HDAC8 Regulates a Stress Response Pathway in Melanoma to Mediate Escape from BRAF Inhibitor Therapy Michael F

Published OnlineFirst April 15, 2019; DOI: 10.1158/0008-5472.CAN-19-0040

Cancer Translational Science Research

HDAC8 Regulates a Stress Response Pathway in Melanoma to Mediate Escape from BRAF Inhibitor Therapy Michael F. Emmons1, Fernanda Faiao-Flores~ 1, Ritin Sharma2, Ram Thapa3, Jane L. Messina4, Jurgen C. Becker5, Dirk Schadendorf5, Edward Seto6, Vernon K. Sondak4, John M. Koomen2, Yian A. Chen3, Eric K. Lau1,4, Lixin Wan2, Jonathan D. Licht7, and Keiran S.M. Smalley1,4

Abstract

Melanoma cells have the ability to switch to a Low HDAC8 activity dedifferentiated, invasive phenotype in response to multiple stimuli. Here, we show that exposure of c-Jun Ras melanomas to multiple stresses including BRAF– BRAFi ac RTK RAF ac ERK MEK inhibitor therapy, hypoxia, and UV irradia- ac tion leads to an increase in histone deacetylase 8 (HDAC8) activity and the adoption of a drug- TRE Cell death resistant phenotype. Mass spectrometry–based High HDAC8 activity phosphoproteomics implicated HDAC8 in the reg- p ulation of MAPK and AP-1 signaling. Introduction RTK Ras of HDAC8 into drug-na€ve melanoma cells con- BRAFi c-Jun RAF ac (EGFR) in vitro in vivo ERK veyed resistance both and . HDAC8- ac mediated BRAF inhibitor resistance was mediated HDAC8 TRETR Survival and via receptor tyrosine kinase activation, leading to invasion MAPK signaling. Although HDACs function at the Increased HDAC8 activity deacetylates c-JUN, leading to increased EGFR signaling and BRAF inhibitor resistance

histone level, they also regulate nonhistone sub- © 2019 American Association for Cancer Research strates, and introduction of HDAC8 decreased the acetylation of c-Jun, increasing its transcriptional activity and enriching for an AP-1 gene signature. Mutation of the putative c-Jun acetylation site at lysine 273 increased transcriptional activation of c-Jun in melanoma cells and conveyed resistance to BRAF inhibition. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with both nonselective and HDAC8-specific inhibitors enhancing the durability of BRAF inhibitor therapy. Our studies demonstrate that HDAC8-specific inhibitors limit the adaptation of melanoma cells to multiple stresses including BRAF–MEK inhibition.

Signiﬁcance: This study provides evidence that HDAC8 drives transcriptional plasticity in melanoma cells in response to a range of stresses through direct deacetylation of c-Jun. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2947/F1.large.jpg.

Introduction position 600 mutations in BRAF (1). Despite this, most Use of BRAF inhibitors and BRAF–MEK inhibitor combina- patients ultimately fail therapy, and cures remain rare (1, 2). tions is associated with impressive therapeutic responses and Although much is now known about the genetic mediators of increased overall survival in patients whose melanomas harbor acquired BRAF and BRAF–MEK inhibitor resistance, there is

1The Department of Tumor Biology, The Moffitt Cancer Center and Research Note: Supplementary data for this article are available at Cancer Research Institute, Tampa, Florida. 2The Department of Molecular Oncology, The Online (http://cancerres.aacrjournals.org/). Moffitt Cancer Center and Research Institute, Tampa, Florida. 3Department Corresponding Author: Keiran S.M. Smalley, H. Lee Moffitt Cancer Center and of Bioinformatics and Biostatistics, The Moffitt Cancer Center and Research Research Institute, 12902 Magnolia Drive, Tampa, FL 33612. Phone: 813-745- Institute, Tampa, Florida. 4The Department of Cutaneous Oncology, The 8725; Fax: 813-449-8260; E-mail: keiran.smalley@moffitt.org Moffitt Cancer Center and Research Institute, Tampa, Florida. 5Department of Translational Skin Cancer Research, German Cancer Consortium (DKTK), Cancer Res 2019;79:2947–61 6 University Hospital Essen, Essen, Germany. George Washington University, doi: 10.1158/0008-5472.CAN-19-0040 Washington, D.C. 7The University of Florida Health Cancer Center, Gaines- ville, Florida. 2019 American Association for Cancer Research.

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still an urgent need to better understand the mechanisms phospho-EphA2 (D9A1, 6347), EphA2 (D4A2, 6997), phospho- underlying treatment failure, particularly at the earliest AKT (D9E, 4060), AKT (9272), phospho-c-Jun (54B3, 2361), stages, so that new therapeutic strategies and drug combina- c-Jun (60A8, 9165), and acetyl (9441) were purchased from Cell tions can be developed (2–5). The process of early adaptation Signaling Technology. Anti-HDAC6 (H-300, sc-11420) was pur- to therapy remains poorly defined but appears to involve the chased from Santa Cruz Biotechnologies. Anti-HDAC11 adoption of a slow-growing "persister" state that is marked by (ab47036) was purchased from Abcam. Anti-Vinculin (G8796) dedifferentiation, phenotypic plasticity and some recovery of and anti-GAPDH (V9131) were purchased from Millipore MAPK signaling (6). This early rebound in MAPK signaling Sigma. Ac-SMC3 was a kind gift from Forma Therapeutics. Phos- is frequently mediated through increased receptor tyrosine pho-RTKs were measured with the Human Phospho-Receptor kinase (RTK) signaling, with a number of studies now impli- Tyrosine Kinase Array Kit (R&D Biosystems). Activated and total catingrolesforIGF1R,EGFR,Axl,c-MET,PDGFR,and Ras were measured with the Active Ras Pull-Down and Detection EphA2 (7–10). Kit (ThermoFisher). For each experiment, all antibodies were In our previous studies, we used comprehensive mass probed on the same blot. In cases where bands were similar, the spectrometry–based phosphoproteomics to identify a ligand- blots were washed with Restore Western Blot Stripping Buffer for independent EphA2-driven signaling network as a driver of 10 minutes before a new antibody was used. an aggressive, epithelial–mesenchymal transition (EMT)-like phenotype in melanoma cells with acquired BRAF inhibitor Cell death assays resistance (11). This S897-EphA2–driven signaling network Cells were treated with drugs (72 hours), harvested, and incu- was dependent upon continuous MAPK pathway inhibition bated with Annexin V APC (BD Biosciences). Fluorescence was and was reversed following drug withdrawal for >3 weeks read on a FACSCalibur (BD Biosciences) and analyzed using (11). The plasticity of this drug-induced phenotype suggested Flowjo software. To measure cell death following induction of these changes could be epigenetically mediated (11). In the stress, 300 cells were counted for cell death by trypan exclusion present study, we asked whether a common transcriptional using a 0.4% trypan blue solution (Millipore Sigma). state that emerged when melanoma cells were subjected to stress allowed melanoma cells to survive diverse insults. Our Colony formation assay work identified a novel role for HDAC8 as a mediator of Cells were treated with drug for 28 days before being stained phenotype switching and the therapeutic adaptation of mel- with a 0.5% Crystal Violet solution. Colonies were quantified anoma cells to BRAF inhibition. Unexpectedly, we found that using ImageJ software. HDAC8 regulates BRAF inhibitor sensitivity and acquired drug resistance through direct effects upon c-Jun acetylation, leading to transcriptional rewiring and increased RTK and Immunohistochemistry MAPK signaling. Together, these results point to a new role Samples from melanoma patients pre- and post-BRAF and for the histone deacetylases in regulating the cell signaling BRAF–MEK inhibitor therapy were collected from the Univer- networks at the protein acetylation level that mediates ther- sity Hospital Essen under a written-informed consent proto- apeutic escape. col. Formalin-fixed, paraffin-embedded (FFPE) slides were stained for HDAC8 expression using the Ventana Discovery XT automated system and an anti-HDAC8 antibody (Novus Materials and Methods Biologicals) at a 1:100 concentration with 60-minute incuba- Cell culture tion. Staining was detected using the Ventana ChromoMap The 1205Lu, WM164, and SKMEL-28 cell lines were a gen- Red Kit, and slides were counterstained with hematoxylin. For erous gift from Dr. Meenhard Herlyn (The Wistar Institute, mouseIHC,FFPEslideswerestainedforphospho-c-Jun Philadelphia, PA). The dual BRAF and MEK inhibitor–resistant (Abcam, ab32385) for 1 hour at a 1:100 concentration, and (RR) lines 1205LuRR, SKMEL28RR, and WM164RR were estab- slides were uploaded into an Aperio AT2 scanner (Leica lished as previously described (12). Panobinostat, PCI-34051, Biosystems) and visualized using Aperio Imagescape 12.3.3 and erlotinib were from Selleckchem. Hypoxia was achieved via (Leica Biosystems). an oxygen control glove box (Coy Labs) for 24 hours in conditions containing 94% N2,1%O2,and5%CO2. All cells Proteomics were tested for Mycoplasma contamination every 3 months Cells were lysed in an urea lysis buffer (20 mmol/L using the Plasmotest-Mycoplasma Detection Test (Invivogen). HEPES, pH 8.0, 9 mol/L urea, 1 mmol/L sodium orthovana- Last test date was March 18, 2019. Each cell line was authen- date, 2.5 mmol/L sodium pyrophosphate, and 1 mmol/L ticated using the Human STR human cell line authentication b-glycerophosphate), and protein concentration of the lysate service (ATCC), and frozen stocks of cells were discarded after wasmeasuredbyBradfordassay.Extractedproteins(10mg) 10 passages. were digested by trypsin and enriched for phospho-tyrosine and phospho-serine/threonine as previously described (11). Extracted Western blotting proteins from each condition [empty vector (EV) or HDAC8] were Lysates were acquired and processed for Western blot trypsin digested, and 2 equal aliquots of tryptic peptides (100 mg) and immunoprecipitation as previously described (11). The were labeled by distinct Tandem Mass Tags (TMT six-plex reagents, anti-HDAC3 and anti-HDAC8 antibodies were described in ThermoFisher), combined, and subjected to offline high pH refs. 13 and 14. The antibodies against HDAC1 (2062), HDAC2 Reverse Phase fractionation (15). Each of the fractions was (2540), BIM (2933), Mcl-1 (4572), phospho-ERK (9101), ERK enriched for phosphopeptides using a Phos-SELECT Iron (9102), phospho-CRAF (56A6, 9427), CRAF (D4B3J, 53745), Affinity Gel (Millipore Sigma; ref. 16). Mass spectrometry data

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were acquired on a QExactive mass spectrometer coupled to a PCR U3000 RSLCnano system (ThermoFisher) as described previous- EGFR mRNA expression was measured by quantitative ly (16). Two technical replicates were performed for the immune- RT-PCR. EGFR and GAPDH primers were purchased from enriched phosphotyrosine samples as well as each of the immo- Applied Biosystems (AB, Thermo). cDNA was made from bilized metal affinity chromatography (IMAC)-enriched fractions. isolated RNA with the High Capacity cDNA Reverse Transcrip- Label-free quantitation was performed for phosphotyrosine sam- tase Kit (AB), and 100 mg of cDNA was run on a 7900HT Fast ples, whereas MS2-reporter ion quantitation was performed for Real-Time PCR System for 40 cycles using Taqman master IMAC-enriched samples using MaxQuant (1.2.2.5; ref. 17). Data mix (AB). Samples were normalized to control. are available in PRIDE (PXD012813 and PXD012812). Promoter assay RNA sequencing To assess ATF2 and c-JUN transcriptional activity, we imple- Isolated RNA was cleaned using an RNAeasy minicleanup kit mented a dual secreted luciferase assay as previously described (Qiagen) and screened for quality on an Agilent BioAnalyzer. The (20). At 48 hours after transfection, the cells were treated with fi samples were then processed for RNA sequencing (RNA-seq) speci ed drugs, and at the indicated times, media samples using the NuGen Ovation Human FFPE RNA-Seq Multiplex containing secreted lucifase were harvested and measured for System. The libraries were then sequenced on the Illumina luciferase activity using the Pierce Gaussia Luciferase Glow NextSeq 500 sequencer with a 2 75-base paired-end run in Assay Kit per the manufacturer's instructions (ThermoFisher). order to generate 40 to 50 million read pairs per sample. Data DNA-binding assay are available in GEO (GSE127564). Binding of c-Jun and c-Jun mutant cells to the consensus JUN DNA sequence was performed using the Mouse/Human/ Analysis of sequencing and proteomic data Rat JUN/c-Jun DNA Binding ELISA Kit (LSBio) per the Combat was used to normalize phosphotyrosine profiles manufacturer's instructions. Samples were read on a plate before further analyses (18). Log transformation was applied 2 reader at 450 nm. to all three datasets (RNA-seq, and both phosphorylation t experiments). Moderated statistics were used to compare the Mutagenesis RNA expression between baseline (EV) and HDAC8 overex- The following primers were ordered from Integrated DNA pression (HDAC8) samples in RNA-seq data for each of 18,542 Technologies: 268 mutant: 50 gcatcgctgc ctccagatgc cgaaaaagga genes using the limma package in R (19). In phosphotyrosine agctggagag aatcg 30 and 50 cgatt ctctccagct tcctttttcg gcatctggag residue data, 172 phosphopeptides were used for assessing gcagcgatgc 30; 271 mutant: 50 gcatcgctgc ctccaagtgc cgaagaagga differential expression in HDAC8 versus EV samples. In serine/ agctggagag aatcg 30 and 50 cgatt ctctccagct tccttcttcg gcacttggag threonine phosphopeptide data, 1,976 phosphopeptides were gcagcgatgc 30; and 273 mutant: 50 gcatcgctgc ctccaagtgc used in assessing differential expression in HDAC8 versus cgaaaaagga gactggagag aatcg 30 and 50 cgatt ctctccagtc tcctttttcg fi EV samples. Volcano plots with signi cant phosphopeptides gcacttggag gcagcgatgc 30. Mutant DNA constructs were made by P denoted by fold change > 2and value < 0.05 in the a site-directed mutagenesis kit (ThermoFisher) against a WT contrast between EV and HDAC8 samples were also used for c-Jun plasmid (Origene Technologies) per the manufacturer's visualization. instructions. Mutant constructs were sequenced (Genewiz) Normalized phosphoproteomic data were combined and ana- using plasmid DNA and c-Jun primer sequence cgtttggagtcgtt- lyzed using GeneGO software (Metacore, Thomson Reuters). gaagttg (IDT). DNA was stably transfected into cells using fi Signi cant interactions between genes were determined with a lipofectamine 2000, and clones were selected for further study. P cutoff value of < 0.05. Normalized pY and pS/T proteomic data After selection, endogenuous levels of c-Jun were knocked were uploaded and analyzed by STRING. The most stringent down using a 30 shRNA for JUN (SHCLNV-NM_002228, fi fi interaction threshold of 0.9 was used to nd the most signi cant TRCN0000039588, Millipore Sigma). interactions upregulated in HDAC8-expressing cells. Significant interactions exported from GeneGO were organized into a global In vivo studies scid signaling hub using Cytoscape software. RNA-seq data were Cells were injected into the hind flank of NOD.CB17-Prkdc /J analyzed by Gene Signature Enrich Analysis (GSEA). The data mice (Taconic) in a solution containing 50% L-15 media (Ther- were analyzed for significant transcription factors using an FDR moFisher) with 1 mmol/L HEPES (Millipore Sigma) and 50% cutoff of 0.05. Matrigel (BD Biosciences). Ten tumors were used for each group in each experiment. All studies were approved by the University of Transfection and infection South Florida's Institutional Animal Care and Use Committee Cells were placed in OPTI-MEM media in the presence of (#IS00004987). PLX 4720 was given using formulated chow the plasmid or siRNA and lipofectamine 2000. Mcl-1 (ON (Research Diets), whereas panobinostat and PCI-34051 were Target SMART pool) siRNA and nontargeting control siRNA given by i.p. injections for the duration of the experiment. Weight were purchased from ThermoFisher. The EV plasmids were and tumor size were measured with calipers and were monitored purchased from Origene Technologies Inc. For infection of 3 times weekly. Millipore Sigma shRNA viral particles, infection was performed per the manufacturer's protocol. After 24 hours, the Statistical analysis media were removed and replaced with media containing 1 For all experiments, significance was determined between mg/mL puromycin (Millipore Sigma). shRNA against HDAC8 groups using a one-way ANOVA followed by a post hoc t test. For (SHCLNV-NM_018486, TRCN0000004851) was purchased all in vitro experiments, 3 independent experiments with an n from Millipore Sigma. of 3 were used for an overall n of 9 with a representative

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Figure 1. HDAC8 protein expression is upregulated in BRAF inhibitor–resistant cell lines and confers resistance to BRAF inhibitors. A, HDAC inhibition reduces phospho- AKT and phospho(S897)-EphA2 expression in BRAF–MEK inhibitor-resistant 1205LuRR, SKMEL28RR, and WM164RR melanoma cells. Cells were treated with 100 nmol/L of panobinostat or DMSO vehicle control (VC) for 24 hours and probed for phospho-EphA2, EphA2, phospho-AKT, and AKT by Western blot. B, HDAC inhibition restores BRAF inhibitor–induced apoptosis. 1205Lu, 1205LuRR, WM164, and WM164RR cells were treated with vemurafenib (3 mmol/L for 1205Lu and 1 mmol/L for WM164) alone or in combination with 20 nmol/L panobinostat for 72 hours. Apoptosis was measured by Annexin V staining using ﬂow cytometry. C, BRAF–MEK inhibitor resistance is associated with increased expression of multiple HDACs. A Western blot shows expression of HDACs in matched-sensitive and -resistant cell lines. Densitometry values for expression normalized to GAPDH are shown under each blot. (Continued on the following page.)

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experiment shown. For in vivo studies, an n of 10 was used for irradiation or hypoxia (Fig. 1F and G). The clinical relevance each group. of these findings was investigated through IHC staining of a cohort of matched pre- and post-BRAF inhibitor–treated melanoma patient specimens (Supplementary Table S1). It was found that HDAC8 was either highly expressed at baseline Results (6/8) and did not change on therapy or showed increased The BRAF and BRAF–MEK inhibitor-adapted state is reversible expression posttherapy (2/8 cases; Fig. 1H). Collectively, these and sensitive to HDAC inhibition data demonstrated that HDAC8 was induced under multiple In previous studies, we identified an S897-EphA2–driven stress conditions, including BRAF–MEK inhibitor therapy, signaling interactome that emerged under continuous BRAF and that expression of HDAC8 could provide protection to therapy, which was readily reversible following drug with- melanoma cells. Continuous drug exposure was required to drawal (11). We reasoned that this network, and therefore maintain the HDAC8-driven adapted state, with drug removal BRAF inhibitor resistance, may be in part epigenetically reg- for >3 weeks leading to reduced expression of HDAC8 (Sup- ulated. To explore this mechanism, we treated BRAF–MEK plementary Fig. S4). inhibitor-resistant melanoma cell lines (designated RR) with the broad-spectrum HDAC inhibitor panobinostat and found HDAC8 mediates BRAF inhibitor tolerance that it decreased both S897-EphA2 and pAKT signaling and We next generated stable HDAC8-expressing clones of drug- restored vemurafenib sensitivity in apoptosis assays (Fig. 1A na€ve WM164 and 1205Lu melanoma cells, that had protein and B). We next asked whether acquired BRAF inhibitor expression levels equivalent to that induced by continuous resistance was associated with an increased expression of BRAF inhibitor therapy (Fig. 2A). The introduction of HDAC8 specific HDAC genes or proteins by microarray and Western increased the tolerance of melanoma cells to BRAF inhibitor blot analysis, respectively. It was determined that Class I therapy in 4-week colony formation assays (Fig. 2B and C) and HDACs (HDAC1, HDAC2, HDAC3, and HDAC8), Class IIb ledtoasignificant reduction in vemurafenib-induced apopto- HDACs (HDAC6), and Class IV HDACs (HDAC11) were sis (Fig. 2D), which was not associated with increased cell consistently expressed (Supplementary Fig. S1A). Although proliferation (Supplementary Fig. S5). These effects were also many HDACs showed alteration following the acquisition of observed following administration with a combination of BRAF (designated R) and BRAF–MEK inhibitor resistance, BRAF–MEK inhibitors (Supplementary Fig. S6A and S6B). HDAC8 expression was consistently increased (>2-fold) in the Conversely, it was found that the silencing of HDAC8 reversed 5 of 5 drug-resistant melanoma cell lines (Fig. 1C; Supple- resistance to vemurafenib in colony formation assays (Fig. 2E– mentary Fig. S1B). Increased expression of HDAC6 (>2-fold) G)andrestoredapoptosislevelstothoseofthedrug-na€ve cell was also noted in 4 of 5 of the cell lines (M229R, SKMEL-28RR, lines (Fig. 2H). We next determined the functional conse- 1205LuRR, and WM164RR; Fig. 1C; Supplementary Fig. S1B). quences of modulating HDAC8 expression in terms of apo- Expression of HDAC8 and c-JUN was also noted in melanoma ptosis regulation. We focused upon BIM and Mcl-1, as (1) both cells with intrinsic BRAF inhibitor resistance, whereas those of these proteins are regulated by mutant BRAF in melanoma with initial BRAF inhibitor sensitivity expressed little c-JUN cells and are (2) important regulators of the apoptotic response and an HDAC8 doublet (Supplementary Fig. S2; ref. 21). A role following BRAF inhibition (23, 24). HDAC8 introduction, for HDAC8 in the restoration of drug sensitivity was suggested followed by BRAF inhibitor treatment, was associated with a by the ability of an HDAC8 inhibitor (PCI-34051), but not an suppression of proapoptotic BIM expression (Fig. 2I) and the HDAC6 inhibitor (tubastatin), to restore the sensitivity of maintenance of Mcl-1 levels (Fig. 2I), while silencing HDAC8 BRAF inhibitor–resistant melanoma cell lines to vemurafenib increased BIM expression (Supplementary Fig. S7A–S7C). The (Fig. 1D; Supplementary Fig. S3). As increased expression is critical role of Mcl-1 maintenance in the prosurvival effects of not always indicative of increased enzymatic activity, we also HDAC8 overexpression was demonstrated through the siRNA probed for the validated HDAC8 target, acetylated-SMC3 (22), silencing of Mcl-1, which restored the sensitivity of the and noted a decrease in acetylation of SMC3 in the resistant HDAC8-overexpressing cells to vemurafenib (Fig. 2J and K). cell lines (Fig. 1E). To explore whether increased HDAC8 activity was a common response of melanoma cells to stress, we next treated Mass spectrometry–based phosphoproteomic analyses reveal 1205Lu melanoma cells with either UV radiation (254 nm: a direct role for HDAC8 in regulating MAPK and JUN 2 3.75 J/m ) or hypoxia (1% O2 for 24 hours). Exposure to both signaling in BRAF-mutant melanoma of these stresses induced HDAC8 expression, with overexpres- We reasoned that the introduction of HDAC8 increased sion of HDAC8 leading to reduced cell death following UV melanoma cell survival under stress by rewiring the signaling

(Continued.) D, HDAC8 inhibition restores BRAF inhibitor sensitivity. 1205Lu, 1205LuRR, WM164, and WM164RR cells were treated with vemurafenib (3 mmol/L for 1205Lu and 1 mmol/L for WM164) alone or in combination with 5 mmol/L PCI-34051 for 72 hours. Apoptosis was measured by Annexin V staining and flow cytometry. E, BRAF–MEK inhibitor resistance is associated with increased HDAC8 activity. A Western blot shows expression of HDAC8 and the HDAC8 substrate acetylated SMC3 (acSMC3) in matched-sensitive and -resistant cell lines. Densitometry for expression over basal levels is shown under each blot. F, HDAC8 2 induction is a generalized response to stress. 1205Lu cells were treated with either UV (254 nm: 3.75 J/m ) or hypoxia (1% O2, 24 hours) before being probed for HDAC8 expression by Western Blot. G, HDAC8 protects from UV and hypoxia-induced cell death. Cells were treated with either UV or hypoxia (as described for F) and cell death measured 24 hours later by trypan exclusion. H, HDAC8 expression is increased in melanoma patient samples post BRAF inhibitor therapy. Pre and posttreatment specimens were stained for HDAC8 by IHC. All experiments were performed in triplicate, and significance was determined with a one-way ANOVA, followed by a post hoc t test with #, P > 0.05 (nonsignificant); , P < 0.05; , P < 0.01.

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Figure 2. HDAC8 confers resistance to MAPK-targeted therapies in melanoma. A, An EV or HDAC8 construct was introduced into 1205Lu and WM164 cells. A Western blot shows levels of HDAC8 expression. Densitometry values for expression normalized to GAPDH are shown under each blot. B and C, HDAC8 increases vemurafenib tolerance in colony formation assays. Isogenic (EV and HDAC8) 1205Lu and WM164 cells were treated with vemurafenib continuously (28 days; 3 mmol/L for 1205Lu and 1 mmol/L for WM164) before being stained with crystal violet. A representative experiment is shown in B, and results were quantiﬁed in C using ImageJ. D, HDAC8 introduction increases cell survival following BRAF inhibition. Isogenic (EV and HDAC8) 1205Lu and WM164 cells were treated with vemurafenib (72 hours; 3 mmol/L for 1205Lu and 1 mmol/L for WM164) and apoptosis measured by Annexin V binding and ﬂow cytometry. E, HDAC8 was knocked down by shRNA (shHDAC8) or nonsilencing control (shNS) in BRAF–MEK (Continued on the following page.)

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network. To explore this, we utilized mass spectrometry– We next turned our attention to RTKs and used RTK arrays to based phosphoproteomics to map the entire signaling net- demonstrate that HDAC8 introduction altered the basal phos- work. The introduction of HDAC8 into drug-na€ve BRAF- phorylation of multiple RTKs including EGFR, c-MET, and FGFR3 mutant melanoma cell lines led to significant increases in (Fig. 4E and F; Supplementary Fig. S8A–S8D). Among the RTKs the tyrosine phosphorylation of 5 peptides and the serine/ identified, EGFR appeared critical for the increased MAPK signal- threonine phosphorylation of 113 peptides (Fig. 3A). These ing associated with HDAC8, with studies showing that erlotinib data demonstrated HDAC8 overexpression enriched for net- resensitized HDAC8-expressing melanoma cells to BRAF inhib- works associated with the adoption of an EMT, as well as itor–mediated apoptosis (Fig. 4G). Use of the c-MET inhibitor, MAPK and AP-1 transcription factor signaling (Fig. 3B). These crizotinib, or the FGFR inhibitor, BGJ398, also resensitized findings with HDAC8 mirrored those reported previously by HDAC8-expressing melanoma cells to BRAF inhibition (Supple- our group on melanomas with acquired BRAF inhibitor resis- mentary Fig. S9A and S9B). Together, these data indicate that tance (11). Grouping of the proteomic data into cellular increased HDAC8 activity contributes to stress tolerance through processes using STRING analysis demonstrated HDAC8 to be maintenance of survival signaling. involved in ribosomal function, RNA binding, cell-cycle regulation, ERK signaling, and organization of the cytoskeleton HDAC8 increases MAPK activity in melanoma cells through (Fig. 3C). Analysis of individual phosphopeptides identified deacetylation of c-Jun the emergence of a signaling interactome that was dependent We next performed RNA-seq analyses on our isogenic (EV and upon MAPK1 and c-Jun (Fig. 3D). Other members of the HDAC8 introduced) cell lines (Fig. 5A) and used GSEA to identify HDAC8-driven signaling network included MAPK pathway transcriptional programs associated with HDAC8 expression. members (p38 MAPKa and p38MAPKg), cytoskeleton regu- One of the top hits was an AP-1 gene signature, indicative of lators (FAK, paxillin, stathmin, LIMA1, PTRF, and MARCKS), c-Jun transcriptional activity (Fig. 5B). Unbiased kinome array cell cycle/spindle regulators (CDK1, ASPM, and TPX2), tran- analysis showed HDCA8 introduction to be associated with scriptional initiation (EIF6 and EEF1D), and PKC signaling increased c-JUN, p53, AKT, and HSP60 phosphorylation (Sup- (PRKCD). plementary Fig. S10A and S10B). Functional studies showed HDAC8 introduction led to increased c-Jun phosphorylation following BRAF inhibitor treatment (Fig. 5C) and enhanced c-Jun HDAC8 enhances therapeutic escape through increased transcriptional activity both immediately following and at 4 RTK-mediated MAPK signaling hours after BRAF inhibitor treatment (Fig. 5D). A role for Our phosphoproteomic studies identified MAPK1 as a major increased c-Jun expression/activity in BRAF inhibitor tolerance HDAC8-regulated signaling hub. We next used two isogenic cell was indicated by the observation that c-Jun silencing restored line pairs transduced with either EV or HDAC8 to evaluate its vemurafenib sensitivity to HDAC8-expressing melanoma cells role in MAPK signaling. HDAC8 introduction increased base- (Supplementary Fig. S11A and S11B). line phospho-ERK levels in both cell lines, and MAPK signaling Previous studies have demonstrated that c-Jun is acetylated at was maintained in the presence of a BRAF inhibitor, i.e., the Lys268, Lys271, and Lys273 (25). We performed immunopre- drug never inhibited the pathway by >50% (Fig. 4A and B). cipitation studies and demonstrated that the introduction of These effects were also seen following administration of a HDAC8 led to the deacetylation of c-Jun (Fig. 5E). A structural combination of BRAF–MEK inhibitors (Supplementary Fig. analysis revealed that the three potential acetylation sites (268, S6A and S6B). Conversely, shRNA knockdown of HDAC8 271, and 273) are located within the DNA-binding domain of reduced MAPK signaling in the presence of a BRAF inhibitor c-Jun (Fig. 5F). A series of acetylation-deficient K!Rc-Jun (Supplementary Fig. S7A–S7C). The increased MAPK signaling mutants were generated at each of the three individual lysines we observed occurred upstream of ERK, with a more pro- (K268R, K271R, and K273R; Supplementary Fig. S12), along nounced and rapid induction of phospho-CRAF signaling with the silencing of the endogenous protein through a 30- being noted in the HDAC8-expressing cells compared with the UTR–directed shRNA. Mutating lysine 273 led to a reduction of EV controls (Fig. 4C). Ras-GTP pulldown experiments demon- BRAF inhibitor sensitivity by both apoptosis (Fig. 5G) and strated that HDAC8 overexpression increased the level of Ras- colony formation assays (Supplementary Fig. S13A and GTP loading, indicating the reactivation of signaling upstream S13B). Introduction of K273R c-JUN also limited the proapop- of RAF (Fig. 4D). totic effects of combined HDAC8 and BRAF inhibition in

(Continued.) inhibitor-resistant 1205LuRR and WM164RR cells. A Western blot shows levels of HDAC8 expression, and densitometry was performed as in A. F and G, A colony formation assay demonstrates silencing HDAC8 reverses BRAF inhibitor resistance. Isogenic (shNS and shHDAC8) 1205LuRR and WM164RR cells were treated with vemurafenib continuously (28 days; 5 mmol/L for 1205LuRR and 3 mmol/L for WM164RR) before being stained with crystal violet. A representative experiment is shown in F, and results were quantified in G using ImageJ. H, Silencing of HDAC8 increases vemurafenib-induced apoptosis. Isogenic cell line pairs (shNS or shHDAC8) were treated with vemurafenib (72 hours; 5 mmol/L for 1205LuRR and 3 mmol/L for WM164RR). Apoptosis was measured by Annexin V staining and flow cytometry. I, Introduction of HDAC8 suppresses BIM and stabilizes Mcl-1 following vemurafenib treatment. Isogenic 1205Lu cell lines (EV or HDAC8) were treated with vemurafenib (3 mmol/L, 0–48 hours), and expression of BIM and Mcl-1 was assessed by Western blot. J and K, Silencing of Mcl-1 restores BRAF inhibitor sensitivity to HDAC8-expressing melanoma cells. J, A Western blot was probed for Mcl-1 following transfection of WM164 EV and HDAC8 cells with Mcl-1 siRNA. K, Analysis of vemurafenib-mediated apoptosis following Mcl-1 silencing. Isogenic 1205Lu and WM164 (EV and HDAC8) cells were silenced for Mcl-1 and treated with vemurafenib (72 hours; 3 mmol/L for 1205Lu and 1 mmol/L for WM164). Apoptosis was measured by Annexin V staining and flow cytometry. Experiments were performed in triplicate, and significance was determined with a one-way ANOVA, followed by a post hoc t test with #, P > 0.05 (nonsignificant); , P < 0.05; , P < 0.01; , P < 0.001. VC, vehicle control.

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Figure 3. Systems level proteomics identified c-Jun and MAPK1 as key HDAC8-regulated signaling hubs. A, Key proteins including MAPK1 and c-Jun are significantly upregulated following increased HDAC8 expression in BRAF inhibitor–na€ve cells. A volcano plot analysis was performed on a phosphoproteomics study (serine/ threonine/tyrosine phosphorylation) comparing isogenic (EV and HDAC8) 1205Lu cells. Significant changes are denoted by fold change > 2 and a P value < 0.05 and are shown in blue. B, Key signaling pathways including MAPK, AP-1, and EGFR are upregulated following increased HDAC8 expression in BRAF inhibitor–

na€ve cells. Significantly changed proteins in A were analyzed using GeneGo software. Shown are the most significantly changed pathways, along with the log10 of the P value. C, Key pathways with significantly altered protein/protein interactions, including interactions involving ERK signaling and cell migration, were changed following increased HDAC8 expression. STRING analysis identified key protein signaling hubs changed in HDAC8-expressing 1205Lu cells. Protein interactions in STRING surpassing the most stringent interaction threshold of 0.9 were exported and visualized by Gephi visualization software using the OpenORD algorithm. D, A global analysis of significantly changed protein/protein interactions determined MAPK1 and c-Jun to be the most altered proteins when HDAC8 is expressed in 1205Lu cells. Significantly altered protein/protein interactions were determined by GeneGO analysis following input of significantly changed proteins following introduction of HDAC8 into cells. Interactions were visualized using Cytoscape.

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Figure 4. HDAC8 modulates BRAF inhibitor sensitivity through regulation of RTK/RAS/RAF/MEK/ERK signaling. A, Introduction of HDAC8 increases basal phospho-ERK levels in melanoma cells and MAPK signaling under BRAF inhibitor therapy. Control (EV) and HDAC8-expressing melanoma cells 1205Lu (3 mmol/L) and WM164 (1 mmol/L) were treated with vemurafenib for increasing periods of time (0–48 hours) before being subjected to Western blotting for phospho-ERK (pERK) and total ERK expression. B, Quantification of phospho-ERK levels relative to total ERK levels. C, HDAC8 introduction is associated with increased CRAF phosphorylation following BRAF inhibition. WM164 cells were treated with vemurafenib (1 mmol/L) for increasing periods of time (0–48 hours) and probed for phospho-CRAF (S338, pCRAF) and CRAF expression. D, HDAC8 introduction increases Ras signaling. Isogenic (EV and HDAC8 expressing) WM164 (1 mmol/L) and 1205Lu (3 mmol/L) cells were treated for vemurafenib for 24 hours and probed for activated Ras (GTP-bound) and total Ras. E and F, Introduction of HDAC8 increases baseline RTK signaling. Isogenic pairs of 1205Lu (E) and WM164 (F) cells were analyzed using a phospho-RTK array. Resulting membranes were scanned, and densitometry was performed. Data show the increase in phosphorylation of the RTKs EGFR (pEGFR), c-MET (p-c-MET), and FGFR3 (p- FGFR3) following HDAC8 introduction. G, EGFR inhibition restores BRAF inhibitor sensitivity in cell lines that express HDAC8. Isogenic 1205Lu and WM164 melanoma cells were treated with vehicle, vemurafenib (BRAFi; 3 mmol/L for 1205Lu, 1 mmol/L for WM164), erlotinib (EGFRi; 1 mmol/L), or both drugs in combination (BRAFi/EGFRi) for 72 hours, and apoptosis was measured by Annexin V staining and flow cytometry. Significance was determined with a one-way ANOVA, followed by a post hoc t test with #, P > 0.05 (nonsignificant); , P < 0.01. ND, none detected; VC, vehicle control.

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Figure 5. HDAC8 deacetylates the transcription factor c-Jun at lysine 273, leading to increased transcriptional activity, increased phospho-ERK signaling, and BRAF inhibitor resistance. A, HDAC8 introduction is associated with a unique gene signature. Heatmap of an RNA-seq analysis of WM164 and 1205Lu melanoma cells introduced with either EV or HDAC8. B, GSEA analysis identiﬁes an AP-1 gene signature is upregulated in melanoma cells expressing HDAC8. C, HDAC8 expression increases phospho-c-Jun levels following BRAF inhibition. Isogenic WM164 cells were treated (Continued on the following page.)

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apoptosis assays (Supplementary Fig. S13C). Functionally, drugs significantly reduced tumor growth compared with either these effects were associated with increased levels of ERK agent alone and was associated with durable responses in these phosphorylation in addition to decreased levels of BIM expres- model systems (Fig. 6E and F). sion following BRAF inhibition (Fig. 5H). Mutating lysine 273 also increased the binding of c-Jun to the consensus JUN/c-Jun Discussion DNA sequence as determined by ELISA (Fig. 5I) and significantly increased levels of EGFR mRNA as measured by qRT-PCR Adaptation to therapy is a major factor that limits the long- (Fig. 5J). These results were supported by kinome and RTK term responses of BRAF-mutant melanoma patients to BRAF arrays that demonstrated K273R introduction to be also asso- inhibitor monotherapy and BRAF–MEK inhibitor combina- ciated with increased EGFR phosphorylation and enhanced tion therapy (3, 6, 26). Despite this, relatively little is known p53, AKT, STAT3, WNK1, and HSP60 signaling (Supplementary about the early events that permit limited numbers of cells to Figs. S14A, S14B, S15A, and S15B). evade the effects of BRAF inhibition. Work from our group and others has demonstrated that diverse therapeutic interventions, including targeted therapy and immune therapy, Cotargeting of BRAF and HDAC8 suppresses therapeutic induce a dedifferentiated state that is reminiscent of an escape EMT (11, 27–31). Melanoma cells that have undergone this As the final step, we asked whether HDAC8 inhibition transition typically exhibit increased invasion and resistance to improved BRAF inhibitor responses in vivo.Fortheinitial most therapies (11, 27–31). Previous studies from our lab studies, we injected isogenic WM164 and 1205Lu melanoma showed this phenotype to be reversible upon drug withdrawal cells that expressed EV, had HDAC8 expression (HDAC8), or and possibly epigenetically mediated (11). Given the postu- were stably knocked down (shRNA) for HDAC8 into the flanks lated links between stress, phenotype switching, and drug of NSG mice. When tumor volumes reached 25 to 40 mm3, resistance in melanoma, we asked whether there was a unifying treatment with the BRAF inhibitor PLX4720 was initiated. In cellular program that regulated the response of melanoma cells these xenografts, melanoma cells expressing HDAC8 showed to multiple stresses. resistance to BRAF inhibitor treatment, whereas the melanoma We began by demonstrating that the drug-adapted, EMT-like with HDAC8 stably knocked down "crashed" following initi- state (here marked by increased S897-EphA2 signaling) could ation of BRAF inhibitor treatment (Fig. 6A and B). At the be reversed following treatment with HDAC inhibitors such as completion of the experiments, the HDAC8 shRNA knockdown panobinostat. HDACs are enzymes that catalyze the hydrolysis tumors were significantly smaller than both the HDAC8-expres- of acetyl groups from acetylated proteins. The HDACs have sing and EV control cells. Western blot studies confirmed the many targets, both nuclear and cytoplasmic, with the best increased expression of HDAC8 and showed this to be asso- characterized of these being the N-terminal tails of his- ciated with a suppression of BIM expression under BRAF tones (32). Our studies revealed that HDAC8 was frequently inhibitor therapy (Fig.6C).Increasednuclear accumulation of upregulated in melanoma cells with acquired BRAF and BRAF– phospho-c-JUN was also seen in the tumors with HDAC8 MEK inhibitor resistance and that introduction of exogenous expression (Fig. 6D). It was not possible to analyze the HDAC8 HDAC8 conveyed resistance to MAPK-targeted therapies. shRNA tumors by Western blot due to very low tumor volumes HDAC8 is a poorly characterized Class I HDAC found in after BRAF inhibitor therapy. We then determined whether both the nucleus and cytoplasm (14, 33). As well as its nuclear similar results could be achieved with small-molecule HDAC activity as a histone deacetylase, HDAC8 also has a number inhibitors. Here, we used two HDAC inhibitors (the broad- of nonhistone substrates including p53, cortactin, and spectrum HDAC inhibitor panobinostat and the HDAC8-spe- SMC3 (22, 34, 35). HDAC8 was not the only HDAC whose cific inhibitor PCI-34051) in combination with the BRAF expression was altered upon chronic BRAF inhibitor treatment, inhibitor PLX4720. For these studies, the animals received a with increased HDAC6 expression being observed in some of lead-in dose of each of the HDAC inhibitors (to mimic the the resistant cultures. Despite this, inhibition of HDAC6 did effects of having the HDACs silenced prior to initiating BRAF not restore sensitivity to BRAF inhibition, suggesting that this inhibitor therapy) before continuing treatment with the com- HDAC played a minor role in the escape from BRAF inhibitor bination of HDAC and BRAF inhibitors. Cotreatment with both therapy.

(Continued.) with vemurafenib (1 mmol/L; 0–48 hours) and probed for phospho-c-Jun (p-c-Jun) and c-JUN by Western blot. D, HDAC8 expression leads to increased c-Jun transcriptional activation following BRAF inhibition. Isogenic 1205Lu cells were transiently transfected with c-Jun– (TRE) or ATF2- (JUN2) targeted promoter luciferase constructs and treated for 4 hours with vehicle (VC) or vemurafenib (3 mmol/L, BRAFi). Luciferase levels were measured and quantified. E, HDAC8 decreases c-Jun acetylation. Total c-JUN was immunoprecipitated (ip) from isogenic 1205Lu (EV) and 1205Lu-HDAC8 cells and blotted for total protein acetylation (ac-c-Jun). c-Jun was used as an input control. F, Structure of c-Jun identifying three potential acetylation sites (at lysine residues) in the DNA-binding domain. G, Acetylation-deficient c-Jun at residue 273 increases melanoma cell survival following BRAF inhibition. 1205Lu cells expressing acetyl mutants of c-Jun (K268R, K271R, or K273R) in addition to WT c-Jun were treated with vemurafenib (3 mmol/L; 72 hours) before being analyzed for Annexin V positivity by flow cytometry. H, Acetylation-deficient c-Jun at residue 273 leads to increased phospho-ERK (pERK) with decreased levels of BIM. Isogenic 1205Lu cells expressing WT, K268R, K271R, and K273R constructs of c-Jun were treated with vemurafenib (3 mmol/L; 0–24 hours) and probed for c-Jun, HDAC8, phospho-ERK, ERK, and BIM expression by Western blot. I, Mutating lysine 273 of c-Jun confers increased binding to DNA. Isogenic 1205Lu cells expressing WT, K268R, K271R, and K273R constructs of c-Jun as well as a negative control (NC) and positive control (PC) were incubated with a plate-bound consensus DNA sequence of c-Jun and read at a wavelength of 450 nm. J, Mutating lysine 273 of c-Jun increases mRNA expression of EGFR. qRT-PCR was performed on samples using primers for EGFR. Readings were normalized to GAPDH control. Experiments were performed in triplicate, and significance was determined with a one- way ANOVA, followed by a post hoc t test with , P < 0.05; , P < 0.001.

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Figure 6. HDAC8 inhibition improves the duration of BRAF inhibitor therapy. A, Responses to vemurafenib in vivo are HDAC8 dependent. Isogenic 1205Lu cell lines (EV control, HDAC8 expressing, and HDAC8 shRNA silenced) were xenografted into NOD.CB17-Prkdcscid/J mice. Tumors were allowed to engraft for 10 days and then were treated with 10 mg/kg PLX 4720 (BRAFi) orally. Data show mean tumor volume SE mean. B, Isogenic WM164 cells were xenografted and treated as in A. C, HDAC8-expressing tumors exhibited increased levels of HDAC8 and lower levels of BIM by Western blot. HDAC8 shRNA tumors were too small to analyze. D, HDAC8-expressing xenografts have increased nuclear phospho-c-JUN expression. Data show IHC analysis of HDAC8-expressing, EV control, and shHDAC8 containing 1205Lu and WM164 tumors. E, Broad-spectrum HDAC inhibition limits BRAF inhibitor failure in treatment-na€ve melanoma cells. Xenografts of 1205Lu melanoma cells were treated with vehicle, panobinostat alone (10 mg/kg every 5 days), vemurafenib alone (10 mg/kg daily), or the two drugs in combination. Tumor volumes were measured three times per week. F, HDAC8 inhibition limits BRAF inhibitor failure. Drug-na€ve 1205Lu cells were xenografted into NSG mice. After 10 days, mice were treated with vehicle, PCI-34051 alone (30 mg/kg daily), vemurafenib alone (10 mg/kg daily), or the two drugs in combination. Experiments were performed with an n of 10 for each condition. Significance was determined in A and B with t tests comparing vehicle control (VC) and BRAFi treatment for each experiment, with , P < 0.01. For E and F, significance was determined with a one-way ANOVA, followed by a post hoc t test, with #, P > 0.05 (nonsignificant); , P < 0.05; , P < 0.01.

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To better understand how HDAC8 regulates signaling in therapy. Treatment of HDAC8-silenced melanoma xenografts melanoma cells, we performed phosphoproteomic analyses of with the BRAF inhibitor PLX4720 showed them to be unable to isogenic melanoma cell line pairs and noted the emergence of a adapt to therapy. In contrast, introduction of HDAC8 into the signaling network dependent upon MAPK1 and c-Jun following same melanoma cells made them BRAF inhibitor tolerant, and the the introduction of HDAC8. These findings closely mirrored tumors grew rapidly in the presence of drug. To determine if these our previous proteomic studies that identified an EGFR, c-JUN effects could be recapitulated by small-molecule HDAC inhibi- signaling network being associated with acquired BRAF inhib- tors, we performed two experiments in which drug-na€ve mela- itor resistance (11). In functional terms, introduction of noma cells were cotreated with either a broad spectrum HDAC HDAC8 was associated with increased baseline levels of phos- inhibitor (panobinostat) and PLX4720 or an HDAC8-specific pho-ERK and the maintenance of MAPK signaling following inhibitor (PCI-03451) and PLX4720. In both cases, the combi- BRAF inhibitor treatment. It is likely that the shallower level of nation of the BRAF inhibitor and the HDAC inhibitor out per- ERK inhibition associated with HDAC8 introduction reduces formed either single agent, with particularly striking effects being drug efficacy. In the clinical setting, >90% ERK inhibition is seen for the broad-spectrum HDAC inhibitor/BRAF inhibitor required for responses in melanoma patients (36). The combination. Although significantly improved responses were HDAC8-mediated adaptation occurred upstream, at the level seen for the HDAC8 inhibitor plus the BRAF inhibitor, these were of RTK signaling, with increases noted in Ras-GTP loading and not as impressive as with panobinostat or HDAC8 silencing. phosphorylation of CRAF at S338. Ultimately, the increased Possible explanations for this difference include the potential level MAPK signaling throughput prevented the melanoma minor contribution of other HDACs to the process of therapeutic cells from being primed for cell death through a mechanism escape, or the failure of PCI-03451 to inhibit HDAC8 to the same including reduced levels of BIM expression and maintenance of extent as the HDAC8 shRNA silencing in vivo. Nevertheless, these prosurvival Mcl-1 levels (24, 37, 38). Both BIM and Mcl-1 are findings provide a strong rationale to pursue the development of known to be regulated through the MAPK pathway, with BIM in more selective and potent HDAC8 inhibitors for future evaluation particular being rapidly targeted for degradation following its as drugs that can limit phenotype switching and therapeutic phosphorylation by MAPK at Ser69 (23). Mcl-1 exerts its escape in melanoma. In support of this goal, there is already antiapoptotic activity by binding to and blocking the function evidence that melanomas with acquired BRAF–MEK inhibitor of BIM-EL and through inhibition of proapoptotic Bak/Bax. In resistance exhibit sensitivity to the broad-spectrum HDAC inhib- melanoma, Mcl-1 conveys resistance to anoikis, and its down- itor vorinostat (46), and that HDAC inhibitors can restore expres- regulation is required for the cytotoxic activity of the HSP90 sion of BIM and BMF in melanomas with acquired BRAF inhibitor inhibitor XL888 (24, 39). resistance (47). As both our proteomics and RNA-seq analyses suggested that In summary, we have shown that HDAC8 is a critical driver of a HDAC8 expression was associated with c-Jun signaling and cellular program that allows melanoma cells to rapidly adapt to Jun/AP-1–driven transcription, we next asked whether HDAC8 multiple cellular stresses, including BRAF inhibitor therapy. The mediated its effects via direct c-Jun regulation. c-Jun is a key mechanism of this adaptation is complex and involves the dea- transcriptional regulator of melanoma cells that has been cetylation of c-Jun leading to a transcriptional program that implicated in melanoma progression, phenotype switching, allows melanoma cells to rewire their signaling to maintain MAPK and therapy resistance (40–42). The expression and activation pathway activity. To date, attempts to therapeutically target c-Jun of c-Jun is subject to complex regulation at both the transcrip- and, indeed, phenotype switching in melanoma have proven to tional and the posttranslational levels. In BRAF- and NRAS- be difficult. The development of potent HDAC8 inhibitors is a mutant melanoma cells, c-Jun activation occurs as a result of a promising strategy to limit this plasticity in melanoma cells complex signaling loop dependent upon ERK-mediated GSK3 allowing therapeutic responses to be improved. and CREB phosphorylation (40). Other recent studies have tied the activation of c-Jun to decreased expression of the ERK fl target gene SPRY-4, following BRAF inhibition (43). Work in Disclosure of Potential Con icts of Interest other systems has suggested that Jun transcriptional activity J.C. Becker is advisor at 4SC, eTheRNA, and CureVac; reports receiving commercial research grant from Alcedis and IQVIA; reports receiving honoraria can be regulated through acetylation at Lys268 (25). Through from the speakers' bureau of Amgen, Sanofi,Pfizer, and Merck; and is a immunoprecipitation and site-directed mutagenesis studies, consultant/advisory board member for Sanofi, Amgen, Merck, and Pfizer. D. we here demonstrated that HDAC8 was required for deacetyla- Schadendorf reports receiving honoraria from the speakers' bureau of Novartis, tion of c-Jun at Lys273 and that the introduction of K273R BMS, MSD, Sanofi, Roche, and Pierre-Fabre and is a consultant/advisory board mutant of c-Jun mimicked the effects of HDAC8. Mechanisti- member for Novartis, BMS, Merck-EMD, Immunocore, Array, MSD, Roche, fi fl cally, it was found that the introduction of the K273R acetyl Pierre-Fabre, Sano , Philogen, In arx, SunPharma, and Hexal. E. Seto reports receiving honoraria from the speakers' bureau of Otsuka Pharmaceutical and is a mutant of c-Jun led to increased transcription of EGFR, the consultant/advisory board member for Otsuka Pharmaceutical, Institute of maintenance of ERK signaling, and the escape of the melano- Biological Chemistry, and EMD Millipore. V.K. Sondak is a consultant/advisory ma cells from BRAF inhibitor therapy. There is already evidence board member for Array, Bristol Myers Squibb, Regeneron, Polynoma, Pfizer, that c-Jun transcriptional activity can be induced in response to Genentech Roche, Novartis, and Merck. K.S.M. Smalley reports receiving com- stresses such as UV (44). Our work provides the first evidence mercial research grant from Forma Therapeutics. No potential conflicts of that HDAC8 activity is increased in responses to multiple, interest were disclosed by the other authors. diverse cellular stresses and that this turn initiates a transcriptional program that is associated with increased melanoma cell Authors' Contributions survival (29, 43, 45). Conception and design: M.F. Emmons, J.C. Becker, V.K. Sondak, J.D. Licht, In vivo models were then used to demonstrate the requirement K.S.M. Smalley for HDAC8 in the adaptation of melanomas to BRAF inhibitor Development of methodology: M.F. Emmons, J.C. Becker, K.S.M. Smalley

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Acquisition of data (provided animals, acquired and managed patients, This study was supported by SPORE grant P50 CA168536 (to K.S.M. provided facilities, etc.): M.F. Emmons, F. Fai~ao-Flores, R. Sharma, Smalley, V.K. Sondak, J.L. Messina, and Y.A. Chen), NCI R21 CA216756 (to J.L. Messina, J.C. Becker, D. Schadendorf, K.S.M. Smalley K.S.M. Smalley), Florida Department of Health 8BC03 (to K.S.M. Smalley Analysis and interpretation of data (e.g., statistical analysis, biostatistics, and J.D. Licht), and Forma Therapeutics (to K.S.M. Smalley). This work has computational analysis): M.F. Emmons, F. Fai~ao-Flores, R. Thapa, J.L. Messina, been supported in part by the Proteomics and Metabolomics Core, the J.C. Becker, V.K. Sondak, Y.A. Chen, J.D. Licht Biostatistics and Bioinformatics Core, the Tissue Core, and Flow Cytometry Writing, review, and/or revision of the manuscript: M.F. Emmons, F. Fai~ao- Core Facility at the Mofﬁtt Cancer, an NCI-designated Comprehensive Flores, R. Sharma, R. Thapa, J.L. Messina, J.C. Becker, D. Schadendorf, Cancer Center (P30-CA076292). V.K. Sondak, J.M. Koomen, Y.A. Chen, E.K. Lau, K.S.M. Smalley Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.F. Emmons, E. Seto, V.K. Sondak, The costs of publication of this article were defrayed in part by the J.M. Koomen, L. Wan payment of page charges. This article must therefore be hereby marked Study supervision: M.F. Emmons, V.K. Sondak, K.S.M. Smalley advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Other (experimental/technical guidance): E.K. Lau this fact. Acknowledgments We would like to thank Bin Fang, Ph.D., for his assistance with the phosphoproteomic experiments and Divya Bhat for her assistance with the in vivo Received January 3, 2019; revised March 1, 2019; accepted April 10, 2019; experiments. published ﬁrst April 15, 2019.

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www.aacrjournals.org Cancer Res; 79(11) June 1, 2019 2961

HDAC8 Regulates a Stress Response Pathway in Melanoma to Mediate Escape from BRAF Inhibitor Therapy

Michael F. Emmons, Fernanda Faião-Flores, Ritin Sharma, et al.

Cancer Res 2019;79:2947-2961. Published OnlineFirst April 15, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-0040

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