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The soybean cyst nematode (SCN; Heterodera glycines Ichinohe) is a significant agricultural pest that causes extensive damage to soybean production worldwide. Second-stage juveniles (J2s) of the SCN migrate through the soil and infest the roots of host plants in response to certain chemical substances secreted from the host roots. Therefore, controlling SCN chemotaxis could be an effective strategy for its management. In the present study, we identified the Hg-gpa-3d gene, which encodes the G protein alpha subunit, as a key regulator of SCN chemotaxis. Gene silencing of Hg-gpa-3d reduced the attraction of SCN J2s to host roots, as well as to nitrate ions, a chemoattractant recognized through a mechanism different from that of host recognition. However, silencing of Hg-gpa-3d did not affect avoidance behavior towards unpleasant temperatures or stylet protrusion. These results suggest that Hg-gpa-3d is a crucial gene in the regulation of SCN chemotaxis and provide new insights into the chemotactic mechanisms of the SCN., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

While it is known that temperature sensors trigger calcium (Ca 2+ ) signaling to confer cold tolerance in cells, less is known about sensors that couple with other secondary messengers. Here, we identify a cold sensor complex of CHILLING-TOLERANCE DIVERGENCE 6 (COLD6) and osmotin-like 1 (OSM1), which triggers 2',3'-cyclic adenosine monophosphate (2',3'-cAMP) production to enhance cold tolerance in rice. COLD6, which is encoded by a major quantitative trait locus (QTL) gene, interacts with the rice G protein α subunit (RGA1) at the plasma membrane under normal conditions. Upon exposure to chilling, cold-induced OSM1 binds to COLD6, kicking out RGA1 from interaction. This triggers an elevation of 2',3'-cAMP levels for enhancing chilling tolerance. Genetic data show that COLD6 negatively regulates cold tolerance and functionally depends on OSM1 in chilling stress. COLD6 alleles were selected during rice domestication. Knockout and natural variation of COLD6 in hybrid rice enhanced chilling tolerance, hinting design potential for breeding. This highlighted a module triggering 2',3'-cAMP to improve chilling tolerance in crops., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The heterotrimeric G-protein α subunit, Gα olf , acts to transduce extracellular signals through G-protein coupled receptors (GPCRs) and stimulates adenylyl cyclase mediated production of the second messenger cyclic adenosine monophosphate. Numerous mutations in the GNAL gene, which encodes Gα olf , have been identified as causative for an adult-onset dystonia. These mutations disrupt GPCR signaling cascades in in vitro assays through several mechanisms, and this disrupted signaling is hypothesized to lead to dystonic motor symptoms in patients. However, the cells and circuits that mutations in GNAL corrupt are not well understood. Published patterns of Gα olf expression outside the context of the striatum are sparse, conflicting, often lack cell type specificity, and may be confounded by expression of the close GNAL homolog of GNAS. Here, we use RNAScope in-situ hybridization to quantitatively characterize Gnal mRNA expression in brain tissue from wildtype C57BL/6J adult mice. We observed widespread expression of Gnal puncta throughout the brain, suggesting Gα olf is expressed in more brain structures and neuron types than previously accounted for. We quantify transcripts at a single cell level, and use neuron type specific markers to further classify and understand patterns of GNAL expression. Our data suggests that brain regions classically associated with motor control, initiation, and regulation show the highest expression of GNAL, with Purkinje Cells of the cerebellum showing the highest expression of any neuron type examined. Subsequent conditional Gnal knockout in Purkinje cells led to markedly decreased intracellular cAMP levels and downstream cAMP-dependent enzyme activation. Our work provides a detailed characterization of Gnal expression throughout the brain and the biochemical consequences of loss of Gα olf signaling in vivo in neurons that highly express Gnal., 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 B.V. All rights reserved.)

G protein-coupled receptors (GPCRs) couple to heterotrimeric G proteins, comprised of α and β γ subunits, to convert extracellular signals into activation of intracellular signaling pathways. Canonically, GPCR-mediated activation results in the exchange of GDP for GTP on G protein α subunits (G α ) and the dissociation of G α -GTP and G protein βγ subunits (G βγ ), both of which can regulate a variety of signaling pathways. Hydrolysis of bound GTP by G α returns the protein to G α -GDP and allows reassociation with G βγ to reform the inactive heterotrimer. Naturally occurring mutations in G α have been found at conserved glutamine and arginine amino acids that disrupt the canonical G protein cycle by inhibiting GTP hydrolysis, rendering these mutants constitutively active. Interestingly, these dysregulated G α mutants are found in many different cancers due to their ability to sustain aberrant signaling without a need for activation by GPCRs. This review will highlight an increased recognition of the prevalence of such constitutively activating G α mutations in cancers and the signaling pathways activated. In addition, we will discuss new knowledge regarding how these constitutively active G α are regulated, how different mutations are biochemically distinct, and how mutationally activated G α are unique compared with GPCR-activated G α Lastly, we will discuss recent progress in developing inhibitors directly targeting constitutively active G α mutants. SIGNIFICANCE STATEMENT: Constitutively activating mutations in G protein α subunits (G α ) widely occur in and contribute to the development of many human cancers. To develop ways to inhibit dysregulated, oncogenic signaling by these mutant G α , it is crucial to better understand mechanisms that lead to constitutive G α activation and unique mechanisms that regulate mutationally activated G α in cells. The prevalence of activating mutations in G α in various cancers makes G α proteins compelling targets for the development of therapeutics., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

During routine dermatologic examination, a 77-year-old male was noted to have a firm blue subcutaneous nodule on his right lateral upper back. His past medical history included metastatic melanoma of unknown primary involving right and left axillary lymph nodes, treated with ipilimumab/nivolumab with complete response, and subsequent primary uveal melanoma. The subcutaneous nodule was located near his previous right axillary scar for metastatic melanoma. Excision of the nodule showed a plexiform neoplasm involving mid and deep dermis composed of spindle and epithelioid atypical cells admixed with numerous melanophages. Central necrosis was present. Immunohistochemical studies revealed the tumor cells to be diffusely positive for HMB45, with retained expression of BAP1 and p16. The tumor cells were negative for PRAME, nuclear expression of β-catenin, LEF1, and BRAF V600E. Molecular studies demonstrated BAP1 and GNA11 somatic mutations, a profile different from that exhibited by his prior melanoma. Collectively, these data were interpreted as a metastasis from uveal melanoma and not a recurrence of his metastatic likely cutaneous melanoma after complete response to immunotherapy. This case emphasizes the importance of molecular studies for definitive diagnosis in challenging clinical situations, especially when there is discordance among histopathological, immunohistochemical, and molecular studies. Integration of clinical, histopathological, and molecular features is warranted., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aims: The Gα subunit is a major component of heterotrimeric G proteins, which play a crucial role in the development and pathogenicity of several model fungi. However, its detailed function in the causal agent of pear black spot (Alternaria alternata) is unclear. Our aim was to understand the characteristics and functions of AaGA1 in A. alternata., Methods and Results: AaGA1 was cloned from A. alternata in this study, which encodes 353 amino acids and has a "G-alpha" domain. Mutant ΔAaGA1 resulted in reduced vegetative growth, conidiation, and spore germination. Especially, mutant ΔAaGA1 produced only fewer conidia on the V8A medium, and spore formation-related genes AbaA, BrlA, and WetA were significantly downregulated. More tolerance against cell wall-inhibiting agents was observed after the deletion of AaGA1. Moreover, AaGA1 deletion led to a significant reduction in melanin and toxin production. Interestingly, deletion of AaGA1 resulted in defective appressorium-like formations, complete loss of the ability to penetrate cellophane, and decreased infection on non-wound inoculated tobacco leaves. Cell wall-degrading enzyme-related genes PME, CL, Cut2, and LC were significantly downregulated in mutant ΔAaGA1 mutant, significantly reducing virulence on wound-inoculated pear fruits., Conclusions: The G protein alpha subunit AaGA1 is indispensable for fungal development, appressorium-like formations, and pathogenicity in A. alternata., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

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.)

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.

Cooperative interactions in protein-protein interfaces demonstrate the interdependency or the linked network-like behavior and their effect on the coupling of proteins. Cooperative interactions also could cause ripple or allosteric effects at a distance in protein-protein interfaces. Although they are critically important in protein-protein interfaces, it is challenging to determine which amino acid pair interactions are cooperative. In this work, we have used Bayesian network modeling, an interpretable machine learning method, combined with molecular dynamics trajectories to identify the residue pairs that show high cooperativity and their allosteric effect in the interface of G protein-coupled receptor (GPCR) complexes with Gα subunits. Our results reveal six GPCR:Gα contacts that are common to the different Gα subtypes and show strong cooperativity in the formation of interface. Both the C terminus helix5 and the core of the G protein are codependent entities and play an important role in GPCR coupling. We show that a promiscuous GPCR coupling to different Gα subtypes, makes all the GPCR:Gα contacts that are specific to each Gα subtype (Gαs, Gαi, and Gαq). This work underscores the potential of data-driven Bayesian network modeling in elucidating the intricate dependencies and selectivity determinants in GPCR:G protein complexes, offering valuable insights into the dynamic nature of these essential cellular signaling components., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by 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.)

Trimeric G proteins transduce signals from a superfamily of receptors and each G protein controls a wide range of cellular and systemic functions. Their highly conserved alpha subunits fall in five classes, four of which have been well investigated (Gs, Gi, G12, Gq). In contrast, the function of the fifth class, Gv is completely unknown, despite its broad occurrence and evolutionary ancient origin (older than metazoans). Here we show a dynamic presence of Gv mRNA in several organs during early development of zebrafish, including the hatching gland, the pronephros and several cartilage anlagen, employing in situ hybridisation. Next, we generated a Gv frameshift mutation in zebrafish and observed distinct phenotypes such as reduced oviposition, premature hatching and craniofacial abnormalities in bone and cartilage of larval zebrafish. These phenotypes could suggest a disturbance in ionic homeostasis as a common denominator. Indeed, we find reduced levels of calcium, magnesium and potassium in the larvae and changes in expression levels of the sodium potassium pump atp1a1a.5 and the sodium/calcium exchanger ncx1b in larvae and in the adult kidney, a major osmoregulatory organ. Additionally, expression of sodium chloride cotransporter slc12a3 and the anion exchanger slc26a4 is altered in complementary ways in adult kidney. It appears that Gv may modulate ionic homeostasis in zebrafish during development and in adults. Our results constitute the first insight into the function of the fifth class of G alpha proteins., (© 2024. The Author(s).)

Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants., Competing Interests: Conflict of interest Joseph M. Jez is an associate editor and Sona Pandey is an editorial board member. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Selective activation of individual subtypes of muscarinic receptors is a promising way to safely alleviate a wide range of pathological conditions in the central nervous system and the periphery as well. The flexible G-protein interface of muscarinic receptors allows them to interact with several G-proteins with various efficacy, potency, and kinetics. Agonists biased to the particular G-protein mediated pathway may result in selectivity among muscarinic subtypes and, due to the non-uniform expression of individual G-protein alpha subunits, possibly achieve tissue specificity. Here, we demonstrate that novel tetrahydropyridine-based agonists exert specific signalling profiles in coupling with individual G-protein α subunits. These signalling profiles profoundly differ from the reference agonist carbachol. Moreover, coupling with individual Gα induced by these novel agonists varies among subtypes of muscarinic receptors which may lead to subtype selectivity. Thus, the novel tetrahydropyridine-based agonist can contribute to the elucidation of the mechanism of pathway-specific activation of muscarinic receptors and serve as a starting point for the development of desired selective muscarinic agonists., (© 2024. The Author(s).)

Understanding how signaling proteins like G proteins are allosterically activated is a long-standing challenge with significant biological and medical implications. Because it is difficult to directly observe such dynamic processes, much of our understanding is based on inferences from a limited number of static snapshots of relevant protein structures, mutagenesis data, and patterns of sequence conservation. Here, we use computer simulations to directly interrogate allosteric coupling in six G protein α-subunit isoforms covering all four G protein families. To analyze this data, we introduce automated methods for inferring allosteric networks from simulation data and assessing how allostery is conserved or diverged among related protein isoforms. We find that the allosteric networks in these six G protein α subunits are largely conserved and consist of two pathways, which we call pathway-I and pathway-II. This analysis predicts that pathway-I is generally dominant over pathway-II, which we experimentally corroborate by showing that mutations to pathway-I perturb nucleotide exchange more than mutations to pathway-II. In the future, insights into unique elements of each G protein family could inform the design of isoform-specific drugs. More broadly, our tools should also be useful for studying allostery in other proteins and assessing the extent to which this allostery is conserved in related proteins.

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.)

Double somatic mutations in CTNNB1 and GNA11/Q have recently been identified in a small subset of aldosterone-producing adenomas (APAs). As a possible pathogenesis of APA due to these mutations, an association with pregnancy, menopause, or puberty has been proposed. However, because of its rarity, characteristics of APA with these mutations have not been well characterized. A 46-year-old Japanese woman presented with hypertension and hypokalemia. She had two pregnancies in the past but had no history of pregnancy-induced hypertension. She had regular menstrual cycle at presentation and was diagnosed as having primary aldosteronism after endocrinologic examinations. Computed tomography revealed a 2 cm right adrenal mass. Adrenal venous sampling demonstrated excess aldosterone production from the right adrenal gland. She underwent right laparoscopic adrenalectomy. The resected right adrenal tumor was histologically diagnosed as adrenocortical adenoma and subsequent immunohistochemistry (IHC) revealed diffuse immunoreactivity of aldosterone synthase (CYP11B2) and visinin like 1, a marker of the zona glomerulosa (ZG), whereas 11β-hydroxylase, a steroidogenic enzyme for cortisol biosynthesis, was mostly negative. CYP11B2 IHC-guided targeted next-generation sequencing identified somatic CTNNB1 (p.D32Y) and GNA11 (p.Q209H) mutations. Immunofluorescence staining of the tumor also revealed the presence of activated β-catenin, consistent with features of the normal ZG. The expression patterns of steroidogenic enzymes and related proteins indicated ZG features of the tumor cells. PA was clinically and biochemically cured after surgery. In conclusion, our study indicated that CTNNB1 and GNA11 -mutated APA has characteristics of the ZG. The disease could occur in adults with no clear association with pregnancy or menopause., Competing Interests: KN received a research grant from AstraZeneca, which is unrelated to the current work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nanba, Blinder, Udager, Hirokawa, Miura, Okuno, Moriyoshi, Yamazaki, Sasano, Yasoda, Satoh-Asahara, Rainey and Tagami.)

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) is the most common primary intraocular malignancy in the adult eye. Despite the aggressive local management of primary UM, the development of metastases is common with no effective treatment options for metastatic disease. Genetic analysis of UM samples reveals the presence of mutually exclusive activating mutations in the Gq alpha subunits GNAQ and GNA11. One of the key downstream targets of the constitutively active Gq alpha subunits is the protein kinase C (PKC) signaling pathway. Herein, we describe the discovery of darovasertib (NVP-LXS196), a potent pan-PKC inhibitor with high whole kinome selectivity. The lead series was optimized for kinase and off target selectivity to afford a compound that is rapidly absorbed and well tolerated in preclinical species. LXS196 is being investigated in the clinic as a monotherapy and in combination with other agents for the treatment of uveal melanoma (UM), including primary UM and metastatic uveal melanoma (MUM).

Papillary hemangioma (PH) is a small, primarily dermal lesion occurring predominantly in the head and neck in both children and adults. Its signature characteristics are dilated thin-walled channels containing papillary clusters of mainly capillary-sized vessels and endothelial cytoplasmic eosinophilic inclusions. Given certain histopathologic similarities to congenital hemangioma which harbor mutations in GNAQ and GNA11 , we investigated whether similar mutations are present in PH. Seven PH specimens were studied. All presented in the first 4 years of life, with one being noted at birth. With the exception of one lesion, all were in the head and neck. Lesions were bluish and ranged in size from 0.5 to 2.8 cm. Four samples had GNA11 p.Q209L and 3 had GNAQ p.Q209L missense mutations. Mutations in GNA11 and GNAQ are associated with other types of somatic vascular lesions including capillary malformation, congenital hemangioma, anastomosing hemangioma, thrombotic anastomosing hemangioma, and hepatic small cell neoplasm. Shared mutations in GNA11 and GNAQ may account for some overlapping clinical and pathologic features in these entities, perhaps explicable by the timing of the mutation or influence of the germline phenotype., Competing Interests: Conflicts of Interest and Source of Funding: Supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Award Number F32HD107878. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Key Message: Nitrate-responsive transcriptomic, phenotypic and physiological analyses of rice RGA1 mutant revealed many novel RGA1-regulated genes/processes/traits related to nitrogen use efficiency, and provided robust genetic evidence of RGA1-regulation of NUE. Nitrogen (N) use efficiency (NUE) is important for sustainable agriculture. G-protein signalling was implicated in N-response/NUE in rice, but needed firm genetic characterization of the role of alpha subunit (RGA1). The knock-out mutant of RGA1 in japonica rice exhibited lesser nitrate-dose sensitivity than the wild type (WT), in yield and NUE. We, therefore, investigated its genomewide nitrate-response relative to WT. It revealed 3416 differentially expressed genes (DEGs), including 719 associated with development, grain yield and phenotypic traits for NUE. The upregulated DEGs were related to photosynthesis, chlorophyll, tetrapyrrole and porphyrin biosynthesis, while the downregulated DEGs belonged to cellular protein metabolism and transport, small GTPase signalling, cell redox homeostasis, etc. We validated 26 nitrate-responsive DEGs across functional categories by RT-qPCR. Physiological validation of nitrate-response in the mutant and the WT at 1.5 and 15 mM doses revealed higher chlorophyll and stomatal length but decreased stomatal density, conductance and transpiration. The consequent increase in photosynthesis and water use efficiency may have contributed to better yield and NUE in the mutant, whereas the WT was N-dose sensitive. The mutant was not as N-dose-responsive as the WT in shoot/root growth, productive tillers and heading date, but equally responsive as WT in total N and protein content. The RGA1 mutant was less impacted by higher N-dose or salt stress in terms of yield, protein content, photosynthetic performance, relative water content, water use efficiency and catalase activity. PPI network analyses revealed known NUE-related proteins as RGA1 interactors. Therefore, RGA1 negatively regulates N-dose sensitivity and NUE in rice., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.)

Background: Astragalus grows mainly in drought areas. Cycloastragenol (CAG) is a tetracyclic triterpenoid allelochemical extracted from traditional Chinese medicine Astragalus root. Phospholipase C (PLC) and Gα-submit of the heterotrimeric G-protein (GPA1) are involved in many biotic or abiotic stresses. Nitric oxide (NO) is a crucial gas signal molecule in plants., Results: In this study, using the seedlings of Arabidopsis thaliana (A. thaliana), the results showed that low concentrations of CAG induced stomatal closure, and high concentrations inhibited stomatal closure. 30 µmol·L -1 CAG significantly increased the relative expression levels of PLC1 and GPA1 and the activities of PLC and GTP hydrolysis. The stomatal aperture of plc1, gpa1, and plc1/gpa1 was higher than that of WT under CAG treatment. CAG increased the fluorescence intensity of NO in guard cells. Exogenous application of c-PTIO to WT significantly induced stomatal aperture under CAG treatment. CAG significantly increased the relative expression levels of NIA1 and NOA1. Mutants of noa1, nia1, and nia2 showed that NO production was mainly from NOA1 and NIA1 by CAG treatment. The fluorescence intensity of NO in guard cells of plc1, gpa1, and plc1/gpa1 was lower than WT, indicating that PLC1 and GPA1 were involved in the NO production in guard cells. There was no significant difference in the gene expression of PLC1 in WT, nia1, and noa1 under CAG treatment. The gene expression levels of NIA1 and NOA1 in plc1, gpa1, and plc1/gpa1 were significantly lower than WT, indicating that PLC1 and GPA1 were positively regulating NO production by regulating the expression of NIA1 and NOA1 under CAG treatment., Conclusions: These results suggested that the NO accumulation was essential to induce stomatal closure under CAG treatment, and GPA1 and PLC1 acted upstream of NO., (© 2023. The Author(s).)

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.

β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., β-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate β-arrestins, thereby limiting the ability to distinguish G protein from β-arrestin-mediated signaling events. We used β2-adrenergic receptor (β2AR) and its β2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gα s , β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gα s and β-arrestins in controlling gene expression. We found that Gα s is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gα s dictates the GPCR kinase isoforms involved in β-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of β2AR in wildtype and β-arrestin1/2-KO cells but absent in Gα s -KO cells. These results were validated by re-expressing Gα s in the corresponding KO cells and silencing β-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas β-arrestins initiate signaling events modulating Gα s -driven nuclear transcriptional activity., Competing Interests: Conflict of interest J. S. G. is consultant for Domain Therapeutics, Pangea Therapeutics, and io9, and founder of Kadima Pharmaceuticals, outside the submitted work. M. B. is the president of Domain Therapeutics Scientific Advisory Board. 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.)

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.)

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.

Uveal melanoma is the most common intraocular tumor in adults, representing approximately 5% of all melanoma cases. Up to 50% of uveal melanoma patients develop metastases that are resistant to most of the commonly used antineoplastic treatments. Virtually all uveal melanoma tumors harbor activating mutations in GNAQ or GNA11 , encoding Gαq and Gα11, respectively. Constant activity of these proteins causes deregulation of multiple downstream signaling pathways including PKC, MAPK and YAP1/TAZ. While the importance of YAP1 signaling for the proliferation of uveal melanoma has recently been demonstrated, much less is known about the paralog of YAP1 transcriptional coactivator, named TAZ; however, similar to YAP1, TAZ is expected to be a therapeutic target in uveal melanoma. We performed a small-scale drug screen to discover a compound synergistically inhibiting uveal melanoma proliferation/survival in combination with YAP1/TAZ inhibition. We found that the combination of genetic depletion of YAP1/TAZ together with Mcl-1 inhibition demonstrates a synergistic inhibitory effect on the viability of uveal melanoma cell lines. Similarly, indirect attenuation of the YAP1/TAZ signaling pathway with an inhibitor of the mevalonate pathway, that is, the geranyl-geranyl transferase inhibitor GGTI-298, synergizes with Mcl-1 inhibition. This combination could be potentially used as a treatment for metastatic uveal melanoma., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2) are due to loss- and gain-of-function mutations, respectively, of the GNA11 gene that encodes the G protein subunit Gα11, a signaling partner of the calcium-sensing receptor (CaSR). To date, four probands with FHH2-associated Gα11 mutations and eight probands with ADH2-associated Gα11 mutations have been reported. In a 10-year period, we identified 37 different germline GNA11 variants in >1200 probands referred for investigation of genetic causes for hypercalcemia or hypocalcemia, comprising 14 synonymous, 12 noncoding, and 11 nonsynonymous variants. The synonymous and noncoding variants were predicted to be benign or likely benign by in silico analysis, with 5 and 3, respectively, occurring in both hypercalcemic and hypocalcemic individuals. Nine of the nonsynonymous variants (Thr54Met, Arg60His, Arg60Leu, Gly66Ser, Arg149His, Arg181Gln, Phe220Ser, Val340Met, Phe341Leu) identified in 13 probands have been reported to be FHH2- or ADH2-causing. Of the remaining nonsynonymous variants, Ala65Thr was predicted to be benign, and Met87Val, identified in a hypercalcemic individual, was predicted to be of uncertain significance. Three-dimensional homology modeling of the Val87 variant suggested it was likely benign, and expression of Val87 variant and wild-type Met87 Gα11 in CaSR-expressing HEK293 cells revealed no differences in intracellular calcium responses to alterations in extracellular calcium concentrations, consistent with Val87 being a benign polymorphism. Two noncoding region variants, a 40bp-5'UTR deletion and a 15bp-intronic deletion, identified only in hypercalcemic individuals, were associated with decreased luciferase expression in vitro but no alterations in GNA11 mRNA or Gα11 protein levels in cells from the patient and no abnormality in splicing of the GNA11 mRNA, respectively, confirming them to be benign polymorphisms. Thus, this study identified likely disease-causing GNA11 variants in <1% of probands with hypercalcemia or hypocalcemia and highlights the occurrence of GNA11 rare variants that are benign polymorphisms. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)., (© 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).)

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.)

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.

Identification of novel therapeutic oncotargets for human glioma is extremely important. Here we tested expression, potential functions and underlying mechanisms of G protein inhibitory α subunit 2 (Gαi2) in glioma. Bioinformatics analyses revealed that Gαi2 expression is significantly elevated in human glioma, correlating with poor patients' survival, higher tumor grade and wild-type IDH status. Moreover, increased Gαi2 expression was also in local glioma tissues and different glioma cells. In primary and immortalized (A172) glioma cells, Gαi2 shRNA or knockout (KO, by Cas9-sgRNA) potently suppressed viability, proliferation, and mobility, and induced apoptosis. Ectopic Gαi2 overexpression, using a lentiviral construct, further augmented malignant behaviors in glioma cells. p65 phosphorylation, NFκB activity and expression of NFκB pathway genes were decreased in Gαi2-depleted primary glioma cells, but increased following Gαi2 overexpression. There was an increased binding between Gαi2 promoter and Sp1 (specificity protein 1) transcription factor in glioma tissues and different glioma cells. In primary glioma cells Gαi2 expression was significantly reduced following Sp1 silencing, KO or inhibition. In vivo studies revealed that Gαi2 shRNA-expressing AAV intratumoral injection hindered growth of subcutaneous glioma xenografts in nude mice. Moreover, Gαi2 KO inhibited intracranial glioma xenograft in nude mice. Gαi2 depletion, NFκB inhibition and apoptosis induction were observed in subcutaneous and intracranial glioma xenografts with Gαi2 depletion. Together, overexpressed Gαi2 is important for glioma cell growth possibly by promoting NFκB cascade activation., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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.

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.)

Objective: To analyze the genetic features of melanocytomas and melanomas of the anterior uvea and assess the value of molecular testing for diagnosis and prognostication., Design: Retrospective case-control study., Subjects: Patients with melanocytoma (n = 16) and melanoma (n = 19) of the anterior uvea., Methods: Targeted next-generation sequencing was performed on formalin-fixed, paraffin-embedded tumor tissue from anterior uveal melanocytic tumors and correlated with clinicopathologic features., Main Outcome Measures: Presence or absence of accompanying oncogenic alterations beyond GNAQ/GNA11 and their association with histologic features and local recurrence., Results: Hotspot missense mutations in GNAQ/GNA11 were identified in 91% (32/35) of all cases. None of the melanocytomas with or without atypia demonstrated chromosomal imbalances or additional oncogenic variants beyond GNAQ mutation, and none recurred over a median follow-up of 36 months. Additional alterations identified in a subset of melanomas include mutations in BAP1 (n = 3), EIF1AX (n = 4), SRSF2 (n = 1), PTEN (n = 1), and EP300 (n = 1); monosomy 3p (n = 6); trisomy 6p (n = 3); trisomy 8q (n = 2); and an ultraviolet mutational signature (n = 5). Local recurrences were limited to melanomas, all of which demonstrated oncogenic alterations in addition to GNAQ/GNA11 (n = 5). A single melanoma harboring GNAQ and BAP1 mutations and monosomy 3 was the only tumor that metastasized., Conclusions: In this study, anterior segment uveal melanocytomas did not display oncogenic alterations beyond GNAQ/GNA11. Therefore, they are genetically similar to uveal nevi rather than uveal melanoma based on their molecular features known from the literature. Molecular testing can be performed on borderline cases to aid risk stratification and clinical management decisions., (Published by Elsevier Inc.)

Respiratory syncytial virus (RSV) is a major pathogen that can cause acute respiratory infectious diseases of the upper and lower respiratory tract, especially in children, elderly individuals, and immunocompromised people. Generally, following viral infection, respiratory epithelial cells secrete cytokines and chemokines to recruit immune cells and initiate innate and/or adaptive immune responses. However, whether chemokines affect viral replication in nonimmune cells is rarely clear. In this study, we detected that chemokine CCL5 was highly expressed, while expression of its receptor, CCR1, was downregulated in respiratory epithelial cells after RSV infection. When we overexpressed CCR1 on respiratory epithelial cells in vivo or in vitro , viral load was significantly suppressed, which can be restored by the neutralizing antibody for CCR1. Interestingly, the antiviral effect of CCR1 was not related to type I interferon (IFN-I), apoptosis induction, or viral adhesion or entry inhibition. In contrast, it was related to the preferential recruitment and activation of the adaptor Gαi, which promoted inositol 1,4,5-triphosphate receptor type 3 (ITPR3) expression, leading to inhibited STAT3 phosphorylation; explicitly, phosphorylated STAT3 (p-STAT3) was verified to be among the important factors regulating the activity of HSP90, which has been previously reported to be a chaperone of RSV RNA polymerase. In summary, we are the first to reveal that CCR1 on the surface of nonimmune cells regulates RSV replication through a previously unknown mechanism that does not involve IFN-I induction. IMPORTANCE Our results revealed a novel mechanism by which RSV escapes innate immunity. That is, although it induces high CCL5 expression, RSV might attenuate the binding of CCL5 by downregulating the expression of CCR1 in respiratory epithelial cells to weaken the inhibitory effect of CCR1 on HSP90 activity and thereby facilitate RSV replication in nonimmune cells. This study provides a new target for the development of co-antiviral inhibitors against other components of the host and co-molecular chaperone/HSP90 and provides a scientific basis for the search for effective broad-spectrum antiviral drugs.

Aims: Blue naevi are benign melanocytic lesions that typically occur in the dermis. Melanoma arising in blue naevus is rare, and shows a molecular profile distinct from conventional forms of cutaneous melanoma and more similar to uveal melanoma and central nervous system (CNS) melanocytomas. In contrast to conventional cutaneous melanoma, these tumour types typically show activating driver mutations in GNAQ or GNA11, a low mutational burden without evidence of a UV signature and a reproducible pattern of chromosomal copy number changes. Blue naevi can also occur at extracutaneous sites. Here we report two cases of melanoma arising in extracutaneous blue naevus and compare their molecular features to cohorts of melanoma arising in cutaneous blue naevus (five patients) and uveal melanoma (six patients)., Methods and Results: We describe the clinical, histomorphological, immunohistochemical and molecular findings in these two cases of melanoma arising in extracutaneous blue naevus. We compare their molecular profiles to melanomas arising in cutaneous blue naevus and uveal melanoma using a targeted next-generation DNA sequencing platform and find striking similarities between all three groups., Conclusions: The close relationship between blue naevus-associated melanomas, regardless of their anatomical site, supports and validates the concept of melanoma arising in extracutaneous blue naevus and suggests that the two groups share common pathogenic mechanisms. The similarity of both groups to uveal melanoma in turn supports the close relationship between blue naevus-associated melanoma, uveal melanoma and CNS melanocytoma, and their distinction from conventional UV-associated melanoma. These findings have important implications for prognosis and therapy., (© 2022 John Wiley & Sons Ltd.)

Background and Objectives: Cutaneous capillary malformations (CMs) describe a group of vascular birthmarks with heterogeneous presentations. CMs may present as an isolated finding or with other associations, including glaucoma and leptomeningeal angiomatosis (i.e., Sturge-Weber syndrome) or pigmentary birthmarks (i.e., phakomatosis pigmentovascularis). The use of targeted genetic sequencing has revealed that postzygotic somatic variations in GNAQ and GNA11 at codon 183 are associated with CMs. We report five patients with early-onset hypertension and discuss possible pathogenesis of hypertension., Methods: Twenty-nine patients with CMs, confirmed GNAQ/11 postzygotic variants, and documented past medical history were identified from a multi-institutional vascular anomalies study. Early-onset hypertension was defined as hypertension before the age of 55 years. Clinical data were reviewed for evidence of hypertension, such as documentation of diagnosis or elevated blood pressure measurements., Results: Five of the 29 patients identified as having GNAQ/11 postzygotic variants had documented early-onset hypertension. Three individuals harbored a GNAQ p.R183Q variant, and two individuals harbored a GNA11 p.R183C variant. All individuals had extensive cutaneous CMs involving the trunk and covering 9%-56% of their body surface area. The median age of hypertension diagnosis was 15 years (range 11-24 years), with three individuals having renal abnormalities on imaging., Conclusions: Early-onset hypertension is associated with extensive CMs harboring somatic variations in GNAQ/11. Here, we expand on the GNAQ/11 phenotype and hypothesize potential mechanisms driving hypertension. We recommend serial blood pressure measurements in patients with extensive CMs on the trunk and extremities to screen for early-onset hypertension., (© 2022 The Authors. Pediatric Dermatology published by Wiley Periodicals LLC.)

Background and Purpose: Sturge-Weber syndrome (SWS) is a neurocutaneous disorder characterized by clinical manifestations involving the brain, eye and skin. SWS is commonly caused by somatic mutations in G protein subunit Alpha Q (GNAQ). Five cases of subunit Alpha 11 (GNA11) mutations have been reported. We studied phenotypic features of GNA11-SWS and compared them with those of classic SWS., Methods: Within two European multidisciplinary centers we looked for patients with clinical characteristics of SWS and a GNA11 mutation. Clinical and radiological data were collected retrospectively and prospectively., Results: We identified three patients with SWS associated with a somatic GNA11 mutation. All had disseminated capillary malformation (CM) and hyper- or hypotrophy of an extremity. At birth, the CMs of the face, trunk and limbs were pink and patchy, and slowly darkened with age, evolving to a purple color. Two of the patients had glaucoma. All had neurological symptoms and moderate brain atrophy with a lower degree of severity than that classically associated with SWS. Susceptibility-weighted imaging (SWI) and contrast-enhanced fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging demonstrated the best sensitivity to reveal the pial angiomas., Conclusions: We have differentiated two distinct clinical/radiological phenotypes of SWS; GNAQ- and GNA11-SWS. The classic GNAQ-SWS is characterized by a homogeneous dark-red CM, commonly associated with underlying soft tissue hypertrophy. The CM in GNA11-SWS is more reticulate and darkens with time, and the neurological picture is milder. SWI and post-contrast FLAIR sequences appear to be necessary to demonstrate leptomeningeal angiomatosis. Anti-epileptic medication or future targeted therapies may be useful, as in classic SWS., (© 2022 European Academy of Neurology.)

Uveal melanoma (UM) is a rare cancer arising from melanocytes in the uveal tract of the eye. Despite effective primary treatment, there is no approved therapy for metastatic UM and prognosis and survival remain poor. Over 90% of UM are driven by mutations affecting the Gα subunits encoded by the GNAQ and GNA11 genes. These mutations activate downstream and targetable signaling pathways, including the protein kinase C (PKC) cascade. PKC inhibitors have been used in clinical trials for metastatic UM but have shown limited efficacy. In this study, we examined the signaling and functional effects of two PKC inhibitors (AEB071 and IDE196) in a panel of UM cell models. In response to PKC inhibition, all UM cell lines showed potent suppression of PKC activity, but this was not sufficient to predict PKC inhibitor sensitivity and only two UM cell lines showed substantial PKC inhibitor-induced cell death. The differences in UM cell responses to PKC inhibition were not attributable to the degree or timing of PKC suppression or inhibition of the downstream mitogen-activated protein kinase (MAPK) or phosphatidylinositol-3-kinase (PI3K) pathways. Instead, UM cell show complex, PKC-independent signaling pathways that contribute to their survival and resistance to targeted therapies., (© 2022. The Author(s).)

Phytomelatonin is a newly identified plant hormone, and its primary functions in plant growth and development remain relatively poorly appraised. Phytomelatonin is a master regulator of reactive oxygen species (ROS) signaling and acts as a darkness signal in circadian stomatal closure. Plants exhibit at least three interrelated patterns of interaction between phytomelatonin and ROS production. Exogenous melatonin can induce flavonoid biosynthesis, which might be required for maintenance of antioxidant capacity under stress, after harvest, and in leaf senescence conditions. However, several genetic studies have provided direct evidence that phytomelatonin plays a negative role in the biosynthesis of flavonoids under non-stress conditions. Phytomelatonin delays flowering time in both dicot and monocot plants, probably via its receptor PMTR1 and interactions with the gibberellin, strigolactone, and ROS signaling pathways. Furthermore, phytomelatonin signaling also functions in hypocotyl and shoot growth in skotomorphogenesis and ultraviolet B (UV-B) exposure; the G protein α-subunit (Arabidopsis GPA1 and rice RGA1) and constitutive photomorphogenic1 (COP1) are important signal components during this process. Taken together, these findings indicate that phytomelatonin acts as a darkness signal with important regulatory roles in circadian stomatal closure, flavonoid biosynthesis, flowering, and hypocotyl and shoot growth., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To identify therapeutic targets and correlate with clinical outcomes from mutation profiling of metastatic uveal melanoma (UM) using next-generation sequencing (NGS)., Methods: Melanoma cases that were tested using DNA-based NGS panels of 25 and/or 214 genes were evaluated retrospectively (263 cases) and identified 27 UM cases. BAP1 expression was examined by immunohistochemistry., Results: Mutations in GNA11 (14) and GNAQ (12) were found in 96% (n = 27) of cases of UM, and most had coexisting BAP1 (17) or SF3B1 (4) mutations. Coexisting GNAQ/11-SF3B1 mutations correlated with a longer average time to first metastasis compared with GNAQ/11-BAP1 mutations (99.7 vs 38.5 months, P = .047). Three patients with BAP1 mutations received trametinib; two are still alive (15 months; 23 months), and one died (32 months). In non-UMs, only 4.2% (n = 236) had BAP1 and 3.8% had SF3B1 mutations; none had coexisting GNAQ/11 mutations., Conclusions: Coexisting BAP1/SF3B1 and GNAQ/11 mutations were unique to UM. SF3B1 mutations were reported to be UM-specific in melanoma and associated with rare/no metastasis. The finding of mutated SF3B1 in 14.8% (n = 27) of UMs suggests its role should be further evaluated. The correlation of BAP1/SF3B1 mutation with survival also warrants investigation., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hydrogen (H 2 ) is a new signaling molecule that regulates stomatal closure via stimulating the generation of reactive oxygen species (ROS) and nitric oxide (NO) in Arabidopsis thaliana. GPA1 is the sole heterotrimeric G protein canonical α subunit found in Arabidopsis genome and functions in stomatal closure. Here, we estimated a possible role of Arabidopsis GPA1 in hydrogen-rich water (HRW)-induced stomatal closure. Our data indicated that HRW induced significant stomatal closure as well as the generation of ROS and NO in the Col-0 guard cells. However, the production of ROS and NO and stomatal closure induced by HRW were absent in the gpa1-4 mutant lacking the expression of AtGPA1. By contrast, overexpression of AtGPA1 in gpa1-4 (AtGPA1-HA/gpa1-4) restored stomatal closure and the generation of NO and ROS in the presence of HRW. Taken together, our results suggest that GPA1 is necessary for HRW-induced stomatal closure in Arabidopsis., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

GNAL mutations (DYT25) have lately been identified as the firstly proven cause of focal adult-onset dystonia. We report here a new mutation in the GNAL gene in two siblings with dystonia. The new mutation is called NM 001,142,339:c.97C > T. Our research emphasizes the possible effects of new mutation on disease risk and the significance of genetic tests for GNAL mutations in confirming the molecular diagnosis., (© 2022. Fondazione Società Italiana di Neurologia.)

Background and Aim of the Study: Mutations in the Gα-genes GNAQ and GNA11 are found in 85-90% of uveal melanomas (UM). Aim of the study is to understand whether the mutations in both genes differentially affect tumor characteristics and outcome and if so, to identify potential mechanisms., Methods: We analyzed the association between GNAQ and GNA11 mutations with disease-specific survival, gene expression profiles, and cytogenetic alterations in 219 UMs. We used tandem-affinity-purification, mass spectrometry and immunoprecipitation to identify protein interaction partners of the two G-proteins and analyzed their impact on DNA-methylation., Results: GNA11 mutation was associated with: i) an increased frequency of loss of BRCA1-associated protein 1 (BAP1) expression (p = 0.0005), ii) monosomy of chromosome 3 (p < 0.001), iii) amplification of chr8q (p = 0.038), iv) the combination of the latter two (p = 0.0002), and inversely with v) chr6p gain (p = 0.003). Our analysis also showed a shorter disease-specific survival of GNA11-mutated cases as compared to those carrying a GNAQ mutation (HR = 1.97 [95%CI 1.12-3.46], p = 0.02). GNAQ and GNA11 encoded G-proteins have different protein interaction partners. Specifically, the Tet Methylcytosine Dioxygenase 2 (TET2), a protein that is involved in DNA demethylation, physically interacts with the GNAQ protein but not with GNA11, as confirmed by immunoprecipitation analyses. High-risk UM cases show a clearly different DNA-methylation pattern, suggesting that a different regulation of DNA methylation by the two G-proteins might convey a different risk of progression., Conclusions: GNA11 mutated uveal melanoma has worse prognosis and is associated with high risk cytogenetic, mutational and molecular tumor characteristics that might be determined at least in part by differential DNA-methylation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Heterotrimeric G-proteins modulate multiple signaling pathways in many eukaryotes. In plants, G-proteins have been characterized primarily from a few model angiosperms and a moss. Even within this small group, they seem to affect plant phenotypes differently: G-proteins are essential for survival in monocots, needed for adaptation but are nonessential in eudicots, and are required for life cycle completion and transition from the gametophytic to sporophytic phase in the moss Physcomitrium (Physcomitrella) patens. The classic G-protein heterotrimer consists of three subunits: one Gα, one Gβ and one Gγ. The Gα protein is a catalytically active GTPase and, in its active conformation, interacts with downstream effectors to transduce signals. Gα proteins across the plant evolutionary lineage show a high degree of sequence conservation. To explore the extent to which this sequence conservation translates to their function, we complemented the well-characterized Arabidopsis Gα protein mutant, gpa1, with Gα proteins from different plant lineages and with the yeast Gpa1 and evaluated the transgenic plants for different phenotypes controlled by AtGPA1. Our results show that the Gα protein from a eudicot or a monocot, represented by Arabidopsis and Brachypodium, respectively, can fully complement all gpa1 phenotypes. However, the basal plant Gα failed to complement the developmental phenotypes exhibited by gpa1 mutants, although the phenotypes that are exhibited in response to various exogenous signals were partially or fully complemented by all Gα proteins. Our results offer a unique perspective on the evolutionarily conserved functions of G-proteins in plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Neurofibromatosis 1 (NF1) is caused by germline mutations in the NF1 gene and manifests as proliferation of various tissues, including plexiform neurofibromas. The plexiform neurofibroma phenotype varies from indolent to locally aggressive, suggesting contributions of other modifiers in addition to somatic loss of NF1. In this study, we investigated a life-threatening plexiform neurofibroma in a 9-month-old female infant with NF1. Germline mutations in two RASopathy-associated genes were identified using whole-exome sequencing-a de novo pathogenic variant in the NF1 gene, and a known pathogenic variant in the LZTR1 gene. Somatic analysis of the plexiform neurofibroma revealed NF1 loss of heterozygosity and a variant in GNAZ, a gene encoding a G protein-coupled receptor. Cells expressing mutant GNAZ exhibited increased ERK 1/2 activation compared to those expressing wild-type GNAZ. Taken together, we suggest the variants in NF1, LZRT1 and GNAZ act synergistically in our patient, leading to MAPK pathway activation and contributing to the severity of the patient's plexiform neurofibromatosis. After treatment with the MEK inhibitor, trametinib, a prominent clinical improvement was observed in this patient. This case study contributes to the knowledge of germline and somatic non-NF1 variants affecting the NF1 clinical phenotype and supports use of personalized, targeted therapy., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia and inappropriately low PTH concentrations. ADH type 2 (ADH2) is caused by a heterozygous gain-of-function mutation in GNA11 that encodes the subunit of G11, the principal G protein that transduces calcium-sensing receptor signaling in the parathyroid. Clinical features related to hypocalcemia in ADH2 range from asymptomatic to tetany and seizures. We report the clinical and molecular analysis of an infant with ADH2. Exome sequencing identified a de novo heterozygous missense variant, c. G548C (p. Arg183Pro) in GNA11. This is the youngest Korean case to be diagnosed with ADH 2. In addition, we summarized the literature related to eight mutations in GNA11 from 10 families., (© 2022 by the Association of Clinical Scientists, Inc.)

Cell surface protein trafficking is regulated in response to nutrient availability, with multiple pathways directing surface membrane proteins to the lysosome for degradation in response to suboptimal extracellular nutrients. Internalized protein and lipid cargoes recycle back to the surface efficiently in glucose-replete conditions, but this trafficking is attenuated following glucose starvation. We find that cells with either reduced or hyperactive phosphatidylinositol 3-kinase (PI3K) activity are defective for endosome to surface recycling. Furthermore, we find that the yeast Gα subunit Gpa1, an endosomal PI3K effector, is required for surface recycling of cargoes. Following glucose starvation, mRNA and protein levels of a distinct Gα subunit Gpa2 are elevated following nuclear translocation of Mig1, which inhibits recycling of various cargoes. As Gpa1 and Gpa2 interact at the surface where Gpa2 concentrates during glucose starvation, we propose that this disrupts PI3K activity required for recycling, potentially diverting Gpa1 to the surface and interfering with its endosomal role in recycling. In support of this model, glucose starvation and overexpression of Gpa2 alter PI3K endosomal phosphoinositide production. Glucose deprivation therefore triggers a survival mechanism to increase retention of surface cargoes in endosomes and promote their lysosomal degradation.

The prognostic value of mutations in G-protein genes GNAQ and GNA11 in patients with intracranial and orbital melanocytomas is unknown. The authors present a case of GNA11 mutation (GNA11Q209L) in a 32-year-old male suffering from a meningeal melanocytoma with orbital involvement and ipsilateral Nevus of Ota. The patient underwent gamma knife stereotactic radiosurgery without biopsy and later partial transcranial resection of the melanocytic tumor that was subject to immunohistochemical and molecular analysis. A 50-gene next-generation sequencing panel revealed a 626A>T mutation in the GNA11 gene. One year later, intracranial extension of the melanocytoma necessitated a ventriculoperitoneal shunt and immunotherapy. Future work is needed to determine how GNA11 mutations in melanocytomas influence prognosis and monitoring strategies., Competing Interests: The authors have no financial or conflicts of interest to disclose., (Copyright © 2021 The American Society of Ophthalmic Plastic and Reconstructive Surgery, Inc.)