Somatic RIT1 delins in arteriovenous malformations hyperactivate RAS-MAPK signaling amenable to MEK inhibition.

Arteriovenous malformations (AVM) are benign vascular anomalies prone to pain, bleeding, and progressive growth. AVM are mainly caused by mosaic pathogenic variants of the RAS-MAPK pathway. However, a causative variant is not identified in all patients. Using ultra-deep sequencing, we identified novel somatic RIT1 delins variants in lesional tissue of three AVM patients. RIT1 encodes a RAS-like protein that can modulate RAS-MAPK signaling. We expressed RIT1 variants in HEK293T cells, which led to a strong increase in ERK1/2 phosphorylation. Endothelial-specific mosaic overexpression of RIT1 delins in zebrafish embryos induced AVM formation, highlighting their functional importance in vascular development. Both ERK1/2 hyperactivation in vitro and AVM formation in vivo could be suppressed by pharmacological MEK inhibition. Treatment with the MEK inhibitor trametinib led to a significant decrease in bleeding episodes and AVM size in one patient. Our findings implicate RIT1 in AVM formation and provide a rationale for clinical trials with targeted treatments.

R(+) Propranolol decreases lipid accumulation in hemangioma-derived stem cells.

Background: Infantile hemangioma (IH) is a benign vascular tumor that undergoes an initial rapid growth phase followed by spontaneous involution. A fibrofatty residuum remains in many tumors and often necessitates resection. We recently discovered that R(+) propranolol, the non-ß blocker enantiomer, inhibits blood vessel formation of IH patient-derived hemangioma stem cells (HemSC) xenografted in mice. HemSC are multipotent cells with the ability to differentiate into endothelial cells, pericytes, and adipocytes. Objectives: We investigated how R(+) propranolol affects HemSC adipogenic differentiation and lipid accumulation, in vitro and in a preclinical murine model for IH. Methods: We conducted a 10-day adipogenesis assay on 4 IH patient-derived HemSCs. Oil Red O (ORO) staining was used to identify the onset and level of lipid accumulation in HemSC while quantitative real-time polymerase chain reaction was conducted to determine the temporal expression of key factors implicated in adipogenesis. 5-20µM R(+) propranolol treatment was added to HemSC induced to undergo adiogenesis for 4 and 8 days, followed by quantification of lipid-stained areas and transcript levels of key adipogenic factors. We immunostained for lipid droplet-associated protein Perilipin 1 (PLIN1) in HemSC-xenograft sections from mice treated with R(+) propranolol and quantified the area using ImageJ. Results: We found that different patient-derived HemSC exhibit a robust and heterogenous adipogenic capacity when induced for adipogenic differentiation in vitro. Consistently across four IH patient-derived HemSC isolates, R(+) propranolol reduced ORO-stained areas and lipoprotein lipase (LPL) transcript levels in HemSC after 4 and 8 days of adipogenic induction. In contrast, R(+) propranolol had no significant inhibitory effect on transcript levels encoding adipogenic transcription factors. In a pre-clinical HemSC xenograft model, PLIN1-positive area was significantly reduced in xenograft sections from mice treated with R(+) propranolol, signifying reduced lipid accumulation. Conclusions: Our findings suggest a novel regulatory role for the R(+) enantiomer of propranolol in modulating lipid accumulation in HemSC. This highlights a novel role of R(+) propranolol in the involuting phase of IH and a strategy to reduce fibrofatty residua in IH. What is already known about this topic?: Propranolol is the mainstay treatment for infantile hemangioma (IH), the most common tumor of infancy, but its use can be associated with concerning ß-blocker side effects.R(+) propranolol, the enantiomer largely devoid of ß-blocker activity, was recently shown to inhibit endothelial differentiation of hemangioma-derived stem cells (HemSC) in vitro and reduce blood vessel formation in a HemSC-derived xenograft murine model of IH. What does this study add?: R(+) propranolol inhibits lipid accumulation in HemSC in vitro.R(+) propranolol does not affect mRNA transcript levels of key adipogenic transcription factors in differentiating HemSC in vitro.R(+) propranolol reduces lipid accumulation in a pre-clinical xenograft murine model of IH. What is the translational message?: The R(+) enantiomer of propranolol could be advantageous in terms of reduction in ß-adrenergic side effects and fibrofatty tissue formation in the involuting phase of IH.Less fibrofatty residua might reduce the need for surgical resection.Disfigurement and associated psychosocial impacts might be improved in this young patient cohort.

Extracranial Vascular Anomalies Driven by RAS/MAPK Variants: Spectrum and Genotype-Phenotype Correlations.

BACKGROUND: We aimed to correlate alterations in the rat sarcoma virus (RAS)/mitogen-activated protein kinase pathway in vascular anomalies to the clinical phenotype for improved patient and treatment stratification. METHODS AND RESULTS: This retrospective multicenter cohort study included 29 patients with extracranial vascular anomalies containing mosaic pathogenic variants (PVs) in genes of the RAS/mitogen-activated protein kinase pathway. Tissue samples were collected during invasive treatment or clinically indicated biopsies. PVs were detected by the targeted sequencing of panels of genes known to be associated with vascular anomalies, performed using DNA from affected tissue. Subgroup analyses were performed according to the affected genes with regard to phenotypic characteristics in a descriptive manner. Twenty-five vascular malformations, 3 vascular tumors, and 1 patient with both a vascular malformation and vascular tumor presented the following distribution of PVs in genes: Kirsten rat sarcoma viral oncogene (n=10), neuroblastoma ras viral oncogene homolog (n=1), Harvey rat sarcoma viral oncogene homolog (n=5), V-Raf murine sarcoma viral oncogene homolog B (n=8), and mitogen-activated protein kinase kinase 1 (n=5). Patients with RAS PVs had advanced disease stages according to the Schobinger classification (stage 3-4: RAS, 9/13 versus non-RAS, 3/11) and more frequent progression after treatment (RAS, 10/13 versus non-RAS, 2/11). Lesions with Kirsten rat sarcoma viral oncogene PVs infiltrated more tissue layers compared with the other PVs including other RAS PVs (multiple tissue layers: Kirsten rat sarcoma viral oncogene, 8/10 versus other PVs, 6/19). CONCLUSIONS: This comparison of patients with various PVs in genes of the RAS/MAPK pathway provides potential associations with certain morphological and clinical phenotypes. RAS variants were associated with more aggressive phenotypes, generating preliminary data and hypothesis for future larger studies.