Whole genome sequencing of AVM endothelial and non-endothelial cell fractions
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ABSTRACT:
PROVIDER: EGAS00001006719 | EGA |
REPOSITORIES: EGA
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Project description:We previously identified somatic activating KRAS mutations in a majority of human arteriovenous malformations (AVMs), using whole exome sequencing. We have now performed targeted sequencing on AVMS, investigating genes in the RAS-MAPK pathway, angiogenesis and brain vascular disorders among others.
| EGAS00001006729 | EGA
Project description:Rationale – We previously identified somatic activating mutations in the KRAS gene in the endothelium of the majority of human sporadic brain arteriovenous malformations (bAVMs); a disorder characterized by direct connections between arteries and veins. However, whether this genetic abnormality alone is sufficient for lesion formation, as well as how active KRAS signaling contributes to AVM formation, remains unknown. Objective – To establish the first in vivo models of somatic KRAS gain of function in the endothelium in both mice and zebrafish in order to directly observe the phenotypic consequences of constitutive KRAS activity at a cellular level in vivo, and to test potential therapeutic interventions for AVMs. Methods and Results – Using both postnatal and adult mice, as well as embryonic zebrafish, we demonstrate that endothelial-specific gain of function mutations in Kras (G12D or G12V) are sufficient to induce bAVMs. Active KRAS signaling leads to altered endothelial cell morphogenesis and increased cell size, ectopic sprouting, expanded vessel lumen diameter, and direct connections between arteries and veins. Furthermore, we show that these lesions are not associated with altered endothelial growth dynamics or a lack of proper arteriovenous identity, but instead appear to feature exuberant angiogenic signaling. Finally, we demonstrate that KRAS-dependent AVMs in zebrafish are refractory to inhibition of the downstream effector PI3K, but instead require active MEK signaling. Conclusions – We demonstrate that active KRAS expression in the endothelium is sufficient for bAVM formation, even in the setting of uninjured adult vasculature. Furthermore, the finding that KRAS-dependent lesions are reversible in zebrafish suggests that MEK inhibition may represent a promising therapeutic treatment for AVM patients.
2020-07-01 | GSE150426 | GEO
Project description:Brain arteriovenous malformation (bAVM) is a disease characterized by the formation of tangled blood vessels with direct connections between arteries and veins. There is currently limited understanding of the underlying molecular mechanisms that are responsible for the development of bAVM. Our previous work identified the presence of somatic activating KRAS mutations in endothelial cells (ECs) isolated from clinical bAVM tissue. As such, we performed this proteomics study to profile the molecular impact of mutant KRAS expression on endothelial biology. We engineered a human EC model with doxycycline-inducible expression of mutant KRAS G12V. As mutant KRAS ECs preferentially activated the MAPK/ERK signaling cascade with no change to the activities of PI3K/AKT or MAPK/p38 pathways, we also included a MEK inhibitor (SL327) in this study to delineate MEK-dependent versus MEK-independent proteome changes.
2025-12-14 | PXD062375 | Pride
Project description:Vascular malformations are congenital lesions caused by somatic and germline mutations that disrupt developmental signaling pathways. Capillary malformations (CMs) typically present as port-wine stains in the skin and can also affect ocular and cerebral tissues in Sturge Weber Syndrome (SWS), leading to aesthetic, neurological, and ophthalmic complications. CMs are caused by a somatic mosaic mutation in the GNAQ gene in endothelial cells, leading to an activating p.R183Q substitution in the Gαq protein. The underlying mechanisms of Gαq-R183Q-driven CMs formation still remain unclear. To address this, we generated CRISPR/Cas9-engineered human dermal microvascular endothelial cells lacking endogenous Gαq, whilst expressing the Gαq-R183Q mutant instead. The Gαq-R183Q mutation strongly impaired endothelial cell migration and angiogenic sprouting capacity compared to wild-type controls. Next, using SILAC-based quantitative proteomics, we investigated the Gαq-R183Q-induced changes in the endothelial phosphoproteome. These analyses revealed prominent activation of calcineurin-NFAT signaling pathway in Gαq-R183Q-expressing endothelial cells, leading to dephosphorylation of NFAT1 and NFAT2 and the selective expression of their transcriptional target DSCR1.4. Immunofluorescence of patient-derived skin biopsies confirmed deregulation of NFAT1/2 and the expression of DSRC1 in endothelial cells, validating their potential importance in CMs. We further demonstrate that pharmacological inhibition of calcineurin with tacrolimus (FK506) could partially restore NFAT signaling, collective cell migration and sprouting in Gαq-R183Q endothelial cells. Intriguingly, the genetic depletion of the NFAT target DSCR1 in Gαq-R183Q cells fully rescued calcineurin/NFAT signaling as well as key endothelial functions. In summary, we uncovered a calcineurin-NFAT-DSCR1.4 signal transduction axis that is driven by Gαq-R183Q and established its importance for endothelial angiogenic properties. These findings highlight that calcineurin/NFAT signaling represents a promising therapeutic target to restore endothelial function in CMs.
2026-02-04 | PXD064187 | Pride
Project description:Brain arteriovenous malformation (bAVM) is a disease characterized by the formation of tangled blood vessels with direct connections between arteries and veins. There is currently limited understanding of the underlying molecular mechanisms that are responsible for the development of bAVM. Our previous work identified the presence of somatic activating KRAS mutations in endothelial cells (ECs) isolated from clinical bAVM tissue. Given the association between mutant KRAS, metabolic reprogramming and EC angiogenesis, we aimed to investigate if altered endothelial metabolism is involved in the pathogenesis of bAVM to drive abnormal vascular remodeling. As such, we engineered a human EC model with inducible expression of mutant KRAS G12V. We observed a significant increase in glucose metabolism upon mutant KRAS expression. This elevation in EC glycolysis contributes to the angiogenic behaviours that are observed in our mutant KRAS EC models. To further explore the transcriptional alterations underlying these changes, we performed this RNA-sequencing study with various glycolysis inhibitors and a MEK inhibitor (the major signaling pathway altered in mutant KRAS ECs) to profile the impact of glucose metabolism changes on endothelial biology.
2025-12-15 | GSE292538 | GEO
Project description:<p>KRAS mutations are prevalent in lung cancer, but KRAS G12C inhibitors exhibit limited efficacy, partly due to metabolic adaptations, such as enhanced glutathione metabolism and increased glycolysis. Glutathione S-Transferase Zeta 1 (GSTZ1) is a metabolic enzyme that regulates cell metabolism. However, its role in KRAS-driven lung cancer remains underexplored. We recently reported that targeting GSTZ1 significantly enhances the efficacy of FDA-approved KRAS G12C inhibitors in non-small cell lung cancer cells. Untargeted metabolomics now revealed significant alterations in glutathione and glycolytic pathways, marked by lowered lactate levels and increased oxidized glutathione following GSTZ1 ablation. Moreover, pharmacological inhibition of glutathione synthesis and glucose uptake mimicked the sensitizing effects of GSTZ1 targeting. These metabolic shifts were accompanied by increased AMPK phosphorylation and reduced AKT phosphorylation, two key mediators of the response to KRAS G12C inhibition. Our findings establish GSTZ1-induced metabolic and signaling alterations as a novel mechanism of sensitization, positioning GSTZ1 as a potential therapeutic target.</p>
2026-05-15 | MTBLS12791 | MetaboLights
Project description:Brain arteriovenous malformation (BAVM) is a high-flow vascular shunt that predisposes to intracranial hemorrhage and represents a leading cause of stroke in young adults. Somatic mutations in KRAS or BRAF drive endothelial dysfunction and lesion formation, yet the contribution of non-endothelial cells within the neuro-glial-vascular unit remains incompletely understood.
2026-04-27 | GSE326421 | GEO
Project description:Sturge-Weber syndrome (SWS) is a sporadic, congenital, neuro-cutaneous disorder characterized by a mosaic, capillary malformation. SWS and isolated capillary malformations are caused by a somatic activating mutation in GNAQ encoding the G protein subunit alpha-q protein. The missense mutation R183Q is the sole GNAQ mutation identified thus far in affected tissues of 90% of SWS patients. In this study, we sequenced skin biopsies of affected capillary malformations from 9 patients. We identified the R183Q mutation in nearly all samples, but one sample exhibited a Q209R mutation. This new mutation occurs at the same residue as the constitutively-activating Q209L mutation, commonly seen in tumors. However, Q209R is a rare variant in this gene. To compare the effect of the Q209R mutation on downstream signaling, we performed reporter assays with a GNAQ-responsive reporter co-transfected with either GNAQ WT, R183Q, Q209L, Q209R, or C9X (representing a null allele). Q209L showed the highest reporter activation, with R183Q and Q209R showing significantly lower activation. To determine whether these mutations had similar or different downstream consequences we performed RNAseq analysis in microvascular endothelial cells (HMEC-1) electroporated with the same GNAQ variants. The R183 and Q209 missense variants caused extensive dysregulation of a broad range of transcripts compared to the WT or null allele, confirming that these are all activating mutations. However, the missense variants exhibited very few differentially expressed genes (DEGs) when compared to each other. These data suggest that these activating GNAQ mutations differ in magnitude of activation but have similar downstream effects.
2022-07-28 | GSE199978 | GEO
Project description:86%–90% of pancreatic ductal adenocarcinoma (PDAC) harbor activating mutations in KRAS, which regulate multiple carcinogenic signaling pathways. In order to elucidate the mechanisms underlying KRAS mutation-driven development and progression of PDAC, quantitative proteomics mass-spectrometry(MS) analysis of differential proteins in KRAS-mutant tissues was performed.
2024-11-29 | PXD058402 |