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

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Author Manuscript Published OnlineFirst on June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3382 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

HDAC inhibition enhances the in vivo efficacy of MEK inhibitor therapy in uveal melanoma

Fernanda Faião-Flores1, Michael F. Emmons1, Michael A. Durante2, Fumi Kinose3, Biswarup Saha1, Bin Fang4, John M. Koomen4, Srikumar Chellappan1, Silvya Stuchi Maria-Engler5, Uwe Rix3, Jonathan D. Licht6, J. William Harbour2, Keiran S.M. Smalley1*

1The Department of Tumor Biology, The Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL, USA. 2Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL, USA. 3Department of Drug Discovery, The Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL, USA 4Department of Molecular Oncology, The Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL, USA. 5Department of Clinical Chemistry and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil. 6Division of Hematology & Oncology, Department of Medicine, University of Florida Health Cancer Center, University of Florida, Gainesville, FL, USA.

*To whom correspondence should be addressed Tel: 813-745-8725 Fax: 813-449-8260 e-mail: [email protected]

Keywords: uveal melanoma, MEK, HDAC, endothelin, adaptation, resistance.

Running title: HDAC-MEK inhibition in uveal melanoma

Conflict of interest: Dr. Harbour is a paid consultant for Castle Biosciences, licensee of intellectual property related to uveal melanoma, and he receives royalties from its commercialization. All other authors declare no conflict of interest

Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3382 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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

Statement of translational relevance: At this time there are no effective therapies for advanced uveal melanoma. One of the most thoroughly explored targeted therapies for uveal melanoma are small molecule inhibitors of MEK. Despite initial clinical responses to MEK inhibition, levels of progression-free survival are very short and the majority of patients fail within 3 months. Here, we used three unbiased platforms (proteomics, RNA-Seq, drug screens) to define the mechanisms by which uveal melanoma cells escaped MEK inhibitor therapy. Our studies identified a complex adaptive response involving G-protein coupled receptor (GPCR)-driven YAP activation and increased receptor tyrosine kinase (RTK)-driven AKT signaling, both of which were suppressed by the pan-HDAC inhibitor panobinostat. The combination of the MEK and HDAC inhibitor was highly effective at limiting therapeutic escape and led to durable anti-tumor responses in both subcutaneous xenograft and liver metastasis models of uveal melanoma. Together our results provide the rationale for the clinical co-targeting MEK and HDACs in advanced uveal melanoma.

Introduction Uveal melanoma is a highly aggressive tumor derived from the melanocytes of the eye, with a tendency to metastasize to the liver. Although few patients show signs of disseminated disease at diagnosis (~4%), up to half will eventually succumb to metastatic disease despite successful treatment of the primary tumor (1). The majority of uveal melanomas harbor activating mutations in the small G-proteins GNAQ and GNA11. These mutations (most commonly at Q209L/P) disable the intrinsic GTPase activity, leading to constitutive activation (2, 3). The major downstream signaling target of GNAQ and GNA11 is phospholipase-C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate to the second messengers: inositol triphosphate (IP3) and diacyl glycerol. Protein kinase C (PKC) is activated by these second messengers in GNAQ/GNA11 mutant melanomas (4). Recent work has shown that PKC and the small G-protein RasGRP3 are required for the GNAQ/GNA11-driven activation of the mitogen activated protein kinase (MAPK) pathway and that the majority of uveal melanomas have constitutive MAPK signaling that contributes to cell growth (5, 6). As a single agent, MEK inhibition has some activity against uveal melanoma cell lines, and is associated with reduced cell proliferation in vitro (7, 8). In light of this promising data, and the FDA-approval of MEK inhibitors for BRAF-mutant cutaneous melanoma, a number of clinical trials were undertaken to evaluate MEK inhibitors in uveal melanoma. In an open-label phase II clinical trial of uveal melanoma patients with no history of prior dacabarzine treatment, use of the MEK inhibitor selumetinib was associated with an increase in PFS from 7 to 16 weeks (9). These initially promising findings led to the initiation of a phase III double-blind clinical trial of selumetinib plus dacarbazine, which unfortunately failed to show any increase in PFS compared to dacarbazine alone (10). Despite these disappointing results, current strategies continue to focus upon combination therapies that include MEK inhibition as the backbone. There is promising preclinical data that indicates the combination of a MEK and a PKC inhibitor potently induces apoptosis and suppresses tumor growth in mouse xenograft models (5). Multiple other signal transduction cascades are also activated in uveal melanoma including the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway, which has been implicated in survival and cell migration (11, 12) and the Hippo tumor suppressor pathway, which plays key roles in tissue homeostasis and organ size (13). Under normal physiological conditions, the MST1/2 and LATS1/2 kinases phosphorylate and inactivate

YAP and TAZ, two transcriptional co-activators implicated in oncogenic transformation (13, 14). In uveal melanoma, GNAQ stimulates YAP through a Hippo-independent mechanism that is initiated through actin polymerization (15). Silencing of GNAQ/GNA11 in uveal melanoma cells led to decreased nuclear accumulation of YAP, with further studies showing that the YAP inhibitor verteporfin abrogates GNAQ/GNA11 driven tumor growth in an orthotopic uveal melanoma ocular xenograft model (15, 16). At this time, little is known about the systems level signaling adaptations of uveal melanoma cells to MEK inhibition. In the present study we used affinity-based protein profiling (ABPP) and RNA-Seq to identify key proteins involved in the adaptation of uveal melanoma cells to MEK inhibition, and identified novel drug combinations to overcome this adaptation.

METHODS Reagents RPMI culture medium was purchased from Corning (Corning, NY). Fetal bovine serum (FBS) was purchased from Sigma Chemical Co. (St. Louis, MO). Trypsin, pen/strep antibiotics, and puromycin were purchased from Gibco (Grand Island, NY). Trametinib (MEK inhibitor), Panobinostat (pan-HDAC inhibitor), Pictilisib (PI3K inhibitor), Bosentan Hydrate (EDNRB inhibitor), Verteporfin (YAP inhibitor), Entinostat (HDAC1/2/3 inhibitor), and Tubastatin A (HDAC 6 inhibitor) were purchased from Selleckchem (Houston, TX). PCI-34051 (HDAC8 inhibitor) was purchased from Cayman Chemical (Ann Arbor, MI). Endothelin-3 was purchased from Sigma Chemical Co. (St. Louis, MO). WNT5A was purchased from R&D Systems (Minneapolis, MN, USA). Antibodies for Western Blot and immunochemistry were purchased from Cell Signaling Technology (Danvers, MA), Sigma Chemical Co. (St. Louis, MO), Millipore (Bedford, MA) and Abcam (Cambridge, MA). The phospho-Receptor Tyrosine Kinase and phospho-Kinase array were purchased from R&D Systems (Minneapolis, MN, USA). Opti‐MEM medium, Lipofectamine 2000 and Live/Dead viability stain were purchased from Invitrogen/Life Technologies Corp). siRNA for ROR1/2, IGF-1R and YAP were purchased from Dharmacon RNA Technologies (Lafayette, CO). Nontargeting siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Endothelin-3 Assay Kit was purchased from IBL (Takasaki, Japan).

Uveal melanoma cell lines

The uveal melanoma cell lines 92.1, Mel270, OMM1, MP41 AND MM28 were used as previously described (17). All uveal melanoma cell lines were cultured in RPMI-1640 supplemented with 10% FBS, L-glutamine and antibiotics at 5% CO2. All cells were tested for mycoplasma contamination every month using the Plasmotest-Mycoplasma Detection Test (Invivogen, San Diego, CA). Last test date: 4/18/19. Each cell line was authenticated using the Human STR human cell line authentication service (ATCC) and frozen stocks of cells were discarded after 10 passages.

Cell viability assay (MTT assay) Uveal melanoma cells were plated in triplicate wells (1 × 103 cells per well) and treated with increasing concentrations of MEK inhibitor (trametinib) for 72 h. Cell viability was determined using the MTT assay as described previously (18).

Colony formation assay 1×103 cells were plated and allowed to attach overnight. The medium and drug/vehicle was replaced every two days for 4 weeks. After the specific treatments for each experiment, colonies were stained with crystal violet dye, as previously described (18).

Flow cytometry for apoptosis analysis 1×105 cells were plated and allowed to attach overnight. After the specific treatments for each experiment, Annexin V staining quantification was performed using FlowJo software as previously described (19).

Activity-based Protein Profiling (ABPP) ABPP experiments were carried out as previously published (20). Briefly, 92.1 and Mel270 cells were treated with MEKi (25nM for 24h) and then solubilized with lysis buffer. A 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 MaxQuant (v. 1.2.2.5), the values were log2 transformed and normalized (21). Signaling pathways, protein interaction and process network analysis were carried out using MetaCore™ (GeneGO, St Joseph, MI). Data are available in PRIDE (PXD013988).

RNA sequencing (RNA-Seq)

RNA was extracted from 92.1 and Mel270 uveal melanoma cell lines using Qiagen’s Rneasy Mini Kit (Qiagen, Hilden, Germany), and screened for quality on an Agilent BioAnalyzer. The samples were then processed for RNA-sequencing using the NuGen Ovation Human FFPE RNA-Seq Multiplex System. Briefly, 100 ng of RNA was used to generate cDNA, and a strand-specific library following the manufacturer’s protocol (NuGEN Technologies, Inc., San Carlos, CA). Quality control steps including analysis on the BioAnalyzer RNA chip and quantitative RT-PCR for library quantification were performed. The libraries were then sequenced on the Illumina NextSeq 500 sequencer with a 2 X 75-base paired-end run in order to generate 40-50 million read pairs per sample. RNAseq data were preprocessed for quality assessments before aligning to the human genome hs37d5 using Tophat 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, and differential expression was analyzed using a Wald statistic test implemented in the DESeq function in the DESeq2 Bioconductor package, which performs serial dispersion estimation and negative binomial generalized linear model fitting procedure. A Benjamini-Hochberg adjusted p-value of less than 0.05 was used as a cutoff to determine significantly differentially expressed genes. Data are available in GEO (GSE127948).

Gene set enrichment analysis (GSEA) Gene set enrichment analysis (GSEA)(22) was conducted utilizing recommended parameters (http://software.broadinstitute.org/gsea/doc/GSEAUserGuideFrame.Html) with gene sets obtained from the Molecular Signatures Database (23) and custom gene sets obtained from the GSEA database.

Phospho-Receptor Tyrosine Kinase and Phospho-Kinase array analysis A Human Phospho-RTK Array Kit (ARY001B) and a Human Phospho-kinase Array Kit (ARY003B) were used to measure the relative level of different RTKs and kinases. 92.1 and Mel270 uveal melanoma cell lines were treated with MEKi (10nM for 48h), the lysed, and 300 µg protein were incubated with the array according to the manufacturer’s protocol.

Immunoblotting

Cells were plated and allowed to attach overnight. After the specific treatments for each experiment, proteins were extracted and blotted as previously described(24). Total and phospho-proteins were analyzed (Supplementary Table S1) and then the membranes were stripped and reprobed for GAPDH/vinculin/β-actin.

Quantitative real-time PCR Total RNA was isolated using Qiagen’s Rneasy Mini Kit (Qiagen, Hilden, Germany). TaqMan Gene Expression Assay primer/probes were used as shown in Supplementary Table S2. GAPDH was used to normalize the genes of interest. Quantitative reverse transcriptase–PCR (RT-PCR) reactions were carried out as described previously (18).

siRNA (Small interfering RNA) Uveal melanoma cell lines cells were plated and allowed to attach overnight in complete RPMI medium with 10% FBS. After 24h, this medium was replaced with Opti-MEM and the cells were transfected with 50nm siRNA for ROR1 (Dharmacon SMARTpool; L- 003171-00-0005), 50 nm siRNA for ROR2 (Thermo-Fisher; #4390824), or 50nm siRNA for IGF-1R (Dharmacon SMARTpool; L-003012-00-0005) in complex with Lipofectamine 2000 overnight. Nontargeting siRNA was added as a siRNA control (Santa Cruz; sc- 37007). After 24h of the transfection, medium was replaced by complete RPMI medium with 10% FBS and treated with MEKi (10nM) for 72h.

Cell proliferation assay 5×104 cells were plated and allowed to attach overnight. After the specific treatments for each experiment, cells were counted using trypan blue reagent. The percentage of total cells was normalized to the percentage of control cells as previously described (25).

Transfections and luciferase assays We have used a YAP/TAZ-responsive synthetic promoter driving reporter plasmid, named 8xGTIIC-luciferase (Addgene, #34615) for the assay. Overnight seeded 92.1 and Mel270 cells (120 X 103 cells per well in a 6-well plate) were transfected with the construct using FuGENE (Promega; #31985-070) for 8h. Media was changed for another 12h prior to the drug(s) treatment with MEKi (10nM), HDACi (10nM) or both for 72h. Harvested cells were washed once with PBS and luciferase assay was performed

according to the manufacturer protocol and plotted the values normalized against Control, without any inhibitor treatment.

Immunofluorescence 3×103 cells were seeded in 8-Well Lab-Tek II Chamber Slides (Naperville, IL) and allowed to attach overnight. On the next day, cells were treated, then fixed, permeabilized and stained using YAP antibody (#14729). The information about the immunofluorescence antibodies can be found in Supplementary Table S3. Slides were mounted with ProLong Antifade with DAPI in accordance with a previously described protocol (26). Glass slides were observed with a Leica TCS SP8 AOBS laser scanning confocal microscope through a 63X/1.4NA oil immersion Plan Apochromat objective. Laser line at 405nm was used to excite the DAPI fluorophores. Images were captured at 200Hz scan speed with photomultiplier detectors using LAS X software version 3.1.5. Images were analyzed using the Definiens Tissue Studio v4.7 (Definiens AG, Munich, Germany) software suite. Cells were segmented by the nuclear stain DAPI (blue) and phalloidin (red) channel was used as cytoplasm marker for cell simulation. The image was analyzed as an 8 bit image and intensity of each RGB channel was measured from 0-255 grayscale fluorescent units. The cells were then quantified for green and red intensity per field for nucleus and for cytoplasm.

Endothelin-3 Assay Kit 92.1, Mel270, MP41 and OMM1 uveal melanoma cells were seeded in 96-well plates at 1.0×104 cells/well. After 24h, cells were treated with MEKi (10nM), HDACi (10nM) or MEKi+HDACi (10nM each one) for 24-72h. Cell supernatent was collect and incubated according to the manufacturer’s protocol.

Drug screening 92.1, Mel270, MP41 and OMM1 uveal melanoma cells were seeded in 384-well plates at 1.0×103 cells/well. All compounds of the library were diluted to 0.5 or 2.5μM, and the experiment was performed in duplicate. A total of 289 compounds from an in-house library were tested. Compounds were aliquoted by a Biotek Precision Pipetting robot. Cell viability was measured by Cell-Titer-Glo (Promega G7572) at 72h post treatment.

Animal experiments

Subcutaneous xenograft model Eight week old female CBySmn.CB17-Prdkc scid/j mice (Stock No: 001803 - Jax) were subcutaneously injected with 1.0 ×106 92.1 or MP41 uveal melanoma cells per mouse. The tumors were allowed to grow for 3 weeks and mice were randomly separated with similar average initial tumor volumes, with a total of three mice per group. Liver metastasis model Eight week old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (Stock No: 005557 - Jackson Laboratory) were injected with 2.0 ×105 MP41 uveal melanoma cells per mouse into the tail vein i. The tumors were allowed to grow for 4 weeks and mice were randomly separated with similar average initial tumor volumes, with a total of three mice per group. The mice were imaged on 7T horizontal magnet (Agilent ASR 310) and (Bruker Biospin, Inc. BioSpec AV3HD), using a 35 mm birdcage coil (m2m imaging corp). Anatomical coronal images, were obtained with a TurboRARE sequence with echo time/repetition time (TR/TE) = 1585 / 15 ms, 33 slices, slice thickness of 0.6 mm, field of view (FOV) = 30 x 30 mm2, image size 256 X 256. Respiration gating was used to minimize motion artifacts. Liver metastases were manually contoured on all MR slices using ImageJ (https://imagej.nih.gov/ij/index.html). A custom program was written in MATLAB (MATLAB 2018a, The MathWorks, Natick, 2018.) to extract voxels within the manually drawn contours and compute total metastases volume burden and individual metastases volumes, in mm3. In both protocols, mice were treated with MEKi (Trametinib – 1mg/kg – gavage - daily), HDACi (Panobinostat – 20mg/kg – IP – three times a week) or the combination of both agents for 30 days (xenograft model) or for 21 days (liver metastasis model). The control group received both vehicles (for Trametinib: 0.5% methylcellulose + 0.5% Tween-80 molecular grade sterile water; for Panobinostat: 5% dextrose in water). Mouse weight and tumor volumes (½ x L x W2) were measured every 72 hours. All animal experiments were carried out in agreement with ethical regulations and protocols approved by the University of South Florida Institutional Animal Care and by The Institutional Animal Care and Use Committee (IACUC number IS00002983). The IACUC protocol did not permit survival to be an experimental endpoint.

Statistical analysis Results are expressed as mean ± standard deviation of a triplicate of at least three independent experiments. One-way analysis of variance (ANOVA) was used followed by a TUKEY-KRAMER posttest to test for multiple comparisons with a given significance

level of p<0.05. Significant differences between the control and treated groups are indicated by ***/&p<0.001, **/&p<0.01 and */&p<0.05.

RESULTS Affinity based proteomic profiling (ABPP) identifies signaling pathways implicated in the escape of uveal melanoma cells from MEK inhibitor therapy We began by characterizing the MEK inhibitor response of a panel of GNAQ/GNA11 mutant uveal melanoma cell lines that were derived from primary and metastatic lesions (92.1, Mel270, MP41: primary, OMM1 and MM28: metastatic). It was found that although the MEK inhibitor trametinib (MEKi) inhibited the growth of all of the uveal melanoma cell lines, these reductions were modest (Figure 1A; Supplementary Table S4), and associated with regrowth of colonies in all cases (Figure 1B,C). Levels of MEKi (10-25 nM trametinib)-induced apoptosis were also minor compared to those seen in cutaneous melanoma (27) (Figure 1D). Little apoptosis induction was observed at 24 h. To better understand the process of adaptation that occurs when uveal melanoma cells are treated with a MEKi, we treated two GNAQ-mutant uveal melanoma cell lines (92.1 and Mel270) with trametinib (25nM, 24h) and performed activity-based protein profiling (ABPP) (20) (Figure 1E). This method, which uses mass spectrometry to quantify ATP uptake levels of proteins through transfer of a desthiobiotin tag to lysine in the active site of enzymes and kinases, allows signaling activity to be mapped in a comprehensive manner (Figure 1E) (20). The ABPP studies demonstrated that MEK inhibition increased the ATP uptake of 128 proteins and 98 proteins in the 92.1 and Mel270 cells, respectively (Figure 1F). Use of STRING analysis allowed us to identify an enrichment for activated proteins implicated in proliferation and survival, ribosome function, metabolism and the cytoskeleton (Figure 1G). The top pathways modulated by MEKi treatment included cell metabolism, cytoskeletal remodeling, apoptosis, AKT signaling, IGF signaling, WNT signaling, FGFR signaling and melanocyte differentiation (Supplementary Figure 1).

Trametinib induces adaptive AKT signaling in uveal melanoma cells We next focused on the potential therapeutic escape pathways identified from our ABPP screen and first considered the PI3K/AKT pathway. GSEA analysis of RNA-Seq data generated from uveal melanoma cells treated with trametinib (25nM, 24 hr) demonstrated an enrichment for genes implicated in PI3K/AKT signaling (Figures 2A;

Supplementary Table 5). This was confirmed in kinome arrays that showed a consistent upregulation of AKT in both cell lines following MEK inhibition (Figure 2B) and an increase in FAK signaling in the Mel270 cells - confirming the link between MEKi and cytoskeletal rearrangement observed in the ABPP data. The AKT data were confirmed by Western Blot, with MEKi found to increase phosphorylation of AKT at T308 (Figure 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 inhibitor pictilisib (PI3Ki) to significantly increase the apoptotic response to trametinib (MEKi) (Figure 2D). Although there was some evidence that the PI3Ki also suppressed the outgrowth of MEK inhibitor treated uveal melanoma cells in colony formation assays, the effects were incomplete and tumor cells were still able to evade therapy (Figure 2E,F).

IGF1R and ROR1/2 activate AKT signaling following MEK inhibition As adaptive AKT signaling frequently results from increased RTK signaling we returned to our RNA-Seq data and identified an increase in receptor protein kinase (ES score 0.41) and receptor tyrosine kinase (ES score 0.44) expression (Figure 3A; Supplementary Tables 6,7). These findings were confirmed by RTK arrays, with MEKi being found to increase the phosphorylation of multiple RTKs including IGF-1R (in the 92.1 cells), as well as ROR1 and ROR2 (in both the 92.1 and Mel270 cell lines) (Figures 3B). q-RT-PCR and Western Blot analyses demonstrated increases in IGF-1R, ROR1 and ROR2 mRNA and protein expression following MEKi (Figure 3C,D). Although the link between IGF-1R and AKT signaling is well known, less is known about whether ROR1 and ROR2 activate AKT signaling. Treatment of 92.1 uveal melanoma cells with WNT5A, the ligand for ROR1 and ROR2 receptors (28), confirmed a time-dependent increase in AKT phosphorylation (Figure 3E). Silencing of ROR1/2 (Figure 3F) in the 92.1 cells inhibited the increases in AKT phosphorylation observed following MEKi treatment (Figure 3G). Silencing of IGF-1R in combination with the MEKi was found to increase cell death and decrease the numbers of 92.1 cells, but not Mel270 cells, a result consistent with the increased IGF-1R signaling seen only in the 92.1 cell line (Figure 3F,H,I). In contrast, silencing of ROR1/2 enhanced the effects of MEKi in terms of decreased cell survival and apoptosis induction in both of the cell lines evaluated (Figures 3F,H,I).

MEK inhibition increases YAP activity leading to increased cell survival As inhibition of PI3K/AKT did not fully abrogate escape from MEK inhibition we next turned our attention to other possible pathways. One signal transduction cascade known to be critical for uveal melanoma progression, frequently upregulated following cytoskeletal rearrangement is the pro-oncogenic mediator of Hippo signaling, YAP (15, 29). Analysis of the RNA-seq data by GSEA showed a significant gene enrichment (ES score 0.27) for Hippo pathway targets with a positive correlation among the genes overexpressed after MEKi treatment (10nM, 24h) (Figure 4A; Supplementary Table 8). Although YAP is constitutively activated in uveal melanoma cells, MEKi was noted to further stimulate YAP transcriptional activity in a reporter assay (Figure 4B) and this was accompanied by an increase in the levels of nuclear YAP accumulation (Figure 4C,D). The increase in mRNA levels of a number of YAP pathway transcriptional target including YAP, connective tissue growth factor (CTGF), amphiregulin (AREG) and cysteine rich angiogenic inducer 61 (CYR61) also occurred following MEKi treatment (Figure 4E). Increased expression of two of the main transcriptional targets of YAP/TAZ activity including CTGF and AREG was also seen by Western Blot after MEKi treatment (10nM – 72h) (Figure 4F). The role of YAP signaling in therapeutic escape was demonstrated by the ability of the YAP inhibitor verteporfin (YAPi) to decrease colony formation in response to MEK inhibition compared to either drug alone (Figure 4G,H). Additionally, siRNA knockdown of YAP (Figure 4I) increased the level of MEKi -induced apoptosis in multiple uveal melanoma cell lines (Figure 4J).

Adaptive GPCR signaling increases YAP activity following MEK inhibition G-protein coupled receptors (GPCRs) are known to be strong activators of YAP signaling. In line with this, it was found that MEK inhibition led to a strong induction of GPCR expression in our GSEA analysis (Figure 5A; Supplementary Table 9). Multiple GPCRs showed increased expression including GPR158, GP133 and endothelin- receptor B (EDRNB) (Figure 5B). A potential role for EDRNB signaling in the adaptive YAP signaling was confirmed through studies in which exogenous endothelin-3 (ET-3) was found to increase both YAP reporter activity, nuclear localization of YAP and induction of YAP-target genes in four uveal melanoma cell lines (Figures 5C,D and Supplemental Figures 2A-C). In each case, the EDNRB antagonist bosentan was found to block the ET-3-mediated increases in YAP activation (Figures 5C,D: Supplementary Figures 2A-C). Mechanistically, it was noted that MEKi treatment led to the release of

ET-3 from the uveal melanoma cell lines by ELISA (Figure 5E) and that the MEKi- mediated increase in YAP reporter activity could be abrogated by the EDNRB antagonist (Figure 5F).Together these results suggested that MEK inhibition led to ET-3 release from the uveal melanoma cells and this functioned in an autocrine manner to activate YAP through EDNRB. No increases in YAP activitity were found following treatment with ET-1 (Supplementary Figure 3).

Histone deacetylase inhibitors increase the effects of MEK inhibition in uveal melanoma Our studies identified both RTK-mediated AKT and GCPR-driven YAP signaling as pathways utilized by uveal melanoma cells to escape MEKi therapy. As there are no known drugs that target both AKT and YAP we performed an unbiased screen of 289 compounds to identify potential drug combination partners that could limit escape from MEK inhibition (drugs listed in Supplementary Table 10). The drug library used covers all major target classes, including kinases, receptor tyrosine kinases, phosphatases, receptor agonists, proteases/proteasome, PARP1, epigenetic enzymes, Hedgehog, HSP90 and Notch, and reflects the current landscape of targeted agents approved for use or have been considered for clinical development (Figure 6A). Among these, several drugs were identified with some activity against one or more uveal melanoma cell lines including PI3K inhibitors (GSK2126458, idelalisib), two kinesin inhibitors (Ispinesib, SB743921), CDK inhibitors (dinaciclib), H3K27 histone demethylase (GSK-J4), and mTOR (Sapanisertib) (Figure 6B). The drug class with the most prominent effect across all four cell lines were the HDAC inhibitors (HDACi) (Figure 6B). To further determine whether other epigenetic inhibitors could also enhance the effects of MEKi (trametinib) we evaluated inhibitors of DOTL1 (EPZ5676), EZH2 (Tazemetostat), LSD1 (GSK 2879552), DNMT (decitabine), HAT (anacardic acid) and HDACi (panobinostat) alone and in combination with trametinib. These studies demonstrated that the pan-HDACi panobinostat was the most effective at enhancing the anti-proliferative effects of MEKi (Supplementary Figure 4). We next turned our attention to more specific HDACis, including entinostat (HDAC1/2/3i), tubastatin (HDAC6i) and PCI-34051 (HDAC8i) and noted that panobinostat (HDACi) was the most effective among all of these across all four cell lines in both MTT and colony formation assays (Figure 6C-E). Further support for the potential role of HDAC activity in the escape of uveal melanoma cells from MEKi therapy was suggested by the increase in global protein deacetylation observed in our

uveal melanoma cell lines following MEKi treatment (Supplementary Figure 5). It was found that co-treatment of multiple uveal melanoma cell lines, including 92.1, MP41, Mel270 and MM28 with the MEKi-HDACi (trametinib-panobinostat) combination was associated with significantly (P<0.05) higher levels of apoptosis compared to either single agent (Figure 6F). These effects were specific to uveal melanoma cells, with no apoptosis seen in 3 different primary uveal melanocyte cell lines (Supplementary Figure 6). The apoptotic response was paralleled by an increased induction of cleaved caspase-7 and cleaved PARP in uveal melanoma cell lines (Figure 6G).

Trametinib plus panobinostat induces uveal melanoma regression in vivo. We next asked whether the enhanced therapeutic efficacy of MEKi and HDACi resulted from the combined inhibition of YAP and AKT signaling. It was found that co-treatment of the uveal melanoma cells with panobinostat and trametinib effectively suppressed the adaptive AKT and YAP signaling and the release of ET-3 seen following MEK inhibitor treatment (Figure 7A,B; Supplementary Figure 7). From a mechanistic standpoint, we identified the PI3K/AKT pathway as being significantly downregulated by GSEA analysis of our RNA-Seq dataset (Supplementary Figure 8A). A pairwise comparison of the effects of each drug demonstrated the MEKi-HDACi combination to also strongly induce PTEN at the mRNA and protein level (Supplementary Figures 8B,C; Supplementary Table S11). To validate the MEKi-HDACi combination in vivo we first generated xenografts of two human uveal melanoma models (92.1 and MP41). After formation of a palpable tumor in the subcutaneous model (around 14-21 days; 100–200 mm3), mice were treated with vehicle, MEKi (trametinib, 1mg/kg, PO, daily), HDACi (panobinostat, 20mg/kg, IP 3X week) or combination of both agents for 30 days. The control group received both vehicles. The combination of MEKi-HDACi led to a significant and durable suppression of uveal melanoma growth compared to either drug alone. (Figures 7C-D and Supplementary Figure 9A,B). Although single-agent MEKi was more effective against MP41 uveal melanoma cells than 92.1 cells, its effects in both models were relatively short lived and the tumors re-initiated growth. IHC staining confirmed that single agent MEKi was associated with increased levels of pAKT and YAP/TAZ and that the combination of MEKi-HDACi simultaneously suppressed pAKT, and YAP/TAZ expression (Figure 7E). Advanced uveal melanoma is typically associated with the development of liver metastases. To explore the effectiveness of the MEKi-HDACi combination in this setting, we injected MP41 uveal melanoma cells into the tail veins of

mice and allowed liver metastases to form (around 28 days; 5-10 mm3). Once the presence of liver metastases was confirmed by MRI imaging, treatment with MEKi (trametinib, 1mg/kg, PO, daily), HDACi (panobinostat, 20mg/kg, IP 3X week) or combination of both agents for 21 days) was initiated. It was noted that although the MEKi was associated with some reduction in liver tumor burden, the MEKi-HDACi combination was associated with more profound and durable anti-tumor responses than either drug alone (Figure 7F,G; Supplementary Figure 10). Together these results confirmed our in vitro findings and demonstrated that the addition of a pan-HDACi could inhibit the pathways involved in the escape from MEK inhibitor therapy, limiting uveal melanoma growth at both subcutaneous and at clinically-relevant liver metastasis sites.

DISCUSSION Although significant progress has been made in the development of systemic therapies for the treatment of advanced cutaneous melanoma, little improvement has been made in the management of metastatic uveal melanoma. Unlike cutaneous melanoma, uveal melanoma has proven extremely resistant to immunotherapy, with anti-CTLA-4 therapy being associated with responses of <7% and no appreciable improvement in overall survival (30, 31). The anti-PD-1 and anti-PD-L1 antibodies have proven similarly ineffective, with the largest clinical trial to date demonstrating response rates of ~4% and a limited level of disease control (32). It has been speculated that the low mutational burden, and therefore the low level of neoantigen expression, of uveal melanoma vs. cutaneous melanoma may underlie the lack of immunotherapy response. Another strategy to treat uveal melanoma is targeted therapy, in which kinase inhibitors are used to selectively target the oncogenic drivers responsible for tumor growth and progression. This strategy has been very successful in the treatment of cancers with strong oncogene addiction such as BRAF-mutant melanoma and EGFR and ALK-mutant lung cancers (33-35). Uveal melanomas typically harbor activating mutations in the small G-proteins GNAQ and GNA11 which are not kinases and therefore not easily tractable to drug development (2). Instead, the development of targeted therapies in uveal melanoma has focused upon kinases and pathways downstream of GNAQ/GNA11. The most extensively explored targeted therapy in uveal melanoma to date are the MEK inhibitors. This class of drugs are FDA-approved in the single agent setting, and in combination with BRAF inhibitors, for the treatment of advanced BRAF-mutant cutaneous melanoma (17, 36). Most MEK inhibitor studies in uveal melanoma to date

have focused upon selumetinib (AZD6244). In a phase II open-label clinical trial of advanced uveal melanoma, selumetinib treatment yielded an improved progression-free survival compared to either dacarbazine or temozolomide (9). Despite these initially encouraging results, a subsequent phase III double-blinded trial of selumetinib plus dacarbazine showed no improvement in PFS compared to dacarbazine alone (10). Although disappointing, these findings fit with our growing understanding of how cancer cells respond to MEK inhibition, with multiple studies demonstrating that initial MEK inhibitor responses are followed by adaptive signaling and transcriptional changes that lead to therapeutic escape (37, 38). The goal of the present study was to define the patterns of adaptive signaling in uveal melanoma cells treated with MEK inhibitor therapy and to identify combination partners that limited therapeutic escape. To achieve this, we used a mass spectrometry-based ABPP approach to comprehensively map global protein ATP uptake following MEK inhibitor treatment (20). Changes in multiple pathways were identified, with some of the most prominent being those associated with the organization of the cytoskeleton, PI3K/AKT signaling and RTK signaling. Inhibition of RAF and MEK is known to trigger a rapid transcriptional reprogramming that is associated with increased RTK expression. This phenomenon was first described for breast cancer, in which chronic MEK inhibitor treatment led to widespread increase in RTK expression that allowed for recovery of signaling through MAPK and other pathways (37, 39). Similar findings have been 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 through multiple RTKs including IGF-1R, EGFR, ERBB3, EphA2 and c-MET (14, 40-43). There is good evidence that targeting these compensatory pathways improves the response to MAPK-targeted drugs in both in vivo mouse models and in clinical settings (14, 36). To investigate whether this also occurred in GNAQ-mutant uveal melanoma cell lines, we performed RTK arrays and identified increased IGF1-R and ROR1/2 activity following MEK inhibition. In BRAF-mutant melanoma, RAF and MEK inhibition typically leads to recovery of MAPK signaling, and in some cell lines, adaptive AKT signaling(27, 39). Here, we found that MEK inhibition in uveal melanoma cells led to increased AKT and FAK signaling and that was mediated through IGF-1R and ROR receptors. Although the combination of MEK and PI3K-AKT-mTOR inhibition was suggested to be superior to MEK inhibition alone in multiple preclinical uveal

melanoma models (11, 12), our results demonstrated that resistance to the MEK-PI3K inhibitor combination still occurred. YAP is a transcriptional co-activator and tumor promoter, whose nuclear localization and activity is regulated by the Hippo pathway. In GNAQ/GNA11 mutant uveal melanoma cells, YAP is activated by the guanine nucleotide exchange factor Trio leading to YAP activation via Rho and Rac (15, 29). Increased signaling through Rho and Rac leads to increased actin dynamics and the release of YAP from its inhibitory complex with the actin-associated protein angiomotin (15). Once free of this complex, YAP is free to migrate to the nucleus and initiate transcription. Although there is good evidence that YAP is a driver of uveal melanoma progression, this pathway has yet to be implicated in the escape of uveal melanoma cells from MEK inhibitor therapy. Our results herein demonstrate that treatment with MEK inhibitors increased YAP activity further and likely constituted an important therapy escape mechanism. YAP signaling is known to be activated through GPCRs, with our RNA-seq studies identifying a whole series of candidate receptors that were upregulated following MEK inhibition. Among these, was endothelin receptor B (EDNRB), a GPCR activated by all three members of the endothelin family. There is good evidence that EDNRB signaling is involved in melanocyte development, with studies showing severe deficits in melanocyte numbers in mice that are null for EDNRB (44, 45). EDNRB signaling is also implicated in melanoma with levels of expression being correlated with melanoma progression and the increased development of melanoma brain metastases in in vivo models (46, 47). There is also evidence from cutaneous melanoma that EDNRB antagonists reduce melanoma growth in vitro and in in vivo xenograft models (48, 49). Other work in BRAF-mutant melanoma demonstrated that BRAF inhibition often leads to increased EDNRB receptor expression and that this confers enhanced sensitivity to the BRAF-Endothelin receptor antagonist combination (50). Further evidence suggests that autocrine Endothelin-1 might also regulate melanoma heterogeneity following BRAF inhibition and could mediate the switch to an Axl-high/MITF-low (drug resistant) phenotype (51). We here demonstrate that uveal melanoma cells released ET-3 in response to MEK inhibition and that the resulting increase in EDNRB signaling activates YAP signaling, leading to increased cell survival. Although it is likely that EDNRB plays a role in the increased YAP signaling observed following MEK inhibition, it is unlikely to be only GCPR involved, and it is possible that different uveal melanomas may have unique GPCR dependencies. One potential strategy to target multiple G-proteins (and GPCRs) simultaneously could be

through allosteric inhibitors of GDP/GTP exchange with recent studies demonstrating that GTP exchange inhibitors such as the depsipeptide FR900359 have activity against GNAQ-mutant uveal melanoma cell lines (52). The increased GPCR expression noted following MEK inhibition might be expected to increase the adhesion of uveal melanoma cells to the extracellular matrix, potentially decreasing their metastatic potential (53). As our goal was to develop novel therapeutic strategies that limited adaptive signaling, we undertook a drug screen to identify potential combination partners for the MEK inhibitors. Our initial analysis identified HDAC inhibitors as a class of drugs with significant single agent activity. The HDACs constitute a family of enzymes that catalyze the hydrolysis of acetyl groups from acetylated proteins, the best characterized of which being the N-terminal tails of histones (54). Inhibition of multiple HDACs, using the pan- HDAC inhibitor panobinostat was found to be superior to multiple other epigenetic inhibitors including EZH2, DOTL1, HATs and LSD1 in enhancing the cytotoxic activity of MEK inhibition. There is already good evidence that HDAC inhibitors, including the class III inhibitor tenovin, and a number of pan-HDAC inhibitors (TSA, depsipeptide butyrate) have activity against uveal melanoma cell lines, through affects upon FAS, p21,p27, p53, c-JUN and -catenin expression (55-57). In cutaneous melanoma there is also evidence that HDAC inhibition can restore sensitivity to BRAF inhibition following the onset of resistance (58-60). At the mechanistic level HDAC inhibition was noted to suppress both AKT and YAP signaling following MEK inhibition, with the effects on AKT mediated in part by increased expression of the PI3K/AKT pathway suppressor PTEN. To our knowledge this is the first demonstration that HDAC inhibitors inhibit YAP signaling. The effectiveness of the panobinostat-trametinib combination was demonstrated in two in vivo uveal melanoma subcutaneous xenograft models, with IHC analysis showing the addition of panobinostat to inhibit AKT and YAP signaling. Of clinical significance, the MEKi-HDACi combination also had good anti-tumor activity against uveal melanoma liver metastases, the major site of disseminated disease. Panobinostat is an HDAC inhibitor that was FDA-approved in 2015 for the treatment of relapsed multiple myeloma. Our finding that a clinically approved pan-HDAC inhibitor was effective at simultaneously limiting YAP and AKT signaling in uveal melanoma cells suggests this could be a good candidate for future clinical development. At this time, the only agent with proven anti-YAP activity is verteporfin, and while it is FDA-approved for local photodynamic therapy in the treatment of macular degeneration, it is unlikely to have much utility as a systemic therapy for metastatic uveal melanoma. Indeed, even

preclinical studies in xenograft models of uveal melanoma have resorted to multiple strategies to improve efficacy, such as mixing liposome-encapsulated verteporfin with uveal melanoma cells prior to xenografting (29). We therefore believe that the combination of trametinib and panobinostat is worthy of future investigation in patients with metastatic uveal melanoma.

ACKNOWLEDGEMENTS This work is supported by the Bankhead-Coley Program of the State of Florida 7BC05, and the National Institutes of Health R21 CA216756. It has been also supported in part by the SAIL Core Facility, the IRAT Core Facility, the Flow Cytometry Core Facility, the Analytic Microscopy Core Facility, the Proteomics and Metabolomics Core Facility, the Molecular Genomics Core and the Tissue Core Facility at the H. Lee Moffitt Cancer Center & Research Institute, an NCI designated Comprehensive Cancer Center (P30- CA076292). This work has also been supported in part by Fapesp (grant number 2013/05172-4 and 2015/10821-7). The authors would like to thank Dr. Manali Phadke for technical assistance and support.

References

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Figure legends

Figure 1: MEK inhibition rewires the signaling network of uveal melanoma cells. A) A) MTT assay showing the anti-proliferative 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 1nM 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, 10-25nM, 24, 72 h) 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 25nM, 24h) 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.

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 cell were treated with MEKi (trametinib 10nM, 48 hr) 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 hr, 10nM) 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, 10nM) and a PI3Ki (pictlisib, 3M) leads to enhanced apoptosis. Cells were treated with vehicle, MEKi alone, PI3Ki alone or the combination for 72h. 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 (1nM), PI3Ki alone (300nM) or the combination for 4 weeks before being stained with Crystal Violet. F) Quantification of the experiment from E).

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 (10nM, 48 h) before being analyzed by RTK array. MEK inhibition (10nM, 72 h) increases IGF-1R, ROR1 and ROR2 C) mRNA expression by RT-PCR and D) protein expression in 92.1 and Mel270 cells. E) WNT5A activates AKT signaling in 92.1 cells. Cell cultures were treated with WNT5A (200ng/mL, 0-120 min) 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 (10nM, 72h) and apoptosis was measured by Annexin-V binding and flow

cytometry. Values are expressed as mean ±s.d. Significance is indicated by *p<0.05, **p<0.01 and ***p<0.001 when comparing Control vs 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.

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. B) MEKi (trametinib 10nM, 48 hr) induces YAP activity in 92.1 and Mel270 cells in a reporter assay and C) enhances YAP nuclear accumulation in 92.1 cells. 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 shows q-RT-PCR for 92.1 and Mel270 cells following treatment with MEKi (10nM, 48 h) 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 (1nM), YAPi (Verteporfin, 100nM) 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 (10nM) for 72h and apoptosis quantified by Annexin-V binding and flow cytometry. Analysis was performed with one-way analysis of variance (ANOVA) followed by Tukey– Kramer post hoc analysis. Values are expressed as mean ± s.d. Significance is indicated by *p<0.05, **p<0.01 and ***p<0.001. For the siRNA experiments significance is indicated by &p<0.05 and &&p<0.01.

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 (100nM, 1 hr), EDNRB antagonist (bosentan, pre-treatment with 80M, 1h) or ET-3 + bosentan (100nM and 80M, respectively). D) ET-3 increases expression of YAP target mRNAs in 92.1, Mel270, MP41 and OMM1 cells. Data shows q-RT-PCR analysis of YAP, CTGF and CYR61. E) MEKi leads to ET-3 release from uveal melanoma cells. Cells were treated with MEKi (10nM 0-72 h) and the supernatant analyzed by ELISA. F)

The EDNRB antagonist bosentan blocks MEKi-mediated YAP activation. Uveal melanoma cells were treated with vehicle, MEKi (10nM, 48h), EDRNBi (bosentan, pre- treatment with 80M, 1h) and the combination for 48 h before being subjected to the YAP reporter assay. Analysis was performed with one-way analysis of variance (ANOVA) followed by Tukey–Kramer post hoc analysis. Values are expressed as mean ± s.d. Significance is indicated by *p<0.05, **p<0.01 and ***p<0.001.

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 2.5μM of drug relative to vehicle. B) Detailed view of the responses of the drugs selected for follow-up in the cell line panel. Data shows the inhibition of growth per cell line at 0.5 and 2.5μM of drug relative to vehicle. C) HDACi increase 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, 10nM) alone and in combination with inhibitors of HDAC1/2/3 (etinostat), HDAC6 (tubastatin), HDAC8 (PCI- 03451) and pan-HDAC (panobinostat) for 72 hr 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 to either drug alone. 92.1, Mel270, MP41, MM28 cells were treated with MEKi (trametinib, 10nM), HDACi (panobinostat, 10nM) or combination of both for 72h 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 with MEKi (trametinib, 10nM), HDACi (panobinostat, 10nM) or combination of both for 48h.

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, 10nM), HDACi (panobinostat, 10nM) or a combination of the two for 0-72 and probed for phospho-AKT, AKT and GAPDH expression. B) HDAC inhibition limits MEKi-induced YAP activity. Data

shows 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, 1mg/kg po daily), HDACi (Panobinostat, 20mg/kg, IP, 3X week) or the combination for 31 days. Data shows 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 x100 in all images. Scale bar= 5mm for the whole images and scale bar= 500µm 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 analysis of variance (ANOVA) followed by Tukey–Kramer post hoc analysis. Values are expressed as mean ±s.d. Significance is indicated by *p<0.05, **p<0.01 and ***p<0.001.

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.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-3382

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2019/06/21/1078-0432.CCR-18-3382.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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