Search

Melanoma arising in association with a blue nevus (BN) is rare but has molecular similarities to uveal melanoma (UM), including GNAQ/11 mutations. Tebentafusp was recently approved for UM based on improved overall survival in a phase 3 study. We hypothesized that tebentafusp may be active in BN-associated melanoma. Here, we present a case of metastatic BN-associated melanoma with rapid response and ~1 year of disease control on tebentafusp. We also explore molecular and histological features of secondary resistance. Our case highlights that PD-1-resistant melanomas should be screened for GNAQ/11 mutations, as tebentafusp may be a treatment option in this extremely rare disease., Competing Interests: Competing interests: ANS reports grants and personal fees from Bristol-Myers Squibb, Immunocore, Novartis, and Castle Biosciences, and institutional grants from Bristol-Myers Squibb, Immunocore, Novartis, Targovax, Pfizer, Mural Oncology, Checkmate Pharmaceuticals, Foghorn Therapeutics, Linnaeus Therapeutics, Prelude Therapeutics, Iovance Biotherapeutics, Obsidian Therapeutics. PF reports equity in GE Healthcare, Iovance, Gilead. JDW is a consultant for Apricity, Ascentage Pharma, Bicara Therapeutics, Daiichi Sankyo, Dragonfly, Imvaq, Larkspur, Takeda, Tizona, Trishula Therapeutics, Immunocore (on their Data Safety Board), Scancell. JDW received grant/research support from Bristol Myers Squibb, Enterome. JDW has equity in Apricity, Arsenal IO/CellCarta, Ascentage, Imvaq, Linneaus, Larkspur, Georgiamune, Maverick, Tizona Therapeutics, Xenimmune. JDW is an inventor on the following patents: Xenogeneic DNA Vaccines, Newcastle Disease viruses for Cancer Therapy, Myeloid-derived suppressor cell (MDSC) assay, Prediction of Responsiveness to Treatment with Immunomodulatory Therapeutics and Method of Monitoring Abscopal Effects During Such Treatment, Anti-PD1 Antibody, Anti-CTLA4 antibodies, Anti-GITR antibodies and methods of use thereof. The remaining authors declare no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

G protein alpha q subunit (Gαq) can binds to the G protein-coupled receptor (GPCR) for signaling and is closely related to lipid metabolism. Endurance exercise is an effective means of combating acquired obesity and its complications, but the mechanisms by which endurance exercise modulates hereditary obesity and its complications are unknown. In this study, we achieved knockdown of Gαq in drosophila adipose tissue and skeletal muscle by constructing the Gαq-UAS-RNAi/Ppl-Gal4 and Gαq-UAS-RNAi/Mef2-GAl4 systems. Drosophila were subjected a three-week endurance exercise intervention, and changes in relevant indicators were detected and observed by RT-PCR, ELISA, oil red staining, immunofluorescence staining, and transmission electron microscopy. The results showed that knockdown of Gαq in both adipose tissue and skeletal muscle induced a significant increase in triglycerides accompanied by a decrease in rapid climbing ability, a decrease in Superoxide Dismutase (SOD) activity level, and a decrease in Mitochondrial respiratory chain complexI (MRCC I) content in Drosophila whole body and skeletal muscle, and down-regulated the expression of the G protein alpha q subunit (Gαq), the skeletal muscle myosin heavy chain expression gene (Mhc), mitochondrial biogenesis gene Spargal(the PGC-1alpha homologue in Drosophila). Endurance exercise significantly improved the triglyceride levels in the whole body and skeletal muscle of drosophila with Gαq knockdown in adipose tissue and skeletal muscle, as well as their ability to climb, increased SOD activity level and MRCCI content level, and up-regulated the expression of Gαq, Mhc, and Spargal(Srl). Thus, the present findings suggest that genetic defects in the Gαq gene in adipose and skeletal muscle tissues induce hereditary obesity and skeletal muscle dysfunction, and that endurance exercise attenuates this hereditary obesity and concomitant skeletal muscle dysfunction in drosophila by improving skeletal muscle fiber contractile proteins, mitochondrial function and function, and antioxidant capacity via mediating the Gαq/Mhc, Gαq/Srl/MRCC-I, and Gαq/SOD pathways., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Capillary malformations (CM) are congenital vascular irregularities of capillary and venous blood vessels that appear in the skin, leptomeninges of the brain, and the choroid of the eye in the disorder known as Sturge Weber Syndrome (SWS). More common are non-syndromic CM found only in the skin, without brain or ocular involvement. A somatic activating mutation in GNAQ (p.R183Q) is found in ~ 90% of syndromic and non-syndromic CM specimens and is present in CD31 pos endothelial cells isolated from brain and skin CM specimens. Endothelial expression of the GNAQ p.R183Q variant is sufficient to form CM-like vessels in mice. Given the distinct features and functions of blood vessels in the brain versus the skin, we examined the features of CM vessels in both tissues to gain insights into the pathogenesis of CM. Herein, we present morphologic characteristics of CM observed in specimens from brain and skin. The GNAQ p.R183Q variant allelic frequency in each specimen was determined by droplet digital PCR. Sections were stained for endothelial cells, tight junctions, mural cells, and macrophages to assess the endothelium as well as perivascular constituents. CM blood vessels in brain and skin were enlarged, exhibited fibrin leakage and reduced zona occludin-1 and claudin-5, and were surrounded by MRC1 pos /LYVE1 pos macrophages. In contrast, the CMs from brain and skin differ in endothelial sprouting activity and localization of mural cells. These characteristics might be helpful in the development of targeted and/or tissue specific therapies to prevent or reverse non-syndromic and syndromic CM., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Polycystic ovarian syndrome (PCOS) is a prevalent endocrinological disorder affected by ghrelin. This study aimed to investigate the molecular mechanisms underlying the effects of ghrelin on PCOS manifestations in mice and to assess the therapeutic potential of ghrelin. Female C57BL/6 mice were subcutaneously injected with 6 mg/100 g dehydroepiandrosterone (DHEA) for 20 days to induce PCOS. Alterations in reproductive cycles, ovarian morphology, serum sex hormone levels, and related signaling markers were examined. Furthermore, ghrelin-induced effects on granulosa cells and the role of ghrelin/Gq/11/ Yes-associated protein (YAP) signaling were studied by silencing Gαq/11 or YAP using si-RNAs. Finally, we evaluated the therapeutic potential of anti-ghrelin antibodies in DHEA-induced PCOS mice. DHEA administration led to significant PCOS-associated changes including weight gain, disrupted estrous cycles, ovarian morphological alterations, and hormonal imbalances in mice, with elevated Gαq/11 and acylated ghrelin expression, which was also noted in PCOS patients. However, treatment with anti-ghrelin antibodies effectively managed DHEA-induced damage in PCOS mice. In vitro, ghrelin exposure resulted in granulosa cell injury and modulated estrogen receptors alpha (ERα) and YAP protein levels, whereas silencing YAP and Gαq/11 reversed ghrelin-induced detrimental effects and up-regulated ERα expression. This study revealed that DHEA-induced PCOS traits in mice could be improved by anti-ghrelin antibodies, with the ghrelin/Gq/11/YAP signaling pathway identified as a crucial mediator in granulosa cells, affecting ERα transcription to regulate PCOS. These findings suggest a potential therapeutic strategy for the treatment of PCOS., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

Phosphatidylinositol is a precursor of various phosphoinositides, which play crucial roles in intracellular signaling and membrane dynamics and have impact on diverse aspects of cell physiology. Phosphoinositide synthesis and turnover occur in the cytoplasmic leaflet of the organellar and plasma membranes. P4-ATPases (lipid flippases) are responsible for translocating membrane lipids from the exoplasmic (luminal) to the cytoplasmic leaflet, thereby regulating membrane asymmetry. However, the mechanism underlying phosphatidylinositol translocation across cellular membranes remains elusive. Here, we discovered that the phosphatidylcholine flippases ATP8B1, ATP8B2, and ATP10A can also translocate phosphatidylinositol at the plasma membrane. To explore the function of these phosphatidylinositol flippases, we used cells depleted of CDC50A, a protein necessary for P4-ATPase function and ATP8B1 and ATP8B2, which express in HeLa cells. Upon activation of the Gq-coupled receptor, depletion of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was accelerated in CDC50A knockout (KO) and ATP8B1/8B2 double KO cells compared with control cells, suggesting a decrease in PtdIns(4,5)P2 levels within the plasma membrane of the KO cells upon stimulation. These findings highlight the important role of P4-ATPases in maintaining phosphoinositide homeostasis and suggest a mechanism for asymmetry of phosphatidylinositol in the cytoplasmic leaflet of the plasma membrane., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cβ (PLCβ) isozymes to increase cytosolic Ca 2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca 2+ . By combining CRISPR/Cas9 genome editing to delete Gα s , the adenylyl cyclase isoforms 3 and 6, or the PLCβ1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gβγ as driver of a PLCβ2/3-mediated cytosolic Ca 2+ release module. This module does not require but crosstalks with Gα s -dependent cAMP, demands Gα q to release PLCβ3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCβ3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs., (© 2024. The Author(s).)

Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate an optogenetic tool that disrupts Gα q signaling through membrane recruitment of a minimal regulator of G protein signaling (RGS) domain. This approach, Photo-induced Gα Modulator-Inhibition of Gα q (PiGM-Iq), exhibited potent and selective inhibition of Gα q signaling. Using PiGM-Iq we alter the behavior of Caenorhabditis elegans and Drosophila with outcomes consistent with GPCR-Gα q disruption. PiGM-Iq changes axon guidance in cultured dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. Furthermore, by altering the minimal RGS domain, we show that this approach is amenable to Gα i signaling. Our unique and robust optogenetic Gα inhibiting approaches complement existing neurobiological tools and can be used to investigate the functional effects neuromodulators that signal through GPCR and trimeric G proteins., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Primary aldosteronism (PA) is a common cause of human hypertension. Somatic mutations in KCNJ5 , CACNA1D , ATP1A1 , and ATP2B3 are found in at least 80% of aldosterone-producing adenomas, which cause unilateral PA in humans. Somatic mutations have been identified infrequently in 7 other genes; few of these were known to play a role in aldosterone secretion before the discovery of their mutations. Interrogating somatic mutations in the domestic cat, in which spontaneous PA is also known to occur, might improve the understanding of normal adrenal gland physiology and the pathophysiology of PA., Methods: DNA and RNA extracted from tissue from 13 cats with unilateral aldosterone-secreting tumors, including 8 carcinomas and 5 adenomas, underwent whole genome sequencing, targeted Sanger sequencing, and RNA sequencing. Single-nucleotide substitution variants were filtered to select those with a predicted deleterious effect on protein function and a suspected role in aldosterone secretion., Results: Probable functional somatic single-nucleotide polymorphisms (n=8) were found in 3 adenomas and 2 carcinomas. Mutations with predicted significant effects were identified in 2 genes also mutated in human PA; GNAQ and CTNNB1 , and in a residue of CACNA1C analogous to a common CACNA1D mutation. In contrast to humans, CACNA1C expression was much greater than CACNA1D in both feline tumor and nontumor adrenal tissue. No mutations were identified in KCNJ5 , CACNA1D , ATP1A1 , or ATP2B3., Conclusions: Similar mutations were identified in cats to those found in humans. It is, therefore, likely that both species have shared underlying selection pressures for mutations that increase aldosterone secretion., Competing Interests: None.

Background: The Gα family plays a crucial role in the complex reproductive regulatory network of teleosts. However, the characterization and function of Gα family members, especially Gαq, remain poorly understood in teleosts. To analyze the characterization, expression, and function of grass carp (Ctenopharyngodon idella) Gαq, we identified the Gα family members in grass carp genome, and analyzed the expression, distribution, and signal transduction of Gαq/gnaq. We also explored the role of Gαq in the reproductive regulation of grass carp., Results: Our results showed that the grass carp genome contains 27 Gα genes with 46 isoforms, which are divided into four subfamilies: Gαs, Gαi/o, Gαq/11, and Gα12/13. The expression level of Cignaq in the testis was the highest and significantly higher than in other tissues, followed by the hypothalamus and brain. The luteinizing hormone receptor (LHR) was mainly localized to the nucleus in grass carp oocytes, with signals also present in follicular cells. In contrast, Gαq signal was mainly found in the cytoplasm of oocytes, with no signal in follicular cells. In the testis, Gαq and LHR were co-localized in the cytoplasm. Furthermore, the grass carp Gαq recombinant protein significantly promoted Cipgr expression., Conclusions: These results provided preliminary evidence for understanding the role of Gαq in the reproductive regulation of teleosts., (© 2024. The Author(s).)

Extra-axial cavernous hemangiomas (ECHs) are complex vascular lesions mainly found in the spine and cavernous sinus. Their removal poses significant risk due to their vascularity and diffuse nature, and their genetic underpinnings remain incompletely understood. Our approach involved genetic analyses on 31 tissue samples of ECHs employing whole-exome sequencing and targeted deep sequencing. We explored downstream signaling pathways, gene expression changes, and resultant phenotypic shifts induced by these mutations, both in vitro and in vivo. In our cohort, 77.4% of samples had somatic missense variants in GNA14, GNAQ, or GJA4. Transcriptomic analysis highlighted significant pathway upregulation, with the GNAQ c.626A>G (p.Gln209Arg) mutation elevating PI3K-AKT-mTOR and angiogenesis-related pathways, while GNA14 c.614A>T (p.Gln205Leu) mutation led to MAPK and angiogenesis-related pathway upregulation. Using a mouse xenograft model, we observed enlarged vessels from these mutations. Additionally, we initiated rapamycin treatment in a 14-year-old individual harboring the GNAQ c.626A>G (p.Gln209Arg) variant, resulting in gradual regression of cutaneous cavernous hemangiomas and improved motor strength, with minimal side effects. Understanding these mutations and their pathways provides a foundation for developing therapies for ECHs resistant to current therapies. Indeed, the administration of rapamycin in an individual within this study highlights the promise of targeted treatments in treating these complex lesions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Certain amino acid sites of 5-HT 2A R play crucial roles in interacting with various G proteins. Hallucinogens and non-hallucinogens both act on 5-HT 2A R but mediate different pharmacological effects, possibly due to the coupling of different G proteins. Therefore, this study identified the binding sites of hallucinogens and non-hallucinogens with 5-HT 2A R through molecular docking. We conducted site mutation to examine the impact of these sites on G proteins, in order to find out the sites that can distinguish the pharmacological effects of hallucinogens and non-hallucinogens. Our results indicate that I4.60A and S3.39A did not affect the ability of hallucinogens to activate G q signaling, but significantly reduced G s signaling activation by hallucinogens. These results suggest that S3.39 and I4.60 are important for the activation of G s signaling by hallucinogens., (© 2024 Federation of European Biochemical Societies.)

Purpose: Cell lines are being used in preclinical uveal melanoma (UM) research. Because not all cell lines harbor typical GNAQ or GNA11 hotspot mutations, we aimed at better classifying them and determining whether we could find genetic causes to explain the protein and mRNA expression profiles of the cell lines., Methods: We studied protein and mRNA expression of 14 UM cell lines and determined the presence of single nucleotide variants and small insertions and deletions with next-generation sequencing and copy number alterations with a single nucleotide polymorphism array. The lists of differentially expressed proteins and genes were merged, and shared lists were created, keeping only terms with concordant mRNA and protein expression. Enrichment analyses were performed on the shared lists., Results: Cell lines Mel285 and Mel290 are separate from GNA-mutated cell lines and show downregulation of melanosome-related markers. Both lack typical UM mutations but each harbors four putatively deleterious variants in CTNNB1, PPP1R10, LIMCH1, and APC in Mel285 and ARID1A, PPP1R10, SPG11, and RNF43 in Mel290. The upregulated terms in Mel285 and Mel290 did not point to a convincing alternative origin. Mel285 shows loss of chromosomes 1p, 3p, partial 3q, 6, and partial 8p, whereas Mel290 shows loss of 1p and 6. Expression in the other 12 cell lines was related to BAP1 expression., Conclusions: Although Mel285 and Mel290 have copy number alterations that fit UM, multi-omics analyses show that they belong to a separate group compared to the other analyzed UM cell lines. Therefore, they may not be representative models to test potential therapeutic targets for UM.

Recent pan-cancer genomic analyses have identified numerous oncogenic driver mutations that occur in a cell-type and tissue-specific distribution. For example, oncogenic mutations in Braf and Nras genes arise predominantly in melanocytic neoplasms of the epidermis, while oncogenic mutations in Gnaq/11 genes arise mostly in melanocytic lesions of the dermis or the uvea. The mechanisms promoting cell-type and tissue-specific oncogenic events currently remain poorly understood. Here, we report that Gnaq/11 hotspot mutations occur as early oncogenic drivers during the evolution of primary melanomas in Hgf-Cdk4 mice. Additional single base substitutions in the Trp53 gene and structural chromosomal aberrations favoring amplifications of the chromosomal region containing the Met receptor gene accumulate during serial tumor transplantation and in cell lines established in vitro. Mechanistically, we found that the Gnaq Q209L mutation transactivates the Met receptor. Overexpression of oncogenic Gnaq Q209L in the immortalized melanocyte cell line promoted in vivo growth that was enhanced by transgenic Hgf expression in the tumor microenvironment. This cross-signaling mechanism explains the selection of oncogenic Gnaq/11 in primary Hgf-Cdk4 melanomas and provides an example of how oncogenic driver mutations, intracellular signaling cascades, and microenvironmental cues cooperate to drive cancer development in a tissue-specific fashion., (© 2024. The Author(s).)

Staff and animals in livestock buildings are constantly exposed to fine particulate matter (PM2.5), which affects their respiratory health. However, its exact pathogenic mechanism remains unclear. Regulator of G-protein signaling 2 (RGS2) has been reported to play a regulatory role in pneumonia. The aim of this study was to explore the therapeutic potential of RGS2 in cowshed PM2.5-induced respiratory damage. PM2.5 was collected from a cattle farm, and the alveolar macrophages (NR8383) of the model animal rat were stimulated with different treatment conditions of cowshed PM2.5. The RGS2 overexpression vector was constructed and transfected it into cells. Compared with the control group, cowshed PM2.5 significantly induced a decrease in cell viability and increased the levels of apoptosis and proinflammatory factor expression. Overexpression of RGS2 ameliorated the above-mentioned cellular changes induced by cowshed PM2.5. In addition, PM2.5 has significantly induced intracellular Ca 2+ dysregulation. Affinity inhibition of Gq/11 by RGS2 attenuated the cytosolic calcium signaling pathway mediated by PLCβ/IP 3 R. To further investigate the causes and mechanisms of action of differential RGS2 expression, the possible effects of oxidative stress and TLR2/4 activation were investigated. The results have shown that RGS2 expression was not only regulated by oxidative stress-induced nitric oxide during cowshed PM2.5 cells stimulation but the activation of TLR2/4 had also an important inhibitory effect on its protein expression. The present study demonstrates the intracellular Ca 2+ regulatory role of RGS2 during cellular injury, which could be a potential target for the prevention and treatment of PM2.5-induced respiratory injury., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Capillary malformations (CM) (port-wine stains) are congenital skin lesions that are characterized by dilated capillaries and postcapillary venules. CMs are caused by altered functioning of the vascular endothelium. Somatic genetic mutations have predominantly been identified in the endothelial cells of CMs, providing an opportunity for the development of targeted therapies. However, there is currently limited in-depth mechanistic insight into the pathophysiology and a lack of preclinical research approaches. In a monocenter exploratory study of 17 adult patients with CMs, we found somatic sequence variants in the GNAQ (p.R183Q, p.R183G, or p.Q209R) or GNA11 (p.R183C) genes. We applied an endothelial-selective cell isolation protocol to culture primary endothelial cells from skin biopsies from these patients. We successfully expanded patient-derived cells in culture in 3 of the 17 cases while maintaining endothelial specificity as demonstrated by vascular endothelial-cadherin immunostainings. In addition, we tested the angiogenic capacity of endothelial cells from a patient with a GNAQ (p.R183G) sequence substitution. These proof-of-principle results reveal that primary cells isolated from CMs may represent a functional research model to investigate the role of endothelial somatic mutations in the etiology of CMs, but improved isolation and culture methodologies are urgently needed to advance the field., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

G protein-coupled receptors (GPCRs) are sophisticated signaling machines able to simultaneously elicit multiple intracellular signaling pathways upon activation. Complete (in)activation of all pathways can be counterproductive for specific therapeutic applications. This is the case for the serotonin 2 A receptor (5-HT 2A R), a prominent target for the treatment of schizophrenia. In this study, we elucidate the complex 5-HT 2A R coupling signature in response to different signaling probes, and its physiological consequences by combining computational modeling, in vitro and in vivo experiments with human postmortem brain studies. We show how chemical modification of the endogenous agonist serotonin dramatically impacts the G protein coupling profile of the 5-HT 2A R and the associated behavioral responses. Importantly, among these responses, we demonstrate that memory deficits are regulated by G αq protein activation, whereas psychosis-related behavior is modulated through G αi1 stimulation. These findings emphasize the complexity of GPCR pharmacology and physiology and open the path to designing improved therapeutics for the treatment of stchizophrenia., (© 2024. The Author(s).)

Objectives: We aimed to investigate the clinicopathologic features of and genetic changes in Sturge-Weber syndrome (SWS) in patients with refractory epilepsy., Methods: Clinical data were retrospectively analyzed. H&E and immunohistochemistry were performed to assess pathologic changes. Targeted amplicon sequencing was applied to investigate the somatic GNAQ (c.548G>A) mutation. The potential predictors of seizure outcomes were estimated by univariate and multivariate statistical analyses., Results: Forty-eight patients with SWS and refractory epilepsy were enrolled. According to the imaging data and pathologic examination, ipsilateral hippocampal sclerosis (HS), calcification of leptomeningeal arteries, and focal cortical dysplasia were found in 14 (29.2%), 31 (64.6%), and 37 (77.1%) patients, respectively. A high frequency of GNAQ alteration was detected in both cerebral cortex (57.7%) and ipsilateral hippocampus (50.0%) from patients with SWS. During follow-up, 43 of 48 patients (85.4%) had achieved seizure control (Engel class I). Statistically, HS signs on imaging were found to be independent predictors of unfavorable seizure outcomes (P = .015)., Conclusions: Calcification of leptomeningeal arteries, focal cortical dysplasia, and GNAQ alteration are common features in SWS pathology. Patients with refractory epilepsy caused by SWS can achieve satisfactory seizure control after surgery, but seizure control was compromised in patients with comorbid HS., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Mosaic mutations in genes GNAQ or GNA11 lead to a spectrum of diseases including Sturge-Weber syndrome and phakomatosis pigmentovascularis with dermal melanocytosis. The pathognomonic finding of localized "tramlining" on plain skull radiography, representing medium-sized neurovascular calcification and associated with postnatal neurological deterioration, led us to study calcium metabolism in a cohort of 42 children. In this study, we find that 74% of patients had at least one abnormal measurement of calcium metabolism, the commonest being moderately low serum ionized calcium (41%) or high parathyroid hormone (17%). Lower levels of ionized calcium even within the normal range were significantly associated with seizures, and with specific antiepileptics despite normal vitamin D levels. Successive measurements documented substantial intrapersonal fluctuation in indices over time, and DEXA scans were normal in patients with hypocalcemia. Neurohistology from epilepsy surgery in five patients revealed not only intravascular, but perivascular and intraparenchymal mineral deposition and intraparenchymal microvascular disease in addition to previously reported findings. Neuroradiology review clearly demonstrated progressive calcium deposition in individuals over time. These findings and those of the adjoining paper suggest that calcium deposition in the brain of patients with GNAQ/GNA11 mosaicism may not be a nonspecific sign of damage as was previously thought, but may instead reflect the central postnatal pathological process in this disease spectrum., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Mosaic variants in genes GNAQ or GNA11 lead to a spectrum of vascular and pigmentary diseases including Sturge-Weber syndrome, in which progressive postnatal neurological deterioration led us to seek biologically targeted therapeutics. Using two cellular models, we find that disease-causing GNAQ/11 variants hyperactivate constitutive and G-protein coupled receptor ligand-induced intracellular calcium signaling in endothelial cells. We go on to show that the aberrant ligand-activated intracellular calcium signal is fueled by extracellular calcium influx through calcium-release-activated channels. Treatment with targeted small interfering RNAs designed to silence the variant allele preferentially corrects both the constitutive and ligand-activated calcium signaling, whereas treatment with a calcium-release-activated channel inhibitor rescues the ligand-activated signal. This work identifies hyperactivated calcium signaling as the primary biological abnormality in GNAQ/11 mosaicism and paves the way for clinical trials with genetic or small molecule therapies., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Sturge-Weber syndrome (SWS), a neurocutaneous disorder, is characterized by capillary malformations (CM) in the skin, brain, and eyes. Patients may suffer from seizures, strokes, and glaucoma, and only symptomatic treatment is available. CM are comprised of enlarged vessels with endothelial cells (ECs) and disorganized mural cells. Our recent finding indicated that the R183Q mutation in ECs leads to heightened signaling through phospholipase Cβ3 and protein kinase C, leading to increased angiopoietin-2 (ANGPT2). Furthermore, knockdown of ANGPT2, a crucial mediator of pro-angiogenic signaling, inflammation, and vascular remodeling, in EC-R183Q rescued the enlarged vessel phenotype in vivo. This prompted us to look closer at the microenvironment in CM-affected vascular beds. We analyzed multiple brain histological sections from patients with GNAQ-R183Q CM and found enlarged vessels devoid of mural cells along with increased macrophage-like cells co-expressing MRC1 (CD206, a mannose receptor), CD163 (a scavenger receptor and marker of the monocyte/macrophage lineage), CD68 (a pan macrophage marker), and LYVE1 (a lymphatic marker expressed by some macrophages). These macrophages were not found in non-SWS control brain sections. To investigate the mechanism of increased macrophages in the perivascular environment, we examined THP1 (monocytic/macrophage cell line) cell adhesion to EC-R183Q versus EC-WT under static and laminar flow conditions. First, we observed increased THP1 cell adhesion to EC-R183Q compared to EC-WT under static conditions. Next, using live cell imaging, we found THP1 cell adhesion to EC-R183Q was dramatically increased under laminar flow conditions and could be inhibited by anti-ICAM1. ICAM1, an endothelial cell adhesion molecule required for leukocyte adhesion, was strongly expressed in the endothelium in SWS brain histological sections, suggesting a mechanism for recruitment of macrophages. In conclusion, our findings demonstrate that macrophages are an important component of the perivascular environment in CM suggesting they may contribute to the CM formation and SWS disease progression., (© 2024. The Author(s).)

Fibroblast growth factor 23 (FGF23) production has recently been shown to increase downstream of Gαq/11-PKC signaling in osteocytes. Inactivating mutations in the gene encoding Gα11 (GNA11) cause familial hypocalciuric hypercalcemia (FHH) due to impaired calcium-sensing receptor signaling. We explored the effect of Gα11 deficiency on FGF23 production in mice with heterozygous (Gna11+/-) or homozygous (Gna11-/-) ablation of Gna11. Both Gna11+/- and Gna11-/- mice demonstrated hypercalcemia and mildly raised parathyroid hormone levels, consistent with FHH. Strikingly, these mice also displayed increased serum levels of total and intact FGF23 and hypophosphatemia. Gna11-/- mice showed augmented Fgf23 mRNA levels in the liver and heart, but not in bone or bone marrow, and also showed evidence of systemic inflammation with elevated serum IL-1β levels. Furin gene expression was significantly increased in the Gna11-/- liver, suggesting enhanced FGF23 cleavage despite the observed rise in circulating intact FGF23 levels. Gna11-/- mice had normal renal function and reduced serum levels of glycerol-3-phosphate, excluding kidney injury as the primary cause of elevated intact FGF23 levels. Thus, Gα11 ablation caused systemic inflammation and excess serum FGF23 in mice, suggesting that patients with FHH - at least those with GNA11 mutations - may be at risk for these complications.

Uveal melanoma (UM) is a neuroectodermal tumor that results from malignant transformation of melanocytes in the eye uvea, including the iris, the ciliary body, and the choroid. UM accounts for 5% of all melanoma cases and is extremely aggressive with half of the UM patients developing metastases within the first 1-2 years after tumor development. Molecular mechanisms of UM carcinogenesis are poorly understood, but are known to differ from those of skin melanoma. Activating mutations of the GNAQ and GNA11 genes, which code for the large G protein subunits Gq and G11, respectively, are found in 90% of UM patients. The Gaq/PKC/MAPK signaling pathway is a main signaling cascade that leads to the transformation of melanocytes of the uveal tract, and major regulators of the cascade provide targets for the development of drugs. Metastatic UM (MUM) is most often associated with mutations of BAP1, EIF1AX, GNA11, GNAQ, and SF3B1. A combination of a commercial expression test panel of 15 genes and a mutation panel of 7 genes, supplemented with data on the size of the primary tumor, is highly efficient in predicting the risk of metastasis. The risk of metastasis determines the choice of therapy and the patient follow-up regimen. However, no systemic therapy for MUM has been developed to date. New drugs undergoing clinical trials are mostly targeted drugs designed to inhibit the protein products of mutant genes or immunotherapeutic agents designed to stimulate the immune response against specific antigens. In addition to these approaches, potential therapeutic targets of epigenetic regulation of UM development are considered in the review.

Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients' tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers., (© 2024. The Author(s).)

Uveal melanoma (UM) represents the predominant ocular malignancy among adults, exhibiting high malignancy and proclivity for liver metastasis. GNAQ and GNA11 encoding Gαq and Gα11 proteins are key genes to drive UM, making the selective inhibition of Gαq/11 proteins to be a potential therapeutic approach for combating UM. In this study, forty-six quinazoline derivatives were designed, synthesized, and assessed for their ability to inhibit Gαq/11 proteins and UM cells. Compound F33 emerged as the most favorable candidate, and displayed moderate inhibitory activity against Gαq/11 proteins (IC 50  = 9.4 μM) and two UM cell lines MP41 (IC 50  = 6.7 μM) and 92.1 (IC 50  = 3.7 μM). Being a small molecule inhibitor of Gαq/11 proteins, F33 could effectively suppress the activation of downstream signaling pathways in a dose-dependent manner, and significantly inhibits UM in vitro.F33 represents a promising lead compound for developing therapeutics for UM by targeting Gαq/11 proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Circumscribed choroidal hemangioma (CCH) and early non-pigmented choroidal melanoma (CM) have similar clinical, ultrasound and morphometric features, which in some cases makes their differential diagnosis difficult. There are few studies in the literature devoted to a comparative analysis of the molecular genetic features of CCH and non-pigmented CM, and the results of those studies are contradictory., Purpose: This study attempts to develop a method of non-invasive molecular genetic differential diagnostics of CCH and non-pigmented CM., Material and Methods: Based on the results of clinical and instrumental examination methods, 60 patients (60 eyes) with CCH ( n =30) and non-pigmented CM ( n =30) were included in this prospective study. The control group consisted of 30 individuals without intraocular tumors. Mutations in the GNAQ / GNA11 genes were determined by real-time PCR using the analysis of genomic circulating tumor DNA isolated from peripheral blood plasma. The average follow-up period was 12.1±1.8 months., Results: The study revealed a significant association of mutations in exons 4 and 5 of the GNAQ / GNA11 genes with the presence of non-pigmented CM (27/30; 90%). These mutations were not detected in the group of patients with CCH. Mutations in exons 4 and 5 of the GNAQ / GNA11 genes were also not detected in the control group of healthy individuals., Conclusion: This study proposes a method of non-invasive and low-cost differential diagnostics based on molecular genetic analysis and detection of mutations in exons 4 and 5 of the GNAQ and GNA11 genes, which are specific for CM (90%).

Most uveal melanoma cases harbor activating mutations in either GNAQ or GNA11. Despite activation of the mitogen-activated protein kinase (MAPK) signaling pathway downstream of Gαq/11, there are no effective targeted kinase therapies for metastatic uveal melanoma. The human genome encodes numerous understudied kinases, also called the "dark kinome". Identifying additional kinases regulated by Gαq/11 may uncover novel therapeutic targets for uveal melanoma. In this study, we treated GNAQ-mutant uveal melanoma cell lines with a Gαq/11 inhibitor, YM-254890, and conducted a kinase signaling proteomic screen using multiplexed-kinase inhibitors followed by mass spectrometry. We observed downregulated expression and/or activity of 22 kinases. A custom siRNA screen targeting these kinases demonstrated that knockdown of microtubule affinity regulating kinase 3 (MARK3) and serine/threonine kinase 10 (STK10) significantly reduced uveal melanoma cell growth and decreased expression of cell cycle proteins. Additionally, knockdown of MARK3 but not STK10 decreased ERK1/2 phosphorylation. Analysis of RNA-sequencing and proteomic data showed that Gαq signaling regulates STK10 expression and MARK3 activity. Our findings suggest an involvement of STK10 and MARK3 in the Gαq/11 oncogenic pathway and prompt further investigation into the specific roles and targeting potential of these kinases in uveal melanoma., Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A. E. A. has an ownership interest in patent number 9880150 and a pending patent, PCT/US22/76492. No potential conflicts of interest were disclosed by the other authors. M. A. D. has been a consultant to Roche/Genentech, Array, Pfizer, Novartis, BMS, GSK, Sanofi-Aventis, Vaccinex, Apexigen, Eisai, Iovance, and ABM Therapeutics, and he has been the PI of research grants to MD Anderson by Roche/Genentech, GSK, Sanofi-Aventis, Merck, Myriad, Oncothyreon, ABM Therapeutics, and LEAD Pharma. J. S. G. has been a consultant for Domain Pharmaceuticals, Pangea Therapeutics, and io9, and is the founder of Kadima Pharmaceuticals, all unrelated to the current study. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM., Competing Interests: Declaration of interests J.S.G. reports consulting fees from Domain Pharmaceuticals, Pangea Therapeutics, and io9 and is founder of Kadima Pharmaceuticals, all unrelated to the current study. J.S.G. and N.A. hold patent US11679113B2 related in part to this work. The Krogan Laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche, and Rezo Therapeutics. N.J.K. has financially compensated consulting agreements with the Icahn School of Medicine at Mount Sinai, New York, Maze Therapeutics, Interline Therapeutics, Rezo Therapeutics, Gen1E Lifesciences, Inc., and Twist Bioscience Corp. He is on the Board of Directors of Rezo Therapeutics and is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics, and Interline Therapeutics. D.L.S. has a consulting agreement with Maze Therapeutics. J.B. is a consultant for Rocket Pharma. J.A.P. and S.C. are employees of Verastem, which has not influenced this study. Other authors declare no competing financial interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Uveal melanoma (UM) is a rare ocular tumor characterized by high metastasis risk and poor prognosis. The in-depth characterization of UM's molecular profile is critical for better disease classification and prognosis. Furthermore, the development of detection tools to monitor UM evolution upon treatment is of great interest for designing optimal therapeutic strategies. However, commonly used techniques, such as ddPCR or NGS, are costly, and they involve sophisticated equipment and complex experimental design. The development of alternative sensing methods that are fast, simple, and inexpensive would be of great benefit to improve UM's diagnosis and management, especially when combined with liquid biopsy. Samples from liquid biopsy can be obtained with minimal invasiveness, and the detection of circulating tumor DNA (ctDNA) in UM patients' plasma has proven useful for the diagnosis of metastasis, prognosis prediction, and disease monitoring. In this context, CRISPR/Cas12a-derived molecular sensors, thanks to their high specificity and sensitivity and their potential for point of care diagnosis, are particularly interesting. Here, we developed a CRISPR/Cas12a-based approach for the specific detection of the UM-related mutation GNAQ Q209P that relies on the design of highly specific crRNAs. Coupled with allele-specific PCR, it constitutes a sensitive platform for liquid biopsy detection, capable of sensing GNAQ Q209P in plasma samples with a low ctDNA concentration and fractional abundance. Finally, our method was validated using plasma samples from metastatic UM patients.

Metastatic uveal melanoma (UM) patients typically survive only 2 to 3 years because effective therapy does not yet exist. Here, to facilitate the discovery of therapeutic targets in UM, we have identified protein kinase signaling mechanisms elicited by the drivers in 90% of UM tumors: mutant constitutively active G protein α-subunits encoded by GNAQ (Gq) or GNA11 (G11). We used the highly specific Gq/11 inhibitor FR900359 (FR) to elucidate signaling networks that drive proliferation, metabolic reprogramming, and dedifferentiation of UM cells. We determined the effects of FR on the proteome and phosphoproteome of UM cells as indicated by bioinformatic analyses with CausalPath and site-specific gene set enrichment analysis. We found that inhibition of oncogenic Gq/11 caused deactivation of PKC, Erk, and the cyclin-dependent kinases CDK1 and CDK2 that drive proliferation. Inhibition of oncogenic Gq/11 in UM cells with low metastatic risk relieved inhibitory phosphorylation of polycomb-repressive complex subunits that regulate melanocytic redifferentiation. Site-specific gene set enrichment analysis, unsupervised analysis, and functional studies indicated that mTORC1 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 drive metabolic reprogramming in UM cells. Together, these results identified protein kinase signaling networks driven by oncogenic Gq/11 that regulate critical aspects of UM cell biology and provide targets for therapeutic investigation., Competing Interests: Conflicts of interest M. D. O. and K. J. B. are listed as co-inventors on a provisional patent application on TARGETED PHARMACOLOGICAL THERAPEUTICS IN UVEAL MELANOMA that is owned by Washington University in St. Louis. All other authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Uveal melanoma (UM) is the most common ocular malignancy in adults. Nearly 95% of UM patients carry the mutually exclusive mutations in the homologous genes GNAQ (amino acid change Q209L/Q209P) and GNA11 (aminoacid change Q209L). UM is located in an immunosuppressed organ and does not suffer immunoediting. Therefore, we hypothesize that driver mutations in GNAQ/11 genes could be recognized by the immune system. Genomic and transcriptomic data from primary uveal tumors were collected from the TCGA-UM dataset ( n  = 80) and used to assess the immunogenic potential for GNAQ/GNA11 Q209L/Q209P mutations using a variety of tools and HLA type information. All prediction tools showed stronger GNAQ/11 Q209L binding to HLA than GNAQ/11 Q209P. The immunogenicity analysis revealed that Q209L is likely to be presented by more than 73% of individuals in 1000 G databases whereas Q209P is only predicted to be presented in 24% of individuals. GNAQ/11 Q209L showed a higher likelihood to be presented by HLA-I molecules than almost all driver mutations analyzed. Finally, samples carrying Q209L had a higher immune-reactive phenotype. Regarding cancer risk, seven HLA genotypes with low Q209L affinity show higher frequency in uveal melanoma patients than in the general population. However, no clear association was found between any HLA genotype and survival. Results suggest a high potential immunogenicity of the GNAQ/11 Q209L variant that could allow the generation of novel therapeutic tools to treat UM like neoantigen vaccinations., Competing Interests: Dr Josep M Piulats has acted as a consultant or advisor for Roche, Novartis, Bristol Meyers Squibb, MSD Merck Serono, AstraZeneca, Clovis, VCN Biosciences, Janssen, Astellas, Bayer, Sanofi Genzyme, and Pfizer; received research funding from Pfizer, Merck Serono, MSD, Bristol Myers Squibb, Incyte, VCN Biosciences, Astellas, and Janssen; and received financial support for attending symposia from Roche, Janssen, and Ipsen., (© 2023 IDIBELL. Published with license by Taylor & Francis Group, LLC.)

Single-nucleotide polymorphisms in G protein subunits are linked to an increased risk of cardiovascular events among the general population. We assessed the effects of GNB3 c.825C > T, GNAQ -695/-694GC > TT, and GNAS c.393C > T polymorphisms on the risk of cardiovascular events among 454 patients undergoing renal replacement therapy. The patients were followed up for a median of 4.5 years after the initiation of dialysis. Carriers of the TT/TT genotype of GNAQ required stenting because of coronary artery stenosis ( p = 0.0009) and developed cardiovascular events involving more than one organ system ( p = 0.03) significantly earlier and more frequently than did the GC/TT or GC/GC genotypes. Multivariate analysis found that the TT/TT genotype of GNAQ was an independent risk factor for coronary artery stenosis requiring stent (hazard ratio, 4.5; p = 0.001), cardiovascular events (hazard ratio, 1.93; p = 0.04) and cardiovascular events affecting multiple organs (hazard ratio, 4.9; p = 0.03). In the subgroup of male patients left ventricular dilatation with abnormally increased LVEDD values occurred significantly more frequently in TT genotypes of GNB3 than in CT/CC genotypes ( p = 0.007). Our findings suggest that male dialysis patients carrying the TT genotype of GNB3 are at higher risk of left ventricular dilatation and that dialysis patients carrying the TT/TT genotype of GNAQ are prone to coronary artery stenosis and severe cardiovascular events.

Purpose: Uveal melanoma (UM) is a tumor of the eye that metastasizes in approximately half of cases. Prognostic testing requires accessibility to tumor tissue, which is usually not available with eye-preserving therapies. Noninvasive approaches to prognostic testing that provide valuable information for patient care are therefore needed. The aim of this study was to evaluate the use of circulating cell-free plasma DNA analysis in UM patients undergoing brachytherapy., Methods: The study recruited 26 uveal melanoma patients referred to the department between February and October 2020. Blood samples were collected at various time points before, during, and after treatment, and deep amplicon sequencing was used to identify oncogenic variant alleles of the GNAQ and GNA11 genes, which serve as indicators for the presence of circulating tumor DNA (ctDNA)., Results: The results showed that all patients were ctDNA negative before brachytherapy. In 31% of patients, ctDNA was detected during therapy. The variant allele fraction of GNAQ or GNA11 alleles in ctDNA positive samples ranged from 0.24% to 2% and correlates with the largest basal diameter and thickness of the tumor., Conclusions: The findings suggest that brachytherapy increases the presence of tumor DNA in the plasma of UM patients. Thus ctDNA analysis may offer a noninvasive approach for prognostic testing. However, efforts are still required to lower the limit of detection for tumor-specific genetic alterations.

Background: Arteriovenous Malformations (AVMs) are complex vascular anomalies that are usually sporadic and can have a variable clinical course. Treatment of AVMs can lead to severe sequeale and require thorough decision-making. There is a lack of standardized treatment protocols showing a growing need for pharmacological targeted therapies, specially in the most severe cases where surgery may not be feasible. Current knowledge in molecular pathways and genetic diagnosis have shed light in the pathophysiology of AVMs, opening possibilities for personalized treatment strategies., Methods: We performed a retrospective review of patients with head and neck AVMs treated in our department between 2003 and 2021 and performed a complete physical examination and imaging with ultrasound and angio-CT or MRI. Patients underwent genetic testing on AVMs' tissue samples and/or peripheral blood samples. Patients were grouped according to the genetic variant and a correlation between phenotype and genotype was studied., Results: 22 patients with head and neck AVMs were included. We found eight patients with varians in MAP2K1, four patients with pathogenic variants in KRAS, six patients with pathogenic variants in RASA1, one patient with a pathogenic variant in BRAF, one patient with a pathogenic variant in NF1, another patient with a pathogenic variant in CELSR1 and one patient with pathogenic variants in PIK3CA and GNA14. Patients with MAP2K1 variants were the biggest group, with a moderate clinical course. Patients with KRAS mutations showed the most aggressive clinical course and a high rate of recurrence and osteolysis. Patients with RASA1 variants showed a characteristic phenotype with an ipsilateral capillary malformation in the neck., Conclusion: We found a correlation between genotype and phenotype in this group of patients. The genetic diagnosis of AVMs is recommended in order to stablish a personalized treatment strategy. Targeted therapies are currently being investigated with promising results and may be recommended in addition to conventional surgical or embolization procedures, specially in the most complex cases., Level of Evidence: Level IV., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Loss of cognitive function with age is devastating. EGL-30/GNAQ and G αq signaling pathways are highly conserved between C. elegans and mammals, and murine Gnaq is enriched in hippocampal neurons and declines with age. We found that activation of EGL-30 in aged worms triples memory span, and GNAQ gain of function significantly improved memory in aged mice: GNAQ(gf) in hippocampal neurons of 24-month-old mice (equivalent to 70- to 80-year-old humans) rescued age-related impairments in well-being and memory. Single-nucleus RNA sequencing revealed increased expression of genes regulating synaptic function, axon guidance, and memory in GNAQ-treated mice, and worm orthologs of these genes were required for long-term memory extension in worms. These experiments demonstrate that C. elegans is a powerful model to identify mammalian regulators of memory, leading to the identification of a pathway that improves memory in extremely old mice. To our knowledge, this is the oldest age at which an intervention has improved age-related cognitive decline., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Episcleral vasculature malformation is a significant feature of Sturge-Weber syndrome (SWS) secondary glaucoma, the density and diameter of which are correlated with increased intraocular pressure. We previously reported that the GNAQ R183Q somatic mutation was located in the SWS episclera. However, the mechanism by which GNAQ R183Q leads to episcleral vascular malformation remains poorly understood. In this study, we investigated the correlation between GNAQ R183Q and episcleral vascular malformation via surgical specimens, human umbilical vein endothelial cells (HUVECs), and the HUVEC cell line EA.hy926. Our findings demonstrated a positive correlation between episcleral vessel diameter and the frequency of the GNAQ R183Q variant. Furthermore, the upregulation of genes from the Notch signaling pathway and abnormal coexpression of the arterial marker EphrinB2 and venous marker EphB4 were demonstrated in the scleral vasculature of SWS. Analysis of HUVECs overexpressing GNAQ R183Q in vitro confirmed the upregulation of Notch signaling and arterial markers. In addition, knocking down of Notch1 diminished the upregulation of arterial markers induced by GNAQ R183Q. Our findings strongly suggest that GNAQ R183Q leads to malformed episcleral vasculatures through Notch-induced aberrant arteriovenous specification. These insights into the molecular basis of episcleral vascular malformation will provide new pathways for the development of effective treatments for SWS secondary glaucoma., (© 2023 Federation of American Societies for Experimental Biology.)

Mammalian type opsin 5 (Opn5m), a UV-sensitive G protein-coupled receptor opsin highly conserved in vertebrates, would provide a common basis for UV sensing from lamprey to humans. However, G protein coupled with Opn5m remains controversial due to variations in assay conditions and the origin of Opn5m across different reports. Here, we examined Opn5m from diverse species using an aequorin luminescence assay and Gα-KO cell line. Beyond the commonly studied major Gα classes, Gαq, Gα11, Gα14, and Gα15 in the Gq class were individually investigated in this study, as they can drive distinct signaling pathways in addition to a canonical calcium response. UV light triggered a calcium response via all the tested Opn5m proteins in 293T cells, which was abolished by Gq-type Gα deletion and rescued by cotransfection with mouse and medaka Gq-type Gα proteins. Opn5m preferentially activated Gα14 and close relatives. Mutational analysis implicated specific regions, including α3-β5 and αG-α4 loops, αG and α4 helices, and the extreme C terminus, in the preferential activation of Gα14 by Opn5m. FISH revealed co-expression of genes encoding Opn5m and Gα14 in the scleral cartilage of medaka and chicken eyes, supporting their physiological coupling. This suggests that the preferential activation of Gα14 by Opn5m is relevant for UV sensing in specific cell types., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the content of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Activating non-inherited mutations in the guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) gene family have been identified in childhood vascular tumors. Patients experience extensive disfigurement, chronic pain and severe complications including a potentially lethal coagulopathy termed Kasabach-Merritt phenomenon. Animal models for this class of vascular tumors do not exist. This has severely hindered the discovery of the molecular consequences of GNAQ mutations in the vasculature and, in turn, the preclinical development of effective targeted therapies. Here we report a mouse model expressing hyperactive mutant GNAQ in endothelial cells. Mutant mice develop vascular and coagulopathy phenotypes similar to those seen in patients. Mechanistically, by transcriptomic analysis we demonstrate increased mitogen activated protein kinase signaling in the mutant endothelial cells. Targeting of this pathway with Trametinib suppresses the tumor growth by reducing vascular cell proliferation and permeability. Trametinib also prevents the development of coagulopathy and improves mouse survival., (© 2023. The Author(s).)

There is no published data regarding the molecular alterations of Polish patients with primary uveal melanoma. We performed whole exome sequencing of 20 primary uveal melanomas (UMs), 10 metastasizing and 10 non-metastasizing cases to identify significant molecular alterations. We detected mutations and copy number variants in the BAP1 gene in 50% (10 cases) of the cases. GNA11 mutations were detected in 50% (10 cases) including nine p.Q209L and one p.R183C. GNAQ mutations gene were detected in 40% (8 cases) and all were p.Q209P. SF3B1, EIF1AX, PLCB4 , and PALB2 mutations were detected in one case each. Genetic aberrations of FBXW7 were detected in 55% of cases, with copy number loss of 10 and missense mutation in one. Gain or loss of copy number was observed in 60%, 60%, and 10% of cases in MYC, MLH1 , and CDKN2A genes, respectively. BAP1 and GNAQ tumor suppressor genes are more often mutated in UM with metastasis, while GNA11 mutations are more frequently detected in non-metastasizing tumors. MYC copy gain was present twice as frequently (80% versus 40%) in cases with versus those without metastases. BAP1 mutation correlated with worse overall survival; while GNA11 mutation and CDKN2A loss correlated with better and worse progression-free survival, respectively. We have confirmed BAP1 prognostic potential and documented frequent MYC amplification in metastasizing cases. Although GNA11 mutation and CDKN2A loss significantly correlated with progression-free survival in our study, our sample size is small. The prognostic significance of GNAQ/GNA11 mutation and CDKN2A loss would require further investigation., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Sturge-Weber Syndrome (SWS) is a rare vascular malformation disorder characterized by abnormal blood vessels in the brain, skin, and eye. SWS is most commonly caused by a somatic mosaic GNAQ-p.Arg183Gln variant. In this series, 12 patients presented for clinical evaluation of SWS but were noted to have atypical features, and therefore germline and/or somatic genetic testing was performed. Atypical features included extensive capillary malformation on the body as well as the face, frontal bossing, macrocephaly, telangiectasia, overgrowth of extremities, absence of neurologic signs and symptoms, and family history of vascular malformations. Five patients had a somatic GNAQ or GNA11 pathogenic variant, one patient had a somatic mosaic likely-pathogenic variant in PIK3CA, and another one had a somatic mosaic deletion that disrupted PTPRD. The other five patients had germline variants in RASA1, EPHB4, or KIT. Our findings suggest that patients presenting for SWS evaluation who have atypical clinical characteristics may have pathogenic germline or somatic variants in genes other than GNAQ or GNA11. Broad germline and somatic genetic testing in these patients with atypical findings may have implications for medical care, prognosis, and trial eligibility., (© 2023 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Purpose: NCI-MATCH is a precision medicine trial using genomic testing to allocate patients with advanced malignancies to targeted treatment subprotocols. This report combines two subprotocols evaluating trametinib, a MEK1/2 inhibitor, in patients with Neurofibromatosis 1 ( NF1 [S1] or GNA11/Q [S2]) altered tumors., Methods: Eligible patients had tumors with deleterious inactivating NF1 or GNA11/Q mutations by the customized Oncomine AmpliSeq panel. Prior MEK inhibitor treatment was excluded. Glioblastomas (GBMs) were permitted, including malignancies associated with germline NF1 mutations (S1 only). Trametinib was administered at 2 mg once daily over 28-day cycles until toxicity or disease progression. Primary end point was objective response rate (ORR). Secondary end points included progression-free survival (PFS) at 6 months, PFS, and overall survival. Exploratory analyses included co-occurring genomic alterations and PTEN loss., Results: Fifty patients were eligible and started therapy: 46 with NF1 mutations (S1) and four with GNA11 mutations (S2). In the NF1 cohort, nonsense single-nucleotide variants were identified in 29 and frameshift deletions in 17 tumors. All in S2 had nonuveal melanoma and GNA11 Q209L variant. Two partial responses (PR) were noted in S1, one patient each with advanced lung cancer and GBM for an ORR of 4.3% (90% CI, 0.8 to 13.1). One patient with melanoma in S2 had a PR (ORR, 25%; 90% CI, 1.3 to 75.1). Prolonged stable disease (SD) was also noted in five patients (four in S1 and one in S2) with additional rare histologies. Adverse events were as previously described with trametinib. Comutations in TP53 and PIK3CA were common., Conclusion: Although these subprotocols did not meet the primary end point for ORR, significant responses or prolonged SD noted in some disease subtypes warrants further investigation.

Heterotrimeric G protein stimulation via G protein-coupled receptors promotes downstream proliferative signaling. Mutations can occur in Gα proteins which prevent GTP hydrolysis; this allows the G proteins to signal independently of G protein-coupled receptors and can result in various cancers, such as uveal melanoma (UM). Most UM cases harbor Q209L, Q209P, or R183C mutations in Gα q/11 proteins, rendering the proteins constitutively active (CA). Although it is generally thought that active, GTP-bound Gα subunits are dissociated from and signal independently of Gβγ, accumulating evidence indicates that some CA Gα mutants, such as Gα q/11 , retain binding to Gβγ, and this interaction is necessary for signaling. Here, we demonstrate that disrupting the interaction between Gβγ and Gα q is sufficient to inhibit aberrant signaling driven by CA Gα q . Introduction of the I25A point mutation in the N-terminal α helical domain of CA Gα q to inhibit Gβγ binding, overexpression of the G protein Gα o to sequester Gβγ, and siRNA depletion of Gβ subunits inhibited or abolished CA Gα q signaling to the MAPK and YAP pathways. Moreover, in HEK 293 cells and in UM cell lines, we show that Gα q -Q209P and Gα q -R183C are more sensitive to the loss of Gβγ interaction than Gα q -Q209L. Our study challenges the idea that CA Gα q/11 signals independently of Gβγ and demonstrates differential sensitivity between the Gα q -Q209L, Gα q -Q209P, and Gα q -R183C mutants., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

G proteins and G protein-coupled receptors activate a diverse array of signal transduction pathways that promote cell growth and survival. Indeed, hot spot-activating mutations in GNAQ/GNA11, encoding Gαq proteins, are known to be driver oncogenes in uveal melanoma (UM), for which there are limited effective therapies currently available. Focal adhesion kinase (FAK) has been recently shown to be a central mediator of Gαq-driven signaling in UM, and as a result, is being explored clinically as a therapeutic target for UM, both alone and in combination therapies. Despite this, the repertoire of Gαq/FAK-regulated signaling mechanisms have not been fully elucidated. Here, we used a whole-genome CRISPR screen in GNAQ-mutant UM cells to identify mechanisms that, when overactivated, lead to reduced sensitivity to FAK inhibition. In this way, we found that the PI3K/AKT signaling pathway represented a major resistance driver. Our dissection of the underlying mechanisms revealed that Gαq promotes PI3K/AKT activation via a conserved signaling circuitry mediated by FAK. Further analysis demonstrated that FAK activates PI3K through the association and tyrosine phosphorylation of the p85 regulatory subunit of PI3K and that UM cells require PI3K/AKT signaling for survival. These findings establish a novel link between Gαq-driven signaling and the stimulation of PI3K as well as demonstrate aberrant activation of signaling networks underlying the growth and survival of UM and other Gαq-driven malignancies., Competing Interests: Conflict of interest O.H. is an employee of Zentalis Pharmaceuticals, D.N. is an employee of TwinStrand Biosciences, unrelated to this study. P.M. is a scientific cofounder of Shape Therapeutics, Boundless Biosciences, Navega Therapeutics, and Engine Biosciences. The terms of these arrangements have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. J.S.G. is consultant for Domain Therapeutics, Pangea Therapeutics, and io9, and founder of Kadima Pharmaceuticals, outside the submitted work. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Uveal melanoma (UM) is the most common intraocular tumor in adults, and up to 50% of patients develop metastatic disease, which remains uncurable. Because patients with metastatic UM have an average survival of less than 1 year after diagnosis, there is an urgent need to develop new treatment strategies. Although activating mutations in Gαq or Gα11 proteins are major drivers of pathogenesis, the therapeutic intervention of downstream Gαq/11 targets has been unsuccessful in treating UM, possibly due to alternative signaling pathways and/or resistance mechanisms. Activation of the insulin-like growth factor 1 (IGF1) signaling pathway promotes cell growth, metastasis, and drug resistance in many types of cancers, including UM, where expression of the IGF1 receptor (IGF1R) correlates with a poor prognosis. In this article, we show that direct inhibition of Gαq/11 by the cyclic depsipeptide YM-254890 in combination with inhibition of IGF1R by linsitinib cooperatively inhibits downstream signaling and proliferation of UM cells. We further demonstrate that a 2-week combination treatment of 0.3 to 0.4 mg/kg of YM-254890 administered by intraperitoneal injection and 25 to 40 mg/kg linsitinib administered by oral gavage effectively inhibits the growth of metastatic UM tumors in immunodeficient NOD scid gamma (NSG) mice and identifies the IGF1 pathway as a potential resistance mechanism in response to Gαq/11 inhibition in UM. These data suggest that the combination of Gαq/11 and IGF1R inhibition provides a promising therapeutic strategy to treat metastatic UM., (©2022 American Association for Cancer Research.)

Objective: Primary anorectal melanomas (AMs) are uncommon neoplasms with aggressive behavior. Molecular profile and clinicopathologic features of AMs are still not well established. In this study, we aimed to investigate BRAF, NRAS, KIT, TERT, and GNAQ/GNA11 mutation status and clinicopathologic features of AMs., Material and Method: All diagnostic slides of 15 AMs were reviewed. Histopathological and follow-up information were documented. Mutations in exon 15 of the BRAF gene; exons 2 and 3 of the NRAS gene; exons 9, 11, 13, 17, and 18 of the KIT gene; and exons 4 and 5 of the GNAQ/GNA11 genes and mutations in the promoter region of the TERT gene (chr.5, 1,295,228C > T and 1,295,250C > T) were analyzed., Results: BRAF(V600E) and KIT(V555I and K642E) mutations were observed in one (7%) and two cases (14%), respectively. NRAS, TERT and GNAQ/GNA11 mutations were not detected. The mean age was 65. Patients presented with rectal mass, rectal bleeding, pain, and weight loss. 73% of the lesions were macroscopically polypoid. The most common tumor cell type was epithelioid. Mean tumor thickness was 10.4 mm. One third of the cases lacked pigmentation. In situ melanoma was present in one third of the cases. Among 14 patients with follow-up data, 12 succumbed to disease. The mean overall survival was 36 months., Conclusion: AMs are uncommon tumors with dismal survival, usually occurring in the elderly in various gross and microscopic appearances. In terms of molecular profile, BRAF and KIT mutations are rarely detected. Profiling of larger cohorts is required to elucidate the pathogenesis and to identify potential molecular indicators that may contribute to the development of individualized targeted therapies.

Loss-of-function mutations of the arginine vasopressin receptor 2 gene (AVPR2) cause Nephrogenic diabetes insipidus (NDI). AVPR2 is a kind of G protein coupled receptor (GPCR) and mainly couples with Gαs protein leading to cAMP accumulation in the cell as a secondary messenger. Recent studies showed that some AVPR2 mutations could cause biased Gαq/11 protein coupling rather than Gαs. Investigation into the characterization of biased receptors may give insights into the relationship between the conformational change of the receptor because of the mutation and related downstream signaling. In this study, R68W and V162A were analyzed to whether they show a bias to Gαs or Gαq/11 proteins. Their functionality in terms of cAMP production via Gαs protein coupling was decreased compared to the wild-type receptor. On the other hand, they showed the ability to couple with Gαq/11 protein and make Ca 2+ mobilization at different levels in the cell. R68W showed bias to coupling with Gαq/11 protein rather than V162A and wild-type receptor. Studies about the Gα protein coupling bias of mutant AVPR2s may broaden our understanding of the relationship between the changed conformation of the receptor and consequently activated signaling pathways, and also may shed light on the development of more effective new therapeutics.

Uveal melanoma is a rare malignancy affecting 5.1 patients/million per year with definitive treatment options of enucleation or radiation therapy to the primary tumor. Unfortunately, no FDA-approved systemic therapies exist for patients in the adjuvant or metastatic setting. Molecular profiling over the past decade has helped define uveal melanomas by characteristic mutations: GNAQ, GNA11, BAP1, SF3B1, and EIF1AX mutations. GNAQ/11 mutations are present in over 90% of patients with uveal melanoma and lead to signal transduction through G-protein coupled receptors to downstream growth factors. PKC inhibition has been an active area of investigation targeting this pathway specific to uveal melanoma. Several molecules have been developed and evaluated in clinical trials. Responses have been noted but clinical development has also yielded multiple toxicities and pathways of resistance limiting both breadth and durability of responses leading to combination therapy approaches. PKC inhibition remains an active and encouraging area of research to determine effective therapies for patients with uveal melanoma., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)