Review article 525

GNAQ and GNA11 mutations in uveal melanoma Alexander N. Shoushtaria and Richard D. Carvajalb G-protein-coupled receptors signal through heterotrimeric G proteins, Gα and G-βγ, to manage numerous aspects of physiologic homeostasis. Many neoplastic processes harbor alterations in G-protein-coupled receptors and/or G-α proteins, best exemplified by recurrent activating mutations in GNAQ or GNA11 in uveal melanomas. This review will discuss the multiple activated signaling targets downstream of mutant GNAQ and GNA11 in uveal melanoma, including MEK, PI3-kinase/Akt, protein kinase C, and YAP. This knowledge has led to the rapid expansion of clinical trials that are specific to patients with uveal melanoma and promises future breakthroughs in therapies. Melanoma Res 24:525–534 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.

G-protein-coupled receptor (GPCR) proteins represent a large, diverse family of transmembrane receptors that function as signal transducers from the extracellular environment to the cellular interior. They are the largest group of cell surface receptors, with ∼ 4% of the genome dedicated to encoding the greater than 800 different GPCRs. They encompass a wide variety of physiologic functions, from sensory functions such as vision, olfaction, and taste to neurologic and endocrine signaling, as well as normal organ development [1]. Given the critical importance and the variety of GPCR signaling to normal homeostatic function, it is perhaps not surprising that alterations and changes in the activity of GPCRs and their downstream effectors are frequently implicated in the pathogenesis of neoplasms. This topic was recently the subject of an excellent review [1]. Nearly 20% of human tumors harbor mutations in a GPCR. Specific tumors have an even higher prevalence of activating mutations in G-alpha (Gα) subunits that signal to downstream growth signals. Several years ago, seminal work by Van Raamsdonk and colleagues identified that the majority of uveal melanomas harbor activating mutations in two highly homologous genes encoding Gα subunits, GNAQ (Gαq) and GNA11 (Gα11) [2,3]. This has led to rapid advancement in the understanding of uveal melanoma pathogenesis, growth signaling, and a variety of novel therapeutic approaches. This review will briefly explain GPCR and Gα protein structure and function, particularly in regard to GNAQ and GNA11 activation. The mechanism by which GNAQ and GNA11 mutations activate downstream signaling in uveal melanoma and other neoplasms will be discussed. The rapidly evolving understanding of 0960-8931 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins

Melanoma Research 2014, 24:525–534 Keywords: G protein, GNA11, GNAQ, MAP kinase, PI3-kinase, protein kinase C, selumetinib, uveal melanoma, YAP a Melanoma and Immunotherapeutics Service and bDepartment of Developmental Therapeutics, Melanoma and Immunotherapeutics Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA

Correspondence to Richard D. Carvajal, MD, Department of Developmental Therapeutics, Melanoma and Immunotherapeutics Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, 300 E 66th St., New York, NY 10065, USA Tel: + 1 646 888 4161; fax: + 646 888 4251; e-mail: [email protected] Received 29 July 2014 Accepted 26 August 2014

signaling downstream of Gαq and Gα11 will be discussed, particularly in relation to the identification of novel therapeutic targets and strategies, some of which are already in clinical trials.

GPCR and G-alpha structure and signaling GPCRs are characterized by the presence of seven αhelical transmembrane segments that span the plasma membrane with an extracellular N terminus and intracellular C terminus. Upon binding of a cognate ligand (e.g. protein, lipid, ion), a conformational change in transmembrane α-helices forms a new binding pocket for the heterotrimeric G proteins, Gα plus the G-βγ dimer, to signal downstream. Gα proteins consist of two domains, a Ras-like GTPase domain and an α-helical bundle, between which lies a pocket for GDP binding. Within the Ras-like domain lie three flexible domains known as Switch I, Switch II, and Switch III that have amino acid residues that interact differently with GDP versus GTP. These residues cause conformational changes within the Gα protein and are responsible for the intrinsic hydrolytic activity of the protein that functions as a key regulator of Gα activity. Mutations in key residues within these switch regions are responsible for much of the aberrant signaling discussed below. Within this general structure, there are at least 17 distinct types of Gα proteins that are classified into four mammalian families: Gαs, Gαi/o, Gαq/11, and Gα11/12 [4]. These families are grouped on the basis of the degree of shared amino acid sequence homology, and they have distinct tissue distribution and signaling roles. For example, GNAQ and GNA11, the genes that encode Gαq and Gα11, are 90% homologous at the amino acid level. DOI: 10.1097/CMR.0000000000000121

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Fig. 1

(a)

(b)

β γ

α

β

GDP

γ

α

GDP

α

GTP

β γ

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(d)

R183 α

R183

PLC α

GTP

Q209

GTP

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Q209

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R183 α

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Schematic of canonical GPCR and heterotrimeric G-protein signaling. (a) In the GDP-bound state, Gα is inactive and bound to Gβγ near the plasma membrane. (b) Upon ligand binding, a conformational change catalyzes GDP exchange for GTP, which alters the conformation of switch I, II, and III regions in red and dissociates Gα from Gβγ. (c) Active Gα and Gβγ signal downstream to multiple effectors. (d) The intrinsic GTPase activity of Gα is accelerated by regulator of G protein signaling (RGS) proteins, which catalyze the hydrolysis of GTP to GDP by the Q209 residue of GNAQ/GNA11. An adjacent R183 residue assists in hydrolysis without RGS binding. This returns the trimer to its membrane-bound, inactive state. GPCR, G-proteincoupled receptor; PLC, phospholipase C.

In the basal state, the heterotrimeric G proteins are bound together in the GDP state and are inactive (Fig. 1a). The Gα-βγ trimer enhances its membrane localization and inhibits the spontaneous dissociation of GDP, both of which limit their basal activity. Upon ligand binding and conformational change into the ‘active’ state, the GPCR binds the trimer and acts as a guanine nucleotide exchange factor to catalyze the dissociation of GDP from Gα for GTP, [5] which is in higher cytoplasmic concentration (Fig. 1b). Once Gα binds GTP, conformational changes in switch regions I–III

cause it to dissociate from G-βγ [6]. Both GTP-boundalpha and G-βγ can then activate downstream signaling cascades via effectors such as adenylyl cyclase, phospholipase C (PLC), diacylglycerol (DAG), Rho/Rac GTPases, and others, as will be discussed below (Fig. 1c). The intrinsic GTPase activity of the Gα subunit is accelerated by proteins from the regulators of G-protein signaling (RGS) family (Fig. 1d), which return the Gα protein to the inactive GDP-bound state, which once again favors the formation of the inactive heterotrimeric protein complex and abates downstream activation [7].

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GNAQ and GNA11 mutations Shoushtari and Carvajal 527

Table 1

Select recurrent mutations in GNAS in COSMIC, version 69

Selected tissue types Fibrous dysplasia of bone Biliary tract Intraductal papillary neoplasm Adenocarcinoma Small intestine, adenoma Pituitary adenoma Pancreas In-situ neoplasm Adenocarcinoma Pheochromocytoma Colon adenocarcinoma Overall mutation frequency

GNAS [n (%)] 126 (70) 28 5 11 228

(39) (3.0) (34) (29)

150 11 3 72 893/17 565

(27) (1.6) (9.6) (4.6) (5.1)

GNAS mutants are more often seen in benign or premalignant conditions rather than in invasive adenocarcinomas. Note the high prevalence in fibrous dysplasias of bone seen in McCune–Albright syndrome.

Additional insights into the mechanisms of GPCR signaling have underscored the complexity of the system. Ligand binding is not a clear on–off signal, as they may function as partial agonists or allosteric regulators. GPCRs are regulated independently of G proteins as well. For example, GPCRs may be activated by oligomerization and regulated by their subcellular location. It has been shown that the arrestin proteins bind to the C terminus of the GPCR and can regulate how quickly the receptors are internalized and recycled back to the plasma membrane on the basis of several factors, including GPCR phosphorylation and arrestin ubiquitination status [8]. These proteins can also serve as scaffolds to directly signal to Src and the MAP kinase pathways. Taken together, the complexity of the GPCR signaling cascade is a reflection of its critical importance in tissue homeostasis and cellular proliferation. Below, we will discuss how alterations in Gα subunits are implicated in various premalignant and neoplastic processes, including uveal melanoma.

Biology of G-alpha mutations As discussed above, Gα protein signaling is largely dependent on the phosphorylation status of the bound guanine nucleotide; GDP is inactive, and GTP is active. Mutations that abrogate the intrinsic GTPase activity of Gα proteins thus increase active downstream signaling, with the severity dependent on the particular mutation. A review of the Catalogue of Somatic Mutations in Cancer (COSMIC) [9] v69 identifies recurrent mutations in select Gα proteins across multiple neoplasms. The genes with the most available data are GNAS, GNAQ, and GNA11. GNAS

GNAS mutations are found in 5.1% of all unique specimens in COSMIC v69 (Table 1). The vast majority (∼90%) of them affect the arginine at codon 201 of exon 8 in the switch I region that hydrolyzes GTP to GDP to either cysteine (R201C) or histidine (R201H). Approximately

10% substitute the glutamine at codon 227 of exon 9, another residue required for GTPase activity, with either leucine (Q227L), arginine (Q227R), or histidine (Q227H). The prevalence of GNAS mutations is highest in benign adenomas or in-situ carcinomas of the pituitary (29%), pancreas (27%), and small intestine (34%); mutations are seen in a minority of colonic adenocarcinomas (4.6%), biliary tract carcinomas (3%), and pancreatic adenocarcinomas (1.6%). Interestingly, many of these neoplasms with wild-type GNAS harbor amplifications, suggesting an alternate method of dysregulated signaling that underscores the importance of this pathway in the pathogenesis of these neoplasms. In-vivo models suggest that GNAS mutations upregulate complementary signaling pathways that cooperate with other mutations to promote neoplasia. For example, a murine model combining the R201C mutation into APC mutant intestinal cells led to the formation of multiple adenomas that showed increased Wnt and MAP kinase signaling [10]. Studies in intraductal papillary mucinous neoplasms of the pancreas have shown that nearly twothirds have mutations in GNAS R201, suggesting that this pathway contributes to the pathogenesis of a subset of pancreatic adenocarcinomas [11,12]. GNAS mutations at R201 underlie the presentation of McCune–Albright syndrome, which is characterized by endocrinopathies such as precocious puberty, polyostotic fibrous dysplasia, and café au lait spots [13]. It is thought that the heterogeneity in the presentation of this disease may be because of postzygotic mosaicism leading to varying allelic frequencies and tissue distributions of the mutation.

GNAQ and GNA11

GNAQ and GNA11 are closely related Gα proteins with 90% amino acid sequence homology. In COSMIC v69, GNAQ and GNA11 mutations are found in 2.3–2.6% of all samples overall (Table 2). The highest prevalence of GNAQ mutations are found in uveal melanomas (33%), blue nevi (32%), and cutaneous melanomas (1.4%). GNA11 mutations are found in 39% of uveal melanomas, 3–5% of blue nevi, and 1.3% of cutaneous melanomas. Select recurrent mutations in GNAQ and GNA11 in COSMIC, version 69

Table 2

Tissue Uveal melanoma Blue nevus, ocular Cutaneous melanoma Blue nevus, cutaneous Meningeal melanocytic lesions Colon adenocarcinoma Overall mutation frequency

GNAQ [n (%)] 294 4 15 75 12 14 431/16 631

(32) (1.1) (1.4) (2.1) (41) (1.4) (2.6)

GNA11 [n (%)] 253 1 13 9 5 15 306/13 488

(39) (3) (1.3) (5.1) (19) (2.0) (2.3)

GNAQ and GNA11 mutations are most frequently found in uveal melanomas and meningeal melanocytic proliferations. Of note, these frequencies are lower than those reported by van Raamsdonk and colleagues, likely owing to the unknown status of exon 4 mutations in COSMIC.

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GNAQ and GNA11 require the glutamine at position 209 (Q209) located in the switch II region encoded by exon 5 for hydrolysis of GTP to GDP. RGS proteins that catalyze and accelerate this process also bind to Q209. Thus, mutations that encode amino acids that disrupt the polar uncharged side chain of Q209 ‘lock’ GNAQ or GNA11 in the active, GTP-bound state [14]. Q209 is a hotspot for mis-sense mutations, with the cyclic ring of proline (Q209P), hydrophobic side chain of leucine (Q209L), or, less commonly, the basic residue arginine (Q209R) resulting in activated Gα signaling. These mutations are thought to completely abrogate the GTPase function of the Gα protein and render it insensitive to RGS proteins. In COSMIC v69, ∼ 90% of all mutations in GNAQ and GNA11 occur at Q209. An additional residue that accelerates the process of GTP hydrolysis is the arginine at position 183 (R183) located in the switch I region encoded by exon 4. In contrast to Q209, this residue is not required for RGS protein binding. As a result, mutations at this site are thought to reduce, but not completely eliminate, GTPase activity. In COSMIC v69, ∼ 5% of all functional GNAQ mutations are found in R183. These alterations in GNAQ and GNA11 are also seen in non-neoplastic proliferative conditions. Recently, wholegenome sequencing was performed in multiple skin and brain samples from patients either with Sturge–Weber syndrome, a neurocutaneous disorder characterized by benign capillary proliferations, or port-wine stains, in the V1 cranial nerve distribution or with nonsyndromic portwine stains or other unrelated vascular malformations [15]. GNAQ R183Q mutations were detected, either in skin or in brain tissue, in 35 of 39 (90%) patients with Sturge–Weber syndrome or nonsyndromic port-wine stains and none of the patients with unrelated cerebrovascular malformations or presumed normal controls. The allelic frequency of the specimens ranged from 1 to 18%. No GNA11 mutations nor GNAQ Q209 mutations were seen, suggesting that GNAQ R183 mutations represent a specific driver for this condition [15]. A Dutch group has described a relatively high frequency of GNAQ Q209 mutations in the spectrum of leptomeningeal melanocytic disease. In their initial investigation of 19 lesions, 39% harbored a GNAQ Q209 lesion [16]. They then demonstrated its utility in differentiating between melanocytomas of low and intermediate grade, which harbor this mutation, from melanotic schwannomas, which have distinct genetic alterations related to Carney’s complex [17,18].

GNAQ and GNA11 mutations in uveal melanoma Van Raamsdonk and colleagues were the first to discover the role that GNAQ and GNA11 had in neoplastic development [2,3]. In these two seminal papers, they reported

that 83% of all uveal melanomas harbored mutations in these two genes, with 95% of the mutations occurring at Q209 and 5% occurring at R183. The mutations were mutually exclusive, underscoring the pathway redundancy in causing downstream growth signal activation. Approximately two-thirds of intradermal melanocytic proliferations known as blue nevi harbored these mutations, including frequent mutations in ocular nevi of Ota that occasionally lead to uveal melanoma. Prospective sequencing efforts with greater depth from ongoing clinical trials have identified even higher proportions of GNAQ or GNA11 mutations of up to 96% [19,20]. Together, these data suggest that GNAQ or GNA11 mutation represents an early event in the development of virtually all uveal melanomas and underscores the distinct genetic nature of these neoplasms compared with those of cutaneous origin. The functional relevance of the Q209 mutation, which abrogates all GTPase activity, versus R183 mutations with some residual GTPase activity, is unclear. Van Raamsdonk et al. [3] found that primary uveal melanomas developed more frequently in nude mice injected with immortalized melan-A cells transduced with GNA11 harboring mutations at Q209 versus R183, which supports the hypothesis that increased GTP-bound Gα proteins lead to greater growth signaling and tumor growth. Importantly, there was no difference in reported survival between patients with Q209 versus R183 mutations, suggesting that other factors influence prognosis. It remains striking, however, that only GNAQ R183Q and not Q209L mutations have been reported in the benign capillary proliferations of Sturge–Weber syndrome [15]. Furthermore, as discussed above, R183 mutations are relatively uncommon in neoplastic tissues compared with Q209. There is some evidence that the specific mutated gene, GNAQ or GNA11, may vary the behavior of uveal melanoma even with the same altered amino acid. In the same mouse model cited above, Van Raamsdonk and colleagues found that GNA11 Q209L mutant melanocytes formed primary tumors in 3–5 weeks, whereas GNAQ Q209L mutant cells took longer, ∼ 5–7 weeks [2,3]. Mice injected with GNA11 mutant cells invariably developed metastases, whereas GNAQ mutant cells occasionally did not. The prevalence of GNAQ versus GNA11 mutations in patient samples mirrors this trend. GNA11 mutations are present in ∼ 7% of blue nevi, 34% of primary uveal melanomas, and 61% of metastatic specimens. In contrast, GNAQ mutations are present in 56% of blue nevi, 48% of primary uveal melanomas, and 27% of metastatic specimens. Although this suggests a different propensity to develop metastasis, their analysis of 81 patients showed no significant difference in either disease-free or overall survival [3]. A smaller study of 30 metastatic lesions, however, found that GNA11 mutations were associated with worse survival from the time of diagnosis [21].

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GNAQ and GNA11 mutations Shoushtari and Carvajal 529

Further work is needed to understand the contributions of GNAQ and GNA11 to primary pathogenesis and metastatic progression of uveal melanoma. Recent work has implicated alterations in additional genes such as BAP1, EIF1AX, SF3B1, and CNKSR3 that likely cooperate with GNAQ and GNA11 alterations to influence metastatic behavior [22–25]. This exciting work lies outside the scope of this review. We will focus on recent advances in understanding how GNAQ and GNA11 signal to downstream effector pathways, as these insights are leading to novel therapeutic strategies in this disease.

Diverse downstream targets in GNAQ and GNA11 mutant uveal melanoma MAPK

The best understood signaling target in uveal melanoma is the mitogen-activated protein kinase (MAPK) pathway (Fig. 2). Shortly after the discovery of BRAF V600E mutations in a significant proportion of cutaneous melanomas, [26,27] investigations into primary and metastatic uveal melanomas corroborated the finding of high levels of phosphorylated MEK and ERK proteins in the absence of BRAF mutations [28–31]. This prompted the search for, and subsequent discovery of, GNAQ and GNA11 mutations discussed above. Mutant GNAQ and GNA11 proteins have long been known to activate downstream signaling through the activation of PLC, which cleaves phophotidylinositol diphosphate into inositol triphosphate, and DAG [32,33]. Inositol triphosphate and DAG then signal through second messengers, including calcium and protein kinase C (PKC) [34]. The phosphorylation of PKC activates the MAPK pathway through sequential phosphorylation of Raf, MEK1 and MEK2, and ERK [35]. These proteins converge on multiple transcription factors that regulate proliferation and apoptosis. Ambrosini et al. [36] were the first to report that an inhibitor of the MAPK pathway, the MEK1/2 inhibitor selumetinib, significantly reduced GNAQ mutant uveal melanoma proliferation. Consistent with the above model of signal transduction, the inhibitory effect of selumetinib was seen in GNAQ mutant, but not in wild type, cell lines [36]. This work represents one of the first to suggest rational clinical interventions. Protein kinase C

PKC refers to a family of at least seven closely related proteins, often divided into classic (α, β, γ), novel (δ, ε, η, θ), and atypical (ζ, λ/ι) isoforms. These varying isoforms are expressed in varying amounts in nearly all human tissues. A hallmark of PKC activation involves subcellular localization, with the inactive enzyme typically in a soluble, free-floating cytoplasmic state and activation requiring association with an organelle, such as the plasma membrane [37]. This has led to the development of multiple types of PKC inhibitors with variable

specificity [38]. Although all isoforms have some degree of dependency on a phospholipid scaffold for activation, the classic isoforms require both products of PLC for activation, DAG plus calcium; novel isoforms are calcium-independent; and atypical isoforms require neither calcium nor DAG [38,39]. Much of the data supporting the notion that mutant GNAQ promotes uveal melanoma proliferation through PKC activation comes from in-vitro experiments using PKC inhibitors of varying specificity. Wu and colleagues found that enzastaurin, which is most selective for classic (β, γ) and novel (δ, ε, θ) isotypes, [40] caused GNAQ mutant cell lines to undergo G1 growth arrest and apoptosis at significantly higher rates than cell lines that are wild type for GNAQ and GNA11 [41]. This effect occurred through decreased MAPK pathway activation, with decreased phosphorylated ERK and Cyclin D1. Further work from their laboratory using sotrastaurin, a more potent inhibitor of both classical and novel isotypes, [42] corroborated the role of PKC in activating ERK. Interestingly, they also identified that PKC inhibition or knockdown via shRNA led to decreased NFkB signaling [43]. This suggests an additional mechanism for proliferation via PKC activation that is independent of the MAPK pathway.

Phosphatidylinositol-3 kinase/Akt

Another well-known growth pathway upregulated in uveal melanoma tissue samples is the kinase cascade of phosphotidylinositol-3 kinase (PI3K) and Akt, which signal through the mammalian target of rapamycin (mTOR) and numerous downstream effectors (Fig. 2) [44,45]. In-vitro studies of GNAQ mutant uveal melanoma cell lines showed that PI3K-alpha and P13K-beta inhibition had only a modest effect on proliferation, suggesting that PI3K was not a sole driver of growth [46]. When a MEK inhibitor was given, however, rebound PI3K/Akt upregulation was seen, suggesting that this pathway contributes to the maintenance of growth in the setting of MAPK inhibition [46]. This has suggested rational combination drug strategies to inhibit uveal melanoma cell growth in vitro. Combined MEK and PI3K/mTOR inhibition with trametinib plus GSK2126456, respectively, led to synergistic inhibition of GNAQ and GNA11 mutant uveal melanoma cell growth and promoted apoptosis [47]. Ho et al. [48] similarly found that the combination of MEK inhibition with selumetinib plus the mTOR inhibitor AZD8055 synergistically inhibited cell growth in GNAQ mutant but not wild-type uveal melanoma cell lines. The latter study by Ho and colleagues found no apoptosis in GNAQ mutant cell lines, suggesting that PI3K inhibition may be a better therapeutic target than mTOR in combination with MAPK inhibitors.

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Fig. 2

AEB071 BYL719

GPCR Active

P

PLC Gα

GNAQ/11 Mutant

Gα

PKC NFkB

PI3K

PIP3

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Trio

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P RAF Rac

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PIP2 PTE N

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G-Actin Inactive

P YAP

Amot

P MEK

YAP

P

F-Actin Akt

GSK2141795

LATS

Active

P

YAP

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Selumetinib trametinib

ERK

Verteporfin mTOR YAP TEAD Cell growth

Simplified signaling cascade in GNAQ and GNA11 mutant uveal melanoma. Mutant GNAQ or GNA11 decreases hydrolysis of GTP to GDP, leading to increased levels of active Gα protein. Gα directly signals to phospholipase C (PLC), which eventually activates protein kinase C (PKC). PKC signals to the MAP kinase and NF kappa B pathways (rectangles, right side of figure). Gα also activates YAP (ovals, center of figure) by signaling to Trio, which triggers Rho/Rac to polymerize globular (G-) actin into filamentous (F-) actin. This favors YAP activation to the nucleus by sequestering angiomotin (Amot), which binds YAP in the cytoplasm. Phosphatidylinositol-3 kinase (PI3K) is also upregulated in UM cells through unclear mechanisms (rectangles, left side of figure). PI3K signals through Akt and mammalian target of rapamycin (mTOR) to affect cell growth. Relevant inhibitors that are currently in clinical development are noted with inhibitory arrows. GPCR, G-protein-coupled receptor; LATS, large tumor suppressor, homolog 1/2; TEAD, TEA domain transcription factor.

YAP and its upstream activators

The Hippo pathway has been implicated in control of organ size, and dysfunction in this pathway has been linked to the development of various malignancies [49]. This pathway is thought to exert its regulatory effects through the regulation of the oncoproteins YAP and TAZ [50], which activate transcription factors in the TEAD and SMAD families to promote cell growth [51]. As first described, Hippo signals through the kinases MST1/2 and Lats1/2 to phosphorylate YAP and sequester it in its inactive state in the cytoplasm [52,53]. Activation of cells with serum and other signals leads to dephosphorylation of YAP and TAZ, with subsequent translocation into the nucleus and leading to proliferation through its downstream effectors. Recently, Yu et al. [54] found that transduction of mutant Gα proteins, including GNAQ

and GNA11, were found to dephosphorylate and activate YAP and TAZ, suggesting a link between GPCR signaling and this oncogenic pathway. Yu and colleagues then investigated uveal melanoma cell lines and patient tissue samples to determine whether GNAQ and GNA11 mutations led to YAP/TAZ activation in physiologic conditions. Indeed, both in-vitro cell lines and human samples with GNAQ or GNA11 Q209 mutations were associated with YAP dephosphorylation and localization to the nucleus at higher levels versus those wild type for GNAQ and GNA11 [55]. Similarly, in-vitro BRAF mutant uveal melanoma cell lines did not display YAP activation. shRNA knockdown of YAP led to decreased tumorigenesis when GNAQ mutant cells were injected in nude mice. This strongly suggests that YAP

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GNAQ and GNA11 mutations Shoushtari and Carvajal 531

is a key contributor to the neoplastic development of GNAQ and GNA11 mutant uveal melanoma in a genotype-specific manner. A parallel investigation by Feng et al. [56] further elucidated the molecular mechanism of YAP activation in a GNAQ Q209 mutant model. Surprisingly, GNAQ signaling led to YAP activation not through the canonical PLC and Hippo pathways described above, but instead through a guanine nucleotide exchange factor, Trio, and its downstream GTPases Rho and Rac. Rho and Rac are well-known regulators of the actin cytoskeleton [57], and they found that YAP may be dynamically regulated via its association with cytoskeleton-associated proteins such as angiomotin, which help sequester it in the cytoplasm [56]. Actin polymerization mediated by Rho/Rac may then sequester angiomotin and increase the ability of YAP to localize to the nucleus. Therefore, GNAQ or GNA11 activating mutations appear to regulate YAPinduced cellular growth at least partially through cytoskeletal regulation (Fig. 2). This raises the intriguing possibility that epigenetic factors that influence the cytoskeleton of GNAQ and GNA11 mutant cells may directly influence YAP activity and neoplastic behavior; in other words, the epigenetic ‘soil’ may influence the growth of the genetic ‘seed’. Recent clinical advances and current trials MEK

The in-vitro efficacy of MEK inhibition spurred the development of a randomized, multicenter phase II study of selumetinib 75 mg twice daily versus the investigator’s choice of cytotoxic chemotherapy in patients with metastatic uveal melanoma. This trial was the first randomized trial to utilize insights from GNAQ and GNA11 mutations and randomized 101 patients to investigate a primary end point of improving progression-free survival (PFS). Patients were stratified by exon 5 mutation status, number of prior therapies, and stage. After an unplanned analysis was undertaken, a significant improvement in median PFS was seen in the selumetinib group over dacarbazine, 16.1 versus 7 weeks [hazard ratio 0.46, 95% confidence interval (CI) 0.3–0.71] [19]. Nearly half of all patients receiving selumetinib (n = 24, 49%) had some degree of tumor shrinkage, and 7/49 (14%) had objective partial responses by RECIST 1.1. Treatment-related adverse events were not uncommon, most commonly acneiform rash, creatinine kinase elevation, fatigue, and AST/ALT elevation. Grade 3 or 4 toxicities by NCTCAE, version 4.0 were seen in 37% of patients, with laboratory abnormalities such as CK elevation (16%), AST/ALT elevation (8–10%), and lymphopenia (6%) being most common. Pharmacodynamic analysis of MEK inhibition via analysis of on-treatment tumor biopsies observed significant inhibition of phosphorylated ERK (48%, P = 0.03) and Cyclin D1 (76%, P = 0.03), consistent with predictions from preclinical models.

This trial represents the first systemic treatment demonstrating clinical efficacy in a randomized manner in patients with metastatic uveal melanoma. It also raises interesting questions regarding the function of GNAQ and GNA11. Prospective genotyping was performed for Q209 mutations only, as R183 mutations had not yet been described at the time of the protocol’s inception. Consistent with Van Raamsdonk’s description, 85/101 patients (85%) had Q209 mutations, with 16 patients having the wild-type form. Retrospectively, additional tissue for R183 mutational analysis was available in only five of 16 wild-type patients, of whom three were R183 mutant. Although it had been hypothesized that selumetinib would prove to be more efficacious in Q209 mutants than wild type, the hazard ratio for PFS in mutant patients (0.55; 95% CI 0.34–0.87) was similar to that seen in the overall population. This may be partially explained by the unknown R183 mutational status in 11 patients. A closer look at mutant genotypes in the selumetinib group raises further questions. Of the 49 patients, 20 (40%) were GNAQ Q209 mutant, 21 (42%) were GNA11 Q209 mutant, three were R183 mutant (two GNAQ, one GNA11), and six were Q209 wild type, R183 unknown. Interestingly, of the 24 patients with some tumor shrinkage, 14 (58%) were GNA11 Q209 mutant, whereas only (21%) were GNAQ Q209 mutant. Among the seven patients with objective responses, five had GNA11 mutations (four Q209, one R183), whereas only two had GNAQ Q209 mutations. Although these subsets are too small to draw conclusions, the disproportionately higher rate of shrinkage in GNA11 mutants versus GNAQ mutants lends support to the notion that GNA11 mutations may rely on the MAPK pathway differently from their homologous GNAQ counterparts. On the basis of the success of this trial, a registration trial is underway (NCT01974752) that randomizes patients 3 : 1 to dacarbazine plus selumetinib plus dacarbazine alone. This trial, which is likely to complete accrual by late 2014, will test the hypothesis that MEK inhibition enhances the cytotoxic effects of chemotherapy. PKC

As discussed above, PKC may signal to both the MAPK and NFkB pathways in GNAQ and GNA11 mutant uveal melanoma. A large, multicenter phase I trial of AEB071, the inhibitor of classical and novel PKC isoforms, recently completed dose escalation [58]. A total of 118 patients were treated, and the maximum tolerated dose was 700 mg twice daily or 800 mg daily in three divided doses. Although only one patient had an objective partial response, 55/118 (47%) had stable disease, with a median PFS of 15.4 weeks (95% CI, 8.3–15.7 weeks). The most common treatment-related side effects were gastrointestinal in nature, with nausea (68%), dysgeusia (58%), constipation (48%), vomiting (42%), and diarrhea (36%) seen frequently [58].

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Combination strategies

Although MEK and PKC inhibition appears to provide clinical benefit in a substantial proportion of patients with uveal melanoma, progression of disease is inevitable. One hypothesis to delay or prevent resistance to single agents is the rational development of combination treatment strategies. Three trials combining targeted kinase inhibitors are either ongoing or nearing launch. The first combination trial to launch was a phase Ib/II study of an MEK inhibitor, MEK162, plus the PKC inhibitor AEB071, which is currently ongoing (NCT01801358). Another approach focuses on suppressing the rebound activation of Akt that is reported with prolonged MEK inhibition. To test the hypothesis that Akt inhibition may improve response rates and delay progression, a phase II trial is currently randomizing patients to therapy with either the MEK inhibitor trametinib alone or trametinib plus the Akt inhibitor GSK2141795 (NCT01979523). A third trial being planned is based on recent observations that adding PI3Kalpha inhibition to PKC inhibition provides synergistic inhibition and apoptosis in GNAQ and GNA11 mutant cell lines [59]. This phase Ib trial will utilize the PKC inhibitor sotrastaurin plus the PI3K-alpha inhibitor BYL719, which is best studied in breast cancers.

Future therapeutic strategies

Both groups that recently reported on YAP/TAZ activation in uveal melanoma have begun preclinical attempts to exploit the ‘addiction’ of GNAQ mutant uveal melanoma cells to this signaling pathway. Verteporfin, a derivative of the porphyrin family, has shown efficacy as a photosensitizer for the treatment of uveal melanoma with photodynamic therapy [60], with the purported mechanism being direct damage to the tumor blood supply. More recently, verteporfin was found to inhibit the interaction of YAP with a transcription factor, TEAD4 [61]. In-vivo studies by both the Guan and Gutkind groups utilized verteporfin to inhibit GNAQ mutant uveal melanoma growth, suggesting a novel therapeutic strategy using this family of inhibitors [55,56]. Further investigation of YAP inhibition is sorely needed to expand our therapeutic options for patients with this disease. An improved knowledge of how GNAQ and GNA11 activate downstream activators is improving our understanding on how to potentially target the myriad effects of activating Gα protein mutations. Waldo et al. [62] discovered a conserved domain in PLC-β3 that was responsible for the rapid assembly of downstream effectors and subsequent hydrolysis of GTP to GDP by GNAQ that inactivates the signal – in essence, a new, rapid on–off switch. Using this understanding of the protein structure of PLC-β3 and an improved reporter for PLC inhibitors [63], high-throughput screening is

ongoing to find molecules that can accelerate the inactivation of mutant GNAQ and GNA11 via PLC. The above approaches to treatment all hinge on the inhibition of downstream signaling events stemming from activating mutations in GNAQ and GNA11. Direct inhibition of GNAQ and GNA11 expression has been limited by its ubiquitous expression throughout tissues and the difficulty in delivering direct inhibitors, such as small hairpin RNAs, to cells to provide durable inhibition. Recently, Rothman et al. [64] reported a novel method to create peptide nucleic acid oligonucleotides, which bind to a DNA sequence of interest and prevent transcription. In-vitro data suggested that PNAs targeting BRAF V600E could inhibit transcription with resulting growth inhibition that was similar to that of vemurafenib. If this can be recapitulated in vivo with acceptable toxicity, it may represent a novel method to directly inhibit not only BRAF but potentially GNAQ, GNA11, and other oncogenic drivers of interest that are currently ‘undruggable’ by other means. Conclusion

Over the past 5 years, enormous progress has been made in understanding the pathogenesis of uveal melanoma. We now know that virtually all uveal melanomas harbor activating mutations in either GNAQ or GNA11, and many groups are improving our understanding of how these mutations promote uveal melanoma growth in a myriad of ways. These have led to rapid development of clinical trials, and MEK inhibition is a promising first step toward offering better treatments to patients with metastatic uveal melanoma. The recent discovery of YAP activation through novel signaling circuits underscores how much more remains to be learned about the behavior of these mutant proteins and offers new avenues for therapeutic intervention. These breakthroughs may eventually lead to an improved understanding of how to treat not only uveal melanoma but also numerous other malignancies with Gα mutations.

Acknowledgements All research described adheres to appropriate standards of ethics. Conflicts of interest

Alexander Shoushtari has received an honorarium for a lecture on the use of selumetinib in uveal melanoma. For the remaining author there are no conflicts of interest.

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