Deciphering the function of the fifth class of Gα proteins: regulation of ionic homeostasis as unifying hypothesis.

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.

[Circumscribed choroidal hemangioma and non-pigmented choroidal melanoma: clinical, instrumental and molecular genetic features]. / Otgranichennaya gemangioma i bespigmentnaya melanoma khorioidei: kliniko-instrumental'nye i molekulyarno-geneticheskie osobennosti.

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%).

G Protein Activation Occurs via a Largely Universal Mechanism.

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.

Agonist-selective activation of individual G-proteins by muscarinic receptors.

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.

Double somatic mutations in CTNNB1 and GNA11 in an aldosterone-producing adenoma.

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 11ß-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 ß-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.

INPP5A phosphatase is a synthetic lethal target in GNAQ and GNA11-mutant melanomas.

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.

Discovery of Darovasertib (NVP-LXS196), a Pan-PKC Inhibitor for the Treatment of Metastatic Uveal Melanoma.

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 Harbors Somatic GNA11 and GNAQ Mutations.

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.

GNAQ/GNA11 Mosaicism Causes Aberrant Calcium Signaling Susceptible to Targeted Therapeutics.

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.

GNAQ/GNA11 Mosaicism Is Associated with Abnormal Serum Calcium Indices and Microvascular Neurocalcification.

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.

Driver mutations in GNAQ and GNA11 genes as potential targets for precision immunotherapy in uveal melanoma patients.

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.

PLC1 mediated Cycloastragenol-induced stomatal movement by regulating the production of NO in Arabidopsis thaliana.

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.

Detection of the Uveal Melanoma-Associated Mutation GNAQ Q209P from Liquid Biopsy Using CRISPR/Cas12a Technology.

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.

Nitrate-responsive transcriptome analysis of rice RGA1 mutant reveals the role of G-protein alpha subunit in negative regulation of nitrogen-sensitivity and use efficiency.

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.

High-throughput chemogenetic drug screening reveals PKC-RhoA/PKN as a targetable signaling vulnerability in GNAQ-driven uveal melanoma.

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.

Release of Cell-Free Tumor DNA in the Plasma of Uveal Melanoma Patients Under Radiotherapy.

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.

Gαs is dispensable for ß-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by ß2-adrenergic receptor.

ß-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether ß-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., ß-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate ß-arrestins, thereby limiting the ability to distinguish G protein from ß-arrestin-mediated signaling events. We used ß2-adrenergic receptor (ß2AR) and its ß2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, ß-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and ß-arrestins in controlling gene expression. We found that Gαs is not required for ß2AR and ß-arrestin conformational changes, ß-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in ß-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of ß2AR in wildtype and ß-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing ß-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of ß2AR. Overall, our results support that Gs is essential for ß2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas ß-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.

Combined Mcl-1 and YAP1/TAZ inhibition for treatment of metastatic uveal melanoma.

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.

Trametinib in Patients With NF1-, GNAQ-, or GNA11-Mutant Tumors: Results From the NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocols S1 and S2.

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.

GNA11 Variants Identified in Patients with Hypercalcemia or Hypocalcemia.

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).