Protein Kinase C Inhibitor AEB071 Targets Ocular Melanoma Harboring GNAQ Mutations Via Effects on the PKC/Erk1/2 and PKC/NF-Kb Pathways

Published OnlineFirst May 31, 2012; DOI: 10.1158/1535-7163.MCT-12-0121

Molecular Cancer Therapeutic Discovery Therapeutics

Protein Kinase C Inhibitor AEB071 Targets Ocular Melanoma Harboring GNAQ Mutations via Effects on the PKC/Erk1/2 and PKC/NF-kB Pathways

Xinqi Wu1,2, Jingjing Li1,2, Meijun Zhu3, Jonathan A. Fletcher3, and F. Stephen Hodi1,2

Abstract Somatic GNAQ mutations at codon 209 have been identiﬁed in approximately 50% of uveal melanomas and have been reported to be oncogenic through activating PLCb/PKC/Erk1/2 pathways. We hypoth- esized that protein kinase C (PKC) may provide new opportunities for therapeutic targeting of uveal melanoma carrying GNAQ mutations. To test this hypothesis, uveal melanoma cells harboring wild-type or mutant GNAQ were treated with the PKC inhibitor AEB071 (sotrastaurin) or infected with lentivirus- expressing short hairpin RNAs (shRNA) targeting PKC isoforms. Notably, AEB071 at low micromolar concentrations signiﬁcantly inhibited the growth of uveal melanoma cells harboring GNAQ mutations

through induction of G1 arrest and apoptosis. However, AEB071 had little effect on uveal melanoma cells carrying wild-type GNAQ. AEB071-mediated cell inhibition in the GNAQ-mutated uveal melanoma was accompanied by inhibition of extracellular signal–regulated kinase (Erk)1/2 phosphorylation, NF-kB, decreased expression of cyclin D1, survivin, Bcl-xL, and XIAP, and increased expression of cyclin- dependent kinase inhibitor p27Kip1. AEB071 suppressed the expression of PKC a, b, d, e,andq in GNAQ-mutated uveal melanoma cells. Our ﬁndings from shRNA-mediated knockdown studies revealed that these PKC isoforms are functionally important for uveal melanoma cells harboring GNAQ mutations. Furthermore, inhibitors of Erk1/2 and NF-kB pathways reduced viability of uveal melanoma cells. Together, our ﬁndings show that AEB071 exerts antitumor action on uveal melanoma cells carrying GNAQ mutations via targeting PKC/Erk1/2 and PKC/NF-kB pathways. Targeted PKC inhibition with drugs such as AEB071 offers novel therapeutic potential for uveal melanoma harboring GNAQ mutations. Mol Cancer Ther; 11(9); 1905–14. 2012 AACR.

Introduction identiﬁed in approximately 50% of uveal melanoma and GNAQ Uveal melanoma is the most common primary intraoc- 83% blue nevi (3–5). The gene encodes for the a ular malignant tumor in adults, with an incidence of 7 subunit of q class of heterotrimeric GTP-binding proteins cases per million annually (1). Approximately half of (G proteins) that are composed of 3 subunits (Ga,Gb, patients with uveal melanoma develop metastases to the and Gg) and transduce signals from 7-transmembrane liver within 15 years of initial diagnosis. With no effective G-protein–coupled receptors (GPCR) to intracellular cas- treatment modality available the median survival time of cades (6). Activation of GPCRs results in exchange of GDP patients with uveal melanoma with metastasis is less than for GTP on the Ga subunit, resulting in the dissociation of a bg a bg 6 months (2). the GTP bound form of G from G . Both G and G The etiology of uveal melanoma has not been fully can then activate downstream cellular signaling path- understood. Mutations in the GNAQ gene have been ways. The signal is terminated when GTP is hydrolyzed to GDP by the intrinsic GTPase activity of the Ga subunit. The majority of GNAQ mutations occur at codon 209 Authors' Afﬁliations: 1Department of Medical Oncology, 2Melanoma within the GTPase catalytic domain, resulting in loss of Disease Center, Dana-Farber Cancer Institute and Harvard Medical School; and 3Department of Pathology, Brigham and Women's Hospital and the intrinsic GTPase activity and constitutively activation Harvard Medical School, Boston, Massachusetts of GNAQ. Expression of mutated GNAQ results in mela- Note: Supplementary data for this article are available at Molecular Cancer nocyte transformation and increased extracellular signal– Therapeutics Online (http://mct.aacrjournals.org/). regulated kinase (Erk)1/2 phosphorylation, indicating GNAQ Corresponding Author: F. Stephen Hodi, Dana-Farber Cancer Institute, that mutant behaves as a dominant acting onco- 450 Brookline Avenue, Boston, MA 02215. Phone: 617-632-5053; Fax: gene (4, 5). Currently, there are no available therapies 617-582-7992; E-mail: [email protected] targeting GNAQ. doi: 10.1158/1535-7163.MCT-12-0121 The protein kinase C (PKC) family is a widely expressed 2012 American Association for Cancer Research. group of serine/threonine kinases comprising multiple

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isoforms that can be divided into 3 structurally and Cell-cycle analysis functionally distinct subgroups (7, 8). These are the Cells were collected by trypsinization and fixed in cold conventional PKCs (PKCa,PKCb,andPKCg), which ethanol. After incubation with RNase A and PI, cell-cycle are activated by diacylglycerol (DAG) and phospholip- distribution was determined by flow cytometric analysis þ id and are Ca2 dependent; the novel PKCs (PKCd, (fluorescence-activated cell sorting, FACS). PKCe,PKCq,andPKCh), which are also activated þ by DAG and phospholipids, but are not Ca2 depen- Analysis of apoptosis dent; and the atypical PKCs, which do not require DAG Apoptotic cells were detected by Annexin V-fluorescein þ or Ca2 for activation. PKCs regulate key biologic isothiocyanate staining and FACS as described pre- processes including cell proliferation, apoptosis, differ- viously (17). Compensation for AEB071 autofluorescence entiation, angiogenesis, tumor development, and was conducted. chemoresistance (7, 9–15). PKCs are involved in GNAQ-mediated activation of the MAPK/Erk1/2 path- Knockdown of PKC isotypes by shRNA ways (6, 16). It has been known that GNAQ transduces The constructs (pLKO.1-puro) containing shRNA tar- signals from GPCRs to phospholipase Cb (PLCb;ref.6). get sequences for PKCa,PKCd,orGFPwereprovided PLCb enzymes catalyze the hydrolysis of phosphatidy- by Dana-Farber Cancer Institute shRNA Core Facility. linositol biphosphate to release inositol trisphosphate Lentivirus expressing PKC shRNA was produced as and DAG that function as second messengers propa- describe previously (17). Cells were infected with virus gating and amplifying the Ga-mediated signal through for 3 days and cell viability was determined using MTS activation of PKCs. Active PKCs further activate Erk1/2 assay. through the RAF/MAPK/Erk1/2 pathway (16). Using short hairpin RNA (shRNA)-mediated downregulation Immunoblotting of PKC isoforms b, e,andq we have recently shown that Preparation of whole-cell lysates and immunoblotting these isoforms are functionally important for GNAQ- have been described previously (19). Antibodies against mutated uveal melanoma cells (17). The oncogenic PKC isoforms were: PKCa (Cell Signaling #2056), PKCd properties of mutant GNAQ and the important (Cell Signaling #2058), PKCq (BD Biosciences #610090); PKC roles in GNAQ-mediated Erk1/2 activation and PKCq Thr538 (Cell Signaling #9377), PKCq/d Ser643/676 GNAQ-mutated uveal melanoma cells (4, 16) suggested (Cell Signaling #9376), and PKCe (BD Biosciences that PKC may provide new opportunities for therapeu- #610085). Antibodies against Akt, phospho-Akt, Erk1/2, tic intervention of uveal melanoma carrying GNAQ phospho-Erk1/2, cyclin D1, Bcl-xL, XIAP, survivin, mutations. To test this hypothesis, uveal melanoma cells HADC1, and glyceraldehyde-3-phosphate dehydroge- carryingwild-typeGNAQorGNAQmutatedatcodon nase (GAPDH) were purchased from Cell Signaling Tech- 209 were treated with the PKC inhibitor AEB071 (sotras- nology. Antibodies against PKCbII, p27Kip1, and RelA taurin), a PKC inhibitor that has potent activity against (p65) were purchased from Santa Cruz Biotechnology. classical and novel PKC isotypes (18). AEB071 selec- Actin antibody was purchased from Sigma-Aldrich. Pro- tively inhibited the growth of uveal melanoma cells tein signal intensity was measured using NIH ImageJ harboring GNAQ mutations by targeting PKC/Erk1/2 software and normalized to that of actin. and PKC/NF-kB pathways. Nuclear extract preparation and electrophoresis mobility shift assay Materials and Methods Nuclear extraction and NF-kB electrophoresis mobility Cell lines shift assay (EMSA) kits purchased from Signosis were The sources and GNAQ mutational status of uveal used to isolate nuclear extracts from uveal melanoma cells melanoma cell lines C918, Ocm1, Ocm3, Mel285, Mel202, and conduct EMSA. The instructions provided by the 92.1, and Omm1.3 have been described previously (19). manufacturer were followed. Uveal melanoma cells were cultured in RPMI-1640 containing 10% FBS, 50 mg/mL penicillin, and 100 mg/mL Measurement of interleukin-6 in cell culture medium streptomycin at 37 C and 5% CO2. These cell lines were Cells were treated with dimethyl sulfoxide (DMSO) or 5 recently authenticated by short tandem repeat PCR anal- mmol/L AEB071 for 72 hours in 6-well plates. Medium ysis at Biosynthesis Inc. Human epidermal melanocytes was collected and centrifuged to remove cells. Interleukin were purchased from Lifeline Cell Technology and grown (IL)-6 in the supernatant was determined using the in the medium provided by the company. Human IL-6 Quantikine ELISA Kit purchased from R&D Systems. Viability assay Cells were seeded in 96-well plates at 2 103 cells per Statistical analysis well and incubated over night followed by treatment with Data are presented as mean SD. Differences between AEB071 (provided by Novartis) for 3 days. Cell viability treated and control groups was analyzed using Student t was measured as previously described (17). test and considered significant with P 0.05.

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Protein Kinase C Inhibition in Uveal Melanoma

Results AEB071 induces G1 arrest in GNAQ-mutated uveal The PKC inhibitor AEB071 selectively inhibits melanoma cells growth of uveal melanoma cells harboring GNAQ To better understand growth inhibitory effect of mutations AEB071 on GNAQ-mutated cells, we investigated wheth- We first evaluated the effect of AEB071 (Fig. 1A) er cell-cycle progression was altered by drug exposure. on viability of a panel of 7 uveal melanoma cell lines AEB071 markedly increased the G1 phase population (Table 1). Sequencing analysis confirmed that GNAQ is whereas decreasing the S-phase population in uveal mel- wild-type in cell lines C918, Ocm1, Ocm3, and Mel285, anoma cells harboring GNAQ mutations (Fig. 2A and B). whereas codon 209 of GNAQ is mutated from CAA There was no significant change in the cell-cycle pattern (glutamine) to CTA (leucine) in cell lines Mel202 and for cell lines carrying wild-type GNAQ (C918, Mel285, 92.1 and to CCA (proline) in cell line Omm1.3. Cell lines and Ocm3). A decrease in the S-phase population with a Ocm1 and Ocm3 have BRAF V600E mutation (19, 20). concomitant increase in the G2–M phase population was AEB071 at low micromolar concentrations significantly observed in Ocm1 cells. In agreement with this G1 arrest, decreased viability of all 3 GNAQ-mutated cell lines AEB071 also significantly increased the accumulation of Kip1 (Fig. 1B). The IC50 value of AEB071 was approximately p27 , while decreasing the expression of cylin D1 in all 3 0.8, 3, and 4 mmol/L for 92.1, Omm1.3, and Mel202 GNAQ-mutated cell lines tested (Fig. 2C). In comparison, cells respectively. AEB071 had little effect on viability the expression of p27Kip1 and cyclin D1 was not signifi- of 3 GNAQ wild-type cells up to 10 mmol/L (Fig. 1B cantly altered by AEB071 in GNAQ wild-type cells. These and Supplementary Fig. S1). In addition, the viability of findings suggest that AEB071 selectively induced G1 human epidermal melanocytes was not reduced by arrest in GNAQ-mutated cells through altering the AEB071 (Fig. 1B) and may have been modestly expression of regulators critical for the G1 to S transition. increased by the inhibitor. Along with decreased viability, microscopic examination found morphologic AEB071 induces apoptosis in GNAQ-mutated uveal alterations in AEB071-treated cells harboring GNAQ melanoma cells mutations but not wild-type GNAQ or normal melano- We next examined whether AEB071 promoted cytes (Fig. 1C). Treated Mel202, 92.1, and Omm1.3 cells apoptosis in uveal melanoma cells. Treatment with 2 and lost spindle shape and became flattened with increased 5 mmol/L AEB071 for 72 hours significantly increased size or round floating dead cells with condensed Annexin V–positive (apoptotic) populations in GNAQ- cytoplasm. mutated 92.1 and Omm1.3 cells (Fig. 3A). Further

A C H N OO Figure 1. Effect of AEB071 on GNAQ wild-type and mutated uveal melanoma cells. A, molecular AEB (μmol/L) structure of AEB071 (sotrastaurin; N 0 25 N N adapted from ref. 43). B, AEB071 H C918 selectively reduced viability of uveal N melanoma cells harboring GNAQ mutations. Cells were treated with Ocm1 N varying amount of AEB071 for 72 WT hours. Data are presented as Ocm3 mean SD of 4 or 5 independent AEB071 experiments. NM, normal B Mel285 melanocytes; WT, GNAQ wild-type; 160 MT, GNAQ mutation. C, AEB071 140 Mel202 caused morphologic alterations in C918 GNAQ-mutated cells. After AEB071 120 Ocm1 treatment for 3 days, Mel202, 92.1, 100 Ocm3 92.1 MT and Omm1.3 cells lost spindle shape WT Mel285 ﬂ 80 and became attened with increased Mel202 Omm1.3 size or round ﬂoating dead cells with 60 92.1 condensed cytoplasm. The Omm1.3 Viability (%) Viability 40 morphology of GNAQ wild-type MT NM NM uveal melanoma cells was not altered 20 by AEB071. 0 0123456 AEB (μmol/L)

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Table 1. Uveal melanoma cell lines used and status, we examined inhibition of PKC isoforms by AEB071 in uveal melanoma cells. Immunoblotting their GNAQ mutational status showed that treatment with AEB071 for 6 hours in the absence of serum resulted in decreased PKCd/qSer643/676 GNAQ Codon 209 Cell lines phosphorylation in Mel202, 92.1, and C918 cells and Thr538 Wild-type CAA (Q) C918, Mel285, PKCq phosphorylation in Mel202 cells (Fig. 4A). It Ocm1, Ocm3 has been reported that expression of PKC isoforms can be Mutation CTA (L) Mel202, 92.1 downregulated by PKC inhibition (21). We thus examined CCA (P) Omm1.3 the expression of PKC isoforms after prolonged AEB071 treatment (5 mmol/L for 24 hours) in the presence of 10% FBS. Uveal melanoma cell lines showed varying reduction demonstration of apoptosis was observed with caspase-3 in PKC expression after AEB071 treatment (Fig. 4B). PKCa cleavage in treated cells (Fig. 3B). For Mel202 cells, only a was reduced in some wild-type (C918 and Ocm1) and in minimal increase in the Annexin V–positive cell popula- GNAQ-mutated (92.1 and Omm1.3) cells, whereas PKCb tion was observed after AEB071 treatment (Fig. 3A), but was reduced only in mutated cells (Mel202 and 92.1). signiﬁcant caspase-3 cleavage was induced (Fig. 3B). In PKCq was decreased in all 3 GNAQ mutated but only in contrast, AEB071 did not increase Annexin V–positive cell one wild-type (Ocm3) cells. PKCe was downregulated in populations or caspase-3 cleavage in uveal melanoma all 3 GNAQ wild-type cell lines and 2 mutated cell lines cells harboring wild-type GNAQ (Fig. 3A and B). AEB071 (Mel202 and 92.1). These ﬁndings suggest that AEB071 further inhibited the expression of the antiapoptotic pro- may have greater overall inhibitory effect on multiple teins survivin, Bcl-xL and XIAP, in a dose-dependent PKC isoforms in uveal melanoma cells with mutated manner in Mel202, 92.1, and Omm1.3 cells (Fig. 3C). GNAQ: PKCa, b, d, e, and/or q expression was sup- Under the same conditions, survivin, Bcl-xL, and XIAP pressed by AEB071 in GNAQ-mutated cells whereas were not affected or were modestly increased in GNAQ PKCa, and PKCd and PKCe expression was affected in wild-type uveal melanoma cells (Fig. 3C). These results GNAQ wild-type cells. indicate that AEB071 selectively induced apoptosis in uveal melanoma cells harboring GNAQ mutations. PKC isoforms are functionally important for uveal melanoma cells harboring GNAQ mutations AEB071 inhibits expression and phosphorylation of As the expression of PKCa and d was suppressed by PKC isoforms in uveal melanoma cells AEB071 in both GNAQ wild-type and mutated uveal To better understand differential responses of uveal melanoma cells, we next investigated whether these melanoma cells to AEB071 based on GNAQ mutational isoforms were of functional importance for uveal

A WT MT C918 Ocm1 Ocm3 Mel285 Mel202 92.1 Omm1.3

DMSO Figure 2. AEB071 induces G1 arrest in uveal melanoma cells harboring GNAQ mutations. A, uveal AEB melanoma cells were treated with DMSO or 5 mmol/L AEB071 for 24 hours and subjected to cell-cycle B analysis. B, bar graphs of 100% percentages of G1, S, and G2–M 80% populations. C, AEB071 selectively G –M 2 increased p27 and decreased 60% S cyclin D1 expression in GNAQ 40% G 1 mutant uveal melanoma cells. Cells 20% were treated with 0, 2, or 5 mmol/L 0% DMSO AEB DMSO AEB DMSO AEB DMSO AEB DMSO AEB DMSO AEB DMSO AEB AEB071 for 72 hours and analyzed by immunoblot. WT, GNAQ wild- C type; MT, GNAQ mutation. WT MT C918 Ocm1 Ocm3 Mel285 Mel20292.1 Omm1.3 Cyclin D1 p27Kip1 GAPDH AEB (μmol/L) 025 025 025 025 025 025 025

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A WT MT C918 Ocm1 Ocm3 Mel285 Mel202 92.1 Omm1.3

DMSO Figure 3. AEB071 induced apoptosis in uveal melanoma cells harboring GNAQ mutations. A, cells were treated with DMSO or 5 mmol/L AEB AEB071 for 72 hours. Apoptotic cells were detected using Annexin V-FITC staining and FACS analysis. B, cells B were treated with DMSO or 5 mmol/L WT MT AEB071 for 72 hours and subjected C918 Ocm1 Ocm3 Mel285 Mel202 92.1 Omm1.3 to immunoblot analysis of cleaved Cleaved caspase-3 caspase-3. Increased caspase-3 cleavage was observed in GNAQ- Actin mutated uveal melanoma cells. C, AEB (μmol/L) 0 5 0 5 0 5 0 5 0 5 0 5 0 5 AEB071 decreased the expression of C antiapoptotic proteins Bcl-xL, XIAP, WT MT and survivin in GNAQ-mutated cells. C918 Ocm1 Ocm3 Mel285 Mel20292.1 Omm1.3 WT, GNAQ wild-type; MT, GNAQ BcL-xL mutation. XIAP GAPDH Survivn GAPDH AEB (μmol/L) 025 025025 025 025025025 melanoma cells by shRNA-mediated knockdown to better mutant cells whereas it had minimal effect on Akt phos- understand the mechanisms for the differential AEB071 phorylation in both GNAQ wild-type and mutated cells response in GNAQ wild-type and mutated cells. PKCa (Fig. 5). Total Akt and Erk1/2 levels were not significantly and PKCd expression was significantly downregulated altered by AEB071 in any of the cell lines examined. These by their shRNA in C918 and Mel202 cells (Fig. 4C). Inter- findings show AEB071-induced selective inhibition of the estingly, knockdown of PKCa or PKCd significantly PKC/MAPK pathway in uveal melanoma cells carrying decreased viability of Mel202 and Omm1.3 cells, but failed GNAQ mutations. We have found that Erk1/2 inhibition to significantly decrease the viability of C918 cells decreased uveal melanoma cell viability (19). Therefore, (Fig. 4D), indicating that PKCa and PKCd are functionally AEB071 may exert its antiproliferative effects in part important in uveal melanoma cells harboring GNAQ through suppression of Erk1/2 activation in GNAQ- mutations. We have previously found that PKC isoforms mutated uveal melanoma cells. b, e, and q are functionally critical for GNAQ-mutated uveal melanomacells (17). These findings togethersuggest AEB071 selectively inhibits NF-kB activity in GNAQ- that AEB071 suppressed growth of GNAQ-mutated uveal mutated uveal melanoma cells melanoma cells via inhibition of multiple PKC isoforms. PKC isoforms are involved in GPCR-mediated NF-kB activation (27–30). In most cells, NF-kB is present as a AEB071 selectively decreases Erk1/2 latent, inactive, IkB-bound complex in the cytoplasm. On phosphorylation in uveal melanoma cells activation, IkB is phosphorylated and targeted to degra- harboring GNAQ mutations dation. The released NF-kB translocates into the nucleus To further define the molecular mechanisms underly- where it drives the expression of its target genes (29). It is ing the antiproliferative action of AEB071, we next well known that aberrant NF-kB activity promotes tumor- assessed downstream effectors of PKC-mediated path- igenesis and metastasis (31). We next examined whether ways that were potentially affected by AEB071. It has been AEB071 altered NF-kB activity in uveal melanoma cells. In previously reported that PKC can activate the phosphoi- the absence of AEB071, nuclear p65 (RelA) levels are nositide 3-kinase (PI3K)/Akt and mitogen-activated pro- more or less similar among wild-type and mutant cell tein kinase (MAPK) pathways as well as GSK3bSer9 phos- lines (Fig. 6A). In contrast, in the presence of AEB071, phorylation. The PKC inhibitor enzastaurin inhibits Akt nuclear p65 levels were significantly decreased in GNAQ- and GSK3bSer9 phosphorylation in some types of cancer mutated uveal melanoma cells whereas they were mini- cells (21–26). Similarly, AEB071 decreased GSK3bSer9 mally altered in wild-type cells (Fig. 6A). Consistent with phsophorylation in all uveal melanoma cell lines studied this, reduced DNA binding activity of NF-kB was here (Supplementary Fig. S2). However, AEB071 only detected only in the nuclear extracts of GNAQ-mutated significantly inhibited Erk1/2 phosphorylation in GNAQ cells exposed to AEB071 (Fig. 6B). In agreement with

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AB WT MT

WT MT C918 Ocm1 Ocm3 Mel202 92.1 Omm1.3 AEB PKCα PKCδ

C918 Ocm1 Ocm3 Mel202 92.1 Omm1.3 Actin AEB PKCβ Ser643/676 pPKCδ/θ PKCε pPKCθThr538 PKCθ Actin Actin C918 Ocm1 Ocm3 Mel20292.1 Omm1.3 PKCδ 0.4 ± 0.1 0.5 ± 0.1 0.7 ± 0.0 0.4 ± 0.1 0.6 ± 0.1 0.4 ± 0.1 PKCε 0.6 ± 0.1 0.7 ± 0.1 0.6 ± 0.1 0.7 ± 0.1 0.8 ± 0.0 0.8 ± 0.3 PKCθ C 0.9 ± 0.2 0.9 ± 0.1 0.7 ± 0.1 0.7 ± 0.0 0.7 ± 0.1 0.7 ± 0.1 C918 Mel202 D shRNA: GFP PKCα PKCδ 120 PKCα WT MT 100 # PKCδ * 80 # Actin 60 # # GFP PKCα PKCδ GFP PKCα PKCδ 40 shRNA Viability (%) 20 0 C918 Mel202 Omm1.3

Figure 4. PKCa and PKCd are functionally important for uveal melanoma cells harboring GNAQ mutations. A, effect of AEB071 on phosphorylation of PKC isoforms in uveal melanoma cells. Cells were treated with 5 mmol/L AEB071 in the absence of serum and analyzed for PKC phosphorylation. B, AEB071 inhibited PKC expression in uveal melanoma cells. Cells were treated with 5 mmol/L AEB071 for 24 hours in the presence of 10% FBS and subjected to immunoblot analysis. PKCd, e, and q levels in AEB-treated cells relative to control (DMSO-treated) cells are also shown. Relative levels (fold changes) are presented as mean SD of 3 to 4 experiments. Except PKCq in C918 and Ocm1 cells and PKCe in Omm1.3 cells the difference between AEB-treated and control cells was statistically signiﬁcant (Student t test, P < 0.05 or 0.01). C, PKCa and PKCd were downregulated by shRNA-mediated knockdown. C918 and Mel202 cells were infected with lentivirus expressing shRNA for PKCa or PKCd for 4 days and protein levels of these isoforms were determined by immunoblot analyses. D, knockdown of PKCa and PKCd signiﬁcantly reduced viability of Mel202 and Omm1.3 cells. Cell viability was determined 4 days after infection with lentivirus expressing PKC shRNA using MTS assay. Results are presented as mean SD of percent viability from 3 independent experiments. #, P < 0.01 versus cells expressing control GFP shRNA.

decreased NF-kB activity, elevated IkBa levels were NF-kB target genes including survivin, Bcl-xL, XIAP, and detected in AEB071-treated GNAQ-mutated cells (Fig. cyclin D1, by AEB071 in GNAQ-mutated cells (Figs. 2C 6C). Besides, the secretion of IL-6, one of the target genes and 3C). of NF-kB, was substantially inhibited by AEB071 in GNAQ-mutated Omm1.3 cells but not GNAQ wild-type NF-kB activity is functionally important for uveal C918 cells (Fig. 6D). These ﬁndings together indicate that melanoma cells AEB071 selectively inhibited NF-kB activation in uveal To determine whether NF-kB activity is functionally melanoma cells harboring GNAQ mutations. This notion important for uveal melanoma cells, both GNAQ wild- is also in agreement with the selective downregulation of type and mutated uveal melanoma cells were treated with an inhibitor of IkB kinase (IKK)1/2 inhibitor that pos- sesses potent NF-kB inhibition activity (32). This treat- WT MT ment resulted in the dramatic decrease in viability of both C918 Ocm1 Ocm3 Mel285 Mel20292.1 Omm1.3 GNAQ wild-type and mutant uveal melanoma cell lines p-Erk1/2 Erk1/2 (Supplementary Fig. S3). These ﬁndings suggest that NF- p-Akt kB activity is critically important for uveal melanoma cells Akt and its suppression contributes to AEB071-induced GAPDH AEB (μmol/L) 025025025025 025025025 growth inhibition of uveal melanoma cells harboring GNAQ mutations.

Figure 5. AEB071 selectively inhibited Erk1/2 phosphorylation in GNAQ- mutated uveal melanoma cells. Cells were treated with AEB071 for 72 Discussion hours and analyzed for the expression of Erk1/2 and Akt and their phosphorylation by immunoblot analyses. WT, GNAQ wild-type; MT, There are currently no drugs available for targeting the GNAQ mutation. oncogenic GNAQ mutations that occur frequently in

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A C WT MT WT MT C918 Ocm1 Ocm3 Mel202 92.1 Omm1.3 C918 Ocm1 Ocm3 Mel202 92.1 Omm1.3 AEB AEB p65 lκBα

HDAC1 Actin B D 200 DMSO AEB WT MT 160

120

80 C918 Ocm1 Ocm3 Mel202 92.1 Omm1.3 AEB 40

NF-κB/DNA Secreted IL-6 (%) 0 C918 Omm1.3 (WT) (MT)

Figure 6. AEB071 selectively inhibited NF-kB activation in uveal melanoma cells harboring GNAQ mutations. A, immunoblot analysis of nuclear RelA (p65) levels of uveal melanoma cells treated with 0 or 5 mmol/L AEB071 for 40 hours. HDAC1 (histone deacetylase 1) was used as loading control for nuclear extracts (56). B, EMSA analysis of NF-kB DNA-binding activity in the same nuclear extracts used in A. NF-kB/DNA complexes were determined as described in Materials and Methods. C, AEB071 increased IkBa protein levels in uveal melanoma cells with mutated GNAQ. Cells were treated with DMSO or 5 mmol/L AEB071 for 20 hours and subjected to immunoblot analysis of IkBa. D, AEB071 reduced IL-6 secretion from Omm1.3 cells. Cells were treated with DMSO or 5 mmol/L AEB071 for 72 hours. Medium was collected for ELISA analysis of IL-6. Data are presented as mean SD of percent secretion of 3 independent experiments. , P < 0.05. primary and metastatic uveal melanoma. In the present The PI3K/Akt and MAPK pathways are frequently study, we describe the ﬁrst small-molecule inhibitor that activated in malignant tumors and are critical for cancer selectively exhibits antiproliferative activity of uveal mel- cell survival and proliferation (35–37). Erk1/2 activation anoma cells harboring GNAQ mutations: the novel PKC is common in uveal melanoma and has been reported inhibitor AEB071 reduced viability of GNAQ-mutated to play a crucial role in uveal melanoma development uveal melanoma cell lines, but had little effect on those (38, 39). AEB071 inhibited Erk1/2 phosphorylation in carrying wild-type GNAQ. AEB071-induced growth inhi- GNAQ mutated but not GNAQ wild-type uveal melano- bition is associated with reduced expression of PKC iso- ma cells, and had minimal impact on Akt phophorylation. forms a, b, d, e, and/or q, accompanied by inhibition of Similarly, we have recently found that PKC inhibitor Erk1/2 phosphorylation, and NF-kB activation. We have enzastaurin inhibits phosphorylation of Erk1/2 but not previously shown that PKCq, PKCb, and PKCe are func- Akt in GNAQ-mutated cell lines (17), although enzas- tionally important for GNAQ-mutated uveal melanoma taurin has been reported to inhibit Akt phosphorylation cells and that inhibition of Erk1/2 by MAP–ERK kinase but not Erk1/2 phosphorylation in other types of cancer (MEK)1/2 inhibitors reduced uveal melanoma cell via- cells (24, 26, 40). The inhibition of Erk1/2 phosphorylation bility (17). Here, we show that PKCa, PKCd, and NF-kB is therefore likely a common mechanism for the antipro- are also functionally important for GNAQ-mutated uveal liferative action of PKC inhibitors in GNAQ-mutated melanoma cells. Together, our ﬁndings suggest that uveal melanoma cells. Further studies are needed to AEB071 may selectively exert antiproliferative activity on identify the PKC isoform(s) that are most crucial for GNAQ-mutated uveal melanoma cells via targeting the Erk1/2 phosphorylation in GNAQ mutant uveal melano- PKC/Erk1/2 and PKC/NF-kB pathways. ma, and whose inhibition by AEB071 leads to decreased AEB071-induced growth suppression of GNAQ mutant Erk1/2 phosphorylation. PKCbII and PKCq are among cells is associated with pronounced G1 arrest and induc- the candidates, because activation of these isoforms tion of apoptosis. The molecular mechanisms for AEB071- triggers several signaling pathways including MAPK induced G1 arrest involve altered expression of positive (9, 13, 17, 41), and siRNA downregulation of PKCbII and negative regulators of transition through G1 phase of decreased Erk1/2 phosphorylation in metastatic hepato- cell cycle, including cyclin D1 and p27Kip1 (33, 34). cellular carcinoma cells (42). AEB071-induced apoptosis is associated with decreased In addition to the PKC/Erk1/2 pathway, activation of expression of antiapoptotic proteins, yet the underlying many GPCRs can trigger the PKC/IKK/NF-kB pathway molecular mechanisms remain to be revealed. through G proteins, including GNAQ (27–30, 43).

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Overexpression of mutant GNAQ(Q209L) leads to con- diverse GPCRs were found in number of primary and stitutive activation of NF-kB, which is mediated by PKCd metastatic cancers including melanoma, breast, prostate, and to a lesser extent PKCa and PKCe in human umbilical non–small cell lung cancer, gastric tumors, head and neck vein endothelial cell (HUVEC; refs. 27, 28). It is not known squamous cell carcinoma, and diffused large B-cell lym- whether the same PKC isoforms have comparable roles in phoma (52, 53). GPCRs can also influence cancer progres- uveal melanoma cells. Importantly, we show that AEB071 sion through cross-talk with growth factor receptors such selectively repressed NF-kB activation in uveal melanoma as EGFR and insulin-like growth factor-1R, G-proteins cells harboring GNAQ mutations and this is accompanied (such as Gaq/11 and Ga12/13), chemokines, Hedgehog by downregulation of multiple PKC isoforms, in partic- and WNT signaling pathways, regulation of the apoptotic ular PKCa, PKCd, and PKCe. The downregulation of response and viral factors. Various GPCRs can be these PKC isoforms may contribute to AEB071 suppres- involved in cancer through activating NF-kB (50). As sion of NF-kB activity. Comparison studies using an IKK more data linking GPCRs to cancer emerge, these recep- inhibitor corroborate that the NF-kB pathway has critical tors are attractive potential targets for tumor therapy. In functional roles in uveal melanoma cells. NF-kB inhibition particular, the GPCR/ERK and GPCR/NF-kB pathways might therefore be another mechanism for the antiproli- could be valuable targets for innovative anticancer drug ferative action of AEB071 on uveal melanoma cells har- discovery. boring GNAQ mutations. The association between PKC We show that PKC inhibitor AEB071 (sotrastaurin) at inhibition and decreased NF-kB activity in GNAQ-mutat- low micromolar concentrations exerts significant antipro- ed uveal melanoma cells suggests that these cells might liferative effect on GNAQ-mutated uveal melanoma cells rely on GNAQ/PKC/NF-kB pathways for NF-kB activa- through targeting the PKC/MAPK and PKC/NF-kB tion. Other pathways presumably regulate NF-kB activa- pathways. Our findings support PKCs as important tar- tion in GNAQ wild-type cells. For example, Ocm1 and gets for therapeutic intervention of uveal melanoma har- Ocm3 cells have been shown to carry the common V600E boring GNAQ mutations. Clinical studies have shown BRAF mutation that constitutively activates the MAPK that a blood concentration of 4 mmol/L was achieved and NF-kB pathways (19, 20, 44–46). High c-Met expres- within 5 hours after an oral dose of 500 mg AEB071 sion has been found in C918 and Mel285 cells (47) and it (18). AEB071 could thus be of therapeutic potential for has been reported that NF-kB can be activated by hepa- uveal melanoma with GNAQ mutations. A clinical trial tocyte growth factor (HGF)/c-Met signaling (48, 49). with AEB071 in patients with uveal melanoma is currently Our data indicate that multiple PKC isoforms including enrolling patients. AEB071 has primarily been studied as a, b, d, e, and q are suppressed by AEB071 in GNAQ- an anti-inflammatory agent for the treatment of diseases mutated uveal melanoma cells whereas only PKCa and such as psoriasis and in preventing solid organ rejection PKCd were affected in GNAQ wild-type cells. The find- following transplant (54, 55). How the immunosuppres- ings from shRNA knockdown studies confirmed the sive effects of this drug may influence the clinical efficacy functional importance of PKCa and PKCd in GNAQ- will have to be determined, and monitoring of changes in mutated uveal melanoma cells. Knockdown of these 2 antitumor immune responses as a function of treatment PKC isoforms had no (PKCa) or little (PKCd) affect on should be helpful to discern such changes. Given that viability of GNAQ wild-type cells, suggesting that these 2 preclinical models for uveal melanoma are difficult and PKC isoforms are less important in GNAQ wild-type cells mostly comprise xenograft studies, much of these ques- than in GNAQ-mutated cells. Similarly, our previous tions will likely be answered by clinical investigation. shRNA-mediated knockdown studies have found that There may be future opportunities to combine AEB071 PKCb, PKCe, and PKCq are functionally more important or other PKC inhibitors with other rational small-mole- for GNAQ-mutated than wild-type uveal melanoma cells cule inhibitors (e.g., MEK or PI3K) or immune therapy (17). Together, these findings suggest that GNAQ-mutat- such as ipilimumab to improve the efficacy and durability ed uveal melanoma cells are more dependent on these of any clinical activity. It would be also of great interest to PKC isoforms than GNAQ wild-type cells and that investigate antitumor effects of other specific PKC inhi- AEB071 exerts antiproliferative action on GNAQ-mutated bitors in melanoma and other cancers with GPCR muta- cells via suppression of these PKC isoforms. These find- tions that may activate MAPK/ERK and/or NF-kB ings also provide a plausible explanation for the differ- pathways. ential response/sensitivity of GNAQ wild-type and mutated uveal melanoma cells to AEB071. Disclosure of Potential Conflicts of Interest GPCRs have a pivotal role in many physiologic func- F.S. Hodi has served as a nonpaid consultant to Novartis and received tions and in multiple diseases, including tumorigenesis clinical trial support from Novartis. No potential conflicts of interest were and metastasis of cancers (50). Frequent somatic muta- disclosed by the other authors. tions in GPCRs have been found in melanoma and mutations in GRM3, which is a glutamate receptor and a Authors' Contributions member of the metabolic GPCRs, activate the MAPK/ Conception and design: X. Wu, F.S. Hodi Development of methodology: X. Wu, J.A. Fletcher, F.S. Hodi ERK pathway, and promote growth and migration of Acquisition of data (provided animals, acquired and managed patients, melanoma cells (51). Furthermore, overexpression of provided facilities, etc.): X. Wu, J. Li, M. Zhu, J.A. Fletcher, F.S. Hodi

1912 Mol Cancer Ther; 11(9) September 2012 Molecular Cancer Therapeutics

Protein Kinase C Inhibition in Uveal Melanoma

Analysis and interpretation of data (e.g., statistical analysis, biostatis- and Emily Mac Naught Fund for Melanoma Research (F.S. Hodi) at tics, computational analysis): X. Wu, M. Zhu, J.A. Fletcher, F.S. Hodi Dana-Farber Cancer Institute, NIH 1P50CA127003-04 (J.A. Fletcher), Writing, review, and/or revision of the manuscript: X. Wu, M. Zhu, J.A. and the Virginia and Daniel K. Ludwig Trust for Cancer Research (J.A. Fletcher, F.S. Hodi Fletcher). Administrative, technical, or material support (i.e., reporting or orga- The costs of publication of this article were defrayed in part by the nizing data, constructing databases): J.A. Fletcher, F.S. Hodi payment of page charges. This article must therefore be hereby marked Study supervision: F.S. Hodi advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant Support This work was supported in part by Sharon Crowley Martin Received February 7, 2012; revised April 18, 2012; accepted May 7, 2012; Memorial Fund for Melanoma Research (F.S. Hodi), the Malcolm published OnlineFirst May 31, 2012.

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1914 Mol Cancer Ther; 11(9) September 2012 Molecular Cancer Therapeutics

Protein Kinase C Inhibitor AEB071 Targets Ocular Melanoma Harboring GNAQ Mutations via Effects on the PKC/Erk1/2 and PKC/NF-κB Pathways

Xinqi Wu, Jingjing Li, Meijun Zhu, et al.

Mol Cancer Ther 2012;11:1905-1914. Published OnlineFirst May 31, 2012.

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