lait macules, feeding difficulties, mild dysmorphic features, psychomotor delay, autism spectrum disorder, thrombocytopenia, hepatosplenomegaly, and recurrent hypertransaminasemia. The identified variant affects an evolutionarily conserved residue located in the RING finger domain, a known mutational hot spot of both germline and somatic mutations. Functional studies documented enhanced EGF-induced ERK phosphorylation in transiently transfected COS1 cells. The present findings further support the association of pathogenic CBL variants with immunological and hematological manifestations in the context of a presentation with only minor findings reminiscent of NS or a clinically related RASopathy.

RAS mutations involving codon 61 are rare in metastatic colorectal cancer (mCRC), accounting for only 1-4%, but they have recently been identified with high frequency in the circulating tumor DNA (ctDNA) of patients with secondary resistance to anti-EGFRs. This retrospective monocentric study aimed to investigate the clinical phenotype and prognostic performance of codon 61 RAS-mutated mCRC. Fifty patients with codon 61 RAS-mutated mCRC treated at our institution between January 2013 and December 2021 were enrolled. Additional datasets of codon 61 RAS wild-type mCRCs (648 patients) were used as comparators. The endpoint for prognostic assessment was overall survival (OS). Metastatic involvement of the peritoneum or ovary was significantly more frequent in codon 61 RAS-mutated mCRC compared to codon 61 RAS wild-type (54 vs. 28.5%), non-codon 61 RAS-mutated (35.6%), BRAF V600E-mutated (25%), and RAS/BRAF wild-type (20.5%) cohorts. At a median follow up of 96.2 months, the median OS for codon 61 RAS-mutated patients was significantly shorter compared to RAS/BRAF wild-type (26.9 vs. 36.0 months, HR 0.56) patients, while no significant difference was observed compared to non-codon 61 RAS-mutated and BRAF V600E-mutated patients. We showed a negative prognostic impact and a statistically significant correlation between codon 61 RAS mutations and metastatic involvement of the peritoneum and ovary.

BACKGROUND: Rasal1 is a Ras GTPase-activating protein which contains C2 domains necessary for dynamic membrane association following intracellular calcium elevation. Membrane-bound Rasal1 inactivates Ras signaling through its RasGAP activity, and through such mechanisms has been implicated in regulating various cellular functions in the context of tumors. Although highly expressed in the brain, the contribution of Rasal1 to neuronal development and function has yet to be explored.
RESULTS: We examined the contributions of Rasal1 to neuronal development in primary culture of hippocampal neurons through modulation of Rasal1 expression using molecular tools. Fixed and live cell imaging demonstrate diffuse expression of Rasal1 throughout the cell soma, dendrites and axon which localizes to the neuronal plasma membrane in response to intracellular calcium fluctuation. Pull-down and co-immunoprecipitation demonstrate direct interaction of Rasal1 with PKC, tubulin, and CaMKII. Consequently, Rasal1 is found to stabilize microtubules, through post-translational modification of tubulin, and accordingly inhibit dendritic outgrowth and branching. Through imaging, molecular, and electrophysiological techniques Rasal1 is shown to promote NMDA-mediated synaptic activity and CaMKII phosphorylation.
CONCLUSIONS: Rasal1 functions in two separate roles in neuronal development; calcium regulated neurite outgrowth and the promotion of NMDA receptor-mediated postsynaptic events which may be mediated both by interaction with direct binding partners or calcium-dependent regulation of down-stream pathways. Importantly, the outlined molecular mechanisms of Rasal1 may contribute notably to normal neuronal development and synapse formation.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is the most common cancer of head and neck cancer. Y-box binding protein-1 (YBX1) has tumor-promoting effects in some types of cancers. However, its role in LSCC remains unknown. This study set out to identify the role of YBX1 in LSCC.
METHODS: Bioinformatics analysis of the Gene Expression Omnibus (GEO) database and our cohort data were used to explore the association of YBX1 expression with clinicopathological factors in LSCC. Then, cells with stably or transiently transfected with plasmid or siRNA were constructed to assess the effect of loss and gain of YBX1 on the biological phenotypes of LSCC cells in vitro. In addition, subcutaneous xenograft and orthotopic liver tumor mouse models were constructed for validation. The interrogated miRNA databases and subsequent luciferase reporter assays were used to confirm the miR-382-5p target of YBX1. At last, KEGG enrichment annotation from TGCA data was used for downstream analyses of miR-382-5p/YBX1 and verified by PCR and Western immunoblotting.
RESULTS: The results showed that significant upregulation of YBX1 in LSCC tumors was correlated with advanced TNM stage and poor prognosis. Knockdown of YBX1 markedly impaired the proliferative, invasive, and migratory activity of Tu212 cells. We confirmed that miR-382-5p targets YBX1 to mediate LSCC progression both in vitro and in vivo. We further confirmed that miR-382-5p/YBX1 modulated the Ras/MAPK signaling axis to regulate the progression of LSCC.
CONCLUSIONS: Together, our results indicated that YBX1 is an important promoter of LSCC progression. And miR-382-5p/YBX1/RAS/MAPK signaling pathway can be perceived as a promising target in the treatment of LSCC.

ARHGAP35, which encodes p190A RhoGAP (p190A), is a major cancer gene. p190A is a tumor suppressor that activates the Hippo pathway. p190A was originally cloned via direct binding to p120 RasGAP (RasGAP). Here, we determine that interaction of p190A with the tight-junction-associated protein ZO-2 is dependent on RasGAP. We establish that both RasGAP and ZO-2 are necessary for p190A to activate large tumor-suppressor (LATS) kinases, elicit mesenchymal-to-epithelial transition, promote contact inhibition of cell proliferation, and suppress tumorigenesis. Moreover, RasGAP and ZO-2 are required for transcriptional modulation by p190A. Finally, we demonstrate that low ARHGAP35 expression is associated with shorter survival in patients with high, but not low, transcript levels of TJP2 encoding ZO-2. Hence, we define a tumor-suppressor interactome of p190A that includes ZO-2, an established constituent of the Hippo pathway, and RasGAP, which, despite strong association with Ras signaling, is essential for p190A to activate LATS kinases.

The Ras (rat sarcoma virus) is a GTP-binding protein that is considered one of the important members of the Ras-GTPase superfamily. The Ras involves several pathways in the cell that include proliferation, migration, survival, differentiation, and fibrosis. Abnormalities in the expression level and activation of the Ras family signaling pathway and its downstream kinases such as Raf/MEK/ERK1-2 contribute to the pathogenic mechanisms of rheumatic diseases including immune system dysregulation, inflammation, and fibrosis in systemic sclerosis (SSc); destruction and inflammation of synovial tissue in rheumatoid arthritis (RA); and autoantibody production and immune complexes formation in systemic lupus erythematosus (SLE); and enhance osteoblast differentiation and ossification during skeletal formation in ankylosing spondylitis (AS). In this review, the basic biology, signaling of Ras, and abnormalities in this pathway in rheumatic diseases including SSc, RA, AS, and SLE will be discussed.

Sprouty-related EVH-1 domain-containing (SPRED) proteins are a family of proteins that negatively regulate the RAS-Mitogen-Activated Protein Kinase (MAPK) pathway, which is involved in the regulation of the mitogenic response and cell proliferation. However, the mechanism by which these proteins affect RAS-MAPK signaling has not been elucidated. Patients with mutations in SPRED give rise to unique disease phenotypes; thus, we hypothesized that distinct interactions across SPRED proteins may account for alternative nodes of regulation. To characterize the SPRED interactome and evaluate how members of the SPRED family function through unique binding partners, we performed affinity purification mass spectrometry. We identified 90-kDa ribosomal S6 kinase 2 (RSK2) as a specific interactor of SPRED2 but not SPRED1 or SPRED3. We identified that the N-terminal kinase domain of RSK2 mediates the interaction between amino acids 123 to 201 of SPRED2. Using X-ray crystallography, we determined the structure of the SPRED2-RSK2 complex and identified the SPRED2 motif, F145A, as critical for interaction. We found that the formation of this interaction is regulated by MAPK signaling events. We also find that this interaction between SPRED2 and RSK2 has functional consequences, whereby the knockdown of SPRED2 resulted in increased phosphorylation of RSK substrates, YB1 and CREB. Furthermore, SPRED2 knockdown hindered phospho-RSK membrane and nuclear subcellular localization. We report that disruption of the SPRED2-RSK complex has effects on RAS-MAPK signaling dynamics. Our analysis reveals that members of the SPRED family have unique protein binding partners and describes the molecular and functional determinants of SPRED2-RSK2 complex dynamics.