HDAC Inhibition Enhances the in Vivo Efficacy of MEK Inhibitor Therapy in Uveal Melanoma

Published OnlineFirst June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3382

Translational Cancer Mechanisms and Therapy Clinical Cancer Research HDAC Inhibition Enhances the In Vivo Efﬁcacy of MEK Inhibitor Therapy in Uveal Melanoma Fernanda Faiao-Flores~ 1, Michael F. Emmons1, Michael A. Durante2, Fumi Kinose3, Biswarup Saha1, Bin Fang4, John M. Koomen4, Srikumar P. Chellappan1, Silvya Stuchi Maria-Engler5, Uwe Rix3, Jonathan D. Licht6, J. William Harbour2, and Keiran S.M. Smalley1

Abstract

Purpose: The clinical use of MEK inhibitors in uveal mel- expression, particularly the endothelin B receptor, and this anoma is limited by the rapid acquisition of resistance. This contributed to therapeutic escape through ET-3–mediated study has used multiomics approaches and drug screens to YAP signaling. A screen of 289 clinical grade compounds identify the pan-HDAC inhibitor panobinostat as an effective identified HDAC inhibitors as potential candidates that sup- strategy to limit MEK inhibitor resistance. pressed the adaptive YAP and AKT signaling that followed Experimental Design: Mass spectrometry–based proteo- MEK inhibition. In vivo, the MEK-HDAC inhibitor combina- mics and RNA-Seq were used to identify the signaling path- tion outperformed either agent alone, leading to a long-term ways involved in the escape of uveal melanoma cells from decrease of tumor growth in both subcutaneous and liver MEK inhibitor therapy. Mechanistic studies were performed to metastasis models and the suppression of adaptive PI3K/AKT evaluate the escape pathways identified, and the efficacy of the and YAP signaling. MEK-HDAC inhibitor combination was demonstrated in mul- Conclusions: Together, our studies have identified GPCR- tiple in vivo models of uveal melanoma. mediated YAP activation and RTK-driven AKT signaling as key Results: We identified a number of putative escape path- pathways involved in the escape of uveal melanoma cells from ways that were upregulated following MEK inhibition, includ- MEK inhibition. We further demonstrate that HDAC inhibi- ing the PI3K/AKT pathway, ROR1/2, and IGF-1R signaling. tion is a promising combination partner for MEK inhibitors in MEK inhibition was also associated with increased GPCR advanced uveal melanoma.

Introduction commonly at Q209L/P) disable the intrinsic GTPase activity, leading to constitutive activation (2, 3). The major downstream Uveal melanoma is a highly aggressive tumor derived from the signaling target of GNAQ and GNA11 is phospholipase-C (PLC), melanocytes of the eye, with a tendency to metastasize to the liver. which hydrolyzes phosphatidylinositol 4,5-bisphosphate to the Although few patients show signs of disseminated disease at second messengers: inositol triphosphate (IP3) and diacyl glyc- diagnosis (4%), up to half will eventually succumb to metastatic erol. Protein kinase C (PKC) is activated by these second mes- disease despite successful treatment of the primary tumor (1). The sengers in GNAQ/GNA11–mutant melanomas (4). majority of uveal melanomas harbor activating mutations in the Recent work has shown that PKC and the small G-protein small G-proteins GNAQ and GNA11. These mutations (most RasGRP3 are required for the GNAQ/GNA11–driven activation of the MAPK pathway and that the majority of uveal melanomas have constitutive MAPK signaling that contributes to cell 1The Department of Tumor Biology, The Moffitt Cancer Center & Research Institute, Tampa, Florida. 2Bascom Palmer Eye Institute, Sylvester Comprehen- growth (5, 6). As a single agent, MEK inhibition has some activity sive Cancer Center and Interdisciplinary Stem Cell Institute, University of Miami against uveal melanoma cell lines, and is associated with reduced Miller School of Medicine, Miami, Florida. 3Department of Drug Discovery, The cell proliferation in vitro (7, 8). In light of this promising data, and Moffitt Cancer Center & Research Institute, Tampa, Florida. 4Department of the FDA approval of MEK inhibitors for BRAF-mutant cutaneous Molecular Oncology, The Moffitt Cancer Center & Research Institute, Tampa, melanoma, a number of clinical trials were undertaken to evaluate 5 Florida. Department of Clinical Chemistry and Toxicological Analysis, School of MEK inhibitors in uveal melanoma. In an open-label phase II Pharmaceutical Sciences, University of Sao~ Paulo, Sao~ Paulo, Brazil. 6Division of Hematology & Oncology, Department of Medicine, University of Florida Health clinical trial of patients with uveal melanoma with no history of Cancer Center, University of Florida, Gainesville, Florida. prior dacabarzine treatment, use of the MEK inhibitor selumetinib was associated with an increase in PFS from 7 to 16 weeks (9). Note: Supplementary data for this article are available at Clinical Cancer fi Research Online (http://clincancerres.aacrjournals.org/). These initially promising ndings led to the initiation of a phase III double-blind clinical trial of selumetinib plus dacarbazine, Corresponding Author: Keiran S.M. Smalley, Moffitt Cancer Center, 12902 which unfortunately failed to show any increase in PFS compared Magnolia Drive, Tampa, FL 33612. Phone: 813-745-8725; Fax: 813-449-8260; E-mail: keiran.smalley@moffitt.org with dacarbazine alone (10). Despite these disappointing results, current strategies continue Clin Cancer Res 2019;XX:XX–XX to focus upon combination therapies that include MEK inhibition doi: 10.1158/1078-0432.CCR-18-3382 as the backbone. There is promising preclinical data that indicates 2019 American Association for Cancer Research. the combination of a MEK and a PKC inhibitor potently induces

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from Cell Signaling Technology, Sigma Chemical Co., Millipore, Translational Relevance and Abcam. The phospho-receptor tyrosine kinase and phospho- At this time, there are no effective therapies for advanced kinase array were purchased from R&D Systems. OptiMEM medi- uveal melanoma. One of the most thoroughly explored tar- um, Lipofectamine 2000, and live/dead viability stain were pur- geted therapies for uveal melanoma is small-molecule inhi- chased from Invitrogen/Life Technologies Corp). siRNA for bitors of MEK. Despite initial clinical responses to MEK ROR1/2, IGF-1R, and YAP were purchased from Dharmacon RNA inhibition, levels of progression-free survival are very short Technologies. Nontargeting siRNA was purchased from Santa and the majority of patients fail within 3 months. Here, we Cruz Biotechnology. The Endothelin-3 Assay Kit was purchased used three unbiased platforms (proteomics, RNA-Seq, drug from IBL. screens) to deﬁne the mechanisms by which uveal melanoma cells escaped MEK inhibitor therapy. Our studies identiﬁed a Uveal melanoma cell lines complex adaptive response involving G-protein coupled The uveal melanoma cell lines 92.1, Mel270, OMM1, MP41, receptor (GPCR)-driven YAP activation and increased receptor and MM28 were used as described previously (17). All uveal tyrosine kinase (RTK)-driven AKT signaling, both of which melanoma cell lines were cultured in RPMI1640 supplemented were suppressed by the pan-HDAC inhibitor panobinostat. with 10% FBS, L-glutamine, and antibiotics at 5% CO2. All cells The combination of the MEK and HDAC inhibitor was highly were tested for Mycoplasma contamination every month using the effective at limiting therapeutic escape and led to durable Plasmotest-Mycoplasma Detection Test (Invivogen). Last test antitumor responses in both subcutaneous xenograft and liver date: April 18, 2019. Each cell line was authenticated using the metastasis models of uveal melanoma. Together, our results Human short-tandem repeat human cell line authentication provide the rationale for the clinical cotargeting MEK and service (ATCC) and frozen stocks of cells were discarded after HDACs in advanced uveal melanoma. 10 passages.

Cell viability assay (MTT assay) Uveal melanoma cells were plated in triplicate wells apoptosis and suppresses tumor growth in mouse xenograft (1 103 cells/well) and treated with increasing concentrations models (5). Multiple other signal transduction cascades are also of MEK inhibitor (trametinib) for 72 hours. Cell viability was activated in uveal melanoma including the PI3K/AKT/mTOR determined using the MTT assay as described previously (18). signaling pathway, which has been implicated in survival and cell migration (11, 12) and the Hippo tumor suppressor pathway, Colony formation assay which plays key roles in tissue homeostasis and organ size (13). A total of 1 103 cells were plated and allowed to attach Under normal physiologic conditions, the MST1/2 and LATS1/2 overnight. The medium and drug/vehicle was replaced every kinases phosphorylate and inactivate YAP and TAZ, two tran- 2 days for 4 weeks. After the specific treatments for each exper- scriptional coactivators implicated in oncogenic transforma- iment, colonies were stained with crystal violet dye, as described tion (13, 14). In uveal melanoma, GNAQ stimulates YAP through previously (18). a Hippo-independent mechanism that is initiated through actin polymerization (15). Silencing of GNAQ/GNA11 in uveal mel- Flow cytometry for apoptosis analysis anoma cells led to decreased nuclear accumulation of YAP, with A total of 1 105 cells were plated and allowed to attach further studies showing that the YAP inhibitor verteporfin abro- overnight. After the specific treatments for each experiment, gates GNAQ/GNA11–driven tumor growth in an orthotopic uveal Annexin V staining quantification was performed using FlowJo melanoma ocular xenograft model (15, 16). At this time, little is software as described previously (19). known about the systems level signaling adaptations of uveal melanoma cells to MEK inhibition. In this study, we used activity- Activity-based protein profiling based protein profiling (ABPP) and RNA-Seq to identify key ABPP experiments were carried out as described previous- proteins involved in the adaptation of uveal melanoma cells to ly (20). Briefly, 92.1 and Mel270 cells were treated with MEKi MEK inhibition, and identified novel drug combinations to (25 nmol/L for 24 hours) and then solubilized with lysis buffer. A overcome this adaptation. total of 1 mg of protein from each sample was prepared for labeling, enrichment, and LC/MS-MS analysis. Protein identification and quantification were performed by Andromeda and Materials and Methods MaxQuant (v. 1.2.2.5), the values were log2-transformed and Reagents normalized (21). Signaling pathways, protein interaction, and RPMI culture medium was purchased from Corning. FBS was process network analysis were carried out using MetaCore purchased from Sigma Chemical Co.. Trypsin, penicillin/strepto- (GeneGO). Data are available in PRIDE (PXD013988). mycin antibiotics, and puromycin were purchased from Gibco. Trametinib (MEK inhibitor), panobinostat (pan-HDAC inhibi- RNA sequencing tor), pictilisib (PI3K inhibitor), bosentan hydrate (EDNRB RNA was extracted from 92.1 and Mel270 uveal melanoma cell inhibitor), verteporfin (YAP inhibitor), entinostat (HDAC1/2/3 lines using Qiagen's Rneasy Mini Kit (Qiagen), and screened for inhibitor), and tubastatin A (HDAC 6 inhibitor) were purchased quality on an Agilent BioAnalyzer. The samples were then pro- from Selleckchem. PCI-34051 (HDAC8 inhibitor) was purchased cessed for RNA-sequencing (RNA-seq) using the NuGen Ovation from Cayman Chemical. Endothelin-3 was purchased from Sigma Human FFPE RNA-Seq Multiplex System. Briefly, 100 ng of RNA Chemical Co. WNT5A was purchased from R&D Systems. Anti- was used to generate cDNA, and a strand-specific library following bodies for Western blot and immunochemistry were purchased the manufacturer's protocol (NuGEN Technologies, Inc.). Quality

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control steps including analysis on the BioAnalyzer RNA chip and Cell proliferation assay qRT-PCR for library quantification were performed. The libraries A total of 5 104 cells were plated and allowed to attach were then sequenced on the Illumina NextSeq 500 sequencer with overnight. After the specific treatments for each experiment, cells a2 75-base paired-end run to generate 40–50 million read pairs were counted using Trypan Blue reagent. The percentage of total per sample. RNAseq data were preprocessed for quality assess- cells was normalized to the percentage of control cells as described ments before aligning to the human genome hs37d5 using Tophat previously (25). v2.0.13 default setting and quantified using htseq-count based on the RefSeq gene model downloaded from USCS Table Browser. Normalized counts were obtained by using the counts function, Transfections and luciferase assays – and differential expression was analyzed using a Wald statistic test We have used a YAP/TAZ responsive synthetic promoter driv- implemented in the DESeq function in the DESeq2 Bioconductor ing reporter plasmid, named 8xGTIIC-luciferase (Addgene, package, which performs serial dispersion estimation and nega- #34615) for the assay. Overnight seeded 92.1 and Mel270 cells 3 tive binomial generalized linear model fitting procedure. A Ben- (120 10 cells/well in a 6-well plate) were transfected with the construct using FuGENE (Promega; #31985-070) for 8 hours. jamini–Hochberg Padj value of less than 0.05 was used as a cutoff to determine significantly differentially expressed genes. Data are Media was changed for another 12 hours prior to the drug(s) available in GEO (GSE127948). treatment with MEKi (10 nmol/L), HDACi (10 nmol/L), or both for 48 hours. Harvested cells were washed once with PBS and Gene set enrichment analysis luciferase assay was performed according to the manufacturer's Gene set enrichment analysis (GSEA; ref. 22) was conducted protocol and plotted the values normalized against Control, utilizing recommended parameters (http://software.broadinsti without any inhibitor treatment. tute.org/gsea/doc/GSEAUserGuideFrame.Html) with gene sets obtained from the Molecular Signatures Database (23) and cus- Immunofluorescence tom gene sets obtained from the GSEA database. A total of 3 103 cells were seeded in 8-Well Lab-Tek II Chamber Slides and allowed to attach overnight. On the next Phospho-receptor tyrosine kinase and Phospho-kinase array day, cells were treated, then fixed, permeabilized and stained analysis using YAP antibody (#14729). The information about the immu- A Human Phospho-RTK Array Kit (ARY001B) and a Human nofluorescence antibodies can be found in Supplementary Table Phospho-kinase Array Kit (ARY003B) were used to measure the S3. Slides were mounted with ProLong Antifade with DAPI in relative level of different RTKs and kinases. 92.1 and Mel270 uveal accordance with a previously described protocol (26). Glass slides melanoma cell lines were treated with MEKi (10 nmol/L for were observed with a Leica TCS SP8 AOBS laser scanning confocal 48 hours), the lysed, and 300-mg protein were incubated with microscope through a 63X/1.4NA oil immersion Plan Apochro- the array according to the manufacturer's protocol. mat objective. Laser line at 405 nm was used to excite the DAPI fluorophores. Images were captured at 200 Hz scan speed with Immunoblotting photomultiplier detectors using LAS X software version 3.1.5. Cells were plated and allowed to attach overnight. After the Images were analyzed using the Definiens Tissue Studio v4.7 specific treatments for each experiment, proteins were extracted (Definiens AG) software suite. Cells were segmented by the and blotted as described previously (24). Total and phospho- nuclear stain DAPI (blue) and phalloidin (red) channel was used proteins were analyzed (Supplementary Table S1) and then the as cytoplasm marker for cell simulation. The image was analyzed membranes were stripped and reprobed for GAPDH/vinculin/ as an 8-bit image and intensity of each RGB channel was measured b-actin. from 0 to 255 grayscale fluorescent units. The cells were then quantified for green and red intensity per field for nucleus and for qRT-PCR cytoplasm. tRNA was isolated using Qiagen's Rneasy Mini Kit (Qiagen). TaqMan Gene Expression Assay primer/probes were used as shown in Supplementary Table S2. GAPDH was used to normal- Endothelin-3 assay kit ize the genes of interest. qRT-PCR reactions were carried out as 92.1, Mel270, MP41, and OMM1 uveal melanoma cells were described previously (18). seeded in 96-well plates at 1.0 104 cells/well. After 24 hours, cells were treated with MEKi (10 nmol/L), HDACi (10 nmol/L), or siRNA MEKi þ HDACi (10 nmol/L each one) for 24–72 hours. Cell Cells from uveal melanoma cell lines were plated and allowed supernatant was collected and incubated according to the man- to attach overnight in complete RPMI medium with 10% FBS. ufacturer's protocol. After 24 hours, this medium was replaced with Opti-MEM and the cells were transfected with 50 nmol/L siRNA for ROR1 (Dharmacon SMARTpool; L-003171-00-0005), 50 nmol/L siRNA Drug screening for ROR2 (Thermo Fisher Scientific; #4390824), or 50 nmol/L 92.1, Mel270, MP41, and OMM1 uveal melanoma cells were siRNA for IGF-1R (Dharmacon SMARTpool; L-003012-00-0005) seeded in 384-well plates at 1.0 103 cells/well. All compounds in complex with Lipofectamine 2000 overnight. Nontargeting of the library were diluted to 0.5 or 2.5 mmol/L, and the exper- siRNA was added as a siRNA control (Santa Cruz Biotechnology; iment was performed in duplicate. A total of 289 compounds sc-37007). After 24 hours of the transfection, medium was from an in-house library were tested. Compounds were aliquoted replaced by complete RPMI medium with 10% FBS and treated by a Biotek Precision Pipetting robot. Cell viability was measured with MEKi (10 nmol/L) for 72 hours. by Cell-Titer-Glo (Promega G7572) at 72 hours posttreatment.

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Animal experiments growth of all of the uveal melanoma cell lines, these reductions Subcutaneous xenograft model. Eight-week-old female CBySmn. were modest (Fig. 1A; Supplementary Table S4), and associated CB17-Prdkc scid/j mice (Stock No: 001803 - Jax) were subcuta- with regrowth of colonies in all cases (Fig. 1B and C). Levels of neously injected with 1.0 106 92.1 or MP41 uveal melanoma MEKi (10–25 nmol/L trametinib)-induced apoptosis were also cells per mouse. The tumors were allowed to grow for 3 weeks and minor compared with those seen in cutaneous melanoma mice were randomly separated with similar average initial tumor (Fig. 1D; ref. 27). Little apoptosis induction was observed at volumes, with a total of 3 mice per group. 24 hours. To better understand the process of adaptation that occurs when uveal melanoma cells are treated with a MEKi, we Liver metastasis model. Eight-week-old female NOD.Cg- treated two GNAQ-mutant uveal melanoma cell lines (92.1 and Prkdcscid Il2rgtm1Wjl/SzJ (Stock No: 005557, Jackson Labo- Mel270) with trametinib (25 nmol/L, 24 hours) and performed ratory) were injected with 2.0 105 MP41 uveal melanoma activity-based protein profiling (ABPP; Fig. 1E; ref. 20). This cells per mouse into the tail vein. The tumors were allowed to method, which uses mass spectrometry to quantify ATP uptake grow for 4 weeks and mice were randomly separated with levels of proteins through transfer of a desthiobiotin tag to lysine similar average initial tumor volumes, with a total of 3 mice in the active site of enzymes and kinases, allows signaling activity per group. to be mapped in a comprehensive manner (Fig. 1E; ref. 20). The The mice were imaged on 7T Horizontal Magnet (Agilent ASR ABPP studies demonstrated that MEK inhibition increased the 310) and (Bruker Biospin, Inc. BioSpec AV3HD), using a 35 mm ATP uptake of 128 proteins and 98 proteins in the 92.1 and birdcage coil (m2m imaging corp). Anatomical coronal images Mel270 cells, respectively (Fig. 1F). Use of STRING analysis were obtained with a TurboRARE sequence with echo time/ allowed us to identify an enrichment for activated proteins repetition time (TR/TE) ¼ 1,585/15 ms, 33 slices, slice thickness implicated in proliferation and survival, ribosome function, of 0.6 mm, field of view (FOV) ¼ 30 30 mm2, image size 256 metabolism, and the cytoskeleton (Fig. 1G). The top pathways 256. Respiration gating was used to minimize motion artifacts. modulated by MEKi treatment included cell metabolism, cyto- Liver metastases were manually contoured on all MR slices using skeletal remodeling, apoptosis, AKT signaling, IGF signaling, ImageJ (https://imagej.nih.gov/ij/index.html). A custom pro- WNT signaling, FGFR signaling, and melanocyte differentiation gram was written in MATLAB (MATLAB 2018a, The MathWorks, (Supplementary Fig. S1). Natick, 2018.) to extract voxels within the manually drawn con- tours and compute total metastases volume burden and individ- Trametinib induces adaptive AKT signaling in uveal melanoma ual metastases volumes, in mm3. In both protocols, mice were cells treated with MEKi (trametinib, 1 mg/kg gavage, daily), HDACi We next focused on the potential therapeutic escape pathways (panobinostat, 20 mg/kg i.p., three times a week), or the combi- identified from our ABPP screen and first considered the PI3K/ nation of both agents for 30 days (xenograft model) or for 21 days AKT pathway. GSEA analysis of RNA-Seq data generated from (liver metastasis model). The control group received both vehicles uveal melanoma cells treated with trametinib (25 nmol/L, (for trametinib: 0.5% methylcellulose þ 0.5% Tween-80 molec- 24 hours) demonstrated an enrichment for genes implicated in ular grade sterile water; for panobinostat: 5% dextrose in water). PI3K/AKT signaling (Fig. 2A; Supplementary Table S5). This was 1 fi Mouse weight and tumor volumes ( /2 L W2) were measured con rmed in kinome arrays that showed a consistent upregula- every 72 hours. All animal experiments were carried out in tion of AKT in both cell lines following MEK inhibition (Fig. 2B) agreement with ethical regulations and protocols approved by and an increase in FAK signaling in the Mel270 cells—confirming the University of South Florida Institutional Animal Care and by the link between MEKi and cytoskeletal rearrangement observed The Institutional Animal Care and Use Committee (IACUC num- in the ABPP data. The AKT data were confirmed by Western blot, ber IS00002983). The IACUC protocol did not permit survival to with MEKi found to increase phosphorylation of AKT at T308 be an experimental endpoint. (Fig. 2C shows fold-increase by densitometry). The potential role of rebound AKT signaling in the escape of the melanoma cells from MEKi therapy was validated by the ability of the PI3K Statistical analysis inhibitor pictilisib (PI3Ki) to significantly increase the apoptotic Results are expressed as mean SD of a triplicate of at least response to trametinib (MEKi; Fig. 2D). Although there was some three independent experiments. One-way ANOVA was used fol- evidence that the PI3Ki also suppressed the outgrowth of MEK lowed by a TUKEY-KRAMER posttest to test for multiple compar- inhibitor–treated uveal melanoma cells in colony formation isons with a given significance level of P < 0.05. Significant assays, the effects were incomplete and tumor cells were still able differences between the control and treated groups are indicated to evade therapy (Fig. 2E and F). by , P < 0.001; , P < 0.01; , P < 0.05. IGF1R and ROR1/2 activate AKT signaling following MEK inhibition Results As adaptive AKT signaling frequently results from increased Activity-based proteomic profiling identifies signaling RTK signaling, we returned to our RNA-Seq data and identified an pathways implicated in the escape of uveal melanoma cells increase in receptor protein kinase (ES score 0.41) and receptor from MEK inhibitor therapy tyrosine kinase (ES score 0.44) expression (Fig. 3A; Supplemen- We began by characterizing the MEK inhibitor response of a tary Tables S6 and S7). These findings were confirmed by RTK panel of GNAQ/GNA11–mutant uveal melanoma cell lines that arrays, with MEKi being found to increase the phosphorylation of were derived from primary and metastatic lesions (92.1, Mel270, multiple RTKs including IGF-1R (in the 92.1 cells), as well as MP41: primary, OMM1 and MM28: metastatic). It was found that ROR1 and ROR2 (in both the 92.1 and Mel270 cell lines; Fig. 3B). although the MEK inhibitor trametinib (MEKi) inhibited the qRT-PCR and Western blot analyses demonstrated increases in

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Figure 1. MEK inhibition rewires the signaling network of uveal melanoma cells. A, MTT assay showing the antiproliferative activity of MEKi (trametinib) against five uveal melanoma cell lines (92.1, MP41, Mel270, MM28, and OMM1). B, MEKi does not suppress uveal melanoma cell growth in long-term colony formation assays. Cells were treated with 1 nmol/L of MEKi for 4 weeks and colonies visualized using Crystal Violet. C, Quantification of experiment from B. D, MEKi is associated with limited apoptosis in uveal melanoma cells. Cells were treated with MEKi (trametinib, 10–25 nmol/L, 24, 72 hours) and apoptosis measured by Annexin-V binding and flow cytometry. E, An overview of the ABPP method to comprehensively map adaptive signaling following MEK inhibition. F, 92.1 and Mel270 uveal melanoma cells were treated with MEKi (trametinib 25 nmol/L, 24 hours) and analyzed by ABPP. Volcano plot shows the decrease in protein expression (blue) and the increase in protein expression (red) after the treatment. G, STRING analysis highlights the key cellular processes and proteins showing increased ATP uptake following MEKi treatment. ns, not significant.

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Figure 2. AKT is a major escape pathway following MEK inhibition. A, GSEA analysis of RNA-Seq experiments of MEKi-treated uveal melanoma cells shows an enrichment for PI3K/AKT signaling. B, MEK inhibition increases T308 AKT phosphorylation in kinome arrays. 92.1 and Mel270 cells were treated with MEKi (trametinib 10 nmol/L, 48 hours) and were subjected to kinome array analysis. Bar graph shows densitometry from scanned array. C, MEKi increases AKT signaling in uveal melanoma cells. 92.1, Mel270, and MP41 cells were treated with trametinib (0–24 hours, 10 nmol/L) before being subject to Western blot for phospho-AKT, total AKT, and GAPDH. Numbers indicate the fold protein increase relative to control, normalized to the loading control. D, Combined treatment with MEKi (trametinib, 10 nmol/L) and a PI3Ki (pictlisib, 3 mmol/L) leads to enhanced apoptosis. Cells were treated with vehicle, MEKi alone, PI3Ki alone, or the combination for 72 hours. Apoptosis was measured by Annexin-V binding and ﬂow cytometry. E, Combined MEK-PI3Ki treatment partly limits therapeutic escape in colony formation assays. 92.1, Mel270, and MP41 cells were treated with vehicle, MEKi alone (1 nmol/L), PI3Ki alone (300 nmol/L), or the combination for 4 weeks before being stained with Crystal Violet. F, Quantiﬁcation of the experiment from E.

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Figure 3. IGF1R and ROR1/2 activate AKT escape signaling following MEK inhibition. A, GSEA analysis of RNA-Seq experiments of MEKi- treated uveal melanoma cells shows an enrichment for receptor protein kinase activity and receptor tyrosine kinases. B, MEKi increases ROR1/2 and IGF-1R signaling activity. 92.1 and Mel270 uveal melanoma cells were treated with MEKi (10 nmol/L, 48 hours) before being analyzed by RTK array. MEK inhibition (10 nmol/L, 72 hours) increases IGF-1R, ROR1, and ROR2 mRNA expression by RT-PCR (C) and protein expression in 92.1 and Mel270 cells (D). E, WNT5A activates AKT signaling in 92.1 cells. Cell cultures were treated with WNT5A (200 ng/mL, 0–120 minutes) and probed for pAKT expression. Numbers indicate the fold protein increase relative to time 0, normalized to the loading control. F, Validation of ROR1/2 and IGF-1R knockdown by Western blot. G, ROR1/2 knockdown prevents MEKi-mediated AKT signaling. ROR1/2 was silenced by siRNA and cells treated with MEKi. pAKT was measured by Western blot. H, Knockdown of ROR1/2 or IGF-1R sensitizes uveal melanoma cells to MEKi-induced cell death. Data show total number of 92.1 and Mel270 cells by Trypan Blue exclusion. I, Silencing of ROR1/2 enhances MEKi- induced apoptosis in 92.1 and Mel270 cells. Following siRNA knockdown cells were treated with MEKi (10 nmol/L, 72 hours), and apoptosis was measured by Annexin-V binding and flow cytometry. Values are expressed as mean SD. Significance is indicated by , P < 0.05; , P < 0.01; , P < 0.001 when comparing Control versus MEKi group and is indicated by &, P < 0.05 and &&, P < 0.01 when comparing shControl treated with MEKi with shIGFR-1R or sh ROR1/2 treated with MEKi. ns, not significant.

IGF-1R, ROR1, and ROR2 mRNA and protein expression follow- observed following MEKi treatment (Fig. 3G). Silencing of IGF-1R ing MEKi (Fig. 3C and D). Although the link between in combination with the MEKi was found to increase cell death IGF-1R and AKT signaling is well known, less is known about and decrease the numbers of 92.1 cells, but not Mel270 cells, a whether ROR1 and ROR2 activate AKT signaling. Treatment of result consistent with the increased IGF-1R signaling seen only in 92.1 uveal melanoma cells with WNT5A, the ligand for ROR1 and the 92.1 cell line (Fig. 3F, H, and I). In contrast, silencing of ROR1/ ROR2 receptors (28), conﬁrmed a time-dependent increase in 2 enhanced the effects of MEKi in terms of decreased cell survival AKT phosphorylation (Fig. 3E). Silencing of ROR1/2 (Fig. 3F) in and apoptosis induction in both of the cell lines evaluated the 92.1 cells inhibited the increases in AKT phosphorylation (Fig. 3F, H, and I).

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Figure 4. Activation of YAP signaling following MEK inhibition. A, GSEA analysis of RNA-Seq experiments of MEKi-treated uveal melanoma cells shows an enrichment for Hippo pathway activity. MEKi (trametinib 10 nmol/L, 48 hours) induces YAP activity in 92.1 and Mel270 cells in a reporter assay (B) and enhances YAP nuclear accumulation in 92.1 cells (C). D, The nuclear and cytoplasmic fluorescence intensity of YAP was analyzed using the Definiens Tissue Studio software suite. E, MEKi increases expression of YAP target genes. Data show qRT-PCR for 92.1 and Mel270 cells following treatment with MEKi (10 nmol/L, 48 hours) for YAP, AREG, CTGF, and CYR61. F, MEKi increases expression of CTGF and AREG in uveal melanoma cells by Western blot. G, YAP inhibition limits MEKi-mediated therapeutic escape. Images show a colony formation assay following treatment with MEKi (1 nmol/L), YAPi (verteporfin, 100 nmol/L), or the combination of both for 4 weeks. H, Quantification of experiment from G. I, Western blot showing YAP silencing. J, Silencing of YAP enhances MEKi-mediated apoptosis. Cells were treated with MEKi (10 nmol/L) for 72 hours and apoptosis quantified by Annexin-V binding and flow cytometry. Analysis was performed with one-way ANOVA followed by Tukey–Kramer post hoc analysis. Values are expressed as mean SD. Significance is indicated by , P < 0.05; , P < 0.01; , P < 0.001. For the siRNA experiments, significance is indicated by &, P < 0.05 and &&, P < 0.01. ns, not significant.

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MEK inhibition increases YAP activity leading to increased cell target both AKT and YAP, we performed an unbiased screen of 289 survival compounds to identify potential drug combination partners that As inhibition of PI3K/AKT did not fully abrogate escape from could limit escape from MEK inhibition (drugs listed in Supple- MEK inhibition, we next turned our attention to other possible mentary Table S10). The drug library used covers all major target pathways. One signal transduction cascade known to be critical classes, including kinases, receptor tyrosine kinases, phospha- for uveal melanoma progression, frequently upregulated follow- tases, receptor agonists, proteases/proteasome, PARP1, epigenetic ing cytoskeletal rearrangement is the prooncogenic mediator of enzymes, Hedgehog, HSP90 and Notch, and reflects the current Hippo signaling, YAP (15, 29). Analysis of the RNA-seq data by landscape of targeted agents approved for use or have been GSEA showed a significant gene enrichment (ES score 0.27) for considered for clinical development (Fig. 6A). Among these, Hippo pathway targets with a positive correlation among several drugs were identified with some activity against one or the genes overexpressed after MEKi treatment (10 nmol/L, more uveal melanoma cell lines including PI3K inhibitors 24 hours; Fig. 4A; Supplementary Table S8). Although YAP is (GSK2126458, idelalisib), two kinesin inhibitors (ispinesib, constitutively activated in uveal melanoma cells, MEKi was noted SB743921), CDK inhibitors (dinaciclib), H3K27 histone to further stimulate YAP transcriptional activity in a reporter assay demethylase (GSK-J4), and mTOR (sapanisertib; Fig. 6B). The (Fig. 4B) and this was accompanied by an increase in the levels of drug class with the most prominent effect across all four cell lines nuclear YAP accumulation (Fig. 4C and D). The increase in mRNA was the HDAC inhibitors (HDACi; Fig. 6B). To further determine levels of a number of YAP pathway transcriptional target includ- whether other epigenetic inhibitors could also enhance the effects ing YAP, connective tissue growth factor (CTGF), amphiregulin of MEKi (trametinib), we evaluated inhibitors of DOTL1 (AREG), and cysteine rich angiogenic inducer 61 (CYR61) also (EPZ5676), EZH2 (tazemetostat), LSD1 (GSK 2879552), DNMT occurred following MEKi treatment (Fig. 4E). Increased expres- (decitabine), HAT (anacardic acid), and HDACi (panobinostat) sion of two of the main transcriptional targets of YAP/TAZ activity alone and in combination with trametinib. These studies dem- including CTGF and AREG was also seen by Western blot after onstrated that the pan-HDACi panobinostat was the most effec- MEKi treatment (10 nmol/L, 72 hours; Fig. 4F). The role of YAP tive at enhancing the antiproliferative effects of MEKi (Supple- signaling in therapeutic escape was demonstrated by the ability of mentary Fig. S4). We next turned our attention to more specific the YAP inhibitor verteporfin (YAPi) to decrease colony formation HDACis, including entinostat (HDAC1/2/3i), tubastatin in response to MEK inhibition compared with either drug alone (HDAC6i), and PCI-34051 (HDAC8i) and noted that panobino- (Fig. 4G and H). In addition, siRNA knockdown of YAP (Fig. 4I) stat (HDACi) was the most effective among all of these across all increased the level of MEKi-induced apoptosis in multiple uveal four cell lines in both MTT and colony formation assays (Fig. 6C– melanoma cell lines (Fig. 4J). E). Further support for the potential role of HDAC activity in the escape of uveal melanoma cells from MEKi therapy was suggested Adaptive GPCR signaling increases YAP activity following MEK by the increase in global protein deacetylation observed in our inhibition uveal melanoma cell lines following MEKi treatment (Supple- G-protein–coupled receptors (GPCR) are known to be strong mentary Fig. S5). It was found that cotreatment of multiple uveal activators of YAP signaling. In line with this, it was found that melanoma cell lines, including 92.1, MP41, Mel270, and MM28 MEK inhibition led to a strong induction of GPCR expression with the MEKi–HDACi (trametinib–panobinostat) combination in our GSEA analysis (Fig. 5A; Supplementary Table S9). was associated with significantly (P < 0.05) higher levels of Multiple GPCRs showed increased expression including GPR158, apoptosis compared with either single agent (Fig. 6F). These GP133, and endothelin-receptor B (EDNRB; Fig. 5B). A potential effects were specific to uveal melanoma cells, with no apoptosis role for EDNRB signaling in the adaptive YAP signaling was seen in 3 different primary uveal melanocyte cell lines (Supple- confirmed through studies in which exogenous endothelin-3 mentary Fig. S6). The apoptotic response was paralleled by an (ET-3) was found to increase both YAP reporter activity, nuclear increased induction of cleaved caspase-7 and cleaved PARP in localization of YAP, and induction of YAP-target genes in four uveal melanoma cell lines (Fig. 6G). uveal melanoma cell lines (Fig. 5C and D; Supplementary Fig. S2A–S2C). In each case, the endothelin receptor B (EDNRB) Trametinib plus panobinostat induces uveal melanoma antagonist bosentan was found to block the ET-3–mediated regression in vivo increases in YAP activation (Fig. 5C and D; Supplementary Fig. We next asked whether the enhanced therapeutic efficacy of S2A–S2C). Mechanistically, it was noted that MEKi treatment led MEKi and HDACi resulted from the combined inhibition of YAP to the release of ET-3 from the uveal melanoma cell lines by ELISA and AKT signaling. It was found that cotreatment of the uveal (Fig. 5E) and that the MEKi-mediated increase in YAP reporter melanoma cells with panobinostat and trametinib effectively activity could be abrogated by the EDNRB antagonist (Fig. 5F). suppressed the adaptive AKT and YAP signaling and the release Together, these results suggested that MEK inhibition led to ET-3 of ET-3 seen following MEK inhibitor treatment (Fig. 7A and B; release from the uveal melanoma cells and this functioned in an Supplementary Fig. S7). From a mechanistic standpoint, we autocrine manner to activate YAP through EDNRB. No increases identified the PI3K/AKT pathway as being significantly down- in YAP activity were found following treatment with ET-1 regulated by GSEA analysis of our RNA-Seq dataset (Supplemen- (Supplementary Fig. S3). tary Fig. S8A). A pairwise comparison of the effects of each drug demonstrated the MEKi–HDACi combination to also strongly Histone deacetylase inhibitors increase the effects of MEK induce PTEN at the mRNA and protein level (Supplementary inhibition in uveal melanoma Fig. S8B and S8C; Supplementary Table S11). To validate the Our studies identified both RTK-mediated AKT and GPCR- MEKi–HDACi combination in vivo,wefirst generated xenografts driven YAP signaling as pathways utilized by uveal melanoma of two human uveal melanoma models (92.1 and MP41). After cells to escape MEKi therapy. As there are no known drugs that formation of a palpable tumor in the subcutaneous model

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Figure 5. Adaptive YAP signaling results from MEKi-mediated release of ET-3. A, GSEA analysis of RNA-Seq experiments of MEKi-treated uveal melanoma cells shows an enrichment for GPCR activity.

B, Graph showing the log2 fold change in expression of individual GPCRs following MEKi treatment. C, Endothelin-3 (ET-3) activates YAP reporter activity in 4 uveal melanoma cell lines. Cells were treated with either ET-3 (100 nmol/L, 1 hour), EDNRB antagonist (bosentan, pretreatment with 80 mmol/L, 1 hour), or ET-3 þ bosentan (100 nmol/L and 80 mmol/L, respectively). D, ET-3 increases expression of YAP target mRNAs in 92.1, Mel270, MP41, and OMM1 cells. Data show qRT-PCR analysis of YAP, CTGF, and CYR61. E, MEKi leads to ET-3 release from uveal melanoma cells. Cells were treated with MEKi (10 nmol/L, 0–72 hours) and the supernatant analyzed by ELISA. F, The EDNRB antagonist bosentan blocks MEKi- mediated YAP activation. Uveal melanoma cells were treated with vehicle, MEKi (10 nmol/L, 48 hours), EDNRBi (bosentan, pretreatment with 80 mmol/L, 1 hour), and the combination for 48 hours before being subjected to the YAP reporter assay. Analysis was performed with one-way ANOVA followed by Tukey–Kramer post hoc analysis. Values are expressed as mean SD. Signiﬁcance is indicated by , P < 0.05; , P < 0.01; , P < 0.001. ns, not signiﬁcant.

(around 14–21 days; 100–200 mm3), mice were treated with were relatively short-lived and the tumors reinitiated growth. IHC vehicle, MEKi (trametinib, 1 mg/kg, orally, daily), HDACi (pano- staining conﬁrmed that single-agent MEKi was associated with binostat, 20 mg/kg, i.p. 3 week), or combination of both agents increased levels of pAKT and YAP/TAZ and that the combination for 30 days. The control group received both vehicles. The com- of MEKi and HDACi simultaneously suppressed pAKT, and YAP/ bination of MEKi and HDACi led to a signiﬁcant and durable TAZ expression (Fig. 7E). Advanced uveal melanoma is typically suppression of uveal melanoma growth compared with either associated with the development of liver metastases. To explore drug alone. (Fig. S7C and S7D; Supplementary Fig. S9A and S9B). the effectiveness of the MEKi–HDACi combination in this setting, Although single-agent MEKi was more effective against MP41 we injected MP41 uveal melanoma cells into the tail veins of uveal melanoma cells than 92.1 cells, its effects in both models mice and allowed liver metastases to form (around 28 days;

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Figure 6. Identification of HDAC inhibitors as a strategy to limit escape from MEK inhibition. A, Response of individual uveal melanoma cell lines to each drug in the panel of 289 compounds. Scale indicates the percentage growth inhibition at 0.5 and 2.5 mmol/L of drug relative to vehicle. B, Detailed view of the responses of the drugs selected for follow-up in the cell line panel. Data show the inhibition of growth per cell line at 0.5 and 2.5 mmol/L of drug relative to vehicle. C, HDACi increases the cytotoxic effects of MEKi. Data show heatmaps showing the inhibition of the growth of uveal melanoma cell lines (92.1, MP41, Mel270, and OMM1) treated with MEKi (trametinib, 10 nmol/L) alone and in combination with inhibitors of HDAC1/2/3 (etinostat), HDAC6 (tubastatin), HDAC8 (PCI-03451), and pan- HDAC (panobinostat) for 72 hours before being subjected to the MTT assay. D, HDACi prevents escape from MEK inhibitor therapy. 92.1, Mel270, MP41, and OMM1 cells were treated with vehicle, MEKi alone, HDACi alone, or the combination for 4 weeks before being stained with Crystal Violet. E, Quantification of data from D. F, The MEK–HDACi combination shows increased apoptosis compared with either drug alone. 92.1, Mel270, MP41, and MM28 cells were treated with MEKi (trametinib, 10 nmol/L), HDACi (panobinostat, 10 nmol/L), or combination of both for 72 hours, and apoptosis was measured by Annexin-V binding and flow cytometry. G, The MEK–HDACi combination is associated with decreased BCL-2 and increased expression of cleaved caspase-7 and PARP by Western blot. Cells were treated with MEKi (trametinib, 10 nmol/L), HDACi (panobinostat, 10 nmol/L), or combination of both for 48 hours.

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Figure 7. The MEK-HDAC inhibitor is effective against subcutaneous xenograft and liver metastasis models of uveal melanoma through combined YAP and AKT inhibition. A, The MEKi–HDACi combination inhibits adaptive AKT signaling in uveal melanoma cells. Cells were treated with vehicle, MEKi (trametinib, 10 nmol/L), HDACi (panobinostat, 10 nmol/L), or a combination of the two for 0–72 hours and probed for phospho-AKT, AKT, and GAPDH expression. B, HDAC inhibition limits MEKi-induced YAP activity. Data show YAP activity in uveal melanoma cells following treatment with vehicle, MEKi, HDACi, or a combination of the two drugs. After the formation of palpable tumors, mice were treated with vehicle (Control group), MEKi (trametinib, 1 mg/kg orally daily), HDACi (panobinostat, 20 mg/kg, i.p., 3 week), or the combination for 31 days. Data show tumor volume. C, The MEK–HDACi combination delivers durable responses in the 92.1 uveal melanoma subcutaneous xenograft model. D, The MEK–HDACi combination delivers durable responses in the MP41 uveal melanoma subcutaneous xenograft model. Data show the mean SD. E, The combination of MEKi and HDACi suppresses pAKT and YAP/TAZ in uveal melanoma xenografts. Representative images of pAKT and YAP/TAZ expression by IHC. Magnification 100 in all images. Scale bar, 5 mm for the whole images and scale bar, 500 mm for inserts on the right side. Brown staining indicates positivity for either YAP/TAZ or pAKT. F, The combination of MEKi and HDACi suppresses growth of uveal melanoma liver metastases. Panel shows representative MRI images of representative mice at day 21 of treatment. The red circles indicate individual liver metastases. G, Mean liver metastasis volumes following 0–21 days of treatment with vehicle, MEKi, HDACi, and the drug combination. Analysis was performed with one-way ANOVA followed by Tukey–Kramer post hoc analysis. Values are expressed as mean SD. Significance is indicated by , P < 0.05; , P < 0.01; , P < 0.001. ns, not significant.

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5–10 mm3). Once the presence of liver metastases was confirmed apeutic escape. To achieve this, we used a mass spectrometry– by MRI imaging, treatment with MEKi (trametinib, 1 mg/kg, based ABPP approach to comprehensively map global protein orally, daily), HDACi (panobinostat, 20 mg/kg, i.p. 3 week), ATP uptake following MEK inhibitor treatment (20). Changes in or combination of both agents for 21 days was initiated. It was multiple pathways were identified, with some of the most prom- noted that although the MEKi was associated with some reduction inent being those associated with the organization of the cyto- in liver tumor burden, the MEKi–HDACi combination was asso- skeleton, PI3K/AKT signaling, and RTK signaling. Inhibition of ciated with more profound and durable antitumor responses than RAF and MEK is known to trigger a rapid transcriptional repro- either drug alone (Fig. 7F and G; Supplementary Fig. S10). gramming that is associated with increased RTK expression. This Together these results confirmed our in vitro findings and dem- phenomenon was first described for breast cancer, in which onstrated that the addition of a pan-HDACi could inhibit the chronic MEK inhibitor treatment led to widespread increase in pathways involved in the escape from MEK inhibitor therapy, RTK expression that allowed for recovery of signaling through limiting uveal melanoma growth at both subcutaneous and at MAPK and other pathways (37, 39). Similar findings have been clinically relevant liver metastasis sites. also reported in many other cancers including BRAF and NRAS- mutant melanoma; where BRAF and MEK inhibition frequently leads to a relief of feedback inhibition and increased signaling Discussion through multiple RTKs including IGF-1R, EGFR, ERBB3, EphA2, Although significant progress has been made in the develop- and c-MET (14, 40–43). There is good evidence that targeting ment of systemic therapies for the treatment of advanced cuta- these compensatory pathways improves the response to MAPK- neous melanoma, little improvement has been made in the targeted drugs in both in vivo mouse models and in clinical management of metastatic uveal melanoma. Unlike cutaneous settings (14, 36). To investigate whether this also occurred in melanoma, uveal melanoma has proven extremely resistant to GNAQ-mutant uveal melanoma cell lines, we performed RTK immunotherapy, with anti–CTLA-4 therapy being associated with arrays and identified increased IGF1-R and ROR1/2 activity fol- responses of <7% and no appreciable improvement in overall lowing MEK inhibition. In BRAF-mutant melanoma, RAF and survival (30, 31). The anti–PD-1 and anti–PD-L1 antibodies have MEK inhibition typically leads to recovery of MAPK signaling, and proven similarly ineffective, with the largest clinical trial to date in some cell lines, adaptive AKT signaling (27, 39). Here, we found demonstrating response rates of approximately 4% and a limited that MEK inhibition in uveal melanoma cells led to increased AKT level of disease control (32). It has been speculated that the low and FAK signaling and that was mediated through IGF-1R mutational burden, and therefore the low level of neoantigen and ROR receptors. Although the combination of MEK and expression, of uveal melanoma versus cutaneous melanoma may PI3K-AKT-mTOR inhibition was suggested to be superior to MEK underlie the lack of immunotherapy response. inhibition alone in multiple preclinical uveal melanoma mod- Another strategy to treat uveal melanoma is targeted therapy, in els (11, 12), our results demonstrated that resistance to the MEK- which kinase inhibitors are used to selectively target the oncogenic PI3K inhibitor combination still occurred. drivers responsible for tumor growth and progression. This strat- YAP is a transcriptional coactivator and tumor promoter, whose egy has been very successful in the treatment of cancers with strong nuclear localization and activity is regulated by the Hippo path- oncogene addiction such as BRAF-mutant melanoma and EGFR way. In GNAQ/GNA11–mutant uveal melanoma cells, YAP is and ALK-mutant lung cancers (33–35). Uveal melanomas typi- activated by the guanine nucleotide exchange factor Trio leading cally harbor activating mutations in the small G-proteins GNAQ to YAP activation via Rho and Rac (15, 29). Increased signaling and GNA11, which are not kinases and therefore not easily through Rho and Rac leads to increased actin dynamics and the tractable to drug development (2). Instead, the development of release of YAP from its inhibitory complex with the actin- targeted therapies in uveal melanoma has focused upon kinases associated protein angiomotin (15). Once free of this complex, and pathways downstream of GNAQ/GNA11. The most exten- YAP is free to migrate to the nucleus and initiate transcription. sively explored targeted therapy in uveal melanoma to date are the Although there is good evidence that YAP is a driver of uveal MEK inhibitors. This class of drugs are FDA-approved in the melanoma progression, this pathway has yet to be implicated in single-agent setting, and in combination with BRAF inhibitors, the escape of uveal melanoma cells from MEK inhibitor therapy. for the treatment of advanced BRAF-mutant cutaneous melano- Our results herein demonstrate that treatment with MEK inhibi- ma (17, 36). Most MEK inhibitor studies in uveal melanoma to tors increased YAP activity further and likely constituted an date have focused upon selumetinib (AZD6244). In a phase II important therapy escape mechanism. YAP signaling is known open-label clinical trial of advanced uveal melanoma, selumeti- to be activated through GPCRs, with our RNA-seq studies iden- nib treatment yielded an improved progression-free survival tifying a whole series of candidate receptors that were upregulated compared with either dacarbazine or temozolomide (9). Despite following MEK inhibition. Among these was EDNRB, a GPCR these initially encouraging results, a subsequent phase III double- activated by all three members of the endothelin family. There is blinded trial of selumetinib plus dacarbazine showed no good evidence that EDNRB signaling is involved in melanocyte improvement in PFS compared with dacarbazine alone (10). development, with studies showing severe deficits in melanocyte Although disappointing, these findings fit with our growing numbers in mice that are null for EDNRB (44, 45). EDNRB understanding of how cancer cells respond to MEK inhibition, signaling is also implicated in melanoma with levels of expression with multiple studies demonstrating that initial MEK inhibitor being correlated with melanoma progression and the increased responses are followed by adaptive signaling and transcriptional development of melanoma brain metastases in in vivo mod- changes that lead to therapeutic escape (37, 38). els (46, 47). There is also evidence from cutaneous melanoma The goal of this study was to define the patterns of adaptive that EDNRB antagonists reduce melanoma growth in vitro and in signaling in uveal melanoma cells treated with MEK inhibitor in vivo xenograft models (48, 49). Other work in BRAF-mutant therapy and to identify combination partners that limited ther- melanoma demonstrated that BRAF inhibition often leads to

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increased EDNRB receptor expression and that this confers clinically approved pan-HDAC inhibitor was effective at simul- enhanced sensitivity to the BRAF–endothelin receptor antagonist taneously limiting YAP and AKT signaling in uveal melanoma combination (50). Further evidence suggests that autocrine cells suggests this could be a good candidate for future clinical endothelin-1 might also regulate melanoma heterogeneity fol- development. At this time, the only agent with proven anti-YAP lowing BRAF inhibition and could mediate the switch to an activity is verteporfin, and while it is FDA-approved for local Axl-high/MITF-low (drug resistant) phenotype (51). We here photodynamic therapy in the treatment of macular degeneration, demonstrate that uveal melanoma cells released ET-3 in response it is unlikely to have much utility as a systemic therapy for to MEK inhibition and that the resulting increase in EDNRB metastatic uveal melanoma. Indeed, even preclinical studies in signaling activates YAP signaling, leading to increased cell surviv- xenograft models of uveal melanoma have resorted to multiple al. Although it is likely that EDNRB plays a role in the increased strategies to improve efficacy, such as mixing liposome- YAP signaling observed following MEK inhibition, it is unlikely to encapsulated verteporfin with uveal melanoma cells prior to be only GPCR involved, and it is possible that different uveal xenografting (29). We therefore believe that the combination of melanomas may have unique GPCR dependencies. One potential trametinib and panobinostat is worthy of future investigation in strategy to target multiple G-proteins (and GPCR) simultaneously patients with metastatic uveal melanoma. could be through allosteric inhibitors of GDP/GTP exchange with recent studies demonstrating that GTP exchange inhibitors such as Disclosure of Potential Conflicts of Interest the depsipeptide FR900359 have activity against GNAQ-mutant J.D. Licht reports receiving commercial research grants from Celgene. uveal melanoma cell lines (52). The increased GPCR expression J.W. Harbour holds ownership interest (including patents) in Castle Biosciences noted following MEK inhibition might be expected to increase the and is a consultant/advisory board member for Castle Biosciences, Aura adhesion of uveal melanoma cells to the extracellular matrix, Biosciences, and Immunocore. No potential conflicts of interest were disclosed potentially decreasing their metastatic potential (53). by the other authors. As our goal was to develop novel therapeutic strategies that limited adaptive signaling, we undertook a drug screen to identify Authors' Contributions potential combination partners for the MEK inhibitors. Our initial Conception and design: F. Fai~ao-Flores, M.F. Emmons, K.S.M. Smalley ~ analysis identified HDAC inhibitors as a class of drugs with Development of methodology: F. Faiao-Flores, M.F. Emmons, B. Saha, B. Fang, S.P. Chellappan, U. Rix, K.S.M. Smalley significant single-agent activity. The HDACs constitute a family Acquisition of data (provided animals, acquired and managed patients, of enzymes that catalyze the hydrolysis of acetyl groups from provided facilities, etc.): F. Fai~ao-Flores, F. Kinose, B. Saha, B. Fang, acetylated proteins, the best characterized of which being the N- S.P. Chellappan, J.W. Harbour terminal tails of histones (54). Inhibition of multiple HDACs, Analysis and interpretation of data (e.g., statistical analysis, biostatistics, using the pan-HDAC inhibitor panobinostat was found to be computational analysis): F. Fai~ao-Flores, M.F. Emmons, M.A. Durante, B. Fang, superior to multiple other epigenetic inhibitors including EZH2, J.W. Harbour, K.S.M. Smalley Writing, review, and/or revision of the manuscript: F. Fai~ao-Flores, DOTL1, HATs, and LSD1 in enhancing the cytotoxic activity of M.F. Emmons, M.A. Durante, B. Saha, B. Fang, J.M. Koomen, U. Rix, MEK inhibition. There is already good evidence that HDAC J.D. Licht, J.W. Harbour, K.S.M. Smalley inhibitors, including the class III inhibitor tenovin, and a number Administrative, technical, or material support (i.e., reporting or organizing of pan-HDAC inhibitors (TSA, depsipeptide butyrate) have activ- data, constructing databases): F. Fai~ao-Flores, J.M. Koomen ity against uveal melanoma cell lines, through affects upon FAS, Study supervision: F. Fai~ao-Flores, S.S. Maria-Engler, K.S.M. Smalley p21,p27, p53, c-JUN, and b-catenin expression (55–57). In cutaneous melanoma, there is also evidence that HDAC inhibition can Acknowledgments restore sensitivity to BRAF inhibition following the onset of The authors would like to thank Dr. Manali Phadke for technical assistance resistance (58–60). At the mechanistic level, HDAC inhibition and support. This work is supported by the Bankhead-Coley Program of the was noted to suppress both AKT and YAP signaling following MEK State of Florida 7BC05, and the NIH R21 CA216756. It has been also supported, in part, by the SAIL Core Facility, the IRAT Core Facility, the Flow Cytometry inhibition, with the effects on AKT mediated, in part, by increased Core Facility, the Analytic Microscopy Core Facility, the Proteomics and Meta- expression of the PI3K/AKT pathway suppressor PTEN. To our bolomics Core Facility, the Molecular Genomics Core and the Tissue Core knowledge, this is the first demonstration that HDAC inhibitors Facility at the H. Lee Moffitt Cancer Center & Research Institute (Tampa, FL), an inhibit YAP signaling. The effectiveness of the panobinostat– NCI designated Comprehensive Cancer Center (P30-CA076292). This work has trametinib combination was demonstrated in two in vivo uveal also been supported, in part, by Fapesp (grant no. 2013/05172-4 and 2015/ melanoma subcutaneous xenograft models, with IHC analysis 10821-7). showing the addition of panobinostat to inhibit AKT and YAP The costs of publication of this article were defrayed in part by the payment of signaling. Of clinical significance, the MEKi–HDACi combination page charges. This article must therefore be hereby marked advertisement in also had good antitumor activity against uveal melanoma liver accordance with 18 U.S.C. Section 1734 solely to indicate this fact. metastases, the major site of disseminated disease. Panobinostat is an HDAC inhibitor that was FDA-approved in 2015 for the Received October 15, 2018; revised February 1, 2019; accepted June 17, 2019; treatment of relapsed multiple myeloma. Our finding that a published first June 21, 2019.

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OF16 Clin Cancer Res; 2019 Clinical Cancer Research

HDAC Inhibition Enhances the In Vivo Efficacy of MEK Inhibitor Therapy in Uveal Melanoma

Fernanda Faião-Flores, Michael F. Emmons, Michael A. Durante, et al.

Clin Cancer Res Published OnlineFirst June 21, 2019.

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