Inhibition of Mutant GNAQ Signaling in Uveal Melanoma Induces AMPK-Dependent Autophagic Cell Death

Published OnlineFirst February 26, 2013; DOI: 10.1158/1535-7163.MCT-12-1020

Molecular Cancer Cancer Therapeutics Insights Therapeutics

Grazia Ambrosini1, Elgilda Musi1, Alan L. Ho1, Elisa de Stanchina2, and Gary K. Schwartz1

Abstract Oncogenic mutations in GNAQ and GNA11 genes are found in 80% of uveal melanoma. These mutations result in the activation of the RAF/MEK signaling pathway culminating in the stimulation of ERK1/2 mitogen- activated protein kinases. In this study, using a siRNA strategy, we show that mutant GNAQ signals to both MEK and AKT, and that combined inhibition of these pathways with the MEK inhibitor selumetinib (AZD6244) and the AKT inhibitor MK2206 induced a synergistic decrease in cell viability. This effect was genotype dependent as autophagic markers like beclin1 and LC3 were induced in GNAQ-mutant cells, whereas apoptosis was the mechanism of cell death of BRAF-mutant cells, and cells without either mutation underwent cell-cycle arrest. The inhibition of MEK/ATK pathways induced activation of AMP-activated protein kinase (AMPK) in the GNAQ-mutant cells. The downregulation of AMPK by siRNA or its inhibition with compound C did not rescue the cells from autophagy, rather they died by apoptosis, deﬁning AMPK as a key regulator of mutant GNAQ signaling and a switch between autophagy and apoptosis. Furthermore, this combination treatment was effective in inhibiting tumor growth in xenograft mouse models. These ﬁndings suggest that inhibition of MEK and AKT may represent a promising approach for targeted therapy of patients with uveal melanoma. Mol Cancer Ther; 12(5); 768–76. 2013 AACR.

a a a a a Introduction include G q (GNAQ), G 11, G s,G i/o and G 12/13. Mutant GNAQ and GNA11 are constitutive active and They transduce external signals from G protein-coupled have oncogenic properties. Activating mutations at codon seven transmembrane domain receptors (GPCR) to intracellular signaling pathways (12). GNAQ stimulation leads 209, and less frequently at codon 183, have been recently b described in uveal melanoma (1–3), whereas BRAF muta- to activation of phospholipase C- , which, through the tions are relatively rare (4, 5). Uveal melanoma represents production of inositol trisphosphate and diacylglycerol, the most common intraocular malignancy. Approximate- elevates intracellular calcium levels and protein kinase C ly 95% of ocular melanomas are posterior uveal (ciliary (PKC) activity. It has been reported that GNAQ binds and body and choroid) in origin (6), and accounts for a sig- signals to phosphoinositide-3 kinase (PI3K; refs. 13–15), nificant rate of deaths. The development of metastasis and that targeting PI3K/AKT pathway while inhibiting occurs in 50% of patients with uveal melanomas within 15 MEK/ERK is an effective approach to reduce the prolif- years of initial diagnosis, even after treatment and remov- eration of uveal melanoma (8, 16, 17). However, the al of the primary tumor (7). We and other groups have mechanisms involved in these processes are still unclear. reported that GNAQ-mutant uveal melanoma cells are Autophagy is a regulated process of degradation and relatively less sensitive to MEK inhibition than BRAF- recycling of cellular constituents, participating in the mutant uveal or cutaneus melanoma (8, 9). Furthermore, bioenergetic management of starvation (18). During mechanism of resistance with MEK inhibitors have been autophagy, parts of the cytoplasm are sequestered into described in some cancers (10, 11) and more effective double-membraned vesicles, called autophagosomes, therapies are needed for the treatment of uveal melanoma. which then fuse with lysosomes and degrading their G alpha protein subunits, classified into subfamilies by content. Autophagy is considered cytoprotective, as it can their sequence homology and downstream signals, rescue cells under conditions of starvation (19), whereas recent studies confirmed that autophagy also plays a role in cell death (20–22). Here, we showed that mutant GNAQ signals to both ERK and AKT pathways, and that dual Authors' Affiliations: 1Laboratory of New Drug Development, Department of Medicine and 2Department of Molecular Pharmacology and Chemistry, inhibition of MEK and AKT induced synergistic inhibition Memorial Sloan-Kettering Cancer Center, New York, New York of cell viability of GNAQ-mutant uveal melanoma in vitro Corresponding Author: Grazia Ambrosini, Memorial Sloan-Kettering Can- and in xenograft tumors. This effect was accompanied by cer Center, 1275 York Avenue, Box 128, New York, NY 10065. Phone: 646- the activation of AMPK and induction of cell death by 888-2184; Fax: 646-422-0631; E-mail: [email protected]. autophagy. These findings indicate that the combined doi: 10.1158/1535-7163.MCT-12-1020 inhibition of MEK and AKT is a valid therapeutic 2013 American Association for Cancer Research. approach for the treatment of GNAQ-mutant uveal

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Targeting MEK and AKT Induces AMPK in Uveal Melanoma Cells

melanoma, and that AMPK is a key regulator in these fragmented chromatin using a Nikon Eclipse TE2000-U tumor cells. microscope (Nikon Instruments Inc).

Materials and Methods RNAi-mediated gene knockdown Cell culture Small-interfering RNA (siRNA) against GNAQ (Gq1, Mel202, Mel270, Mel290, 92.1 and OCM1A are pri- sc-35429), AMPK (sc-45312), and control siRNA (sc-37007) mary choroidal uveal melanoma and Omm1.3 cells are were purchased from Santa Cruz Biotechnology. The liver metastasis from Mel270 (23). Omm1.3, Mel202 and SMARTPool GNAQ siRNA (Gq2) was from Dharmacon Mel270 have been kindly provided by Dr. Bruce Ksan- (L-008562). Single sequence siRNA was: CAAUAAGG- der (Harvard Medical School, Boston, MA). OCM1A CUCAUGCACAA (Gq3, Life Technologies, s5886). They and 92.1 were from Dr. William Harbour (Washington were transfected using Lipofectamine RNAiMAX reagent University, St. Louis, MO). Mel290 and C918 were from (Invitrogen) following the manufacturer’s instructions. Robert Folberg (University of Illinois, Chicago, IL). Uveal melanoma cell lines have been sequenced for the GFP-LC3 transfections presence of activating mutations in codons 209 (exon 5) GFP-LC3 was generated as described (24). Cells were and 183 (exon 4) of GNAQ and GNA11 and for BRAF grown on slides and transiently transfected with the GFP- mutations. Omm1.3 and 92.1 cell lines were also authen- LC3 plasmid using FuGENE 6 (Roche Applied Science). ticated by cytology analysis. Cells were cultured in Treated and untreated cells were visualized using wide- RPMI medium supplemented with 10% FBS, 100 U/mL field fluorescence microscopic images with a Nikon penicillin, and 100 mg/mL streptomycin, and main- Eclipse TE2000-U microscope (Nikon Instruments Inc). tained at 37C in 5% CO2. Cells were treated with selumetinib (AZD6244, ARRY-142866, AstraZeneca) Immunoblotting and MK2206 (supplied by MERCK). Compound C was Cells were lysed in RIPA buffer supplemented with purchased from Sigma. protease inhibitor cocktail tablets (Complete Mini, Roche Diagnostics) and 1 mmol/L NaVO3. Equal amounts of Cell viability assays and combination index analysis protein were loaded on 4%–12% PAGE gels (Invitrogen). Cells were plated in 96-well plates, and treated with the Polyvinylidene difluoride membranes were blocked with indicated concentrations of drugs. Viability was assessed 5% nonfat dried milk in PBS buffer containing 0.1% after 5 days of treatment using the Cell Counting Kit 8 Tween-20 (PBST) and probed with pERK, ERK, pAkt, (CCK8) from Dojindo Molecular Technologies according AKT, pAMPK, AMPK, BIJ, pBAD, p70S6K, mTor, pS6, to the manufacturer’s instructions. Survival is expressed beclin1, tubulin (Cell Signaling), and LC3 (Novus Biolo- as a percentage of untreated cells. For the combination gicals) antibodies. index (CI) analysis, dose–effect curve parameters were used to calculate CI values using the CompuSyn software Animal studies (ComboSyn) where CI < 1, ¼ 1, and >1 indicate synergism, Severe combined immunodeficient mice (SCID) mice additive effect, and antagonism, respectively (Chou TC, were purchased from Taconic, and used when they were 2010). 8-weeks old. A total of 15 106 92.1 cells were inoculated subcutaneously into the right flanks of the nude mice. Flow cytometry When tumors reached a volume of approximately 100 After treatments, the cells were collected and resus- mm3 diameter, animals were administered (5/group) vehi- pended in 5 mg/mL propidium iodide containing cle, selumetinib 25 mg/kg orally, MK2206 150 mg/kg 50 mg/ml RNAse A (Sigma). Samples were analyzed orally, alone and in combination. The treatment duration on a FACScan (Becton Dickinson), and data were ana- was 3 weeks and the tumor size was measured twice a lyzed for DNA content using Flowjo software. week. After the fifth treatment, 2 animals from each cohort were sacrificed and the tumors were collected for Western Colony assay blot analysis. Experiments were carried out under an For each condition, 1,000 cells were plated and treated Institutional Animal Care and Use Committee–approved for 24 hours with selumetinib, MK2206, or the combina- protocol, and Institutional guidelines for the proper and tion. After treatments, drug-containing medium was humane use of animals were followed. Statistical signifi- removed, and cells were cultured for further 12 or more cance was determined by 2-sample Student t tests. days to allow cells to form colonies. Colony formation was calculated as a percentage of untreated control. Results Depletion of mutant GNAQ inhibits multiple Apoptosis assay pathways The nuclear morphology of the cells was determined by Uveal melanoma cells with GNAQ mutations are sen- staining nuclear chromatin with 40,60-diamidino-2-pheny- sitive to the MEK inhibitor selumetinib, showing lindole (DAPI, Sigma). The percentage of apoptosis was decreased pERK and cell-cycle arrest (9). To further determined by counting the cells with condensed and explore signaling pathways downstream of mutant

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GNAQ in uveal melanoma cells, GNAQ was downregu- itor MK2206 (structures shown in Fig. 1C) and measured lated using 2 specific pools of siRNA (Fig. 1). Western blot cell viability of uveal melanoma cell lines with GNAQ analysis confirmed decreased levels of GNAQ in all the mutation, in comparison with cells carrying a BRAF muta- cell lines, which correlated with a decrease in pERK and tion, which also activates the mitogen-activated protein pAKT only in the GNAQ-mutant cells, compared to kinase (MAPK) pathway, or cells without either muta- nonspecific control siRNA (Fig. 1A). The downregulation tion (WT). Combinatorial treatments inhibited cell viabi- of pERK and pAKT was observed also using single siRNA lity more effectively than monotherapy in GNAQ and sequences at different time points up to 72 hours (Sup- BRAF-mutant cells (Fig. 2A–C). The treatments were less plementary Fig. S1). No changes in pERK and pAKT were effective in the WT cells as higher doses of the drugs detected in cell lines with wild-type GNAQ, and the levels were required for the inhibition of cell viability (Fig. 2D). of total ERK and AKT did not change in any cell line (Fig. The combination of selumetinib þ MK2206 at increasing 1A). Furthermore, downregulation of GNAQ decreased doses consistently reduced cell viability in the mutant cell viability of the mutant cells only (Fig. 1B). cells in a synergistic manner by more than 50% "fractional activity" (Fa > 0.5) and with CI values < 1 (Supplementary Combined MEK and AKT inhibition synergistically Fig. S2; ref. 25). suppresses cell viability and induces autophagy in The effect of either drug alone or in combination on cell GNAQ-mutant cells cycle and apoptosis was evaluated by flow cytometry. All We hypothesized that combined inhibition of both the cell lines showed an arrest in G1 of the cell cycle with pathways downstream of mutant GNAQ would induce selumetinib treatment (Fig. 3A). MK2206 as single agent enhanced antiproliferative effects. We used the MEK induced a modest G2 arrest, and the combination was inhibitor selumetinib in combination with the AKT inhib- similar to the effects of selumetinib alone. Furthermore, no

A GNAQWT GNAQMUT

C918 Mel290 Omm1.3 92.1 Mel270 Mel202 C Gq1 Gq2 C Gq1 Gq2 C Gq1 Gq2 C Gq1 Gq2 C Gq1 Gq2 C Gq1 Gq2 (siRNA)

GNAQ

pERK

ERK

pAKT(S473)

AKT

Tubulin

BCGNAQWT GNAQMUT 120 Selumetinib (AZD6244) H HO N O 100 siCtr O CI H siGq1 N 80 siGq2 N F Br 60 N

% Viability MK2206 40 NH2 N 20 HCI N O 0 HN N C918 Mel290 Mel202 92.1 Omm1.3 Mel270

Figure 1. Mutant GNAQ knockdown inhibits multiple pathways. A, uveal melanoma cells were transfected with a nonspeciﬁc control siRNA (C) or two independent pools of GNAQ siRNAs (Gq1, Gq2). Immunoblots show downregulation of GNAQ in all the cell lines, and speciﬁc inhibition of pERK and pAKT only in the mutant cell lines. B, cells transfected with siRNAs were plated in 96-well plates and assayed for viability after 3 days from transfection. Columns, mean of 3 independent experiments conducted in triplicates. C, chemical structure of selumetinib (top) and MK2206 (bottom).

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AB 120 100 Selumetinib 92.1Gq Selumetinib Omm1.3Gq 100 MK2206 MK2206 80 Selumetinib+MK2206 80 Selumetinib+MK2206 60 60 40 40 20 20 % Inhibition of cell viability % Inhibition of cell viability 0 0 25 50 100 1,000 2,500 25 50 100 1,000 2,500 [nmol/L] [nmol/L] C D 120 BRAF Selumetinib 100 OCM1A Mel290WT 100 MK2206 Selumetinib 80 MK2206 80 Selumetinib+MK2206 Selumetinib+MK2206 60 60

40 40

20 20 % Inhibition of cell viability % Inhibition of cell viability

0 25 50 100 1,000 2,500 0 [nmol/L] 25 50 100 1,000 2,500 [nmol/L]

Figure 2. Combined inhibition of MEK and AKT induces synergistic antiproliferative effects in mutant GNAQ cells. Uveal melanoma cells with mutant GNAQ (A and B), BRAFV600E (C), and WT (D) were treated with increasing concentrations of selumetinib and MK2206 (0, 50, 100, 1,000, 2,500 nmol/L) alone or in combination for ﬁve days. Inhibition of cell viability is expressed as percentage of untreated controls. Mean and SEM of 3 independent experiments conducted in triplicates are shown.

significant sub-G1 peak (apoptosis) was detected in the Bad integrates survival signaling by MAPK and PI3K/ GNAQ-mutant cells with any treatment. A small apopto- Akt kinase pathways (27). Phosphorylation at ser-136 of tic sub-G1 population was present in the BRAF-mutant Bad was inhibited by MK2206 in the BRAF-mutant and cells treated with the combination. The WT cells had also a wild-type cells, but not in the GNAQ-mutant cells. Only small sub-G1 population that did not change with the the combination therapy was effective in decreasing pBad treatments. Overall, the decreases in cell growth with the in these cells (Fig. 3C). However, the induction of Bim and combination therapy could not be accounted for by the downregulation of pBad were insufficient to induce changes in cell-cycle distribution. apoptosis in the GNAQ-mutant cells, as no PARP cleav- The combination treatment was also the most effective age was detected at 24 hours (Fig. 3C), or 48 hours (data in decreasing colony survival of the GNAQ- and BRAF- not shown). In contrast, cleaved PARP was detected in the mutant cells (Fig. 3B). BRAF-mutant cell line with the combination therapy, Western blot analysis was used to assess drug-mediat- suggesting possible alternative ways of cell death in the ed target inhibition and the effect on downstream signal- GNAQ-mutant cells. ing pathways. Selumetinib alone or in combination inhib- We then assessed the mTOR signaling pathway, which ited pERK in either GNAQ- or BRAF-mutant cell lines is important in the induction of autophagy. Phospho- (Fig. 3C). In contrast, the WT cell line did not show a p70S6K and p-pS6 were inhibited by selumetinib and the significant decrease in pERK levels. MK2206 inhibited combination with MK2206 in the mutant BRAF while they pAKT in all the cell lines. Cyclin D1 was downregulated were inhibited only by the combination therapy in the in all the cells by selumetinib and the combination treat- GNAQ-mutant cells. Also, the energy sensor AMPK was ment. To determine whether the treatments affected proa- induced in the GNAQ-mutant cells, as well as the autop- poptotic proteins, we examined expression levels of Bim hagy marker beclin1. In addition, the levels of LC3-II were and Bad. As previously reported (26), selumetinib markedly elevated in the GNAQ-mutant cells, especially induced Bim expression in all the cell lines, especially in with the combination therapy, suggesting that autophagy OCM1A and Omm1.3. may represent the major mechanism of cell death in these

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A 92.1Gq Omm1.3Gq OCM1ABRAF Mel290WT C 100% Sub-G 1 92.1Gq Omm1.3Gq Mel290WT OCM1ABRAF S 80% G Selum. – + – + – + – + – + – + – + – + 2 MK2206 – – + + – – + + – – + + – – + + 60% G1 pERK 40% % Cells ERK 20% pAKT(S473) 0% AKT

MK2206 MK2206 MK2206 MK2206 Untreated Untreated Untreated Untreated Selumetinib Selumetinib Selumetinib Selumetinib Cyclin D1 Selum+MK2206 Selum+MK2206 Selum+MK2206 Selum+MK2206 Bim B pBAD(S136) ND 120 Selumetinib PARP MK2206 100 p-p70 S6K Selumetinib+MK2206 80 p-pS6

60 pAMPK

40 Beclin1 % Colony survival % Colony 20 LC3-I LC3-II 0 Tubulin 92.1 Omm1.3 OCM1A Mel290 (Gq) (Gq) (BRAF) (WT)

Figure 3. Selumetinib þ MK2206 induce G1 arrest and inhibit colony formation of mutant cells. A, ﬂow cytometry analysis of GNAQ-mutant cell lines (92.1, Omm1.3), BRAF-mutant (OCM1A), and WT cells (Mel290) after treatments with 250 nmol/L selumetinib and 2.5 mmol/L MK2206 alone or in combination for 24 h. Each bar indicates the percentage of cells in each phase of the cell cycle. B, colony formation was calculated as a percentage of untreated control. Bars are means SD of 3 independent experiments conducted in duplicates. C, inhibition of pERK and pAKT induces the expression autophagic markers in cells with mutant GNAQ. After 24 hours of treatments, cell lysates were prepared from cells with the indicated mutational status, and protein expression was detected by immublotting for pERK, ERK, pAKT, AKT, cyclin D1 and other indicated proteins.

cells. Induction of autophagy was further measured by the (Fig. 5A and Supplementary Fig. S3), showing the regu- expression of a GFP-LC3 fusion protein, which localizes lation of AMPK by the inhibition of mutant GNAQ sig- to autophagosomal membranes during autophagy. In naling, and excluding drug unrelated effects. Also, beclin1 control untreated cells (ND) or cells treated with single and LC3 were upregulated by GNAQ silencing, whereas agents, GFP-LC3 localized evenly throughout the cyto- no PARP cleavage was detected (Fig. 5A). plasm with little or no punctuations per cell (Fig. 4A). Both Activation of AMPK occurs in various cellular stress 92.1 and Omm1.3 GNAQ-mutant cells treated with conditions when the AMP/ATP ratio is elevated, and it selumetinib þ MK2206 showed increased punctate autop- has been recently implicated in the induction of autophagosomal structures, whereas WT cells did now show hagy (28). To elucidate the role of AMPK in GNAQ- GFP-LC3 condensation under any condition (Fig. 4A). The mutant cells under energetic stress, AMPK expression percentage of cells that contained 2 or more punctuations was silenced by siRNA. Upon treatment with selumetinib is shown in Fig. 4B, indicating that 62 4.0 to 74 4.5% þ MK2206, the AMPK-depleted cells showed induction of of the cells undergo autophagy with the combination apoptosis by 2 independent methods, PARP cleavage (Fig. therapy. 5B) and quantitative fluorescent microscopy (Fig. 5E), in both 92.1 and Omm1.3 cell lines, with a concomitant Activation of AMPK is essential in the autophagic decrease in beclin1 expression, suggesting that AMPK response of GNAQ-mutant cells activation is mainly responsible for the induction of Induction of autophagy in combination-treated GNAQ- beclin1 and autophagy. The effect of AMPK downregula- mutant cells was accompanied by an increase in pAMPK, tion was also assessed by flow cytometry (Supplemen- LC3 and beclin1. This was confirmed in mutant cells after tary Fig. S4). AMPK silencing caused an increase from GNAQ depletion by siRNA (Fig. 5A). pAMPK was upre- 51.5% to 76.0% of cells in G1, suggesting a role of AMPK gulated in mutant cells but not in the wild-type cell line in cell-cycle regulation. With the combination treatment,

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Targeting MEK and AKT Induces AMPK in Uveal Melanoma Cells

A ND Selumetinib MK2206 Selumet+MK2206

Mel290WT

Figure 4. Effect of combined inhibition of MEK and AKT on Omm1.3Gq autophagosome formation in GNAQ-mutant and WT cells. A, Cells expressing GFP fused LC3 were treated with each drug as single agent or in combination for 24 hours. The accumulation of GFP-LC3 in autophagosomes in cells was 92.1Gq assessed by fluorescence microscopy by the appearance of GFP-LC3 punctate dots (magnification, 400). B, cells with 3 B or more punctuations were counted 100 ND under a microscope and reported as Selumetinib percentage of untreated cells. At 80 least three fields for each condition MK2206 were counted. Bars, SD. Selumetinib+MK2206 60

40 % Cells with >3 GFP-L C3 puncta 20

0 Mel209 Omm1.3 92.1 (WT) (Gq) (Gq)

there was a further increase in G1 and the induction of a The combination of selumetinib with MK2206 sub-G1 population (Supplementary Fig. S4). inhibits growth of xenograft tumors Next, we used compound C, an inhibitor of AMPK, To test the effects of selumetinib and MK2206 in xeno- alone or together with selumetinib þ MK2206. Compound graft mouse models, the GNAQ-mutant cell line 92.1 was C inhibited treatment-induced phosphorylation of AMPK subcutaneously injected in mice. Animals were treated and its specific substrate acetyl-CoA carboxylase (Fig. 5C). with vehicle, selumetinib, and MK2206 alone or in com- It also decreased the expression of beclin1 and LC3 and bination for 5 days each week for a total of 3 weeks. By day induced PARP cleavage when combined with selumetinib 21, the combination of selumetinib þ MK2206 resulted in þ MK2206. Furthermore, the direct suppression of beclin1 significant lower tumor volumes than vehicle or single with siRNA induced PARP cleavage in GNAQ-mutant– agents (Fig. 6A). Tumor samples from each group were treated cells (Fig. 5D) and increased apoptotic cells (Fig. analyzed by immunoblotting. Tumors from treated mice 5E). The suppression of both GNAQ and AMPK by siRNA showed inhibition of pERK and pAKT, and only speci- also resulted in induction of PARP cleavage (Supplemen- mens from mice treated with the combination selumetinib tary Fig. S5). þ MK2206 showed induction of pAMPK, beclin1, and LC3 AMPK activation thus serves to protect the cells from (Fig. 6B), confirming the induction of autophagic markers, apoptosis by inducing autophagy. Nevertheless, autop- as in the in vitro experiments. hagy is the mechanisms of cell death when inhibiting MEK and AKT in GNAQ-mutant cells. Altogether, these results show that AMPK phosphorylation is induced in Discussion response to mutant GNAQ signaling inhibition and it Most uveal melanoma harbor mutually exclusive muta- mediates an autophagic response, whereas its inhibition tions of the GNAQ or GNA11 proteins, which lead to the favors cell-cycle arrest in G1 and apoptosis under these activation of RAF/MEK/ERK pathway. In fact, these conditions. tumor cells are sensitive to MEK inhibition (2, 9).

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A 92.1Gq Omm1.3Gq BC92.1Gq Omm1.3Gq – – + + Selumet+MK2206 – + – + Comp. C GqCtr Ctr Gq (siRNA) siCtr siAMPK siCtr siAMPK –+–+ –+–+Selumet PARP GNAQ +MK2206 pAMPK PARP pAMPK pACC AMPK Beclin1 Beclin1 Beclin1 LC3-I LC3-I LC3-II LC3-II Tubulin Tubulin PARP

Tubulin E 35 siCtr ** D 30 siAMPK ** Gq 92.1Gq Omm1.3Gq 25 siBeclin * Omm1.3 siCtr sibeclin siCtr sibeclin 20 92.1Gq * – +–+ –+–+Selumet +MK2206 15 Beclin1 10

PARP % Apoptotic Nuclei 5

Tubulin 0 DMSO Selumet. DMSO Selumet. +MK2206 +MK2206

Figure 5. Inhibition of AMPK induces apoptosis in GNAQ-mutant cells treated with selumetinib þ MK2206. A, GNAQ knockdown in mutant cells induces pAMPK, LC3, beclin1, and no PARP cleavage. B, GNAQ-mutant cell lines were transfected with siRNA against AMPK or a nontargeting control siRNA (Ctr) and after 24 hours were exposed to the drugs. C, The GNAQ-mutant cell line 92.1 was pretreated with 1 mmol/L compound C for 30 minutes before addition of selumetinib þ MK2206 and incubated for further 24 hours. D, GNAQ-mutant cells were transfected with control or beclin1 siRNA and treated with the combination selumetinib þ MK2206 for immunoblot detection of beclin1 and cleaved PARP. E, 92.1 and Omm1.3 were transfected with AMPK or beclin1 siRNA, then treated with selumetinib þ MK2206 for 24 hours. The apoptotic nuclei were counted under a microscope, shown as percent of untreated controls. , P < 0.002; , P < 0.001, comparing treated AMPK and beclin1 siRNA versus treated control cells for 92.1 cells; and , P < 0.003, , P < 0.001 for Omm1.3 cells.

However, MEK inhibitors cause cell-cycle arrest without possibly through PI3K direct binding (29) or cross talk signiﬁcant cell death. The PI3K/AKT and mTOR/p70S6K between GPCR and receptor tyrosine kinase (RTK)-medi- pathways are critical for tumorigenesis, and the targeting ated signaling pathways (30, 31). We report that inhibition of PI3K while inhibiting ERK has been shown to reduce of both MEK and AKT induced a synergistic decrease in the proliferation of uveal melanoma cells (8, 16, 17). Inter- cell viability and this is associated with autophagic cell estingly, we have shown that the constitutively active death. This is mediated by the activation of AMPK, a key mutant GNAQ signals not only to ERK but also to AKT, regulator of energy homeostasis. In contrast, the blockade

Figure 6. Combined MEK and AKT inhibition results in antitumor ABeffects in mouse xenografts. A, GNAQ-mutant cells (92.1) were 120 Selumetinib transplanted into the right ﬂank of VehicleSelumetinibMK2206 + MK2206 8-week-old nu/nu SCID male mice. 100 pERK When subcutaneus tumors were approximately 100 mm3 diameter, pAKT(S473) mice were treated with vehicle, 80 MK2206 (150 mg/kg/d), and pAMPK selumetinib (25 mg/kg/d) as single 60 agents or in combination, Beclin1 5 days/week for 3 weeks. Tumor volumes are presented for day 21 40 * as percent tumor volume relative to

Volume (% control) Volume LC3 control vehicle SEM of 5 mice in 20 each group. , P < 0.05, comparing AKT the combination treatment versus each single agent. B, xenograft 0 ERK tumors were lysed at the end of the Vehicle MK2206 Selumetinib Selumetinib treatments and analyzed by + MK2206 GAPDH Western blotting with the indicated antibodies.

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of AMPK by siRNA or using compound C in the setting of treatment with inhibitors of PI3K and MEK induced inhibition of GNAQ signaling caused downregulation of apoptosis in GNAQ-mutant uveal melanoma cells, even beclin1, converting autophagy into apoptosis. Recent though pAKT was not inhibited by GNAQ knockdown studies have shown that activation of AMPK induced by (17). It seems that the direct inhibition of AKT and MEK by metformin disrupted crosstalk between insulin/IGF-1 specific inhibitors or GNAQ downregulation results in an receptor and GPCR signaling and inhibited cell growth energy depletion signal rather than an apoptotic stimulus of pancreatic cancer cells (32). AMPK seems to be at the like PI3K inhibition, and further studies are needed to crossroad of multiple signaling pathways, and its activa- characterize these differences. tion in uveal melanoma coincided with induction of The energy sensor AMPK was activated together with autophagy. autophagic markers in GNAQ-mutant cells when AKT AMPK is activated under conditions of low intracellular and MEK were inhibited, whereas its inhibition favored ATP following stresses such as nutrient deprivation, or apoptosis. Although autophagy is considered a protective hypoxia (33), and numerous reports showed that AMPK is mechanism, it still mediated a synergistic decrease in cell involved in the regulation of autophagic proteolysis viability of GNAQ-mutant cells, confirming that MEK and (28, 34, 35). One of the major downstream signaling path- AKT inhibition is a valid strategy for the treatment of ways regulated by AMPK is the mTOR pathway, through GNAQ-mutant cells. The combination therapy selumeti- the phosphorylation of TSC2 and inhibition of Raptor (36), nib þ MK2206 is currently in a phase I clinical trial and is thereby impeding protein synthesis (37). In GNAQ- well tolerated in patients with advanced metastatic solid mutant cells, AMPK was activated by the inhibition of tumors (43). both AKT and MEK, with downstream inhibition of The preliminary results of the single agent phase II p70S6K and only partial downregulation of p-pS6, where- study with selumetinib in patients with metastatic uveal as LC3 and beclin1 were upregulated. Low levels of melanoma appear promising, with inhibition of pERK, autophagy are considered a survival mechanism as suppression of cyclin D1 and partial radiologic responses they allow removal of damaged proteins and mitochon- (44). Therefore, these results would indicate that combin- dria and help maintain metabolic homeostasis. Converse- ing MEK and AKT inhibitors could represent the next step ly, high levels of autophagic activity can lead to cell death in developing a novel therapeutic approach for the treat- and cooperate with apoptosis (20). In addition, excessive ment of patients with this disease. stimulation of autophagy through overexpression of beclin1 has been associated with suppression of tumori- Disclosure of Potential Conflicts of Interest genesis, and eventually with cell death (38, 39). A.L. Ho has a commercial research grant and is a consultant/advisory The induction of autophagy in uveal melanoma was board member of AstraZeneca. No potential conflicts of interest were genotype-dependent as only GNAQ-mutant cells showed disclosed by the other authors. an increase in autophagic markers, as opposed to BRAF- mutant cells and cells without either mutation. Our group Authors' Contributions also reported that the effect of combined MEK and mTOR Conception and design: G. Ambrosini, E. de Stanchina, G.K. Schwartz Development of methodology: G. Ambrosini, G.K. Schwartz kinase inhibition in uveal melanoma cells depended on Acquisition of data (provided animals, acquired and managed patients, genotype, as the BRAF-mutant cells were particularly provided facilities, etc.): G. Ambrosini, E. Musi, E. de Stanchina, G.K. Schwartz susceptible to these treatments compared to GNAQ- Analysis and interpretation of data (e.g., statistical analysis, biostatis- mutant cells (40). This might be due to the complexity of tics, computational analysis): G. Ambrosini, E. Musi, A.L. Ho, G.K. GPCR and G protein-mediated signaling. G proteins have Schwartz Writing, review, and/or revision of the manuscript: G. Ambrosini, A.L. multiple downstream binding partners and various reg- Ho, G.K. Schwartz ulatory scaffolding/adaptor and effector proteins that are Administrative, technical, or material support (i.e., reporting or orga- often cell type specific (41). nizing data, constructing databases): G. Ambrosini, G.K. Schwartz Study supervision: G.K. Schwartz The combination therapy selumetinib þ MK2206 has been reported to have synergistic effects on the inhibition of lung cancer cells with RAS mutation, compared with The costs of publication of this article were defrayed in part by the single drug treatment, by the enhancement of Bim expres- payment of page charges. This article must therefore be hereby marked sion and induction of apoptosis (42). Despite a great advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. increase in Bim expression and inhibition of pBAD in our cells, autophagy was the prevalent pathway of cell death Received October 19, 2012; revised February 21, 2013; accepted February in GNAQ-mutant cells. It has been reported that the 21, 2013; published OnlineFirst February 26, 2013.

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776 Mol Cancer Ther; 12(5) May 2013 Molecular Cancer Therapeutics

Inhibition of Mutant GNAQ Signaling in Uveal Melanoma Induces AMPK-Dependent Autophagic Cell Death

Grazia Ambrosini, Elgilda Musi, Alan L. Ho, et al.

Mol Cancer Ther 2013;12:768-776. Published OnlineFirst February 26, 2013.

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